Seputar Kesehatan

Anaphylaxis
Definition

The life-threatening anaphylactic response of a sensitized human appears within minutes after administration of specific antigen and is manifested by respiratory distress, laryngeal edema, and/or intense bronchospasm, often followed by vascular collapse or by shock without antecedent respiratory difficulty. Cutaneous manifestations exemplified by pruritus and urticaria uwith or without angioedema are characteristic of such systemic anaphylactic reactions. Gastrointestinal manifestations include nausea, vomiting, crampy abdominal pain, and diarrhea

Predisposing Factors and Etiology

There is no convincing evidence that age, sex, race, or geographic location predisposes a human to anaphylaxis except through exposure to some immunogen. According to most studies, atopy does not predispose individuals to anaphylaxis from penicillin therapy or venom of a stinging insect but is a risk factor for allergens in food or latex.

The materials capable of eliciting the systemic anaphylactic reaction in humans include the following: heterologous proteins in the form of hormones (insulin, vasopressin, parathormone); enzymes (trypsin, chymotrypsin, penicillinase, streptokinase); pollen extracts (ragweed, grass, trees); nonpollen extracts (dust mites, dander of cats, dogs, horses, and laboratory animals); food (peanuts, milk, eggs, seafood, nuts, grains, beans, gelatin in capsules); monoclonal antibodies; occupation-related products (latex rubber products); and Hymenoptera venom (yellow jacket, yellow and baldfaced hornets, paper wasp, honey bee, imported fire ants); polysaccharides such as dextran and thiomersal as a vaccine preservative; drugs such as protamine; antibiotics (penicillins, cephalosporins, amphotericin B, nitrofurantoin, quinolones); chemotherapy agents (carboplatin, paclitaxel, doxorubicin); local anesthetics (procaine, lidocaine); muscle relaxants (suxamethonium, gallamine, pancuronium); vitamins (thiamine, folic acid); diagnostic agents (sodium dehydrocholate, sulfobromophthalein); and occupation-related chemicals (ethylene oxide), which are considered to function as haptens that form immunogenic conjugates with host proteins. The conjugating hapten may be the parent compound, a nonenzymatically derived storage product, or a metabolite formed in the host.

Pathophysiology and Manifestations

Individuals differ in the time of appearance of symptoms and signs, but the hallmark of the anaphylactic reaction is the onset of some manifestation within seconds to minutes after introduction of the antigen, generally by injection or less commonly by ingestion. There may be upper or lower airway obstruction or both. Laryngeal edema may be experienced as a “lump” in the throat, hoarseness, or stridor, while bronchial obstruction is associated with a feeling of tightness in the chest and/or audible wheezing. Patients with bronchial asthma are predisposed to severe involvement of the lower airways. Flushing with diffuse erythema and a feeling of warmth may occur. A characteristic feature is the eruption of well-circumscribed, discrete cutaneous wheals with erythematous, raised, serpiginous borders and blanched centers. These urticarial eruptions are intensely pruritic and may be localized or disseminated. They may coalesce to form giant hives, and they seldom persist beyond 48 h. A localized, nonpitting, deeper edematous cutaneous process, angioedema, may also be present. It may be asymptomatic or cause a burning or stinging sensation.

In fatal cases with clinical bronchial obstruction, the lungs show marked hyperinflation on gross and microscopic examination. The microscopic findings in the bronchi, however, are limited to luminal secretions, peribronchial congestion, submucosal edema, and eosinophilic infiltration, and the acute emphysema is attributed to intractable bronchospasm that subsides with death. The angioedema resulting in death by mechanical obstruction occurs in the epiglottis and larynx, but the process is also evident in the hypopharynx and to some extent in the trachea; on microscopic examination there is wide separation of the collagen fibers and the glandular elements; vascular congestion and eosinophilic infiltration are also present. Patients dying of vascular collapse without antecedent hypoxia from respiratory insufficiency have visceral congestion with a presumptive loss of intravascular blood volume. The associated electrocardiographic abnormalities, with or without infarction, noted in some patients may reflect a primary cardiac event mediated by mast cells or be secondary to a critical reduction in blood volume.

The angioedematous and urticarial manifestations of the anaphylactic syndrome have been attributed to release of endogenous histamine. A role for the cysteinyl leukotrienes in causing marked bronchiolar constriction seems likely. Vascular collapse without respiratory distress in response to experimental challenge with the sting of a hymenopteran was associated with marked and prolonged elevations in blood histamine and intravascular coagulation and kinin generation. The finding that patients with systemic mastocytosis and episodic vascular collapse excrete large amounts of PGD2 metabolites in addition to histamine suggests that PGD2 is also of importance in the hypotensive anaphylactic reactions. The actions of the array of mast cell–derived mediators are likely additive or synergistic at the target tissues.

Diagnosis

The diagnosis of an anaphylactic reaction depends on a history revealing the onset of the symptoms and signs within minutes after the responsible material is encountered. It is appropriate to rule out a complement-mediated immune complex reaction, an idiosyncratic response to a nonsteroidal anti-inflammatory drug (NSAID), or the direct effect of certain drugs or diagnostic agents on mast cells. Intravenous administration of a chemical mast cell–degranulating agent, including opiate derivatives and radiographic contrast media, may elicit generalized urticaria , angioedema, and a sensation of retrosternal oppression with or without clinically detectable bronchoconstriction or hypotension. Aspirin and other NSAIDs such as indomethacin, aminopyrine, and mefenamic acid may precipitate a life-threatening episode of obstruction of upper or lower airways, especially in patients with asthma, that is clinically indistinguishable from anaphylaxis but is not associated with the presence of specific IgE or elevation of blood tryptase. This syndrome, which is commonly associated with nasal polyposis, is due to inhibition of PGHS-1 with corresponding unregulated, amplified generation of the cysteinyl leukotrienes via the 5-LO/LTC4 synthase pathway. In the transfusion anaphylactic reaction that occurs in patients with IgA deficiency, the responsible specificity resides in IgG or IgE anti-IgA; the mechanism of the reaction mediated by IgG anti-IgA is presumed to be complement activation with secondary mast cell participation.

The presence of specific IgE in the blood of patients with systemic anaphylaxis has been demonstrated by passive transfer of the serum intradermally into a normal recipient, followed in 24 h by antigen challenge into the same site, with subsequent development of a wheal and flare, the Prausnitz-Küstner reaction. To avoid the hazards of transferring hepatitis or other infections, it is preferable to use the serum to seek passive sensitization of a human leukocyte suspension enriched with basophils for subsequent antigen-induced histamine release. Furthermore, radioimmunoassays using purified antigens can demonstrate specific IgE in serum of patients with anaphylactic reactions. Elevations of tryptase levels in serum implicate mast cell activation in an adverse systemic reaction and are particularly informative with episodes of hypotension during general anesthesia or when there has been a fatal outcome.

Anaphylaxis: Treatment

Early recognition of an anaphylactic reaction is mandatory, since death occurs within minutes to hours after the first symptoms. Mild symptoms such as pruritus and urticaria can be controlled by administration of 0.3 to 0.5 mL of 1:1000 (1.0 mg/mL) epinephrine SC or IM, with repeated doses as required at 5- to 20-min intervals for a severe reaction. If the antigenic material was injected into an extremity, the rate of absorption may be reduced by prompt application of a tourniquet proximal to the reaction site, administration of 0.2 mL of 1:1000 epinephrine into the site, and removal without compression of an insect stinger, if present. An IV infusion should be initiated to provide a route for administration of 2.5 mL epinephrine, diluted 1:10,000, at 5- to 10-min intervals, volume expanders such as normal saline, and vasopressor agents such as dopamine if intractable hypotension occurs. Replacement of intravascular volume due to postcapillary venular leakage may require several liters of saline. Epinephrine provides both - and -adrenergic effects, resulting in vasoconstriction, bronchial smooth-muscle relaxation, and attenuation of enhanced venular permeability. When epinephrine fails to control the anaphylactic reaction, hypoxia due to airway obstruction or related to a cardiac arrhythmia, or both, must be considered. Oxygen alone via a nasal catheter or with nebulized albuterol may be helpful, but either endotracheal intubation or a tracheostomy is mandatory for oxygen delivery if progressive hypoxia develops. Ancillary agents such as the antihistamine diphenhydramine, 50 to 100 mg IM or IV, and aminophylline, 0.25 to 0.5 g IV, are appropriate for urticaria- angioedema and bronchospasm, respectively. Intravenous glucocorticoids, 0.5–1.0 mg/kg of medrol, are not effective for the acute event but may alleviate later recurrence of bronchospasm, hypotension, or utticaria

Prevention

Prevention of anaphylaxis must take into account the sensitivity of the recipient, the dose and character of the diagnostic or therapeutic agent, and the effect of the route of administration on the rate of absorption. Beta blockers are relatively contraindicated in persons at risk for anaphylactic reactions, especially those sensitive to Hymenoptera venom or those undergoing immunotherapy for respiratory system allergy. If there is a definite history of a past anaphylactic reaction, even though mild, it is advisable to select a structurally unrelated agent. A knowledge of cross-reactivity among agents is critical since, for example, cephalosporins have a cross-reactive ring structure with the penicillins. A prick or scratch skin test should precede an intradermal skin test, since the latter has a higher risk of causing anaphylaxis. These tests should be performed before the administration of certain materials that are likely to elicit anaphylactic reactions, such as allergenic extracts. Skin testing for antibiotics or chemotherapeutic agents should be performed only on patients with a positive clinical history consistent with an IgE-mediated reaction and in imminent need of the antibiotic in question; skin testing is of no value for non-IgE-mediated eruptions. With regard to penicillin, two-thirds of patients with a positive reaction history and positive skin tests to benzylpenicilloyl-polylysine (BPL) and/or the minor determinant mixture (MDM) of benzylpenicillin products experience allergic reactions with treatment, and these are almost uniformly of the anaphylactic type in those patients with minor determinant reactivity. Even patients without a history of previous clinical reactions have a 2–6% incidence of positive skin tests to the two test materials, and about 3 per 1000 with a negative history experience anaphylaxis with therapy, with a mortality of about 1 per 100,000.

Desensitization with most antibiotics and other classes of therapeutic agents can proceed by the IV, SC, or oral route. Typically, graded quantities of the drug are given by the selected route using double doses until a therapeutic dosage is achieved. Due to the risk of systemic anaphylaxis during the course of desensitization, such a procedure should be performed only in a setting in which resuscitation equipment is at hand and an IV line is in place. It is critical to give the therapeutic agent at regular intervals throughout the treatment period to prevent the reestablishment of a significant pool of sensitized cells.

A different form of protection involves the development of blocking antibody of the IgG class, which is protective against Hymenoptera venom–induced anaphylaxis by interacting with antigen so that less reaches the sensitized tissue mast cells. The maximal risk for systemic anaphylactic reactions in persons with Hymenoptera sensitivity occurs in association with a currently positive skin test. Although there is only low-grade cross-reactivity between honey bee and yellow jacket venoms, there is a high degree of cross-reactivity between yellow jacket venom and the rest of the vespid venoms (yellow or baldfaced hornets and wasps). Prevention involves modification of outdoor activities to exclude bare feet, wearing perfumed toiletries, eating in areas attractive to insects, clipping hedges or grass, and hauling away trash or fallen fruit. As with each anaphylactic sensitivity, the individual should wear an informational bracelet and have immediate access to an unexpired auto-injectable epinephrine kit. The limitations of lifestyle and the psychological duress can be addressed by venom immunotherapy. Although it has been recommended that venom therapy be continued indefinitely or until the skin and specific serum IgE tests are unremarkable, there is evidence that 5 years of treatment induces a state of resistance to sting reactions that is independent of serum levels of specific IgG or IgE. For children with a systemic reaction limited to skin, the likelihood of progression to more serious respiratory or vascular manifestations is low, and thus immunotherapy is not recommended

copyright by Horrison Principles

urticaria.

Pengertian

Anaphylaxis (Yunani, Ana = jauh dari dan phlah reaksi alergi umum dengan efek pada beberapa sistem organ terutama kardiovaskularylaxis = perlindungan). Anafilaksis berarti Menghilangkan perlindungan. Anafilaksis ada, respirasi, kutan dan gastro intestinal yang merupakan reaksi imunologis yang didahului dengan terpaparnya alergen yang sebelumnya sudah tersensitisasi. Syok anafilaktik(= shock anafilactic ) adalah reaksi anafilaksis yang disertai hipotensi dengan atau tanpa penurunan kesadaran. Reaksi Anafilaktoid adalah suatu reaksi anafilaksis yang terjadi tanpa melibatkan antigen-antibodi kompleks. Karena kemiripan gejala dan tanda biasanya diterapi sebagai anafilaksis.

Penyebab

  • Alergen
  • makanan, obat-obatan, bisa atau racun serangga dan alergen lain yang tidak bisa di golongkan.
  • Allergen penyebab Anafilaksis Makanan
  • Krustasea: Lobster, udang dan kepiting
  • Moluska : kerang Ikan Kacang-kacangan dan biji-bijian Buah beri Putih telur Susu
  • Obat Hormon : Insulin, PTH, ACTH, Vaso-presin, Relaxin
  • Enzim : Tripsin,Chymotripsin, Penicillinase, As-paraginase Vaksin dan Darah
  • Toxoid : ATS, ADS, SABU Ekstrak alergen untuk uji kulit Dextran
  • Antibiotika: Penicillin, Streptomisin, Cephalosporin, Tetrasiklin, Ciprofloxacin, Amphotericin B, Nitrofurantoin.
  • Agent diagnostik-kontras: Vitamin B1, Asam folat Agent
  • anestesi: Lidocain, Procain,

Lain-lain: Barbiturat, Diazepam, Phenitoin, Protamine, Aminopyrine, Acetil cystein , Codein, Morfin, Asam salisilat dan HCT Bisa serangga Lebah Madu, Jaket kuning, Semut api Tawon (Wasp). Lain-lain Lateks, Karet, Glikoprotein

Tanda dan gejala

Anafilaksis merupakan reaksi sistemik, gejala yang timbul juga menyeluruh.

Gejala permulaan: Sakit Kepala, Pusing, Gatal dan perasaan panas Sistem Organ Gejala Kulit Eritema, urticaria, angioedema, conjunctivitis, pallor dan kadang cyanosis Respirasi Bronkospasme, rhinitis, edema paru dan batuk, nafas cepatdan pendek, terasa tercekik karena edema epiglotis, stridor, serak, suara hilang, wheezing, dan obstruksi komplit. Cardiovaskular Hipotensi, diaphoresis, kabur pandangan, sincope, aritmia dan hipoksia Gastrintestinal Mual, muntah, kram perut, diare, disfagia, inkontinensia urin SSP, Parestesia, konvulsi dan kom Sendi Arthralgia Haematologi darah, trombositopenia, DIC

Patofisiologi

Anafilaksis merupakan reaksi sistemik, gejala yang timbul juga menyeluruh.

Gejala permulaan: Sakit Kepala, Pusing, Gatal dan perasaan panas Sistem Organ Gejala Kulit Eritema, urticaria, angioedema, conjunctivitis, pallor dan kadang cyanosis Respirasi Bronkospasme, rhinitis, edema paru dan batuk, nafas cepatdan pendek, terasa tercekik karena edema epiglotis, stridor, serak, suara hilang, wheezing, dan obstruksi komplit. Cardiovaskular Hipotensi, diaphoresis, kabur pandangan, sincope, aritmia dan hipoksia Gastrintestinal Mual, muntah, kram perut, diare, disfagia, inkontinensia urin SSP, Parestesia, konvulsi dan kom Sendi Arthralgia Haematologi darah, trombositopenia, DIC

Syok Anafilaktik secara garis besar

Jika seseorang sensitif terhadap suatu antigen dan kemudian terjadi kontak lagi terhadap antigen tersebut, akan timbul reaksi hipersensitivitas. Antigen yang bersangkutan terikat pada antibodi dipermukaan sel mast sehingga terjadi degranulasi, pengeluaran histamin, dan zat vasoaktif lain. Keadaan ini menyebabkan peningkatan permeabilitas dan dilatasi kapiler menyeluruh. Terjadi hipovolemia relatif karena vasodilatasi yang mengakibatkan syok, sedangkan peningkatan permeabilitas kapiler menyebabkan udem. Pada syok anafilaktik, bisa terjadi bronkospasme yang menurunkan ventilasi.

Penatalaksanaan

  1. Hentikan obat/identifikasi obat yang diduga menyebabkan reaksi anafilaksis
  2. Segera baringkan penderita pada alas yang keras. Kaki diangkat lebih tinggi dari kepala untuk meningkatkan aliran darah balik vena, dalam usaha memperbaiki curah jantung dan menaikkan tekanan darah.
  3. Penilaian A, B, C dari tahapan resusitasi jantung paru, yaitu:

1) Airway ‘penilaian jalan napas’. Jalan napas harus dijaga tetap bebas, tidak ada sumbatan sama sekali. Untuk penderita yang tidak sadar, posisi kepala dan leher diatur agar lidah tidak jatuh ke belakang menutupi jalan napas, yaitu dengan melakukan ekstensi kepala, tarik mandibula ke depan, dan buka mulut.

2) Breathing support, segera memberikan bantuan napas buatan bila tidak ada tanda-tanda bernapas, baik melalui mulut ke mulut atau mulut ke hidung. Pada syok anafilaktik yang disertai udem laring, dapat mengakibatkan terjadinya obstruksi jalan napas total atau parsial. Penderita yang mengalami sumbatan jalan napas parsial, selain ditolong dengan obat-obatan, juga harus diberikan bantuan napas dan oksigen. Penderita dengan sumbatan jalan napas total, harus segera ditolong dengan lebih aktif, melalui intubasi endotrakea, krikotirotomi, atau trakeotomi.

3) Circulation support, yaitu bila tidak teraba nadi pada arteri besar (arteri karotis, atau aarteri. femoralis), segera lakukan kompresi jantung luar.

  1. Adrenalin 1:1000, 0,3–0,5 ml SC/IM lengan atas , paha, sekitar lesi pada venom, Dapat diulang 2-3 x dengan selang waktu 15-30 menit, Pemberian IV pada stadium terminal /pemberian dengan dosis1 ml gagal , 1:1000 dilarutkan dalam 9 ml garam faali diberikan 1-2 ml selama 5-20 menit (anak 0,1 cc/kg BB)
  2. Diphenhidramin IV ( ± 20 detik ), IM ( 1 – 2 mg/kg BB ) 50 – 100 mg
  3. Dalam hal terjadi spasme bronkus di mana pemberian adrenalin kurang memberi respons, dapat ditambahkan aminofilin 5–6 mg/kgBB intravena dosis awal yang diteruskan 0.4–0.9 mg/kgBB/menit dalam cairan infus.
  4. Dapat diberikan kortikosteroid, misalnya hidrokortison 100 mg atau deksametason 5–10 mg intravena sebagai terapi penunjang untuk mengatasi efek lanjut dari syok anafilaktik atau syok yang membandel.
  5. Bila tekanan darah tetap rendah, diperlukan pemasangan jalur intravena untuk koreksi hipovolemia akibat kehilangan cairan ke ruang ekstravaskular sebagai tujuan utama dalam mengatasi syok anafilaktik. Pemberian cairan akan meningkatkan tekanan darah dan curah jantung serta mengatasi asidosis laktat. Pemilihan jenis cairan antara larutan kristaloid dan koloid tetap merupakan perdebatan didasarkan atas keuntungan dan kerugian mengingat terjadinya peningkatan permeabilitas atau kebocoran kapiler. Pada dasarnya, bila memberikan larutan kristaloid, maka diperlukan jumlah 3–4 kali dari perkiraan kekurangan volume plasma. Biasanya, pada syok anafilaktik berat diperkirakan terdapat kehilangan cairan 20–40% dari volume plasma. Sedangkan bila diberikan larutan koloid, dapat diberikan dengan jumlah yang sama dengan perkiraan kehilangan volume plasma. Tetapi, perlu dipikirkan juga bahwa larutan koloid plasma protein atau dextran juga bisa melepaskan histamin. Pemberian vasopresor pada hipotensi menetap
  6. Dalam keadaan gawat, sangat tidak bijaksana bila penderita syok anafilaktik dikirim ke rumah sakit, karena dapat meninggal dalam perjalanan. Kalau terpaksa dilakukan, maka penanganan penderita di tempat kejadian sudah harus semaksimal mungkin sesuai dengan fasilitas yang tersedia dan transportasi penderita harus dikawal oleh dokter. Posisi waktu dibawa harus tetap dalam posisi telentang dengan kaki lebih tinggi dari jantung.
  7. Kalau syok sudah teratasi, penderita jangan cepat-cepat dipulangkan, tetapi harus diawasi/diobservasi dulu selama kurang lebih 4 jam. Sedangkan penderita yang telah mendapat terapi adrenalin lebih dari 2–3 kali suntikan, harus dirawat di rumah sakit semalam untuk observasi.

Penanggulangan syok anafilaktik memerlukan tindakan cepat sebab penderita berada pada keadaan gawat. Sebenarnya, pengobatan syok anafilaktik tidaklah sulit, asal tersedia obat-obat emerjensi dan alat bantu resusitasi gawat darurat serta dilakukan secepat mungkin. Hal ini diperlukan karena kita berpacu dengan waktu yang singkat agar tidak terjadi kematian atau cacat organ tubuh menetap.

Kalau terjadi komplikasi syok anafilaktik setelah kemasukan obat atau zat kimia, baik peroral maupun parenteral, maka tindakan yang perlu dilakukan, adalah:

Pencegahan

  • Mencegah reaksi ulang
  • Anamnesa penyakit alergi px sebelum terapi diberikan (obat,makanan,atopik)
  • Lakukan skin test sebelum memberikan obat injeksi
  • Encerkan obat bila pemberian dengan SC//IM/IV dan observasi selama pemberian
  • Catat obat pasien pada status yang menyebabkan alergi
  • Hindari obat-obat yang sering menyebabkan syok anafilaktik.
  • Edukasi pasien supaya menghindari makanan atau obat yang menyebabkan alergi
  • Bersiaga selalu bila melakukan injeksi dengan emergency kit

 

DaftarPustaka

Rab, Prof.Dr. H tabrani. Pengatasan shock, EGC Jakarta 2000, 153-161

Panduan Gawat Darurat, Jilid I, FKUI, Penerbit FKUI Jakarta 2000, 17-18

Ho, Mt, Luce JM, Trunkey, DD, Salber PR, Mills J, Resusitasi KardioPulmoner dan Syok,

EGC Jakarta 1990 : 76-78

Purwadianto, A, Sampurna, B, Kedaruratan Medik, Bina Rupa Aksara, Jakarta 2000, 56-57

Effendi, C, Anaphylaxis dalam PKB XV , Lab. Ilmu Penyakit Dalam FKUA/ RSUD Dr. Soetomo, 2000 : 91-99

Fauci,dkk,HARRISON,Principles of INTERNAL MEDICINE,17th Editiion

 

 

HYPOPHYSEOTROPIC HORMONES AND NEUROENDOCRINE AXES

Kronenberg: Williams Textbook of Endocrinology, 11th ed.
Copyright © 2008 Saunders, An Imprint of Elsevier

HYPOPHYSEOTROPIC HORMONES AND NEUROENDOCRINE AXES

With the demonstration by the first half of the 1900s that pituitary secretion was controlled by hypothalamic hormones released into the portal circulation, the search was on for the hypothalamic-releasing factors. The search for hypothalamic neurohormones with anterior pituitary regulating properties focused on extracts of stalk median eminence, neural lobe, and hypothalamus from sheep and pigs. To give some idea of the Herculean nature of this effort, approximately 250,000 hypothalamic fragments were required to purify and characterize the first such factor, TRH.[9] Such hypophyseotropic substances were initially called releasing factors but are now more commonly called releasing hormones.

All of the hypothalamic-pituitary regulating hormones are peptides with the notable exception of dopamine, which is a biogenic amine and the principal prolactin-inhibiting factor (PIF) (see Table 7-2 ). All are available for clinical investigations or diagnostic tests, and therapeutic analogues for dopamine, GnRH, and somatostatin are widely prescribed.

In addition to regulating hormone release, some hypophyseotropic factors control pituitary cell differentiation and proliferation and hormone synthesis. Somatostatin and dopamine are inhibitory, and some act on more than one pituitary hormone. For example, TRH is a potent releaser of prolactin (PRL) and of TSH, and under some circumstances releases corticotropin (adrenocorticotropic hormone [ACTH]) and growth hormone (GH). GnRH releases both LH and follicle-stimulating hormone (FSH). Somatostatin inhibits the secretion of GH, TSH, and a wide variety of nonpituitary hormones. The principal inhibitor of PRL secretion, dopamine, also inhibits secretion of TSH, gonadotropins and, under certain conditions, GH. Dual control is exerted by the interaction of inhibitory and stimulatory hypothalamic hormones. For example, somatostatin interacts with growth hormone–releasing hormone (GHRH) and TRH to control secretion of GH and TSH, respectively, and dopamine interacts with prolactin-releasing factors (PRFs) to regulate PRL secretion. Some hypothalamic hormones act synergistically; for example, CRH and vasopressin act together to regulate the release of pituitary ACTH.

Secretion of the releasing hormones in turn is regulated by neurotransmitters and neuropeptides released by a complex array of neurons synapsing with hypophyseotropic neurons. Control of secretion is also exerted through feedback control by hormones such as glucocorticoids, gonadal steroids, thyroid hormone, anterior pituitary hormones (short-loop feedback control), and hypophyseotropic factors themselves (ultrashort-loop feedback control).

The distribution of the hypophyseotropic hormones is not limited to the hypothalamus. Most are produced in nonhypophyseotropic hypothalamic neurons, in extrahypothalamic regions of the brain, and in peripheral organs where they mediate functions unrelated to pituitary regulation (e.g., effects on behavior or homeostasis). A majority of the peptides, hormones, and neurotransmitters involved in the regulation of hypothalamic-pituitary control transduce their signals through members of the extensive G protein–coupled receptor family ( Table 7-3 ).

TABLE 7-3 — RECEPTORS FOR NEUROTRANSMITTERS AND NEUROPEPTIDES INVOLVED IN HYPOTHALAMIC-PITUITARY CONTROL AND NEUROENDOCRINE HOMEOSTASIS
Group and Ligand Receptor Family Receptor Protein[*] Receptor Gene Mode of Action[+]
Classic Neurotransmitters
Catecholamines (NE, E) α1-Adrenoreceptors ADA1A (α1A) ADRA1A 7-TM, Gq/11
ADA1B (α1B) ADRA1B 7-TM, Gq/11
ADA1D (α1D) ADRA1D 7-TM, Gq/11
α2-Adrenoreceptors ADA2A (α2A) ADRA2A 7-TM, Gi/o
ADA2B (α2B) ADRA2B 7-TM, Gi/o
ADA2C (α2C) ADRA2C 7-TM, Gi/o
β-Adrenoreceptors ADRB1 (β1) ADRB1 7-TM, GS
ADRB3 (β2) ADRB2 7-TM, GS
ADRB3 (β3) ADRB3 7-TM, GS
Serotonin (5-OH-tryptamine) 5-HT1 receptors 5HT1A (5HT1A-α) HTR1A 7-TM, Gi/o
5HT1B (5HT1D-β) HTR1B 7-TM, Gi/o
5HT1D (5HT1D-α) HTR1D 7-TM, Gi/o
5HT1E HTR1E 7-TM, Gi/o
5-HT2 receptors 5HT2A HTR2A 7-TM, Gq/11
5HT2B HTR2B 7-TM, Gq/11
5HT2C (5HT1C) HTR2C 7-TM, Gq/11
5-HT3 receptors 5HT3 Pentamer Cation flux
Subunit genes: HTR3A, HTR3B
5-HT4 receptors 5HT4R HTR4 7-TM, GS
Dopamine Dopamine receptors DRD1 (D1-R, D1A) DRD1 7-TM, GS
DRD2 (D2-R) DRD2 7-TM, Gi/o
DRD3 (D3-R) DRD3 7-TM, Gi/o
DRD4 (D4-R, D2C) DRD4 7-TM, Gi/o
DRD5 (D5-R, D1B) DRD5 7-TM, GS
Histamine Histamine receptors HRH1 (H1-R) HRH1 7-TM, Gq/11
HRH2 (H2-R) HRH2 7-TM, GS
HRH3 (H3-R) HRH3 7-TM, Gi/o
Melatonin Melatonin receptors MT1RA (Mel1AR, MT1) MTNR1A 7-TM, Gi/o, PLC-β
MT1RB (Mel1BR, MT2) MTNR1B 7-TM, Gi/o, Gq/11
MT3 (quinone reductase 2) NQO2 cytosolic enzyme
Trace amines Trace amine receptor TAAR1 (TaR-1) TAAR1 7-TM, ••
Acetylcholine Muscarinic receptors ACM1 (M1) CHRM1 7-TM, Gq/11
ACM2 (M2) CHRM2 7-TM, Gq/11
ACM3 (M3) CHRM3 7-TM, Gq/11
ACM4 (M4) CHRM4 7-TM, Gi/o
ACM5 (M5) CHRM5 7-TM, Gq/11
Nicotinic receptors ACHA-P, ACH1-7 Pentamer Cation flux
Subunit genes: CHRNA, CHRNB
Glutamate Ionotropic receptors NMDA (NR1, NR2A-D) Oligomer Cation flux
NMZ1 subunit gene: GRIN1 (NMDAR1)
AMPA (GluR1-4) Oligomer Cation flux
GRIA1 subunit gene: GRIA1 (GLUR1)
Kainate (GluR5-7, KA-1/2) Oligomer Cation flux
LK1 subunit gene: GRIK1 (GLUR5)
Metabotropic receptors MGR1 (mGluR1) GRM1 7-TM, Gq/11
MGR2 (mGluR2) GRM2 7-TM, Gi/o
MGR3 (mGluR3) GRM3 7-TM, Gi/o
MGR4 (mGluR4) GRM4 7-TM, Gi/o
MGR5 (mGluR5) GRM5 7-TM, Gq/11
MGR6 (mGluR6) GRM6 7-TM, Gi/o
MGR7 (mGluR7) GRM7 7-TM, Gi/o
γ-aminobutyric acid (GABA) Ionotropic GAA-E (GABA-A-R) Pentamer [Cl-] ion flux
GAA1 (α1) subunit gene GABRA1
Heterodimeric GABR1 (GABA-B-R1) GABBR1 7-TM, Gi/o
GABR2 (GABA-B-R2) GABBR2 7-TM, Gi/o
Neuropeptides
Neurohypophyseal hormones
Vasopressin Vasopressin receptors V1AR (V1a) AVPR1A 7-TM, Gq/11
V1BR (V1b, V3) AVPR1B 7-TM, Gq/11
V2R (ADH-R) AVPR2 7-TM, GS
Oxytocin Oxytocin receptor OXYR (OT-R) OXTR 7-TM, Gq/11
Hypophyseotropic hormones
TRH TRH receptor TRFR (TRH-R) TRHR 7-TM, Gq/11
GHRH GHRH receptor GHRHR (GRFR) GHRHR 7-TM, GS
GHRP/Ghrelin GHS receptor GHSR (GHRP-R) GHSR 7-TM, Gq/11
GnRH GnRH receptor GNRHR (GnRH-R) GNRHR 7-TM, Gq/11
GRH/Urocortin CRH receptors CRFR1 (CRH-R1) CRHR1 7-TM, GS
CRFR2 (CRH-R2) CRHR2 7-TM, GS
Somatostatin/Cortistatin Somatostatin receptors SSR1 (SS1R, SRIF-2) SSTR1 7-TM, Gi/o
SSR2 (SS2R, SRIF-1) SSTR2 7-TM, Gi/o
SSR3 (SS3R, SSR-28) SSTR3 7-TM, Gi/o
SSR4 (SS4R) SSTR4 7-TM, Gi/o
SSR5 (SS5R) SSTR5 7-TM, Gi/o
Endogenous opioid peptides
β-endorphin Mu opioid receptor OPRM (μ, MOR-1) OPRM1 7-TM, Gi/o
Enkephalin Delta opioid receptor OPRD (δ, DOR-1) OPRD1 7-TM, Gi/o
Dynorphin Kappa opioid receptor OPRK (κ, KOR-1) OPRK1 7-TM, Gi/o
Nociceptin/OFQ OFQ opioid receptor OPRX (KOR-3) OPRL1 7-TM, Gi/o
Melanocortin peptides
MSH MSH receptor MSHR (MC1-R) MC1R 7-TM, GS
ACTH ACTH receptor ACTHR (MC2-R) MC2R 7-TM, GS
γMSH, MSH Melanocortin receptor 3 MC3R (MC3-R) MC3R 7-TM, GS
MSH, βMSH Melanocortin receptor 4 MC4R (MC4-R) MC4R 7-TM, GS
MSH Melanocortin receptor 5 MC5R (MC5-R) MC5R 7-TM, GS
Tachykinins (neurokinins)
Substance P Neurokinin receptors NK1R (SPR) TACR1 7-TM, Gi/o
Substance K NK2R (SKR) TACR2 7-TM, Gi/o
Neurokinin B NKR3 (NKR) TACR3 7-TM, Gi/o
Vasoactive peptides
Angiotensin II Angiotensin receptors AGTR1 (AT1) AGTR1 7-TM, Gq/11
AGTR2 (AT2) AGTR2 7-TM, Gi/o
Atrial natriuretic peptide ANP receptors ANPRA (NPR-A) NPR1 cGMP, 1-TM
ANPRB (NPR-B) NPR2 cGMP, 1-TM
Endothelin Endothelin receptors ENDRA (ETA-R) EDNRA 7-TM, Gq/11
ENDRB (ETB-R) EDNRB 7-TM, Gq/11
Miscellaneous neuropeptides
CART No receptor identified •• 7-TM, Gi/o
Orexin/hypocretin Orexin receptors OX1R (HCRTR-1) HCRTR1 7-TM, many
OX2R (HCRTR-2) HCRTR2 7-TM, many
Melanin-concentrating hormone MCH receptor MCHR1 (GPCR24) MCHR1 7-TM, Gi/q
Prolactin-releasing peptide PRP receptor PRLHR (GPCR10) PRLHR 7-TM, Gi/o/q
Kisspeptins/Metastin Kisspeptin receptor KISSR (GPCR54) KISS1R 7-TM, Gq/11
Neuromedin U Neuromedin receptors NMUR1 (GPCR66) NMUR1 7-TM, Gq/11
NUMR2 NMUR2 7-TM, Gq/11
Neurotensin Neurotensin receptor NTR1 (NTRH) NTSR1 7-TM, Gq/11
PACAP PACAP receptor PACR (PACAP-R-1) ADCYAR1R1 7-TM, GS
Vasoactive intestinal peptide VIP receptors VIPR1 (PACAP-R-2) VIPR1 7-TM, GS
VIPR2 (PACAP-R-3) VIPR3 7-TM, GS
Galanin/GALP Galanin receptors GALR1 (GAL1-R) GALR1 7-TM, Gi/o
GALR2 (GAL2-R) GALR2 7-TM, Gi/o
GALR3 (GAL3-R) GALR3 7-TM, Gi/o
Glucagon-like peptide GLP receptor GLP1R GLP1R 7-TM, GS
CCK/Gastrin CCK receptors CCKAR (CCK1-R) CCKAR 7-TM, Gq/11
GASR (CCK2-R) CCKBR 7-TM, Gq/11
Neuropeptide Y NPY/PYY/PP receptors NPY1R (NPY-Y1) NPY1R 7-TM, Gi/o
PYY (3-32) NPY2R (NPY-Y2) NPY2R 7-TM, Gi/o
Pancreatic polypeptide NPY4R (PP1) PPYR1 7-TM, Gi/o
Neuropeptide Y NPY5R (NPY-Y5) NPY5R 7-TM, Gi/o
Other
Cannabinoid Cannabinoid receptor CNR1 (CB1) CNR1 7-TM, Gi/o

AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazdeproprionic acid; CART, cocaine and amphetamine responsive transcript; CCK, cholecystokinin; CRH, corticotropin-releasing hormone; E, epinephrine; GALP, galanin-like peptide; GHRH, growth hormone-releasing hormone; GnRH, gonadotropin-releasing hormone; NE, norepinephrine; NMDA, N-methyl-d-aspartate; OFQ, orphanin FQ; PACAP, pituitary adenylyl cyclase activating peptide; PYY, peptide YY; TRH, thyrotropin-releasing hormone.

* Receptors cited are human. Swiss-Prot identifiers and alternative names (in parentheses) are provided for each receptor and were obtained with the use of the GPCRDB information system (http://www.gpcr.org/7tm/) described in: Horn F, Bettler E, Oliveira L, Campagne F, Cohen FE, Vriend G (2003) GPCRDB information system for G protein-coupled receptors. Nucleic Acids Res. 31:294-297.
+ The mode of action designation is oversimplified. It is common for seven transmembrane (7-TM) G-protein coupled receptors (GPCR) to interact with multiple different G-protein complexes depending on the specific cell. Gi/o, GPCR coupled to the Gi/o family, inhibits adenylyl cyclase and decreases intracellular cAMP, opens K+ channels and closes Ca2+ channels; Gq/11, GPCR coupled to the Gq/11 family, stimulates phosphoinositol cascade; GS, GPCR coupled to the GS family, stimulates adenylyl cyclase and increases intracellular cAMP; PLC-ß, GPCR coupled to G protein that activates phospholipase Cß (PLC-ß); cGMP, Guanylate cyclase activity intrinsic to these 1 trans-membrane pass receptors.

Feedback Concepts in Neuroendocrinology

In order to understand the regulation of each hypothalamic-pituitary-target organ axis, it is important to understand some basic concepts of homeostatic systems. A simplified account of feedback control in relation to neuroendocrine regulation is presented in this section. [83] [84] [85] Hormonal systems form part of a feedback loop in which the controlled variable (generally the blood hormone level or some biochemical surrogate of the hormone) determines the rate of secretion of the hormone. In negative feedback systems, the controlled variable inhibits hormone output, and in positive feedback control systems, the controlled variable increases hormone secretion. Both negative and positive endocrine feedback control systems can be part of a closed loop, in which regulation is entirely restricted to the interacting regulatory glands, or an open loop, in which the nervous system influences the feedback loop. All pituitary feedback systems have nervous system inputs that either alter the set-point of the feedback control system or introduce open-loop elements that can influence or override the closed-loop control elements.

In engineering formulations of feedback, three controlled variables can be identified: a sensing element that detects the concentration of the controlled variable, a reference input that defines the proper control levels, and an error signal that determines the output of the system. The reference input is the set-point of the system.

Hormonal feedback control systems resemble engineering systems in that the concentration of the hormone in the blood (or some function of the hormone) regulates the output of the controlling gland. Hormonal feedback differs from engineering systems in that the sensor element and the reference input element are not readily distinguishable. The set-point of the controlled variable is determined by a complex cascade beginning with the kinetics of binding to a receptor and the activities of successive intermediate messengers. Sophisticated models incorporating control elements, compartmental analysis, and hormone production and clearance rates exist for many systems.

Endocrine Rhythms

Virtually all functions of living animals (regardless of their position on the evolutionary scale) are subject to periodic or cyclic changes, many of which are influenced mainly by the nervous system (see Table 7-4 for definitions). [86] [87] [88] [89] Most periodic changes are free-running; that is, they are intrinsic to the organism, independent of the environment, and driven by a biologic “clock.”

TABLE 7-4 — TERMS USED TO DESCRIBE CYCLIC ENDOCRINE PHENOMENA
Period Length of the cycle
Circadian Around a day (24 h)
Diurnal Exactly a day
Ultradian Less than a day, i.e., minutes or hours
Infradian Longer than a day, i.e., month or year
Mean Arithmetic mean of all values within a cycle
Range Difference between the highest and lowest values
Nadir Minimal level (inferred from mathematical curve fitting calculations)
Acrophase Time of maximal levels (inferred from curve fitting)
Zeitgeber “Time-giver” (German), the external cue, usually the light-dark cycle that synchronizes endogenous rhythms
Entrainment The process by which an endogenous rhythm is regulated by a zeitgeber
Phase shift Induced change in an endogenous rhythm
Intrinsic clock Neural structures that possess intrinsic capacity for spontaneous rhythms; for circadian rhythms these are located in the suprachiasmatic nucleus

Adapted from Van Cauter E, Turek FW. Endocrine and other biological rhythms. In DeGroot LJ, ed. Endocrinology, 3rd ed. Philadelphia: WB Saunders, 1995:2497-2548.

Most free-running rhythms are coordinated (entrained) by external signals (cues), such as light-dark changes, meal patterns, cycles of the lunar periods, or the ratio of the length of day to the length of night. External signals of this type (zeitgeber or “time givers”) do not bring about the rhythm but provide the synchronizing time cue. Many endogenous rhythms have a period of approximately 24 hours (circadian [around a day] or diurnal rhythms). Circadian changes follow an intrinsic program that is about 24 hours long, whereas diurnal rhythms can be either circadian or dependent on shifts in light and dark. Rhythms that occur more frequently than once a day are ultradian. Infradian rhythms have a period longer than 1 day, as in the approximately 27-day human menstrual cycle and the yearly breeding patterns of some animals.

Most endocrine rhythms are circadian ( Fig. 7-7 ). The secretion of GH and PRL in humans is maximal shortly after the onset of sleep, and that of cortisol is maximal between 2 and 4 am. TSH secretion is lowest in the morning between 9 am and 12 noon and maximal between 8 pm and midnight. Gonadotropin secretion in adolescents is increased at night. Superimposed on the circadian cycle are ultradian bursts of hormone secretion. LH secretion during adolescence is characterized by rapid, high-amplitude pulsations at night, whereas in sexually mature individuals secretory episodes are lower in amplitude and occur throughout the 24 hours. GH, ACTH, and PRL are also secreted in brief, fairly regular pulses. The short-term fluctuations in hormonal secretion have important functional significance. In the case of LH, the normal endogenous rhythm of pituitary secretion reflects the pulsatile release of GnRH. The period of approximately 90 minutes between the peak of pulses corresponds to the optimal timing to induce maximal pituitary stimulation. Episodic secretion of GH also enhances its biopotency, but for many rhythms, the function is not clear. Most homeostatic activities are also rhythmic, including body temperature, water balance, blood volume, sleep, and activity. [90] [91]

Assessment of endocrine function must take into account the variability of hormone levels in the blood, and appropriately obtained samples at different times of day or night may provide useful dynamic indicators of hypothalamic-pituitary function. For example, the loss of diurnal rhythm of GH and ACTH secretion may be an early sign of hypothalamic dysfunction. Furthermore, the optimal timing for the administration of glucocorticoids to suppress ACTH secretion (as in therapy for congenital adrenal hyperplasia) must take into account the varying suppressibility of the axis at different times of day.

The best understood neural structures responsible for circadian rhythms are the SCN, paired structures in the anterior hypothalamus above the optic chiasm. [87] [91] In addition to the retinohypothalamic projection from the retina described earlier, the SCN receives neuronal input from many nuclei. Individual cells of the SCN have an intrinsic capacity to oscillate in a circadian pattern,[92] and the nucleus is organized to permit many reciprocal neuron-neuron interactions through direct synaptic contacts. It is especially rich in neuropeptides, including somatostatin, VIP, NPY, and neurotensin, and microinjections of pancreatic polypeptide into the SCN reset the timing cycle of some circadian rhythms in hamsters. The SCN also responds to the pineal hormone melatonin through melatonin receptors. [64] [71] Interestingly, recent studies have indicated that intrinsic pacemaker function is not unique to neurons of the SCN. Circadian oscillators are also found in multiple peripheral tissues.[91]

Metabolic changes in the SCN, such as increased uptake of 2-deoxyglucose and an increased level of VIP, accompany circadian rhythms. This nucleus projects to the pineal gland indirectly via the PVH and the autonomic nervous system (see earlier section on the pineal gland) and regulates its activity.[87] However, the bulk of SCN outflow occurs in a trunk coursing dorsal-laterally through the ventral subparaventricular zone and terminating in the dorsal medial nucleus of the hypothalamus. Polysynaptic pathways involving these latter structures are responsible for the actions of the SCN to produce the circadian rhythms in thermoregulation, glucocorticoid secretion, sleep, arousal, and feeding.[87]

Circadian rhythms during fetal life are regulated by maternal circadian rhythms.[93] Circadian changes can be detected 2 to 3 days before birth, and SCN from fetuses of this age display spontaneous rhythmicity in vitro. Maternal regulation of fetal circadian rhythms may be mediated by circulating melatonin or by cyclic changes in the food intake of the mother. The timing of the circadian pacemaker can be shifted in humans by the administration of triazolam, a short-acting benzodiazepine, or melatonin, as described earlier, or by altered patterns of intense illumination.[76]

Thyrotropin-Releasing Hormone

Chemistry and Evolution

TRH, the smallest known peptide-releasing hormone, is the tripeptide pyroGlu-His-Pro-NH2. Six copies of the TRH peptide sequence are encoded within the human TRH pre-prohormone gene ( Fig. 7-8 ).[94] The rat pro-TRH precursor contains five TRH peptide repeats flanked by dibasic residues (Lys-Arg or Arg-Arg), along with seven or more non-TRH peptides.[95] Two prohormone convertases, PC1 and PC2, cleave the TRH tripeptides at the dibasic residues within the regulated secretory pathway. Carboxypeptidase E then removes the dibasic residues, leaving the sequence Gln-His-Pro-Gly. This peptide is then amidated at the C-terminus by peptidylglycine α-amidating monooxygenase (PAM), with Gly acting as the amide donor. The amino-terminal pyro-Glu residue results from cyclization of the Gln.

Although the TRH tripeptide is the only established hormone encoded within its large prohormone, rat pro-TRH yields seven additional peptides that have unique tissue distributions.[96] Several biologic activities of these peptides have been observed: pro-TRH(160-169) may be a hypophyseotropic factor because it is released from hypothalamic slices and potentiates the TSH-releasing effects of TRH. Pro-TRH(178-199) is also released from the median eminence and appears to inhibit ACTH release. TRH is a phylogenetically ancient peptide, which has been isolated from primitive vertebrates such as the lamprey, and even invertebrates such as the snail. TRH is widely expressed in both the CNS and periphery in amphibians, reptiles, and fishes but does not stimulate TSH release in these poikilothermic vertebrates. Thus, TRH has multiple peripheral and central activities and was co-opted as a hypophyseotropic factor midway during the evolution of vertebrates, perhaps specifically as a factor needed for coordinated regulation of temperature homeostasis.

Effects on the Pituitary Gland and Mechanism of Action

After intravenous injection of TRH in humans, serum TSH levels rise within a few minutes,[97] followed by a rise in serum triiodothyronine (T3) levels; there is an increase in thyroxine (T4) release as well, but a change in blood levels of T4 is usually not demonstrable because the pool of circulating T4 (most of which is bound to carrier proteins) is so large. The clinical applications of TRH testing are discussed later in this chapter and in Chapter 10 . TRH action on the pituitary is blocked by previous treatment with thyroid hormone, which is a crucial element in feedback control of pituitary TSH secretion.

TRH is also a potent PRF.[97] The time course of response of blood PRL levels to TRH, the dose-response characteristics, and the suppression by thyroid hormone pretreatment (all of which parallel changes in TSH secretion) suggest that TRH may be involved in the regulation of PRL secretion. Moreover, TRH is present in the hypophyseal-portal blood of lactating rats. However, it is unlikely to be a physiologic regulator of PRL secretion because the PRL response to nursing in humans is unaccompanied by changes in plasma TSH levels[98] and mice lacking TRH have normal lactotrophs and basal prolactin secretion.[99] Nevertheless, TRH may occasionally cause hyperprolactin-emia (with or without galactorrhea) in patients with hypothyroidism.

In normal individuals, TRH has no influence on the secretion of pituitary hormones other than TSH and PRL, but it enhances the release of GH in acromegaly and of ACTH in some patients with Cushing’s disease. Furthermore, prolonged stimulation of the normal pituitary with GHRH can sensitize it to the GH-releasing effects of TRH. TRH also causes the release of GH in some patients with uremia, hepatic disease, anorexia nervosa, and psychotic depression and in children with hypothyroidism.[97] TRH inhibits sleep-induced GH release through its actions in the CNS (see later in the section on extrapituitary actions of TRH).

Stimulatory effects of TRH are initiated by binding of the peptide to specific receptors on the plasma membrane of the thyrotroph.[100] Neither thyroid hormone nor somatostatin, both of which antagonize the effects of TRH, interfere with its binding. TRH action is mediated mainly through hydrolysis of phosphatidylinositol, with phosphorylation of key protein kinases and an increase in intracellular free Ca2+ as the crucial step in postreceptor activation (see Chapter 5 ).[101] TRH effects can be mimicked by exposure to a Ca2+ ionophore and are partially abolished by a Ca2+-free medium. TRH stimulates the formation of mRNAs coding for TSH and PRL in addition to regulating their secretion and stimulates the mitogenesis of thyrotrophs.

TRH is degraded to acid TRH and to the dipeptide histidylprolineamide, which cyclizes nonenzymatically to histidylproline diketopiperazine (cyclic His-Pro). Acid TRH has some behavioral effects in rats that are similar to those of TRH but no other proven actions. Cyclic His-Pro is reported to act as a PRF and to have other neural effects, including reversal of ethanol-induced sleep (TRH is also effective in this system), elevation of brain cyclic guanosine monophosphate levels, an increase in stereotypical behavior, modification of body temperature, and inhibition of eating behavior. Some of the effects of TRH may be mediated through cyclic His-Pro, but the fact that cyclic His-Pro is abundant in some areas and is not proportional to the amount of TRH suggests that the peptide may not be derived solely from TRH. This latter assertion appears to be confirmed by the detection of substantial amounts of the dipeptide in brains of TRH knockout mice.[99]

Extrapituitary Function

TRH is present in virtually all parts of the brain: cerebral cortex, circumventricular structures, neurohypophysis, pineal gland, and spinal cord.[102] TRH is also found in pancreatic islet cells and in the gastrointestinal tract. Although it exists in low concentration, the total amount in extrahypothalamic tissues exceeds the amount in the hypothalamus.

The extensive extrahypothalamic distribution of TRH, its localization in nerve endings, and the presence of TRH receptors in brain tissue suggest that TRH serves as a neurotransmitter or neuromodulator outside the hypothalamus. TRH is a general stimulant and induces hyperthermia on intracerebroventricular injection, suggesting a role in central thermoregulation.[102] Studies in TRH knockout mice are expected to further clarify the nonhypophyseotropic actions of TRH.[99]

Clinical Applications

The use of TRH for the diagnosis of hyperthyroidism is less common since the development of ultrasensitive assays for thyroid-stimulating hormone (TSH)[97] (see Chapter 10 ); its use to discriminate between hypothalamic and pituitary causes of TSH deficiency has also declined because of the test’s poor specificity,[97] but the application of ultrasensitive assays in conjunction with the TRH test has not been fully evaluated. TRH testing also is not of value in the differential diagnosis of causes of hyperprolactinemia but is useful for the demonstration of residual abnormal somatotropin-secreting cells in acro-megalic patients who release hGH in response to TRH before treatment.

Studies of the effect of TRH on depression have shown inconsistent results, possibly because of poor blood-brain barrier penetration.[102] Intrathecal administration of TRH may improve responses in depressed patients, but its clinical utility is unknown.[103] Although a role for TRH in depression is not established, many depressed patients have a blunted TSH response to TRH and changes in TRH responsiveness correlate with the clinical course. The mechanism by which blunting occurs is unknown.

TRH has been evaluated for the treatment of diverse neurobiologic disorders (for review, see reference 102 ) including spinal muscle atrophy and amyotrophic lateral sclerosis; transient improvement in strength was reported in both disorders, but the combined experience at many centers using a variety of treatment protocols including long-term intrathecal administration failed to confirm efficacy. TRH administration also reduces the severity of experimentally induced spinal and ischemic shock; preliminary studies in humans suggest that TRH treatment may improve recovery after spinal cord injury and head trauma. TRH has been used to treat children with neurologic disorders including West’s syndrome, Lennox-Gastaut syndrome, early infantile epileptic encephalopathy, and intractable epilepsy.[104] TRH has been proposed to be an analeptic agent. Sleeping or drug-sedated animals were awakened by the administration of TRH, TRH reportedly reversed sedative effects of ethanol in humans, and TRH is said to have awakened a patient with a profound sleep disorder caused by a hypothalamic and midbrain eosinophilic granuloma.[102]

Regulation of TSH Release

The secretion of TSH is regulated by two interacting elements: negative feedback by thyroid hormone and open-loop neural control by hypothalamic hypophyseotropic factors ( Fig. 7-9 ). TSH secretion is also modified by other hormones, including estrogens, glucocorticoids, and possibly GH, and is inhibited by cytokines in the pituitary and hypothalamus. [97] [105] Aspects of the pituitary-thyroid axis are also considered in Chapter 10 .

Feedback Control: Pituitary-Thyroid Axis

In the context of a feedback system, the level of thyroid hormone in blood or of its unbound fraction is the controlled variable and the set-point is the normal resting level of plasma thyroid hormone. Secretion of TSH is inversely regulated by the level of thyroid hormone so that deviations from the set-point of control lead to appropriate changes in the rate of TSH secretion ( Fig. 7-10 ). Factors that determine the rate of TSH secretion required to maintain a given level of thyroid hormone include the rate at which TSH and thyroid hormone disappear from the blood (turnover rate) and the rate at which T4 is converted to its more active form, T3.

Thyroid hormones act on both the pituitary and the hypothalamus. Feedback control of the pituitary by thyroid hormone is remarkably precise. Administration of small doses of T3 and T4 inhibited the TSH response to TRH, and barely detectable decreases in plasma thyroid hormone levels were sufficient to sensitize the pituitary to TRH. TRH stimulates TSH secretion within a few minutes through its action on a membrane receptor, whereas thyroid hormone actions, mediated by intranuclear receptors, require several hours to take effect (see Chapter 10 ).

The secretion of hypothalamic TRH is also regulated by thyroid hormone feedback. Systemic injections of T3 or implantations of tiny T3 pellets in the PVH of hypothyroid rats[106] ( Fig. 7-11A and B ) reduced the concentration of TRH mRNA and TRH prohormone in TRH-secreting cells. Thyroid hormone also suppressed TRH secretion into hypophyseal-portal blood in sheep.

T4 in the blood gains access to TRH-secreting neurons in the hypothalamus by way of the CSF. The hormone is taken up by epithelial cells of the choroid plexus of the lateral ventricle of the brain, bound within the cell to locally produced transthyretin (T4-binding prealbumin), and then secreted across the blood-brain barrier.[107] Within the brain, T4 is converted to T3 by type II deiodinase, and T3 interacts with subtypes of the thyroid hormone receptor, TRα1, TRβ1, and TRβxf2, in the PVH and other brain cells (see Chapter 10 ). Thereby the set-point of the pituitary-thyroid axis is determined by thyroid hormone levels within the brain.[108] T3 in the circulation is not transported into brain in this manner but presumably gains access to the paraventricular TRH neurons across the blood-brain barrier. The brain T4 transport and deiodinase system account for the fact that higher blood levels of T3 are required to suppress pituitary-thyroid function after administration of T3 than after administration of T4. [108] [109]

Transthyretin is present in the brain of early reptiles and in addition is synthesized by the liver in warm-blooded animals.[107] During embryogenesis in mammals, transthyretin is first detected when the blood-brain barrier appears, ensuring thyroid hormone access to the developing nervous system.

Neural Control

The hypothalamus determines the set-point of feedback control around which the usual feedback regulatory responses are elicited. Lesions of the thyrotropic area lower basal thyroid hormone levels and make the pituitary more sensitive to inhibition by thyroid hormone, and high doses of TRH raise TSH and thyroid hormone levels. Synthesis of TRH in the paraventricular nuclei is regulated by feedback actions of thyroid hormones.[108] The hypothalamus can override normal feedback control through an open-loop mechanism involving neuronal inputs to the hypophyseotropic TRH neurons (see Fig. 7-9 ). For example, cold exposure causes a sharp increase in TSH release in animals and in human newborns. Circadian changes in TSH secretion are another example of brain-directed changes in the set-point of feedback control, but if thyroid hormone levels are sufficiently elevated, as in hyperthyroidism, TRH cannot overcome the inhibition.

Hypothalamic regulation of TSH secretion is also influenced by two inhibitory factors, somatostatin and dopamine. Antisomatostatin antibodies increase basal TSH levels and potentiate the response to stimuli that normally induce TSH release in the rat, such as cold exposure and TRH administration.[110] Thyroid hormone in turn inhibits the release of somatostatin, implying coordinated, reciprocal regulation of TRH and somatostatin by thyroid hormone. GH stimulates hypothalamic somatostatin synthesis and can inhibit TSH secretion. The role of somatostatin in the regulation of TSH secretion in humans is uncertain.

Dopamine has modest effects on TSH secretion, and blockade of dopamine receptors (in the human) stimulates TSH secretion slightly. Changes in the metabolism of thyroid hormone also influence T3 homeostasis within the brain. In states of thyroid hormone deficiency, brain T3 levels are maintained by an increase in the deiodinase that converts T4 to T3.[41]

The pineal gland has been reported to inhibit thyroid function in some but not all studies. The pineal gland contains TRH, and in the frog its content changes with the season and with light and dark cycles independently of hypothalamic TRH.

Circadian Rhythm

Plasma TSH in humans is characterized by a circadian periodicity, with a maximum between 9 pm and 5 am and a minimum between 4 pm and 7 pm.[111] Smaller ultradian TSH peaks occur every 90 to 180 minutes, probably because of bursts of TRH release from the hypothalamus, and are physiologically important in controlling the synthesis and glycosylation of TSH. Glycosylation is a determinant of TSH potency.[112]

Temperature

External cold exposure activates and high ambient temperature inhibits the pituitary-thyroid axis in animals, and analogous changes occur in humans under certain conditions.[113] Exposure of infants to cold at the time of delivery causes an increase in blood TSH levels, possibly because of alterations in the turnover and degradation of the thyroid hormones. Blood thyroid hormone levels are higher in the winter than in the summer in individuals in cold climates but not in other climates. However, it is difficult to show that changes in environmental or body temperature in adults influence TSH secretion. For example, exposure to cold ambient temperature or central hypothalamic cooling does not modify TSH levels in young men. Behavioral changes, activation of the sympathetic nervous system, and shivering appear to be more important in temperature regulation in adults than the thyroid response.

The autonomic nervous system and the thyroid axis work together to maintain temperature homeostasis in mammals, and TRH plays a role in both pathways.[113] Hypothalamic TRH release is rapidly (30 to 45 minutes) increased in rats exposed to cold. Rapid inhibition of somatostatin release in the median eminence also has been documented, and both changes appear to play important roles in the rise in plasma TSH induced by cold exposure. TRH mRNA is elevated within an hour of cold exposure (see Fig. 7-11C and D ).[114] The regulation of hypophyseotropic TRH release and expression by cold is largely mediated by catecholamines. Noradrenergic and adrenergic fibers, originating in the brain stem, are found in close proximity to TRH nerve endings in the median eminence, and a rapid rise in TRH release was seen after norepinephrine treatment of hypothalamic fragments containing mainly median eminence. Brain stem adrenergic and noradrenergic fibers also make synaptic contacts with TRH neurons in the PVH (see Fig. 7-9 ), and thus catecholamines are likely to be involved in the regulation of TRH gene expression by cold. TRH neurons in the PVH are densely innervated by NPY terminals,[115] and a portion of the NPY terminals arising from the C1, C2, C3, and A1 cell groups of the brain stem and projecting to the PVH are known to be catecholaminergic. Somatostatin, dopamine, and serotonin also play a variety of roles in the regulation of TRH.

Stress

Stress is another determinant of TSH secretion.[105] In humans, physical stress inhibits TSH release, as indicated by the finding that in the euthyroid sick syndrome low levels of T3 and T4 do not cause compensatory increases in TSH secretion as would occur in normal individuals.[116]

A number of observations demonstrate interactions between the thyroid and adrenal axes. Physiologically, the bulk of evidence suggests that glucocorticoids in humans and rodents act to blunt the thyroid axis through actions in the CNS.[117] Some actions may be direct because the TRH gene (see Fig. 7-8 ) contains the glucocorticoid response element consensus sequence[95] and hypophyseotropic TRH neurons appear to contain glucocorticoid receptors.[118] The diurnal rhythm of cortisol is opposite that of TSH (see Fig. 7-7 ) and acute administration of glucocorticoids can block the nocturnal rise in TSH, but disruption of cortisol synthesis with metyrapone only modestly affects the TSH circadian rhythm.

Several lines of evidence, however, identify conditions in which elevated glucocorticoids are associated with stimulation of the thyroid axis. Human depression is often associated with hypercortisolism and hyperthyroxinemia, and TRH mRNA levels are elevated by glucocorticoids in a number of cell lines as well as in cultured fetal hypothalamic TRH neurons from the rat. Thus, although glucocorticoids probably stimulate TRH production in TRH neurons, their overall inhibitory effect on the thyroid axis results from indirect glucocorticoid negative feedback on structures such as the hippocampus. Disruption of hippocampal suppression of the hypothalamic-pituitary-adrenal (HPA) axis is proposed to be involved in the hypercortisolemia commonly seen in affective illness, and disruption of hippocampal inputs to the hypothalamus have been shown to produce a rise in hypophyseotropic TRH in the rat.[119]

Starvation

The thyroid axis is depressed during starvation, presumably to help conserve energy by depressing metabolism (see Fig. 7-11E to G ). In humans, reduced T3, T4, and TSH are seen during starvation or fasting.[120] There are also changes in the thyroid axis in anorexia nervosa, such as low blood levels of T3 and low normal levels of T4 (see Chapter 10 ). Inappropriately low levels of TSH are found, suggesting defective activation of TRH production by low thyroid hormone levels. During starvation in rodents, reduced TRH release into hypophyseal portal blood and reduced pro-TRH mRNA levels are seen, despite lowered thyroid hormone levels.[121] Reduced basal TSH levels are also usually present.

The hypothyroidism seen in fasting or in the leptin-deficient ob/ob mouse can be reversed by administration of leptin,[122] and the evidence suggests that the mechanism involves leptin’s ability to up-regulate TRH gene expression in the PVH (see Fig. 7-11E to G ).[123] Leptin appears to act both directly through leptin receptors on hypophyseotropic TRH neurons and indirectly through its actions on other hypothalamic cell groups, such as arcuate nucleus POMC and NPY-agouti-related peptide (AgRP) neurons. [124] [125] TRH neurons in the PVH receive dense NPY-AgRP and POMC projections from the arcuate and express NPY and melanocortin-4 receptors (MC4R),[126] and α-MSH administration partially prevents the fasting-induced drop in thyroid hormone levels. [124] [125] Indeed, the TRH promoter contains a signal transducer and activator of transcription (STAT) response element and a cAMP response element that have been demonstrated to mediate induction of TRH gene expression by leptin and α-MSH, respectively, in a heterologous cell system (see Fig. 7-8 ).[126] The regulation of TRH by metabolic state is likely to be under redundant control, however, because, unlike rodents, leptin-deficient children are euthyroid,[127] and both MC4R-deficient rodents and humans are euthyroid.[128]

Infection and Inflammation

The molecular basis of infection- or inflammation-induced TSH suppression is partially established. Sterile abscesses or the injection of interleukin-1β (IL-1β; endogenous pyrogen, a secretory peptide of activated lymphocytes)[129] or of tumor necrosis factor α (TNF-α) inhibits TSH secretion, and IL-1β stimulates the secretion of somatostatin.[130] TNF-α inhibits TSH secretion directly and induces functional changes in the rat characteristic of the “sick euthyroid” state.[131] It is likely that the TSH inhibition in animal models of the sick euthyroid syndrome is due to cytokine-induced changes in hypothalamic and pituitary function.[132] IL-6, IL-1, and TNF-α contribute to the suppression of TSH in the sick euthyroid syndrome.[133]

Corticotropin-Releasing Hormone

Chemistry and Evolution

The HPA axis is the humoral component of an integrated neural and endocrine system that functions to respond to internal and external challenges to homeostasis (stressors). The system comprises the neuronal pathways linked to release of catecholamines from the adrenal medulla (fight-or-flight response) and the hypothalamic-pituitary control of ACTH release in the control of glucocorticoid production by the adrenal cortex. Pituitary ACTH release is stimulated primarily by CRH and to a lesser extent by AVP (see Chapter 8 ). The hypophyseotropic CRH neurons are located in the parvicellular division of the PVH and project to the median eminence (see Figs. 7-3 and 7-4 [3] [4]).

In a broader context, the CRH system in the CNS is also vitally important in the behavioral response to stress. This complex system includes not only nonhypophyseotropic CRH neurons but also three CRH-like peptides (urocortin I, urocortin II or stresscopin-like peptide, and urocortin III or stresscopin), at least two cognate receptors (CRH-R1 and CRH-R2), and a high-affinity CRH-binding protein, each with distinct and complex distributions in the CNS.

The Schally and Guillemin laboratories demonstrated in 1955 that extracts from the hypothalamus stimulated ACTH release from the pituitary. The principal bioactive peptide, CRH, was purified and characterized from the sheep in 1981 by Vale and colleagues. Human CRH is an amidated 41-amino-acid peptide that is cleaved from the carboxyl terminus of a 196-amino-acid pre-prohormone precursor by PC1 and PC2 ( Fig. 7-12 ).[134] In general, the peptide is highly conserved; the human peptide is identical in sequence to the mouse and rat peptides but differs at seven residues from the ovine sequence. Mammalian CRH and urocortin I, II, and III, fish urotensin, anuran sauvagine, and the insect diuretic peptides are members of an ancient family of peptides that evolved from an ancestral precursor early in the evolution of metazoans, approximately 500 million years ago.[135] Comparison of peptide sequences in vertebrates suggests a grouping of the peptides into two families, CRH-urotensin-urocortin-sauvagine and urocortin II-urocortin III ( Fig. 7-13 ).[136] Urocortin and sauvagine appear to represent tetrapod orthologues of fish urotensin. Sauvagine, isolated originally from Phyllomedusa sauvagei, is an osmoregulatory peptide produced in the skin of certain frogs; urotensin is an osmoregulatory peptide produced in the caudal neurosecretory system of the fish. Whereas isolation of CRH required 250,000 ovine hypothalami, the cloning of urocortin II and III was accomplished by computer search of the human genome database.[136]

The CRH peptides signal by binding to CRH-R1 [137] [138] and CRH-R2[139] receptors that couple to Gs and activation of adenylyl cyclase. Two splice variants of the latter that differ in the ex-tracellular amino-terminal domain, CRH-R2α and CRH-R2β, have been found in both rodents and humans,[140] and a third N-terminal splice variant, CRH-R2γ, has been reported in the human.[141]

CRH, urotensin, and sauvagine are all potent agonists of CRH-R1, urocortin is a potent agonist of both receptors, and urocortins II and III are specific agonists of CRH-R2. CRH-activation of the HPA axis is mediated exclusively through CRH-R1 expressed in the corticotroph. The PVH is the site of the majority of CRH neurons projecting to the median eminence, although some CRH neurons projecting to the median emin-ence are found in most hypothalamic nuclei ( Fig. 7-14A ). Some CRH fibers in the PVH also project to the brain stem, and nonhypophyseotropic CRH neurons are abundant elsewhere, primarily in limbic structures involved in processing sensory information and in regulating the autonomic nervous system. Sites include the prefrontal, insular, and cingulate cortices; amygdala; substantia nigra; periaqueductal gray; locus coeruleus; nucleus of the solitary tract; and parabrachial nucleus. In the periphery, CRH is found in human placenta, where it is up-regulated 6- to 40-fold during the third trimester; lymphocytes; autonomic nerves; and gastrointestinal tract. Urocortin is expressed at highest levels in the Edinger-Westphal nucleus, lateral superior olive, and SON of the rodent brain, with additional sites including the substantia nigra, ventral tegmental area, and dorsal raphe (see Fig. 7-14B ). In the human, urocortin is widely distributed with highest levels in the frontal cortex, temporal cortex, and hypothalamus[142] and has also been reported in the Edinger-Westphal and olivary nuclei.[143] In the periphery, urocortin is seen in placenta, mucosal inflammatory cells in the gastrointestinal tract, lymphocytes, and cardiomyocytes. There is limited information concerning the tissue distribution of urocortins II and III, particularly in the brain.

In addition to its expression in pituitary corticotrophs, CRH-R1 is found in the neocortex and cerebellar cortex, subcortical limbic structures, and amygdala, with little to no expression in the hypothalamus ( Fig. 7-14C ). CRH-R1 also is found in a variety of peripheral sites in humans, including ovary, endometrium, and skin. CRH-R2α is found mainly in the brain in rodents, with high levels of expression seen in the ventromedial hypothalamic nucleus and lateral septum (see Fig. 7-14C ).[144] CRH-R2β is seen centrally in cerebral arterioles and peripherally in gastrointestinal tract, heart, and muscle. [139] [145] In contrast, in humans CRH-R2α is seen in brain and periphery, and the β and γ subtypes are primarily central. [140] [141] Little CRH-R2 message is seen in pituitary. Although CRH-R1 appears to be exclusively involved in regulation of pituitary ACTH synthesis and release, both receptors are expressed in the rodent adrenal cortex. Data suggest that this intraadrenal CRH-ACTH system may be involved in fine-tuning of adrenocortical corticosterone release.

The CRH system is also regulated in both brain and periphery by a 37-kd high-affinity CRH-binding protein. [146] [147] [148] This factor was initially postulated from the observation that CRH levels rise dramatically during the second and third trimesters of pregnancy without activating the pituitary-adrenal axis. Among hypophyseotropic factors, CRH is the only one for which a specific binding protein (in addition to the receptor) exists in tissue or blood. The placenta is the principal source of pregnancy-related CRH-binding protein. Human and rat CRH-binding proteins are homologous (85% amino acid identity), but in the rat the protein is expressed only in brain. The binding protein is species specific; bovine CRH, which is almost identical in sequence to rat-human CRH, has a lower affinity of binding to the human binding protein.

The functional significance of the CRH-binding protein is not fully understood. CRH-binding protein does not bind to the CRH receptor but does inhibit CRH action. For this reason CRH-binding protein probably acts to modulate CRH actions at the cellular level. Corticotroph cells in the anterior pituitary have membrane CRH receptors and intracellular CRH-binding protein; conceivably, the binding protein acts to sequester or terminate the action of membrane-bound CRH. CRH-binding protein is present in many regions of the CNS, including cells that synthesize CRH and cells that receive innervation from CRH-containing neurons. The anatomic distribution of the protein, the variability of its location in relation to the presence of CRH, and its relative sparseness in the CRH tuberohypophyseal neuronal system suggest a control system that is as yet poorly understood. Transgenic mouse models with both overexpression and gene deletion of the CRH-binding protein have been produced with little effect on basal or stress-activation of the HPA axis (reviewed in reference 149 ).

Structure-activity relationship studies have demonstrated that C-terminal amidation and an α-helical secondary structure are both important for biologic activity of CRH. The first CRH antagonist described was termed α-helical CRH9-41.[150] A second, more potent antagonist, termed astressin, has the structure cyclo(30-33)(d-Phe12, Nle12, Glu12, Lys12)hCRH12-41.[151] Both peptides are somewhat nonspecific, antagonizing both CRH-R1 and CRH-R2. Because of the anxiogenic activity of CRH and urocortin, a number of pharmaceutical companies have developed small-molecule CRH antagonists; several of the molecules are currently in clinical trials for anxiety and depression (discussed in more detail later). Thus far, this structurally diverse group of small molecule compounds, such as antalarmin, CP-154,526, and NBI27914, are potent antagonists of CRH-R1, with little activity at CRH-R2. The efficacy of these compounds across the entire behavioral, neuroendocrine, and autonomic repertoire of response to stress has been demonstrated in a number of laboratory animal studies. For example, oral administration of antalarmin in a social stress model in the primate (introduction of strange males) reduced behavioral measures of anxiety such as lack of exploratory behavior, decreased plasma ACTH and cortisol, and reduced plasma epinephrine and norepinephrine.[152] Other preclinical studies in rhesus monkeys have compared the pharmacologic profiles of astressin B and antalarmin.[153] A peptide antagonist with 100-fold selectivity for the CRH 2b receptor, (d-Phe,11 His12)sauvagine 11-40 or anti-sauvagine-30, has also been described.[154]

Effects on the Pituitary and Mechanism of Action

Administration of CRH to humans causes prompt release of ACTH into the blood, followed by secretion of cortisol ( Fig. 7-15 ) and other adrenal steroids including aldosterone. Most studies have used ovine CRH, which is more potent and longer acting than human CRH, but human and porcine CRHs appear to have equal diagnostic value. The effect of CRH is specific to ACTH release and is inhibited by glucocorticoids.

As mentioned earlier, CRH acts on the pituitary corticotroph primarily by binding to CRH-R1 and activating adenylyl cyclase. The concentration of cAMP in the tissue is increased in parallel with the biologic effects and is reduced by glucocorticoids. The rate of transcription of the mRNA that encodes the ACTH prohormone POMC is also enhanced by CRH.

Extrapituitary Functions

CRH and the urocortin peptides have a wide range of biologic activities in addition to the hypophyseotropic role of CRH in regulating ACTH synthesis and release. Centrally, these peptides have behavioral activities in anxiety, mood, arousal, locomotion, reward, and feeding [155] [156] and increase sympathetic activation. Many of the nonhypophyseotropic behavioral and autonomic functions of these peptides can be viewed as complementary to activation of the HPA axis in the maintenance of homeostasis under exposure to stress. In the periphery, activities have been reported in immunity, cardiac function, gastrointestinal function, and reproduction.[157]

The CRH and urocortin peptides have a repertoire of behavioral and autonomic actions after central administration that suggests a role for these pathways in mediating the behavioral-autonomic components of the stress response. Hyperactivity of the HPA axis is a common neuroendocrine finding in affective disorders (see Fig. 7-15 ). [155] [158] Furthermore, normalization of HPA regulation is highly predictive of successful treatment. Defective dexamethasone suppression of CRH release, implying defective corticosteroid receptor signaling, is seen not only in depressed patients but also in healthy subjects with a family history of depression.[159] Depressed patients also show elevated levels of CRH in the CSF.[160] Extensive behavioral testing in a variety of mutant mouse models with genetically altered expression of either the CRH ligands or receptors generally supports the hypothesis that activation of central CRH pathways is a critical neurobiologic substrate of anxiety and depressive states. [149] [156]

Central administration of CRH or urocortin activates neuronal cell groups involved in cardiovascular control and increases blood pressure, heart rate, and cardiac output.[161] However, urocortin is expressed in cardiac myocytes, and intravenous administration of CRH or urocortin decreases blood pressure and increases heart rate in most species, including humans.[161] This hypotensive effect is probably mediated peripherally because ganglion blockade did not disrupt the hypotensive effects of intravenous urocortin. Furthermore, high levels of CRH-R2b have been seen in the cardiac atria and ventricles, [139] [145] and knockout of the CRH-R2 gene in the mouse eliminated the hypotensive effects of intravenous urocortin administration. [162] [163]

Cytokines have an important role in extinguishing inflammatory responses through activation of CRH and AVP neurons in the PVH and subsequent elevation of anti-inflammatory glucocorticoids. Interestingly, CRH is generally proinflammatory in the periphery, where it is found in sympathetic efferents, sensory afferent nerves, leukocytes, and in macrophages in some species. [157] [164] CRH also functions as a paracrine factor in the endometrium, where it may play a role in decidualization and implantation and act as a uterine vasodilator.[157]

The relative contributions of each of the CRH-urocortin peptides and receptors to the different biologic functions reported has been the topic of considerable analysis, given the receptor-specific antagonists already described as well as the CRH, CRH-R1, and CRH-R2 knockout mice available for study (reviewed in references 149 and 156 [149] [156]). Examination of three potent stressors—restraint, ether, and fasting—demonstrated that other ACTH secretagogues, such as AVP, oxytocin, and catecholamines, could not replace CRH in its role in mounting the stress response. In contrast, augmentation of glucocorticoid secretion by a stressor after prolonged stress was not defective in the CRH knockout mouse, implicating CRH-independent mechanisms.

Although CRH is a potent anxiogenic peptide, the CRH knockout mouse exhibits normal anxiety behaviors in, for example, conditioned fear paradigms. The nonpeptide CRH-R1 specific antagonist CP-154,526 was anxiolytic in a shock-induced freezing paradigm in both wild-type and CRH knockout mice, suggesting that the anxiogenic activity is a CRH-like peptide acting at the CRH-R1 receptor.

CRH and urocortin peptides also have potent anorexigenic activity, implicating the CRH system in stress-induced inhibition of feeding. Stress-induced inhibition of feeding remained intact, however, in the CRH knockout mouse. Likewise, suppression of the proestrous LH surge by restraint was intact in the CRH knockout mouse. Both CRH-R1 and CRH-R2 knockout strains had normal weight and feeding behavior but were distinctly different from wild-type mice in the anorexigenic response to centrally administered urocortin or CRH. The CRH-R1-deficient mice lacked the acute anorexigenic response (0 to 1.5 hours) to urocortin seen in wild-type mice. Both wild-type and CRH-R1 knockout mice exhibited comparable reduction in feeding 3 to 11 hours after administration. In contrast, the late phase of urocortin responsiveness appeared to depend on the presence of CRH-R2. Thus, signaling through CRH-R1 and CRH-R2 appears to play a complex role in the acute effects of stress on feeding behavior.

Clinical Applications

No approved therapeutic application of CRH or CRH-like peptides exists, although the peptide has been demonstrated to have a number of activities in human and primate studies. For example, intravenous administration of CRH was found to stimulate energy expenditure and has been proposed for use in weight loss. CRH is used diagnostically, often in combination with dexamethasone suppression or inferior petrosal venous sampling, in the evaluation of Cushing’s syndrome to differentiate between pituitary and ectopic sources of ACTH (reviewed in reference 165 ; see Chapters 8 and 14 ).

The development of small molecule, orally available, CRH-R1 antagonists has, however, produced considerable interest in their potential for treatment of anxiety and depression. [166] [167] In particular the compound R121919 was studied in phase I and IIa clinical trials before its discontinuance. These studies of 20 patients demonstrated significant reductions in scores of anxiety and depression, using ratings determined by either patient or clinician, and also demonstrated the compound’s safety and favorable side-effect profile including a lack of effect on endocrine function or body weight gain. [168] [169] [170]

Feedback Control

The administration of glucocorticoids inhibits ACTH secretion; removal of the adrenals (or administration of drugs that impair secretion of glucocorticoids) leads to increased ACTH release. The set-point of pituitary feedback is determined by the hypothalamus acting through hypothalamic-releasing hormones CRH and AVP (see Chapter 8 ). [171] [172] [173] [174] Glucocorticoids act on both the pituitary corticotrophs and the hypothalamic neurons that secrete CRH and AVP. These regulatory actions are analogous to the control of the pituitary-thyroid axis. However, whereas TSH becomes completely unresponsive to TRH when thyroid hormone levels are sufficiently high, severe neurogenic stress and large amounts of CRH can break through the feedback inhibition by glucocorticoids. A still higher level of feedback control is exerted by glucocorticoid-responsive neurons in the hippocampus that project to the hypothalamus; these neurons affect the activity of CRH hypophyseotropic neurons and determine the set-point of pituitary responsiveness to glucocorticoids.[174] A recent comprehensive review of glucocorticoid effects on CRH and AVP and regulation of the HPA axis has emphasized the complexity of this control beyond that of a simple closed-loop feedback.[175]

Glucocorticoids are lipid soluble and freely enter the brain through the blood-brain barrier.[173] In brain and pituitary they can bind to two receptors, type I (the mineralocorticoid receptor, so named because it binds aldosterone and glucocorticoids with high affinity) and type II (glucocorticoid receptor, which has low affinity for mineralocorticoids). [172] [173] [174] Classic glucocorticoid action involves binding of the steroid-receptor complex to regulator sequences in the genome. Type I receptors are saturated by basal levels of glucocorticoids, whereas type II receptors are not saturated under basal conditions but approach saturation during peak phases of the circadian rhythm and during stress. These differences and differences in regional distribution within the brain suggest that type I receptors determine basal activity of the hypothalamic-pituitary axis and that type II receptors mediate stress responses.

In the pituitary, glucocorticoids inhibit secretion of ACTH and the synthesis of POMC mRNA; in the hypothalamus, the secretion of CRH and AVP and the synthesis of their respective mRNAs are inhibited, although with distinct temporal patterns. [173] [174] [175] Neuron membrane excitability and ion transport properties are suppressed by changes in glucocorticoid-directed synthesis of intracellular protein. Recent studies indicate that glucocorticoids can exert additional rapid signaling events in neurons including an endocannabinoid-mediated suppression of synaptic excitation.[176] These rapid events involve membrane-associated complexes and are independent of changes in gene transcription or acute protein translation, but the exact mechanisms and nature of the receptors are still under investigation.[177]

Glucocorticoids block stress-induced ACTH release. The latency of the inhibitory effect is so short (less than 30 minutes) that it is likely that gene regulation is not the sole basis of the response.[177] Long-term suppression (more than 1 hour) clearly acts through genomic mechanisms.

Glucocorticoid receptors are also found outside the hypothalamus in the septum and amygdala, [173] [174] structures that are involved in the psychobehavioral changes in hypercortisolism and hypocortisolism. It is worth noting that in all these areas, apart from CRH neurons of the PVH, glucocorticoids have either a stimulatory or a neutral effect on CRH gene expression.[175] Hippocampal neurons are reduced in number by prolonged elevation of glucocorticoids during chronic stress.[174]

Neural Control

Significant physiologic or psychological stressors evoke an adaptive response that commonly includes activation of both the HPA axis and the sympathoadrenal axis. The end products of these pathways then help to mobilize resources to cope with the physiologic demands in emergency situations, acutely through the fight-or-flight response and over the long term through systemic effects of glucocorticoids on functions such as gluconeogenesis and energy mobilization (see Chapter 33 ). The HPA axis also has unique stress-specific homeostatic roles, the best example being the role of glucocorticoids in down-regulating immune responses after infection and other events that stimulate cytokine production by the immune system.

The PVH is the primary hypothalamic nucleus respon-sible for providing the integrated whole-animal response to stress. [175] [178] [179] This nucleus contains three major types of effector neurons that are spatially distinct from one another within it: (1) magnicellular oxytocin and AVP neurons that project to the posterior pituitary and participate in the regulation of blood pressure, fluid homeostasis, lactation, and parturition; (2) neurons projecting to the brain stem and spinal cord that regulate a variety of autonomic responses including sympathoadrenal activation; and (3) parvicellular CRH neurons that project to the median eminence and regulate ACTH synthesis and release. Many CRH neurons coexpress AVP, which acts as an auxiliary ACTH secretagogue, synergistic with CRH. AVP is regulated quite differently in parvicellular versus magnicellular neurons but is also regulated somewhat differently from CRH by stressors in parvicellular cells expressing both peptides.[175] Different stressors result in different patterns of activation of the three major visceromotor cell groups within the PVH, as measured by the general neuronal activation marker c-Fos ( Fig. 7-16 ). For example, salt loading down-regulates CRH mRNA in parvicellular CRH cells, up-regulates CRH in a small number of magnicellular CRH cells, but only activates magnicellular cells. Hemorrhage activates every division of the PVH, whereas cytokine administration primarily activates parvicellular CRH cells with some minor activation of magnicellular and autonomic divisions.

The synthesis and release of AVP, which regulates renal water absorption and vascular smooth muscle, are controlled mainly by the volume and tonicity of the blood. This information is relayed to the magnicellular AVP cell through the nucleus of the solitary tract and A1 noradrenergic cell group of the ventrolateral medulla and projections from a triad of CVOs lining the third ventricle, the SFO, medial preoptic nucleus (MePO), and OVLT. Oxytocin is primarily involved in reproductive functions, such as parturition, lactation, and milk ejection, although it is cosecreted with AVP in response to osmotic and volume challenges, and oxytocin cells receive direct projections from the nucleus of the solitary tract as well as from the SFO, MePO, and OVLT. In contrast to the neurosecretory neurons functionally defined by the three peptides, CRH, oxytocin, and AVP, PVH neurons projecting to brain stem and spinal cord include neurons expressing each of these peptides.

In the rodent, a wide variety of stressors have been determined to activate parvicellular CRH neurons, including cytokine injection, salt loading, hemorrhage, adrenalectomy, restraint, foot shock, hypoglycemia, fasting, and ether exposure. Thus, in contrast to the simplicity of inputs to magnicellular cells ( Fig. 7-17A ), it is not surprising that parvicellular CRH neurons receive a diverse and complex assortment of inputs ( Fig. 7-18 ; see Fig. 7-17B ). These inputs are divided into three major categories: brain stem, limbic forebrain, and hypothalamus. Because the PVH is not known to receive any direct projections from the cerebral cortex or thalamus, stressors involving emotional or cognitive processing must involve indirect relay to the PVH.

Visceral sensory input to the PVH involves primarily two pathways. The nucleus of the solitary tract, the primary recipient of sensory information from the thoracic and abdominal viscera, sends dense catecholaminergic projections to the PVH, both directly and through relays in the ventrolateral medulla. These brain stem projections account for about half of the NPY fibers present in the PVH. A second major input responsible for transducing signals from blood-borne substances derives from three CVOs adjacent to the third ventricle, the SFO, OVLT, and MePO. These pathways account for activation of CRH neurons by what are referred to as systemic or physiologic stressors.[179]

By contrast, what are termed neurogenic, emotional, or psychological stressors involve, in addition, nociceptive or somatosensory pathways as well as cognitive and affective brain centers. Using elevation of c-Fos as an indicator of neuronal activation, detailed studies have compared PVH-projecting neurons activated by IL-1 treatment (systemic stressor) versus foot shock (neurogenic stressor).[179] Only catecholaminergic solitary tract nucleus and ventrolateral medulla neurons were activated by moderate doses of IL-1. In contrast, foot–shock mediated activation of neurons of the solitary tract nucleus and ventrolateral medulla but also cell groups in the limbic forebrain and hypothalamus. Notably, pharmacologic or mechanical disruption of the ascending catecholaminergic fibers blocked IL-1–mediated activation but not foot–shock–mediated activation of the HPA axis. Data suggest that pathways activated by other neurogenic and systemic stressors may overlap significantly with those activated by foot–shock and IL-1 treatment, respectively. [178] [179]

Except for the catecholaminergic neurons of the nucleus of the solitary tract and ventrolateral medulla, parts of the bed nucleus of the stria terminalis, and the dorsomedial nucleus of the hypothalamus, many inputs to the PVH, such as those deriving from the prefrontal cortex and lateral septum, are thought to act indirectly through local hypothalamic glutamatergic[180] and GABAergic neurons[181] with direct synapses to the CRH neurons. The bed nucleus of the stria terminalis is the only limbic region with prominent direct projections to the PVH. With substantial projections from the amygdala, hippocampus, and septal nuclei, it may thus serve as a key integrative center for transmission of limbic information to the PVH.[178]

Inflammation and Cytokines

Stimulation of the immune system by foreign pathogens leads to a stereotyped set of responses orchestrated by the CNS. These responses are the result of the complex interaction of the immune system and the CNS. This constellation of stereotyped responses is mediated in large part by the hypothalamus, and includes coordinated autonomic, endocrine, and behavioral components with adaptive consequences to restore homeostasis. It is now clear that cytokines produced by white blood cells of the immune system mediate the CNS responses. Early evidence supporting this hypothesis was provided by the seminal observations that cytokines such as IL-1β can activate the HPA axis. [182] [183] [184] Neuroimmunology, the discipline arising from the study of reciprocal interactions between the neuroendocrine and immune systems, and particularly the role of cytokines in mediating cachexia is covered fully in Chapter 34 .

This section focuses on cytokines and activation of the HPA axis. The resultant glucocorticoid secretion acts as a classic negative feedback to the immune system to dampen its response. In general, glucocorticoids inhibit most limbs of the immune response, including lymphocyte proliferation, production of immunoglobulins, cytokines, and cytotoxicity. These inhibitory reactions form the basis of the antiinflammatory actions of glucocorticoids.

Glucocorticoid feedback on immune responses is regulatory and beneficial because loss of this function makes animals with adrenal insufficiency vulnerable to inflammation. Moreover, this feedback response can have pathophysiologic consequences, as chronic activation of the HPA axis can certainly be detrimental. [185] [186] Indeed, it is well established that chronic stress can lead to immunosuppression. The fact that products of inflammation such as IL-1β can activate the HPA axis suggests the operation of a negative feedback control loop to regulate the intensity of inflammation. The role of the hypothalamus in regulating pituitary-adrenal function is an excellent example of neuroimmunomodulation. Proposed models to explain how immune system signals might act upon the CNS to modulate homeostatic circuits by the integration of vagal input, peripheral cytokine interactions with receptors in the CVOs and cerebral blood vessels, and local production of cytokines within the CNS are explored in Chapter 34 .

Other Factors Influencing Secretion of Corticotropin

Circadian Rhythms

Levels of ACTH and cortisol (in humans) peak in the early morning, fall during the day to reach a nadir at about midnight, and begin to rise between 1 am and 4 am (see Fig. 7-7 ). Within the circadian cycle, approximately 15 to 18 pulses of ACTH can be discerned, their height varying with the time of day.[187] The set-point of feedback control by glucocorticoids also varies in a circadian pattern. Pituitary-adrenal rhythms are entrained to the light-dark cycle and can be changed over several days by exposure to an altered light schedule. It has long been assumed that the rhythm of ACTH secretion is driven by CRH rhythms, and CRH knockout mice were found to exhibit no circadian rhythm in corticosterone production. Remarkably, however, a diurnal rhythm in corticosterone was restored by a constant infusion of CRH to the CRH knockout mouse,[188] suggesting that CRH is necessary to permit pituitary or adrenal responsiveness to another diurnal rhythm generator.

Corticotropin Release-Inhibiting Factor

Disconnection of the pituitary from the hypothalamus in several species leads to increased basal levels of ACTH, and certain responses to physical stress (in contrast to psychological stress) are retained in such animals. These observations have led several investigators to postulate the existence of an ACTH inhibitory factor analogous to dopamine in the control of PRL secretion and to somatostatin in the control of GH secretion. Candidate hypothalamic peptides to inhibit ACTH release at the level of the pituitary include atrial natriuretic peptide, activins and inhibins, and sequence 178 to 199 of the TRH prohormone.[189] There is not yet a consensus on the existence of a physiologically relevant ACTH release-inhibiting factor or on its identity.

Growth Hormone–Releasing Hormone

Chemistry and Evolution

Evidence for neural control of GH secretion came from studies of its regulation in animals with lesions of the hypothalamus[190] and from the demonstration that hypothalamic extracts stimulate the release of GH from the pituitary. When it was shown that GH is released episodically, follows a circadian rhythm, responds rapidly to stress, and is blocked by pituitary stalk section, the concept of neural control of GH secretion became a certainty. However, it was only with the discovery of the paraneoplastic syndrome of ectopic GHRH secretion by pancreatic adenomas in humans that sufficient starting material became available for peptide sequencing and subsequent cloning of a complementary deoxyribonucleic acid (cDNA). [191] [192] [193] [194]

Two principal molecular forms of GHRH occur in human hypothalamus: GHRH(1-44)-NH2 and GHRH(1-40)-OH ( Fig. 7-19 ).[195] As with other neuropeptides, the various forms of GHRH arise from post-translational modification of a larger prohormone. [191] [196] The NH2-terminal tyrosine of GHRH (or histidine in rodent GHRHs) is essential for bioactivity, but a COOH-terminal NH2 group is not. Fragments as short as (1-29)-NH2 are active, but GHRH(1-27)-NH2 is inactive. A circulating type IV dipeptidylpeptidase potently inactivates GHRH to its principal and more stable metabolite, GHRH(3-44)-NH2,[197] which accounts for most of the immunoreactive peptide detected in plasma. As in the case of GnRH, there are species differences among GHRHs; the peptides from seven species range in sequence homology with the human peptide from 93% in the pig to 67% in the rat.[195] The COOH-terminal end of GHRH exhibits the most sequence diversity among species, consistent with the exon arrangement of the gene and dispensability of these residues for GHRH receptor binding.

Despite its importance for the elucidation of GHRH structure, ectopic secretion of the peptide is a rare cause of acromegaly. Fewer than 1% of acromegalic patients have elevated plasma levels of GHRH (see Chapter 8 ).[198] Approximately 20% of pancreatic adenomas and 5% of carcinoid tumors contain immunoreactive GHRH, but most are clinically silent. [199] [200]

In addition to expression in the hypothalamus, the GHRH gene is expressed eutopically in human ovary, uterus, and placenta,[201] although its function in these tissues is not known. Studies in rat placenta indicate that an alternative transcriptional start site 10 kilobases upstream from the hypothalamic promoter is utilized together with an alternatively spliced exon 1a.[202]

Growth Hormone–Releasing Hormone Receptor

The GHRH receptor is a member of a subfamily of G protein–coupled receptors that includes receptors for VIP, pituitary adenylyl cyclase-activating peptide, secretin, glucagon, glucagon-like peptide 1, calcitonin, parathyroid hormone or parathyroid hormone–related peptide, and gastric inhibitory polypeptide. [203] [204] GHRH elevates intracellular cAMP by its receptor coupling to a Gs, which activates adenylyl cyclase, increases intracellular free Ca2+, releases preformed GH, and stimulates GH mRNA transcription and new GH synthesis (see Chapter 8 ).[205] GHRH also increases pituitary phosphatidylinositol turnover. Nonsense mutations in the human GHRH receptor gene are the cause of rare familial forms of GH deficiency [206] [207] and indicate that no other gene product can fully compensate for the specific receptor in pituitary.

Effects on the Pituitary and Mechanism of Action

Intravenous administration of GHRH to individuals with normal pituitaries caused a prompt, dose-related increase in serum GH that peaked between 15 and 45 minutes, followed by a return to basal levels by 90 to 120 minutes ( Fig. 7-20 ).[208] A maximally stimulating dose of GHRH is approximately 1 μg/kg, but the response differs considerably between individuals and within the same individual tested on different occasions, presumably because of cosecretagogue and somatostatin tone that exists at the time of GHRH injection. Repeated bolus administration or sustained infusions of GHRH over several hours cause a modest decrease in the subsequent GH secretory response to acute GHRH administration. However, unlike the marked desensitization of the GnRH receptor and decline in circulating gonadotropins that occur in response to continuous GnRH exposure, pulsatile GH secretion and insulin-like growth factor I (IGF-I) production are maintained by constant GHRH in the human.[208] This response suggests the involvement of additional factors that mediate the intrinsic diurnal rhythm of GH, and these factors are addressed in the following sections.

The pituitary effects of a single injection of GHRH are almost completely specific for GH secretion, and there is minimal evidence for any interaction between GHRH and the other classic hypophyseotropic releasing hormones.[208] GHRH has no effect on gut peptide hormone secretion. The GH secretory response to GHRH is enhanced by estrogen administration, glucocorticoids, and starvation. Major factors known to blunt the response to GHRH in humans are somatostatin, obesity, and advancing age.

In addition to its role as a GH secretagogue, GHRH is a physiologically relevant growth factor for somatotrophs. Transgenic mice expressing a GHRH cDNA coupled to a suitable promoter developed diffuse somatotroph hyperplasia and eventually pituitary macroadenomas. [209] [210] The intracellular signal transduction pathways mediating the mitogenic action of GHRH are not known with certainty but probably involve an elevation of adenylyl cyclase activity. Several lines of evidence support this conclusion, including the association of activating mutations of the Gsα polypeptide in many human somatotroph adenomas.[211]

Extrapituitary Functions

GHRH has few known extrapituitary functions. The most important may be its activity as a sleep regulator. The administration of nocturnal GHRH boluses to normal men significantly increased the density of slow wave sleep, as also shown in other species.[212] Furthermore, there is a striking correlation between the age-related declines in slow wave sleep and daily integrated GH secretion in healthy men.[213] These and other data suggest that central GHRH secretion is under circadian entrainment and nocturnal elevations in GHRH pulse amplitude or frequency directly mediate sleep stage and sleep-induced increases in GH secretion.

GHRH has been reported to stimulate food intake in rats and sheep, but the effect is dependent on route of administration, time of administration, and macronutrient composition of the diet.[203] The neuropeptide’s physiologic relevance to feeding in humans is unknown, although a study indicated that GHRH stimulated food intake in patients with anorexia nervosa but reduced it in patients with bulimia or in normal female control subjects.[214]

Growth Hormone–Releasing Peptides

In studies of the opioid control of GH secretion, several peptide analogues of met-enkephalin were found to be potent GH secretagogues. These include the GH-releasing peptide GHRP-6 ( Fig. 7-21 ), hexarelin (His-d2MeTrp-Ala-Trp-dPhe-Lys-NH2), and other more potent analogues including cyclic peptides and modified pentapeptides. [203] [215] Subsequently, a series of nonpeptidyl GHRP mimetics were synthesized with greater oral bioavailability, including the spiropiperidine MK-0677 and the shorter acting benzylpiperidine L-163,540 (see Fig. 7-21 ). Common to all these compounds, and the basis of their differentiation from GHRH analogues in pharmacologic activity screens, is their activation of phospholipase C and inositol 1,4,5-trisphosphate. This property was exploited in a cloning strategy that led to the identification of a G protein–coupled receptor GHS-R that is highly selective for the GH secretagogue class of ligands.[216] The GHS-R is unrelated to the GHRH receptor and is highly expressed in the anterior pituitary gland and multiple brain areas, including the medial basal hypothalamus, the hippocampus, and the mesencephalic nuclei that are centers of dopamine and serotonin production.

Peptidyl and nonpeptidyl GHSs are active when administered by intranasal and oral routes, are more potent on a weight basis than GHRH itself, are more effective in vivo than in vitro, synergize with coadministered GHRH and are almost ineffective in the absence of GHRH, and do not suppress somatostatin secretion. [203] [208] Prolonged infusions of GHRP amplify pulsatile GH secretion in normal men. GHRP administration, like that of GHRH, facilitates slow wave sleep. Patients with hypothalamic disease leading to GHRH deficiency have low or no response to hexarelin; similarly, pediatric patients with complete absence of the pituitary stalk have no GH secretory response to hexarelin.[217]

The potent biologic effects of GHRPs and the identification of the GHS-R suggested the existence of a natural ligand for the receptor that is involved in the physiologic regulation of GH secretion. A probable candidate for this ligand is the acylated peptide ghrelin, produced and secreted into the circulation from the stomach ( Fig. 7-22 ).[13] The effects of ghrelin on GH secretion in humans are identical to or more potent than those of the non-natural GHRPs (see Fig. 7-20 ).[218] In addition, ghrelin acutely increases circulating PRL, ACTH, cortisol, and aldosterone levels.[218] There is debate concerning the extent and localization of ghrelin expression in the brain that must be resolved before the implications of gastric-derived ghrelin in the regulation of pituitary hormone secretion are fully understood. A proposed role for ghrelin in appetite and the regulation of food intake is discussed in Chapter 34 .

Clinical Applications

GHRH stimulates growth in children with intact pituitaries, but the optimal dosage, route, and frequency of administration, as well as possible usefulness by the nasal route, have not been determined. The availability of recombinant hGH (which requires less frequent injections than GHRH) and the development of the more potent GHSs with improved oral bioavailability have reduced enthusiasm for the clinical use of GHRH or its analogues. GHRH is not useful for the differential diagnosis of hypothalamic and pituitary causes of GH deficiency in children. However, in adults a combined GHRH-GHRP challenge test may be ideal for the diagnosis of GH reserve. GH release in response to the combined secretagogues is not influenced by age, gender, or body mass index, and the test has a wider margin of safety than an insulin tolerance test. [219] [220]

The potential clinical applications of GHSs including MK-0677 are still being explored. [203] [215] An area of intense interest is the normal decline in GH secretion with age. GH administration in healthy older individuals has been associated with increased lean body mass, increased muscle strength, and decreased fat mass, although there is a high incidence of adverse side effects. The physiologic GH profile induced by MK-0677 may be better tolerated than GH injections. However, unlike treatment with GHRH, chronic administration of GHSs leads to significant desensitization of the GHS-R and attenuation of the GH response. The release of pituitary hormones other than GH may also limit the applicability of GHS therapy. Finally, apart from actions on GH secretion, both GHRH and GHSs are being investigated for the treatment of sleep disorders commonly associated with aging.

Neuroendocrine Regulation of Growth Hormone Secretion

GH secretion is regulated by hypothalamic GHRH and somatostatin interacting with circulating hormones and additional modulatory peptides at the level of both the pituitary and the hypothalamus (see Fig. 7-22 ). [203] [208] Additional background on somatostatin and its functions other than control of GH secretion are presented in a later section (see Somatostatin).

Feedback Control

Negative feedback control of GH release is mediated by GH itself and by IGF-I, which is synthesized in the liver under control of GH. Direct GH effects on the hypothalamus are produced by short-loop feedback, whereas those involving IGF-I and other circulating factors influenced by GH, including free fatty acids and glucose, are long-loop systems analogous to the pituitary-thyroid and pituitary-adrenal axes. Control of GH secretion thus includes two closed-loop systems (GH and IGF-I) and one open-loop regulatory system (neural).

Although most of the evidence for a direct role of GH in its own negative feedback has been derived from animals, an elegant study in normal men demonstrated that GH pretreatment blocks the subsequent GH secretory response to GHRH by a mechanism that is dependent on somatostatin.[221] The mechanism responsible for GH feedback through the hypothalamus has been largely elucidated in rodent models. GH receptors are selectively expressed on somatostatin neurons in the hypothalamic periventricular nucleus and on NPY neurons in the arcuate nucleus. C-Fos gene expression is acutely elevated in both populations of GH receptor-positive neurons by GH administration, indicating an activation of hypothalamic circuitry that includes these neurons. Similarly, GHRH neurons in the arcuate nucleus are acutely activated by MK-0677 because of their selective expression of the GHS-R. Zheng and colleagues[222] showed in the latter group of neurons that c-Fos induction after MK-0677 administration was blocked by pretreatment of mice with GH ( Fig. 7-23 ). The effect must be indirect because there are no GH receptors on GHRH neurons. However, there are type 2 somatostatin receptors expressed on GHRH neurons, and the somatostatin analogue octreotide also significantly blocked c-Fos activation in the arcuate nucleus by MK-0677. The inhibitory effects of either GH or octreotide pretreatment were abolished in knockout mice lacking the specific somatostatin receptor (see Fig. 7-23 ). Together with data from many other experiments, these results strongly support a model of GH-negative feedback regulation that involves the primary activation of periventricular somatostatin neurons by GH. These tuberoinfundibular neurons then inhibit GH secretion directly by release of somatostatin in the median eminence, but they also indirectly inhibit GH secretion by way of collateral axonal projections to the arcuate nucleus that synapse on and inhibit GHRH neurons (see Fig. 7-22 ). It is probable from evidence in rodents that NPY and galanin also play a part in the short-loop feedback of GH secretion, but a definitive mechanism in humans is not yet established.

IGF-I has a major inhibitory action on GH secretion at the level of the pituitary gland.[203] IGF-I receptors are expressed on human somatotroph adenoma cells and inhibit both spontaneous and GHRH-stimulated GH release. In addition, gene expression of both GH and the pituitary-specific transcription factor PIT1 is inhibited by IGF-I. Conflicting data among species suggest that circulating IGF-I may also regulate GH secretion by actions within the brain. The feedback effects of IGF-I account for the fact that in conditions in which circulating levels of IGF-I are low, such as anorexia nervosa, protein-calorie starvation,[223] and Laron dwarfism (the result of a defect in the GH receptor), serum GH levels are elevated.

Neural Control

The predominant hypothalamic influence on GH release is stimulatory, and section of the pituitary stalk or lesions of the basal hypothalamus cause reduction of basal and induced GH release. When the somatostatinergic component is inactivated (e.g., by antisomatostatin antibody injection in rats), basal GH levels and GH responses to the usual provocative stimuli are enhanced.

GHRH-containing nerve fibers that terminate adjacent to portal vessels in the external zone of the median eminence arise principally from within, above, and lateral to the infundibular nucleus in human hypothalamus, corresponding to rodent arcuate and ventromedial nuclei.[224] Perikarya of the tuberoinfundibular somatostatin neurons are located almost completely in the medial periventricular nucleus and parvocellular component of the anterior PVH. Neuroanatomic and functional evidence suggests a bidirectional synaptic interaction between the two peptidergic systems.[203]

Multiple extrahypothalamic brain regions provide efferent connections to the hypothalamus and regulate GHRH and somatostatin neuronal activity ( Fig. 7-24 ; see Fig. 7-22 ). Somatosensory and affective information is integrated and filtered through the amygdaloid complex. The basolateral amygdala provides an excitatory input to the hypothalamus, and the central extended amygdala, which includes the central and medial nuclei of the amygdala together with the bed nucleus of the stria terminalis, provides a GABAergic inhibitory input. Many intrinsic neurons of the hypothalamus also release GABA, often with a peptide cotransmitter. Excitatory cholinergic fibers arise to a small extent from forebrain projection nuclei but mostly from hypothalamic cholinergic interneurons, which densely innervate the external zone of the median eminence. Similarly, the origin of dopaminergic and histaminergic neurons is local with their cell bodies located in the hypothalamic arcuate and tuberomammillary bodies, respectively. Two important ascending pathways to the medial basal hypothalamus regulate GH secretion and originate from serotoninergic neurons in the raphe nuclei and adrenergic neurons in the nucleus of the tractus solitarius and ventral lateral nucleus of the medulla.

Both GHRH and somatostatin neurons express presynaptic and postsynaptic receptors for multiple neurotransmitters and peptides ( Table 7-5 ). The α2-adrenoreceptor agonist clonidine reliably stimulates GH release, and for this reason a clonidine test was a standard diagnostic tool in pediatric endocrinology. The stimulatory effect is blocked by the specific α2-antagonist yohimbine and appears to involve a dual mechanism of action, inhibition of somatostatin neurons and activation of GHRH neurons. In addition, partial attenuation of the effects of clonidine by mixed 5-HT1 and 5-HT2 antagonists suggests that some of the relevant α2-receptors are located presynaptically on serotoninergic nerve terminals and increase serotonin release. Both norepinephrine and epinephrine play physiologic roles in the adrenergic stimulation of GH secretion. The α1-agonists have no effect on GH secretion in humans, but β2-agonists such as the bronchodilator salbutamol inhibit GH secretion by stimulating the release of somatostatin from nerve terminals in the median eminence. These effects are blocked by propranolol, a nonspecific β-antagonist. Dopamine generally has a net effect to stimulate GH secretion, but the mechanism is not clear because of multiple dopamine receptor subtypes and the apparent activation of both GHRH and somatostatin neurons.

TABLE 7-5 — FACTORS THAT CHANGE GROWTH HORMONE SECRETION IN HUMANS
Physiologic Hormones and Neurotransmitters Pathologic
STIMULATORY FACTORS
Episodic, spontaneous release Insulin hypoglycemia Acromegaly
Exercise 2-Deoxyglucose TRH
Stress Amino acid infusions GnRH
Physical Arginine, lysine Glucose
Psychological Neuropeptides Arginine
Slow wave sleep GHRH Interleukins 1, 2, 6
Postprandial glucose decline Ghrelin Protein depletion
Fasting Galanin Starvation
Opioids (μ-receptors) Anorexia nervosa
Melatonin Renal failure
Classical neurotransmitters Liver cirrhosis
α2-Adrenergic agonists Type 1 diabetes mellitus
β-Adrenergic antagonists
M1-cholinergic agonists
5-HT1D-serotonin agonists
H1-histamine agonists
GABA (basal levels)
Dopamine (? D2 receptor)
Estrogen
Testosterone
Glucocorticoids (acute)
INHIBITORY FACTORS[*]
Postprandial hyperglycemia Glucose infusion Acromegaly
Elevated free fatty acids Neuropeptides l-Dopa
Elevated GH levels Somatostatin D2R DA agonists
Elevated IGF-I (pituitary) Calcitonin Phentolamine
Rapid eye movement (REM) sleep Neuropeptide Y (NPY[†]) Galanin
Senescence, aging CRH[†] Obesity
Classical neurotransmitters Hypothyroidism
α1/2-Adrenergic antagonists Hyperthyroidism
β2-Adrenergic agonists
H1-Histamine antagonists
Serotonin antagonist
Nicotinic cholinergic agonists
Glucocorticoids (chronic)

CRH, Corticotropin-releasing hormone; DA, dopamine; GHRH, growth hormone-releasing hormone; GnRH, gonadotropin-releasing hormone; IGF-I, insulin-like growth factor I; TRH, thyrotropin-releasing hormone.

* In many instances, the inhibition can be demonstrated only as a suppression of GH release induced by a pharmacologic stimulus.
† The inhibitory actions of NPY and CRH on GH secretion are firmly established in the rodent and are secondary to increased somatostatin tone. Contradictory evidence exists in the human for both peptides and further studies are required.

Serotonin’s effect on GH release in humans was difficult to decipher because of the large number of receptor subtypes. However, clinical studies with the receptor-selective agonist sumatriptan clearly implicated the 5-HT1D receptor subtype in the stimulation of basal GH levels.[225] The drug also potentiates the effect of a maximal dose of GHRH, suggesting the recurring theme of GH disinhibition by inhibition of hypothalamic somatostatin neurons in its mechanism of action. Histaminergic pathways acting through H1 receptors play only a minor, conditional stimulatory role in GH secretion in humans.

Acetylcholine appears to be an important physiologic regulator of GH secretion.[226] Blockade of acetylcholinergic muscarinic receptors reduces or abolishes GH secretory responses to GHRH, glucagon and arginine, morphine, and exercise. In contrast, drugs that potentiate cholinergic transmission increase basal GH levels and enhance the GH response to GHRH in normal individuals or in subjects with obesity or Cushing’s disease. In vitro acetylcholine inhibits somatostatin release from hypothalamic fragments, and acetylcholine can act directly on the pituitary to inhibit GH release. There even may be a paracrine cholinergic control system within the pituitary. However, the sum of evidence suggests that the primary mechanism of action of M1 agonists is inhibition of somatostatin neuronal activity or the release of peptide from somatostatinergic terminals. Short-term cholinergic blockade with the M1 muscarinic receptor antagonist pirenzepine reduced the GH excess of patients with poorly controlled diabetes mellitus.[227] However, in the long term, cholinergic blockade did not prevent complications associated with the hypersomatotropic state.

Many neuropeptides in addition to GHRH and somatostatin are involved in the modulation of GH secretion in humans (see Table 7-5 ). [203] [208] Among these, the evidence is most compelling for a stimulatory role of galanin acting in the human hypothalamus by a GHRH-dependent mechanism.[228] Many GHRH neurons are immunopositive for galanin as well as neurotensin and tyrosine hydroxylase. Galanin’s actions may be explained, in part, by presynaptic facilitation of catecholamine release from nerve terminals and subsequent direct adrenergic stimulation of GHRH release.[229] Opioid peptides also stimulate GH release, probably by disinhibition of GHRH neurons, but under normal circumstances endogenous opioid tone in the hypothalamus is presumed to be low because opioid antagonists have little acute effect on GH secretion.

A larger number of neuropeptides are known or suspected to inhibit GH secretion in humans, at least under certain circumstances.[208] The list includes NPY, CRH, calcitonin, oxytocin, neurotensin, VIP, and TRH. Inhibitory actions of NPY are well established in the rat. The effect on GH secretion is secondary to stimulation of somatostatin neurons and is of particular interest because of the presumed role in GH autofeedback (discussed earlier) and the integration of GH secretion with regulation of energy intake and expenditure (discussed in a later section see External and Metabolic Signals). Finally, TRH has the well-established paradoxical effect of increasing GH secretion in patients with acromegaly, type 1 diabetes mellitus, hypothyroidism, or hepatic and renal failure.

Factors Influencing Secretion of Growth Hormone

Human Growth Hormone Rhythms

The deciphering of rhythmic GH secretion has relied on a combination of technical innovations in sampling and GH assay, and sophisticated mathematical modeling including deconvolution analysis and the calculation of approximate entropy as a measure of orderliness or regularity in minute-to-minute secretory patterns.[208] At least three distinct categories of GH rhythms, which differ markedly in their time scales, can be considered here. The daily GH secretion rate varies over two orders of magnitude from a maximum of nearly 2.0 mg/day in late puberty to a minimum of 20 μg/day in older or obese adults. The neonatal period is characterized by markedly amplified GH secretory bursts followed by a prepubertal decade of stable, moderate GH secretion of 200 to 600 μg/day. There is a marked increase in daily GH secretion during puberty that is accompanied by a commensurate rise in plasma IGF-I to levels that constitute a state of physiologic hypersomatotropism. This pubertal increase in GH secretion is due to increased GH mass per secretory burst and not to increased pulse frequency. Although the changes are clearly related to the increases in gonadal steroid hormones and can be mimicked by administration of estrogen or testosterone to hypogonadal children, the underlying neuroendocrine mechanisms are not fully understood. One hypothesis is that decreased sensitivity of the hypothalamic-pituitary axis to negative feedback of GH and IGF-I leads to increased GHRH release and action. Young adults have a return of daily GH secretion to prepubertal levels despite continued gonadal steroid elevation. The so-called somatopause is defined by an exponential decline in GH secretory rate with a half-life of 7 years starting in the third decade of life.

GH secretion in young adults exhibits a true circadian rhythm over a 24-hour period, characterized by a greater nocturnal secretory mass that is independent of sleep onset.[230] However, as discussed earlier, GH release is further facilitated when slow wave sleep coincides with the normal circadian peak. Under basal conditions, GH levels are low most of the time, with an ultradian rhythm of about 10 (men) or 20 (women) secretory pulses per 24 hours as calculated by deconvolution analysis.[231] Both sexes have an increased pulse frequency during the nighttime hours, but the fraction of total daily GH secretion associated with the nocturnal pulses is much greater in men. Overall, women have more continuous GH secretion and more frequent GH pulses that are of more uni-form size than men.[231] A complementary study using approximate entropy analysis concluded that the nonpulsatile regularity of GH secretion is also significantly different in men and women.[232] These sexually dimorphic patterns in the human are actually quite similar to those in the rat, although the sex differences are not as extreme in humans. [208] [232] The neuroendocrine basis for sex differences in the ultradian rhythm of GH secretion is not fully understood. Gonadal sex steroids play both an organizational role during development of the hypothalamus and an activational role in the adult, regulating expression of the genes for many of the peptides and receptors central to GH regulation. [203] [208] In the human, unlike the rat, the hypothalamic actions of testosterone appear to be predominantly due to its aromatization to 17β-estradiol and interaction with estrogen receptors. Hypothalamic somatostatin appears to play a more prominent role in men than in women in the regulation of pulsatile GH secretion, and this difference is postulated to be a key factor in producing the sexual dimorphism. [231] [233] [234]

External and Metabolic Signals

The various peripheral signals that modulate GH secretion in humans are summarized in Table 7-5 (also see Figs. 7-22 and 7-24 [22] [24]). Of particular importance are factors related to energy intake and metabolism because they provide a common signal between the peripheral tissues and hypothalamic centers regulating nonendocrine homeostatic pathways in addition to the classical hypophyseotropic neurons. It is also in this complex arena that species-specific regulatory responses are particularly prominent, making extrapolations between rodent experimental models and human GH regulation less reliable. [203] [208]

Important triggers of GH release include the normal decrease in blood glucose level after intake of a carbohydrate-rich meal, absolute hypoglycemia, exercise, physical and emotional stress, and high intake of protein (mediated by amino acids). Some of the pathologic causes of elevated GH represent extremes of these physiologic signals and include protein-calorie starvation, anorexia nervosa, liver failure, and type 1 diabetes mellitus. A critical concept is that many of these GH triggers work through the same final common mechanism of somatostatin withdrawal and consequent disinhibition of GH secretion. In contrast, postprandial hyperglycemia, glucose infusion, elevated plasma free fatty acids, type 2 diabetes mellitus (with obesity and insulin resistance), and obesity are all associated with inhibition of GH secretion. The role of leptin in mediating either increases or decreases in GH release is complicated by its multiple sites of action and coexistent secretory environment. Similarly, other members of the cytokine family including IL-1, IL-2, IL-6, and endotoxin have been inconsistently shown to stimulate GH in humans.

The actions of steroid hormones on GH secretion are complex because of their multiple loci of action within the proximal hypothalamic-pituitary components in addition to secondary effects on other neural and endocrine systems. Glucocorticoids in particular produce opposite responses that are dependent on the chronicity of administration. Moreover, glucocorticoid effects follow an inverted U-shaped dose-response curve. Both low and high glucocorticoid levels reduce GH secretion, the former because of decreased GH gene expression and somatotroph responsiveness to GHRH and the latter because of increased hypothalamic somatostatin tone and decreased GHRH. Similarly, physiologic levels of thyroid hormones are necessary to maintain GH secretion and promote GH gene expression. Excessive thyroid hormone is also inhibitory to the GH axis, and the mechanism is speculated to be a combination of increased hypothalamic somatostatin tone, GHRH deficiency, and suppressed pituitary GH production.

Somatostatin

Chemistry and Evolution

A factor that potently inhibited GH release from pituitary in vitro was unexpectedly identified during early efforts to isolate GHRH from hypothalamic extracts.[235] Somatostatin, the peptide responsible for this inhibition of GH secretion and the inhibition of insulin secretion by a pancreatic islet extract, was eventually isolated from hypothalamus and sequenced by Brazeau and colleagues in 1973.[236] The term somatostatin was originally applied to a cyclic peptide containing 14 amino acids (somatostatin-14 [SST-14]; Fig. 7-25 ). Subsequently, a second form, N-extended somatostatin-28 (SST-28), was identified as a secretory product. Both forms of somatostatin are derived by independent cleavage of a common prohormone by prohormone convertases.[237] In addition, the isolation of SST-28(1-12) in some tissues suggests that SST-14 can be secondarily processed from SST-28. SST-14 is the predominant form in the brain (including the hypothalamus), whereas SST-28 is the major form in the gastrointestinal tract, especially the duodenum and jejunum.

The name somatostatin is descriptively inadequate because the molecule also inhibits TSH secretion from the pituitary and has nonpituitary roles including activity as a neurotransmitter or neuromodulator in the central and peripheral nervous systems and as a regulatory peptide in gut and pancreas. As a pituitary regulator, somatostatin is a true neurohormone, that is, a neuronal secretory product that enters the blood (hypophyseal-portal circulation) to affect cell function at remote sites. In the gut, somatostatin is present in both the myenteric plexus, where it acts as a neurotransmitter, and epithelial cells, where it influences the function of adjacent cells as a paracrine secretion. Somatostatin can influence its own secretion from delta cells (an autocrine function) in addition to acting as a paracrine factor in pancreatic islets. Gut exocrine secretion can be modulated by intraluminal action, so it is also a lumone. Because of its wide distribution, broad spectrum of regulatory effects, and evolutionary history, this peptide can be regarded as an archetypical pan-system modulator.

The genes that encode somatostatin in humans[238] (see Fig. 7-25 ) and a number of other species exhibit striking sequence homology, even in primitive fish such as the anglerfish. Furthermore, the amino acid sequence of SST-14 is identical in all vertebrates. Formerly, it was accepted that all tetrapods have a single gene encoding both SST-14 and SST-28 whereas teleost fish have two nonallelic pre-prosomatostatin genes (PPSI and PPSII), each of which encodes only one form of the mature somatostatin peptides. This situation implied that a common ancestral gene underwent a duplication event after the split of teleosts from the descendants of tetrapods.

However, both lampreys and amphibians, which predate and postdate the teleost evolutionary divergence, respectively, have now been shown to have at least two PPS genes.[239] A more distantly related gene has been identified in mammals that encodes cortistatin, a somatostatin-like peptide that is highly expressed in cortex and hippocampus. [240] [241] Cortistatin-14 differs from SST-14 by three amino acid residues but has high affinity for all known subtypes of somatostatin receptors (see next section). The human gene sequence predicts a tripeptide-extended cortistatin-17 and a further N-terminally extended cortistatin-29.[242] A revised evolutionary concept of the somatostatin gene family is that a primordial gene underwent duplication at or before the advent of chordates and the two resulting genes under-went mutation at different rates to produce the distinct pre-prosomatostatin and pre-procortistatin genes in mammals.[239] A second gene duplication probably occurred in teleosts to generate PPSI and PPSII from the ancestral somatostatin gene.

Apart from its expression in neurons of the periventricular and arcuate hypothalamic nuclei and involvement in GH secretion discussed earlier, somatostatin is highly expressed in the cortex, lateral septum, extended amygdala, reticular nucleus of the thalamus, hippocampus, and many brain stem nuclei. Cortistatin is present in the brain at a small fraction of the levels of somatostatin and in a more limited distribution primarily confined to cortex and hippocampus. The molecular mechanisms underlying the developmental and hormonal regulation of somatostatin gene transcription have been most extensively studied in pancreatic islet cells. [243] [244] [245] Less is known concerning the regulation of somatostatin gene expression in neurons except that activation is strongly controlled by binding of the phosphorylated transcription factor cAMP response element-binding protein to its cognate cAMP response element contained in the promoter sequence. [246] [247] Enhancer elements in the somatostatin gene promoter that bind complexes of homeodomain-containing transcription factors (PAX6, PBX, PREP1) and up-regulate gene expression in pancreatic islets may actually represent gene silencer elements in neurons (see Fig. 7-25 , promoter elements TSEII and UE-A).[245] Conversely, another related cis element in the somatostatin gene (see Fig. 7-25 , promoter element TSEI) apparently binds a homeodomain transcription factor PDX1 (also called STF1/IDX1/IPF1) that is common to developing brain, pancreas, and foregut and regulates gene expression in both the CNS and gut.[248]

The function of somatostatin in GH and TSH regulation is considered earlier in this chapter. Its actions in the extrahypothalamic brain and diagnostic and therapeutic roles are considered in the remainder of this section and in Chapter 8 . An additional function of somatostatin in pancreatic islet cell regulation is described in Chapter 33 , and the manifestations of somatostatin excess as in somatostatinoma are described in Chapter 38 .

Somatostatin Receptors

Five somatostatin receptor subtypes (SSTR1 to SSTR5) have been identified by gene cloning techniques and one of these (SSTR2) is expressed in two alternatively spliced forms.[249] These subtypes are encoded by separate genes located on different chromosomes, are expressed in unique or partially overlapping distributions in multiple target organs, and differ in their coupling to second messenger signaling molecules and therefore in their range and mechanism of intracellular actions. [249] [250] The subtypes also differ in their binding affinity to specific somatostatin analogues. Certain of these differences have important implications for the use of somatostatin analogues in therapy and in diagnostic imaging.

All SSTR subtypes are coupled to pertussis toxin–sensitive G proteins and bind SST-14 and SST-28 with high affinity in the low nanomolar range, although SST-28 has a uniquely high affinity for SSTR5. SSTR1 and SSTR2 are the two most abundant subtypes in brain and probably function as presynaptic autoreceptors in the hypothalamus and limbic forebrain, respectively, in addition to their postsynaptic actions. SSTR4 is most prominent in hippocampus. All the subtypes are expressed in pituitary, but SSTR2 and SSTR5 are the most abundant on somatotrophs. These two subtypes are also the most physiologically important in pancreatic islets, with SSTR5 responsible for inhibition of insulin secretion from bata cells and SSTR2 responsible for inhibition of glucagon from alpha cells.[251]

Binding of somatostatin to its receptor leads to activation of one or more plasma membrane-bound inhibitory G proteins (Gi/o), which in turn inhibit adenylyl cyclase activity and lower intracellular cAMP. Other G protein–mediated actions common to all SSTRs are activation of a vanadate-sensitive phosphotyrosine phosphatase and modulation of mitogen-activated protein kinase (MAPK). Different subsets of SSTRs are also coupled to inwardly rectifying K+ channels, voltage-dependent Ca2+ channels, a Na+/H+ exchanger, α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)-kainate glutamate receptors, phospholipase C, and phospholipase A2.[249] The lowering of intracellular cAMP and Ca2+ is the most important mechanism for the inhibition of hormone secretion, and actions on phosphotyrosine phosphatase and MAPK are postulated to play a role in somatostatin’s antiproliferative effect on tumor cells.

Effects on Target Tissues and Mechanism of Action

In the pituitary, somatostatin inhibits secretion of GH and TSH and, under certain conditions, of PRL and ACTH as well. It exerts inhibitory effects on virtually all endocrine and exocrine secretions of the pancreas, gut, and gallbladder ( Table 7-6 ). Somatostatin inhibits secretion by the salivary glands and, under some conditions, the secretion of parathyroid hormone and calcitonin. Somatostatin blocks hormone release in many endocrine-secreting tumors, including insulinomas, glucagonomas, VIPomas, carcinoid tumors, and some gastrinomas.

TABLE 7-6 — BIOLOGIC ACTIONS OF SOMATOSTATIN OUTSIDE THE CENTRAL NERVOUS SYSTEM
Inhibits Hormone Secretion by Inhibits Other Gastrointestinal Actions
Pituitary gland GH, thyrotropin, ACTH, prolactin

Gastrointestinal tract Gastrin
Secretin
Gastrointestinal polypeptide
Motilin
Glicentin (enteroglucagon)
VIP

Pancreas Insulin
Glucagon
Somatostatin

Genitourinary tract Renin

Gastric acid secretion
Gastric and jejunal fluid secretion
Gastric emptying
Pancreatic bicarbonate secretion
Pancreatic enzyme secretion
(Stimulates intestinal absorption of water and electrolytes)
Gastrointestinal blood flow
AVP-stimulated water transport
Bile flow
Extragastrointestinal Actions
Inhibits the function of activated immune cells
Inhibition of tumor growth

ACTH, Adrenocorticotropic hormone; AVP, arginine vasopressin; GH, growth hormone; VIP, vasoactive intestinal peptide.

The physiologic actions of somatostatin in extrahypothalamic brain remain the subject of investigation.[252] In the striatum, somatostatin increases the release of dopamine from nerve terminals by a glutamate-dependent mechanism. It is widely expressed in GABAergic interneurons of limbic cortex and hippocampus, where it modulates the excitability of pyramidal neurons. Temporal lobe epilepsy is associated with a marked reduction in somatostatin-expressing neurons in the hippocampus consistent with a putative inhibitory action on seizures.[253] A wealth of correlative data has linked reduced forebrain and CSF concentrations of somatostatin with Alzheimer’s disease, major depression, and other neuropsychiatric disorders, raising speculation about the role of somatostatin in modulating neural circuits underlying cognitive and affective behaviors.[254]

Clinical Applications of Somatostatin Analogues

An extensive pharmaceutical discovery program has produced somatostatin analogues with receptor subtype selectivity and improved pharmacokinetics and oral bioavailability compared with the native peptide. Initial efforts focused on the rational design of constrained cyclic peptides that incorporated D-amino acid residues and included the Trp8-Lys9 dipeptide of somatostatin, which was shown by structure-function studies to be necessary for high-affinity binding to its receptor (see Fig. 7-25 ). Many such analogues have been studied in clinical trials including octreotide, lanreotide, vapreotide, and the hexapeptide MK-678. These compounds are agonists with similarly high-affinity binding to SSTR2 and SSTR5, moderate binding to SSTR3, and no (or low) binding to SSTR1 and SSTR4. A combinatorial chemistry approach has led to a new generation of nonpeptidyl somatostatin agonists that bind selectively and with subnanomolar affinity to each of the five SSTR subtypes. [255] [256] In contrast to the marked success in development of potent and selective somatostatin agonists, there is a relative paucity of useful antagonists.[249]

The actions of octreotide (SMS 201-995 or Sandostatin) illustrate the general potential of somatostatin analogues in therapy. [257] [258] Octreotide controls excess secretion of GH in acromegaly in most patients and shrinks tumor size in about one third. It is also indicated for the treatment of TSH-secreting adenomas that recur after surgery. It is used to treat other functioning metastatic neuroendocrine tumors, including carcinoid, VIPoma, glucagonoma, and insulinoma, but is seldom of use for the treatment of gastrinoma. Octreotide is also useful in the management of many forms of diarrhea (acting on salt and water excretion mechanisms in the gut) and in reducing external secretions in pancreatic fistulae (thus permitting healing). A decrease in blood flow to the gastrointestinal tract is the basis for its use in bleeding esophageal varices, but it is not effective in the treatment of bleeding from a peptic ulcer.

The only major undesirable side effect of octreotide is reduction of bile production and of gallbladder contractility, leading to “sludging” of bile and an increased incidence of gallstones. Other common adverse effects including nausea, abdominal cramps, diarrhea secondary to malabsorption of fat, and flatulence usually subside spontaneously within 2 weeks of continued treatment. Impaired glucose tolerance is not associated with long-term octreotide therapy, despite an inhibitory effect on insulin secretion, because of compensating reductions in carbohydrate absorption and GH and glucagon secretion that are caused by the drug.

Somatostatin analogues labeled with a radioactive tracer have been used as external imaging agents for a wide range of disorders. [257] [258] A 111In-labeled analogue of octreotide (OctreoScan) has been approved for clinical use in the United States and several other countries ( Fig. 7-26 ). The majority of neuroendocrine tumors and many pituitary tumors that express somatostatin receptors are visualized by external imaging techniques after administration of this agent; a variety of nonendocrine tumors and inflammatory lesions are also visualized, all of which have in common the expression of somatostatin receptors. Such tumors include non–small cell cancer of the lung (100%), meningioma (100%), breast cancer (74%), and astrocytomas (67%). Because activated T cells of the immune system display somatostatin receptors, inflammatory lesions that take up the tracer include sarcoidosis, Wegener’s granulomatosis, tuberculosis, and many cases of Hodgkin’s disease and non-Hodgkin’s lymphoma. Although the tracer lacks specificity in differential diagnosis, its ability to identify the presence of abnormality and the extent of the lesion provides important information for management, including tumor staging. The use of a small handheld radiation detector in the operating room makes it possible to ensure the completeness of removal of medullary thyroid carcinoma metastases.[259] New developments in the synthesis of tracers chelated to octreotide for positron emission tomography have allowed the sensitive detection of meningiomas only 7 mm in diameter and located beneath osseous structures at the base of the skull.[260]

The ability of somatostatin to inhibit the growth of normal and some neoplastic cell lines and to reduce the growth of experimentally induced tumors in animal models has stimulated interest in somatostatin analogues for the treatment of cancer. Somatostatin’s tumoristatic effects may be a combination of direct actions on tumor cells related to inhibition of growth factor receptor expression, inhibition of MAPK, and stimulation of phosphotyrosine phosphatase. SSTR1, SSTR2, SSTR4, and SSTR5 can all promote cell cycle arrest associated with induction of the tumor suppressor retinoblastoma and p21, and SSTR3 can trigger apoptosis accompanied by induction of the tumor suppressor p53 and the proapoptotic protein Bax.[249] In addition, somatostatin has indirect effects on tumor growth by its inhibition of circulating, paracrine, and autocrine tumor growth-promoting factors and it can modulate the activity of immune cells and influence tumor blood supply. Despite this promise, the therapeutic utility of octreotide as an antineoplastic agent remains controversial.

Two new treatment approaches in preclinical trials may yet effectively utilize somatostatin receptors in the arrest of cancer cells.[257] The first is receptor-targeted radionuclide therapy using octreotide chelated to a variety of γ- or β-emitting radioisotopes. Theoretical calculations and empirical data suggest that radiolabeled somatostatin analogues can deliver a tumoricidal radiotherapeutic dose to some tumors after receptor-mediated endocytosis. A variation on this theme is the chelation of a cytotoxic chemotherapeutic agent to a somatostatin analogue. A second approach involves somatic cell gene therapy to transfect SSTR-negative pancreatic cancer cells with an SSTR gene.[261] Therapeutic results can be obtained with the creation of autocrine or paracrine inhibitory growth effects or the addition of targeted radionuclide treatments.

Prolactin-Regulating Factors

Dopamine

It is well known that PRL secretion, unlike the secretion of other pituitary hormones, is primarily under tonic inhibitory control by the hypothalamus ( Fig. 7-27 ).[262] Destruction of the stalk median eminence or transplantation of the pituitary gland to ectopic sites causes a marked constitutive increase in PRL secretion, in contrast to a decrease in the release of GH, TSH, ACTH, and the gonadotropins. Many lines of evidence indicate that dopamine is the principal, physiologic prolactin-inhibiting factor (PIF) released from the hypothalamus.[263] Dopamine is present in hypophyseal-portal vessel blood in sufficient concentration to inhibit PRL release,[264] dopamine inhibits PRL secretion from lactotrophs both in vivo and in vitro,[265] and dopamine D2 receptors are expressed on the plasma membrane of lactotrophs. [266] [267] Mutant mice with a targeted disruption of the D2 receptor gene uniformly developed lactotroph hyperplasia, hyperprolactinemia, and eventually lactotroph adenomas, further emphasizing the importance of dopamine in the physiologic regulation of lactotroph proliferation in addition to hormone secretion.[268]

The intrinsic dopamine neurons of the medial-basal hypothalamus constitute a dopaminergic population with regulatory properties that are distinct from those in other areas of the brain. Notably, they lack D2 autoreceptors but express PRL receptors, which are essential for positive feedback control as discussed in detail later (see Feedback Control). In the rat, these neurons are subdivided by location into the A12 group within the arcuate nucleus and the A14 group in the anterior periventricular nucleus. The caudal A12 dopamine neurons are further classified as tuberoinfundibular (TIDA) because of their axonal projections to the external zone of the median eminence.

Tuberohypophyseal (THDA) neuronal soma are located more rostrally in the arcuate nucleus and project to both the neural lobe and intermediate lobe through axon collaterals that are found in the internal zone of the median eminence. Finally, the A14 periventricular hypophyseal (PHDA) neurons send their axons only to the intermediate lobe of the pituitary gland.

Although the TIDA neurons are generally considered to be the major source of dopamine to the anterior lobe through the long portal vessels originating in the median eminence, dopamine can also reach the anterior lobe from the neural and intermediate lobes by the interconnecting short portal veins.[269] Consistent with this pathway for dopamine access to the anterior lobe, surgical removal of the neurointermediate lobe in rats caused a significant increase in basal PRL levels.[270] In addition to direct actions of dopamine on lactotrophs, central dopamine can indirectly affect PRL secretion by altering the activity of inhibitory interneurons that in turn synapse on the TIDA neurons. These effects are complicated by opposing intracellular signaling pathways linked to D1 and D2 receptors located on different populations of interneurons.[271]

The binding of dopamine or selective agonists such as bromocriptine to the D2 receptor has multiple effects on lactotroph function. D2 receptors are coupled to pertussis toxin-sensitive G proteins and inhibit adenylyl cyclase and decrease intracellular cAMP levels. Other effects include activation of an inwardly rectifying K+ channel, increase of voltage-activated K+ currents, decrease of voltage-activated Ca2+ currents, and inhibition of inositol phosphate production. Together, this spectrum of intracellular signaling events decreases free Ca2+ concentrations and inhibits exocytosis of PRL secretory granules.[272] Dopamine also has a modest effect on thyrotrophs to inhibit the secretion of TSH.

There is continuing debate concerning the mechanism by which D2 receptor activation inhibits transcription of the PRL gene. Likely pathways involve the inhibition of MAPK or protein kinase C, with a resultant reduction in the phosphorylation of Ets family transcription factors. Ets factors are important for the stimulatory responses of TRH, insulin, and epidermal growth factor on PRL expression [273] [274] [275] and they interact cooperatively with the pituitary-specific POU protein Pit1, which is essential for cAMP-mediated PRL gene expression.[276]

The second messenger pathways used by the D2 receptor to inhibit lactotroph cell division are also unsettled. A study using primary pituitary cultures from rats demonstrated that forskolin treatment, which activates protein kinase A and elevates intracellular cAMP, or insulin treatment, which activates a potent receptor tyrosine kinase, were both effective mitogenic stimuli for lactotrophs. Bromocriptine competitively antagonized the proliferative response caused by elevated cAMP. Furthermore, inhibition of MAPK signaling by PD98059 markedly suppressed the mitogenic action of both insulin and forskolin, suggesting an interaction of MAPK and protein kinase A signaling.[277]

Another study used immortalized mammosomatotroph tumor cells that were transfected with a D2 receptor expression vector and concluded that stimulation of a phosphotyrosine phosphatase activity was an important component of dopamine’s antiproliferative action.[278] Therefore, it is clear that dopamine actions on lactotrophs involve multiple different intracellular signaling pathways linked to activation of the D2 receptor, but different combinations of these pathways are relevant for the inhibitory effects on PRL secretion, PRL gene transcription, and lactotroph proliferation.

The other major action of dopamine in the pituitary is the inhibition of hormone secretion from the POMC-expressing cells of the intermediate lobe, although, as noted earlier, the adult human differs from most other mammals in the rudimentary nature of this lobe. THDA and PHDA axon terminals provide a dense plexus of synaptic-like contacts on melanotrophs. Dopamine release from these terminals is inversely correlated with serum MSH levels[279] and also regulates POMC gene expression and melanotroph proliferation.[280]

Other hypothalamic factors probably play a role secondary to that of dopamine as additional PIFs.[262] The primary reason to conjecture the existence of these PIFs is the frequent inconsistency between portal dopamine levels and circulating PRL in different rat models. GABA is the strongest candidate and most likely acts through GABAA inotropic receptors in the anterior pituitary. Melanotrophs, like lactotrophs, are inhibited by both dopamine and GABA but with the principal involvement of G protein–coupled, metabotropic GABAB receptors.[281] Because basal dopamine tone is high, the measurable inhibitory effects of GABA on PRL release are generally small under normal circumstances. Other putative PIFs include somatostatin and calcitonin.

Prolactin-Releasing Factors

Although tonic suppression of PRL release by dopamine is the dominant effect of the hypothalamus on PRL secretion, a number of stimuli promote PRL release, not merely by disinhibition of PIF effects but by causing release of one or more neurohormonal PRFs (see Fig. 7-27 ). The most important of the putative PRFs are TRH, oxytocin, and VIP, but vasopressin, angiotensin II, NPY, galanin, substance P, bombesin-like peptides, and neurotensin can also trigger PRL release under different physiologic circumstances.[262] TRH is discussed in a previous section of this chapter. In humans there is an imperfect correlation between pulsatile PRL and TSH release, suggesting that TRH cannot be the sole physiologic PRF under basal conditions.[282]

Like TRH, oxytocin, vasopressin, and VIP fulfill all the basic criteria for a PRF. They are produced in paraventricular hypothalamic neurons that project to the median eminence. Concentrations of the hormones in portal blood are much higher than in the peripheral circulation and are sufficient to stimulate PRL secretion in vitro. Moreover, there are functional receptors for each of the neurohormones in the anterior pituitary gland and either pharmacologic antagonism or passive immunization against each hormone can decrease PRL secretion, at least under certain circumstances. [283] [284] [285] [286] [287]

Vasopressin is released during stress and hypovolemic shock, as is PRL, suggesting a specific role for vasopressin as a PRF in these contexts. Similarly, another candidate PRF, peptide histidine isoleucine, may be specifically involved in the secretion of PRL in response to stress. Peptide histidine isoleucine and the human homologue PHM are structurally related to VIP and synthesized from the same prohormone precursor in their respective species.[288] Both peptides are coexpressed with CRH in parvocellular paraventricular neurons and presumably released by the same stimuli that cause release of CRH into the hypophyseal-portal vessels.[289]

There is evidence suggesting that dopamine itself may also act as a PRF, in contrast to its predominant function as a PIF.[262] At concentrations three orders of magnitude lower than that associated with maximal inhibition of PRL secretion, dopamine was shown to be capable of stimulating secretion from primary cultures of rat pituitary cells.[290] These studies were extended to an in vivo model by Arey and colleagues,[291] who demonstrated that low-dose dopamine infusion in cannulated rats caused a further increase in circulating PRL above the already elevated baseline produced by pharmacologic blockade of endogenous dopamine biosynthesis. The physiologic relevance of these findings to humans has yet to be established.

Finally, reports of “new” PRFs continue to be published. Much excitement was generated by the isolation of a mammalian RFamide peptide from bovine hypothalamus named prolactin-releasing peptide (PrRP). [292] [293] PrRP binds with high affinity to its G protein–coupled receptor expressed specifically in human pituitary and selectively stimulates PRL release from rat pituitary cells with a potency similar to that of TRH. However, PrRP is expressed predominantly in a subpopulation of noradrenergic neurons in the medulla and a small population of nonneurosecretory neurons of the VMH, raising the serious question of whether PrRP reaches the anterior pituitary and actually causes PRL secretion. Subsequent studies found no direct evidence for release of PrRP in the arcuate nucleus-median eminence, further suggesting that the peptide is not a hypophyseotropic neurohormone. However, PrRP probably does function as a neuromodulator within the CNS at sites expressing its receptor and may be involved in the neural circuitry mediating satiety.[293]

Intrapituitary Regulation of Prolactin Secretion

Probably more than that of any other pituitary hormone, the secretion of PRL is regulated by autocrine-paracrine factors within the anterior lobe and by neurointermediate lobe factors that gain access to venous sinusoids of the anterior lobe by way of the short portal vessels. The wealth of local regulatory mechanisms within the anterior lobe has been reviewed extensively [262] [294] and is also discussed in Chapter 8 . Galanin, VIP, endothelin-like peptides, angiotensin II, epidermal growth factor, basic fibroblast growth factor, GnRH, and the cytokine IL-6 are among the most potent local stimulators of PRL secretion. Locally produced inhibitors include PRL itself, acetylcholine, transforming growth factor β, and calcitonin. Although none of these stimulatory or inhibitory factors plays a dominant role in the regulation of lactotroph function and much of the research in this area has not been directly confirmed in human pituitary, it seems apparent that the local milieu of autocrine and paracrine factors plays an essential modulatory role in determining the responsiveness of lactotrophs to hypothalamic factors in different physiologic states.

As noted earlier, a proportion of the inhibitory dopamine tone to the anterior lobe lactotrophs is derived from the neurointermediate lobe. It was therefore unanticipated that surgical removal of this structure in rats would block suckling-induced PRL release over the moderate basal increase attributed to partial dopamine disinhibition.[295] Further studies showed that exposure of the anterior pituitary to intermediate lobe extracts (devoid of VIP, vasopressin, and other known PRFs) stimulated PRL secretion. At least two kinds of PRF activity have been isolated from intermediate lobe tumors of the mouse, but the specific molecules involved have yet to be identified.[296] Other researchers have suggested a more passive role for the neurointermediate lobe in the regulation of PRL secretion. Melanotroph-derived N-acetylated MSH appears to act as a lactotroph responsiveness factor by recruiting nonsecretory cells to an active state and sensitizing secreting lactotrophs to the actions of other direct PRFs.[297] However, the relevance of the neurointermediate lobe for PRL regulation in primates (including humans) is not clear because of its attenuated structure in these species.

Neuroendocrine Regulation of Prolactin Secretion

Secretion of PRL, like that of other anterior pituitary hormones, is regulated by hormonal feedback and neural influences from the hypothalamus. [262] [263] [298] Feedback is exerted by PRL itself at the level of the hypothalamus. PRL secretion is regulated by many physiologic states including the estrous and menstrual cycles, pregnancy, and lactation. Furthermore, PRL is stimulated by several exteroceptive stimuli including light, ultrasonic vocalization of rodent pups, olfactory cues, and various modalities of stress. Expression and secretion of PRL are also influenced strongly by estrogens at the level of both the lactotrophs and TIDA neurons[299] (see Fig. 7-27 ) and by paracrine regulators within the pituitary such as galanin and VIP.

Feedback Control

Negative feedback control of PRL secretion is mediated by a unique short-loop mechanism within the hypothalamus.[300] PRL activates PRL receptors, which are expressed on all three subpopulations of A12 and A14 dopamine neurons, leading to increased tyrosine hydroxylase expression and dopamine synthesis and release. [299] [301] Ames dwarf mice that secrete virtually no PRL, GH, or TSH have decreased numbers of arcuate dopamine neurons and this hypoplasia can be reversed by neonatal administration of PRL, suggesting a trophic action on the neurons.[302] However, another mouse model of isolated PRL deficiency generated by gene targeting appears to have normal numbers of hypofunctioning dopamine neurons secondary to the loss of PRL feedback.[303]

Neural Control

Lactotrophs have spontaneously high secretory activity, and therefore the predominant effect of the hypothalamus on PRL secretion is tonic suppression, which is mediated by regulatory hormones synthesized by tuberohypophyseal neurons. Secretory bursts of PRL are caused by the acute withdrawal of dopamine inhibition, stimulation by PRFs, or combinations of both events. At any given moment, locally produced autocrine and paracrine regulators further modulate the responsiveness of individual lactotrophs to neurohormonal PIFs and PRFs.

Multiple neurotransmitter systems impinge on the hypothalamic dopamine and PRF neurons to regulate their neurosecretion[262] (see Fig. 7-27 ). Nicotinic cholinergic and glutamatergic afferents activate TIDA neurons, whereas histamine, acting predominantly through H2 receptors, inhibits these neurons. An inhibitory peptidergic input to TIDA neurons of major physiologic significance is that associated with the endogenous opioid peptides enkephalin and dynorphin and their cognate μ- and κ-receptor subtypes.[304] Opioid inhibition of dopamine release has been associated with increased PRL secretion under virtually all physiologic conditions, including the basal state, different phases of the estrous cycle, lactation, and stress.

Ascending serotoninergic inputs from the dorsal raphe nucleus are the major activator of PRF neurons in the PVH.[305] There is still debate concerning the identity of the specific 5-HT receptors involved in this activation.

The PRL regulatory system and its monoaminergic control have been scrutinized in detail because of the frequent occurrence of syndromes of PRL hypersecretion (see Chapter 8 ). Both the pituitary and the hypothalamus have dopamine receptors, and unfortunately the response to dopamine receptor stimulation and blockade does not distinguish between central and peripheral actions of the drug. Many commonly used neuroleptic drugs influence PRL secretion. Reserpine (a catecholamine depletor) and phenothiazines such as chlorpromazine and haloperidol enhance PRL release by disinhibition of dopamine action on the pituitary, and the PRL response is an excellent predictor of the antipsychotic effects of phenothiazines because of its correlation with D2 receptor binding and activation.[306] The major antipsychotic neuroleptic agents act on brain dopamine receptors in the mesolimbic system and in the pituitary-regulating tuberoinfundibular system. Consequently, treatment of such patients with dopamine agonists such as bromocriptine can reverse the psychiatric benefits of such drugs. A report of three patients with psychosis and concomitant prolactinomas recommended the combination of clozapine and quinagolide as the treatment of choice to manage both diseases simultaneously.[307]

Factors Influencing Secretion

Circadian Rhythm

PRL is detectable in plasma at all times during the day but is secreted in discrete pulses superimposed on basal secretion and exhibits a diurnal rhythm with peak values in the early morning hours.[308] There is a true circadian rhythm in humans because it is maintained in a constant environment independently of the sleep rhythm.[309] The combined body of data examining TIDA neuronal activity, dopamine concentrations in the median eminence, and manipulations of the SCN suggests that endogenous diurnal alterations in dopamine tone that are entrained by light constitute the major neuroendocrine mechanism underlying the circadian rhythm of PRL secretion.

External Stimuli

The suckling stimulus is the most important physiologic regulator of PRL secretion. Within 1 to 3 minutes of nipple stimulation, PRL levels rise and remain elevated for 10 to 20 minutes.[310] This reflex is distinct from the milk let-down, which involves oxytocin release from the neurohypophysis and contraction of mammary alveolar myoepithelial cells. These reflexes provide a mechanism by which the infant regulates both the production and the delivery of milk. The nocturnal rise in PRL secretion in nursing women and in nonnursing women may have evolved as a mechanism of milk maintenance during prolonged nonsuckling periods at night.

Pathways involved in the suckling reflex arise in nerves innervating the nipple, enter the spinal cord by way of spinal afferent neurons, ascend the spinal cord through spinothalamic tracts to the midbrain, and enter the hypothalamus by way of the median forebrain bundle (see Fig. 7-27 ). In most of the pathway, neurons regulating the oxytocin-dependent milk let-down response accompany those involved in PRL regulation and then separate at the level of the paraventricular nuclei. The suckling reflex brings about an inhibition of PIF activity and a release of PRFs, although the identity of an undisputed suckling-induced PRF is unsettled.

Although the significance for PRL regulation in humans is not certain, environmental stimuli from seasonal changes in light duration and auditory and olfactory cues are clearly of great importance to many mammalian species.[262] Seasonal breeders, such as the sheep, exhibit a reduction in PRL secretion in response to shortened days. The specific ultrasound vocalization of rodent pups is among the most potent stimuli for PRL secretion in lactating and virgin female rats. Olfactory stimuli from pheromones also have potent actions in rodents. A prime example is the Bruce effect or spontaneous abortion induced by exposure of a pregnant female rat to an unfamiliar male. It is mediated by a well-studied neural circuitry involving the vomeronasal nerves, corticomedial amygdala, medial preoptic area of the hypothalamus, and finally activation of TIDA neurons and a reduction in circulating PRL that is essential for maintenance of luteal function in the first half of pregnancy.

Stress in many forms dramatically affects PRL secretion, although the teleologic significance is uncertain. It may be related to actions of PRL on cells of the immune system or some other aspect of homeostasis. Different stressors are associated with either a reduction or an increase in PRL secretion, depending on the local regulatory environment at the time of the stress. However, whereas well-documented changes in PRL are associated with relatively severe forms of stress in laboratory animal models, a study of academic stress in college students failed to show any significant correlation among the time periods before, during, or after final examinations and diurnal PRL levels.[311]

Gonadotropin-Releasing Hormone and Control of the Reproductive Axis

Chemistry and Evolution

The hypothalamic neuropeptide that controls the function of the reproductive axis is GnRH. GnRH is a 10-amino-acid peptide that is synthesized as part of a larger precursor molecule and is then enzymatically cleaved to remove a signal peptide from the N-terminus and GnRH-associated peptide (GAP) from the C-terminus ( Fig. 7-28 ).[312] All forms of the decapeptide have a pyroGlu at the N-terminus and Gly-amide at the C-terminus, indicating the functional importance of the terminal regions throughout evolutionary biology.

Within mammals, two genes encoding GnRH have been identified. [313] [314] The first encodes a 92-amino-acid precursor protein. This form of GnRH is now referred to as GnRH-I and is the form found in hypothalamic neurons that serves as a releasing factor to regulate pituitary gonadotroph function.[315] The second GnRH gene, GnRH-II, encodes a decapeptide that differs from the first by three amino acids.[316] This form of GnRH is found in the midbrain region and serves as a neurotransmitter rather than as a pituitary releasing factor. Both GnRH-I and GnRH-II are found in phylogenetically diverse species, from fish to mammals, suggesting that these multiple forms of GnRH diverged from one another early in vertebrate evolution.[315] A third form of GnRH, GnRH-III, has been identified in neurons of the telencephalon in teleost fish. GnRH is also found in cells outside the brain. The roles of GnRH peptides produced outside the brain are not well understood but are an area of current investigation.

All GnRH genes have the same basic structure, with the pre-prohormone mRNA encoded in four exons. Exon 1 contains the 5′ untranslated region of the gene; exon 2 contains the signal peptide, GnRH, and the N-terminus of GAP; exon 3 contains the central portion of GAP; and exon 4 contains the C-terminus of GAP and the 3′ untranslated region (see Fig. 7-28 ).[315] Among species, the nucleotide sequences encoding the GnRH decapeptide are highly homologous. In this chapter, we focus on the hypothalamic GnRH that is derived from GnRH-1 mRNA and that plays an important role in the regulation of the hypothalamic-pituitary-gonadal axis.

Two transcriptional start sites have been identified in the rat GnRH-1 gene at +1 and -579, with the +1 promoter being active in hypothalamic neurons and the other promoter active in placenta. The first 173 base pairs of the promoter are highly conserved among species. In the rat, this promoter region has been shown to contain two OCT1 binding sites; three regions that bind the POU domain family of transcription factors, SCIP, OCT6, and TST1; and three regions that can bind the progesterone receptor.[317] In addition, a variety of hormones and second messengers have been shown to regulate GnRH gene expression, and the majority of the cis-acting elements thus far characterized for hormonal control of GnRH transcription have been localized to the proximal promoter region. [318] [319] The 5′ flanking region of the rodent and human GnRH-1 genes also contain a distal 300-base-pair enhancer region that is 1.8 or 0.9 kb, respectively, upstream of the transcription start site. [319] [320] Recent studies implicate the homeodomain transcription factors OCT1, MSX, and DLX in the specification of neuron expression and developmental activation. [320] [321]

Anatomic Distribution

GnRH neurons are small, diffusely located cells that are not concentrated in a discrete nucleus. They are generally bipolar and fusiform in shape, with slender axons projecting predominantly to the median eminence and infundibular stalk. The location of hypothalamic GnRH neurons is species-dependent. In the rat, hypothalamic GnRH neurons are concentrated in rostral areas including the medial preoptic area, the diagonal band of Broca, the septal areas, and the anterior hypothalamus. In humans and nonhuman primates, the majority of hypothalamic GnRH neurons are located more dorsally in the medial basal hypothalamus, the infundibulum, and periventricular region. Throughout the hypothalamus, neurohypophyseal GnRH neurons are interspersed with nonneuroendocrine GnRH neurons, which extend their axons to other regions of the brain including other hypothalamic regions and various regions of the cortex. GnRH secreted from nonneuroendocrine neurons has been implicated in the control of sexual behavior in rodents but not in higher primates.[322]

Embryonic Development

GnRH neuroendocrine neurons are an unusual neuronal population in that they originate outside the CNS, from the epithelial tissue of the nasal placode (reviewed in reference 323 ). During embryonic development, GnRH neurons migrate across the surface of the brain and into the hypothalamus, with the final hypothalamic location differing somewhat among species. Migration is dependent on a scaffolding of neurons and glial cells along which the GnRH neurons move, with neural cell adhesion molecules playing a critical role in guiding the migration process. In contrast to this widely accepted view of GnRH development, recent data have suggested an alternative embryonic origin of GnRH neurons from the anterior pituitary placode and cranial neural crest.[29]

Failure of GnRH neurons to migrate properly leads to a clinical condition, Kallmann’s syndrome, in which GnRH neuroendocrine neurons do not reach their final destination and thus do not stimulate pituitary gonadotropin secretion.[324] Patients with Kallmann’s syndrome do not enter puberty spontaneously. X-linked Kallmann’s syndrome results from a deficiency of the KAL1 gene, which encodes a putative protein of 680 amino acids and contains four fibronectin type III repeats and a four-disulfide core motif. Loss of function mutations in the fibroblast growth factor receptor type 1 gene (FGFR1) produce an autosomal dominant form of Kallman’s syndrome. However, these known genetic mechanisms together account for a minority of cases, and other lesions are yet to be characterized.[325] Administration of exogenous GnRH effectively treats this form of hypothalamic hypogonadism. Patients with Kallmann’s syndrome often have other congenital midline defects, including anosmia, which results from hypoplasia of the olfactory bulb and tracts.

Action at the Pituitary

Receptors

GnRH binds to a membrane receptor on pituitary gonadotrophs and stimulates both LH and FSH synthesis and secretion. The GnRH receptor is a seven-transmembrane-domain G protein–coupled receptor, but it lacks a typical intracellular C-terminal cytoplasmic domain.[319] Under physiologic conditions, GnRH receptor number varies and is usually directly correlated with the gonadotropin secretory capacity of pituitary gonadotrophs. For example, across the rat estrous cycle, a rise in GnRH receptors is seen just before the surge of gonadotropins that occurs on the afternoon of proestrus. GnRH receptor message levels are regulated by a variety of hormones and second messengers including steroid hormones (estradiol can both suppress and stimulate, and progesterone suppresses), gonadotropins (which suppress), and calcium and protein kinase C (which stimulate).[319]

Gq/11 is the primary guanosine triphosphate–binding protein mediating GnRH responses; however, there is evidence that GnRH receptors can couple to other G proteins including Gs and Gi.[319] With activation, the GnRH receptor couples to a phosphoinositide-specific phospholipase C, which leads to increases in calcium transport into gonadotrophs and calcium release from internal stores through a diacylglycerol-protein kinase C pathway. Increased calcium entry is a critical step in GnRH-stimulated release of gonadotropin secretion. However, the MAPK cascade is also stimulated by GnRH.

When there is a decline in GnRH stimulation to the pituitary, as occurs in a variety of physiologic conditions including states of lactation, undernutrition, or seasonal periods of reproductive quiescence, the number of GnRH receptors on pituitary gonadotrophs declines dramatically. Subsequent exposure of the pituitary to pulses of GnRH restores receptor number by a Ca2+-dependent mechanism that requires protein synthesis.[326] The effect of GnRH to induce its own receptor is termed up-regulation or self-priming. Only certain physiologic frequencies of pulsatile GnRH can augment GnRH receptor production, and these frequencies appear to differ among species.[327] Up-regulation of GnRH receptors after a period of low GnRH stimulation to the pituitary can take hours to days of exposure to pulsatile GnRH, depending on the duration and extent of the prior decrease in GnRH. The self-priming effect of GnRH to up-regulate its own receptors also plays a crucial role in the production of the gonadotropin surge that occurs at midcycle in females of spontaneously ovulating species and triggers ovulation. Just before the gonadotropin surge, two factors, the increased frequency of pulsatile GnRH release and a sensitization of the pituitary gonadotrophs by rising levels of estradiol, make the pituitary exquisitely sensitive to GnRH and allow an output of LH that is an order of magnitude greater than the release seen during the rest of the female reproductive cycle. This surge of LH triggers the ovulatory process at the ovary.

In contrast to up-regulation of GnRH receptors by pulsatile regimens of GnRH, continuous exposure to GnRH leads to down-regulation of GnRH receptors and an accompanying decrease in LH and FSH synthesis and secretion, termed desensitization.[328] Down-regulation does not require calcium mobilization or gonadotropin secretion. It involves a rapid uncoupling of receptor from G proteins and sequestration of the receptors from the plasma membrane, followed by internalization and proteolytic degradation of the receptors.

The concept of down-regulation has a number of clinical applications. For example, the most common current therapy for precocious puberty of hypothalamic origin (i.e., precocious GnRH secretion) is to treat the child with a long-acting GnRH agonist, which down-regulates pituitary GnRH receptors and effectively turns off the reproductive axis. [327] [329] Children with precocious puberty can be maintained with long-acting GnRH agonists for years to suppress the premature activation of the reproductive axis, and at the normal age of puberty agonist treatment can be withdrawn, allowing a reactivation of pituitary gonadotrophs and a downstream increase in gonadal steroid hormone production (also see Chapter 24 ). Long-acting GnRH agonists are also used in the treatment of forms of breast cancer that are estrogen-dependent as well as other gonadal steroid-dependent cancers.[327] Long-acting antagonists of GnRH have been developed that can also be used for these therapies.[330] Antagonists have the advantage of not having a flare effect, that is, an acute stimulation of gonadotropin secretion that is seen during the initial treatment of individuals with superagonists.

Pulsatile Gonadotropin-Releasing Hormone Stimulation

Because a single pulse of GnRH stimulates the release of both LH and FSH and chronic exposure of the pituitary to pulsatile GnRH supports the synthesis of both LH and FSH, it is generally believed that there is only one releasing factor regulating the synthesis and secretion of LH and FSH. However, in a number of physiologic conditions there are divergent patterns of LH and FSH secretion, and thus a second FSH-releasing peptide has been proposed, but such a peptide has not been isolated to date. Other mechanisms, discussed in more detail later, are likely to account for the differential regulation of LH and FSH release.

The ensemble of GnRH neurons in the hypothalamus that send axons to the portal blood system in the median eminence fire in a coordinated, repetitive, episodic manner, producing distinct pulses of GnRH in the portal bloodstream.[331] The pulsatile nature of GnRH stimulation to the pituitary leads to the release of distinct pulses of LH into the peripheral bloodstream. In experimental animals, in which it is possible to collect blood samples simultaneously from the portal and peripheral bloodstream, GnRH and LH pulses have been found to correspond in about a one-to-one ratio at most physiologic rates of secretion.[332] Because the portal bloodstream is generally inaccessible in humans, the collection of frequent blood samples from the peripheral bloodstream is used to define the pulsatile nature of LH secretion (i.e., frequency and amplitude of LH pulses), and pulsatile LH is used as an indirect measure of the activity of the GnRH secretory system. Indirect assessment of GnRH secretion by monitoring the rate of pulsatile LH secretion also is used in many animal studies examining the factors that govern the regulation of the pulsatile activity of the reproductive neuroendocrine axis. Unlike LH secretion, FSH secretion is not always pulsatile, and even when it is pulsatile, there is only partial concordance between LH and FSH pulses.

It is possible to place multiple unit recording electrodes in the medial basal hypothalamus of monkeys and other species and find spikes of electrical activity that are concordant with the pulsatile secretion of LH secretion.[333] It is unknown, however, whether these bursts of electrical activity reflect the activity of GnRH neurons themselves or the activity of neurons that impinge on GnRH neurons and govern their firing. With the development of mice in which the gene for green fluorescent protein has been put under the regulation of the GnRH promoter, it has been possible to identify GnRH neurons in hypothalamic tissue slices using fluorescence microscopy and record from them intracellularly.[14] These studies have shown that many, but not all, GnRH neurons show a bursting pattern of electrical activity. A central, unsolved question in the field of reproductive neuroendocrinology is what causes GnRH neurons to pulse in a coordinated manner. Studies using a line of clonal GnRH neurons have shown that these neurons grown in culture can release GnRH in a pulsatile pattern, suggesting that the pulse-generating capacity of GnRH neurons may be intrinsic.[334] The term GnRH pulse generator is often used to acknowledge the fact that GnRH secretion occurs in pulses and to refer to the central mechanisms responsible for pulsatile GnRH release.

A critical factor governing LH and FSH secretion and release is the rate of pulsatile GnRH stimulation of the gonadotrophs. Experimental studies in which the hypothalamus was lesioned and GnRH was replaced by pulsatile administration of exogenous GnRH showed that different frequencies of GnRH can lead to differential ratios of LH to FSH secretion from the pituitary. Fig. 7-29 shows that in a monkey with a hypothalamic lesion, replacement of one pulse of GnRH per hour led to a relatively low ratio of FSH to LH secretion. Subsequent institution of a slower pulse frequency of one pulse of GnRH every 3 hours led to a decrease in LH secretion but an increase in FSH secretion such that the ratio of FSH to LH secretion was greatly elevated. It is likely that this effect of pulse frequency on the ratio of FSH to LH secretion accounts, at least in part, for the clinical finding that at times when the GnRH pulse generator is just turning on, such as at the onset of puberty and during recovery from chronic undernutrition, the ratio of FSH to LH is higher than when it is measured in adults experiencing regular reproductive function. As discussed subsequently, steroid hormones act at both the hypothalamus and pituitary to influence strongly the rate of pulsatile GnRH release and amount of LH and FSH secreted from the pituitary.

GnRH pulse frequency not only influences the rate of pulsatile gonadotropin release and the ratio of FSH to LH secretion but also plays an important role in modulating the structural makeup of the gonadotropins. LH and FSH are structurally similar glycoprotein hormones. Each of these hormones is made up of an α and a β subunit. LH, FSH, and TSH share a common α subunit, and each has a unique β subunit that conveys receptor specificity to the intact hormone. Before secretion of gonado-tropins, terminal sugars are attached to each gonadotropin molecule.[112] The sugars include sialic acid, galactose, N-acetylglucosamine, and mannose, but the most important is sialic acid. The extent of glycosylation of LH and FSH is important for the physiologic function of these hormones.[112] Forms of gonadotropin with more sialic acid have a longer half-life because they are protected from degradation by the liver. Forms of gonadotropin with less sialic acid can have more potent effects at their biologic receptors. Both the rate of GnRH stimulation and ovarian hormone feedback at the level of the pituitary regulate the degree of LH and FSH glycosylation. For example, slow frequencies of GnRH, seen during follicular development, are associated with greater degrees of FSH glycosylation, which would provide sustained FSH support to growing follicles. In contrast, faster frequencies of GnRH, seen just before the midcycle gonadotropin surge, are associated with lesser degrees of FSH glycosylation, providing a more potent but shorter lasting form of FSH at the time of ovulation.[335]

Regulatory Systems

Many neurotransmitter systems from the brain stem, limbic system, and other areas of the hypothalamus convey information to GnRH neurons ( Fig. 7-30 ). These afferent systems include neurons that contain norepinephrine, dopamine, serotonin, GABA, glutamate, endogenous opiate peptides, NPY, galanin, and a number of other peptide neurotransmitters. Glutamate and norepinephrine play important roles in providing stimulatory drive to the reproductive axis, whereas GABA and endogenous opioid peptides provide a substantial portion of the inhibitory drive to GnRH neurons. Influences of specific neurotransmitter systems are discussed where appropriate in later sections on the physiologic regulation of GnRH neurons.

GnRH neurons are surrounded by glial processes, and only a small percentage of their surface area is available to receive dendritic contacts from afferent neurons. Changes in the steroid hormone milieu influence the degree of glial sheathing and may play important roles in regulating afferent input to GnRH neurons by this mechanism. [41] [43] Some glial cells also secrete substances including transforming growth factor α and PGE2 that can modulate the activity of GnRH neurons.

Feedback Regulation

Steroid hormone receptors are abundant in the hypothalamus and in many neural systems that impinge on GnRH neurons, including noradrenergic, serotoninergic, β-endorphin–containing, and NPY neurons. Early studies identifying regions of the brain that bound labeled estrogens showed that in rodents the preoptic area and ventromedial hypothalamus had the highest concentrations of estrogen receptors in the brain. Further localization studies, identifying estrogen receptors by immunocytochemistry or in situ hybridization, confirmed the abundance of estrogen receptors in the hypothalamus and in brain areas with strong connections to the hypothalamus, including the amygdala, septal nuclei, bed nucleus of the stria terminalis, medial part of the nucleus of the solitary tract, and lateral portion of the parabrachial nucleus.[336] In 1986, a new member of the steroid hormone receptor superfamily with high sequence homology to the classical estrogen receptor (now referred to as estrogen receptor α) was isolated from rat prostate and named estrogen receptor β. This novel estrogen receptor was shown to bind estradiol and to activate transcription by binding to estrogen response elements.[337]

In situ hybridization studies examining the localization of estrogen receptor β mRNA have shown that these receptors are present throughout the rostral-caudal extent of the brain, with a high level of expression in the preoptic area, bed nucleus of the stria terminalis, paraventricular and supraoptic nuclei, amygdala, and laminae II to VI of the cerebral cortex.[338] Specific receptors for progesterone are induced by estrogen in hypothalamic regions of the brain, including the preoptic area, the ventromedial and ventrolateral nuclei, and the infundibular-arcuate nucleus, although there is also evidence for constitutive expression of progesterone receptors in some regions.[339] Androgen receptor mapping studies have shown considerable overlap in the distribution of androgen and estrogen receptors throughout the brain. The highest density of androgen receptors was found in hypothalamic nuclei known to participate in the control of reproduction and sexual behaviors, including the arcuate nucleus, PVH, medial preoptic nucleus, VMH, and brain regions with strong connections to the hypothalamus including the amygdala, nuclei of the septal region, bed nucleus of the stria terminalis, nucleus of the solitary tract, and lateral division of the parabrachial nucleus.[336] The anterior pituitary also contains receptors for all of the gonadal steroid hormones.

Steroid hormones can dramatically alter the pattern of pulsatile release of GnRH and of the gonadotropins through actions at both the hypothalamus and the pituitary. At the hypothalamus, estradiol, progesterone, and testosterone can all act to slow the frequency of GnRH release into the portal bloodstream as part of a closed negative feedback loop.[340] Because GnRH neurons have generally been shown to lack steroid hormone receptors, it is likely that the effects of steroid hormones on the firing rate of GnRH neurons are mediated by steroid hormone actions on other neural systems that provide afferent input to GnRH neurons. For example, progesterone-mediated negative feedback on GnRH secretion in primates appears to be regulated by β-endorphin-containing neurons in the hypothalamus, acting primarily through μ-opioid receptors. If a μ-receptor antagonist, such as naloxone, is administered along with progesterone, the negative feedback action of progesterone on GnRH secretion can be blocked.

Negative feedback of steroid hormones can also occur directly at the level of the pituitary. For example, estradiol has been shown to be capable of binding to the pituitary, decreasing LH and FSH synthesis and release, and decreasing the sensitivity of pituitary gonadotrophs to the actions of GnRH such that less LH and FSH are released when a pulse of GnRH stimulates the pituitary. Evidence for such a direct pituitary action of estradiol came from studies with rhesus monkeys that had been rendered deficient in endogenous GnRH by a lesion in the arcuate nucleus and showed a decline in endogenous gonadotropin secretion. When these monkeys received a pulsatile regimen of GnRH gonadotropin secretion, subsequent estradiol infusions dramatically suppressed the responsiveness of the pituitary to GnRH and suppressed the gonadotropin secretion that was being driven by the pulsatile administration of GnRH.[341] Steroid hormones can have direct negative feedback actions at the pituitary; however, the extent of hypothalamic versus pituitary negative feedback actions is species-specific. In primate species including humans, there is considerable feedback of estradiol at the pituitary, but most of the progesterone and testosterone negative feedback occurs at the level of the hypothalamus.[340]

Most of the time, the hypothalamic-pituitary axis is under the negative feedback influence of gonadal steroid hormones. If the gonads are removed surgically or their normal secretion of steroid hormones is suppressed pharmacologically, there is a dramatic increase (10- to 20-fold) in circulating levels of LH and FSH secretion.[340] This type of “castration response” occurs normally at the menopause in women, when ovarian follicular development and thus ovarian production of large quantities of estradiol and progesterone decrease and eventually cease.

In addition to negative feedback, estradiol can have a positive feedback action at the level of the hypothalamus and pituitary to lead to a massive release of LH and FSH from the pituitary. This massive release of gonadotropins occurs once each menstrual cycle and is referred to as the LH-FSH surge. The positive feedback action of estradiol occurs as a response to the rising tide of estradiol that is produced during the process of dominant follicle development in the late follicular phase of the menstrual cycle. In women, elevated estradiol levels are generally maintained at about 300–500 pg/mL for about 36 hours prior to stimulation of the gonadotropin surge.

Experiments have shown that both a critical concentration of plasma estradiol and a critical duration of elevated estradiol are necessary to achieve positive feedback and a resulting gonadotropin surge. Moreover, the duration of estrogen elevation that is required to trigger a surge depends on the concentration of circulating estrogen. If supraphysiologic doses of estradiol are administered, the surge can occur as early as 18 hours after their administration. Because the ovary is responsible for the production of estradiol and the time course and magnitude of estradiol release control the rate of positive feedback, the ovary has been referred to as the zeitgeber of the menstrual cycle. The dependence of the positive feedback system on the magnitude of estradiol production helps explain the fact that the portion of the menstrual cycle that varies most in length is the follicular phase. Production of higher levels of estradiol by a dominant follicle in one cycle would lead to a more rapid positive feedback action with earlier ovulation and thus a shorter follicular phase compared with a cycle in which the dominant follicle produced lower levels of estradiol.

As with negative feedback in response to estradiol, the positive feedback actions of estradiol occur both at the hypothalamus, to increase GnRH secretion, and at the pituitary, to enhance greatly pituitary responsiveness to GnRH. At the pituitary, estradiol increases pituitary sensitivity to GnRH by increasing the synthesis of new GnRH receptors and by enhancing the responsiveness to GnRH at a postreceptor site of action. At the level of the hypothalamus in rodent species, estradiol appears to act at a “surge center” to induce the ovulatory surge of GnRH. Lesions in areas adjacent to the medial preoptic area, near the anterior commissure and septal complex, block the ability of estradiol to induce a surge in these species without blocking negative feedback effects of estradiol.[342] In primate species, there does not appear to be a separate surge center mediating the positive feedback actions of estradiol.

The cellular mechanisms that mediate the switch from negative to positive feedback of estrogen are not fully understood, but there is support for the concept that estrogen induction of various transcription factors and receptors (notably progesterone receptors) may play an important role in mediating this switch.[343] Alternatively, estrogen has been shown to have biphasic actions on hypothalamic GABAergic neurons that impinge on GnRH neurons and are strong regulators of their activity, with the switch in action dependent on the duration of estradiol exposure. The molecular mechanisms by which estradiol influences GnRH gene expression are not well understood, but it is likely that these influences also occur through actions of neural systems afferent to GnRH neurons.

Regulation of the Ovarian Cycle

Cyclic activity in the ovary is controlled by an interplay between steroid hormones produced by the ovary and the hypothalamic-pituitary neuroendocrine components of the reproductive axis. The duration of each phase of the ovarian cycle is species-dependent, but the general mechanisms controlling the cycle are similar in all species that have spontaneous ovarian cycles. In the human menstrual cycle, day 1 of the cycle is designated as the first day of menstrual bleeding. At this time, small and medium-sized follicles are present in the ovaries and only small amounts of estradiol are produced by the follicular cells. As a result, there is a low level of negative feedback to the hypothalamic-pituitary axis, LH pulse frequency is relatively fast (one pulse about every 60 minutes), and FSH concentrations are slightly elevated compared with much of the rest of the cycle ( Fig. 7-31 ). FSH acts at the level of the ovarian follicles to stimulate development and causes an increase in follicular estradiol production, which in turn provides increased negative feedback to the hypothalamic-pituitary unit.

A result of the increased negative feedback is a slowing of pulsatile LH secretion over the course of the follicular phase to a rate of about one pulse every 90 minutes. However, as the growing follicle (or follicles, depending on the species) secretes more estradiol, a positive feedback action of estradiol is triggered that leads to an increase in GnRH release and a surge release of LH and FSH. The surge of gonadotropins acts at the fully developed follicle to stimulate the dissolution of the follicular wall and leads to ovulation of the matured ovum into the nearby fallopian tube, where fertilization takes place if sperm are present. Ovulation results in a reorganization of the cells of the follicular wall, which undergo hypertrophy and hyperplasia and start to secrete large amounts of progesterone and some estradiol. Progesterone and estradiol have a negative feedback effect at the level of the hypothalamus and pituitary, and thus LH pulse frequency becomes very slow during the luteal phase of the menstrual cycle. The corpus luteum has a fixed life span, and without additional stimulation in the form of chorionic gonadotropin from a developing embryo, the corpus luteum regresses spontaneously after about 14 days and progesterone and estradiol secretion diminishes. This reduces the negative feedback signals to the hypothalamus and pituitary and allows an increase in FSH and LH secretion. The fall in progesterone is also a withdrawal of steroid hormone support to the endometrial lining of the uterus, and as a result the endometrium is shed as menses and a new cycle begins.

In other species, the interplay between the neuroendocrine and ovarian hormones is similar but the timing of events is different and other factors, such as circadian and seasonal regulatory factors, play a role in regulating the cycle. The rat has a 4- or 5-day ovarian cycle with no menses (the endometrial lining is absorbed rather than shed). The rat also shows strong circadian rhythmicity in the timing of the LH-FSH surge, with the surge always occurring in the afternoon of the day of proestrus. Sheep are an example of a species that has a strongly seasonal pattern of ovarian cyclicity. During the breeding season they have 15-day cycles, with a very short follicular phase and an extended luteal phase; during the nonbreeding season signals relaying information about day length through the visual system, pineal, and SCN cause a dramatic suppression of GnRH neuronal activity, and cyclic ovarian function is prevented by a decrease in trophic hormonal support from the pituitary.

Early Development and Puberty

Neuroendocrine stimulation of the reproductive axis is initiated during fetal development, and in primates in midgestation circulating levels of LH and FSH reach values similar to those in castrated adults.[344] Later in gestational development, gonadotropin levels decline, restrained by rising levels of circulating gonadal steroids. The steroids that have this effect are probably placental in origin in that after parturition there is a rise in circulating gonadotropin levels that is apparent for variable periods of the first year of life, depending on the species.[345] The decline in reproductive hormone secretion in the postnatal period appears to be due to a decrease in GnRH stimulation of the reproductive axis because it occurs even in the castrated state and gonadotropin and gonadal steroid secretion can be supported by administration of pulses of GnRH.[346]

Pubertal reawakening of the reproductive axis occurs in late childhood and is marked initially by nighttime elevations in gonadotropin and gonadal steroid hormone levels. [346] [347] The mechanisms controlling the pubertal reawakening of the GnRH pulse generator have been an area of intense investigation for the past 2 decades.[346] Although the mechanisms are not fully understood, significant progress has been made in identifying central changes in the hypothalamus that appear to play a role in this process. There appear to be both a decrease in transsynaptic inhibition to the GnRH neuronal system at puberty and an increase in stimulatory input to GnRH neurons at this time.[346] One of the major inhibitory inputs to the GnRH system is provided by GABAergic neurons. Studies in rhesus monkeys have shown that hypothalamic levels of GABA decrease during early puberty and that blocking GABAergic input before puberty, by intrahypothalamic administration of antisense oligodeoxynucleotides against the enzymes responsible for GABA synthesis, results in premature activation of the GnRH neuronal system.

It has been suggested, on the basis of findings that a subset of glutamate receptors (i.e., kainate receptors) increase in the hypothalamus at puberty, that the pubertal decrease in GABA tone may be caused by an increase in glutamatergic transmission. Further evidence for a role for glutamate comes from studies showing that administration of N-methyl-dl-aspartic acid (NMDA) to prepubertal rhesus monkeys can drive the reawakening of the reproductive axis.[348] Increased stimulatory drive to the GnRH neuronal system also appears to come from increases in norepinephrine and NPY at the time of puberty.[346] Furthermore, as discussed earlier, there is evidence that growth factors act through release of prostaglandin from glial cells at puberty to play a role in stimulating GnRH neurons.[349]

Despite an increased understanding of the neural changes occurring at puberty, the question of what signals trigger the pubertal awakening of the reproductive axis is unanswered at this time.[350] However, recent descriptions of two novel neuropeptide systems have shed further light on this area. The first was the isolation of a novel mammalian RFamide peptide named kisspeptin or metastin that is the natural ligand for the orphan G protein–coupled receptor GPR54.[293] Loss of function mutations in GPR54 cause hypogonadotropic hypogonadism, kisspeptin is expressed in subpopulations of arcuate and anteroventral periventricular neurons that project to GnRH neurons, kisspeptin expression is regulated by estradiol and testosterone and is upregulated at the time of puberty, and intracerebroventricular administration of kisspeptin causes the secretion of GnRH and gonadotropins. [351] [352] The second discovery was galanin-like peptide (GALP), which is expressed specifically in the arcuate nucleus and binds with high affinity to galanin receptors. GALP is a potent central stimulator of gonadotropin release and sexual behavior in the rat and can reverse the decreased reproductive function associated with diabetes mellitus and hypoinsulinemia.[353] Both kisspeptin and GALP neurons are targets of leptin and are hypothesized to be involved in the well-known modulation of puberty and reproductive function by food availability and nutritional status (see the following section).

Reproductive Function and Stress

Many forms of physical stresses, such as energy restriction, exercise, temperature stress, infection, pain, and injury, as well as psychological stresses, such as being subordinate in a dominance hierarchy or being acutely psychologically stressed, can suppress the activity of the reproductive axis. [354] [355] If the stress exposure is brief, there may be acute suppression of circulating gonadotropins and gonadal steroid hormones and in females disruption of normal menstrual cyclicity, but fertility is unlikely to be impaired.[354] In contrast, prolonged periods of significant stress exposure can lead to complete impairment of reproductive function, also characterized by low circulating levels of gonadotropins and gonadal steroids.[355] Stress appears to decrease the activity of the reproductive axis by decreasing GnRH drive to the pituitary because in all cases in which it has been examined, administration of exogenous GnRH can reverse the effects of the stress-induced decline in reproductive hormone secretion. Although we do not know the neural circuits through which many forms of stress suppress GnRH neuronal activity, some forms of stress-induced suppression of reproductive function are better understood.

In the case of foot shock stress in rats[356] and immune stress (i.e., injection of IL-1α) in primates,[357] the suppression of gonadotropin secretion that occurs has been shown to be reversible by administration of a CRH antagonist, implying that endogenous CRH secretion mediates the effects of these stresses on GnRH neurons. In other studies, naloxone, a μ-opiate receptor antagonist, has been shown to be capable of reversing restraint stress-induced suppression of gonadotropin secretion in monkeys; however, naloxone is ineffective in reversing the suppression of gonadotropin secretion that occurs during insulin-induced hypoglycemia. [358] [359] In the case of metabolic stresses, multiple regulators appear to mediate changes in the neural drive to the reproductive axis.

Various metabolic fuels including glucose and fatty acids can regulate the function of the reproductive axis, and blocking cellular utilization of these fuels can lead to suppression of gonadotropin secretion and decreased gonadal activity.[360] Leptin, a hormone produced by fat cells, can also modulate the activity of the reproductive axis. Mutant mice deficient in leptin or leptin receptors are infertile, and fertility can be restored to ob/ob mice by administration of leptin.[361] Moreover, leptin administration has been shown to reverse the suppressive effects of undernutrition on the reproductive axis in some situations.[362] Leptin receptors are found in several populations that are known to have a strong influence on the reproductive axis, particularly NPY and kisspeptin neurons.

In summary, it appears that a number of neural circuits can mediate effects of stress on the GnRH neuronal system and that the neural systems involved are at least somewhat specific to the type of stress that is experienced.

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PINEAL GLAND

Kronenberg: Williams Textbook of Endocrinology, 11th ed.
Copyright © 2008 Saunders, An Imprint of Elsevier

PINEAL GLAND

Historically, the functional significance of the pineal gland has been obscure. For example, Descartes called the pineal gland the “seat of the soul.” The pineal gland is both an endocrine and a circumventricular organ; it is derived from cells from the roof of the third ventricle and lies above the posterior commissure near the level of the habenular complex and the sylvian aqueduct. The pineal gland is composed of two cell types, pinealocytes and interstitial (glial-like) cells. Histologic studies suggest that the pineal gland cells are secretory in nature, and indeed the pineal is the principal source of melatonin in mammals. As discussed subsequently, the pineal gland integrates information encoded by light into coordinated secretions that underlie biologic rhythmicity. [63] [64]

The pineal is an epithalamic structure and consists of primordial photoreceptive cells. The pineal retains its light sensitivity in lower vertebrates such as fish and amphibians but lacks photosensitivity in mammals and has evolved as a strictly secretory organ in higher vertebrates. However, neuroanatomic studies have established that light-encoded information is relayed to the pineal by a polysynaptic pathway. This series of synapses ultimately results in innervation of the gland by noradrenergic sympathetic nerve terminals that are critical regulators of melatonin production and release. Specifically, the retina provides direct innervation to the SCN of the hypothalamus through the retinohypothalamic tract.[65] The SCN in turn provides input to the dorsal parvicellular PVH, a key cell group in neuroendocrine and autonomic control. This input is provided through direct and indirect pathways by intrahypothalamic projections. [66] [67] The PVH in turn provides direct innervation to sympathetic preganglionic neurons in the intermediolateral cell column of the thoracic regions of the spinal cord.[68] Sympathetic preganglionic neurons innervate postganglionic neurons in the superior cervical ganglion,[69] which in turn provide the noradrenergic innervation to the pineal (see Hypothalamic-Pituitary Unit). This rather circuitous pathway represents the anatomic substrate for light to regulate the secretion of melatonin. It is important to note that in the absence of light input, the pineal gland rhythms persist but are not entrained to the external light-dark cycle.

The Pineal Is the Source of Melatonin

The predominant hormone secreted by the pineal gland is melatonin. However, the pineal contains other biogenic amines, peptides, and GABA. Pineal-derived melatonin is synthesized from tryptophan, through serotonin, with the rate-limiting step catalyzed by the enzyme arylalkylamine N-acetyltransferase (AANAT) ( Fig. 7-6 ). [70] [71] Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the final step of melatonin synthesis. These enzymes are expressed in a pineal specific manner; however, HIOMT is also expressed in the retina and red blood cells. Melatonin plays a key role in regulating a myriad of circadian rhythms, and a fundamental principle of circadian biology is that the synthesis of melatonin is exquisitely controlled.[63] This control is exerted at several levels. AANAT mRNA levels, AANAT activity, and melatonin synthesis and release are regulated in a circadian fashion and are entrained by the light-dark cycle, with darkness thought to be the most important signal. [64] [70] [71] For example, melatonin and AANAT levels are highest during the dark and decrease sharply with the onset of light. Melatonin is not stored to any significant degree and thus is released into blood or CSF directly after its biosynthesis in proportion to AANAT activity.

The CNS control of melatonin secretion during the dark is mediated by the neuroanatomic pathway outlined above. Lack of light ultimately results in the release of norepinephrine from postganglionic sympathetic nerve terminals that act on β-adrenergic receptors in pinealocytes, resulting in an increase in adenylyl cyclase activity and synthesis of cyclic adenosine monophosphate (cAMP) from ATP.[70] Increased levels of intracellular cAMP activate downstream signal transduction cascades, including the catalytic subunits of protein kinase A and phosphorylation of cAMP response element-binding protein. Notably, cAMP response elements have been identified in the promoter of AANAT. [70] [72] Thus, light (or lack of it) acting through the sympathetic nervous system induces an increase in cAMP, representing a fundamental regulator of AANAT transcription and melatonin synthesis that ultimately results in a dramatic change of melatonin levels across the day.

Physiologic Roles of Melatonin

One of the best characterized roles of melatonin is the regulation of the reproductive axis, including gonadotropin secretion[73] and the timing and onset of puberty (see Gonadotropin-Releasing Hormone and Control of the Reproductive Axis). The potent regulation of the reproductive axis by melatonin is established in rodents and domestic animals such as the sheep. It was observed experimentally with the demonstration that removal of the pineal leads to precocious puberty and ameliorates the effects of constant darkness to induce gonadal involution. In addition, male rats exposed to constant darkness or blinded by enucleation display testicular atrophy and decreased levels of testosterone. These profound effects are normalized by removal of the pineal gland.[74] The physiologic significance of melatonin is probably most important in species referred to as seasonal breeders. Indeed, the role of melatonin in regulating reproductive capacity in species such as the sheep and the horse is now established. This type of reproductive strategy probably evolved to synchronize the length of day with the gestational period of the species to ensure that the offspring are born at favorable times of the year and maximize the viability of the young. Interestingly, although there is a strong and consistent correlation between altered melatonin secretion, day length, and seasonal breeding in diverse species, the valence of the signal can be either positive or negative dependent on the ecologic niche for each species.

Despite the potent effects of day length on reproduction in these species, exact mechanisms of melatonin regulation of GnRH release are unsettled. However, melatonin inhibits LH release from the rat pars tuberalis.[73] The role of the pineal in human reproduction is even less understood.[75] Earlier onset of menarche in blind women has been reported. In addition, a decline in melatonin at puberty has been described in some, but not other studies.

Interspecies comparative studies of melatonin’s physiologic function must be tempered by knowledge of key differences between rodent and human melatonin regulation. Significantly more light, as much as 4 log units, is required in humans to produce an equivalent nocturnal suppression of melatonin[76] and the control of AANAT is largely posttranscriptional in humans rather than transcriptional.[71]

Melatonin Receptors

Melatonin mediates its effects by acting on a family of G protein–coupled receptors, which have been characterized by pharmacologic, neuroanatomic, and molecular approaches. [63] [64] [71] The first member of the family, MT1 (Mel1a), is a high-affinity receptor that was isolated originally from Xenopus melanophores. The second, MT2 (Mel1b), has approximately 60% homology with MT1. A third receptor in mammals, MT3, is not a GPCR but instead a high-affinity binding site on the cytosolic enzyme quinone reductase 2 that is involved in cellular detoxification and might account for some of melatonin’s effects as an antioxidant. [64] [71]

The mechanisms for melatonin’s effects on regulating and entraining circadian rhythms are becoming increasingly understood. For example, melatonin inhibits the activity of neurons in the SCN of the hypothalamus, the master circadian pacemaker in the mammalian brain. [63] [77] [78] Melatonin can entrain several mammalian circadian rhythms, probably by the inhibition of neurons in the SCN. Neuroanatomic evidence suggests that many of the effects of melatonin on circadian rhythms involve actions on MT1 receptors in that the distribution of MT1 mRNA overlaps with radiolabeled melatonin-binding sites in the relevant brain regions. These sites include the SCN, the retina, and the pars tuberalis of the adenohypophysis. The MT2 receptor is also expressed in retina and brain, particularly the SCN, but evidently at much lower levels. [63] [71] [77]

Genetic studies in mice have also helped illuminate the relative roles of each melatonin receptor in mediating the effects of this hormone. Targeted deletion (knockout) of the MT1 but not the MT2 receptor abolished the ability of melatonin to inhibit the activity of SCN neurons. [78] [79] Several studies have suggested that the inhibition of SCN neurons by melatonin is of great physiologic significance. For example, Reppert and colleagues have suggested that elevations of melatonin at night could decrease the responsiveness of the SCN to activity-related stimuli that could result in phase shifts. As noted, light potently inhibits melatonin synthesis and release. Thus, melatonin may underlie the mechanism by which light induces phase shifts. However, it should be noted that lack of the MT1 gene does not block the ability of melatonin to induce phase shifts. These unexpected and somewhat confusing results have resulted in the hypothesis that MT2 is involved in melatonin-induced phase shifts, because this receptor may be expressed in the SCN in human brain.[71]

Melatonin Therapy in Humans

Melatonin is purported to exert multiple beneficial functions that include slowing or reversing the progression of aging, protecting against ischemic damage after vascular reperfusion, and enhancing immune function. [64] [71] However, the most studied and established role of melatonin in humans is that of phase shifting and resetting circadian rhythms. In this context, melatonin has been used to treat jet lag and may be effective in treating circadian-based sleep disorders.[80] In addition, melatonin administration has been shown to regulate sleep in humans. Specifically, melatonin has a hypnotic effect at relatively low doses. Melatonin therapy has also been suggested as a way to treat seasonal affective disorders. However, two recent metaanalyses of the published reports on melatonin for the treatment of either primary or secondary sleep disorders concluded that there is limited evidence for significant clinical efficacy, but melatonin is safe with short-term use (≤3 months). [81] [82] Because melatonin is now available over the counter and without a prescription throughout the United States, it is important that further controlled clinical studies be conducted to assess fully the therapeutic potential and safety of long-term melatonin use in humans.

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CIRCUMVENTRICULAR ORGANS

Kronenberg: Williams Textbook of Endocrinology, 11th ed.
Copyright © 2008 Saunders, An Imprint of Elsevier

CIRCUMVENTRICULAR ORGANS

A fundamental principle of physiology and neuropharmacology is that the brain, including the hypothalamus, resides in an environment that is protected from humoral signals. [41] [45] [46] The exclusion of macromolecules is due to the structural vascular specializations that make up the blood-brain barrier. These specializations include tight junctions of brain vascular endothelial cells that preclude the free passage of polarized macromolecules including peptides and hormones. In addition, astrocytic foot processes and perivascular microglial cells contribute to the integrity of the blood-brain barrier.[46] However, to exert homeostatic control, the brain must assess key sensory information from the bloodstream including hormone levels, metabolites, and potential toxins. For example, to monitor key signals, the brain has “windows on the circulation” or circumventricular organs (CVOs) that serve as a conduit of peripheral cues into key neuronal cell groups that maintain homeostasis. [45] [46]

As the name implies, CVOs are specialized structures that lie on the midline of the brain along the third and fourth ventricles. These structures include the OVLT, subfornical organ (SFO), median eminence, neurohypophysis (posterior pituitary), subcommissural organ, and the area postrema (see Fig. 7-5 ). Unlike the vasculature in the rest of the brain, the blood vessels in CVOs have fenestrated capillaries that allow relatively free passage of molecules such as proteins and peptide hormones. Thus, neurons and glial cells that reside within the CVOs have access to these macromolecules. In addition to the distinct nature of the vessels themselves, the CVOs have an unusually rich blood supply, allowing them to act as integrators at the interface of the blood-brain barrier. As discussed in more detail in the following subsections, several of the CVOs have major projections to hypothalamic nuclear groups that regulate homeostasis. Thus, the CVOs serve as a critical link between peripheral metabolic cues, hormones, and potential toxins and cell groups within the brain that regulate coordinated endocrine, autonomic, and behavioral responses. Detailed discussion of the physiologic roles of individual CVOs is beyond the scope of this chapter, but several in-depth reviews have assessed the function of each. [45] [46] [47] [48]

Median Eminence

The median eminence and neurohypophysis contain the neurosecretory axons that control pituitary function. The role of the median eminence as a link between the hypothalamus and the pituitary gland is detailed in other sections of this chapter (see Figs. 7-2 and 7-4 [2] [4] and see Hypothalamic-Pituitary Unit). However, it is important to understand that the anatomic location of the median eminence places it in a position to serve as an afferent sensory organ as well. Specifically, the median eminence is located adjacent to several neuroendocrine and autonomic regulatory nuclei at the tuberal level of the hypothalamus (see Fig. 7-3 ). These nuclear groups include the arcuate, ventromedial, dorsomedial, and paraventricular nuclei.[34]

A role of hypothalamic nuclei surrounding the median eminence as afferent sensory centers is supported by several observations. For example, toxins such as monosodium glutamate and gold thioglucose damage neurons in cell groups overlying the median eminence, resulting in obesity and hyperphagia. Experimental evidence suggests that the median eminence is a portal of entry for hormones such as leptin. Indeed, administration of radiolabeled peptides or hormones, such as α-MSH or leptin, led to their accumulation around the median eminence. [49] [50] Moreover, leptin receptor messenger ribonucleic acid (mRNA) and leptin-induced gene expression are densely localized in the arcuate, ventromedial, dorsomedial, and ventral premammillary hypothalamic nuclei.[51] Leptin is an established mediator of body weight and neuroendocrine function that acts on several cells in the hypothalamus including POMC neurons that reside in the arcuate nucleus. [15] [51] [52] Notably, POMC neurons are also found embedded within the median eminence. Thus, it is likely that the median eminence is involved in conveying information from humoral factors such as leptin to key hypothalamic regulatory neurons in the medial basal hypothalamus.[41]

Organum Vasculosum of the Lamina Terminalis and the Subfornical Organ

The OVLT and the SFO are located at the front wall of the third ventricle, the lamina terminalis. The OVLT and SFO lie at the ventral and dorsal boundaries of the third ventricle, respectively (see Fig. 7-5 ).[45] Because it lies at the rostral and ventral tip of the third ventricle, the OVLT is surrounded by cell groups of the preoptic region of the hypothalamus. Like other CVOs, the OVLT is composed of neurons, glial cells, and tanycytes. It is also noteworthy that axon terminals containing several neuropeptides and neurotransmitters including GnRH, somatostatin, angiotensin, dopamine, norepinephrine, serotonin, acetylcholine, oxytocin, AVP, and TRH innervate the OVLT. In the rodent, neurons that contain GnRH surround the OVLT. In addition, the OVLT in the rat brain contains estrogen receptors and the application of estrogen or electric stimulation at this site is capable of stimulating ovulation through GnRH-containing neurons that project to the median eminence, suggesting that the region regulates sexual behavior in the rat.[43]

The region of the hypothalamus that immediately surrounds the OVLT regulates a diverse array of autonomic processes. However, as the OVLT is potentially involved in the maintenance of so many processes, definitive studies ascribing specific functions to the OVLT are inherently difficult. For example, lesions of the OVLT and surrounding preoptic area led to altered febrile responses after immunologic stimulation and disruptions in fluid and electrolyte balance, blood pressure, reproduction, and thermoregulation. Indeed, large lesions of the OVLT attenuated lipopolysaccharide (LPS)-induced fever.[53] Consistent with this finding, it has been demonstrated that receptors for prostaglandin E2 (PGE2) are located within and immediately surrounding the OVLT.[54] Because PGE2 is thought to be an obligate endogenous pyrogen, the OVLT may be a critical regulator of febrile responses.

The OVLT is also likely to be involved in sensing serum osmolality because lesions of the OVLT attenuate vasopressin and oxytocin secretion in response to osmotic stimuli. In addition, hypertonic saline administration to rats induced c-Fos (a marker of neuronal activation) in OVLT neurons.[55] The efferent projections of the OVLT are not well defined because of the inherent difficulty of injecting this small structure with specific neuroanatomic tracers without contaminating surrounding preoptic nuclei. However, the neurons in the OVLT apparently have a remarkably restricted range of projections that include the paraventricular and supraoptic nuclei, the dorsomedial hypothalamic nucleus, and the lateral hypothalamic area (Elmquist JK, Sherin JE, and Saper CB, unpublished observations.)

The SFO is located in the roof of the third ventricle below the fornix. This CVO critically regulates fluid homeostasis and contributes to blood pressure regulation.[45] Consistent with these functions, the SFO has receptors for angiotensin II and atrial natriuretic peptide. [47] [56] In addition to expressing these key receptors, the SFO is thought to regulate fluid homeostasis because of its specific and massive projections to key hypothalamic regulatory sites. Notable among these are the inputs to oxytocin and AVP magnicellular neurons in the supraoptic and paraventricular nuclei. Parvicellular neurons in the PVH concerned with neuroendocrine and autonomic control also receive innervation from the SFO. In addition, the SFO densely innervates the paramedian preoptic region of the hypothalamus (often known as the anteroventral third ventricular region) and other hypothalamic sites including the perifornical area of the lateral hypothalamus. A major cell group within the anteroventral third ventricular region is the median preoptic nucleus, which receives dense innervation from the SFO.[57] Several neuroanatomic studies have demonstrated that the median preoptic nucleus is a major source of afferents to the magnicellular neuroendocrine neurons in the paraventricular and supraoptic hypothalamic nuclei.

In addition to the preceding neuroanatomic findings, physiologic evidence suggests that the SFO is critical in maintaining fluid balance. For example, Simpson and Routtenberg demonstrated that substances such as angiotensin II induced drinking behavior only when the SFO was intact. Specifically, they found that low doses of angiotensin II when injected into the SFO elicited drinking.[58] Later studies demonstrated that SFO neurons have electrophysiologic responses to angiotensin II.[47] In addition, stimulation of the SFO elicited vasopressin secretion. Like the OVLT, the SFO expressed c-Fos after stimulation by hypertonic saline administration.[55] Thus, the SFO provides dense direct and indirect innervation to the magnicellular neuroendocrine neurons in the paraventricular and supraoptic nuclei that are critical in the maintenance of fluid balance and blood pressure.

Area Postrema

The area postrema lies at the caudal end of the fourth ventricle adjacent to the nucleus of the solitary tract (see Fig. 7-5 ). In experimental animals such as the rat and mouse, it is a midline structure lying above the nucleus of the solitary tract. [46] [59] However, in humans the area postrema is a bilateral structure. As the area postrema overlies the nucleus of the solitary tract, it also receives direct visceral afferent input from the glossopharyngeal nerve (including the carotid sinus nerve) and the vagus nerve. In addition, the area postrema receives direct input from several hypothalamic nuclei. The efferent projections of the area postrema include projections to the nucleus of the solitary tract, ventral lateral medulla, and the parabrachial nucleus. Consistent with its role as a sensory organ, the area postrema is enriched with receptors for several neuropeptides including glucagon-like peptide-I and cholecystokinin (CCK). [60] [61] It also contains chemosensory neurons that include osmoreceptors.[45] Notably, the area postrema is thought to be critical in the detection of potential toxins and can induce vomiting in response to foreign substances. In fact, the area postrema is often referred to as the chemoreceptor trigger zone.[59]

The best-described physiologic role of the area postrema is the coordinated control of blood pressure. [45] [46] For example, the area postrema contains binding sites for angiotensin II, AVP, and atrial natriuretic peptide. Moreover, lesions of the area postrema in rats blunt the rise in blood pressure induced by angiotensin II.[62] Finally, administration of angiotensin II induces the expression of c-Fos in neurons of the area postrema. The area postrema has also been hypothesized to play a role in responding to inflammatory cytokines during the acute febrile response.

Subcommissural Organ

The subcommissural organ (SCO) is located near the junction of the third ventricle and cerebral aqueduct below the posterior commissure and the pineal gland (see Fig. 7-5 ).[45] It is composed of specialized ependymal cells that secrete a highly glycosylated protein of unknown function. The secretion of this protein leads to aggregation and formation of the so-called Reissner’s fibers.[48] The glycoproteins are extruded through the aqueduct, the fourth ventricle, and the spinal cord lumen to terminate in the caudal spinal canal. In humans, intracellular secretory granules are identifiable in the SCO but Reissner’s fibers are absent. The SCO secretion in humans is therefore presumed to be more soluble and to be absorbed directly from the CSF. Compared with other CVOs, the physiologic role of the SCO is largely unknown. Hypothesized roles for the SCO include clearance of substances from the CSF.[48]

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HYPOTHALAMIC-PITUITARY UNIT

Kronenberg: Williams Textbook of Endocrinology, 11th ed.
Copyright © 2008 Saunders, An Imprint of Elsevier

HYPOTHALAMIC-PITUITARY UNIT

The hypothalamus is one of the most evolutionarily conserved and essential regions of the mammalian brain. Indeed, the hypothalamus is the ultimate brain structure that allows mammals to maintain homeostasis, and destruction of the hypothalamus is not compatible with life. Hypothalamic control of homeostasis stems from the ability of this collection of neurons to orchestrate coordinated endocrine, autonomic, and behavioral responses. A key principle is that the hypothalamus receives sensory inputs from the external environment (e.g., light, pain, temperature, odorants) and information regarding the internal environment (e.g., blood pressure, blood osmolality, blood glucose levels). In addition, of particular relevance to neuroendocrine control, hormones (e.g., glucocorticoids, estrogen, testosterone, thyroid hormone) exert both negative and positive feedback directly on the hypothalamus.

The hypothalamus integrates diverse sensory and hormonal inputs and provides coordinated responses through motor outputs to key regulatory sites. These include the anterior pituitary gland, posterior pituitary gland, cerebral cortex, premotor and motor neurons in the brain stem and spinal cord, and parasympathetic and sympathetic preganglionic neurons. The patterned hypothalamic outputs to these effector sites ultimately result in coordinated endocrine, behavioral, and autonomic responses that maintain homeostasis. The focus of this section, the hypothalamic–pituitary unit, is an exquisitely controlled system and underlies the ability of mammals to coordinate endocrine functions that are necessary for survival.

Development and Differentiation of Hypothalamic Nuclei

Tremendous advances in our knowledge of the molecular and genetic basis for embryonic development of the hypothalamic-pituitary unit have occurred in the past 2 decades as a result of the genome sequencing projects and use of transgenic model systems.[26] Pituitary development is discussed in detail in Chapter 8 . Therefore, only a few key points most relevant to the physiology and pathophysiology of the neuroendocrine hypothalamus are presented in this section.

There has been considerable debate concerning the extent to which developmental studies in the rodent hypothalamic-pituitary system are applicable to the human. However, accumulating data suggest that the similarities outweigh the differences. Ontogenic analyses of the organization of the human hypothalamus utilizing a battery of neurochemical markers have reinforced its homologies to the better studied rat brain.[27] The cytoarchitectonic boundaries of hypothalamic nuclei are much more easily discerned in fetal human brain than in the adult’s, and for the most part correspond to homologous structures in the rat hypothalamus. This finding has important implications for the validity of interspecies comparative analyses. Two examples further illustrate this point. First, the ventromedial nucleus (VMH) of the hypothalamic core, which plays a role in energy balance and in female sexual behavior, differentiates from neuroblasts at a time-point intermediate to the earlier differentiation of lateral hypothalamic nuclei and later differentiation of the midline nuclei (including the suprachiasmatic [SCN], arcuate, and paraventricular nuclei [PVH]) in both humans and rodents. [27] [28] Expression of the transcription factor SF1 has been shown to be restricted both temporally and spatially to cells in the VMH and knockout of the SF1 gene in mice alters VMH development by influencing the migration of cells and hence their ultimate location.[28] A second example of interspecies homologies in hypothalamic development is the migration of gonadotropin-releasing hormone neurons from their origins in rostral neuroepithelium to the anterior hypothalamus.[29] As discussed in later sections of this chapter, spontaneous and inherited mutations in genes that affect the migration of these neurons are an important cause of Kallman’s syndrome or hypogonadotropic hypogonadism associated with anosmia.

A growing list of genes in addition to SF1 and those associated with Kallman’s syndrome, primarily encoding transcription factors, have been implicated in human neuroendocrine disorders and characterized experimentally in rodent models.[30] This list includes the homeobox transcription factor OTP and the heterodimeric complex formed by the helix-loop-helix (bHLH) factors SIM1 and ARNT2. These factors are required for the proper development of the PVH and supraoptic nucleus (SON) and expression of many key hypophyseotropic neuropeptide genes. The physiologic importance of SIM1 is illustrated by the development of an obesity phenotype in both mice and humans with a haploinsufficiency of SIM1 expression.[30]

Two key concepts involved in CNS development, which also apply to the hypothalamus, are the balance between neurogenesis and cell death in the establishment of nuclei and the role of circulating hormones in providing organizational signals that regulate cell number and synaptic remodeling. The most thoroughly characterized examples are the effects of sex steroid hormones on the developing brain that result in key sexual dimorphisms of functional importance in later reproductive behaviors.[31] This principle has been extended recently to include organizational effects of other classes of hormones. Notably, leptin plays an important role in the development of medial-basal hypothalamic circuits important for energy homeostasis by mediating axonal projections between hypothalamic nuclei.[32]

Anatomy of the Hypothalamic-Pituitary Unit

The pituitary gland is regulated by three interacting elements: hypothalamic inputs (releasing factors or hypophyseotropic hormones), feedback effects of circulating hormones, and paracrine and autocrine secretions of the pituitary itself. In humans, the pituitary gland (hypophysis) can be divided into two major parts, the adenohypophysis and the neurohypophysis, which are easily distinguishable from each other by T1-weighted magnetic resonance imaging (MR T1WI) ( Fig. 7-2 ).[33] The adenohypophysis in turn can be subdivided into three distinct lobes, the pars distalis (anterior lobe), pars intermedia (intermediate lobe), and pars tuberalis. Whereas a well-developed intermediate lobe is found in most mammals, only rudimentary vestiges of the intermediate lobe are detectable in adult humans with the bulk of intermediate lobe cells being dispersed in the anterior and posterior lobes.

The neurohypophysis is composed of the pars nervosa (also known as the neural or posterior lobe), the infundibular stalk, and the median eminence. The infundibular stalk is surrounded by the pars tuberalis, and together they constitute the hypophyseal stalk. The pituitary gland lies in the sella turcica (the Turkish saddle) of the sphenoid bone and underlies the base of the hypothalamus. This anatomic location explains the hypothalamic damage described by Fröhlich.[1] In humans, the base of the hypothalamus forms a mound called the tuber cinereum, the central region of which gives rise to the median eminence (see Fig. 7-2 ).[34]

The anterior and intermediate lobes of the pituitary derive from a dorsal invagination of the pharyngeal epithelium, called Rathke’s pouch, in response to inductive signals from the overlying neuroepithelium of the ventral diencephalon. Precursor cells within the pouch undergo steps of organ determination and cell fate commitment to a pituitary phenotype, proliferation, and migration during development.[26] The intermediate lobe is in direct contact with the neural lobe and is the least prominent of the three lobes. With age, the human intermediate lobe decreases in size to leave a small, residual collection of POMC cells. In nonprimate species, these cells are responsible for secreting the POMC-derived product α-melanocyte–stimulating hormone (a-MSH).[35]

The major component of the neural lobe is a collection of axon terminals arising from magnicellular secretory neurons located in the PVH and SON of the hypothalamus (see Fig. 7-1 ; Fig. 7-3 ). These axon terminals are in close association with a capillary plexus, and they secrete substances including AVP and oxytocin into the hypophyseal veins and into the general circulation ( Table 7-1 ). The blood supply to the neurohypophysis arises from the inferior hypophyseal artery (a branch of the internal carotid artery). Glial-like cells called pituicytes are scattered among the nerve terminals. As the source of AVP to the general circulation, the PVH and SON and their axon terminals in the neural lobe are the effector arms of the central regulation of blood osmolality, fluid balance, and blood pressure (see Chapter 9 ).

TABLE 7-1 — NEUROTRANSMITTERS AND NEUROMODULATORS IN THE PARAVENTRICULAR NUCLEUS AND THE ARCUATE NUCLEUS OF THE HYPOTHALAMUS
Paraventricular Nucleus Arcuate Nucleus
Magnicellular Division
Angiotensin II
Cholecystokinin (CCK)
Dynorphins
Acetylcholine
γ-Aminobutyric acid (GABA)
Agouti-related peptide (AGRP)
Cocaine- and amphetamine-regulated transcript (CART)

Nitric oxide (NO)
Oxytocin
Vasopressin
Dopamine
Endocannabinoids
Enkephalins

Parvicellular Divisions
γ-Aminobutyric acid (GABA)
Angiotensin II
Atrial natriuretic factor (ANF)
Bombesin-like peptides
Cholecystokinin (CCK)
Galanin
Galanin-like peptide (GALP)
Glutamate
Gonadotropin-releasing hormone (GnRH)
Growth hormone–releasing hormone (GHRH)
Kisspeptins

Corticotropin-releasing hormone (CRH)
Dopamine
Endocannabinoids
Enkephalins
Galanin
Glutamate
Interleukin-1 (IL-1)
Neuropeptide Y (NPY)
Neurotensin
Neuromedin U
Neuropeptide Y (NPY)
Neurotensin
Nociceptin/orphanin FQ (OFQ)
Pancreatic polypeptide
Prolactin
Pro-opiomelanocortin
Melanocortins (ACTH, α-MSH, β-MSH, γ-MSH)

Nitric Oxide (NO)
RFRP (RF amide-related peptides)
Somatostatin
Opioids (β-endorphin)
QRFP (pyro-glutamyl-RFamide peptide)
Somatostatin

Thyrotropin-releasing hormone (TRH)
Vasopressin
Vasoactive intestinal peptide (VIP)
Substance P

The secretion of oxytocin by magnicellular neurons is critical at parturition, resulting in uterine myometrial contraction. In addition, the secretion of oxytocin is regulated by the classic milk let-down reflex.[36] Although the exact neuroanatomic substrate underlying this response is still unclear, apparently mechanosensory information from the nipple reaches the magnicellular neurons, directly or indirectly, from the dorsal horn of the spinal cord, resulting in release of oxytocin into the general circulation.[37] Oxytocin acts on receptors on myoepithelial cells in the mammary gland acini, leading to release of milk into the ductal system and ultimately the release of milk from the mammary gland.

The Median Eminence and Hypophyseotropic Neuronal System

The median eminence is the functional link between the hypothalamus and the anterior pituitary gland, lies in the center of the tuber cinereum, and is composed of an extensive array of blood vessels and nerve endings (see Fig. 7-2 ; Fig. 7-4 ). [17] [34] [38] Its extremely rich blood supply arises from the superior hypophyseal artery (a branch of the internal carotid artery), which sends off many small branches that form capillary loops. The small capillary loops extend into the internal and external zones (see next paragraph), form anastomoses, and drain into sinusoids that become the pituitary portal veins that enter the vascular pool of the pituitary gland. [38] [39] [40] The flow of blood in these short loops is thought to be predominantly (if not exclusively) in a hypothalamic-to-pituitary direction.[40] This well-developed plexus results in a tremendous increase in the vascular surface area. In addition, the vessels are fenestrated, allowing diffusion of the peptide-releasing factors to their site of action in the anterior pituitary gland. This vascular complex in the base of the hypothalamus and its “arteriolized” venous drainage to the pituitary compose a circulatory system analogous to the portal vein system of the liver, hence the term hypophyseal-portal circulation.

Three distinct compartments of the median eminence are recognized: the innermost ependymal layer, the internal zone, and the external zone (see Fig. 7-4 ).[38] Ependymal cells form the floor of the third ventricle and are unique in that they have microvilli rather than cilia. Tight junctions at the ventricular pole of the ependymal cells prevent the diffusion of large-molecular-weight substances between the cerebrospinal fluid (CSF) and the extracellular space within the median eminence. The ependymal layer also contains specialized cells called tanycytes that send processes into the other layers of the median eminence.[41] Tight junctions between tanycytes at the lateral edges of the median eminence likely prevent the diffusion of releasing factors back into the medial basal hypothalamus.

The internal zone of the median eminence is composed of axons of passage of the supraoptic and paraventricular magnicellular neurons en route to the posterior pituitary (see Fig. 7-4C ) and the axons of the hypophyseotropic neurons destined for the external layer of the median eminence (see Fig. 7-4A and B ). In addition, supportive cells populate this layer.

Finally, the external zone of the median eminence represents the exchange point of the hypothalamic-releasing factors and the pituitary portal vessels.[38] Two general types of tuberohypophyseal neurons project to the external zone: (1) peptide-secreting (peptidergic) neurons including thyrotropin-releasing hormone (TRH), corticotropin-releasing hormone (CRH), and gonadotropin-releasing hormone (GnRH) (see Fig. 7-1 ) and (2) neurons containing monoamines (e.g., dopamine and serotonin). Although the secretion of these substances into the portal circulation is an important control mechanism, some peptides and neurotransmitters in nerve endings are not released into the hypophyseal-portal circulation but instead function to regulate the secretion of other nerve terminals.[42] The anatomic relationships of nerve endings, basement membranes, interstitial spaces, fenestrated (windowed) capillary endothelia, and glia in the median eminence are similar to those in the neural lobe. As in the case of neurohormone secretion from the neurohypophysis, depolarization of hypothalamic cells leads to the release of neuropeptides and monoamines at the median eminence.

Nonneuronal supporting cells in the hypothalamus also play a dynamic role in hypophyseotropic regulation. For example, nerve terminals in the neurohypophysis are enveloped by pituicytes; when the gland is inactive they surround the nerve endings, whereas they retract to expose the terminals when AVP secretion is enhanced as in states of dehydration. Within the median eminence, GnRH nerve endings are enveloped by the tanycytes, which also cover or uncover neurons with changes in functional status. [41] [43] Thus, supporting elements, with their own sets of receptors, can change the neuroregulatory milieu within the hypothalamus, median eminence, and pituitary.

The site of production, the genetics, and the regulation of synthesis and release of individual peptide-releasing factors are discussed in detail in later sections. Briefly, the cell groups in the hypothalamus that contain releasing factors that are secreted into the pituitary portal circulation are located in several cell groups of the medial hypothalamus ( Table 7-2 ). [34] [44] These cell groups include the arcuate (infundibular) nucleus (see Fig. 7-3D ), the PVH (see Fig. 7-3A and C ), the periventricular nucleus, and a group of cells in the medial preoptic area near the organum vasculosum of the lamina terminalis (OVLT) ( Fig. 7-5 ). As discussed earlier, magnicellular neurons in the SON and PVH send axons that predominantly traverse the median eminence to terminate in the neural lobe of the pituitary. In addition, a smaller number of magnicellular axons project directly to the external zone of the median eminence, but their functional significance is unknown.

TABLE 7-2 — STRUCTURAL FORMULAS OF PRINCIPAL HUMAN HYPOTHALAMIC PEPTIDES DIRECTLY RELATED TO PITUITARY SECRETION
Vasopressin

Oxytocin

Thyrotropin-Releasing Hormone
pGlu-His-Pro-NH2 (MW = 362.42)

Gonadotropin-Releasing Hormone
pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (MW = 1182.39)

Corticotropin-Releasing Hormone
Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH2 (MW = 4758.14)

Growth Hormone–Releasing Hormone (GHRH 1-40; 1-44-NH2, Human)
Tyr-Ala-Asp-Ala-Ile-Phe-Thr-Asn-Ser-Tyr-Arg-Lys-Val-Leu-Gly-Gln-Leu-Ser-Ala-Arg-Lys-Leu-Leu-Gln-Asp-Ile-Met-Ser-Arg-Gln-Gln-Gly-Glu-Ser-Asn-Gln-Glu-Arg-Gly-Ala (MW = 4544.73); [-Arg-Ala-Arg-Leu-NH2] (MW = 5040.4)

Somatostatin

Somatostatin-28

Somatostatin-28 (1-12)
Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu (MW = 1244.49)

Vasoactive Intestinal Peptide
His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn-NH2 (MW = 3326.26)

Prolactin-Releasing-Peptide (PrRP31; PrRP20)
[Ser-Arg-Thr-His-Arg-His-Ser-Met-Glu-Ile-Arg]-Thr-Pro-Asp-Ile-Asn-Pro-Ala-Trp-Tyr-Ala-Ser-Arg-Gly-Ile-Arg-Pro-Val-Gly-Arg-Phe-NH2 = (MW 3665.16; 2273.58)

Ghrelin
Gly-Ser-Ser-Phe-Leu-Ser-Pro-Glu-His-Gln-Arg-Val-Gln-Gln-Arg-Lys-Glu-Ser-Lys-Lys-Pro-Pro-Ala-Lys-Leu-Gln-Pro-Arg (MW = 3314.9) [Ser 3 is n-octanoylated]

Disulfide bonds between pairs of cystines that produce cyclization of the peptides are indicated by brackets above the sequences.

MW, Molecular weight; pGlu, pyro-glutamyl.

The third structure often grouped as a component of the median eminence is the pars tuberalis. The pars tuberalis is a subdivision of the adenohypophysis and is a thin sheet of glandular tissue that lies around the infundibulum and pituitary stalk. In some animals, the epithelial component may make up as much as 10% of the total glandular tissue of the anterior pituitary. The pars tuberalis contains cells making pituitary tropic hormones including luteinizing hormone (LH) and thyrotropin (thyroid stimulating hormone [TSH]). A definitive physiologic function of the pars tuberalis is not established, but melatonin receptors are expressed in the pars tuberalis.

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NEURAL CONTROL OF GLANDULAR SECRETION

Kronenberg: Williams Textbook of Endocrinology, 11th ed.
Copyright © 2008 Saunders, An Imprint of Elsevier

NEURAL CONTROL OF GLANDULAR SECRETION

A fundamental principle of neuroendocrinology encompasses the regulated secretion of hormones, neurotransmitters, or neuromodulators by specialized cells.[16] Endocrine cells and neurons are prototypical secretory cells. Both have electrically excitable plasma membranes and specific ion conductances that regulate exocytosis of their signaling molecules from storage vesicles. In addition, secretory cells are broadly classified by their topographic mechanisms of secretion. For example, endocrine cells secrete their contents directly into the bloodstream, allowing these substances to act globally as hormones. Cells classified as paracrine secrete their contents into the extracellular space and predominantly affect the function of closely neighboring cells. Similarly, autocrine secretory cells affect their own function by the local actions of their secretions. In contrast, secretory cells within exocrine glands secrete proteinaceous substances including enzymes into the lumen of ductal systems.

Neurosecretion

Neurons are secretory cells that send their axons throughout the nervous system to release their neurotransmitters and neuromodulators predominantly at specialized chemical synapses. Neurohumoral or neurosecretory cells constitute a unique subset of neurons whose axon terminals are not associated with synapses. Two examples of neurosecretory cells are neurohypophyseal and hypophyseotropic cells. The prototypical neurohypophyseal cells are the magnicellular neurons of the paraventricular and supraoptic nuclei in the hypothalamus. Hypophyseotropic cells are neurons that secrete their products into the pituitary portal vessels at the median eminence ( Fig. 7-1 ).

In the most basic sense, neurosecretory cells are neurons that secrete substances directly into the bloodstream to act as hormones. The theory of neurosecretion evolved from the seminal work of Scharrer and Scharrer, [16] [17] who used morphologic techniques to identify stained secretory granules in the supraoptic and paraventricular hypothalamic neurons. They found that cutting the pituitary stalk led to an accumulation of these granules in the hypothalamus. These findings led them to hypothesize that the source of substances secreted by the neural lobe (posterior pituitary) was hypothalamic neurons. Although the Scharrers’ concept initially raised great skepticism among contemporary researchers, we now know that the axon terminals in the neural lobe arise from the supraoptic and paraventricular magnicellular neurons that contain oxytocin and arginine vasopressin (AVP).

The modern definition of neurosecretion has evolved to include the release of any neuronal secretory product from a neuron. Indeed, a fundamental tenet of neuroscience is that all neurons in the CNS, including neurons that secrete AVP and oxytocin in the neural lobe, receive multiple synaptic inputs largely onto their dendrites and cell bodies. In addition, neurons have the basic ability to detect and integrate input from multiple neurons through specific receptors. They in turn fire action potentials that result in the release of neurotransmitters and neuromodulators into synapses formed with postsynaptic neurons. The vast majority of communications between neurons is accomplished by “classical” fast-acting neurotransmitters (e.g., glutamate, γ-aminobutyric acid [GABA], acetylcholine) and neuromodulators (e.g., neuropeptides) acting at chemical synapses. [11] [18] Thus, neurosecretion represents a fundamental concept in understanding the mechanisms used by the nervous system to control behavior and maintain homeostasis.

In the era of the elucidation of the human genome, the importance of these early observations often is not fully appreciated. However, accounts of these early studies are illuminating. Moreover, it is not an overstatement that the confirmation of the neurosecretion hypothesis represented one of the major advances in the field of neuroscience and neuroendocrinology. Indeed, this and other early experiments, including the pioneering work of Geoffrey Harris, [7] [19] led to the fundamental concept that the hypothalamus releases hormones directly into the bloodstream (neurohypophyseal cells). These observations provided the principles on which the modern discipline of neuroendocrinology is built.

The Autonomic Nervous System’s Contribution to Endocrine Control

Another major precept of neuroendocrinology is that the nervous system controls or modifies the function of both endocrine and exocrine glands. The exquisite control of the anterior pituitary gland is accomplished by the action of releasing factor hormones (see Hypophyseotropic Hormone, and Neuroendocrine Axes). Other endocrine and exocrine organs (e.g., pancreas, adrenal, pineal, salivary glands) are also regulated through direct innervation from the cholinergic and noradrenergic inputs from the autonomic nervous system. Although it is beyond the scope of this chapter, an appreciation of the functional anatomy and pharmacology of the parasympathetic and sympathetic nervous systems is fundamental in understanding the neural control of endocrine function.

The efferent arms of the autonomic nervous system comprise the sympathetic and parasympathetic systems. Each system has a similar wiring diagram characterized by a preganglionic neuron that innervates a postganglionic neuron that in turn targets an end organ.[20] Preganglionic and postganglionic parasympathetic neurons are cholinergic. In contrast, preganglio-nic sympathetic neurons are cholinergic and postganglionic neurons are noradrenergic (except for those innervating sweat glands, which are cholinergic). Another basic concept is that autonomic neurons coexpress several neuropeptides. This coexpression is a common feature of neurons in the central and peripheral nervous systems. [11] [18] [21] For example, postganglionic noradrenergic neurons coexpress somatostatin and neuropeptide Y (NPY). Postganglionic cholinergic neurons coexpress neuropeptides including vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP).

The majority of sympathetic preganglionic neurons lie in the intermediolateral cell column in the thoracolumbar regions of the spinal cord.[20] Most postganglionic neurons are located in sympathetic ganglia lying near the vertebral column (e.g., sympathetic chain and superior cervical ganglia). Postganglionic fibers, in turn, innervate target organs. Thus, as a rule, sympathetic preganglionic fibers are relatively short and the postganglionic fibers are long. In contrast, the parasympathetic preganglionic neurons lie in the midbrain (Edinger-Westphal nucleus of the third cranial nerve), the medulla oblongata (e.g., dorsal motor nucleus of the vagus and nucleus ambiguus), and the sacral spinal cord. Postganglionic neurons that innervate the eye and salivary glands arise from the ciliary, pterygopalatine, submandibular, and otic ganglia. Postganglionic parasympathetic neurons in the thorax and abdomen typically lie within the target organs including the gut wall and pancreas.[20] Consequently, parasympathetic preganglionic fibers are relatively long and the postganglionic fibers are short.

A dual autonomic innervation of the pancreas illustrates the importance of coordinated neural control of endocrine organs. The endocrine pancreas receives sympathetic (noradrenergic) and parasympathetic (cholinergic) innervation. [20] [22] The latter activity is provided by the vagus nerve (dorsal motor nucleus of the vagus) and is an excellent example of neural modulation because cholinergic tone of β cells affects their secretion of insulin. For example, vagal input is thought to modulate insulin secretion before (cephalic phase), during, and after ingestion of food.[23] In addition, noradrenergic stimulation of the endocrine pancreas can alter the secretion of glucagon and inhibits insulin release.[22] It should be noted, of course, that a major regulator of insulin secretion is the extracellular concentration of glucose.[24] In fact, glucose can induce insulin secretion in the absence of neural input. However, the exquisite control by the nervous system is illustrated by the fact that populations of neurons in the brain stem and hypothalamus, like the β cell, have the ability to sense glucose levels in the bloodstream.[25] This information is integrated by the hypothalamus and ultimately results in alterations in the activity of the autonomic nervous system innervating the pancreas. Thus, neural control of the endocrine pancreas probably contributes to the physiologic control of insulin secretion and may contribute to the pathophysiology of disorders such as diabetes mellitus. Certainly, an increased understanding of this complex interplay between the CNS and endocrine function is necessary to diagnose and clinically manage endocrine disorders.

Email to Colleague Print Version
Copyright © 2008 Elsevier Inc. All rights reserved. – http://www.mdconsult.com

Kronenberg: Williams Textbook of Endocrinology, 11th ed.
Copyright © 2008 Saunders, An Imprint of Elsevier

NEURAL CONTROL OF GLANDULAR SECRETION

A fundamental principle of neuroendocrinology encompasses the regulated secretion of hormones, neurotransmitters, or neuromodulators by specialized cells.[16] Endocrine cells and neurons are prototypical secretory cells. Both have electrically excitable plasma membranes and specific ion conductances that regulate exocytosis of their signaling molecules from storage vesicles. In addition, secretory cells are broadly classified by their topographic mechanisms of secretion. For example, endocrine cells secrete their contents directly into the bloodstream, allowing these substances to act globally as hormones. Cells classified as paracrine secrete their contents into the extracellular space and predominantly affect the function of closely neighboring cells. Similarly, autocrine secretory cells affect their own function by the local actions of their secretions. In contrast, secretory cells within exocrine glands secrete proteinaceous substances including enzymes into the lumen of ductal systems.

Neurosecretion

Neurons are secretory cells that send their axons throughout the nervous system to release their neurotransmitters and neuromodulators predominantly at specialized chemical synapses. Neurohumoral or neurosecretory cells constitute a unique subset of neurons whose axon terminals are not associated with synapses. Two examples of neurosecretory cells are neurohypophyseal and hypophyseotropic cells. The prototypical neurohypophyseal cells are the magnicellular neurons of the paraventricular and supraoptic nuclei in the hypothalamus. Hypophyseotropic cells are neurons that secrete their products into the pituitary portal vessels at the median eminence ( Fig. 7-1 ).

In the most basic sense, neurosecretory cells are neurons that secrete substances directly into the bloodstream to act as hormones. The theory of neurosecretion evolved from the seminal work of Scharrer and Scharrer, [16] [17] who used morphologic techniques to identify stained secretory granules in the supraoptic and paraventricular hypothalamic neurons. They found that cutting the pituitary stalk led to an accumulation of these granules in the hypothalamus. These findings led them to hypothesize that the source of substances secreted by the neural lobe (posterior pituitary) was hypothalamic neurons. Although the Scharrers’ concept initially raised great skepticism among contemporary researchers, we now know that the axon terminals in the neural lobe arise from the supraoptic and paraventricular magnicellular neurons that contain oxytocin and arginine vasopressin (AVP).

The modern definition of neurosecretion has evolved to include the release of any neuronal secretory product from a neuron. Indeed, a fundamental tenet of neuroscience is that all neurons in the CNS, including neurons that secrete AVP and oxytocin in the neural lobe, receive multiple synaptic inputs largely onto their dendrites and cell bodies. In addition, neurons have the basic ability to detect and integrate input from multiple neurons through specific receptors. They in turn fire action potentials that result in the release of neurotransmitters and neuromodulators into synapses formed with postsynaptic neurons. The vast majority of communications between neurons is accomplished by “classical” fast-acting neurotransmitters (e.g., glutamate, γ-aminobutyric acid [GABA], acetylcholine) and neuromodulators (e.g., neuropeptides) acting at chemical synapses. [11] [18] Thus, neurosecretion represents a fundamental concept in understanding the mechanisms used by the nervous system to control behavior and maintain homeostasis.

In the era of the elucidation of the human genome, the importance of these early observations often is not fully appreciated. However, accounts of these early studies are illuminating. Moreover, it is not an overstatement that the confirmation of the neurosecretion hypothesis represented one of the major advances in the field of neuroscience and neuroendocrinology. Indeed, this and other early experiments, including the pioneering work of Geoffrey Harris, [7] [19] led to the fundamental concept that the hypothalamus releases hormones directly into the bloodstream (neurohypophyseal cells). These observations provided the principles on which the modern discipline of neuroendocrinology is built.

The Autonomic Nervous System’s Contribution to Endocrine Control

Another major precept of neuroendocrinology is that the nervous system controls or modifies the function of both endocrine and exocrine glands. The exquisite control of the anterior pituitary gland is accomplished by the action of releasing factor hormones (see Hypophyseotropic Hormone, and Neuroendocrine Axes). Other endocrine and exocrine organs (e.g., pancreas, adrenal, pineal, salivary glands) are also regulated through direct innervation from the cholinergic and noradrenergic inputs from the autonomic nervous system. Although it is beyond the scope of this chapter, an appreciation of the functional anatomy and pharmacology of the parasympathetic and sympathetic nervous systems is fundamental in understanding the neural control of endocrine function.

The efferent arms of the autonomic nervous system comprise the sympathetic and parasympathetic systems. Each system has a similar wiring diagram characterized by a preganglionic neuron that innervates a postganglionic neuron that in turn targets an end organ.[20] Preganglionic and postganglionic parasympathetic neurons are cholinergic. In contrast, preganglio-nic sympathetic neurons are cholinergic and postganglionic neurons are noradrenergic (except for those innervating sweat glands, which are cholinergic). Another basic concept is that autonomic neurons coexpress several neuropeptides. This coexpression is a common feature of neurons in the central and peripheral nervous systems. [11] [18] [21] For example, postganglionic noradrenergic neurons coexpress somatostatin and neuropeptide Y (NPY). Postganglionic cholinergic neurons coexpress neuropeptides including vasoactive intestinal polypeptide (VIP) and calcitonin gene-related peptide (CGRP).

The majority of sympathetic preganglionic neurons lie in the intermediolateral cell column in the thoracolumbar regions of the spinal cord.[20] Most postganglionic neurons are located in sympathetic ganglia lying near the vertebral column (e.g., sympathetic chain and superior cervical ganglia). Postganglionic fibers, in turn, innervate target organs. Thus, as a rule, sympathetic preganglionic fibers are relatively short and the postganglionic fibers are long. In contrast, the parasympathetic preganglionic neurons lie in the midbrain (Edinger-Westphal nucleus of the third cranial nerve), the medulla oblongata (e.g., dorsal motor nucleus of the vagus and nucleus ambiguus), and the sacral spinal cord. Postganglionic neurons that innervate the eye and salivary glands arise from the ciliary, pterygopalatine, submandibular, and otic ganglia. Postganglionic parasympathetic neurons in the thorax and abdomen typically lie within the target organs including the gut wall and pancreas.[20] Consequently, parasympathetic preganglionic fibers are relatively long and the postganglionic fibers are short.

A dual autonomic innervation of the pancreas illustrates the importance of coordinated neural control of endocrine organs. The endocrine pancreas receives sympathetic (noradrenergic) and parasympathetic (cholinergic) innervation. [20] [22] The latter activity is provided by the vagus nerve (dorsal motor nucleus of the vagus) and is an excellent example of neural modulation because cholinergic tone of β cells affects their secretion of insulin. For example, vagal input is thought to modulate insulin secretion before (cephalic phase), during, and after ingestion of food.[23] In addition, noradrenergic stimulation of the endocrine pancreas can alter the secretion of glucagon and inhibits insulin release.[22] It should be noted, of course, that a major regulator of insulin secretion is the extracellular concentration of glucose.[24] In fact, glucose can induce insulin secretion in the absence of neural input. However, the exquisite control by the nervous system is illustrated by the fact that populations of neurons in the brain stem and hypothalamus, like the β cell, have the ability to sense glucose levels in the bloodstream.[25] This information is integrated by the hypothalamus and ultimately results in alterations in the activity of the autonomic nervous system innervating the pancreas. Thus, neural control of the endocrine pancreas probably contributes to the physiologic control of insulin secretion and may contribute to the pathophysiology of disorders such as diabetes mellitus. Certainly, an increased understanding of this complex interplay between the CNS and endocrine function is necessary to diagnose and clinically manage endocrine disorders.

Email to Colleague Print Version
Copyright © 2008 Elsevier Inc. All rights reserved. – http://www.mdconsult.com

Thyroid

Kronenberg: Williams Textbook of Endocrinology, 11th ed.
Copyright © 2008 Saunders, An Imprint of Elsevier

Thyroid

THYROID PHYSIOLOGY AND DIAGNOSTIC EVALUATION OF PATIENTS WITH THYROID DISORDERS

P. Reed Larsen Terry F. Davies Martin-Jean Schlumberger Ian D. Hay

▪ Phylogeny, Embryology, and Ontogeny, 299
▪ Anatomy and Histology, 300
▪ Iodine and the Synthesis and Secretion of Thyroid Hormones, 301
▪ Thyroid Hormones in Peripheral Tissues, 305
▪ Regulation of Thyroid Function, 312
▪ Physical Evaluation of the Thyroid Gland, 318
▪ Laboratory Assessment of Thyroid Status, 319
▪ Quantitation of Serum Thyroid Hormone Concentrations, 320

Dysfunction and anatomic abnormalities of the thyroid are among the most common diseases of the endocrine glands. This chapter provides an up-to-date physiologic and biochemical background and describes the various tests for evaluating patients with suspected thyroid disease based on the pathophysiology of these conditions.

PHYLOGENY, EMBRYOLOGY, AND ONTOGENY

Phylogeny

The phylogeny, embryogenesis, and certain aspects of thyroid function are closely interlinked with the gastrointestinal tract. The capacity of the thyroid to metabolize iodine and incorporate it into a variety of organic compounds occurs widely throughout the animal and plant kingdoms. Monoiodotyrosine (3′-monoiodo-l-tyrosine [MIT]) and diiodotyrosine (3,5′-diiodo-l-tyrosine [DIT]) are present in a variety of invertebrate species, including mollusks, crustaceans, coelenterates, annelids, insects, and certain marine algae ( Fig. 10-1 ). In these lower forms, however, no recognizable thyroid tissue is present. Thyroid tissue is confined to, and is present in, all vertebrates. A close link to the thyroid of higher vertebrates is evident in the ammocoete, the larval form of the lamprey. Here the endostyle is capable of carrying out iodinations, but prior to metamorphosis, a protease is expressed in the endostyle that can hydrolyze the iodoprotein formed. Presumably this permits the endostyle to lose its connection with the pharynx during metamorphosis and to assume its adult function as an endocrine organ that secretes iodothyronines, including 3,5,3′,5′-tetraiodo-L-thyronine (thyroxine, T4) and 3,5,3′-triiodo-l-thyronine(T3) (see Fig. 10-1 ).

The phylogenetic association of the thyroid gland and the gastrointestinal tract is evident in several functions. The salivary and gastric glands, like the thyroid, are capable of concentrating iodide in their secretions, although iodide transport in thesesites is not responsive to stimulation by thyrotropin (TSH). The salivary gland contains enzymes that are capable of iodinating tyrosine in the presence of hydrogen peroxide, although it forms insignificant quantities of iodoproteins under normal circumstances.

Structural Embryology

The human thyroid anlage is first recognizable at E16-17. The primordium begins as a thickening of epithelium in the pharyngeal floor, which later forms a diverticulum adjacent to the developing myocardial cells. With continuing development, the median diverticulum is displaced caudally following the myocardial cells in their descent. The primitive stalk connecting the primordium with the pharyngeal floor elongates into the thyroglossal duct. During its caudal displacement, the primordium assumes a bilobate shape, coming into contact and fusing with the ventral aspect of the fourth pharyngeal pouch when it reaches its final position at about E50. Normally the thyroglossal duct undergoes dissolution and fragmentation by about the second month after conception, leaving at its point of origin a small dimple at the junction of the middle and posterior thirds of the tongue, the foramen caecum. Cells of the lower portion of the duct differentiate into thyroid tissue, forming the pyramidal lobe of the gland. At this time the lobes contact the ultimobranchial glands, leading to the incorporation of C cells into the thyroid. Concomitantly, histologic alterations occur throughout the gland. Complex interconnecting cordlike arrangements of cells interspersed with vascular connective tissue replace the solid epithelial mass and become tubule-like structures at about the third month of fetal life; shortly thereafter, follicular arrangements devoid of colloid appear, and by 13 to 14 weeks the follicles begin to fill with colloid. Investigations of thyroid gland development in mice using gene targeting techniques are beginning to identify the critical factors which are required for normal thyroid gland development. [1] [2] The role of these various homeobox proteins is currently being evaluated with respect to the potential for defects in the synthesis or formation of the thyroid gland (see Chapter 12 ).

Functional Ontogeny

The ontogeny of thyroid function and its regulation in the human fetus are fairly well defined.[3] Future follicular cells acquire the capacity to form thyroglobulin (Tg) as early as the 29th day of gestation, whereas the capacities to concentrate iodide and synthesize thyroxine (T4) are delayed until about the 11th week. Radioactive iodine inadvertently given to the mother would be accumulated by the fetal thyroid soon thereafter. Early growth and development of the thyroid do not seem to be TSH-dependent, because the capacity of the pituitary to synthesize and secrete TSH is not apparent until the 14th week. Subsequently, rapid changes in pituitary and thyroid function take place. Probably as a consequence of hypothalamic maturation and increasing secretion of thyrotropin-releasing hormone (TRH), the serum TSH concentration increases between 18 and 26 weeks’ gestation, after which levels remain higher than those in the mother. [3] [4] The higher levels may reflect a higher set-point of the negative feedback control of TSH secretion during fetal life than at maturity. Thyroxine-binding globulin (TBG), the major thyroid hormone–binding protein in plasma, is detectable in the serum by the 10th gestational week and increases in concentration progressively to term. This increase in TBG concentration accounts in part for the progressive increase in the serum T4 concentration during the second and third trimesters, but increased secretion of T4 must also play a role because the concentration of or free T4 also rises.

Several aspects of thyroid development are of note from the clinical standpoint.[5] Rarely, thyroid tissue may develop from remnants of the thyroglossal duct near the base of the tongue. Such lingual thyroid tissue may be the sole functioning thyroid present and, thus, its surgical removal will lead to hypothyroidism. More commonly, elements of the thyroglossal duct may persist and later give rise to thyroglossal duct cysts, or ectopic thyroid tissue may be present at any location in the mediastinum or, rarely, even in the heart.

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POSTERIOR PITUITARY

Kronenberg: Williams Textbook of Endocrinology, 11th ed.
Copyright © 2008 Saunders, An Imprint of Elsevier

POSTERIOR PITUITARY

Alan G. Robinson Joseph G. Verbalis

▪ Anatomy, 263
▪ Synthesis and Release of Neurohypophyseal Hormones, 264
▪ Physiology of Secretion of Vasopressin and Thirst, 264
▪ Diabetes Insipidus, 268
▪ Treatment of Diabetes Insipidus, 273
▪ The Syndrome of Inappropriate Antidiuretic Hormone Secretion, 275
▪ Oxytocin, 286
ANATOMY

Normal

The posterior pituitary is neural tissue and consists only of the distal axons of the hypothalamic magnocellular neurons that make up the neurohypophysis. The perikarya (cell bodies) of these axons are located in paired supraoptic nuclei and the paired paraventricular nuclei of the hypothalamus. During embryogenesis,[1] neuroepithelial cells of the lining of the third ventricle mature into magnocellular neurons while migrating laterally to and above the optic chiasm to form the supraoptic nucleus and to the walls of the third ventricle to form the paraventricular nuclei. In the posterior pituitary, the axon terminals of the magnocellular neurons contain neurosecretory granules, membrane-bound packets of hormones stored for subsequent release. The blood supply for the anterior pituitary is via the hypothalamic-pituitary portal system, but the posterior pituitary blood supply is directly from the inferior hypophyseal arteries, which are branches of the posterior communicating and internal carotid arteries. The drainage is into the cavernous sinus and internal jugular vein.

The hormones of the posterior pituitary—oxytocin and vasopressin—are synthesized in individual hormone-specific magnocellular neurons. The supraoptic nucleus is relatively simple with 80% to 90% of the neurons producing vasopressin[2] and virtually all axons projecting to the posterior pituitary.[1] The organization of the paraventriculer nucleus (PVN), however, is much more complex and varies among species. In the human there are five subnuclei[2] and parvocellular (smaller cells) divisions that synthesize other peptides, such as corticotropin-releasing hormone (CRH), thyrotropin-releasing hormone (TRH), and somatostatin,[3] and opioids.[4] The parvocellular neurons project to the median eminence, brain stem, and spinal cord[5] where they play a role in a variety of neuroendocrine autonomic functions. The suprachiasmatic nucleus, which is located in the midline at the base of and anterior to the third ventricle, also synthesizes vasopressin and controls circadian rhythms as well as seasonal rhythms.[2]

Numerous neurotransmitters have been described in the pathway to stimulation or inhibition of secretion of vasopressin and oxytocin. The major stimulatory input is glutamate, with noradrenergic stimulatory inputs acting by stimulation of glutamate. [6] [7] Glutamate receptors account for 25% of synapsis on magnocellular neurons.[7] The major inhibitory input is γ-aminobutyric acid (GABA), which accounts for 50% of the synaptic input to the magnocellular neurons.[6] Steroid hormone actions on the magnocellular neurons are mediated by GABA or glutamate receptors.[8]

One of the most remarkable aspects of the magnocellular system is the plasticity of the system in response to prolonged stimulation. Plasticity is demonstrated in animals by prolonged osmotic stimulation with hypertonic saline but are probably most often of import in humans during parturition and lactation.[6] During prolonged stimulation the perikarya themselves enlarge and the glia retracts, diminishing astrocyte coverage of the neurons. These two events interact to increase the contact between cells, which enhances the synchrony and the pulsatile secretion, which for oxytocin is especially important during parturition and milk let-down of lactation. Secretion of oxytocin or vasopressin by dendrites of the respective neurons produces a positive feedback in a paracrine/autocrine fashion to enhance the further secretion of that hormone. Neurotransmitter numbers may increase on the neuron and the astrocytes themselves contribute to the plasticity, not just by altering their shape but also because they function to clear neurotransmitters from the extracellular space and release active substances themselves. This structural plasticity during stimulation is enabled by the continued presence in the supraoptic and paraventricular nuclei of cytoskeleton proteins and cell adhesion molecules that are present elsewhere in the brain only during embryogenesis.[9]

Ectopic Posterior Pituitary

With the development of magnetic resonance imaging (MRI) scans of the brain, it was discovered that T1-weighted images with MRI produced a bright signal in the posterior pituitary.[10] This new diagnostic imaging technology (described in detail later) allowed the identification of a group of patients in whom there was abnormal anatomy of the posterior pituitary and the “bright spot” was recognized in the base of the hypothalamus. These cases are referred to as ectopic posterior pituitary. Most of these cases are recognized in children with growth retardation and anterior pituitary deficiency rather than posterior pituitary deficiency. The degree of anterior pituitary deficit depends on the persistence of a pituitary stalk and a retained portal vasculature from the hypothalamus to the anterior pituitary. [11] [12] [13] Most authors believe this is not traumatic but represents a congenital abnormality with an “undescended”[13] posterior pituitary that may be at any level along the pituitary stalk.[13]

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ANTERIOR PITUITARY
Kronenberg: Williams Textbook of Endocrinology, 11th ed.
Copyright © 2008 Saunders, An Imprint of Elsevier

ANTERIOR PITUITARY

Shlomo Melmed David Kleinberg

▪ Development, Anatomy, and Overview of Control of Hormone Secretion, 155
▪ Pituitary Masses, 159
▪ Physiology and Disorders of Pituitary Hormone Axes, 180
▪ Pituitary Failure, 235
DEVELOPMENT, ANATOMY, AND OVERVIEW OF CONTROL OF HORMONE SECRETION

The pituitary gland, situated within the sella turcica, derives its name from the Greek ptuo and Latin pituita, “phlegm,” reflecting its nasopharyngeal origin. Galen hypothesized that nasal phlegm originated from the brain and drained via the pituitary gland. It is now clear that together with the hypothalamus, the pituitary orchestrates the structural integrity and function of endocrine glands including the thyroid, adrenal, and gonads, in addition to target tissues including cartilage and breast. The pituitary stalk serves as an anatomic and functional connection to the hypothalamus. Preservation of the hypothalamic-pituitary unit is critical for integration of anterior pituitary control of sexual function and fertility, linear and organ growth, lactation, stress responses, energy, appetite, and temperature regulation and secondarily for carbohydrate and mineral metabolism.

Integration of vital body functions by the brain was first proposed by Descartes in the 17th century. In 1733, Morgagni recorded the absence of adrenal glands in an anencephalic neonate, providing early evidence for a developmental and functional connection between the brain and the adrenal glands. In 1849, Claude Bernard set the stage for the subsequent advances in neuroendocrinology by demonstrating that central lesions to the area of the fourth ventricle resulted in polyuria.[1] Subsequent studies led to the identification and chemical isolation of pituitary hormones, and astute clinical observations led to the realization that pituitary tumors were associated with functional hypersecretory syndromes, including acromegaly and Cushing’s disease. [2] [3] [4] In 1948, Geoffrey Harris, the father of modern neuroendocrinology, in reviewing anterior pituitary gland hormone control, proposed their hypothalamic regulation, predicting the subsequent discovery of specific hypothalamic regulating hormones.[5]

Anatomy

The pituitary gland comprises the predominant anterior lobe, the posterior lobe, and a vestigial intermediate lobe ( Fig. 8-1 ). The gland is situated within the bony sella turcica and is overlaid by the dural diaphragma sella, through which the stalk connects to the median eminence of the hypothalamus. The adult pituitary weighs about 600 mg (range 400-900 mg) and measures about 13 mm in the longest transverse diameter, 6 to 9 mm vertically, and about 9 mm anteroposteriorly. Structural variation can occur in multiparous women, and gland volume also changes during the menstrual cycle. During pregnancy these measurements may be increased in either dimension, with pituitary weight increasing up to 1 gram. Recently, normal pituitary hypertrophy without evidence for the presence of an adenoma was described in seven eugonadal women with pituitary height greater than 9 mm and a convex upper gland boundary observed on magnetic resonance imaging (MRI).[6]

The sella turcica, located at the base of the skull, forms the thin bony roof of the sphenoid sinus. The lateral walls comprising, either bony or dural tissue, abut the cavernous sinuses, which are traversed by the third, fourth, and sixth cranial nerves and the internal carotid arteries ( Fig. 8-2 ). Thus, the cavernous sinus contents are vulnerable to increased intrasellar expansion. The dural roofing protects the gland from compression by fluctuant cerebrospinal fluid (CSF) pressure. The optic chiasm, located anterior to the pituitary stalk, is directly above the diaphragma sella. The optic tracts and central structures are therefore vulnerable to pressure effects by an expanding pituitary mass, which likely follows the path of least tissue resistance by lifting the diaphragma sella ( Fig. 8-3 ). The intimate relationship of the pituitary and chiasm is borne out in optic chiasmic hypoplasia associated with developmental pituitary dysfunction seen in patients with septo-optic dysplasia. The posterior pituitary gland, in contrast to the anterior pituitary, is directly innervated by supraopticohypophyseal and tuberohypophyseal nerve tracts of the posterior stalk. Hypothalamic neuronal lesions, stalk disruption, or direct systemically derived metastases therefore are often associated with attenuated vasopressin (AVP) (diabetes insipidus) or oxytocin secretion, or both.

The hypothalamus contains nerve cell bodies that synthesize hypophysiotropic releasing and inhibiting hormones, as well as the neurohypophyseal hormones of the posterior pituitary (AVP and oxytocin). Five distinct hormone-secreting cell types are present in the mature anterior pituitary gland. Corticotroph cells express pro-opiomelanocortin (POMC) peptides includ-ing adrenocorticotropic hormone (ACTH); somatotroph cells express growth hormone (GH); thyrotroph cells express the common glycoprotein α-subunit and the specific thyroid-stimulating hormone (TSH) β-subunit; gonadotrophs express the α and β subunits for both follicle-stimulating hormone (FSH) and luteinizing hormone (LH); the lactotroph expresses prolactin (PRL). Each cell type is under highly specific signal controls, which regulate their respective differentiated gene expression.

Pituitary Development

The pituitary gland arises from within the rostral neural plate. Rathke’s pouch, a primitive ectodermal invagination anterior to the roof of the oral cavity, is formed by the fourth to fifth week of gestation and gives rise to the anterior pituitary gland ( Fig. 8-4 ). [7] [8] The pouch is directly connected to the stalk and hypothalamic infundibulum, and it ultimately becomes distinct from the oral cavity and nasopharynx. Rathke’s pouch proliferates toward the third ventricle, where it fuses with the diverticulum, and subsequently obliterates its lumen, which sometimes persists as Rathke’s cleft. The anterior lobe is formed from Rathke’s pouch, and the diverticulum gives rise to the adjacent posterior lobe. Remnants of pituitary tissue can persist in the nasopharyngeal midline, and they rarely give rise to functional ectopic hormone-secreting tumors in the nasopharynx. The neurohypophysis arises from neural ectoderm associated with third ventricle development.[9]

Functional development of the anterior pituitary cell types involves complex spatiotemporal regulation of cell lineage–specific transcription factors expressed in pluripotential pituitary stem cells, as well as dynamic gradients of locally acting soluble factors. [10] [11] [12] Critical neuro-ectodermal signals for organizing the dorsal gradient of pituitary morphogenesis include infundibular bone morphogenetic protein 4 (BMP4) required for the initial pouch invagination,[8] fibroblast growth factor 8 (FGF-8), Wnt 5, and Wnt 4. Subsequent ventral developmental patterning and transcription factor expression is determined by spatial and graded expression of BMP2 and sonic hedgehog protein (shh) which appears critical for directing early patterns of cell proliferation.[13]

The human fetal Rathke’s pouch is evident at 3 weeks, and the pituitary grows rapidly in utero. By 7 weeks, the anterior pituitary vasculature begins to develop, and by 20 weeks, the entire hypophyseal-portal system is already established. The anterior pituitary undergoes major cellular differentiation during the first 12 weeks, by which time all the major secretory cell compartments are structurally and functionally intact, except for lactotrophs. Totipotential pituitary stem cells give rise to acidophilic (mammosomatotroph, somatotroph, and lactotroph) and basophilic (corticotroph, thyrotroph, and gonadotroph) differentiated pituitary cell types, which appear at clearly demarcated developmental stages. At 6 weeks, corticotroph cells are morphologically identifiable, and immunoreactive ACTH is detectable by 7 weeks. At 8 weeks, somatotroph cells are evident, with abundant immunoreactive cytoplasmic GH expression. Glycoprotein hormone–secreting cells express a common α-subunit for TSH, and at 12 weeks, differentiated thyrotrophs and gonadotrophs express immunoreactive β-subunits LH and FSH, respectively. Interestingly, in female fetuses, LH- and FSH-expressing gonadotrophs are equally distributed, whereas in the male fetus, LH-expressing gonadotrophs predominate.[14] Fully differentiated PRL-expressing lactotrophs are only evident late in gestation (after 24 weeks). Before then, immunoreactive PRL is only detectable in mixed mammosomatotrophs, also expressing GH, reflecting the common genetic origin of these two hormones.[15]

Pituitary Transcription Factors

Determination of anterior pituitary cell type lineages results from a temporally regulated cascade of homeodomain transcription factors. Although most pituitary developmental information has been acquired from murine models,[16] histologic and pathogenetic observations in human subjects have largely corroborated these developmental mechanisms (see Fig. 8-4 ). Early cell differentiation requires intracellular Rpx and Ptx expression. Rathke’s pouch expresses several transcription factors of the LIM homeodomain family, including Lhx3, Lhx4, and IsI-1,[17] which are early determinants of functional pituitary development. Pitx1 is expressed in the oral ectoderm, and subsequently in all pituitary cell types, particularly those arising ventrally.[18] Rieger’s syndrome, characterized by defective eye, tooth, umbilical cord, and pituitary development, is caused by defective related Pitx2. [19] [20]

Ptx behaves as a universal pituitary regulator and activates transcription of the α-glycoprotein subunit (a-GSU), POMC, and LHb (Ptx1) and GH (Ptx2). Lhx3 determines GH-, PRL-, and TSH-cell diffentiation, and Prop-1 behaves as a prerequisite for Pit-1, which activates GH, PRL, TSH, and growth hormone–releasing hormone (GHRH) receptor transcription. TSH and gonadotropin-expressing cells share a common α-subunit (aGSU) expression under developmental control of GATA-2.[11] These specific anterior pituitary transcription factors participate in a highly orchestrated cascade leading to the commitment of the five differentiated cell types (see Fig. 8-4 ). The major proximal determinant of pituitary cell lineage derived from a totipotential stem cell is thus Prop-1 expression, which determines subsequent development of PIT-1–dependent and gonadotroph cell lineages.[21]

POU1F1, the renamed Pit-1, is a POU-homeodomain transcription factor, which determines development and appropriate temporal and spatial expression of cells committed to GH-, PRL-, TSH-, and GHRH-receptor expression. POU1F1 binds to specific DNA motifs and activates and regulates somatotroph, lactotroph, and thyrotroph development and mature secretory function. Signal-dependent coactivating factors also cooperate with Pit-1 to determine specific hormone expression. Thus, in POU1F1-containing cells, high estrogen receptor levels induce a commitment to express PRL, whereas thyrotroph embryonic factor (TEF) favors TSH expression. Selective pituitary cell-type specificity is also perpetuated by binding of POU1F1 to its own DNA regulatory elements as well as those contained within the GH, PRL, and TSH genes. Steroidogenic factor (SF-1) and DAX-1 determine subsequent gonadotroph development. [22] [23] Corticotroph cell commitment, although occurring earliest during fetal development, is independent of POU1F1-determined lineages, and T-pit protein appears to be a prerequisite for POMC expression.[24] Hereditary mutations arising within these transcription factors can result in isolated or combined pituitary hormone failure syndromes (see later).

Pituitary Blood Supply

The pituitary gland enjoys an abundant blood supply derived from several sources (see Fig. 8-1 ). The superior hypophyseal arteries branch from the internal carotid arteries to supply the hypothalamus, where they form a capillary network in the median eminence, external to the blood-brain barrier. Both long and short hypophyseal portal vessels originate from infundibular plexuses and the stalk, respectively. These vessels form the hypothalamic-portal circulation, the predominant blood supply to the anterior pituitary gland. They deliver hypothalamic releasing and inhibiting hormones to the trophic hormone-producing cells of the adenohypophysis, without significant systemic dilution, allowing the pituitary cells to be sensitively regulated by timed hypothalamic hormone secretion. Vascular transport of hypothalamic hormones is also locally regulated by a contractile internal capillary plexus (gomitoli) derived from stalk branches of the superior hypophysial arteries.[25] Retrograde blood flow toward the median eminence also occurs, facilitating bidirectional functional hypothalamic-pituitary interactions.[26] Systemic arterial blood supply is maintained by inferior hypophysial arterial branches, which predominantly supply the posterior pituitary. Disruption of stalk integrity can lead to compromised pituitary portal blood flow, depriving the anterior pituitary cells of hypothalamic hormone access.

Pituitary Control

Three levels of control subserve the regulation of anterior pituitary hormone secretion ( Fig. 8-5 ). Hypothalamic control is mediated by adenohypophysiotropic hormones, which are secreted into the portal system and impinge directly upon anterior pituitary cell surface receptors. G-protein–linked cell surface membrane binding sites are highly selective and specific for each of the hypothalamic hormones, and they elicit positive or negative signals mediating pituitary hormone gene transcription and secretion. Peripheral hormones also participate in mediating pituitary cell function, predominantly by negative feedback regulation of trophic hormones by their respective target hormones. Intrapituitary paracrine and autocrine soluble growth factors and cytokines act to locally regulate neighboring cell development and function.

The net result of these three tiers of complex intracellular signals is the controlled pulsatile secretion of the six pituitary trophic hormones, ACTH, GH, PRL, TSH, FSH, and LH, through the cavernous sinus, petrosal veins, and ultimately the systemic circulation via the superior vena cava ( Fig. 8-6 ). The temporal and quantitative control of pituitary hormone secretion is critical for physiologic integration of peripheral hormonal systems, such as the menstrual cycle, which relies on complex and precisely regulated pulse control.

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Hypothalamus and Pituitary
Kronenberg: Williams Textbook of Endocrinology, 11th ed.
Copyright © 2008 Saunders, An Imprint of Elsevier
Hypothalamus and Pituitary

NEUROENDOCRINOLOGY

Malcolm J. Low

▪ Historical Perspective, 85
▪ Neural Control of Glandular Secretion, 86
▪ Hypothalamic-Pituitary Unit, 88
▪ Circumventricular Organs, 92
▪ Pineal Gland, 94
▪ Hypophyseotropic Hormones and Neuroendocrine Axes, 96
▪ Neuroendocrine Disease, 136
HISTORICAL PERSPECTIVE

The field of neuroendocrinology has expanded from its original focus on the control of pituitary hormone secretion by the hypothalamus to encompass multiple reciprocal interactions between the central nervous system (CNS) and endocrine systems in the control of homeostasis and physiologic responses to environmental stimuli. Although many of these concepts are relatively recent, the intimate interaction of the hypothalamus and the pituitary gland was recognized more than a century ago. For example, at the end of the nineteenth century, clinicians including Alfred Fröhlich described an obesity and infertility condition referred to as adiposogenital dystrophy in patients with sellar tumors.[1] This condition subsequently became known as Fröhlich’s syndrome and was most often associated with the accumulation of excessive subcutaneous fat, hypogonadotrophic hypogonadism, and growth retardation.

Whether this syndrome was due to injury to the pituitary gland itself or to the overlying hypothalamus was extremely controversial. Several leaders in the field of endocrinology, including Cushing and his colleagues, argued that the syndrome was due to disruption of the pituitary gland.[2] However, experimental evidence began to accumulate that the hypothalamus was somehow involved in the control of the pituitary gland. For example, Aschner demonstrated in dogs that the precise removal of the pituitary gland without damage to the overlying hypothalamus did not result in obesity.[3] Later, seminal studies by Hetherington and Ranson demonstrated that stereotaxic destruction of the medial basal hypothalamus with electrolytic lesions, which spared the pituitary gland, resulted in morbid obesity and neuroendocrine derangements similar to those of the patients described by Fröhlich.[4] This and subsequent studies clearly established that an intact hypothalamus is required for normal endocrine function. However, the mechanisms by which the hypothalamus was involved in endocrine regulation remained unsettled for years to come. We now know that the phenotypes of Fröhlich’s syndrome and the ventromedial hypothalamic lesion syndrome are probably due to dysfunction or destruction of key hypothalamic neurons that regulate pituitary hormone secretion and energy homeostasis.

The field of neuroendocrinology took a major step forward when several groups, especially Ernst and Berta Scharrer, recognized that neurons in the hypothalamus were the source of the axons that constitute the neural lobe (see Neurosecretion). The hypothalamic control of the anterior pituitary gland remained unclear, however. For example, Popa and Fielding identified the pituitary portal vessels linking the median eminence of the hypothalamus and the anterior pituitary gland.[5] Although they appreciated the fact that this vasculature provided a link between hypothalamus and pituitary gland, they hypothesized at the time that blood flowed from the pituitary up to the brain. Anatomic studies by Wislocki and King supported the concept that blood flow was from the hypothalamus to the pituitary.[6] Later studies, including the seminal work of Geoffrey Harris, established the flow of blood from the hypothalamus at the median eminence to the anterior pituitary gland.[7] This supported the concept that the hypothalamus controlled anterior pituitary gland function indirectly and led to the now accepted hypophyseal-portal chemotransmitter hypothesis.

Subsequently, several important studies, especially those from Schally and colleagues and the Guillemin group, established that the anterior pituitary is tightly controlled by the hypothalamus. [8] [9] Both groups identified several putative peptide hormone-releasing factors (see later sections). These fundamental studies resulted in the awarding of the Nobel Prize in Medicine in 1977 to Andrew Schally and Roger Guillemin. [10] [11] Of course, we now know that these releasing factors are the fundamental link between the central nervous system (CNS) and the control of endocrine function. Furthermore, these neuropeptides are highly conserved across species and are essential for reproduction, growth, and metabolism. The anatomy, physiology, and genetics of these factors constitute a major portion of this chapter.

Over the past 2 decades, work in the field of neuroendocrinology has continued to advance across several fronts. Cloning and characterization of the specific G protein–coupled receptors used by the hypothalamic-releasing factors have helped define signaling mechanisms utilized by the releasing factors. Characterization of the distribution of these receptors has universally demonstrated receptor expression in the brain and in peripheral tissues other than the pituitary, arguing for multiple physiologic roles for the neuropeptide releasing factors. Finally, the last 2 decades have also seen tremendous advances in our understanding of both regulatory neuronal and humoral inputs to the hypophyseotropic neurons.

The adipostatic hormone leptin, discovered in 1994,[12] is an example of a humoral factor that has profound effects on multiple neuroendocrine circuits. Reduction in circulating leptin is responsible for suppression of the thyroid and reproductive axes during the starvation response. The subsequent discovery of ghrelin,[13] a stomach peptide that regulates appetite and also acts on multiple neuroendocrine axes, demonstrates that much remains to be learned regarding the regulation of the hypothalamic-releasing hormones. Traditionally, it has been extremely difficult to study releasing factor gene expression or the specific regulation of the releasing factor neurons because of their small numbers and, in some cases, diffuse distribution. Transgenic experiments have produced mice in which expression of fluorescent marker proteins has been specifically targeted to gonadotropin-releasing hormone (GnRH) neurons[14] and arcuate pro-opiomelanocortin (POMC) neurons[15] among others. This technology will allow detailed study of the electrophysiologic properties of hypothalamic neurons in the more native context of slice preparations or organotypic cultures.

As just described, much of the field of neuroendocrinology has focused on hypothalamic-releasing factors and their control of reproduction, growth, development, fluid balance, and the stress response through their control of pituitary hormone production. More broadly, however, neuroendocrinology has become a rubric to define the study of interaction of the endocrine and nervous systems in the regulation of homeostasis. The field of neuroendocrinology has been further expanded, however, because diverse areas of basic research have often been fundamental to understanding the neuroendocrine system and thus championed by its investigators. These areas include studies of neuropeptide structure, function, and mechanism of action; neural secretion; hypothalamic neuroanatomy; G protein–coupled receptor structure, function, and signaling; transport of substances into the brain; and the action of hormones on the brain. Many homeostatic systems involve integrated endocrine, autonomic, and behavioral responses. Thus, many homeostatic systems exist in which the classic neuroendocrine axes are important but not autonomous pathways, such as energy homeostasis and immune function, and these subjects also are studied often in the context of neuroendocrinology.

This chapter first presents the concepts of neural secretion, the neuroanatomy of the hypothalamic-pituitary unit, and the CNS structures most relevant to the control of the neurohypophysis and adenohypophysis. It then covers each classical hypothalamic-pituitary axis, including a consideration of the immune system and its integration with neuroendocrine function. Finally, the chapter reviews the pathophysiology of disorders of neural regulation of endocrine function. The neuroendocrinology of energy homeostasis is fully considered in Chapter 34 .

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MECHANISM OF ACTION OF HORMONES THAT ACT AT THE CELL SURFACE

Kronenberg: Williams Textbook of Endocrinology, 11th ed.
Copyright © 2008 Saunders, An Imprint of Elsevier

MECHANISM OF ACTION OF HORMONES THAT ACT AT THE CELL SURFACE

Allen Spiegel Christin Carter-Su Simeon I. Taylor

▪ Receptors, 47
▪ Hormone Binding, 48
▪ Regulation of Hormone Sensitivity, 48
▪ Receptor Tyrosine Kinases, 48
▪ Downstream Signaling Pathways, 51
▪ Off Signals: Termination of Hormone Action, 53
▪ Mechanisms of Disease, 53
▪ Receptor Serine Kinases, 54
▪ Receptors that Signal through Associated Tyrosine Kinases, 55
▪ G Protein–Coupled Receptors, 58
▪ G Protein–Coupled Receptor Interactions with Other Proteins, 61

Hormones are secreted into the blood and act upon target cells at a distance from the secretory gland. In order to respond to a hormone, a target cell must contain the essential components of a signaling pathway. First, there must be a receptor to bind the hormone. Second, there must be an effector—or example, an enzymatic activity that is regulated when the hormone binds to its receptor. Finally, there must be appropriate downstream signaling pathways to mediate the physiologic responses to the hormone. In fact, this type of mechanism involving receptors, effectors, and downstream signaling pathways is quite general and also functions in nonendocrine systems such as those regulated by neurotransmitters, cytokines, and paracrine and autocrine factors. This chapter reviews several examples of endocrine signaling pathways that begin with activation of receptors located on the surface of target cells, with particular attention to the molecular mechanisms that function in normal physiology and to the molecular pathology causing disease.

Receptors

Definition and Classification

There are two essential functions that define hormone receptors: (1) the ability to bind the hormone and (2) the ability to couple hormone binding to hormone action. Both components of the definition are essential; for example, many hormones bind to binding proteins that are distinct from receptors because the binding proteins do not trigger the signaling pathways that mediate hormone action.

Many classes of receptors are of interest in endocrinology. Some receptors are located within the cell and function as transcription factors (e.g., receptors for steroid and thyroid hormones). Other receptors are located on the cell surface and function primarily to transport their ligands into the cell by a process referred to as receptor-mediated endocytosis (e.g., low-density lipoprotein receptors). In this chapter, we focus upon cell-surface receptors that trigger intracellular signaling pathways. These cell-surface receptors can be classified according to the molecular mechanisms by which they accomplish their signaling function: 1. Ligand-gated ion channels (e.g., nicotinic acetylcholine receptors)
2. Receptor tyrosine kinases (e.g., receptors for insulin and insulin-like growth factor I [IGF-I])
3. Receptor serine/threonine kinases (e.g., receptors for activins and inhibins)
4. Receptor guanylate cyclase (e.g., atrial natriuretic factor receptor)
5. G protein–coupled receptors (e.g., receptors for adrenergic agents, muscarinic cholinergic agents, glycoprotein hormones, glucagon, and parathyroid hormone)
6. Cytokine receptors (e.g., receptors for growth hormone, prolactin, and leptin)

The receptors belonging to classes 1 to 4 are bifunctional molecules that can bind hormone as well as serve as effectors by functioning either as ion channels or as enzymes. In contrast, the receptors belonging to classes 5 and 6 have the ability to bind the hormone but must recruit a separate molecule to catalyze the effector function. For example, as the name implies, G protein–coupled receptors utilize G proteins to regulate downstream effector molecules. Similarly, cytokine receptors recruit cytosolic tyrosine kinases (e.g., Janus family tyrosine kinases, or JAKs) as effectors to trigger downstream signaling pathways.

Hormone Binding

As predicted by the fact that hormones circulate in relatively low concentrations in the plasma, the binding interaction between a hormone and its receptor is characterized by high binding affinity. Furthermore, hormone binding has a high degree of specificity. Generally, the receptor binds its cognate hormone more tightly than it binds other hormones. However, some receptors may bind structurally related hormones with lower affinity. For example, the insulin receptor binds insulin-like growth factors (IGFs) with approximately 100-fold lower affinity than it binds insulin. Similarly, the thyrotropin receptor binds human chorionic gonadotropin with lower affinity than it binds thyrotropin. This phenomenon has been referred to as specificity spillover and provides an explanation of several pathologic conditions, such as hypoglycemia caused by tumors secreting IGF-II and hyperthyroidism associated with choriocarcinoma.[1]

Binding of a hormone (H) to its receptor (R) can be described mathematically as an equilibrium reaction:

At equilibrium, Ka = (HR)/(H)(R), where Ka is the association constant for the formation of the hormone receptor complex (HR). As originally shown by Scatchard, it is possible to rearrange this equation in terms of the total concentration of receptor binding sites, R0 = (R) + (RH), as follows:

A straight line is obtained when (HR)/(H) (i.e., the ratio of bound to free hormone) is plotted as a function of (HR) (the concentration of bound hormone). The slope of the line is -Ka, and the line intercepts the horizontal axis at the point where (HR) = R0 = the total number of binding sites. This type of plot is referred to as a Scatchard plot and has been used as a graphic method to estimate the affinity with which a receptor binds its hormone. Although the binding properties of some receptors are described more or less accurately by these simple equations, other receptors exhibit more complex properties. This simple algebraic derivation of the Scatchard equation implicitly assumes that there is only one class of receptors and that the binding sites on the receptors do not interact with one another. If these assumptions do not apply to the interaction of a particular hormone with its receptor, the Scatchard plot may not be linear.

Several molecular mechanisms may contribute to nonlinearity of the Scatchard plot. For example, there may be more than one type of receptor that binds the hormone (e.g., a high-affinity, low-capacity site and a low-affinity, high-capacity site). Alternatively, some receptors have more than one binding site, and there may be cooperative interactions among the binding sites (e.g., the insulin receptor). In addition, the interaction between a G protein and a G protein–coupled receptor may affect the affinity with which the receptor binds its ligand; moreover, the effect on binding affinity depends on whether guanosine diphosphate (GDP) or guanosine triphosphate (GTP) is bound to the G protein. However, a detailed discussion of these complexities is beyond the scope of this chapter.

Regulation of Hormone Sensitivity

Early in the history of endocrinology, attention was focused on the regulation of hormone secretion as the most important mechanism for regulating physiology. However, it has become apparent that the target cell is not passive. Rather, there are many influences that can alter the sensitivity of the target cell’s response to a given concentration of hormone. For example, the number of receptors can be regulated. All things being equal, hormone sensitivity is directly related to the number of hormone receptors expressed on the cell surface. In addition, posttranslational modifications of the receptor can modify either the affinity of hormone binding or the efficiency of coupling to downstream signaling pathways. Moreover, all of the downstream components in the hormone action pathway are subject to similar types of regulatory influences, which can have a significant impact on the ability of the target cell to respond to hormone.

Just as hormone sensitivity is subject to normal physiologic regulation, pathologic influences can cause disease by targeting components of the hormone action pathway. Multiple etiologic factors can impair the hormone action pathway, such as genetic influences, autoimmune processes, and exogenous toxins. For example, disease mechanisms can alter the functions of cell-surface receptors, effectors such as G proteins, and other components of the downstream signaling pathways. This chapter describes several examples illustrating these principles.

Receptor Tyrosine Kinases

Receptor tyrosine kinases have several structural features in common: an extracellular domain containing the ligand-binding site, a single transmembrane domain, and an intracellular portion that includes the tyrosine kinase catalytic domain ( Fig. 5-1 ). Analysis of the sequence of the human genome suggests that there are approximately 100 receptor tyrosine kinases. The tyrosine kinase domain is the most highly conserved sequence among all the receptors in this family. In contrast, there is considerable variation among the sequences of the extracellular domains. Indeed, the family of receptor tyrosine kinases can be classified into 16 subfamilies, primarily on the basis of the differences in the structure of the extracellular domain.[2] Furthermore, receptor tyrosine kinases mediate the biologic actions of a wide variety of ligands, including insulin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and vascular endothelial cell–derived growth factor. The variation in the sequences of the extracellular domains enables the receptors to bind this structurally diverse collection of ligands.

The EGF receptor was the first cell-surface receptor demonstrated to possess tyrosine kinase activity[4] and it was the first receptor tyrosine kinase to be cloned.[5] Like most receptor tyrosine kinases, the EGF receptor exists primarily as a monomer in the absence of ligand. However, binding of ligand induces receptor dimerization. As discussed later in this chapter, ligand-induced dimerization is central to the mechanism whereby the receptor mediates the biologic activity of EGF. In addition to the ability to form homodimers, the EGF receptor can form heterodimers with other members of the same subfamily of receptor tyrosine kinases. Because a small number of receptors can combine in a large number of pairings, heterodimer formation has the potential to fine-tune the specificity of receptors with respect to both ligand binding and downstream signaling.

The insulin receptor is of special interest to endocrinologists because diabetes is among the most common diseases of the endocrine system. Furthermore, the insulin receptor closely resembles the type 1 receptor for IGFs.[6] This is the receptor that mediates the biologic actions of IGF-I and therefore also plays an important role in the physiology of growth hormone (GH) in vivo. Although the kinase domains of receptors for insulin and IGF-I closely resemble other receptor tyrosine kinases, at least two distinctive features set them apart. First, the receptors are synthesized as proreceptors that undergo proteolytic cleavage into two subunits (α and β). The α subunit contains the ligand-binding site; the β subunit includes the transmembrane and tyrosine kinase domains. Second, both receptors exist as α2β2 heterotetramers that are stabilized by intersubunit disulfide bonds. In contrast to other receptor tyrosine kinases, which are thought to dimerize in response to ligand binding, the insulin receptor exists as a dimer of αβ monomers even in the absence of ligand. The remainder of this section reviews the molecular mechanisms whereby receptor tyrosine kinases mediate biologic action, with special emphasis on the insulin receptor as an illustrative example.

Receptor Activation: Role of Receptor Dimerization

Dimerization plays a central role in the mechanism whereby most receptor tyrosine kinases are activated by their cognate ligands. [2] [7] Although receptor dimerization is a common theme, the detailed molecular mechanisms differ from receptor to receptor. The following are three examples of the mechanisms of receptor dimerization ( Fig. 5-2 ). [8] [9] [10] [11] [12]

Dimeric Ligand

PDGF and vascular endothelial cell-derived growth factor are examples of dimeric ligands (see Fig. 5-2 ). [8] [9] [13] Because each subunit of ligand can bind one receptor molecule, simultaneous binding of two receptor molecules drives receptor dimerization. Direct support for this type of mechanism is provided by the crystal structure of vascular endothelial cell–derived growth factor bound to its receptor (Flt-1).[13]

Two Receptor Binding Sites on a Monomeric Ligand

Although this mechanism is important for many receptor tyrosine kinases, it was first shown rigorously for the GH receptor, which is not a member of the receptor tyrosine kinase family (see Fig. 5-2 ). [10] [11] [12] As illustrated by the crystal structure of GH bound to its receptor, one molecule of ligand can bind two molecules of receptor. In fact, there are two distinct receptor-binding sites on each GH molecule, and this enables the ligand to promote receptor dimerization. This observation has an important implication for pharmacology. By abolishing one of the two receptor-binding sites, it is possible to design mutant ligands that lack the ability to promote receptor dimerization and therefore lack the ability to trigger hormone action. Nevertheless, by binding to receptors, the mutant ligand acquires the ability to inhibit the action of the endogenous hormone. Such mutant GH molecules have been developed as therapeutic agents, for example, in conditions such as acromegaly.

Preexisting Receptor Dimers

The insulin receptor represents a paradox. The insulin receptor exists as a dimer even in the absence of ligand. (Actually, it is an α2β2 heterotetramer, which is a dimer of αβ monomers.) If the receptor is already dimerized, why is it not active? Although the molecular details remain to be elucidated, it seems likely that the two halves of the insulin receptor are not oriented in an optimal way to permit receptor activation in the absence of ligand. Perhaps, insulin binding triggers a conformational change that somehow mimics the effects of dimerization in other receptor tyrosine kinases. In any case, several studies have demonstrated that receptor dimerization is necessary for the ability of insulin to activate its receptor. For example, αβ monomers retain the ability to bind insulin but are not activated in response to insulin binding. [14] [15] Furthermore, indirect evidence suggests that a single insulin molecule binds simultaneously to both α subunits of the insulin receptor [16] [17]; the ability to bind simultaneously to both halves of the dimeric receptor appears to be essential to the ability of insulin to activate its receptor.

Receptor Activation: Conformational Changes in the Kinase Domain

When ligand binds to the extracellular domain, it stimulates the tyrosine kinase activity of the intracellular domain. Although the detailed mechanisms of transmembrane signaling are not completely understood, considerable progress has been made in elucidating the molecular mechanisms of receptor activation. Investigations of the three-dimensional structure of the insulin receptor tyrosine kinase domain help explain why the receptor is maintained in a low-activity state in the absence of insulin ( Fig. 5-3 ). [18] [19] [20] In the inactive form of the insulin receptor kinase, Tyr1162 is located in a position so that it blocks protein substrates from binding to the active site. Furthermore, in the inactive state of the tyrosine kinase domain, the active site assumes a conformation that does not accommodate magnesium adenosine triphosphate (ATP). Thus, the tyrosine kinase is inactive because the active site cannot bind either of its substrates. How does insulin activate the receptor? Insulin binding triggers autophosphorylation of three tyrosine residues (Tyr1158, Tyr1162, and Tyr1163) in the “activation loop.” When the three tyrosine residues in the activation loop become phosphorylated, an important conformational change occurs. As a result of the movement of the activation loop, the active site acquires the ability to bind both ATP and protein substrates. Thus, the conformational change induced by autophosphorylation activates the receptor to phosphorylate other substrates. [21] [22]

It remains unclear how this process is initiated. Because the inactive state of the tyrosine kinase cannot bind ATP, it seems unlikely that phosphorylation of Tyr1162 proceeds by a true autophosphorylation mechanism. Rather, it is likely that Tyr1162 in one β subunit is transphosphorylated by the second β subunit in the α2β2 heterotetramer molecule. [2] [23] However, this proposed mechanism poses a “chicken and egg” problem. It requires that at least one of the β subunits is active before the Tyr residues in the activation loop become phosphorylated. Perhaps the activation loop is somewhat mobile so that some molecules of unphosphorylated tyrosine kinase can assume an active conformation and initiate a chain reaction of transphosphorylation and receptor activation.

Receptor Tyrosine Kinases Phosphorylate Other Intracellular Proteins

Once activated, tyrosine kinases are capable of phosphorylating other protein substrates. Several factors determine which proteins are phosphorylated under physiologic conditions within the cell.

Amino Acid Sequence Context of Tyr Residue

Tyrosine kinases do not exhibit strict specificity with respect to the amino acid sequence of the phosphorylation site. Nevertheless, most tyrosine phosphorylation sites are located in the vicinity of acidic amino acid residues (i.e., Glu or Asp).[2]

Binding to the Tyrosine Kinase

Some protein substrates bind directly to the intracellular domain of the receptor. The binding interaction brings the substrate into close proximity to the kinase, thereby promoting phosphorylation of the substrate. For example, the insulin receptor substrate (IRS) proteins are characterized by a highly conserved phosphotyrosine-binding (PTB) domain that binds to a conserved motif (Asn-Pro-Xaa-pTyr) in the juxtamembrane domain of the insulin receptor. [24] [25] [26] Binding of the PTB domain to the insulin receptor requires phosphorylation of the Tyr residue in the Asn-Pro-Xaa-pTyr motif. This provides another mechanism (in addition to activation of the intrinsic receptor tyrosine kinase) whereby autophosphorylation of the receptor enhances phosphorylation of IRS proteins. Similarly, substrates for some tyrosine kinases contain Src homology 2 (SH2) domains, highly conserved domains that bind phosphotyrosine residues (see Functional Significance of Tyrosine Phosphorylation). For example, the activated PDGF receptor contains a phosphotyrosine residue near its C-terminus that binds the SH2 domain of phospholipase Cγ. This enables the PDGF receptor to phosphorylate and activate phospholipase Cγ. [2] [27]

Subcellular Localization

Because receptor tyrosine kinases are located in the plasma membrane, they are in close proximity to other plasma membrane proteins. This colocalization has the potential to pro-mote phosphorylation. For example, the insulin receptor has been reported to phosphorylate pp120/hepatocyte antigen-4 (HA4). [28] [29] Like the insulin receptor, pp120/HA4 is an integral membrane glycoprotein associated with the plasma membrane of hepatocytes. Similarly, FGF receptor substrate-2 (FRS2), a substrate of the fibroblast-derived growth factor receptor, is targeted to the plasma membrane by an N-terminal myristoylation site.[30]

Functional Significance of Tyrosine Phosphorylation

There are at least two distinct mechanisms whereby tyrosine phosphorylation regulates protein function. First, tyrosine phosphorylation can induce a conformational change in a protein, thereby altering its function. For example, as discussed earlier, phosphorylation of the three Tyr residues in the activation loop of the insulin receptor changes the conformation of the active site, thereby facilitating binding of substrates and activating the receptor tyrosine kinase. [18] [19] [20] However, most of the effects of tyrosine phosphorylation on protein function are mediated indirectly by regulating protein-protein interactions. In order to understand how tyrosine phosphorylation regulates protein-protein interactions, it is useful to review the biochemistry of c-src, the prototype of a nonreceptor tyrosine kinase. When the amino acid sequence of c-src is analyzed, it is apparent that there are three highly conserved domains in the molecule: the kinase catalytic domain and two noncatalytic domains that are referred to as src homology domains 2 and 3 (SH2 and SH3, respectively).

SH2 Domains

SH2 domains consist of conserved sequences (approximately 100 amino acid residues) that are present in many proteins that function in signaling pathways. From a functional point of view, SH2 domains share the ability to bind pTyr residues. However, individual SH2 domains vary with respect to their binding specificity. The binding affinity of an SH2 is determined by the three amino acid residues downstream from the pTyr residue. For example, the SH2 domains of phosphatidylinositol (PI) 3-kinase exhibit a preference for pTyr-(Met/Xaa)-Xaa-Met, whereas the SH2 domain of growth factor receptor binding protein 2 (Grb-2) prefers to bind pTyr-Xaa-Asn-Xaa. Thus, a given SH2 domain binds to a tyrosine-phosphorylated protein if and only if the pTyr residue is located in a context that corresponds to the binding specificity of the SH2 domain.

SH3 Domains

SH3 domains consist of conserved sequences (approximately 50 amino acid residues) that bind to proline-rich sequences. Like SH2 domains, SH3 domains are found in many proteins that function in signaling pathways.

Downstream Signaling Pathways

Receptor tyrosine kinases mediate the action of a wide variety of ligands in a wide variety of cell types. The bewildering complexity of the downstream signaling pathways corresponds to the huge number of physiologic processes that are regulated by receptor tyrosine kinases. Although it is beyond the scope of this chapter to attempt an encyclopedic review of all the downstream signaling pathways, we have selected examples to illustrate general principles.

As discussed earlier, the activated insulin receptor phosphorylates multiple substrates including IRS-1, IRS-2, IRS-3, and IRS-4.[31] Each of these substrates contains multiple tyrosine phosphorylation sites, many of which correspond to consensus sequences for SH2 domains in important signaling molecules. Thus, IRS proteins serve as docking proteins that bind SH2 domain-containing proteins. Among these, two of the most important are PI 3-kinase and Grb-2. Binding of SH2 domains triggers multiple downstream signaling pathways.

Phosphatidylinositol 3-Kinase

The catalytic subunit of PI 3-kinase (p110; molecular mass approximately 110,000) is bound to a regulatory subunit. The classical isoforms of the regulatory subunit (p85; molecular mass approximately 85,000) contain two SH2 domains, both of which bind to pTyr in the context of pTyr-(Met/Xaa)-Xaa-Met motifs. Binding of pTyr residues to both SH2 domains of p85 leads to maximal activation of PI 3-kinase catalytic activity. (Submaximal activation can be achieved with occupancy of a single SH2 domain in p85.) Because all four IRS molecules (IRS-1, IRS-2, IRS-3, and IRS-4) contain multiple tyrosine phosphorylation sites that conform to the Tyr-(Met/Xaa)-Xaa-Met consensus sequence, insulin-stimulated phosphorylation promotes binding of IRS proteins to the SH2 domains in the regulatory subunit PI 3-kinase, thereby increasing the enzymatic activity of the catalytic subunit. [32] [33] [34] [35] Activation of PI 3-kinase triggers activation of a cascade of downstream kinases, beginning with phosphoinositide-dependent kinases 1 and 2. These phosphoinositide-dependent kinases phosphorylate and activate multiple downstream protein kinases including protein kinase B and atypical isoforms of protein kinase C. [36] [37] [38] [39] [40] [41] [42]

A large body of evidence demonstrates that the pathways downstream from PI 3-kinase mediate the metabolic activities of insulin (e.g., activation of glucose transport into skeletal muscle, activation of glycogen synthesis, and inhibition of transcription of the phosphoenolpyruvate carboxykinase gene). Among other lines of evidence, PI 3-kinase inhibitors (e.g., LY294002 and wortmannin) block the metabolic actions of insulin.[43] Similarly, overexpression of dominant negative mutants of the p85 regulatory subunit of PI 3-kinase also inhibits the metabolic actions of insulin.[36] Although it is generally agreed that activation of PI 3-kinase is necessary, it is controversial whether it is sufficient to trigger the metabolic actions of insulin. For example, a second parallel pathway may also be required. The latter pathway involves tyrosine phosphorylation of Cbl, another protein that can be phosphorylated by the insulin receptor in some cell types. [44] [45] [46]

Grb-2 and the Activation of Ras

Grb-2 is a short adaptor molecule that contains an SH2 domain[47] capable of binding to pTyr residues in several signaling molecules, for example, IRS-1 and Shc, another PTB domain-containing protein that is phosphorylated by several receptor tyrosine kinases including the insulin receptor. [48] [49] The SH2 domain of Grb-2 is flanked by two SH3 domains,[47] which bind to proline-containing sequences in mSos (the mammalian homologue of Drosophila son-of-sevenless).[50] mSos is capable of activating Ras, a small G protein that plays an important role in intracellular signaling pathways. mSos activates Ras by catalyzing the exchange of GTP for GDP in the guanine nucleotide-binding site of Ras. This, in turn, triggers the activation of a cascade of serine/threonine-specific protein kinases including Raf, mitogen-activated protein/extracellular signal-regulated kinase (MEK), and mitogen-activated protein (MAP) kinase. These pathways downstream from Ras contribute to the ability of tyrosine kinases to promote cell growth and regulate the expression of various genes.

We have focused on the signaling pathways downstream from the insulin receptor because of the importance of insulin and IGF-I in endocrinology ( Fig. 5-4 ). In many ways, the molecular mechanisms closely resemble those downstream from other receptor tyrosine kinases. However, the insulin signaling pathway is atypical in at least one respect. The insulin receptor phosphorylates docking proteins (e.g., IRS-1), which bind SH2 domain-containing proteins (e.g., PI 3-kinase and Grb-2). In contrast, the intracellular domains of most receptor tyrosine kinases contain binding sites for SH2 domains. For example, the SH2 domain of Grb-2 binds to pTyr716 in the activated PDGF receptor.[2] Similarly, the PDGF receptor contains two Tyr-(Met/Xaa)-Xaa-Met motifs in the kinase insert domain that bind to the two SH2 domains in the p85 subunit of PI 3-kinase. [2] [51] It is not clear why some tyrosine kinases (e.g., the PDGF receptor) activate PI 3-kinase through a direct binding interaction, whereas others (e.g., the insulin receptor) utilize an indirect mechanism involving docking proteins. However, in contrast to PDGF receptors, which are associated with the plasma membrane, IRS proteins appear to be associated with the cytoskeleton.[52] Perhaps this differential subcellular localization contributes to signaling specificity. In other words, if insulin and PDGF receptors trigger translocation of PI 3-kinase to different locations within the cell, this compartmentation may permit two different receptors to elicit different biologic responses even though both responses are mediated by the same signaling molecule (i.e., PI 3-kinase).

Off Signals: Termination of Hormone Action

Just as there are complex biochemical pathways that mediate hormone action, there are also mechanisms to terminate the biologic response. The necessity for these mechanisms is illustrated by the following example. After we eat a meal, the concentration of plasma glucose increases. This elicits an increase in insulin secretion, which in turn leads to a decrease in plasma glucose levels. If these processes went on unchecked, the level of glucose in the plasma would eventually fall so low that it would lead to symptomatic hypoglycemia. How is insulin action terminated? The answers to this question are not yet entirely clear, but several mechanisms contribute to turning off the insulin signaling pathway.

Receptor-Mediated Endocytosis

Insulin binding to its receptor triggers endocytosis of the receptor. Although most of the internalized receptors are recycled to the plasma membrane, some receptors are transported to lysosomes, where they are degraded. [55] [56] As a result, insulin binding accelerates the rate of receptor degradation, thereby down-regulating the number of receptors on the cell surface. Furthermore, endosomes contain proton pumps, which acidify the lumen; the acidic pH within the endosome promotes dissociation of insulin from its receptor. Ultimately, insulin is transported to the lysosome for degradation. In fact, receptor-mediated endocytosis is the principal mechanism whereby insulin is cleared from the plasma.[57] Binding of ligands to other receptor tyrosine kinases also triggers receptor-mediated endocytosis by similar mechanisms.

Protein Tyrosine Phosphatases

Protein phosphorylation is a dynamic process. Tyrosine kinases catalyze the phosphorylation of tyrosine residues, but there are also protein tyrosine phosphatases (PTPases) to remove the phosphates.[2] Thus, PTPases antagonize the action of tyrosine kinases. Studies with knockout mice have demonstrated that the absence of PTPase-1B is associated with increased insulin sensitivity and also protects against weight gain. [58] [59] Nevertheless, the human genome encodes a large number of PTPases, and it is an important goal of research to elucidate their physiologic functions. If one could develop selective inhibitors of the PTPases that oppose the effects of the insulin receptor tyrosine kinase, it is possible that these inhibitors would provide novel therapies for diabetes.

Serine/Threonine Kinases

Most receptor tyrosine kinases, including the insulin receptor, are substrates for phosphorylation by Ser/Thr-specific protein kinases. Interestingly, the Ser/Thr phosphorylation appears to inhibit the action of the tyrosine kinase. Similarly, other phosphotyrosine-containing proteins are subject to inhibitory influences of Ser/Thr phosphorylation resistance. For example, it has been reported that Ser/Thr phosphorylation of IRS-1 may inhibit insulin action, thereby causing insulin resistance. [60] [61] [62] [63] [64]

Mechanisms of Disease

The simplest forms of endocrine disease are caused by either a deficiency or an excess of a hormone. However, hormone resistance syndromes resulting from defects in the signaling pathways can masquerade as hormone deficiency states. Similarly, diseases associated with constitutively activated receptors can mimic states of hormone excess. In some cases, the abnormality in hormone action is genetic in origin, resulting from a mutation in a gene encoding one of the proteins in the signaling pathway. Similar syndromes can also be caused by other mechanisms; for example, there are autoimmune syndromes caused by autoantibodies directed against cell-surface receptors. These clinical syndromes illustrate the principle that understanding the biochemical pathways of hormone action can provide important insights into the pathophysiology of human disease.

Genetic Defects in Receptor Function

At least two distinct major types of genetic defects can cause hormone resistance.[65] First, mutations can lead to a decrease in the number of receptors. For example, in the case of the insulin receptor, mutations have been identified that decrease receptor number by at least three mechanisms: (1) impairing receptor biosynthesis, (2) inhibiting the transport of receptors to their normal location in the plasma membrane, and (3) accelerating the rate of receptor degradation. Second, mutations can impair the intrinsic activities of the receptor. In the case of the insulin receptor, mutations have been reported that decrease the affinity of insulin binding or inhibit receptor tyrosine kinase activity.

Receptor dimerization is known to play a central role in the mechanisms whereby ligands activate many cell-surface receptors. This role has been shown most convincingly in the case of the GH receptor (a member of the family of cytokine receptors) but has also been postulated for receptor tyrosine kinases. The syndromes of multiple endocrine neoplasia types 2A and 2B and familial medullary carcinoma of the thyroid are caused by mutations in the gene encoding the Ret tyrosine kinase (a subunit of the receptor for glial cell–derived growth factor).[66] Ordinarily, cysteine residues in the extracellular domain of Ret participate in the formation of intramolecular disulfide bonds. Mutation of one of the cysteine residues leaves an unpaired cysteine residue that promotes dimerization of Ret molecules, thereby activating the Ret receptor tyrosine kinase ( Fig. 5-5 ). Activation of the Ret tyrosine kinase through this germ line mutation converts Ret into an oncogene.

Autoantibodies Directed against Cell-Surface Receptors

Inhibitory antireceptor autoantibodies were first identified in myasthenia gravis.[69] In this neurologic disease, antibodies to the nicotinic acetylcholine receptor impair neuromuscular transmission, apparently by accelerating receptor degradation. Subsequently, autoantibodies to the insulin receptor were demonstrated to block insulin action in the syndrome of type B extreme insulin resistance.[70] Insulin resistance is caused by at least two mechanisms: (1) the antireceptor antibodies inhibit insulin binding to the receptor[71] and (2) the antibodies accelerate receptor degradation.[72]

Graves’ disease provided the first example of stimulatory antireceptor autoantibodies.[73] In Graves’ disease, there are autoantibodies directed against the thyroid-stimulating hormone (TSH) receptor. These antireceptor antibodies activate the TSH receptor, thereby stimulating growth of the thyroid gland as well as hypersecretion of thyroid hormone. This “experiment of nature” demonstrates that the receptor can be activated by ligands other than the physiologic ligand and that the normal spectrum of biologic actions can be triggered by this unphysiologic ligand (i.e., the antireceptor antibody). Similarly, antibodies to the insulin receptor have been demonstrated to activate the insulin receptor by mimicking insulin action. Although it is more common for a patient with anti–insulin receptor autoantibodies to present with insulin resistance, patients with anti–insulin receptor autoantibodies have also been reported to experience fasting hypoglycemia. [74] [75]

Receptor Serine Kinases

Receptor serine kinases[76] have several features in common with receptor tyrosine kinases. For example, both classes of receptors possess (1) N-terminal extracellular domains, which bind ligand, (2) a single transmembrane domain, and (3) C-terminal intracellular domains, which possess protein kinase activity. However, the two classes of receptors differ with respect to enzymatic specificity. Whereas receptor tyrosine kinases phosphorylate tyrosine residues, receptor serine kinases phosphorylate serine and threonine residues in their protein substrates. There are two types of receptor serine kinases: type I and type II. The human genome contains 12 genes encoding receptor serine kinases—seven type I and five type II receptors—each of which is approximately 500 amino acids in length.

Receptor Activation: Role of Receptor Dimerization

Receptor serine kinases mediate the biologic actions ( Fig. 5-6 ) of a single, large family of ligands: the transforming growth factor (TGF)-β family of ligands, which are characterized by the presence of six conserved cysteine residues.[76] The human genome contains 42 genes encoding cytokines in the TGF-β family, which are divided into two classes: (1) the activin/TGF-β family and (2) the müllerian inhibitory substance (MIS)/bone morphogenic protein (BMP) family. Activin and the related inhibin, as well as MIS, are of particular interest within the field of reproductive endocrinology (see Chapters 8 and 16 ).

When ligands bind to the receptors, this promotes a physical interaction between the type I receptor (RI) and the type II receptor (RII) (see Fig. 5-6 ). As a consequence of this physical interaction, the RII receptor activates the RI receptor by phosphorylating one or more serine residues in the GS domain (TTSGSGSG sequence) of the RI receptor.[76] How does the ligand trigger receptor activation? In the case of the MIS/BMP family of cytokines, the ligand binds to the isolated RI receptor with high affinity and the RII receptor with relatively low affinity. Because the ligand can bind simultaneously to both RI and RII, this provides a ready explanation for the ability to promote a physical interaction between RI with RII. The mechanism is different in the case of the activin/TGF-β family of cytokines. For example, TGF-β binds with high affinity to RII but does not interact directly with RI. However, TGF-β binding appears to induce a conformational change in RII, thereby promoting a direct binding interaction between the intracellular domains of RII and RI. Furthermore, cytokines such as TGF-β exist as dimers, which permits them to bind simultaneously to two RII molecules so that the activated receptor complex probably exists as a heterotetramer: (RI)2(RII)2.

There is an additional level of complexity that contributes to the regulation of receptor serine kinases.[76] The biology related to activin provides several examples. Follistatin is a “ligand trap,” a soluble protein that binds activin, thereby blocking access to RI and RII. Inhibin is a peptide that inhibits activin signaling by binding to the receptor without activating the phosphorylation of RI. Betaglycan is a membrane-anchored protein, which functions as a coreceptor for inhibin by promoting inhibin binding to the activin receptor. Interestingly, betaglycan does not bind activin.

Receptor Serine Kinases Phosphorylate Other Intracellular Proteins

Once activated, receptor serine kinases are capable of phosphorylating other protein substrates. Receptor-regulated Smad proteins (R-Smad) function as the immediate downstream effectors of receptor serine kinases (see Fig. 5-6 ).[76] Smad proteins are the mammalian homologs of the proteins encoded by the drosophila Mad (Mothers against decapentaplegic) gene and the C. elegans Sma genes. There are five human R-Smad proteins. Smad2 and Smad3 mediate the actions of the activin/TGF-β family of cytokines; Smad1, Smad5, and Smad8 mediate the actions of the MIS/BMP family of cytokines.

The mechanism of action of TGF-β has been studied in considerable detail[71] and provides a prototype for the mechanism of action of receptor serine kinases.[76] When RI becomes phosphorylated in its GS domain in response to TGF-β, this increases the binding affinity for R-Smad proteins such as Smad2 (see Fig. 5-6 ). This, in turn, leads to phosphorylation of the two C-terminal serine residues in the SSXS sequence at the C-terminus of Smad2. Receptor-mediated phosphorylation of the R-Smad, Smad2, takes place when Smad 2 is bound to the Smad anchor for receptor activation (SARA), which is located in early endosomes. When the two C-terminal serine residues in Smad2 become phosphorylated, this promotes dissociation of Smad2 from SARA and also promotes binding of Smad2 to the co-mediator (Co-Smad), Smad4. Thus, phosphorylation of the R-Smad promotes the assembly of heteromeric complexes of R-Smad molecules with the Co-Smad.

Smads also contain sites for phosphorylation by other protein kinases, which provides an opportunity for regulatory cross-talk from other cellular signaling systems.

Smad7 is an inhibitory Smad (I-Smad), which binds to activated receptors in competition with R-Smads. Binding of Smad7 promotes receptor ubiquitination and degradation mediated by E3 ubiquitin ligases and Smad ubiquitination regulatory factors (Smurfs). These processes represent a negative feedback system, which contributes to the termination of TGF-β signaling (see Fig. 5-6 ).

Smad Proteins Regulate Gene Expression

Some R-Smads contain lysine-rich nuclear localization signals (KKLKK), which bind to importin, thereby mediating translocation into the nucleus.[76] It is possible that direct binding to components of the nuclear pore complex also contribute to the mechanism whereby Smads are translocated into the nucleus. Most R-Smads (with the exception of Smad2) bind to DNA in a sequence-specific fashion. The minimum Smad binding element is a 4 base pair (bp) sequence: 5′-AGAC-3′. This sequence is quite short, and would not be expected to provide a high degree of specificity. Therefore, it seems likely that other factors also contribute to the specificity of gene regulation.

Receptors that Signal through Associated Tyrosine Kinases

Overview

Members of the cytokine family of receptors resemble receptor tyrosine kinases in their mechanism of action, with one important difference. Instead of the tyrosine kinase being intrinsic to the receptor, enzymatic activity resides in a protein that associates with the cytokine receptor. As with receptor tyrosine kinases, ligand binding to the cytokine receptor activates the associated kinase. The more than 25 known ligands that bind to members of the cytokine receptor family have diverse functions. Three of the ligands are hormones: (1) GH, which is vital for normal body height; (2) prolactin (PRL), which is required for reproduction and lactation; and (3) leptin, which suppresses appetite and stimulates energy expenditure. Other ligands of cytokine receptors, for example, erythropoietin, most interleukins, and interferons α, β, and γ, regulate hematopoiesis or the immune response. A number of genetic diseases can be traced to defects in cytokine receptors. For example, Laron dwarfism is caused by autosomal recessive mutations of the GH receptor[77] and autosomal recessive mutations of the leptin receptor can cause morbid obesity.[78]

Cytokine Receptors Are Composed of Multiple Subunits

Members of the cytokine family of receptors share homology in both the extracellular and cytoplasmic domains. Some cytokine receptors, including the receptors for GH, PRL, and leptin, are thought to be composed of dimers of a single receptor subunit ( Fig. 5-7 ). One ligand is thought to bind to both receptor subunits as discussed earlier for the GH receptor. However, most cytokine receptors are composed of two or more different subunits, with as many as six subunits constituting a single receptor. [79] [80] Some of these receptors are thought to bind ligand dimers. One or more of these receptor subunits is shared by receptors for other cytokines. This phenomenon of “mixing and matching” receptor subunits is an efficient way for the cell to fine-tune its cellular responses and increase the number of ligands a group of receptor subunits can bind. For example, a receptor composed of gp130 and leukemia inhibitory factor receptor β subunit binds leukemia inhibitory factor, a pleiotropic cytokine with multiple functions that appears to serve as a molecular interface between the neuroimmune and endocrine systems.[81] The same receptor subunits, when combined with a ciliary neurotrophic factor receptor subunit, show a preference for ciliary neurotrophic factor, a trophic factor for motor neurons in the ciliary ganglion and spinal cord and a potent appetite suppressor.[82] Combine two gp130 subunits with an interleukin-6 (IL-6) receptor subunit and the new receptor shows a preference for IL-6, an inducer of the acute phase response with additional antiinflammatory properties.[83]

Cytokine Receptors Activate Members of the Janus Family of Tyrosine Kinases

Members of the cytokine family of receptors do not themselves exhibit enzymatic activity. Rather, they bind members of the Janus family of tyrosine kinases (JAKs) via a proline-rich region (see Fig. 5-7 ). There are four known JAKs, designated JAK1, JAK2, JAK3, and TYK2. As do the cytokine receptors themselves, the JAKs mix and match in that some receptors show a strong preference for a single JAK, some require two different JAKs, and others appear to activate multiple JAK family members. For example, GH, PRL, and leptin preferentially activate JAK2. Interferon-γ activates JAK1 and JAK2, and IL-2 activates JAK1 and JAK3. [79] [84]

Binding of ligand to a cytokine receptor activates the appropriate JAK family member or members. In some cases (e.g., PRL), the JAKs appear to be constitutively associated with the cytokine receptor and ligand binding increases their activity.[85] In other cases (e.g., the GH receptor), ligand binding increases both the affinity of JAKs for the cytokine receptor and the activity of the associated JAKs.[86] Activation of JAKs requires receptor oligomerization, presumably to bring two or more JAKs into sufficiently close proximity to transphosphorylate each other on the activating tyrosine in the kinase domain, as described earlier in the chapter for the receptor tyrosine kinases. Both receptor dimerization and ligand-induced changes in receptor conformation appear to be required for receptor activation.[87] Transphosphorylation is believed to cause a conformational change that exposes the ATP- or substrate-binding site, or both. Once the JAKs are activated, they phosphorylate themselves and their associated receptor subunits on multiple tyrosines. JAKs appear to be vital for normal human function. Mutations in the JAK3 gene have been linked to an autosomal recessive form of severe combined immunodeficiency disease.[88] Targeted disruption of the JAK2 gene in mice is embryonic lethal.[89]

Signaling Pathways Initiated by Cytokine Receptor–JAK Complexes

Phosphorylated tyrosines within the cytokine receptor subunits and their associated JAKs form binding sites for various signaling proteins containing phosphotyrosine binding domains, such as SH2 and PTB domains. Each cytokine receptor–JAK complex would be expected to have some tyrosine-containing motifs shared with many other cytokine receptor–JAK complexes (e.g., tyrosines within JAKs) and some ligand-specific tyrosine-containing motifs (e.g., tyrosines within a specific combination of receptor subunits). Thus, ligand binding to cytokine receptors would be expected to initiate some signaling pathways that are shared by many cytokines and some that are more specialized to a particular cytokine receptor. The signaling proteins known to be recruited to subsets of cytokine receptor–JAK complexes are generally the same as those recruited to receptor tyrosine kinases. Examples include the IRS proteins, the adaptor proteins Shc and Grb-2 that lead to activation of the Ras-MAP kinase pathway, phospholipase Cγ, and PI 3-kinase. However, there is one family of signaling proteins that appears to be particularly important for the function of cytokines—signal transducers and activators of transcription (STATs) ( Fig. 5-8 ). STAT proteins are latent cytoplasmic transcription factors. STATs bind, through their SH2 domains, to one or more phosphorylated tyrosines in activated receptor-JAK complexes. Once bound, they themselves are tyrosyl phosphorylated, presumably by the receptor-associated JAKs. STATs then dissociate from the receptor-JAK complexes, homodimerize or heterodimerize with other STAT proteins, move to the nucleus, and bind to gamma-activated sequence–like elements in the promoters of cytokine-responsive genes.[90] The transcriptional response depends on how many STAT binding sites exist in the receptor-JAK complex, with which of the seven known STATs a particular STAT heterodimerizes, to what other proteins a particular STAT binds, the degree of serine or threonine phosphorylation of the STAT, and what other transcription factors are also activated. For example, leukemia inhibitory factor, whose receptor contains seven STAT3 binding motifs (YXXQ, where Y = tyrosine, X = any amino acid, and Q = glutamine) is a particularly potent activator of STATSTAT3.[91] The transcriptional activity of STAT5 is enhanced by its forming a complex with the glucocorticoid, mineralocorticoid, and progesterone receptors but is diminished by its forming a complex with the estrogen receptor.[92] The importance of STAT5b for GH signaling is illustrated by the finding that severe growth failure is associated with point mutations in the STAT5b gene that result in a defective SH2 domain or an unstable, truncated form of STAT5b. [93] [94]

Precise Regulation of the Cytokine Receptors is Required for Normal Function

Ligand binding to cytokine receptors normally activates JAKs rapidly and transiently. Conversely, constitutively activated JAKs and STATs are associated with cellular transformation. For example, a single acquired activating point mutation in JAK2 is present in the majority of patients with a myeloproliferative disorder.[95] Constitutively active JAKs and STATs are also a common characteristic of leukemias,[96] and both JAK2 and STAT5b have been identified as fusion partners in translocations in leukemias. The Tel-JAK2 fusion protein is constitutively active, leading to constitutively active STAT proteins. Thus, an understanding of what turns off cytokine receptor signaling is of utmost importance in understanding normal signaling via cytokine receptors.

As with the receptor tyrosine kinases, several steps have been hypothesized to serve as points of signal termination for cytokine signaling. These include receptor degradation (e.g., through a ubiquination/proteosome pathway) and dephosphorylation of tyrosines within JAK or receptor (e.g., by a tyrosine phosphatase that binds to receptor-JAK complexes). The suppressors of cytokine-signaling (SOCSs) are thought to be particularly important players in the termination or suppression of cytokine signaling pathways. SOCS proteins are an excellent example of an effective negative feedback loop. They are generally synthesized in response to cytokines. The newly synthesized SOCS proteins in turn bind, through their SH2 domain, to phosphorylated tyrosines within the cytokine receptor–JAK complex and inhibit further cytokine signaling. In some cases (i.e., SOCS1), SOCS proteins are thought to bind to phosphotyrosines in the kinase domain of JAK and inhibit kinase activity.[97] In other cases (i.e., SOCS3), SOCS proteins bind to phosphorylated tyrosines in the receptor and inhibit JAK activity.[98] Finally, in some cases (i.e., cytokine-inducible SH2 protein [CIS]), SOCS proteins bind to phosphorylated tyrosines in the receptor and block STAT binding and activation.[99] SOCS proteins can also be synthesized in response to noncytokine receptors, suggesting a mechanism whereby prior exposure to one ligand suppresses subsequent responses to another. For example, SOCS proteins have been implicated in the well-known ability of endotoxin to cause resistance to GH.[100]

Summary

Hormones, growth factors, and cytokines that bind to members of the cytokine family of receptors activate JAK family tyrosine kinases. The activated kinases in turn phosphorylate tyrosines in themselves and associated receptors. The phosphorylated tyrosines form binding sites for other signaling proteins, including STAT proteins and a variety of other phosphotyrosine-binding proteins. STAT proteins promote the regulation of cytokine-sensitive genes, including SOCS proteins that serve a negative feedback function of terminating ligand activation of JAKs or STATs, or both.

Although this gives the general picture, it should be recognized that the picture is becoming much more complex every day. For example, there are reports that members of the Src family of tyrosine kinases can also be activated by some cytokine receptors (e.g., PRL receptor),[101] that some JAK-binding proteins (e.g., SH2-B) are potent activators of JAK2,[102] and that other proteins contribute to the down-regulation of cytokine-signaling pathways, including protein inhibitors of activated STAT (PIAS) proteins that bind to specific STATs and negatively regulate their activity.[103] Cytokine receptors, JAKs, STATs, and/or SOCS proteins have also been shown to interact with or be components of signaling downstream of some receptor tyrosine kinases (e.g., receptors for insulin, IGF-I, epidermal growth factor) as well as several G protein–coupled receptors (e.g., the receptors for angiotensin II, serotonin, α-thrombin, luteinizing hormone). In contrast to their essential role in signaling by cytokine receptors, JAKs do not appear to be the primary signaling mediator with either the receptor tyrosine kinases or the G protein–coupled receptors.

G Protein–Coupled Receptors

Overview

G protein–coupled receptors (GPCRs) are an evolutionarily conserved gene superfamily with members in all eukaryotes from yeast to mammals. They transduce a wide variety of extracellular signals including photons of light; chemical odorants; divalent cations; monoamine, amino acid, and nucleoside neurotransmitters; lipids; and peptide and protein hormones.[104] All members of the GPCR superfamily share a common structural feature, seven membrane-spanning helices, but various subfamilies diverge in primary amino acid sequence and in the domains that serve in ligand binding, G protein coupling, and interaction with other effector proteins ( Fig. 5-9 ).

All GPCRs act as guanine nucleotide exchange factors. In their activated (agonist-bound) conformation, they catalyze exchange of GDP tightly bound to the α subunit of heterotrimeric G proteins for GTP ( Fig. 5-10 ). This in turn leads to activation of the α subunit and its dissociation from the G protein βγ dimer. Both G protein subunits are capable of regulating effector activity.[105] Identified G protein–regulated effectors include enzymes of second messenger metabolism such as adenylyl cyclase and phospholipase C-β and a variety of ion channels. Agonist binding to GPCRs thus alters intracellular second messenger and ion concentrations with resultant rapid effects on hormone secretion, muscle contraction, and a variety of other physiologic functions. Long-term changes in gene expression are also seen as a result of second messenger-stimulated phosphorylation of transcription factors.

Figure 5-10 The G protein guanosine triphosphatase (GTPase) and G protein–coupled receptor (GPCR) desensitization-resensitization cycle. In each panel, the stippled region denotes the plasma membrane with extracellular above and intracellular below. In the basal state, the G protein is a heterotrimer with guanosine diphosphate (GDP) tightly bound to the α subunit. The agonist-activated GPCR catalyzes release of GDP, which permits guanosine triphosphate (GTP) to bind. The GTP-bound α subunit dissociates from the βγ dimer. Arrows from α subunit to effector and from βγ dimer to effector indicate regulation of effector activity by the respective subunits. Arrow from effector to α subunit indicates regulation of its GTPase activity by effector interaction. Under physiologic conditions, effector regulation by G protein subunits is transient and is terminated by the GTPase activity of the α subunit. The latter converts bound GTP to GDP, thus returning the α subunit to its inactivated state with high affinity for the βγ dimer, which reassociates to form the heterotrimer in the basal state. In the basal state, the receptor kinase and arrestin are shown as cytosolic proteins. Dissociation of the GTP-bound α subunit from the βγ dimer permits the latter to facilitate binding of receptor kinase to the plasma membrane (arrow from βγ dimer to receptor kinase). Plasma membrane binding permits the receptor kinase to phosphorylate the agonist-bound GPCR (depicted here as “P” occurring on the carboxyl-terminal tail of the GPCR, but sites on intracellular loops are also possible). GPCR phosphorylation in turn facilitates arrestin binding to GPCR, resulting in desensitization. Endocytic trafficking of arrestin-bound GPCR and recycling to the plasma membrane during resensitization are not depicted here.

The G protein subunits are encoded by three distinct genes. The α subunit binds guanine nucleotides with high affinity and specificity and has intrinsic guanosine triphosphatase (GTPase) activity. The β and γ polypeptides are tightly but noncovalently associated in a functional dimer subunit. The three-dimensional structures of the individual and associated subunits have been determined. [105] [106] There is considerable diversity in G protein subunits, with multiple genes encoding all three subunits and alternative gene splicing resulting in additional polypeptide products. There are at least 16 distinct α subunit genes in mammals. These vary widely in range of expression. Some, such as Gs-α, which couples many GPCRs to stimulation of adenylyl cyclase, are ubiquitous; others, such as Gtl-α, which couples the GPCR rhodopsin to cyclic guanosine monophosphate phos-phodiesterase in retinal rod photoreceptor cells, are highly localized.

Because multiple distinct GPCRs, G proteins, and effectors are expressed within any given cell, the degree and basis for specificity in G protein coupling to GPCRs and to effectors are major subjects of investigation with implications for drug action and disease mechanisms.[106] Since the pioneering work of Rodbell[107] in discovering G proteins and showing that G protein–mediated signal transduction involves three separable components (receptor, G protein, and effector), additional complexity has emerged.

A large new gene family termed RGS (for regulators of G protein signaling) has been identified. RGS proteins bind to a transition state of the GTP-activated G protein α subunit and accelerate its GTPase activity, thus helping deactivate the α subunit. RGS domains have also been found in modular proteins with additional functions, in certain cases linking heterotrimeric G protein signaling with the function of low-molecular-weight GTP-binding proteins in the ras superfamily.[106] Lefkowitz[108] has shown that a family of GPCR kinases and of arrestin proteins is involved in GPCR desensitization after agonist binding. In addition, it is now clear that GPCRs interact directly with a number of other proteins in addition to G proteins. Not only are GPCRs important targets for treatment of many diseases, but also mutations in genes encoding GPCRs have been identified as the cause of a number of endocrine as well as nonendocrine disorders.

G Protein–Coupled Receptor Structure and Function

Structure

Hydropathy analysis of the primary sequence of all GPCRs predicts seven membrane-spanning α helices connected by three intracellular loops and three extracellular loops with an extracellular amino terminus and an intracellular carboxyl terminus (see Fig. 5-9 ). This basic structure has now been verified by x-ray crystallography for rhodopsin.[109] Although there was already evidence that visual transduction in the retina and hormone activation of adenylyl cyclase shared common features, the discovery that the β-adrenergic receptor has the same topographic structure as rhodopsin came as a surprise.[108] Cloning of the complementary deoxyribonucleic acids (cDNAs) for a vast number of GPCRs followed elucidation of the primary sequence of the β-adrenergic receptor, and in every case the same core structure was predicted by hydropathy analysis.

In addition to the predicted core structure, certain other common features (with exceptions in some subsets of the GPCR superfamily) were noted[104]: (1) a disulfide bridge connecting the first and second extracellular loops; (2) one or more N-linked glycosylation sites, usually in the amino terminus but occasionally in extracellular loops; (3) palmitoylation of one or more cysteines in the carboxyl terminus, effectively creating a fourth intracellular loop; (4) potential phosphorylation sites in the carboxyl terminus and occasionally the third intracellular loop. Glycosylation appears to be important for proper folding and trafficking to the plasma membrane rather than for ligand binding. The disulfide bridge may help in proper arrangement of the transmembrane helices.

Superimposed on the basic structure of GPCRs are a number of variations relevant to differences in ligand binding, G protein coupling, and interaction with other proteins.[104] First, there are major differences in amino acid sequence among members of the GPCR superfamily. Sequence alignment, especially of the transmembrane helices, allows one to divide the superfamily into subfamilies (see Fig. 5-9 ). Of these, family 1 is the largest and itself can be subdivided. The largest subset includes opsins; odorant receptors; and monoamine, purinergic, and opiate receptors. These are characterized by a short amino terminus. The next subset includes chemokine, protease-activated, and certain peptide hormone receptors characterized by a slightly longer amino terminus. The last subset comprises receptors for the large glycoprotein hormones, TSH, luteinizing hormone, and follicle-stimulating hormone. These have an approximately 400-residue extracellular amino terminus.

Family 2 shows essentially no sequence homology to family 1 even within the transmembrane helices and is characterized by an approximately 100-residue amino terminus. Members include receptors for a number of peptide hormones such as parathyroid hormone (PTH), calcitonin, vasoactive intestinal peptide, and corticotropin-releasing hormone.

Family 3, in addition to a unique primary sequence, has other unique features such as an approximately 200-residue carboxyl terminus and an approximately 600-residue amino terminus. The latter consists of a putative “Venus flytrap–like” domain and a cysteine-rich domain. Members include the metabotropic glutamate receptors, an extracellular Ca2+-sensing receptor, and putative taste and pheromone receptors.[110] The determination of the three-dimensional crystal structure of part of the extracellular amino terminus of one of the metabotropic glutamate receptors verifies the Venus flytrap structure.[110]

Ligand Binding

Given the diversity of ligands (>1000) that bind to GPCRs, it is not surprising that considerable diversity is evident in both the sequence and structure of presumptive GPCR ligand-binding domains. The opsins are unique among GPCRs in that the ligand, retinal, is covalently bound to a lysine in the seventh transmembrane helix.[109] Ligand binding for other members of family 1 with a short extracellular amino terminus, for example, adrenergic and other monoamine receptors, probably involves a pocket within the transmembrane helices as demonstrated for rhodopsin (see Fig. 5-9 ). For other family 1 GPCRs, the extracellular amino terminus, perhaps together with extracellular loops and portions of the transmembrane helices, is involved in ligand binding. In the case of the glycoprotein hormone receptors, the large extracellular amino terminus plays the principal role in hormone binding. In a model for peptide binding to family 2 receptors, the extracellular amino terminus is responsible for initial binding to the peptide carboxy-terminus followed by peptide amino-terminus binding to the seven-transmembrane domain.[111] For family 3 GPCRs, the three-dimensional structure of the type 1 metabotropic glutamate receptor shows that agonist binding occurs within a cleft between the lobes of the Venus flytrap.[110]

G Protein Coupling

Because the number of potential G proteins to which GPCRs couple is much more limited than the number of ligands that bind GPCRs, more conservation of the domains involved in G protein coupling would be expected. Although GPCRs can be broadly divided into those that couple to Gs, those that couple to the Gq subfamily, and those that couple to the Gi-Go subfamily, the situation is probably more complicated.[106] Specificity of coupling to the most recently identified G proteins, Gl2 and G13, is still uncertain. Also, some GPCRs evidently can couple to both Gs and Gq.

A vast number of studies have been performed to define the sites of ligand binding and G protein coupling of GPCRs. [106] [112] Considerable evidence points to the third intracellular loop (particularly its membrane-proximal portions) and to the membrane-proximal portion of the carboxyl terminus as key determinants of G protein coupling specificity. For example, exchanging only the third intracellular loop between different GPCRs confers the G protein coupling specificity of the exchanged loop upon the recipient GPCR.[113] In contrast, the second intracellular loop, although important for G protein coupling, appears to play a role in the activation mechanism rather than in determining specificity of coupling.[113] A tripeptide motif (D/E, R, Y/W) at the start of the second intracellular loop that is highly conserved in family 1 GPCRs is critical for G protein activation.[112]

Mechanism of Activation

The precise mechanism of activation after agonist binding remains to be defined for most GPCRs, but studies of rhodopsin provide the clearest picture available. In the ground state, retinal covalently bound to the seventh transmembrane helix in rhodopsin holds the transmembrane helices in an inactive conformation. Isomerization of retinal upon absorption of light of the appropriate wavelength converts an antagonist ligand into an agonist. The rhodopsin crystal structure identifies the residues in the transmembrane helices that interact with retinal and suggests a mechanism for movement of the helices upon photoactivation of retinal.[109] Movement of the transmembrane helices in turn leads to changes in conformation of cytoplasmic loops that promote G protein activation.

For family 1 receptors related to rhodopsin, the determination of its three-dimensional structure validates the idea that a change in conformation of transmembrane helices is the direct result of agonist versus antagonist binding to residues within the helices. Further refinements in understanding the mechanism of activation for opsin-related GPCRs should come as additional three-dimensional structures are determined. Until then, molecular modeling by computer on the basis of the rhodopsin structure and then experimental testing offer a useful approach.[114] For other GPCRs whose presumptive site of agonist binding does not involve direct contact with transmembrane helices (families 2 and 3 and the glycoprotein hormone receptors in family 1), much remains to be learned about how agonist binding to the extracellular domain of such GPCRs leads to presumptive changes in conformation of transmembrane helices and receptor activation. Determination of the structure of the extracellular domain of the FSH receptor bound to FSH provides important insights into the general mechanism of glycoprotein hormone binding to their cognate GPCRs, and resultant interactions with the seven-transmembrane domain leading to activation.[115] A general hypothesis of GPCR activation postulates that GPCRs are in equilibrium between an activated state and an inactive state. These states presumably differ in the disposition of the transmembrane helices and, in turn, the cytoplasmic domains that determine G protein coupling. Agonists, according to this ternary complex model, are viewed as stabilizing the activated state. Antagonists may be neutral; that is, they simply compete with agonists for receptor binding but their binding does not influence this equilibrium. Alternatively, they may be “inverse” agonists; that is, their binding stabilizes the inactive state of the receptor.[116] Refinements of the ternary complex model based on kinetic and biophysical studies suggest added complexity.[117]

Dimerization

Members of the tyrosine kinase receptor family have long been known to require dimerization as part of their activation mechanism. It is now apparent that many GPCRs likewise form homodimers and heterodimers.[104] Residues within transmembrane helix 6 may foster dimerization of small family 1 GPCRs,[118] and intermolecular disulfide bonds in the extracellular amino-terminal domain are involved in homodimerization of most family 3 GPCRs. [110] [119] [120] A coiled-coil interaction in the carboxyl terminus of γ-aminobutyric acid B receptor subtypes is responsible for heterodimerization, and this is critical for proper receptor function.[121] Modifications of ligand binding, signaling, and receptor sequestration have been demonstrated upon heterodimerization of angiotensin with bradykinin receptors, of κ with δ opioid receptors, and of opioid with β-adrenergic receptors.[122] Further studies are needed to elucidate the physiologic relevance of GPCR homodimerization and heterodimerization.

G Protein–Coupled Receptor Desensitization

Pharmacologists long ago appreciated that continued exposure to agonist leads to a diminished response, so-called desensitization. This phenomenon has been extensively studied in GPCRs. Two forms are defined: heterologous, in which binding of agonist to one GPCR leads to a diminished response of a different GPCR to its agonist, and homologous, in which desensitization occurs only for the GPCR to which agonist is bound. Both forms of desensitization involve GPCR phosphorylation but by different kinases and at different sites. Stimulation of cyclic adenosine monophosphate formation by agonist binding to a Gs-coupled GPCR leads to activation of protein kinase A, which in turn can phosphorylate and desensitize the GPCR. Such phosphorylation may also alter G protein–coupling specificity.[104] Similarly, protein kinase C activation resulting from GPCR coupling to Gq family members may cause protein kinase C–catalyzed phosphorylation of GPCRs with desensitization.

In retinal photoreceptors, a specific rhodopsin kinase and a protein termed arrestin were implicated in attenuation of the light response. Just as parallels were identified between rhodopsin and GPCR structure, so were parallels identified in this desensitization mechanism. Rhodopsin kinase is but one member of a family of GPCR kinases and arrestin only one of a family of related proteins that function in desensitization of many members of the GPCR superfamily.[108] GPCR kinases preferentially phosphorylate the agonist-bound form of a GPCR, thus ensuring homologous desensitization. Upon GPCR phosphorylation by GPCR kinase, arrestins bind to the third intracellular loop and carboxyl-terminal tail of the GPCR, thereby blocking G protein binding (see Fig. 5-10 ). GPCR kinases and arrestins not only act to desensitize GPCRs but also mediate other functions including receptor internalization and interaction with other effectors (see next section).

G Protein–Coupled Receptor Interactions with Other Proteins

The initial paradigm of GPCR function postulated that G protein activation is the sole outcome of agonist binding to GPCRs. With the identification of GPCR interactions with GPCR kinases and arrestins, this concept was modified to include these proteins involved in GPCR desensitization. Later evidence, however, suggests that GPCR interaction with arrestins may also permit recruitment of other proteins to the GPCR. For example, the src tyrosine kinase may interact with the β-adrenergic receptor, with β-arrestin serving as an adaptor.[123] Arrestins may also recruit proteins involved in endocytosis. GPCR kinases may also serve to recruit additional signaling proteins to the GPCR.[123]

Other classes of proteins may interact with specific GPCRs without recruitment by GPCR kinases and arrestins. These include SH2 domain-containing proteins, small GTP-binding proteins, and PDZ (for postsynaptic density protein-95/discs large/zona occluden-1) domain-containing proteins. Examples of the latter include binding of the Na+/H+ exchanger regulatory factor to the carboxyl terminus of the β-adrenergic receptor.[123] The long carboxyl terminus of family 3 GPCRs such as metabotropic glutamate receptors contains polyproline motifs involved in binding members of the Homer family. The latter can facilitate functional interactions with yet other proteins such as the inositol triphosphate receptor.[124] Receptor activity-modifying proteins (RAMPs), a new family of single-transmembrane-domain proteins, appear to heterodimerize with certain GPCRs, assisting them in proper folding and membrane trafficking.[125] Interestingly, when the calcitonin receptor-like GPCR associates with RAMP1, it forms a calcitonin gene-related peptide receptor, whereas when it associates with RAMP2, it becomes an adrenomedullin receptor. Clearly, this rapidly evolving aspect of GPCR function holds many further interesting developments in store.

G Protein–Coupled Receptors in Disease Pathogenesis and Treatment

Because of their diverse and critical roles in normal physiology, their accessibility on the cell surface, and the ability to synthesize selective agonists and antagonists, GPCRs have long been a major target for drug development. One estimate is that about 65% of prescription drugs are targeted against GPCRs. Drugs targeting GPCRs may act not only as agonists or antagonists, but also as allosteric modulators. So-called calcimimetic drugs inhibit parathyroid hormone release, for example, by binding to the seven-transmembrane domain and acting as positive allosteric modulators of the Ca2+-sensing receptor.[120] With the cloning of GPCR cDNAs, much greater diversity of receptor subclasses became evident than had been anticipated on the basis of pharmacologic studies. For example, five muscarinic receptor subtypes and an even greater number of serotoninergic GPCRs were identified.[112] This has allowed the development of highly specific, subtype-selective drugs that have fewer side effects than those produced by previously available agents.

Another result of the cloning of GPCR cDNAs by homology screening and polymerase chain reaction-based approaches is the identification of “orphan” GPCRs, that is, receptors with the canonical, predicted seven-transmembrane-domain structure of GPCRs but without knowledge of their physiologic agonist. There have been substantial efforts to identify the relevant ligands for such orphan receptors. Krebs cycle intermediates, succinate and α-ketoglutarate, for example, were shown to be the physiologically relevant activators of orphan GPCRs, GPR91 and GPR99, respectively, and thereby to regulate renin release and blood pressure.[126] GPR40, another orphan receptor selectively expressed in β cells, is activated by free fatty acids and may play a role in linking obesity to type 2 diabetes.[127] In addition to revealing new physiologic and pathophysiologic mechanisms, GPCR “deorphanization” provides novel targets for drug development.

Beyond drug development, defects in GPCRs are an important cause of a wide variety of human diseases.[128] GPCR mutations can cause loss of function by impairing any of several steps in the normal GPCR-GTPase cycle (see Fig. 5-10 ). These include failure to synthesize GPCR protein altogether, failure of synthesized GPCR to reach the plasma membrane, failure of GPCR to bind or be activated by agonist, and failure of GPCR to couple to or activate G protein. Because in most cases clinically significant impairment of signal transduction requires loss of both alleles of the GPCR gene, most such diseases are inherited in autosomal recessive fashion ( Table 5-1 ).

TABLE 5-1 — DISEASES CAUSED BY G PROTEIN–COUPLED RECEPTOR LOSS-OF-FUNCTION MUTATIONS
Receptor Disease Inheritance
V2 vasopressin Nephrogenic diabetes insipidus X-linked
ACTH Familial ACTH resistance Autosomal recessive
GHRH Familial GH deficiency Autosomal recessive
GnRH Hypogonadotropic hypogonadism Autosomal recessive
GPR54 Hypogonadotropic hypogonadism Autosomal recessive
FSH Hypergonadotropic ovarian dysgenesis Autosomal recessive
LH Male pseudohermaphroditism Autosomal recessive
TSH Familial hypothyroidism Autosomal recessive
Ca2+ sensing Familial hypocalciuric hypercalcemia, neonatal severe primary hyperparathyroidism Autosomal dominant autosomal recessive
Melanocortin 4 Obesity Autosomal recessive
PTH/PTHrP Blomstrand chondrodysplasia Autosomal recessive

ACTH, Adrenocorticotropic hormone; FSH, follicle-stimulating hormone; GH, growth hormone; GHRH, growth hormone–releasing hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; PTH, parathyroid hormone; PTHrP, parathyroid hormone–related protein; TSH, thyroid-stimulating hormone.

Most of these diseases are manifested as resistance to the action of the normal agonist and thus mimic deficiency of the agonist. For example, TSH receptor loss-of-function mutations cause a form of hypothyroidism mimicking TSH deficiency, but serum TSH is actually elevated in such cases, reflecting resistance to the hormone’s action caused by defective receptor function. Interestingly, hypogonadotropic hypogonadism may be caused by loss of function mutations of either the GnRH receptor or of the orphan GPCR, GPR54. In the former, there is resistance to the action of GnRH. The latter may be due to failure to release GnRH, but the precise mechanism has not been defined.[129] Nephrogenic diabetes insipidus (renal vasopressin resistance) is caused by loss-of-function mutations in the V2 vasopressin receptor gene located on the X chromosome. Thus, males with a single copy of the gene experience the disease when they inherit a mutant gene, whereas most females do not show overt disease because random X inactivation leaves them with an average 50% of normal gene function. Most V2 vasopressin receptor mutations associated with nephrogenic diabetes insipidus cause loss of function by impairing normal synthesis or folding of the receptor, or both. A novel mechanism for receptor loss of function elucidated for a V2 vasopressin receptor missense mutation associated with nephrogenic diabetes insipidus involves constitutive arrestin-mediated desensitization.[130]

The extracellular Ca2+-sensing receptor appears to be an interesting exception to the association between GPCR loss-of-function mutations and hormone resistance. Loss-of-function mutations of the Ca2+-sensing receptor mimic a hormone hypersecretion state, primary hyperparathyroidism. In fact, Ca2+-sensing receptor loss-of-function mutations do cause hormone resistance, but in this case extracellular Ca2+ is the hormonal agonist that acts through this receptor to inhibit PTH secretion. A loss-of-function mutation of one copy of the receptor gene typically causes mild resistance to extracellular Ca2+ manifested as familial hypocalciuric hypercalcemia. If two defective copies are inherited, extreme Ca2+ resistance causing neonatal severe primary hyperparathyroidism results (see Table 5-1 ). In some cases, a heterozygous receptor loss-of-function mutation may be associated with neonatal severe primary hyperparathyroidism, perhaps reflecting a dominant negative effect caused by dimerization of wild-type and mutant receptors.[131]

GPCR gain-of-function mutations ( Table 5-2 ) are also an important cause of disease.[128] Given the dominant nature of activating mutations, most such diseases are inherited in an autosomal dominant manner. Activating TSH receptor mutations may be inherited in autosomal dominant fashion and cause diffuse thyroid enlargement in familial nonautoimmune hyperthyroidism, or they may occur as somatic mutations causing focal, sporadic hyperfunctional thyroid nodules.[132] Likewise, activating, germline LH receptor mutations cause familial male precocious puberty due to LH-independent Leydig cell hyperfunction, while somatic LH receptor mutations may cause focal Leydig cell tumors.[132]

TABLE 5-2 — DISEASES CAUSED BY G PROTEIN–COUPLED RECEPTOR GAIN-OF-FUNCTION MUTATIONS
Receptor Disease Inheritance
LH Familial male precocious puberty Autosomal dominant
TSH Sporadic hyperfunctional thyroid nodules Noninherited (somatic)
TSH Familial nonautoimmune hyperthyroidism Autosomal dominant
Ca2+ sensing Familial hypocalcemic hypercalciuria Autosomal dominant
PTH/PTHrP Jansen’s metaphyseal chondrodysplasia Autosomal dominant
V2 vasopressin Nephrogenic inappropriate antidiuresis Autosomal dominant

LH, Luteinizing hormone; PTH, parathyroid hormone; PTHrP, parathyroid hormone–related protein; TSH, thyroid-stimulating hormone.

Unlike loss-of-function mutations, which may be missense as well as nonsense or frameshift mutations that truncate the normal receptor protein, GPCR gain-of-function mutations are almost always missense mutations. The location and nature of naturally occurring, disease-causing mutations offer important insights into GPCR structure and function. The basis for defective receptor function is clear with mutations that truncate receptor synthesis prematurely. More subtle missense mutations may impair function if they involve highly conserved residues in transmembrane helices critical for normal protein folding. Activating missense mutations often involve residues within or bordering transmembrane helices and are thought to disrupt normal inhibitory constraints that maintain the receptor in its inactive conformation.[133] Mutations disrupting these constraints mimic the effects of agonist binding and shift the equilibrium toward the activated state of the receptor. A striking example is the activating, missense mutations in the V2 vasopressin receptor of arginine 137, part of the “DRY” motif at the intracellular border of transmembrane helix 3 conserved in most family 1 GPCRs, leading to the syndrome of nephrogenic inappropriate antidiuresis.[134]

Clinically, diseases caused by activating GPCR mutations therefore mimic states of agonist excess, but direct measurement shows that agonist concentrations are actually low, re-flecting normal negative feedback mechanisms. Again, the Ca2+-sensing receptor is an apparent exception, with activating mutations causing functional hypoparathyroidism. For most GPCRs, disease-associated gain-of-function mutations cause constitutive, agonist-independent, activation but with rare exceptions, the Ca2+-sensing receptor gain-of-function mutations cause increased sensitivity to extracellular Ca2+ rather than to Ca2+-independent activation.[131]

Naturally occurring animal models of human disease have revealed additional examples of etiologic GPCR mutations. For example, a loss-of-function mutation in the hypocretin (orexin) type 2 receptor gene was identified in canine narcolepsy.[135] Dozens of mouse GPCR gene knockout models have been created, many revealing interesting and in some cases unexpected phenotypes. Characterization of the phenotype resulting from disruption of a mouse GPCR gene may accurately predict the clinical picture resulting from the corresponding mutation in humans, such as with disruption of the melanocortin-4 receptor gene resulting in obesity in mouse[136] and human[137] and disruption of the PTH/PTH-related protein receptor gene impairing normal bone growth and development in mouse[138] and in the human disease Blomstrand chondrodysplasia.[139] Further knockout models and further detailed studies of these models can be expected to increase substantially our understanding of GPCR function and to address questions such as the unique roles of multiple subtypes of various GPCR subclasses, for example, the β3-adrenergic receptor subtype.[140] Availability of mouse knockout models of human diseases should also facilitate testing of novel therapies including gene transfer. For example, aminoglycosides, which are known to suppress premature termination codons, were shown to rescue expression and function in mice with nephrogenic diabetes insipidus caused by a nonsense mutant in the V2 vasopressin receptor.[141] Many disease-causing loss of function mutations in GPCRs lead to defective protein folding and/or protein routing. Novel therapeutic approaches such as use of molecular “chaperones” and modulation of the cell’s “quality control” mechanisms have shown promise in in vitro studies.[142]

Screening of GPCR genes for mutations as the potential cause of additional human disorders may continue to turn up new examples, but it is also becoming clear that variations in GPCR gene sequence can have profound consequences beyond simply causing resistance to, or activation independent of, the cognate hormone agonist. Familial spontaneous ovarian hyperstimulation syndrome occurring in early pregnancy, for example, was shown to be caused by missense mutations in the transmembrane helix domain of the FSH receptor. [143] [144] Such mutations increase receptor basal activity, and permit low affinity binding of hCG to the ectodomain to activate the FSH receptor. Studies are needed to determine whether variable susceptibility to iatrogenic ovarian hyperstimulation occurring in the context of in vitro fertilization might be due to such variations in FSH receptor sequence.[144]

As more polymorphisms are discovered in the human genome, many examples of variations in GPCR gene sequence will be found and the challenge will be to elucidate their possible functional significance. In vitro studies may reveal functional differences, such as differences in G protein coupling seen with a four-amino-acid polymorphism in the third intracellular loop of the α2C-adrenergic receptor,[145] but further studies are required to determine whether such differences are important in individual variation in response to various drugs (pharmacogenomics) or in other subtle physiologic differences that could confer susceptibility to disease (complex disease genes). Given the high proportion of the human genome devoted to GPCR genes, it is clear that studies of this gene superfamily will play a prominent role in the post-human genome sequence era.

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GENETIC CONTROL OF PEPTIDE HORMONE FORMATION

Kronenberg: Williams Textbook of Endocrinology, 11th ed.
Copyright © 2008 Saunders, An Imprint of Elsevier

GENETIC CONTROL OF PEPTIDE HORMONE FORMATION

Joel F. Habener

▪ Evolution of Peptide Hormones and Their Functions, 19
▪ Steps in Expression of a Protein-Encoding Gene, 19
▪ Subcellular Structure of Cells that Secrete Protein Hormones, 21
▪ Intracellular Segregation and Transport of Polypeptide Hormones, 21
▪ Processes of Hormone Secretion, 24
▪ Structure of a Gene Encoding a Polypeptide Hormone, 25
▪ Regulation of Gene Expression, 26
▪ Generation of Biologic Diversification, 30

Advances in the fields of molecular and cellular biology have provided new insights into the mechanistic workings of cells. Recombinant deoxyribonucleic acid (DNA) technology and the sequencing (decoding) of the entire human and mouse genomes now make it possible to analyze the precise structure and function of DNA, the genetic substance that is the basis for life. The discovery of the unique biochemical and structural properties of DNA provided the conceptual framework with which to begin a systematic investigation of the origins, development, and organization of life forms.[1]

The completion of the entire sequences of the human and mouse genomes was accomplished in the years 2003 and 2004, and the annotation of the encoded information is nearing completion. The availability of a complete blueprint of the structure and organization of all expressed genes now provides profound insights into the basis of genetically determined diseases. Within the next decade, genotyping of individuals shortly after birth likely will be possible. Therapeutic approaches for the correction of genetic defects by techniques of gene replacement are likely to become a reality, thereby providing insights into relative risks for developing diseases.

The polypeptide hormones constitute a critically important and diverse set of regulatory molecules encoded by the genome whose functions are to convey specific information among cells and organs. This type of molecular communication arose early in the development of life and evolved into a complex system for the control of growth, development, and reproduction and for the maintenance of metabolic homeostasis. These hormones, including the many chemokines and cytokines primarily involved in the regulation of the immune system, consist of approximately 400 or more small proteins ranging from as few as three amino acids (thyrotropin-releasing hormone, or TRH) to 192 amino acids (growth hormone). In a broader sense, these polypeptides function both as hormones, whose actions on distant organs are mediated by way of their transport through the bloodstream, and as local cell-to-cell communicators ( Fig. 3-1 ). The latter function of the polypeptide hormones is exemplified by their elaboration and secretion within neurons of the central, autonomic, and peripheral nervous systems, where they act as neurotransmitters, and in leukocytes where they modulate immune responses. These multiple modes of expression of the polypeptide hormone genes have aroused great interest in the specific functions of these peptides and the mechanisms of their synthesis and release.

This chapter reviews the diverse structures of genes encoding peptide hormones and the multiple mechanisms that govern their expression. The synthesis of nonpeptide hormones (e.g., catecholamines, thyroid hormones, and steroid hormones) involves the action of multiple enzymes and hence the expression of multiple genes; these are discussed in the individual chapters devoted to such hormones.

Figure 3-1 Different modes of utilization of polypeptide hormones in expression of their biologic actions. The peptide hormones are expressed in at least four ways in fulfilling their functions as cellular messenger molecules: (1) endocrine mode, for purposes of communication among organs (e.g., pituitary-thyroid axis); (2) paracrine mode, for communication among adjacent cells, often located within endocrine organs; (3) neuroendocrine mode, for synthesis and release of peptides from specialized peptidergic neurons for action on distant organs through the bloodstream (e.g., neuroendocrine peptides of hypothalamus); and (4) neurotransmitter mode, for action of peptides in concert with classic amino acid–derived aminergic transmitters in the neuronal communication network. Identical polypeptides are often utilized in the nervous system both as neuroendocrine hormones and as neurotransmitters. In some instances, the same gene product is used in all four modes of expression.

Evolution of Peptide Hormones and Their Functions

Peptide hormones arose early in the evolution of life. Indeed, polypeptides that are structurally similar to mammalian peptides are present in lower vertebrates, insects, yeasts, and bacteria.[2] An example of the early evolution of regulatory peptides is the α-factor (mating pheromone) of yeast, which is similar in structure to gonadotropin-releasing hormone (GnRH).[3] The oldest member of the cholecystokinin-gastrin family of peptides appeared at least 500 million years ago in the protochordate Ciona intestinalis.[4]

Thus, the genes encoding polypeptide hormones, and particularly regulatory peptides, evolved early in the development of life and initially fulfilled the function of cell-to-cell communication to cope with problems concerning nourishment, growth, development, and reproduction. As specialized organs connected by a circulatory system developed during evolution, similar, if not identical, gene products became hormones for purposes of organ-to-organ communication.

Steps in Expression of a Protein-Encoding Gene

The steps involved in transfer of information encoded in the polynucleotide language of DNA to the poly–amino acid language of biologically active proteins involve gene transcription, posttranscriptional processing of ribonucleic acids (RNAs), translation, and posttranslational processing of the proteins. The expression of genes and protein synthesis can be considered in terms of several major processes, any one or more of which may serve as specific control points in the regulation of gene expression ( Fig. 3-2 ).

Figure 3-2 Steps in the cellular synthesis of polypeptide hormones. Steps that take place within the nucleus include transcription of genetic information into a messenger ribonucleic acid (mRNA) precursor (pre-mRNA) followed by posttranscriptional processing, which includes RNA cleavage, excision of introns, and rejoining of exons, resulting in formation of mRNA. Ends of mRNA are modified by addition of methylguanosine caps at the 5′ end and addition of poly(A) tracts at the 3′ ends. The cytoplasmic mRNA is assembled with ribosomes. Amino acids, carried by aminoacylated transfer RNAs (tRNAs), are then polymerized into a polypeptide chain. The final step in protein synthesis is that of posttranslational processing. These processes take place both during growth of the nascent polypeptide chain (cotranslational) and after release of the completed chain (posttranslational), and they include proteolytic cleavages of polypeptide chain (conversion of pre-prohormones or prohormones to hormones), derivatizations of amino acids (e.g., glycosylation, phosphorylation), and cross-linking and assembly of the polypeptide chain into its conformed structure. The diagram depicts posttranslational synthesis and processing of a typical secreted polypeptide, which requires vectorial or unidirectional transport of the polypeptide chain across the membrane bilayer of the endoplasmic reticulum, thus resulting in sequestration of the polypeptide in the cisterna of the endoplasmic reticulum, a first step in the export of proteins destined for secretion from the cell (see Fig. 3-6 ). Most translational processing occurs within the cell as depicted (presecretory) and in some instances outside the cell, when further proteolytic cleavages or modifications of the protein may take place (postsecretory). CHO, carbohydrate.

Rearrangements and Transpositions of DNA Segments.

These processes occur over many years (eons) in evolution, with the exception of uncommon mechanisms of somatic gene rearrangements such as the rearrangements in the immunoglobulin genes during the lifetime of an individual.

Transcription.

Synthesis of RNA results in the formation of RNA copies of the two gene alleles and is catalyzed by the basal RNA polymerase II–associated transcription factors.

Posttranscriptional Processing.

Specific modifications of the RNA include the formation of messenger RNA (mRNA) from the precursor RNA by way of excision and rejoining of RNA segments (introns and exons) and modifications of the 3′ end of the RNA by polyadenylation and of the 5′ end by addition of 7-methylguanine “caps.”

Translation.

Amino acids are assembled by base pairing of the nucleotide triplets (anticodons) of the specific “carrier” aminoacylated transfer RNAs to the corresponding codons of the mRNA bound to polyribosomes and are polymerized into the polypeptide chains.

Posttranslational Processing and Modification.

Final steps in protein synthesis may involve one or more cleavages of peptide bonds, which result in the conversion of biosynthetic precursors (prohormones), to intermediate or final forms of the protein; derivatization of amino acids (e.g., glycosylation, phosphorylation, acetylation, myristoylation); and the folding of the processed polypeptide chain into its native conformation.

Each of the specific steps of gene expression requires the integration of precise enzymatic and other biochemical reactions. These processes have developed to provide high fidelity in the reproduction of the encoded information and to provide control points for the expression of the specific phenotype of cells.

The posttranslational processing of proteins creates diversity in gene expression through modifications of the protein. Although the functional information contained in a protein is ultimately encoded in the primary amino acid sequence, the specific biologic activities are a consequence of the higher order secondary, tertiary, and quaternary structures of the polypeptide. Given the wide range of possible specific modifications of the amino acids, such as glycosylation, phosphorylation, acetylation, amidation, lipidation, and sulfation,[5] any one of which may affect the conformation or function of the protein, a single gene may ultimately encode a wide variety of specific proteins as a result of posttranslational processes.

Polypeptide hormones are synthesized in the form of larger precursors that appear to fulfill several functions in biologic systems ( Fig. 3-3 ), including (1) intracellular trafficking, by which the cell distinguishes among specific classes of proteins and directs them to their sites of action, and (2) the generation of multiple biologic activities from a common genetically encoded protein by regulated or cell-specific variations in the posttranslational modifications ( Fig. 3-4 ).

All the peptide hormones and regulatory peptides studied thus far contain signal or leader sequences at the amino termini; these hydrophobic helical sequences recognize specific sites on the membranes of the rough endoplasmic reticulum, which results in the transport of nascent polypeptides into the secretory pathway of the cell (see Figs. 3-2 and 3-3 [2] [3]).[6] The consequence of the specialized signal sequences of the precursor proteins is that proteins destined for secretion are selected from a great many other cellular proteins for sequestration and subsequent packaging into secretory granules and export from the cell. In addition, most, if not all, of the smaller hormones and regulatory peptides are produced as a consequence of posttranslational cleavages of the precursors within the Golgi complex of secretory cells.

Subcellular Structure of Cells that Secrete Protein Hormones

Cells whose principal functions are the synthesis and export of proteins contain highly developed, specialized subcellular organelles for the translocation of secreted proteins and their packaging into secretory granules. The subcellular pathways utilized in protein secretion have been elucidated largely through the early efforts of Palade[7] (reviewed by Jamieson[8]). Secretory cells contain an abundance of endoplasmic reticulum, Golgi complexes, and secretory granules ( Fig. 3-5 ). The proteins that are to be secreted from the cells are transferred during their synthesis into these subcellular organelles, which transport the proteins to the plasma membrane.

Protein secretion begins with translation of the mRNA encoding the precursor of the protein on the rough endoplasmic reticulum, which consists of polyribosomes attached to elaborate membranous saccules that contain cavities (cisternae). The newly synthesized, nascent proteins are discharged into the cisternae by transport across the lipid bilayer of the membrane. Within the cisternae of the endoplasmic reticulum, proteins are carried to the Golgi complex by mechanisms that are incompletely understood. The proteins gain access to the Golgi complex either by direct transfer from the cisternae, which are in continuity with the membranous channels of the Golgi complex, or by way of shuttling vesicles known as transition elements (see Fig. 3-5 ).

Within the Golgi complex, the proteins are packaged into secretory vesicles or secretory granules by their budding from the Golgi stacks in the form of immature granules. Immature granules undergo maturation through condensation of the proteinaceous material and application of a specific coat around the initial Golgi membrane. On receiving the appropriate extracellular stimuli (regulated pathway of secretion), the granules migrate to the cell surface and fuse to become continuous with the plasma membrane, which results in the release of proteins into the extracellular space, a process known as exocytosis.

The second pathway of intracellular transport and secretion involves the transport of proteins contained within secretory vesicles and immature secretory granules (see Fig. 3-5 ). Although the use of this alternative vesicle-mediated transport pathway remains to be demonstrated conclusively (it is generally considered to be a constitutive, or unregulated, pathway), different extracellular stimuli may modulate hormone secretion differently, depending on the pathway of secretion. For example, in the parathyroid gland and in the pituitary cell line derived from corticotropic cells (AtT-20), newly synthesized hormone is released more rapidly than hormone synthesized earlier. These findings suggest that the newly synthesized hormone may be transported by way of a vesicle-mediated pathway without incorporation into mature storage granules.

Intracellular Segregation and Transport of Polypeptide Hormones

Specific amino acid sequences encoded in the proteins serve as directional signals in the sorting of proteins within subcellular organelles. [6] [9] [10] A typical eukaryotic cell synthesizes an estimated 5000 different proteins during its lifespan. These different proteins are synthesized by a common pool of polyribosomes. However, each of the different proteins is directed to a specific location within the cell, where its biologic function is expressed. For example, specific groups of proteins are transported into mitochondria, into membranes, into the nucleus, or into other subcellular organelles, where they serve as regulatory proteins, enzymes, or structural proteins. A subset of proteins is specifically designed for export from the cell (e.g., immunoglobulins, serum albumin, blood coagulation factors, and protein and polypeptide hormones).

This process of directional transport of proteins involves sophisticated informational signals. Because the information for these translocation processes must reside either wholly or in part within the primary structure or in the conformational properties of the protein, sequential posttranslational modifications may be crucial for determining the specificity of protein function. Continued investigations of protein sorting and trafficking in cells have revealed increased complexities beyond the simple paradigm illustrated in Fig. 3-5 .[11] The sequential sorting of proteins to their final destinations, be they export from the cells (secretion) or targeting to a subcellular compartment or organelle, takes place not only in the Golgi apparatus but also pre-Golgi in the endoplasmic reticulum and post-Golgi in endosomes and tubulosaccules.[12] It is interesting to contemplate that each and every one of the 5000 or so proteins expressed in a given cell contains a specific targeting signal responsible for directing the protein to its final destination. These targeting signals consist of short stretches of amino acids in the proteins that serve as “ZIP codes” to ensure their accurate delivery. Modern approaches using proteomics and bioinformatics are able to predict localization in cells based on the characteristics of these targeting signals.[13]

Signal Sequences in Peptide Prohormone Processing and Secretion

The early processes of protein secretion that result in the specific transport of exported proteins into the secretory pathway are now becoming better understood. [6] [10] [11] [12] [13] [14] [15] Initial clues to this process came from determinations of the amino acid sequences of the proteins programmed by the cell-free translation of mRNAs encoding secreted polypeptides.[16] Secreted proteins are synthesized as precursors that are extended at their NH2 termini by sequences of 15 to 30 amino acids, called signal or leader sequences. Signal sequence extensions, or their functional equivalents, are required for targeting the ribosomal or nascent protein to specific membranes and for the vectorial transport of the protein across the membrane of the endoplasmic reticulum. On emergence of the signal sequence from the large ribosomal subunit, the ribosomal complex specifically makes contact with the membrane, which results in translocation of the nascent polypeptide across the endoplasmic reticulum membrane into the cisterna as the first step in the transport of the polypeptide within the secretory pathway. These observations initially left unanswered the question of how specific polyribosomes that translate mRNAs encoding secretory proteins recognize and attach to the endoplasmic reticulum ( Fig. 3-6 ).

Because microsomal membranes in vitro reproduce the processing activity of intact cells, it was possible to identify macromolecules responsible for processing of the precursor and for translocation activities.[17] The endoplasmic reticulum and the cytoplasm contain an aggregate of molecules, called a signal recognition particle complex, that consists of at least 16 different proteins, including three guanosine triphosphatases to generate energy[18] and a 7S RNA. [6] [10] [19] (Most recently reviewed in reference 20 ). This complex, or particle, binds to the polyribosomes involved in the translation of mRNAs encoding secretory polypeptides when the NH2-terminal signal sequence first emerges from the large subunit of the ribosome.

The specific interaction of the signal recognition particle with the nascent signal sequence and the polyribosome arrests further translation of mRNA. The nascent protein remains in a state of arrested translation until it finds a high-affinity binding protein on the endoplasmic reticulum, the signal recognition particle receptor, or docking protein.[6] On interaction with the specific docking protein, the translational block is released and protein synthesis resumes. The protein is then transferred across the membrane of the endoplasmic reticulum through a proteinaceous tunnel called the translocon.[20]

At some point, near the termination of synthesis of the polypeptide chain, the NH2-terminal signal sequence is cleaved from the polypeptide by a specific signal peptidase located on the cisternal surface of the endoplasmic reticulum membrane. The removal of the hydrophobic signal sequence frees the protein (prohormone or hormone) so that it may assume its characteristic secondary structure during transport through the endoplasmic reticulum and the Golgi apparatus. Interestingly, after its cleavage from the protein by signal peptidase, the signal peptide may sometimes be further cleaved in the endoplasmic reticulum membrane to produce a biologically active peptide. The signal sequence of preprolactin of 30 amino acids, for example, is cleaved by a signal peptide peptidase to give a charged peptide of 20 amino acids that is released into the cytosol, where it binds to calmodulin and inhibits Ca2+-calmodulin-dependent phosphodiesterase.[21]

This sequence in the directional transport of specific polypeptides ensures optimal cotranslational processing of secretory proteins, even when synthesis commences on free ribosomes. The presence of a cytoplasmic form of the signal recognition particle complex that blocks translation guarantees that the synthesis of the presecretory proteins is not completed in the cytoplasm; the efficient transfer of proteins occurs only after contact has been made with the specific receptor or docking protein on the membrane. Although the identification of the signal recognition particle and the docking protein explains the specificity of the binding of ribosomes containing mRNAs encoding the secretory proteins, it does not explain the mode of translocation of the nascent polypeptide chain across the membrane bilayer. Further dissection and analysis of the membrane have identified other macromolecules that are responsible for the transport process.[6]

Cellular Processing of Prohormones

The signal sequences of prehormones and pre-prohormones are involved in the transport of these molecules, but the function of the intermediate hormone precursors (prohormones) is not fully understood. The conversion of prohormones to their final products begins in the Golgi apparatus. For example, the time that elapses between the synthesis of pre-proparathyroid hormone and the first appearance of parathyroid hormone correlates closely with the time required for radioautographic grains to reach the Golgi apparatus.[22] Similarly, the conversion of proinsulin to insulin takes place about an hour after the synthesis of proinsulin is complete, and processing of proinsulin to insulin and C peptide takes place during the transport within the secretory granule.[23] The conversion of prohormones to hormones can also be blocked by inhibitors of cellular energy production such as antimycin A and dinitrophenol[24] and by drugs that interfere with the functions of microtubules (vinblastine, colchicine).[25] Thus, the translocation of the prohormone from the rough endoplasmic reticulum to the Golgi complex depends on metabolic energy and probably involves microtubules.

There is no evidence that sequences that are specific to the prohormone contribute to or are chemically involved in transport of the newly synthesized protein from the rough endoplasmic reticulum to the Golgi apparatus or that they are involved in the packaging of the hormone in the vesicles or granules. Analyses of the structures of the primary products of translation of mRNAs encoding secretory proteins indicate that many of these are not synthesized in the form of prohormone intermediates (see Fig. 3-3 ). It remains puzzling that some secretory proteins (e.g., parathyroid hormone, insulin, serum albumin) are formed by way of intermediate precursors, whereas others (e.g., growth hormone, prolactin, albumin) are not.

Size constraints may be placed on the length of a secretory polypeptide. When the bioactivity of peptides resides at the COOH termini of the precursors (e.g., somatostatin, calcitonin, gastrin), NH2-terminal extensions may be required to provide a sufficient “spacer” sequence to allow the signal sequence on the growing nascent polypeptide chain to emerge from the large ribosome subunit for interaction with the signal recognition particle and to provide adequate polypeptide length to span the large ribosomal subunit and the membrane of the endoplasmic reticulum during vectorial transport of the nascent polypeptide across the membrane (see Fig. 3-6 ). When the final hormonal product is 100 amino acids long or longer (e.g., growth hormone, prolactin, or the α and β subunits of the glycoprotein hormones), there may be no requirement for a prohormone intermediate.

Although the exact functions of prohormones remain unknown, certain details of their cleavages have been established. Unlike the situation with prehormones, in which the amino acids at the cleavage site between the signal sequence and the remainder of the molecule (hormone or prohormone) vary from one hormone to the next, the cleavage sites of the prohormone intermediates consist of the basic amino acid lysine or arginine, or both, usually two to three in tandem. This sequence is preferentially cleaved by endopeptidases with trypsin-like activities.

Specific prohormone-converting enzymes (PCs) consist of a family of at least eight such enzymes. [26] [27] [28] The most studied of the isozymes are PC2 and PC1/3, which are responsible for the cleavages of proinsulin between the A chain/C peptide and B chain/C peptide, respectively. A rare patient missing PC1 presented with childhood obesity, hypogonadotropic hypogonadism, and hypercortisolism and was found to have elevated proinsulin levels and presumably widespread abnormalities in neuropeptide modification.[29] Targeted disruption of the PC2 gene in mice resulted in incomplete processing of proinsulin, leaving the A chain and C peptide intact.[30] Notably, proglucagon in the pancreas remains completely unprocessed, indicating that PC2 is required for the formation of glucagon. As a consequence of defective PC2 activity and low levels of glucagon, the mice have severe chronic hypoglycemia.

After endopeptidase cleavage, the remaining basic residues are selectively removed by exopeptidases with activity resembling that of carboxypeptidase B. In the instances in which the COOH terminal residue of the peptide hormone is amidated, a process that appears to enhance the stability of a peptide by conferring resistance to carboxypeptidase, specific amidation enzymes (peptide amidating monooxygenases) in the Golgi complex work in concert with the cleavage enzymes for modification of the COOH terminal of the bioactive peptides. [31] [32]

All proproteins and prohormones are cleaved by PC enzymatic processes within the Golgi complex of cells of diverse origins. The significance of specific cleavages of specific prohormones remains incompletely understood, as does the reason for the existence of prohormone intermediates in some but not all secretory proteins. As indicated earlier, precursor peptides removed from the prohormones may have intrinsic biologic activities that are as yet unrecognized.

Processes of Hormone Secretion

Specific extracellular stimuli control the secretion of polypeptide hormones. The stimuli consist of changes in homeostatic balance; the hormonal products released in response to the stimuli act on the respective target organs to reestablish homeostasis ( Fig. 3-7 ). Endocrine systems typically consist of closed-loop feedback mechanisms such that, if hormones from organ A stimulate organ B, organ B in turn secretes hormones that inhibit the secretion of hormones from organ A. The concerted actions of both positive and negative hormonal influences thereby maintain homeostasis. An example of such negative feedback regulation is the control of the secretion of adrenocorticotropic hormone (ACTH) by the anterior pituitary gland. Increased ACTH stimulates the adrenal cortex to produce and secrete cortisol, which in turn feeds back to suppress further pituitary secretion of ACTH. These regulatory processes may also include feedback loops in which nonhormonal substances controlled by the target organs regulate hormone secretion. For example, an increase in the concentration of plasma electrolytes as a consequence of dehydration stimulates the release of arginine vasopressin (also called antidiuretic hormone [ADH]) in the neural lobe of the pituitary gland, and vasopressin in turn acts on the kidney to increase the reabsorption of water from the renal tubule, thereby readjusting serum electrolyte concentrations toward normal levels.

In many instances, endocrine regulation is complex and involves the responses of several endocrine glands and their respective target organs. After a meal, the release of a dozen or more hormones is triggered as a result of gastric distention, variations in the pH of the contents of the stomach and duodenum, and increased concentrations of glucose, fatty acids, and amino acids in the blood. The rise in plasma glucose and amino acid levels stimulates the release of insulin and the incretin hormones glucagon-like peptide 1 and glucose-dependent insulinotropic peptide and suppresses the release of glucagon from the pancreas. Both effects promote the net uptake of glucose by the liver; insulin increases cellular transport and uptake of glucose, and the lower blood levels of glucagon decrease the outflow of glucose because of diminished rates of glycogenolysis and gluconeogenesis.

Structure of a Gene Encoding a Polypeptide Hormone

Structural analyses of gene sequences have resulted in at least three major discoveries that are important for understanding the expression of peptide-encoding genes. First, sequences of almost all the known biologically active hormonal peptides are contained within larger precursors that often encode other peptides, many of which are of unknown biologic activity. Second, the transcribed regions of genes (exons) are interrupted by sequences (introns) that are transcribed but subsequently cleaved from the initial RNA transcripts during their nuclear processing and assembly into specific mRNAs. Third, specific regulatory sequences reside in the regions of DNA flanking the structural genes as well as within introns, and these DNA sequences constitute specific targets for the interactions of DNA-binding proteins that determine the level of expression of the gene.

The DNA of higher organisms is wound around proteins forming tightly and regularly packed chromosomal structures called nucleosomes. [33] [34] Nucleosomes are composed of four or five different histone subunits that form a core structure about which approximately 140 base pairs of genomic DNA are wound. The nucleosomes are arranged similarly to beads on a string, and coils of nucleosomes form the fundamental organizational units of the eukaryotic chromosome.

The nucleosomal structure serves several purposes. For example, nucleosomes enable the large amount of DNA (∼2 × 109 pairs) of the genome to be compacted into a small volume. Nucleosomes are involved in the replication of DNA and gene transcription. In addition to histones, other proteins are associated with DNA, and the complex nucleoprotein structure provides specific recognition sites for regulatory proteins and enzymes involved in DNA replication, rearrangements of DNA segments, and gene expression. The acetylation and deacetylation of histone-rich chromatin is involved in the regulation of gene transcription.

The topography of a typical protein-encoding gene consists of two functional units ( Fig. 3-8 ): (1) A transcriptional region and (2) A promoter or regulatory region.

Transcriptional Regions

The transcriptional unit is the segment of the gene that is transcribed into an mRNA precursor. The sequences corresponding to the mature mRNA consist of the exon sequences that are spliced from the primary transcript during the cotranscriptional and posttranscriptional processing of the precursor RNA; these exons contain the code for the mRNA sequence that is translated into protein and for untranslated sequences at the 5′- and 3′-flanking regions. The 5′ sequence typically begins with a methylated guanine residue known as the cap site. The 3′-untranslated region contains within it a short sequence, AATAAA, that signals the site of cleavage of the 3′ end of the RNA and the addition of a poly(A) tract of 100 to 200 nucleotides located approximately 20 bases from the AATAAA sequence. Although the functions of these modifications of the ends of mRNAs are not completely understood, they appear to provide signals for leaving the nucleus; enhance stability, perhaps through providing resistance to degradation by exonucleases; and stimulate initiation of mRNA translation. The protein-coding sequence of the mRNA begins with the codon AUG for methionine and ends with the codon immediately preceding one of the three nonsense, or stop, codons (UGA, UAA, and UAG).

The nature of the enzymatic splicing mechanisms that result in the excision of intron-coded sequences and the rejoining of exon-coded sequences is incompletely understood. Helpful interpretations of the splicing processes are provided in recent reviews. [35] [36] Short “consensus” sequences of nucleotides reside at the splice junctions—for example, the bases GT and AG at the 5′ and 3′ ends of the introns, respectively, are invariant—and a polypyrimidine stretch is found near the AG.[37] Splicing involves a series of cleavage and ligation steps that remove the introns as a lariat structure with its 5′ end ligated near the 3′ end of the introns and ligate the two adjacent exons together. An elaborate machinery (the spliceosome) consisting of five small nuclear RNAs (snRNAs) and roughly 50 proteins direct these steps, guided by base pairing between three of the snRNAs and the mRNA precursor.

Regulatory Regions

The molecular mechanisms involved in the regulation of the expression of genes that encode polypeptides are becoming understood in some detail. As a result of experiments involving the deletions of 5′ sequence segments that reside upstream from structural genes, followed by analyses of the expression of the genes after introduction into cell lines, several insights have been obtained. These regulatory sequences, termed promoters and enhancers, consist of short polynucleotide sequences (see Fig. 3-8 ). They can be divided into at least four groups with respect to their functions and distances from the transcriptional initiation site.

First, the sequence TATAA (TATA, or Goldberg-Hogness, box) is usually present in the more proximal promoter within 25 to 30 nucleotides upstream from the point of transcriptional initiation. The TATA sequence is required to ensure the accuracy of initiation of transcription at a particular site. The TATA box directs the binding of a complex of several proteins, including RNA polymerase II. The proteins, referred to as TATA box transcription factors (TFs), number six or more basal factors (IIA, IIB, IID, IIE, IIF, IIH) and, along with RNA polymerase II, form the general or basal transcriptional machinery required for the initiation of RNA synthesis. [38] [39] [40]

The other three groups of regulatory sequences consist of tissue-specific silencers (TSSs), which function by binding repressor proteins; tissue-specific enhancers (TSEs), which are activated by the binding of transcriptional activator proteins; and metabolic response elements (MREs), which are regulated by the binding of specialized proteins whose transcriptional activities (repressor or activator) are regulated by metabolic signaling, often involving changes in their phosphorylation.

Introns and Exons

Genes encoding proteins and ribosomal RNAs in eukaryotes are interrupted by intervening DNA sequences (introns) that separate them into coding blocks (exons). [35] [36] [41] In bacterial genes, the nucleotide sequences of the chromosomal genes match precisely the corresponding sequences in the mRNAs. Interruption of the continuity of genetic information appears to be unique to nucleated cells. The reasons for such interruption are not completely understood, but introns appear to separate exons into functional domains with respect to the proteins that they encode. An example is the gene for proglucagon, a precursor of glucagon in which five introns separate six exons, three of which encode glucagon and the two glucagon-related peptides contained within the precursor ( Fig. 3-9 ).[42] A second example is the growth hormone gene, which is divided into five exons by four introns that separate the promoter region of the gene from the protein-coding region and the latter into three partly homologous repeated segments, two coding for the growth-promoting activity of the hormone and the third for its carbohydrate metabolic functions.[43] As a rule, the genes for the precursors of hormones and regulatory peptides contain introns at or about the region where the signal peptides join the apoproteins or prohormones, thus separating the signal sequences from the components of the precursor that are exported from the cell as hormones or peptides.

There are exceptions to the one exon, one function theory in mammalian cells. The genes of several precursors of peptide hormones are not interrupted by introns in a manner that corresponds to the separation of the functional components of the precursor. Notable in this regard is the precursor pro-opiomelanocortin, from which the peptides ACTH, α-melanocyte-stimulating hormone, and β-endorphin are cleaved during the posttranslational processing of the precursor. The protein-coding region of the pro-opiomelanocortin gene is devoid of introns. Likewise, no introns interrupt the protein-coding region of the gene for the proenkephalin precursor, which contains seven copies of the enkephalin sequences. It is possible that, in the past, introns separated each of these coding domains and were lost during the course of evolution.

A precedent for the selective loss of introns appears to be exemplified by the rat insulin genes. The rat genome harbors two nonallelic insulin genes: one containing two introns and the other containing a single intron. The most likely explanation is that an ancestral gene containing two introns was transcribed into RNA and spliced; then that RNA was copied back into DNA by a cellular reverse transcriptase and inserted back into the genome at a new site.

Regulation of Gene Expression

The regulation of expression of genes encoding polypeptide hormones can take place at one or more levels in the pathway of hormone biosynthesis ( Fig. 3-10 ) [44] [45] [46]: • DNA synthesis (cell growth and division)
• Transcription
• Posttranscriptional processing of mRNA
• Translation
• Posttranslational processing

Figure 3-10 Diagram of an endocrine cell showing potential control points for regulation of gene expression in hormone production. Specific effector substances bind either to plasma membrane receptors (peptide effectors) or to cytosolic or nuclear receptors (steroids), which leads to initiation of a series of events that couple the effector signal with gene expression. In the illustration shown, peptide effector-receptor complex interactions act initially through activation of adenylate cyclase (AC) coupled with a guanosine triphosphate-binding protein (G). Coupling factors and substances such as glucose, cyclic adenosine monophosphate, and cations activate protein kinases, resulting in a series of phosphorylations of macromolecules. As discussed in the text, specific effectors for various endocrine cells appear to act at one or more of the indicated five levels of gene expression, with the possible exception of posttranslational processing of prohormones, for which no definite examples of metabolic regulation have yet been found.

In different endocrine cells, one or more levels may serve as specific control points for regulation of production of a hormone (see Generation of Biologic Diversification).

Levels of Gene Control

Newly synthesized prolactin transcripts are formed within minutes after exposure of a prolactin-secreting cell line to TRH.[47] Cortisol stimulates growth hormone synthesis in both somatotropic cell lines and pituitary slices through increases in rates of gene transcription and enhancement of the stability of mRNA. [48] [49] The time required for cortisol to enhance transcription of the growth hormone gene is 1 to 2 hours, which is considerably longer than the time required for the action of TRH on prolactin gene transcription. Regulation of proinsulin biosynthesis appears to take place primarily at the level of translation. [50] [51] Within minutes after raising the plasma glucose level, the rate of proinsulin biosynthesis increases 5- to 10-fold. Glucose acts either directly or indirectly to enhance the efficiency of initiation of translation of proinsulin mRNA.[52]

Rapid metabolic regulation at the level of posttranscriptional processing of mRNA precursors is not yet clearly established. However, alternative exon splicing plays a major role in the regulation of the formation of mRNAs during development (see Generation of Biologic Diversification). For example, the primary RNA transcripts derived from the calcitonin gene are alternatively spliced to provide two or more tissue-specific mRNAs that encode chimeric protein precursors with both common and different amino acid sequences, indicating that regulation takes place at the level of processing of the calcitonin gene transcripts.

In many instances, the level of gene expression under regulatory control is optimal for meeting the secretory and biosynthetic demands of the endocrine organ. For example, after a meal there is an immediate requirement for the release of large amounts of insulin. This release depletes insulin stores of the pancreatic β cells within a few minutes, and increasing the translational efficiency of preformed proinsulin mRNA provides additional hormone rapidly.

Tissue-Specific Gene Expression

Differentiated cells have a remarkable capacity for selective expression of specific genes. In one cell type, a single gene may account for a large fraction of the total gene expression, and in another cell type the same gene may be expressed at undetectable levels.

When a gene can be expressed in a particular cell type, the associated chromatin is loosely arranged; when the same gene is never expressed in a particular cell type, the chromatin organization is more compact. Thus, the DNA within the chromatin of expressed genes is more susceptible to cleavage by deoxyribonuclease than is the DNA in tissues in which the genes are quiescent. [53] [54] [55] This looseness may facilitate access of RNA polymerase to the gene for purposes of transcription. In addition, inactive genes appear to have a higher content of methylated cytosine residues than the same genes in tissues in which they are expressed. [56] [57]

Determinants for the tissue-specific transcriptional expression of genes exist in control sequences usually residing within 1000 base pairs of the 5′-flanking region of the transcriptional sequence. Enhancer sequences in animal cell genes were first described for immunoglobulin genes, a finding that extended the earlier observations of enhancer control elements in viral genomes.[58] Historically, the first clear demonstrations of these elements directing transcription to cells of distinct phenotypes came from studies of the comparative expression of two model genes, insulin and chymotrypsin, in the endocrine and exocrine pancreas, respectively.[59] The restricted expression of genes in a cell-specific manner is determined by the assembly of specific combinations of DNA-binding proteins on a predetermined array of control elements of the promoter regions of genes to create a transcriptionally active complex of proteins that includes the components of the general or basal transcriptional apparatus.

Transcription Factors in Developmental Organogenesis of Endocrine Systems

Certain families of transcription factors are critical for organogenesis and the development of the body plan. Among these factors are the homeodomain proteins[60] and the nuclear receptor proteins. [61] [62] [63] The family of homeotic selector, or homeodomain, proteins are highly conserved throughout the animal kingdom from flies to humans. The orchestrated spatial and temporal expression of these proteins and the target genes that they activate determine the orderly development of the body plan of specific tissues, limbs, and organs. Similarly, the actions of families of nuclear receptors (steroid and thyroid hormones, retinoic acid, and others) are critical for normal development to occur. Inactivating mutations in the genes encoding these essential transcription factors predictably result in loss or impairment of the development of the specific organ whose development they direct.

Three examples are described of impaired organogenesis attributable to mutations in essential transcription factors: • Partial anterior pituitary agenesis (Pit-1)
• Adrenal and gonadal agenesis (SF-1, DAX-1)
• Pancreatic agenesis (IPF-1)

Partial Pituitary Agenesis

The transcription factor Pit-1 is a member of a family of pou-homeodomain proteins, which is a specialized subfamily of the larger family of homeodomain proteins.[64] Pit-1 is a key transcriptional activator of the promoters of the growth hormone, prolactin, and thyroid-stimulating hormone β genes, produced in the anterior pituitary somatotrophs, lactotrophs, and thyrotrophs, respectively. Pit-1 is also the major enhancer activating factor for the promoter of the growth hormone-releasing factor receptor gene.[65] Mutations in Pit-1 that impair its DNA-binding and transcriptional activation functions are responsible for the phenotype of the Jackson and Snell dwarf mice.[64]

Mutations in the gene encoding Pit-1 have been found in patients with combined pituitary hormone deficiency in which there is no production of growth hormone, prolactin, or thyroid-stimulating hormone, resulting in growth impairment and mental deficiency.[66] Notably, the production of the other two of the five hormones secreted by the anterior pituitary gland, adrenocorticotropin and the gonadotropin luteinizing hormone (LH) and follicle-stimulating hormone (FSH), is unaffected.[66] In these human Pit-1 mutations, Pit-1 can bind to its cognate DNA control elements but is defective in trans-activating gene transcription. Furthermore, the mutated Pit-1 acts as a dominant negative inhibitor of Pit-1 actions on the unaffected allele.

Pancreatic Agenesis

The homeodomain protein pancreas duodenum homeobox 1 or PDX-1 (somatostatin transcription factor 1 [STF-1], islet duodenum homeobox 1 [IDX-1], insulin promoter factor 1 [IPF-1]) appears to be responsible for the development and growth of the pancreas. Targeted disruption of the PDX-1 gene in mice resulted in a phenotype of pancreatic agenesis.[67] A child born without a pancreas was shown to be homozygous for inactivating mutations in the IPF-1 gene (IPF-1 in the human nomenclature).[68] Notably, the parents and their ancestors who are heterozygous for the affected allele have a high incidence of maturity-onset (type 2) diabetes mellitus, suggesting that a decrease in gene dosage of IPF-1 may predispose to the development of diabetes. The possibility that a mutated IPF-1 allele may be one of several “diabetes genes” is supported by the observation that PDX-1/IPF-1 and the helix-loop-helix transcription factors E47 and β-2 appear to be key up-regulators of the transcription of the insulin gene.[69]

Agenesis of the Adrenal Gland and Gonads

Two nuclear receptor transcription factors have been identified as critical for the development of the adrenal gland, gonads, pituitary gonadotrophs, and the ventral medial hypothalamus. These nuclear receptors are SF-1 (steroidogenic factor 1)[70] and DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, X chromosome).[71] SF-1 binds to half-sites of estrogen response elements that bind estrogen receptors in the promoters of genes. DAX-1 binds to retinoic acid receptor (RAR) binding sites in promoters and inhibits RAR actions. Targeted disruption of SF-1 in mice results in a phenotype of adrenal and gonadal agenesis. In addition, pituitary gonadotrophs are absent and the ventral medial hypothalamus is severely underdeveloped. [72] [73]

X-linked adrenal hypoplasia congenita is an X-linked, developmental disorder of the human adrenal gland that is lethal if untreated. The gene responsible for adrenal hypoplasia congenita has been identified by positional cloning and encodes DAX-1, a member of the nuclear receptor proteins related to RAR.[71] Several inactivating mutations identified in the DAX-1 gene result in the syndrome of adrenal hypoplasia congenita and hypogonadotropic hypogonadism. Thus, genetically defined and transmitted defects in the genes encoding the transcription factors SF-1 and DAX-1 result in profound arrest in the development of the target organs regulated by the hypothalamic-pituitary-adrenal axis involved in steroidogenesis—the adrenal gland (glucocorticoids, mineralocorticoids) and the gonads (estrogens and androgens).

Coupling of Effector Action to Cellular Response

Another mode of gene control consists of the induction and suppression of genes that are normally expressed in a specific tissue. These processes are at work in the minute-to-minute and day-to-day regulation of rates of production of the specific proteins produced by the cells (e.g., production of polypeptide hormones in response to extracellular stimuli).

At least two classes of signaling pathways—protein phosphorylation and activation of steroid hormone receptors by hormone binding—appear to be involved in the physiologic regulation of hormone gene expression. These two pathways mediate the actions of peptide and steroid hormones, respectively. Peptide ligands bind to receptor complexes on the plasma membrane, which results in enzyme activation, mobilization of calcium, formation of phosphorylated nucleotide intermediates, activation of protein kinases, and phosphorylation of specific regulatory proteins such as transcription factors (see Chapter 5 ). [74] [75]

Steroidal compounds, because of their hydrophobic composition, readily diffuse through the plasma membrane, bind to specific receptor proteins, and interact with other macromolecules in the nucleus, including specific domains on the chromatin located in and around the gene that is activated (see Chapter 4 ). [61] [62] [63] Phosphorylated nucleotides such as cyclic adenosine monophosphate (cAMP), adenosine triphosphate, and guanosine triphosphate, as well as calcium, appear to have important functions in secretory processes. In particular, fluxes of calcium from the extracellular fluid into the cell and from intracellular organelles (e.g., endoplasmic reticulum) into the cytosol are closely coupled to secretion. [76] [77]

The cellular signaling pathways that involve protein phosphorylations are multiple and complex. They typically consist of sequential phosphorylations and dephosphorylations of molecules referred to as protein kinase or phosphatase cascades.[78] These cascades are initiated by hormones, sensor molecules known as ligands, that bind to and activate receptors located on the surface of cells, resulting in the generation of small second messenger molecules such as cAMP, diacylglycerol, or calcium ions. These second messengers then activate protein kinases that phosphorylate and thereby activate key target proteins ( Fig. 3-11 ). The final step in the signaling pathways is the phosphorylation and activation of important transcription factors, resulting in gene expression (or repression).

Insight has been gained into the identities of some of the phosphoproteins. As discussed earlier, a specific group of transcription factors, DNA-binding proteins, interacts with cAMP-responsive and phorbol ester-responsive DNA elements to stimulate gene transcription mediated by the cAMP-protein kinase A, diacylglycerol-protein kinase C, and calcium-calmodulin signal transduction pathways (see Fig. 3-11 ). These proteins are encoded by a complex family of genes and bind to the DNA elements in the form of heterodimers or homodimers through a coiled-coil helical structure known as a leucine zipper motif.[79] There is evidence that phosphorylation of these proteins modulates dimerization, DNA recognition and binding, and transcriptional trans-activation activities. Phosphorylation of the protein substrates might change their conformations and activate the proteins, which, in turn, interact with coactivator proteins such as the cAMP response element-binding (CREB) protein and the protein components of the basal transcriptional machinery, thereby allowing RNA polymerase to initiate gene transcription.[80]

Generally, the second messengers activate serine/threonine kinases, which phosphorylate serine or threonine residues, or both, on proteins, whereas the receptor kinases are tyrosine-specific kinases that phosphorylate tyrosine residues. [78] [81] Examples of receptor tyrosine kinases are growth factor receptors such as those for insulin, insulin-like growth factor (IGF), epidermal growth factor, and platelet-derived growth factor. Receptors in the cytokine receptor family, which include leptin, growth hormone, and prolactin, activate associated tyrosine kinases in a variation on the theme.

The different types of signal transduction pathways are described as more or less distinct pathways for semantic purposes. In reality, there is considerable cross-talk among the different pathways that occur developmentally and in cell type-specific settings. An active area of research in endocrine systems is attempting to understand these complex interactions among different signal transduction pathways. Although the growth factor and cytokine receptors are similar in some respects, they differ in other respects. For example, growth factor receptor tyrosine kinases activate transcription factors through cascades that involve both tyrosine phosphorylation and serine/threonine kinases such as mitogen-activated protein kinases, whereas the Janus kinases (JAKs) activated by cytokine receptors directly tyrosine phosphorylate the signal transducer and activator of transcription (STAT) factors. [81] [82]

Generation of Biologic Diversification

In addition to providing control points for the regulation of gene expression, the various steps involved in transfer of information encoded in the DNA of the gene to the final bioactive protein are a means for diversification of information stored in the gene ( Fig. 3-12 ). Five steps in gene expression can be arbitrarily described: (1) gene duplication and copy number, (2) transcription, (3) posttranscriptional RNA processing, (4) translation, and (5) posttranslational processing.

Figure 3-12 Schema indicating levels in expression of genetic information at which diversification of information encoded in a gene may take place. The three major levels of genetic diversification are (1) gene duplication, a process that occurs in terms of evolutionary time; (2) variation in the processing of ribonucleic acid (RNA) precursors, which results in formation of two or more messenger RNAs (mRNAs) by way of alternative pathways of splicing of transcript (see Figs. 3-13 and 3-14 [13] [14]); and (3) use of alternative patterns in processing of protein biosynthetic precursors (polyproteins, or prohormones). These three levels in gene expression provide a means for diversification of gene expression at levels of deoxyribonucleic acid (DNA), RNA, or protein. One or a combination of these processes leads to formation of the final biologically active peptide or hormone. In the diagram, loops depicted in transcripts denote introns; in diagrammatic structures of proteins, the stippled, shaded, and unshaded areas denote exons. See text for details.

Gene Duplications

At the level of DNA, diversification of genetic information comes about by way of gene duplication and amplification. Many of the polypeptide hormones are derived from families of mul-tiple, structurally related genes. Examples include the growth hormone family, consisting of growth hormone, prolactin, and placental lactogen; the glucagon family, consisting of glucagon, vasoactive intestinal peptide, secretin, gastric inhibitory peptide, and growth hormone-releasing hormone; and the glycoprotein hormone family, thyrotropin, luteinizing hormone, follicle-stimulating hormone, and chorionic gonadotropin.

A remarkable example of diversification at the level of gene amplifications is the extraordinarily large number of genes encoding the pheromone and odorant receptors.[83] It is estimated that as many as 1000 such receptor genes may exist in mouse and rat genomes, each receptive to a particular odorant ligand. Over the course of evolution, an ancestral gene encoding a prototypic polypeptide representative of each of these families was duplicated one or more times and, through mutation and selection, the progeny proteins of the ancestral gene assumed different biologic functions. The exonic-intronic structural organization of the genomes of higher animals lends itself to gene recombination and RNA copying of genetic sequences with subsequent reintegration of DNA reverse-transcribed sequences back into the genome, resulting in rearrangement of transcriptional units and regulatory sequences. [84] [85]

Transcription

In addition to duplication of genes and their promoters, another way to create diversity in expression is at the level of gene transcription by providing genes with alternative promoters[86] and by utilizing a large array of cis-regulatory elements in the promoters regulated by complex combinations of transcription factors.

Alternative Promoters

Many of the genes encoding hormones and their receptors utilize more than one promoter during development or when expressed in different tissue types. The employment of alternative promoters results in the formation of multiple transcripts that differ at their 5′ ends ( Fig. 3-13 ). It is presumed that some genes have multiple promoters because they provide flexibility in the control of expression of the genes. For example, in some cases, expression of genes in more than one tissue or developmental stage may require distinct combinations of tissue-specific transcription factors. This flexibility enables genes in different cell types to respond to the same signal transduction pathways or genes in the same cell type to respond to different signal transduction pathways. A single promoter may not be adequate to respond to a complex array of transcription factors and a changing environment of cellular signals.

Figure 3-13 Utilization of alternative promoters in the expression of genes as a means to generate biologic diversification of gene expression. The use of alternative promoters allows a gene to be expressed in a variety of unique contexts that alter the properties of the messenger ribonucleic acid (mRNA) that is expressed. Such alternative promoter usage may render the mRNA more or less stable, affect translational efficiencies, or switch the translation of one protein isoform to another. The use of alternative promoters in genes characteristically occurs during development, or after development is completed, to designate tissue-specific patterns of expression of the gene. Exons are shown as boxes whose protein-coding regions are shaded. Introns are designated by horizontal lines. Dashed lines indicate introns that are spliced out. (Adapted from Ayoubi TAY, Van De Ven WJM. Regulation of gene expression by alternative promoters. FASEB J 1996;10:453-460.)

The organization of alternative promoters in genes is manifested in several patterns within exons or introns in the 5′ noncoding sequence or the coding sequence (see Fig. 3-13 ). The most common occurrence of alternative promoters is within the 5′ noncoding or leader exons. The utilization of different promoters in the 5′ untranslated region of a gene, often accompanied by alternative exon splicing, results in the formation of mRNAs with different 5′ sequences. The alternative usage of promoters in 5′ leader exons can affect gene expression and generate diversity in several different ways. These include the developmental stage-specific and temporal expression of genes, the tissue-type specificity of expression, the levels of expression, the responsivity of gene expression to specific metabolic signals conveyed through signal transduction pathways, the stability of the mRNAs, the efficiencies of translation, and the structures of the amino termini of proteins encoded by the genes.[86]

Examples of genes that use alternative 5′ leader promoters during development are those encoding IGF-I, IGF-II, the retinoic acid receptors, and glucokinase, all of which are regulated by multiple promoters that are active in a variety of embryonic and adult tissues and are subject to developmental and tissue-specific regulation.[86] During fetal development, promoters P2, P3, and P4 of the IGF-II gene are active in the liver. These promoters are shut off after birth, at which time the P1 promoter is activated. The P1 and P2 promoters of the IGF-I gene are differentially responsive to growth hormone: P2 expressed in liver is responsive to growth hormone, whereas P1 expressed in muscle is not.

The retinoic acid receptor exists in three isoforms (RARα, RARβ, and RARγ) encoded by separate genes that give rise to at least 17 different mRNAs generated by a combination of multiple promoters and alternative splicing.[87] The RAR isoforms appear to differ in their specificity for retinoic acid-responsive promoters, in their affinities for ligand isoforms, and in trans-activating capabilities. The different RAR isoforms are expressed at different times in different tissues during development. It has been proposed that the different RAR isoforms provide a means of achieving a diverse set of cellular responses to a single, simple ligand, retinoic acid.[87]

Glucokinase is an example of the alternative use of 5′ leader promoters that have different metabolic responsiveness.[88] Expression of glucokinase in pancreatic beta cells and some other neuroendocrine cells utilizes an upstream promoter (1 β), whereas in liver a promoter (IL) 26 kb downstream of the 1 β promoter is used exclusively. In β cells, expression of the glucokinase gene is apparently not responsive to hormones. In contrast, in liver expression mediated by the IL promoter it is intensely up-regulated by insulin and down-regulated by glucagon.

The α-amylase gene provides an example in which two alternative promoters in the 5′ noncoding exons expressed in two different tissues have dramatically different strengths of expression.[86] A strong upstream promoter directs expression within the parotid gland, contrasting with weak expression directed by an alternative downstream promoter in liver.

Examples of the alternative usage of promoters in the coding regions of genes are the progesterone receptor (PR) and the transcription factor cAMP response element modulator (CREM). In both of these examples, different protein isoforms are produced that have markedly different functional activities. The genes encoding the chicken and human progesterone receptors express two isoforms of the receptor (isoforms A and B).[89] Isoform A initiates translation at a methionine residue located 164 amino acids downstream from the methionine that initiates the translation of the longer form B. Analyses of the mechanisms responsible for the synthesis of two different isoforms revealed that two promoters exist in the human PR gene: one upstream of the 5′ leader exon and the other in the first protein coding exon. The two isoforms of the human PR differ markedly in their capabilities to trans-activate transcription from different progesterone responsive elements (PRE). Both human PR isoforms equivalently activate a canonical PRE. Isoform B is much more efficient than A at activating the PRE in the mouse mammary tumor virus promoter, whereas isoform A, but not B, activates transcription from the ovalbumin promoter.[89]

The utilization of an alternative intronic promoter within the protein coding sequence of a gene is exemplified by the CREM gene.[90] The CREM gene employs a constitutively active, unregulated promoter (P1) that encodes predominantly activator forms of CREM and an internal promoter (P2) located in the fourth intron that is regulated by cAMP signaling and encodes a repressor isoform, ICER (inducible cAMP early response). The remarkable complexity of the alternative mechanisms of expression of the CREM and CREB genes is discussed subsequently.

Diversity of Transcription Factors

Another mechanism to create diversity at the level of gene transcription is that of the interplay of multiple transcription factors on multiple cis-regulatory sequences. The promoters of typical genes may contain 20 or 30 or more cis-acting control elements, either enhancers or silencers. These control elements may respond to ubiquitous transcription factors found in all cell types and to cell type-specific factors.

Unique patterns of control of gene expression can be affected by several different mechanisms acting in concert. The spacing, relative locations, and juxtapositioning of control elements with respect to each other and to the basal transcriptional machinery influences levels of expression. Transcription factors often act in the form of dimers or higher oligomers among factors of the same or different classes. A given transcription factor may act as either an activator or a repressor as a consequence of the existing circumstances. The ambient concentrations of transcription factors within the nucleus in conjunction with their relative DNA-binding affinities and trans-activation potencies may determine the levels of expression of genes.

Posttranscriptional Processing (Alternative Exon Splicing)

Identification of the mosaic structure of transcriptional units encoding polypeptide hormones and other proteins that consist of exons and introns raised the possibility that the use of alternative pathways in RNA splicing could provide informationally distinct molecules. Different proteins could arise either by inclusion or exclusion of specific exonic segments or by utilization of parts of introns in one mRNA as exons in another mRNA. In addition, differences in the splice sites would result in expression of new translational reading frames. Alternative splicing utilizes two distinct mechanisms ( Fig. 3-14 ). One is that of exon skipping or switching in or out of exons. The other mechanism, known as intron slippage, is to include part of an intron in an exon, to splice out part of an exon along with the intron, or to include a “coding” intron.

There are many examples of both mechanisms used to generate diversity in endocrine systems. Included among the genes encoding prohormones in which the pre-mRNAs are alternatively spliced by exon skipping or switching are those for procalcitonin/calcitonin gene-related peptide, prosubstance P/K, and the prokininogens. Alternative processing of the RNA transcribed from the calcitonin gene results in production of an mRNA in neural tissues that is distinct from that formed in the C cells of the thyroid gland.[91] The thyroid mRNA encodes a precursor to calcitonin, whereas the mRNA in the neural tissues generates a neuropeptide known as calcitonin gene-related peptide. Immunocytochemical analyses of the distribution of the peptide in brain and other tissues suggest functions for the peptide in perception of pain, ingestive behavior, and modulation of the autonomic and endocrine systems.

The splicing of the RNA precursor that encodes substance P can take place in at least two ways.[92] One splicing pattern results in the mRNA that encodes substance P and another peptide, called substance K, in a common protein precursor. Other mRNAs are apparently spliced so as to exclude the coding sequence for substance K. An alternative RNA splicing pattern also occurs in the processing of transcripts arising from the gene encoding bradykinin.[93] The high-molecular-weight and low-molecular-weight kininogens are translated from mRNAs that differ by the alternative use of 3′-end exons encoding the COOH termini of the prohormones, a situation similar to that found in the transcription of the calcitonin gene.

Other examples of genetic diversification arise from the programmed flexibility in the choice of splice acceptor sites within coding regions (intron slippage), which allows an array of coding sequences (exons) to be put together in a number of useful combinations. For example, the coding sequences of the growth hormone, lutropin-choriogonadotropin,[94] and leptin receptors[95] can be brought together in two different ways, one to include, the other to exclude, an exonic coding sequence specifying the transmembrane spanning domains of the polypeptide chains that anchor the receptors to the surface of cells. If mRNA splicing excludes the anchor’s peptide sequence, a secreted rather than a surface protein is produced.

Translation

The process of translation provides a fourth level for the creation of diversity of gene expression. As discussed earlier under Regulation of Gene Expression, the rate of translational initiation can be regulated as typified by the proinsulin and prohormone convertase mRNAs, in which translation is augmented by glucose and cAMP. Molecular diversity of translation, however, is generated by the developmentally regulated utilization of alternative translation initiation (start) codons (methionine codons, AUGs). The mechanism of translation initiation involves the assembly of the 40S ribosome subunit on the 5′ methyl guanosine cap of the mRNA.[96] The ribosome subunit then scans 5′ to 3′ along the mRNA until it encounters an AUG sequence in a context of surrounding nucleotides favorable for the initiation of protein synthesis. Upon encountering such a favorable AUG, the subunit pauses and recruits the 60S subunit plus a number of other essential translation initiation factors, allowing the polymerization of amino acids.

The use of an alternative downstream start codon for translation can occur by mechanisms of loose scanning and reinitiation ( Fig. 3-15 ).[97] Loose scanning is believed to occur when the most 5′ AUG codon is not in a strongly favorable context and allows the 40S ribosomal subunit to continue scanning until it encounters a more favorable AUG downstream. Thus, in the loose scanning mechanism, both translational start codons are used. In contrast, the mechanism of translational reinitiation involves the termination of translation followed by the reinitiation of translation at a downstream start codon. Thus, two proteins are encoded from the same mRNA by a start and stop mechanism.

This process of translational reinitiation can occur either by continued scanning of the 40S ribosomal subunit after termination of translation followed by reinitiation, as in loose scanning, or by complete dissociation of the ribosomal subunits at the time of termination followed by complete reassembly at a downstream start codon, referred to as an internal ribosomal entry site (IRES). Such utilization of alternative translation start codons occurs in mRNAs encoding certain classes of transcription factors illustrated by the basic leucine zipper (bZIP) proteins CREB, CREM, and certain of the CCAAT/enhancer binding proteins (C/EBPs), the C/EBPα and C/EBPβ isoforms. In all four of these DNA-binding proteins, the alternative use of internal start codons results in a switch from activators to repressors.

The CREB gene uses translational reinitiation by the somewhat novel mechanism of alternative exon switching that occurs during spermatogenesis.[98] At developmental stages IV and V of the seminiferous tubule of the rat, an exon (exon W) is spliced into the CREB mRNA. Exon W introduces an in-frame stop codon, thereby terminating translation approximately 40 amino acids upstream of the DNA-binding domain. [99] [100] The termination of translation then permits reinitiation of translation at each of two downstream start codons, resulting in the synthesis of two repressor or inhibitor isoforms of CREB known as I-CREBs that are powerful dominant negative inhibitors of activator forms of CREB and CREM because they consist of the DNA-binding domain devoid of any trans-activation domains. [98] [99] [100] The function, if any, of the amino-terminal truncated protein consisting of the activation domains devoid of the DNA-binding domain is unknown. It has been postulated that the role of the alternative splicing of exon W in the CREB pre-mRNA is to interrupt a forward positive feedback loop during spermatogenesis.

CREM, C/EBPα, and C/EBPβ mRNAs utilize alternative downstream start codons to synthesize repressors during development. Like the I-CREBs, these repressors consist of the DNA-binding domains and lack trans-activation domains. The CREM repressor (S-CREM) is expressed during brain development.[90] The C/EBPα-30 and C/EBPα-20 isoforms are expressed during the differentiation of adipoblasts to adipocytes, and the C/EBP repressor liver inhibitory protein (LIP) is expressed during the development of the liver.[90]

Posttranslational Processing

A fifth level of gene expression at which diversification of biologic information can take place is that of posttranslational processing. Many precursors of polypeptide hormones, particularly those encoding small peptides, contain multiple peptides that are cleaved during posttranslational processing of the prohormones.[101] Certain polyprotein precursors, however, contain several copies of the peptide. Examples of prohormones that contain multiple identical peptides are the precursors encoding TRH[102] and the a mating factor of yeast,[103] each of which contains four copies of the respective peptide. Polyproteins that contain several distinct peptides include proenkephalins,[104] pro-opiomelanocortin,[105] and proglucagon.[106]

In many instances, biologic diversification at the level of posttranslational processing occurs in a tissue-specific manner. The processing of pro-opiomelanocortin differs markedly in the anterior compared with the intermediate lobe of the pituitary gland. In the anterior pituitary the primary peptide products are ACTH and β-endorphin, whereas in the intermediate lobe of the pituitary one of the primary products is α-melanocyte-stimulating hormone. The smaller peptides produced are extensively modified by acetylation and phosphorylation of amino acid residues.

The processing of proglucagon in the pancreatic A cells and that in the intestinal L cells are also different (see Fig. 3-15 ).[42] In the pancreatic A cells, the predominant bioactive product of the processing of proglucagon is glucagon itself; the two glucagon-like peptides are not processed efficiently from proglucagon in the A cells and are biologically inactive by virtue of having NH2-terminal and COOH-terminal extensions. On the other hand, in the intestinal L cell, the glucagon immunoreactive product is a molecule, called glicentin, that consists of the NH2-terminal extension of the proglucagon plus glucagon and the small COOH-terminal peptide known as intervening peptide I.

Glicentin has no glucagon-like biologic activity, and therefore the bioactive peptide (or peptides) in the intestinal L cells must be one or both of the glucagon-like peptides. In fact, glucagon-like peptide I in its shortened form of 31 amino acids, GLP-I (7-37), is a potent insulinotropic hormone in its actions of stimulating insulin release from pancreatic beta cells.[107] This peptide is released from the intestines into the bloodstream in response to oral nutrients and appears to be a potent intestinal incretin factor implicated in the augmented release of insulin in response to oral compared with systemic (intravenous) nutrients. This potential for diversification of biologic information provided by the alternative pathways of gene expression is impressive when one considers that these pathways can occur in multiple combinations.

Unexpectedly Low Numbers of Expressed Genes in Genomes of Mammals (Humans and Mice)

A somewhat surprising initial conclusion, heralded in the lay press when the results of the sequencing of the human and mouse genomes were revealed, was that the number of genes in the human and mouse was approximately 30,000. This number was viewed as remarkably low because the number of genes in yeast (Saccharomyces cerevisiae), worm (Caenorhabditis elegans), and fly (Drosophila melanogaster) is about 20,000. However, it seems quite clear from the complexities of the mRNAs expressed in humans and mice, as exemplified by the growing database of expressed sequence tags, that tissue-specific alternative exon splicing and alternative promoter usage occur much more frequently in humans and mice than in yeast, worms, and flies. Considering the as yet incomplete database of expressed genes at the mRNA level, it seems reasonable to extrapolate that the human genome may actually express as many as 100,000 to 200,000 mRNAs that encode proteins with distinct, specific functions. This extrapolation is based on the observation that alternative exon splicing and promoter usage appear to be on the order of 5 to 10 times more frequent in higher vertebrate mammals than in yeasts and flies.

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THE ENDOCRINE PATIENT

Kronenberg: Williams Textbook of Endocrinology, 11th ed.
Copyright © 2008 Saunders, An Imprint of Elsevier

THE ENDOCRINE PATIENT

Daniel D. Federman

▪ General Considerations, 12
▪ Special Features of Endocrine Illness, 12
▪ Unique Features of Reproductive Disorders, 13
▪ Evaluation of Patients with Endocrine Disorders, 13
▪ Imaging, 16
▪ Conveying Results, 16
▪ Some New Features of Clinical Endocrinology, 16
▪ Management, 17

A textbook of medicine is inevitably about disease, but the practice of medicine deals with illness, that is, a person experiencing a disease. This is why the present chapter has been entitled “The Endocrine Patient.” This chapter lays out the general issues and approaches applicable to caring for patients with endocrine disorders. The topics to be discussed include initial evaluation and the nature of referral, the fact-finding required in clinical evaluation, the use of the laboratory and imaging, the formulation of a differential diagnosis, decision making, and management. In each case, the steps are portrayed from the patient’s point of view. Except for acute adrenal insufficiency, endocrine disorders are seldom life-threatening. They have enormous effect on the quality of life, however, and successful intervention can be extremely important to both patient and family.

General Considerations

Many features of being an endocrine patient are common to all experiences of illness. Most often, a perceived change in bodily function, a symptom, gets one to the doctor. Although generations of medical students have described new patients as being “in no acute distress,” most patients are, in fact, worried and anxious when they see a physician, the more so when the physician is not known to them. A few minutes spent in getting to know the patient pays enormous dividends in the accuracy of the history obtained and in setting the stage for further cooperation with testing and treatment.

Inasmuch as most endocrine consultation is elective rather than emergent, I favor asking a few simple questions, such as “Where are you from?” “What do you do?” “How did you come to us?” “Were you referred?” and so on. Almost always, some common experience or acquaintance is discovered that provides the basis for a rapport that does not emerge from formal medical questioning. This approach also immediately conveys that you are interested in the patient as a person and not just the patient’s disease. It also provides reassurance that you have the time to listen to the person, a simple luxury often omitted in the current maelstrom of medicine.

Special Features of Endocrine Illness

Traditionally a consultation begins with either a telephone call or letter from the referring physician. But this invaluable step is today honored more in the breach than the fact.

Discovery through Screening

Numerous special features of endocrine disease make patient presentation quite different from that seen in general medicine. One feature is the discovery of abnormality through screening of asymptomatic individuals, for example, a high serum calcium level discovered through multiphasic screening or a high blood glucose level discovered in a shopping mall kiosk. The very absence of symptoms lends an unreality to the moment and should become an explicit topic of the patient-doctor interaction. In this circumstance, it is worth emphasizing the value of early discovery and prevention of greater morbidity.

Quantitative Rather than Qualitative Abnormalities

A second special feature of endocrine disorders is that they are quantitative, rather than qualitative, departures from normal. No endocrine disorder is due to a novel hormone. Everyone has cortisol circulating as a determining feature of his or her life. Hypercorticism and adrenal insufficiency represent just more or less of the natural hormone. Similarly, all hormones found in excess or in deficiency in disease are physiologic determinants of stature, weight, complexion, hairiness, temperament, and behavior. In contrast, no one has a little pneumonia or a little inflammatory bowel disease as a constitutive status. In addition, most endocrine glands have both a basal and a stimulable or reserve function. It is common to have partial diminution of capacity in which the basal function is adequate but a reserve called upon during part of each day—or, more dramatically, in emergencies—is not available.

Overlap with Other Diseases

The symptoms of endocrine disorders overlap a great range of normal characteristics, including body contour, facial configurations, weight distributions, skin and hair coloring, and muscular capacity. They also overlap with other conditions that are far more common, including depression, obesity, and normal aging. The added adipose tissue of hyperadrenocorticism is more difficult to recognize in a person who is already obese. The nervousness associated with hyperthyroidism is less apparent in a thin, hyperkinetic man than in a person of moderate body weight. The effects of an androgen-producing adrenal tumor are less likely to be noticed in a family of swarthy, hirsute individuals.

Finally, most endocrine disorders evolve gradually over months to years instead of appearing suddenly, such as a heart attack or an acute infection. This combination of varied host background and slow evolution of disease leads to considerable delay in diagnosis: both the patient and primary care physician adapt to the changes as part of the person, and definitive evaluation, now relatively easy for most disorders, is not undertaken. Hypothyroidism and acromegaly are good examples of this phenomenon. Published series for both diseases show a remarkable delay in diagnosis despite sometimes disabling symptoms.

Hormones have more distant effects than local effects. This, of course, reflects their messenger status. Unlike an abscess, a myocardial infarction, or an esophageal cancer, endocrine disorders seldom produce symptoms near the gland of origin. (Subacute thyroiditis and large pituitary tumors, of course, are exceptions.) But because in most endocrinopathies the excess or missing hormone works on several or many systems, the resulting syndrome can be enigmatic.

Several endocrine disorders are important not because of their incidence but because of their curability: Cushing’s disease, acromegaly, and pheochromocytoma are cases in point. Although these disorders enter the differential diagnosis of common problems such as diabetes, their occurrence is so rare that the primary care physician does not easily think of them, thus the maxim: “What you don’t see often you don’t see.”

Unique Features of Reproductive Disorders

Reproductive disorders have symptoms and signs that have no parallel in other areas. This is the one system in which sexual dimorphism is inherent rather than epiphenomenal; it is also the one with the greatest span of developmental change. Once the heart starts beating in the embryo, it goes on doing so until the last moment of life; but puberty, adult sexual functioning, and menopause establish time lines against which all symptoms are to be assessed. Thus, vaginal bleeding has entirely different meanings when it occurs on the first day of life, as a natural appearance at age 12, during pregnancy, as a harbinger of menopause at age 46 years, or as a highly probable symptom of cancer at age 66 years.

Physical appearance and function are important features of self-image. Thus, hirsutism, thinness, obesity, sexual arousal, and erectile capacity bear considerable psychological import to the endocrine patient. The clinician should be constantly aware of both spoken and unspoken thoughts that may be troubling the patient.

The Couple as a Clinical Unit

The ultimate goal of reproductive capacity is, of course, a fertile union. This means that the couple, rather than the individual, is the unit of clinical concern. It is thus the principal area in medicine wherein two people and their interaction, rather than a single person and her capacities, are studied and treated. In addition, there are dimensions of successful sexual function that are important at other times than when fertility is sought. Sex drive, erotic responsiveness, affection, and tenderness are all important aspects of life whether or not fertility is a current issue.

Evaluation of Patients with Endocrine Disorders

I have emphasized previously the belief that establishing an interested and warm relationship is the beginning of excellence in any elective medical interaction. In addition to its affective power, the relationship elicits a more informative history, establishes better cooperation in both testing and treatment, and provides a platform for informed decision making by the patient.

History

As in most areas of medicine, precision of diagnosis and economy of investigation begin with a carefully wrought history. An open-ended question, combined with an attentive silence, allows the patient to provide the background for the clinical moment. After the patient has spoken spontaneously, the physician elicits a guided expansion of the information. Details of timing, sequence, changes of diet or activity, relationship to the menstrual cycle, changes in weight or size, and alterations in mood or sleep pattern—all of these may provide clues to underlying endocrine abnormality.

A good example of the power of the history is the interpretation of irregular periods in a woman of reproductive age. The simple statement, “I’ve never been regular,” points to a presumptive diagnosis of polycystic ovary syndrome in a way that a very convoluted sequence of questions might actually fail to do. That statement is to be contrasted with this one: “I used to be regular, but in the last year or so, I never know when my period is going to come.” If the presenting symptom is irregular periods, the simple invitation, “Tell me about your periods,” is likely to be the key to the diagnosis.

Careful questioning about use of complementary and alternative medicines is an important and, occasionally, a very revealing step.

A thorough family history has become increasingly important as the genetic basis for more and more endocrine diseases becomes established. For practical purposes, I favor diagramming a pedigree of the first-order relatives—parents, siblings, children—of all patients, not just those for whom a genetic disorder is already suspected. Known disorders are readily revealed this way, and unknown conjunctions of clinical and genetic factors may also be disclosed ( Fig. 2-1 ).

Figure 2-1 Simple pedigree of the propositus (arrow) and first-order relatives should be the standard family history in a new patient workup. If the patient has children, their health status should be included as well.

Physical Examination

General Examination

It is said that the history is 80% or more of clinical diagnosis, and that is no less true in endocrine disorders than in general medicine. Yet the physical examination is a critical element in arriving at a diagnosis, and here I call particular attention to the first impression.

Cushing’s syndrome, Addison’s disease, hyperthyroidism, hypothyroidism, acromegaly, polycystic ovary syndrome, hypogonadism, and Turner’s syndrome—these and other endocrine disorders should be considered from the first moment one encounters a new patient. Otherwise, one risks accepting that the appearance of the patient is just that and no more. In other words, as soon as one accepts that the initial impression is what the person looks like naturally, the quantitative departure from normal that is the essence of endocrine disease fails to be impressive. This, incidentally, is why both families and primary care physicians often miss a diagnosis that seems obvious to the consultant endocrinologist.

A quantification of this last point may be helpful. If the signs of hypothyroidism or acromegaly, for example, take 3 years to become striking, the person living with the patient is exposed to 1/1095th of fractional change per day—well below the threshold of just noticeable difference. Similarly, a primary care physician seeing the patient perhaps four times a year for a general checkup and management of hypertension is exposed to 91/1095th fractional change. This can sometimes lead to a diagnosis but often does not. When one sees the patient for the first time, however, the imprint of the disease catches attention and the constitutional appearance is in the background.

Although a consultant participates because of a special area of interest and expertise, he or she is a general physician first and should be alert to all dimensions of the physical examination: What is the height-to-weight ratio? What is the basic degree of muscularity? Is there evidence of heart disease to explain the chest pain and dyspnea one has heard about in the history? What is the degree of hirsutism? Are there signs of liver disease, malnutrition, or poor or excellent physical training? What is the blood pressure with the patient standing as well as lying or sitting? These and many other points of a general examination begin to modify the thinking one has undertaken on the basis of history.

Targeted Examination

The targeted physical examination of any consultant is a dynamic interplay of general and specific goals. Theoretically, any experienced clinician should undertake a general examination and come to all the findings pertinent to an underlying endocrine disorder. In fact, however, the physical examination is greatly influenced by the hypotheses generated in the history. Let us look at a few examples.

If a patient reports weight loss despite a good appetite, there is only a very restricted differential diagnosis, principally malabsorption or hypermetabolism. In doing a physical examination, therefore, I would pay particular attention to signs of malabsorption (muscular wasting, vitamin deficiencies, purpura) and to signs of thyroid disease with its generalized hypermetabolism and localized autoimmune phenomena, including ophthalmopathy.

Similarly, if a patient complains of hirsutism or other signs of androgen excess, one is immediately thrust into a consideration of ethnic hair distribution and quality. Is there temporal recession of the hairline? Does the hair on the abdomen come up over the umbilicus? Is hair present on the back (rare without marked hyperandrogenism)? How much acne is there? Is acanthosis nigricans present? At the extreme, is there evidence of clitoral enlargement?

Finally, and most important, does the patient look like or unlike the other women in her family?

Direct Assessment of Endocrine Glands

Three endocrine glands are palpable—the thyroid, the testis, and the ovary. Specific attention should be given to each of these.

The thyroid gland should be approached first by inspection—while the patient swallows—for size, symmetry, or localized enlargement. Many thyroid nodules are visible, and inspection often calls attention to lesions that would be missed on palpation. The thyroid should then be felt while the patient swallows, from the front with your thumbs or from behind the patient with the index and third fingers. It is crucial to keep your own fingers from moving while the patient is swallowing. The principal observation is whether there is diffuse enlargement of the thyroid gland (most often Graves’ hyperplasia or Hashimoto’s thyroiditis) or one or more nodules. Although the consistency of the gland is to be noted, in fact it is often not concordant with the pathology.

Functioning tumors of the testis may be too small to be felt with the fingers, and most internists and general physicians are not skilled in palpation of the ovaries. For this reason, ultrasound and other forms of imaging have become key features of gonadal evaluation and are discussed later.

The size of one other endocrine gland, the pituitary, can be inferred from physical examination for what Cushing called “neighborhood signs.” As a pituitary tumor or diffuse enlargement proceeds, it pushes up on the optic chiasm from below, producing a bitemporal hemianopsia first manifested in the upper quadrants, often to a blinking or flashing red light. This finding is too subtle for confidence, however, and pituitary assessment depends on formal visual fields and imaging.

Indirect Assessment of Endocrine Status

Many consequences of hormone action can be detected on physical examination; the results combine with the history to produce a highly reliable differential diagnosis and thus an informed basis for laboratory evaluation and imaging. Among the things to be looked for are the eye signs and dermopathy of Graves’ disease, acanthosis nigricans as a clue to insulin resistance, muscular wasting and tremor, changes in the voice due to hypothyroidism or acromegaly, and a general impression of nutrition and its adequacy or excess. Each of these findings is described in more detail with the specific disorder in subsequent chapters.

Laboratory Testing of Endocrine Function

Modern endocrine laboratory evaluation began with the introduction of radioimmunoassay by Berson and Yalow. The precise measurement of hormone concentrations, determined by competitive displacement of specific antibodies, was soon succeeded by competitive binding assays and, more recently, by immunofluorescent and radioluminescent determinations of even greater sensitivity and specificity. It should theoretically be possible to enter the name of a hormone on a laboratory slip and expect to get back a definitive reflection of the status of the patient for that gland. For practical purposes, that has become true of thyroid-stimulating hormone (TSH). Reliable determinations of elevated, normal, and suppressed levels of this hormone by radiochemiluminescent determination have made it the standard of care for thyroid disease and a model for all endocrine laboratory tests. However, it is an exception rather than the rule, and it is worthwhile reviewing why other testing is not as easy and why considerable judgment is required. The following examples illustrate this point.

Pulsatile Hormone Secretion

Many hormones are secreted in pulses rather than steadily. The peaks or valleys of hormones secreted in pulsatile fashion, such as luteinizing hormone or growth hormone, may fall above or below the ostensibly normal range. If such a value is obtained by chance, it can erroneously suggest hypofunction or hyperfunction. Repeating the test with three samples drawn at 30-minute intervals and pooled can clarify this type of problem.

Diurnal Variation

The hypothalamic-pituitary-adrenal axis of cortisol secretion is typically maximal during the day and lower in the evening and night. A plasma cortisol level of 12 μg/dL is normal at 8 am, but the same value at 8 pm reflects a loss of diurnal rhythm resulting from either stress or hypercorticism. A plasma cortisol sample drawn at midnight is an excellent test for evaluation of overactive adrenal function.

Cyclic Variation

The menstrual cycle provides the most extreme “normal variation” of any hormone level. From the first day of a menstrual period, when estrogen levels may be indistinguishable from those of a normal man, the level rises extraordinarily rapidly and at the 14th day can be as high as in early pregnancy. As a consequence, an estrogen level must be evaluated in the light of the stage of the cycle at which it is drawn.

Age

All clinicians are aware that gonadal hormones show marked differences reflective of the individual’s stage of life. It is not as widely known that the adrenal hormone dehydroepiandrosterone (DHEA) is barely secreted during childhood, is actively put out by the adrenal glands from age 8 or so to age 55, and then disappears as mysteriously as it came. At present, there is no clear understanding of the physiologic role of its presence or absence, nor of its control.

Sleep Entrainment

Both prolactin and growth hormone have a sleep-entrained secretory pulse shortly after sleep begins. In people who work at night and sleep during the day, this secretion is clearly related to sleep and not to clock time.

Hormone Antagonism

Certain hormones antagonize the effects of other hormones; it is thus necessary to know the value of each hormone to interpret the clinical phenomenon. The opposite effects of estrogen and androgen on the male breast are a good example. A normal testosterone level combined with an elevated estrogen level, or a normal estrogen level but a decreased androgen level, easily accounts for gynecomastia.

Dynamic Testing

Many endocrine glands have a basal secretory level and a reserve secretion elicited by either a tropic hormone or a change in metabolic or physiologic state. Cortisol secretion can increase 5- to 10-fold in response to stress or adrenocorticotropic hormone (ACTH). Insulin release is stimulated by both glucose and amino acids and by distinct pathways.

Baseline hormone levels can be misleading. The test results in Table 2-1 were obtained from a 30-year-old woman who complained of fatigue and amenorrhea 6 months after a pregnancy during which she had been markedly anemic (hemoglobin, 9 g/dL); she had never been in shock and had received no transfusions.

TABLE 2-1 — BASELINE HORMONAL VALUES
INSULIN IV TRH IV GnRH IV
Time 0 Glucose 80 mg/dL Cortisol 7.7 μg/dL TSH 3.2 mU/L Prolactin 6.6 ng/mL FSH 8.9 mIU/mL LH 6.7 mIU/mL
15 47 8.2 5.6 7.6 5.9 9.3
30 23 8.6
45 30 7.7
60 38 13.0
90 50 9.7
120 63 9.6

FSH, Follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; IV, intravenous; LH, luteinizing hormone; TRH, thyrotropin-releasing hormone; TSH, thyroid-stimulating hormone.

In this study, all basal values are within normal limits. Note, however, that intravenous insulin lowers the blood glucose levels but does not elicit an adequate release of cortisol. Thyrotropin-releasing hormone (TRH) does not induce a normal rise in thyrotropin (TSH) or prolactin. Gonadotropin-releasing hormone (GnRH) evokes a submaximal increase in follicle-stimulating hormone (FSH) and luteinizing hormone (LH).

Hormone and Metabolite Interaction

Insulin is a good example of a hormone whose absolute level is less meaningful than its relationship to the blood glucose level. A plasma insulin of 70 is a normal response to a meal, when the blood glucose level is rising. In contrast, an insulin value of 10 or 12 is abnormal (is not appropriately suppressed) if the glucose level is 40 mg/dL. Indeed, the lower insulin level in a fasting hypoglycemic patient is distinct evidence of spontaneous hyperinsulinism, such as in an islet cell tumor.

Growth hormone represents another instance in which a single random sample cannot be given much meaning. During a day, plasma growth hormone levels vary from values that, if sustained, would be diagnostic of acromegaly to values that, again if sustained, would point to hypopituitarism. In normal people, growth hormone secretion is suppressed by glucose intake. A plasma growth hormone of 8 within an hour of a standard meal containing glucose would be pathologically elevated; it should be less than 2. Similarly, however, a growth hormone value of 2 in a fasting person who had run up a flight of stairs suggests deficient pituitary function.

Most hormones are part of a feedback loop in which an artificial increase, especially by ingestion of the hormone in a medication, decreases endogenous secretion. If a normal person takes 0.l to 0.3 mg of thyroxine (T4), hypothalamic secretion of TRH and pituitary secretion of thyrotropin (TSH) are suppressed. Plasma levels of T4 and triiodothyronine (T3) may not change, but TSH levels would be decreased and reflections of TSH effect, such as radioactive iodine uptake, would similarly be suppressed. Although ultrasensitive TSH testing has replaced tests of suppressibility for the diagnosis of thyroid disease, tests of suppressibility are the standard approach to evaluating growth hormone and ACTH/cortisol regulation.

Protein Binding

Hormones such as T4 and cortisol are compartmentalized into a fraction attached to a transport protein (and thus physiologically unavailable) and a free portion able to diffuse into cells and initiate a hormone effect. It is the free or unbound portion that is physiologically regulated; the level of the binding protein may be increased or decreased without physiologic consequence if the free portion is unchanged. The measurement of free T4 or a free T4 index (FT4I) (see Chapter 10 ) has become the standard second step if a screening TSH value is abnormally high or low.

Testosterone is even more complicated because it is trebly partitioned among sex hormone–binding globulin, albumin, and a free portion. Measurement of the free hormone level is often necessary, particularly when the binding protein level has been artifactually raised or lowered (see Chapter 6 ).

Laboratory Error

Laboratory error may seem too obvious a source of confusion to mention, but it provides a reminder for an important caution about laboratory testing. It is easy to be seduced by numbers and to consider the laboratory report the final arbiter. In fact, it is the history and physical examination, plus the clinician’s judgment, that establish the prior probability of a given diagnosis. Both in choosing and in interpreting laboratory tests, the endocrinologist should establish his or her own expectations before testing. If the physician feels strongly that a particular condition is present, discordant initial laboratory results should not be dissuasive. More detailed testing, as discussed in subsequent chapters, is then appropriate. The clinician’s judgment is still a key component of the process.

Imaging

The extraordinary power of modern imaging, particularly ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI), has enriched endocrinology as it has all of medicine. However, the role of imaging in endocrinology is, to my mind, different from its contribution elsewhere.

For one thing, several endocrine glands (the thyroid, the pituitary, and the adrenals in particular) frequently contain clinically insignificant, nonfunctioning adenomas and cysts. Second, functioning and nonfunctioning lesions other than in the thyroid gland can be difficult to distinguish from each other. Thus, except in an emergency (e.g., suspected pituitary apoplexy), the clinician should define the functional state of the gland before requesting imaging. In other words, one should be clear from hormone measurements, including dynamic testing, whether the gland is overactive, underactive, or normal.

In addition, one should have a clear idea of how the radiologist can be expected to help. Such clarity reduces costs by targeting the selection of imaging and making the radiologic findings a truly complementary element of evaluation. The best imaging modalities for the various glands are discussed in their respective chapters. The approach suggested here, however, is broadly applicable.

Conveying Results

Both sophisticated imaging and thorough laboratory testing produce results after the actual office visit. Patients are understandably anxious about the findings and deserve a prompt response. The best approach depends on the circumstance. A new patient with Cushing’s syndrome or acromegaly should be given an early in-person visit. A patient with hypothyroidism who understands the disease well and just needs a slight change in T4 dose can easily be informed with a telephone call. Someone with negative results can be left a message of reassurance and can be encouraged to call back, both to confirm receipt of the information and to get questions answered.

Some New Features of Clinical Endocrinology

Genetics

The decoding of the human genome promises to change the face of medical practice. Ironically, the first human disease in which cancer was prevented by application of genetic testing in susceptible families was the screening for medullary thyroid carcinoma in pedigrees of multiple endocrine neoplasia type 2 (MEN-2). The screening at that time was done by pentagastrin or calcium provocation of calcitonin release. Now the screening for endocrine manifestations of MEN-2 is secondary to screening families for the RET proto-oncogene defect that is the basis of the disease. Endocrine testing, such as measurement of calcitonin or plasma catecholamines, is restricted to patients who have the genetic abnormality. In the dangerous variant of the MEN-2 syndrome in which medullary thyroid carcinomas appear in the first year of life, aggressive genetic screening is done during that year, and endocrine testing in patients at risk can justify surgical thyroidectomy before the first birthday.

Hereditary predispositions will certainly emerge for other endocrine disorders and will make it crucial for the clinician to take a revealing family history and follow up even minor clues.

The Internet

Never in history has so much medical information been available to patients. I now routinely ask patients what they already know about their condition or their symptoms. A bit sheepishly in some cases, many patients admit to looking up topics on the World Wide Web and are about to compare what I tell them with what they have already read. Much of that information is accurate, but some is nonsense, and it requires patience and clear explanation before such patients go away satisfied.

Electronic Mail

Although opinions differ widely, I find e-mail an extremely useful advance in communicating with patients who have computers, provided there is a doctor-patient relationship already established personally. Patients have access to you between appointments and on a time frame of mutual convenience. The computer thus reduces anxiety on the patient’s part, particularly regarding questions or findings for which they might otherwise hesitate to make an appointment. Reporting laboratory test results is expedited, and accompanying the report with a few sentences of interpretation can be as useful as a telephone call.

It is wise to keep copies of e-mails so that a clear record of the exchange is available. There are, however, several important caveats. Never let an e-mail exchange substitute for a true evaluation, including history and physical examination. I believe that one should not prescribe for a patient whom one has not seen, and one should not provide much interpretation of history or laboratory tests without very fundamental disclaimers. But in an established patient-doctor relationship, e-mail as the patient’s choice can be very helpful.

Managed Care

The effort to control health care costs by limiting reimbursement for physician services, laboratory testing, and imaging has had a profound impact throughout medicine. Without taking on the whole issue, I want to comment on several practical consequences.

“Curbsiding”—the request by a physician for patient guidance without being asked to see the patient—has increased strikingly. Consultants can provide some general help to primary care physicians without seeing the patient; however, effectiveness hinges on the history and physical examination done by the primary care physician. The failure to realize that hyperthyroidism is due to a hot nodule, for example, totally distorts the picture and will lead to an erroneous recommendation for treatment. The failure to distinguish a recent onset of amenorrhea and virilization from a polycystic ovary-like syndrome may hide the presence of a readily curable virilizing tumor. The failure to recognize hypoglycemic unresponsiveness may perpetuate a dangerous degree of overinsulinization and elicit inappropriate advice from the consultant. Thus, the consultant must set boundaries and at some point indicate that it is important for a formal consultation to take place.

Costs

Some endocrine workups can be expensive and invite challenge from third-party insurers. The best approach to this concern is a careful history and physical examination, clear establishment of the prior probabilities of certain diagnoses, and then effective use of screening tests before embarking on an unnecessarily extensive evaluation. For example, in a patient with suspected Cushing’s syndrome, it is mandatory to establish the presence of hypercorticism before embarking on a search for its cause. Once this lethal but curable disorder has been properly diagnosed, however, no cost should deter one from finding the cause and correcting it. One argument I make is that expensive tests and imaging should be amortized rather than considered an extravagant or unnecessary one-time expense. If a young woman age 30, with a life expectancy of 80 years or more, has a husband and two children to whom her life matters, the $3000 evaluation breaks down to $20 per loved one per year of life expectancy. Any plan manager has to see that this is an appropriate cost.

Management

There are few more gratifying experiences in medicine than recognizing and correcting an endocrine disorder. Patients feel that they have been rescued from a mysterious overtaking of their identity. Body contour, facial appearance, temperament, and well-being are restored to the patient’s constitutive status. Deterioration previously attributed to aging or depression or chronic disease is reversed. In brief, something almost miraculous takes place. Even when these goals cannot be achieved, as in diabetes, a major impact on mortality and morbidity can be. Of course, these optimal outcomes require accuracy of diagnosis—but that is only the beginning. A true sharing by patient and physician, based on a sound knowledge of normal physiology, provides the best foundation for choice of therapy and maintenance of a continuing program. The result can be, simply put, wonderful.

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Hormones and Hormone Action

Kronenberg: Williams Textbook of Endocrinology, 11th ed.
Copyright © 2008 Saunders, An Imprint of Elsevier

Hormones and Hormone Action

PRINCIPLES OF ENDOCRINOLOGY

Henry M. Kronenberg Shlomo Melmed P. Reed Larsen Kenneth S. Polonsky

▪ Introduction, 3
▪ The Evolutionary Perspective, 3
▪ Endocrine Glands, 5
▪ Transport of Hormones in Blood, 6
▪ Target Cells as Active Participants, 6
▪ Control of Hormone Secretion, 8
▪ Hormone Measurement, 10
▪ Endocrine Diseases, 10
▪ Therapeutic Strategies, 11
Introduction

Roughly a hundred years ago, Starling coined the term “hormone” to describe secretin, a substance secreted by the small intestine into the bloodstream to stimulate pancreatic secretion. In his Croonian Lectures, Starling considered the endocrine and nervous systems as two distinct mechanisms for coordination and control of organ function. Thus, endocrinology found its first home in the discipline of mammalian physiology.

Work over the next several decades by biochemists, physiologists, and clinical investigators led to the characterization of many hormones secreted into the bloodstream from discrete glands or other organs. These investigators showed that diseases such as hypothyroidism and diabetes could be treated successfully for the first time by replacing specific hormones. These initial triumphs formed the foundation of the clinical specialty of endocrinology.

Advances in cell biology, molecular biology, and genetics over the ensuing years began to explain the mechanisms of endocrine diseases and of hormone secretion and action. Even though these advances have embedded endocrinology in the framework of molecular cell biology, they have not changed the essential subject of endocrinology—the signaling that coordinates and controls the functions of multiple organs and processes. Herein we survey the general themes and principles that underpin the diverse approaches used by clinicians, physiologists, biochemists, cell biologists, and geneticists to understand the endocrine system.

The Evolutionary Perspective

Hormones can be defined as chemical signals secreted into the bloodstream that act on distant tissues, usually in a regulatory fashion. Hormonal signaling represents a special case of the more general process of signaling between cells. Even unicellular organisms, such as baker’s yeast, Saccharomyces cerevisiae, secrete short peptide mating factors that act on receptors of other yeast cells to trigger mating between the two cells. These receptors resemble the ubiquitous family of mammalian 7-transmembrane spanning receptors that respond to ligands as diverse as photons and glycoprotein hormones. Because these yeast receptors trigger activation of heterotrimeric G proteins just as mammalian receptors do, this conserved signaling pathway must have been present in the common ancestor of yeast and humans.

Signals from one cell to adjacent cells, so-called paracrine signals, often trigger cellular responses that use the same molecular pathways used by hormonal signals. For example, the sevenless receptor controls the differentiation of retinal cells in the Drosophila eye by responding to a membrane-anchored signal from an adjacent cell. Sevenless is a membrane-spanning receptor with an intracellular tyrosine kinase domain that signals in a way that closely resembles the signaling by hormone receptors such as the insulin receptor tyrosine kinase. Because paracrine factors and hormones can share signaling machinery, it is not surprising that hormones can, in some settings, act as paracrine factors. Testosterone, for example, is secreted into the bloodstream but also acts locally in the testes to control spermatogenesis. Insulin-like growth factor I (IGF-I) is a hormone secreted into the bloodstream from the liver and other tissues, but is also a paracrine factor made locally in most tissues to control cell proliferation. Furthermore, one receptor can mediate actions of a hormone, such as parathyroid hormone, and of a paracrine factor, such as parathyroid hormone–related protein. In some cases, the paracrine actions of “hormones” have functions quite unrelated to the hormonal functions. For example, macrophages synthesize the active form of vitamin D (1,25(OH)2vitaminD3), which can then bind to vitamin D receptors in the same cells and stimulate production of antimicrobial peptides.[1] The vitamin D 1α-hydroxylase responsible for activating 25(OH)-vitamin D is synthesized in multiple tissues in which it has functions not apparently related to the calcium homeostatic actions of the 1,25(OH)2vitaminD3 hormone. One can speculate that the hormonal actions of vitamin D might have evolved well after the paracrine vitamin D system provided the raw materials for the hormonal system.

Target cells respond similarly to signals that reach them from the bloodstream (hormones) or from the cell next door (paracrine factors); the cellular response machinery does not distinguish the sites of origin of signals. The shared final common pathways used by hormonal and paracrine signals should not, however, obscure important differences between hormonal and paracrine signaling systems ( Fig. 1-1 ). Paracrine signals do not travel very far; consequently, the specific site of origin of a paracrine factor determines where it will act and provides specificity to that action. When the paracrine factor BMP4 is secreted by cells in the developing kidney, BMP4 regulates the differentiation of renal cells; when the same factor is secreted by cells in bone, it regulates bone formation. Thus, the site of origin of BMP4 determines its physiologic role. In contrast, because hormones are secreted into the bloodstream, their sites of origin are often divorced from their functions. We know nothing about thyroid hormone function, for example, that requires that the thyroid gland be in the neck.
Figure 1-1 Comparison of determinants of endocrine and paracrine signaling.

Because the specificity of paracrine factor action is so dependent on its precise site of origin, elaborate mechanisms have evolved to regulate and constrain the diffusion of paracrine factors. Paracrine factors of the hedgehog family, for example, are covalently bound to cholesterol to constrain the diffusion of these molecules in the extracellular milieu. Most paracrine factors interact with binding proteins that block their action and control their diffusion. Chordin, noggin, and many other distinct proteins all bind to various members of the BMP family to regulate their action, for example. Proteases such as tolloid then destroy the binding proteins at specific sites to liberate BMPs so that they can act on appropriate target cells.

Hormones have rather different constraints. Because they diffuse throughout the body, they must be synthesized in enormous amounts relative to the amounts of paracrine factors needed at specific locations. This synthesis usually occurs in specialized cells designed for that specific purpose. Hormones must then be able to travel in the bloodstream and diffuse in effective concentrations into tissues. Therefore, for example, lipophilic hormones bind to soluble proteins that allow them to travel in the aqueous media of blood at relatively high concentrations. The ability of hormones to diffuse through the extracellular space means that the local concentration of hormone at target sites will rapidly decrease when glandular secretion of the hormone stops. Because hormones diffuse throughout extracellular fluid quickly, hormonal metabolism can occur in specialized organs such as the liver and kidney in a way that determines the effective concentration of the hormones in other tissues.

Thus, paracrine factors and hormones use several distinct strategies to control their biosynthesis, sites of action, transport, and metabolism. These differing strategies probably explain partly why a hormone such as IGF-I, unlike its close relative, insulin, has multiple binding proteins that control its action in tissues. As noted earlier, IGF-I has a double life as both a hormone and a paracrine factor. Presumably, the local actions of IGF-I mandate an elaborate binding protein apparatus.

All the major hormonal signaling programs—G protein–coupled receptors, tyrosine kinase receptors, serine/threonine kinase receptors, ion channels, cytokine receptors, nuclear receptors—are also used by paracrine factors. In contrast, several paracrine signaling programs are used only by paracrine factors and are probably not used by hormones. For example, Notch receptors respond to membrane-based ligands to control cell fate, but no blood-borne ligands use Notch-type signaling (at least, none is currently known). Perhaps the intracellular strategy used by Notch, which involves cleavage of the receptor and subsequent nuclear actions of the receptor’s cytoplasmic portion, is too inflexible to serve the purposes of hormones.

The analyses of the complete genomes of multiple bacterial species, the yeast S. cerevisiae, the fruit fly Drosophila melanogaster, the worm Caenorhabitis elegans, the plant Aradopsis thaliana, humans, and many other species have allowed a comprehensive view of the signaling machinery used by various forms of life. As noted already, S. cerevisiae uses G protein–linked receptors; this organism, however, lacks tyrosine kinase receptors and nuclear receptors that resemble the estrogen/thyroid receptor family. In contrast, the worm and fly share with humans the use of each of these signaling pathways, although with substantial variation in numbers of genes committed to each pathway. For example, the Drosophila genome encodes 20 nuclear receptors, the C. elegans genome encodes 270, and the human genome encodes (tentatively) more than 50. These patterns suggest that ancient multicellular animals must have already established the signaling systems that are the foundation of the endocrine system as we know it in mammals.

Even before the sequencing of the human genome, sequence analyses had made clear that many receptor genes are found in mammalian genomes for which no clear ligand or function was known. The analyses of these “orphan” receptors have succeeded in broadening the current understanding of hormonal signaling. For example, the LXR receptor was one such orphan receptor found when searching for unknown nuclear receptors. Subsequent experiments showed that oxygenated derivatives of cholesterol are the ligands for LXR, which regulates genes involved in cholesterol and fatty acid metabolism.[2] The examples of LXR and many others raise the question of what constitutes a hormone. The classic view of hormones is that they are synthesized in discrete glands and have no function other than activating receptors on cell membranes or in the nucleus. Cholesterol, which is converted in cells to oxygenated derivatives that activate the LXR receptor, in contrast, uses a hormonal strategy to regulate its own metabolism. Other orphan nuclear receptors similarly respond to ligands such as bile acids and fatty acids. These “hormones” have important metabolic roles quite separate from their signaling properties, although the hormone-like signaling serves to allow regulation of the metabolic function. The calcium-sensing receptor is an example from the G protein–linked receptor family of receptors that responds to a nonclassic ligand, ionic calcium. Calcium is released into the bloodstream from bone, kidney, and intestine and acts on the calcium-sensing receptor on parathyroid cells, renal tubular cells, and other cells to coordinate cellular responses to calcium. Thus, many important metabolic factors have taken on hormonal properties as part of a regulatory strategy.

Endocrine Glands

Hormone formation may occur either in localized collections of specific cells, the endocrine glands, or in cells that have additional roles. Many protein hormones, such as growth hormone, parathyroid hormone, prolactin, insulin, and glucagon, are produced in dedicated cells by standard protein synthetic mechanisms common to all cells. These secretory cells usually contain specialized secretory granules designed to store large amounts of hormone and to release the hormones in response to specific signals. Formation of small hormone molecules initiates with commonly found precursors, usually in specific glands such as the adrenals, gonads, or thyroid. In the case of the steroid hormones, the precursor is cholesterol, which is modified by various hydroxylations, methylations, and demethylations to form the glucocorticoids, androgens, estrogens, and their biologically active derivatives. In contrast, the precursor of vitamin D, 7-dehydrocholesterol, is produced in skin keratinocytes, again from cholesterol, by a photochemical reaction. Leptin, which regulates appetite and energy expenditure, is formed in adipocytes, thus providing a specific signal reflecting the nutritional state to the central nervous system.

Thyroid hormone synthesis occurs via a unique pathway. The thyroid cell synthesizes a 660,000-kd homodimer, thyroglobulin, which is then iodinated at specific iodotyrosines. Certain of these “couple” to form the iodothyronine molecule within thyroglobulin, which is then stored in the lumen of the thyroid follicle. In order for this to occur, the thyroid cell must concentrate the trace quantities of iodide from the blood and oxidize it via a specific peroxidase. Release of thyroxine (T4) from the thyroglobulin requires its phagocytosis and cathepsin-catalyzed digestion by the same cells.

Hormones are synthesized in response to biochemical signals generated by various modulating systems. Many of these systems are specific to the effects of the hormone product, for example, parathyroid hormone synthesis is regulated by the concentration of ionized calcium. For others, such as gonadal, adrenal, and thyroid hormones, control of hormone synthesis is achieved by the hormonostatic function of the hypothalamic-pituitary axis. Cells in the hypothalamus and pituitary monitor the circulating hormone concentration and secrete tropic hormones, which activate specific pathways for hormone synthesis and release. Typical examples are luteinizing hormone, follicle-stimulating hormone, thyroid-stimulating hormone, and adrenocorticotropic hormone (LH, FSH, TSH, and ACTH, respectively).

These trophic hormones increase rates of hormone synthesis and secretion, and they may induce target cell division, thus causing enlargement of the various target glands. For example, in hypothyroid individuals living in iodine-deficient areas of the world, TSH secretion causes a marked hyperplasia of thyroid cells. In such regions, the thyroid gland may be 20- to 50-fold times its normal size. Adrenal hyperplasia occurs in patients with genetic deficiencies in cortisol formation. Hypertrophy and hyperplasia of parathyroid cells, in this case initiated by an intrinsic response to the stress of hypocalcemia, occurs in patients with renal insufficiency or calcium malabsorption.

Hormones may be fully active when released into the bloodstream (e.g., growth hormone or insulin), or they may require activation in specific cells to produce their biologic effects. These activation steps are often highly regulated. For example, the T4 released from the thyroid cell is a prohormone that must undergo a specific deiodination to form the active 3,5,3′ triiodothyronine (T3). This deiodination reaction can occur in target tissues, such as in the central nervous system, in the thyrotrophs, where T3 provides feedback regulation of TSH production, or in hepatic and renal cells from which it is released into the circulation for uptake by all tissues. A similar post-secretory activation step catalyzed by a 5α-reductase causes tissue-specific activation of testosterone to dihydrotestosterone in target tissues including the male urogenital tract and genital skin, as well as in liver. Vitamin D undergoes hydroxylation at the 25 position in the liver, and in the 1 position in the kidney. Both hydroxylations must occur to produce the active hormone 1,25(OH)2 vitamin D. The activity of the 1α-hydroxylase, but not the 25-hydroxylase, is stimulated by parathyroid hormone and reduced plasma phosphate but is inhibited by calcium, 1,25(OH)2 vitamin D, and FGF23.

Hormones are synthesized as required on a daily, hourly, or minute-to-minute basis with minimal storage, but there are significant exceptions. One is the thyroid gland, which con-tains enough stored hormone to last for about 2 months. This permits a constant supply of this hormone despite significant variations in the availability of iodine. However, if iodine deficiency is prolonged, the normal reservoirs of thyroxine can be depleted.

The various feedback signaling systems exemplified above provide the hormonal homeostasis characteristic of virtually all endocrine systems. Regulation may include the central nervous system or local signal recognition mechanisms in the glandular cells, such as the calcium-sensing receptor of the parathyroid cell. Superimposed, centrally programmed increases and decreases in hormone secretion or activation through neuroendocrine pathways also occur. Examples include the circadian variation in the secretion of ACTH directing the synthesis and release of cortisol. The monthly menstrual cycle exemplifies a system with much longer periodicity that requires a complex synergism between central and peripheral axes of the endocrine glands. Disruption of hormonal homeostasis due to glandular or central regulatory system dysfunction has both clinical and laboratory consequences. Recognition and correction of these are the essence of clinical endocrinology.

Transport of Hormones in Blood

Protein hormones and some small molecules, such as the catecholamines, are water-soluble and are readily transported via the circulatory system. Others are nearly insoluble in water (e.g., the steroid and thyroid hormones) and their distribution presents special problems. Such molecules are bound to 50- to 60-kd carrier plasma glycoproteins such as thyroxine-binding globulin (TBG), sex hormone-binding globulin (SHBG), and corticosteroid-binding globulin (CBG), as well as to albumin. These ligand-protein complexes serve as reservoirs of these hormones, ensure ubiquitous distribution of their water-insoluble ligands, and protect the small molecules from rapid inactivation or excretion in the urine or bile. Without these proteins, it is unlikely that hydrophobic molecules would be transported much beyond the veins draining the glands in which they are formed. The protein-bound hormones exist in rapid equilibrium with the often minute quantities of hormone in the aqueous plasma. It is this “free” fraction of the circulating hormone that is taken up by the cell. It has been shown, for example, that if tracer thyroid hormone is injected into the portal vein in a protein-free solution, it is bound to hepatocytes at the periphery of the hepatic sinusoid. When the same experiment is repeated with a protein-containing solution, there is a uniform distribution of the tracer hormone throughout the hepatic lobule.[3] Despite the very high affinity of some of the binding proteins for their ligands, one specific protein may not be essential for hormone distribution. For example, in humans with a congenital deficiency of TBG, other proteins, transthyretin (TTR) and albumin, subsume its role. Because the affinity of these secondary thyroid hormone transport proteins is several orders of magnitude lower than that of TBG, it is possible for the hypothalamic-pituitary feedback system to maintain free thyroid hormone in the normal range at a much lower total hormone concentration. The fact that the “free” hormone concentration is normal in subjects with TBG deficiency indicates that it is this free moiety that is defended by the hypothalamic-pituitary axis and is the active hormone.[4]

The availability of gene targeting techniques has allowed specific tests of the physiologic role of several hormone-binding proteins. For example, mice with targeted inactivation of the vitamin D–binding protein (DBP) have been generated.[5] While the absence of DBP markedly reduces the circulating concentration of vitamin D, the mice are otherwise normal. However, they do show enhanced susceptibility to a vitamin D–deficient diet because of the reduced reservoir of this sterol. In addition, the absence of DBP markedly reduces the half-life of 25(OH)D2 by accelerating its hepatic uptake, making the mice less susceptible to vitamin D intoxication.

In rodents, transthyretin (TTR) carries retinol-binding protein and is also the principal thyroid hormone–binding protein. This protein is synthesized in the liver and in choroid plexus. It is the major thyroid hormone–binding protein in the cerebrospinal fluid of both rodents and humans and was thought to perhaps serve an important role in thyroid hormone transport into the central nervous system. This hypothesis has been disproved by the fact that mice without TTR have normal concentrations of T4 in the brain as well as of free T4 in the plasma. [6] [7] To be sure, the serum concentrations of vitamin A and total T4 are decreased, but the knockout mice have no signs of vitamin A deficiency or hypothyroidism. Such studies suggest that these proteins primarily serve distributive and reservoir functions.

Protein hormones and some small ligands (e.g., catecholamines) produce their effects by interacting with cell-surface receptors. Others, such as the steroid and thyroid hormones, must enter the cell to bind to cytosolic or nuclear receptors. In the past, it has been thought that much of the transmembrane transport of hormones was passive. Evidence is now in-hand that there are specific transporters involved in cellular uptake of thyroid hormone.[8] This may be found to be the case for other small ligands as well, revealing yet another mechanism for ensuring the distribution of a hormone to its site of action. Studies in mice missing megalin, a large, cell-surface protein in the LDL receptor family, suggest that estrogen and testosterone, bound to SHBG, uses megalin to enter certain tissues while still bound to SHBG.[9] In this case, therefore, the hormone bound to SHBG, rather than “free” hormone, is the active moiety that enters cells. It is unclear how generally this apparent exception to the “free hormone” hypothesis occurs.

Target Cells as Active Participants

Hormones determine cellular target actions by binding with high specificity to receptor proteins. Whether or not a peripheral cell is hormonally responsive, depends to a large extent on the presence and function of specific and selective hormone receptors. Receptor expression thus determines which cells will respond, as well as the nature of the intracellular effector pathways activated by the hormone signal. Receptor proteins may be localized to the cell membrane, cytoplasm, and nucleus. Broadly, polypeptide hormone receptors are cell membrane–associated, while soluble intracellular proteins selectively bind to steroid hormones ( Fig. 1-2 ). This idea of selective localization has, however, recently been challenged, because related sequences can be found in multiple cellular compartments.
Figure 1-2 Hormonal signaling by cell-surface and intracellular receptors. The receptors for the water-soluble polypeptide hormones, LH, and IGF-I are integral membrane proteins located at the cell surface. They bind the hormone-utilizing extracellular sequences and transduce a signal by the generation of second messengers: cyclic adenosine monophosphate for the LH receptor and tyrosine-phosphorylated substrates for the insulin-like growth factor I receptor. Although effects on gene expression are indicated, direct effects on cellular proteins (e.g., ion channels) are also observed. In contrast, the receptor for the lipophilic steroid hormone progesterone resides in the cell nucleus. It binds the hormone and becomes activated and capable of directly modulating target gene transcription.) R, Receptor molecule; TF, transcription factor. (Reproduced from Mayo K. In Conn PM, Melmed S, eds. Endocrinology: Basic and Clinical Principles. Totowa, NJ: Humana Press, 1997:11.)

Membrane-associated receptor proteins usually consist of extracellular sequences that recognize and bind ligand, transmembrane anchoring hydrophobic sequences, and intracellular sequences, which initiate intracellular signaling. Intracellular signaling is mediated by soluble second messengers (e.g., cyclic adenosine monophosphate) or by activation of intracellular signaling molecules (e.g., STAT proteins). Receptor-dependent activation of heterotrimeric G proteins, comprising α, β, and γ subunits, may either induce or suppress effector enzymes or ion channels.

Several growth factors and hormone receptors (e.g., for insulin) behave as intrinsic tyrosine kinases or activate intracellular protein tyrosine kinases. Ligand activation may cause receptor dimerization (e.g., GH) or heterodimerization (e.g., interleukin-6), followed by activation of intracellular phosphorylation cascades. These activated proteins ultimately determine specific nuclear gene expression.

Both the number of receptors expressed per cell, as well as their responses, are also regulated, thus providing a further level of control for hormone action. Several mechanisms account for altered receptor function. Receptor endocytosis causes internalization of cell-surface receptors; the hormone-receptor complex is subsequently dissociated, resulting in abrogation of the hormone signal. Receptor trafficking may then result in recycling back to the cell-surface (e.g., as for insulin), or the internalized receptor may undergo lysosomal degradation. Both these mechanisms triggered by activation of receptors effectively lead to impaired hormone signaling by down-regulation of these receptors. The hormone signaling pathway may also be down-regulated by receptor desensitization (e.g., as for epinephrine); ligand-mediated receptor phosphorylation leads to a reversible deactivation of the receptor. Desensitization mechanisms can be activated by a receptor’s ligand (homologous desensitization) or by another signal (heterologous desensitization), thereby attenuating receptor signaling in the continued presence of ligand. Receptor function may also be limited by action of specific phosphatases (e.g., SHP) or by intracellular negative regulation of the signaling cascade (e.g., SOCS proteins inhibiting JAK-STAT signaling).

Mutational changes in receptor structure can also determine hormone action. Constitutive receptor activation may be induced by activating mutations (e.g., TSH receptor) leading to endocrine organ hyperfunction, even in the absence of hormone. Conversely, inactivating receptor mutations may lead to endocrine hypofunction (e.g., testosterone or vasopressin receptors). These syndromes are well-characterized and are well-described in this volume ( Fig. 1-3 ).

Figure 1-3 Diseases caused by mutations in G-protein–coupled receptors. All are human conditions with the exception of the final two entries, which refer to the mouse. AD, Autosomal dominant; AR, autosomal recessive inheritance. Loss of function refers to inactivating mutations of the receptor, and gain of function to activating mutations. Abbreviations for G-protein–coupled receptors: ACTH, Adrenocorticotropic hormone; LH, luteinizing hormone; TSH, thyroid-stimulating hormone; PTH-PTHrP, parathyroid hormone and parathyroid hormone–related peptide; MSH, melanocyte-stimulating hormone; GHRH, growth hormone–releasing hormone; FSH, follicle-stimulating hormone. (Reproduced from Mayo K. In Conn PM, Melmed S, eds. Endocrinology: Basic and Clinical Principles. Totowa, NJ: Humana Press, 1997:27.)

The functional diversity of receptor signaling also results in overlapping or redundant intracellular pathways. For example, both GH and cytokines activate JAK-STAT signaling, whereas the distal effects of these stimuli clearly differ. Thus, despite common signaling pathways, hormones elicit highly specific cellular effects. Tissue or cell-type genetic programs or receptor-receptor interactions at the cell surface (e.g., dopamine D2 with SRIF receptor hetero-oligonization) may also confer specific cellular response to a hormone and provide an additive cellular effect.[10]

Control of Hormone Secretion

Anatomically distinct endocrine glands are composed of highly differentiated cells that synthesize, store, and secrete hormones. Circulating hormone concentrations are a function of glandular secretory patterns and hormone clearance rates. Hormone secretion is tightly regulated to attain circulating levels that are most conducive to elicit the appropriate target tissue response. For example, longitudinal bone growth is initiated and maintained by exquisitely regulated levels of circulating GH, while mild GH hypersecretion results in gigantism and GH deficiency causes growth retardation. Ambient circulating hormone concentrations are not uniform, and secretion patterns determine appropriate physiologic function. Thus, insulin secretion occurs in short pulses elicited by nutrient and other signals, gonadotrophin secretion is episodic, determined by a hypothalamic pulse generator, and prolactin secretion appears to be relatively continuous with secretory peaks elicited during suckling.

Hormone secretion also adheres to rhythmic patterns. Circadian rhythms serve as adaptive responses to environmental signals and are controlled by a circadian timing mechanism.[11] Light is the major environmental cue adjusting the endogen-ous clock. The retinohypothalamic tract entrains circadian pulse generators situated within hypothalamic suprachiasmatic nuclei. These signals subserve timing mechanisms for the sleep-wake cycle and determine patterns of hormone secretion and action. Disturbed circadian timing results in hormonal dysfunction, and may also be reflective of entrainment or pulse generator lesions. For example, adult GH deficiency due to a damaged hypothalamus or pituitary is associated with elevations in integrated 24-hour leptin concentrations, decreased leptin pulsatility, and yet preserved circadian rhythm of leptin. GH replacement restores leptin pulsatility, followed by loss of body fat mass.[12] Sleep is also an important cue regulating hormone pulsatility. About 70% of overall GH secretion occurs during slow-wave sleep, and increasing age is associated with declining slow-wave sleep and concomitant decline in GH and elevation of cortisol secretion.[13] Most pituitary hormones are secreted in a circadian (day-night) rhythm, best exemplified by ACTH peaks before 9 am, whereas ovarian steroids follow a 28-day menstrual rhythm. Disrupted episodic rhythms are often a hallmark of endocrine dysfunction. Thus, loss of circadian ACTH secretion with high midnight cortisol levels is a feature of Cushing’s disease.

Hormone secretion is induced by multiple specific biochemical and neural signals. Integration of these stimuli results in the net temporal and quantitative secretion of the hormone ( Fig. 1-4 ). Thus, signals elicited by hypothalamic hormones (GHRH, SRIF), peripheral hormones (IGF-I, sex steroids, thyroid hormone), nutrients, adrenergic pathways, stress, and other neuropeptides, all converge on the somatotroph cell, resulting in the ultimate pattern and quantity of GH secretion. Networks of reciprocal interactions allow for dynamic adaptation and shifts in environmental signals. These regulatory systems embrace the hypothalamic pituitary and target endocrine glands, as well as the adipocyte and lymphocyte. Peripheral inflammation and stress elicit cytokine signals that interface with the neuroendocrine system, resulting in hypothalamic-pituitary axis activation. The parathyroid and pancreatic secreting cells are less tightly controlled by the hypothalamus, but their functions are tightly regulated by the effects they elicit. Thus, parathyroid hormone (PTH) secretion is induced when serum calcium levels fall, and the signal for sustained PTH secretion is abrogated by rising calcium levels.
Figure 1-4 Peripheral feedback mechanism and a million-fold amplifying cascade of hormonal signals. Environmental signals are transmitted to the central nervous system, which innervates the hypothalamus, which responds by secreting nanogram amounts of a specific hormone. Releasing hormones are transported down a closed portal system, pass the blood-brain barrier at either end through fenestrations, and bind to specific anterior pituitary cell membrane receptors to elicit secretion of micrograms of specific anterior pituitary hormones. These enter the venous circulation through fenestrated local capillaries, bind to specific target gland receptors, trigger release of micrograms to milligrams of daily hormone amounts, and elicit responses by binding to receptors in distal target tissues. Peripheral hormone receptors enable widespread cell signaling by a single initiating environmental signal, thus facilitating intimate homeostatic association with the external environment. Arrows with a black dot at their origin indicate a secretory process. (Reproduced from Normal AW, Litwack G. Hormones, ed 2. New York: Academic Press, 1997:14.)
Several tiers of control subserve the ultimate net glandular secretion. First, central nervous system signals including stress, afferent stimuli, and neuropeptides signal the synthesis and secretion of hypothalamic hormones and neuropeptides ( Fig. 1-5 ). Four hypothalamic-releasing hormones (GHRH, CRH, TRH, and GnRH) traverse the hypothalamic portal vessels and impinge upon their respective transmembrane trophic hormone-secreting cell receptors. These distinct cells express GH, ACTH, TSH, and gonadotrophins. In contrast, hypothalamic somatostatin and dopamine suppress GH, PRL, or TSH secretion. Trophic hormones also maintain the structural-functional integrity of endocrine organs, including the thyroid and adrenal glands, and the gonads. Target hormones, in turn, serve as powerful negative feedback regulators of their respective trophic hormone; they often also suppress secretion of hypothalamic-releasing hormones. In certain circumstances (e.g., during puberty), peripheral sex steroids may positively induce the hypothalamic-pituitary-target gland axis. Thus, LH induces ovarian estrogen secretion, which feeds back positively to induce further LH release. Pituitary hormones themselves, in a short feedback loop, may also regulate their own respective hypothalamic-controlling hormone. Hypothalamic-releasing hormones are secreted in nanogram amounts, and have short half-lives of a few minutes. Anterior pituitary hormones are produced in microgram amounts and have longer half-lives, while peripheral hormones can be produced in up to milligram amounts daily, with much longer half-lives.

Figure 1-5 Model for regulation of anterior pituitary hormone secretion by three tiers of control. Hypothalamic hormones impinge directly on their respective target cells. Intrapituitary cytokines and growth factors regulate tropic cell function by paracrine (and autocrine) control. Peripheral hormones exert negative feedback inhibition of respective pituitary trophic hormone synthesis and secretion. (Reproduced from Ray D, Melmed S. Pituitary cytokine and growth factor expression and action. Endocrin Rev 1997;18:206-228.)

A further level of secretion control occurs within the gland itself. Thus, intraglandular paracrine or autocrine growth peptides serve to autoregulate pituitary hormone secretion, as exemplified by EGF control of prolactin or IGF-I control of GH secretion. Molecules within the endocrine cell may also subserve an intracellular feedback loop. Thus, corticotrope SOCS-3 induction by gp130-linked cytokines serves to abrogate the ligand-induced JAK-STAT cascade and block POMC transcription and ACTH secretion. This rapid on-off regulation of ACTH secretion provides a plastic endocrine response to changes in environmental signaling and serves to maintain homeostatic integrity.[14]

In addition to the central-neuroendocrine interface mediated by hypothalamic chemical signal transduction, the central nervous system directly controls several hormonal secretory processes. Posterior pituitary hormone secretion occurs as direct efferent neural extensions. Postganglionic sympathetic nerves also regulate rapid changes in renin, insulin, and glucagon secretion, and preganglionic sympathetic nerves signal to adrenal medullary cells eliciting adrenaline release.

Hormone Measurement

Endocrine function can be assessed by measuring levels of basal circulating hormone, evoked or suppressed hormone, or hormone binding proteins. Alternatively, peripheral hormone receptor function can be assessed. Meaningful strategies for timing hormonal measurements vary from system to system. In some cases, circulating hormone concentrations can be measured in randomly collected serum samples. This measurement, when standardized for fasting, environmental stress, age, and gender is reflective of true hormone concentrations only when levels do not fluctuate appreciably. For example, thyroid hormone, prolactin, and IGF-I levels can be accurately assessed in fasting morning serum samples. On the other hand, when hormone secretion is clearly episodic, timed samples may be required over a defined time course to reflect hormone bioavailability. Thus, early morning and late evening cortisol measurements are most appropriate. Twenty-four–hour sampling for GH measurements, with samples collected every 2, 10, or 20 minutes, are expensive and cumbersome, yet may yield valuable diagnostic information. Random sampling may also reflect secretion peaks or nadirs, thus confounding adequate interpretation of results.

In general, confirmation of failed glandular function is made by attempting to evoke hormone secretion by recognized stimuli. Thus, testing of pituitary hormone reserve may be accomplished by injecting appropriate hypothalamic releasing hormones. Injection of trophic hormones, including TSH and ACTH, evokes specific target gland hormone secretion. Pharmacologic stimuli (e.g., metoclopramide for induction of PRL secretion) may also be useful tests of hormone reserve. In contrast, hormone hypersecretion can be diagnosed by suppressing glandular function. Thus, failure to appropriately suppress GH levels after a standardized glucose load implies inappropriate GH hypersecretion.

Radioimmunoassays utilize highly specific antibodies unique to the hormone, or a hormone fragment, to quantify hormone levels. Enzyme-linked immunosorbent assays (ELISAs) employ enzymes instead of radioactive hormone markers, and enzyme activity is reflective of hormone concentration. This sensitive technique has allowed ultrasensitive measurements of physiologic hormone concentrations. Hormone-specific receptors may be employed in place of the antibody in a radioreceptor assay.

Endocrine Diseases

Endocrine diseases fall into four broad categories: (1) hormone overproduction, (2) hormone underproduction, (3) altered tissue responses to hormones, and (4) tumors of endocrine glands.

Hormone Overproduction

Occasionally, hormones are secreted in increased amounts because of genetic abnormalities that cause abnormal regulation of hormone synthesis or release. For example, in glu-cocorticoid-remediable hyperaldosteronism, an abnormal chromosomal crossing over event puts the aldosterone synthetase gene under the control of the ACTH-regulated 11β-hydroxylase gene. More often, diseases of hormone overproduction are associated with an increase in the total number of hormone-producing cells. For example, the hyperthyroidism of Graves’ disease, in which antibodies mimic TSH and activate the TSH receptors on thyroid cells, is associated with dramatic increase in thyroid cell proliferation, as well as with increased synthesis and release of thyroid hormone from each thyroid cell. In this example, the increase in thyroid cell number represents a polyclonal expansion of thyroid cells, in which large numbers of thyroid cells proliferate in response to an abnormal stimulus. However, most endocrine tumors are not polyclonal expansions, but instead represent monoclonal expansions of one mutated cell. Pituitary and parathyroid tumors, for example, are usually monoclonal expansions in which somatic mutations in multiple tumor suppressor genes and proto-oncogenes occur. These mutations lead to an increase in proliferation and/or survival of the mutant cells. Sometimes, this proliferation is associated with abnormal secretion of hormone from each tumor cell as well. For example, mutant Gs α proteins in somatotrophs can lead to both increased cellular proliferation and increased secretion of growth hormone from each tumor cell.

Hormone Underproduction

Underproduction of hormone can result from a wide variety of processes, ranging from surgical removal of parathyroid glands during neck surgery, to tuberculous destruction of adrenal glands, or to iron deposition in β cells in hemochromatosis. A frequent cause of destruction of hormone-producing cells is autoimmunity. Autoimmune destruction of β cells in type 1 diabetes mellitus or of thyroid cells in Hashimoto’s thyroiditis are two of the most common disorders treated by endocrinologists. More uncommonly, a host of genetic abnormalities can also lead to decreased hormone production. These disorders can result from abnormal development of hormone-producing cells (e.g., hypogonadotrophic hypogonadism caused by KAL gene mutations), from abnormal synthesis of hormones (e.g., deletion of the growth hormone gene), or from abnormal regulation of hormone secretion (e.g., the hypoparathyroidism associated with activating mutations of the parathyroid cell’s calcium-sensing receptor).

Altered Tissue Responses

Resistance to hormones can be caused by a variety of genetic disorders. Examples include mutations in the growth hormone receptor in Laron dwarfism and mutations in the Gs α gene in the hypoparathyroidism of pseudohypoparathyroidism type 1a. The insulin resistance in muscle and liver central to the etiology of type 2 diabetes mellitus appears to be polygenic in origin. Type 2 diabetes is also an example of a disease in which end organ insensitivity is worsened by signals from other organs, in this case by signals originating in fat cells. In other cases, the target organ of hormone action is more directly abnormal, as in the PTH resistance of renal failure.

Increased end organ function can be caused by mutations in signal reception and propagation. For example, activating mutations in TSH, LH, and PTH receptors can cause increased activity of thyroid cells, Leydig cells, and osteoblasts, even in the absence of ligand. Similarly, activating mutations in the Gs α protein can cause precocious puberty, hyperthyroidism, and acromegaly in McCune-Albright syndrome.

Tumors of Endocrine Glands

Tumors of endocrine glands, as noted above, often result in hormone overproduction. Some tumors of endocrine glands produce little if any hormone, but cause disease by causing local compressive symptoms or by metastatic spread. Examples include so-called nonfunctioning pituitary tumors, which are usually benign but can cause a variety of symptoms due to compression on adjacent structures, and thyroid cancer, which can spread throughout the body without causing hyperthyroidism.

Therapeutic Strategies

In general, hormones are employed pharmacologically for both their replacement or suppressive effects. Hormones may also be utilized for diagnostic stimulatory effects (e.g., hypothalamic hormones) to evoke target organ responses, or to diagnose endocrine hyperfunction by suppressing hormone hypersecretion (e.g., T3). Ablation of endocrine gland function due to genetic or acquired causes can be restored by hormone replacement therapy. In general, steroid and thyroid hormones are replaced orally, whereas peptide hormones (e.g., insulin, growth hormone) require injection. Gastrointestinal absorption and first pass kinetics determine oral hormone dosage and availability. Physiologic replacement can achieve both appropriate hormone levels (e.g., thyroid) as well as approximate hormone secretory patterns (e.g., GnRH delivered intermittently via a pump). Hormones can also be used to treat diseases associated with glandular hyperfunction. Long-acting depot preparations of somatostatin analogues suppress GH hypersecretion in acromegaly or 5-HIAA hypersecretion in carcinoid syndrome. Estrogen receptor antagonists (e.g., tamoxifen) are useful for some patients with breast cancer, and GnRH analogues may down-regulate the gonadotrophin axis and benefit patients with prostate cancer.

Novel formulations of receptor-specific hormone ligands are now being clinically developed (e.g., estrogen agonists/antagonists, somatostatin receptor subtype ligands), resulting in more selective therapeutic targeting. Modes of hormone injection (e.g., for PTH) may also determine therapeutic specificity and efficacy. Improved hormone delivery systems, including computerized minipumps, intranasal sprays (e.g., for DDAVP), pulmonary inhalations, and depot intramuscular injections, will also allow added patient compliance and ease of administration. Insulin delivered by inhalation has already been approved for use, and inhaled growth hormone is under investigation, for example.

Despite this tremendous progress, some therapies, such as insulin delivery to rigorously control blood sugar, still re-quire tremendous patient involvement and await innovative approaches. Hormones are biologically powerful molecules that exert therapeutic benefit and effectively replace pathologic deficits. They should not be prescribed without clear-cut indications and should not be administered without careful evaluation by an appropriately qualified medical practitioner.

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