Search

Introduction by Editor David Julius In the second century ad, Galen of Pergamon was the first to describe the anatomical connection between the hypothalamus and the pituitary gland. In the 1800s, von Luska, Cajal, and others showed that this connecting structure, or stalk, contains nerve fibers as well as a plexus of capillary vessels. Such observations raised long-standing questions as to the functional nature of these connections and their relationship to neural and/or humoral communication between these two endocrine organs. Hypothalamic nerve fibers projecting to the posterior pituitary were later shown to account for the release of vasopressin and oxytocin from vesicle-rich terminals within this lobe. Up until the mid-1900s, however, it remained unclear as to how the hypothalamus controls the release of corticotropin, thyrotropin, growth hormone, and other key endocrine factors from the anterior lobe. Evidence for direct neural control was lacking, and in the 1940s the British physiologist Geoffrey Harris proposed that the hypothalamus exerts control over the anterior lobe through a humoral mechanism in which blood-borne releasing factors are delivered to the pituitary through capillaries of the interconnecting stalk (the so-called hypothalamo-hypophysial portal system), inducing cells in the anterior lobe to secrete their hormones. This model was not without controversy, and skeptics would not be satisfied until the mythical hypothalamic releasing factors had been biochemically and functionally identified. In 1977, Roger Guillemin and Andrew Schally shared the Nobel Prize in Medicine and Physiology for accomplishing this daunting and far-reaching goal. In doing so, they established the field of neuroendocrinology. In this Perspective, Dr. Guillemin, Distinguished Professor at the Salk Institute, recalls events that diverted him from a career as a country doctor in his native France to one of scientific research, first in Canada and then in the United States and France. The story of the releasing factors is legendary, not only for its scientific import, but also for the great technical challenges that beset these efforts when the advantages of protein microsequencing, molecular cloning, and other such powerful techniques were not yet available. Rather, a scale and persistence of experimentation almost unthinkable (and likely unfundable) by today's standards proved successful. Consequently, key steps in hypothalamic-pituitary signaling were elucidated, and new pharmacological strategies for treating endocrine disorders, including those affecting stature and reproduction, were realized. Following on a recent Annual Review of Physiology (ARP) advent, this Perspective takes the form of an oral history that can be read here in transcript form and viewed online. Dr. Guillemin was interviewed on August 7, 2012, at his ranch in the beautiful hills of Truchas, New Mexico (see Figure 1 ), by Dr. Greg Lemke, Françoise Gilot–Salk Chair at the Salk Institute. We are most grateful to Roger Guillemin and to his wife, Lucienne, for opening their home to ARP for this interview, as well as to Greg Lemke for guiding this lively and fascinating conversation.

Neuroendocrinology had a double origin in the 1940’s and 1950’s with the morphological recognition of neurosecretory cells from invertebrates to the hypothalamus of all mammals, and with demonstration of physiological controls by these neurosecretory cells of various functions including those of both lobes of the pituitary. The characterization of the molecules involved in these relationships, which turned out to be peptides, became a major undertaking with the methodology then available.

The isolation and characterization of TRF (TRH) in 1968 made the hypothesis of the existence of hypothalamic releasing factors into a scientific fact, requiring de facto, the search for other releasing factors involved in controlling the secretion of all (anterior) pituitary hormones. Indeed, LRF (LHRH, GnRH) stimulating the secretion of LH and FSH was characterized in 1970. A releasing factor for growth hormone (GH), postulated since 1960, was next, notwithstanding an early report by Schally’s group, which turned out to be an unfortunate artifact. We then set up a highly sensitive and specific radioimmunoassay for rat growth hormone (Paul Brazeau, a new post-doc in the lab) and started adding crude extracts of sheep hypothalamus to anterior pituitary cells maintained in vitro with the elegant monolayer culture method recently developed by Wylie Vale. To our surprise, in repeated experiments we kept observing an inhibition of the secretion of growth hormone, linearly related to the amounts of hypothalamic extracts added (≥0.001 of a hypothalamic fragment). The results were so consistent that we eventually eliminated the possibility (indeed first considered) of an artifact. There had been, actually, an earlier report (Krulich et al) claiming inhibition or stimulation of growth hormone release by crude extracts of different parts of the hypothalamus, but with borderline statistical significance. So, we decided to isolate and characterize whatever in these crude extracts was inhibiting the secretion of growth hormone (while at the same time, these same crude extracts could be shown to stimulate release of TSH and LH). With our radioimmunoassay for rat growth hormone, we tested aliquots of the whole effluent still available from the isolation of LRF and rapidly identified a zone well separated from LRF with the GH-release inhibiting activity. Several steps of purification later, Roger Burgus, who was in charge of this whole isolation procedure, isolated a single compound accounting for all the GH-release inhibiting activity of the crude extract, showed it to be composed of 14 residues, sequenced it by manual Edman degradation (1 residue per day), Nicholas Ling confirming the sequence of the fragments by mass spectrometry, got a molecular structure that Jean Rivier synthesized by Merrifield method, Wylie Vale and Paul Brazeau showing the biological activity of the synthetic molecule in vitro and in vivo (in the rat). The paper was written, sent to Science on September 2, 1972, accepted on October 20, 1972, and published on January 5, 1973 (v. 179, pp 77–79). There is when I suggested the name somatostatin, preferred over the acronyms SRIF (for Somatotropin-Release-Inhibiting Factor) or GHRIF !. As reported in the Science paper we showed with Sam Yen that our synthetic peptide was highly active in lowering plasma GH-levels in a few acromegalic patients. As a part of my ethics in the lab, we started sending mgs of our synthetic peptide to any colleague asking for some, the only condition being would they please let us know what they observed with it in their own studies. One day, we received a couple of calls from Charlie Gale in Seattle who said that in their studies in baboons they regularly observed the peptide to lower plasma levels of GH but also of glucagon and insulin. We never saw such effects in rats but Sam Yen confirmed the results of the Seattle group in going back to the blood samples of the original patients mentioned earlier. Knowing the plasma half-life and barely detectable levels of the polypeptide in peripheral-rat blood, it occurred to me that perhaps some local source in the pancreas might be responsible, my original idea being possibly the nerve endings of the vagus nerve, since I had just received from Peter Petrusz stunning images of somatostatin-immunoreactive neurons in parts of the brain other than the hypothalamus. The surprise came when Maurice Dubois of the INRA (Institut National de la Recherche Agronomique) in Nouzilly with some antibodies I had sent him and Rolf Luft and Tomas Hokfelt in Stockholm independently showed immunoreactive somatostatin in the delta-cells of the endocrine pancreas, delta-cells for which no activity was known to that day. Then came literally a flood of papers from the group around Reginald Hall, Mike Besser in London on the presence and effects of somatostatin in the upper GI tract, etc. Meanwhile, Jean Rivier had synthesized a large number of analogs, bioassayed by Paul Brazeau and Wylie Vale showing minimal active sequences of the original tetradecapeptide as well as some with relative specificity for the secretion of one of the original three targets. Those were the beginnings. Then things developed with industry (octreotide, lanreotide), molecular biology, the several forms of the precursors, the multiple receptors etc. and all that is to be read in that special issue of the Journal. A wonderful feeling. Cortistatin, which is developing remarkably well, came as a surprise in 1995, from the group around Luis de Lecea, then at The Scripps Research Institute, - next door to the Salk Institute. I met Luis de Lecea for the first time at a meeting on sleep, last February (2007)!

Originally searched for and eventually isolated as factors of hypothalamic origin controlling anterior pituitary secretions, these hypophysiotropic peptides are now a chapter of physiology and medical endocrinology of their own. Defying the concept of ‘neuropeptides’ they and their receptors are now known to be ubiquitous and to have subtle as well as profound effects on a large number of functions of both soma and psyche. This review will be composed of brief essays on current knowledge of each of the original ‘hypothalamic hormones’, TRH, GnRH, somatostatin, GHRH and corticotropin releasing hormone and will close on possible and probable futures.

The first proposal for the existence of a growth hormone releasing substance of hypothalamic origin was probably made by Seymour Reichlin in 1959. Reichlin had shown that rats made obese by some (rather extensive) hypothalamic stereotaxic lesions had shorter long bones than controls and that these bones had narrower epiphyseal cartilages. It was to take 23 years eventually to validate the concept by the isolation, amino acid sequencing and reproduction by total synthesis of the hypothalamic growth hormone releasing factor (GHRF).

A short historical review is presented of the events leading to the discovery of somatostatin, an inhibitor of the secretion of growth hormone, while we were engaged in the search of a releasing factor for growth hormone. Also, of the early days of the recognition of the unexpected biological activity of somatostatin in inhibiting the secretions of insulin and glucagon and its eventual localization in the delta cells of the pancreatic islets.

Summary While showing consistent and easily reproducible results as observed in vitro or in acute in vivo systems, several N α -acylated-des-[Ala 1 -Gly 2 ]-dihydrosomatostatin analogs exhibit variable protracted inhibition of the growth hormone release induced in rats by pentobarbital. These results may reflect variable central nervous system responses to pentobarbital as well as to the somatostatin analogs.

Well-characterized antisera to porcine beta-endorphin were used to localize immunoreactive sites in cryostat sections of formaldehyde-fixed rat brain by indirect immunohistochemistry. Specificity was established by absorption of immune sera with synthetic peptide fragments. Specific immunoreactivity was localized to neuronal perikarya in the basal tuberal hypothalamus, and to varicose nerve fibers which were distributed to midline nuclear areas throughout the diencephalon and anterior pons. These patterns of reactivity were unaffected by preabsorption of the immune sera with millimolar concentrations of Met5- or Leu5-enkephalin or alpha-endorphin. The beta-endorphin immunoreactive structures were morphologically separate from those cells and fibers reported to react with antisera to the enkephalins. One anti-beta-endorphin serum gave additional immunoreactivity with myelinated axons in limbic cortical zones; when absorbed with purified rat myelin basic protein, only the specific patterns of immunoreactivity remained. Thus, discrete beta-endorphin-containing neuronal circuits exist in rat brain and are anatomically distinguishable from enkephalin-containing nerve cell and fiber pathways.

Fragments of the pituitary adenoma of a patient with Cushing's disease were maintained in defined culture medium. Immunoreactive (IR) ACTH, IR alpha-MSH, IR beta-lipotropin (beta-LPH), IR beta-endorphin, and IR gamma-MSHs secreted from the adenoma were studied with gel permeation chromatography and the respective RIAs. The adenoma secreted roughly equimolar quantities of IR beta-LPH plus IR beta-endorphin, IR gamma 3-MSHs, and IR ACTHs. It also secreted IR alpha-MSH as well as IR gamma 1-MSH, although in a much lower concentration than the above four peptides. The secreted gamma 3-MSH-like peptides were found to be glycosylated. The secretion pattern suggests that this particular adenoma processes the pro-opiomelanocortin molecule in pathways which encompass those of both the pars distalis and the pars intermedia.

Human hypothalamic growth hormone-releasing factor (GRF) was purified by gel filtration and reverse-phase HPLC. Bioassay and two radioimmunoassays of different specificity revealed the presence of two major forms of GRF-activity which coelute with human pancreas GRFs, hpGRF-44-NH2 and hpGRF-40 previously characterized in pancreas tumors. The bioactive material coeluting with hpGRF-44-NH2 is recognized by two antibodies which are directed against the amidated COOH-terminal sequence and the central portion of the GRF-44 peptide. The bioactive GRF which coelutes with hpGRF-40 reacts only with the antibody directed against the central portion of hpGRF. These data strongly suggest that the human hypothalamus contains the same major forms of GRF that were identified in pancreas tumors responsible for acromegaly in the absence of a pituitary tumor.

The effects of various substances on the secretion of β-endorphin-like immunoactivities (β- END-LI) or ACTH-like immunoactivities (ACTH-LI) by primary anterior pituitary cell cultures have been studied. Culture medium levels of /J-END-LI and ACTH-LI were estimated by RIAs directed against the LeuH-His27 region of β-endorphin and the N-terminal region of ACTH (ACTH antiserum courtesy of D. Orth). General secretagogues, such as 8Br-AMP, 3-isobutyl methyl xanthine, and elevated medium [K+], stimulate the secretion of both /3-END-LI and ACTH-LI. More specific ACTH secretagogues, such as norepinephrine, vasopressin, and a highly purified preparation of ovine hypothalamic corticotropin-releasing factor (HYPCRF), increase the secretory rates of 8-END-LI as wellas ACTH-LI. The glucocorticoid, dexamethasone-21-PO4, progesterone; and prostaglandin, PGE2, inhibit theCRF-mediated secretion of 0-END-LI and ACTH-LI. Multiple peaks of 0-END-LI and ACTH-LI were resolved by gel filtration of cu ture fluids on Biogel P-60 ...

Compared to littermate controls (C57BL/6J ob/?), body weights of genetically obese (ob/ob) mice are significantly higher at 1-6 months of age; the greatest percentage weight gain of the ob/ob group occurs during the first 3 months of life. Levels of pituitary immunoreactive beta-endorphin and immunoreactive alpha-melanocyte-stimulating hormone are also significantly elevated in ob/ob animals compared to controls. However, these pharmacological differences only emerge at 4-6 months of age--3 months after the appearance of obesity. High levels of immunoreactive endorphin in the pituitary are, therefore, more likely to be a consequence than a cause of obesity. Furthermore, numerous other neurologic abnormalities, which may or may not play a role in the obesity syndrome, are evident in ob/ob mice. Compared to controls, ob/ob total brain, hypothalamus, and pituitary weights are 11%, 16%, and 23% less, respectively. Levels of immunoreactive Leu5-enkephalin in pars nervous are also 200% higher in ob/ob mice; this increase is apparent at 1-6 months of age and is highly correlated with changes in body weight.

A Mr 35,000 protein with follicle-stimulating hormone release-inhibitory activity was isolated from porcine ovarian follicular fluid by heparin-Sepharose affinity chromatography, gel filtration on Sephacryl S-200, and multiple steps of high-performance liquid chromatography. The isolated molecule is highly enriched in cysteines and is composed of a single polypeptide chain. In addition, it has no sequence homology with the previously characterized follicular fluid inhibins, which are heterodimeric proteins of Mr 32,000 with follicle-stimulating hormone release-inhibiting activity. This protein specifically inhibits the basal secretion of follicle-stimulating hormone, but not that of luteinizing hormone, in the rat anterior pituitary monolayer culture system with a half-maximal effective dose of 2.5-6.0 ng/ml. Another form of the molecule of Mr 32,000 present in much lower concentration in follicular fluid was also isolated. It may differ from the Mr 35,000 form in glycosylation or carboxyl-terminal truncation. We suggest that this compound be called "follistatin" to signify its structural difference from inhibin.

The synthetic replicate of a 44 amino acid peptide isolated from a human pancreatic tumor which had caused acromegaly possesses high specific activity to release growth hormone (GH) in anesthetized male rats. The GH secretion induced by this peptide is dose-dependent from 50 ng to 1 μg, with plasma GH concentrations increasing more than 10-fold within 5 min of iv administration at the higher doses. Two enzymatic degradation products of the 44 residue peptide were also isolated and consist of the first 37 and 40 amino acids. All three peptides appear to possess similar potency, on a molar basis, invivo, contrary to invitro results. The specificity of these peptides on GH release was shown by their failure to alter plasma concentrations of prolactin (PRL), thyroid-stimulating hormone (TSH), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and corticosterone. Based on these invivo results, the three peptides with serve as powerful tools with which to investigate the mechanisms of GH secretion.

The brain levels of β-endorphin, α-endorphin and enkephalin were measured by radioimmunoassay after different methods of sacrifice. Microwave irradiation proved not to be better than decapitation followed by boiling of the intact tissue, the latter procedure giving values of β-endorphin 10 fold higher than decapitation alone. Concurrently when decapitation was followed by boiling, α-endorphin was no longer detected. Evaluation in brain tissue of several extraction media--phosphate buffered saline, 5% TCA, HCl methanol, and 1N HOAc--showed the last to be the most satisfactory for both β-endorphin and enkephalin. Since β-endorphin was found to be readily hydrolized by brain homogenates with consequent appearance of α-endorphin, these results indicate that disruption of tissue modifies the content of opioid peptides in brain.

Fibroblast growth factors (FGFs) are potent mitogens for vascular and capillary endothelial cells in vitro and can stimulate the formation of blood capillaries (angiogenesis) in vivo. A crucial event in this process is the invasion of the perivascular extracellular matrix by sprouting endothelial cells. Using a recently developed in vitro model of angiogenesis, we show here that highly purified basic pituitary FGF can induce capillary endothelial cells to invade a three-dimensional collagen matrix and to organize themselves to form characteristic tubules that resemble blood capillaries. We also show that basic FGF concomitantly stimulates endothelial cells to produce a urokinase-type plasminogen activator, a protease that has been implicated in the neovascular response. The results demonstrate that basic FGF can stimulate processes that are characteristic of angiogenesis in vivo, including endothelial cell migration, invasion, and production of plasminogen activator.

The production of inhibin in cultured granulosa cells from immature hypophysectomized, estrogen-treated rats and Sertoli cells from normal animals was determined by a specific radioimmunoassay using an antiserum against a synthetic replicate of [Tyr30]inhibin alpha-chain-(1-30). The amount of immunoreactive inhibin detected in the spent media of these cells is in proportion to the density of cells plated and the concentration of exogenously added follicle-stimulating hormone (FSH). In the presence of the estrogen precursor androstenedione (10(-7) M), FSH, but not luteinizing hormone, produced a dose-dependent increase in inhibin during 2-day culture of granulosa cells. In the absence of the estrogen precursor, similar but somewhat diminished inhibin production in responding to FSH was observed. Exogenously added estrogen potentiated the FSH-mediated release of inhibin in the absence of androstenedione. Neither androstenedione nor estradiol added to the cultured Sertoli cells had effect on inhibin production. A preparation of pure inhibin isolated on the basis of an in vitro bioassay and characterized chemically specifically suppressed serum FSH but not luteinizing hormone, when it was injected (24 micrograms per injection, two injections) into acutely ovariectomized rats. Thus, inhibin secreted by the granulosa and Sertoli cells specifically suppresses the secretion of pituitary FSH, and in turn FSH is primarily responsible for the inhibin production in these gonadal cells, as in a classical negative-feedback relationship.

Purified (rat) hypothalamic growth hormone releasing factor (GRF), native human GRF isolated from an islet cell tumor of the pancreas that had caused acromegaly, and the synthetic replicates of the human material are potent secretagogues of immunoreactive growth hormone (GH) by primary cultures of rat pituitary cells. Native or synthetic peptides give identical dose-response curves, with identical slopes and identical maximal effects. The median effective dose of the tumor-derived GRF is 15 x 10(-12) M. The effect of hypothalamic GRF or of a synthetic replicate of tumor-derived GRF is immediate, being demonstrable in less than or equal to 30 sec after contact in a pituitary cell perifusion system. The effect of hypothalamic GRF or of tumor-derived GRF is highly specific for stimulating release of immunoreactive growth hormone; there is no demonstrable concomitant effect on the secretion of other pituitary hormones. Somatostatin-28 and somatostatin-14 inhibit the release of growth hormone produced by hypothalamic GRF or tumor-derived GRF in typical noncompetitive antagonism. On the basis of the results reported here, hypothalamic GRF and tumor-derived GRF are qualitatively indistinguishable in their ability to stimulate the secretion of immunoreactive growth hormone in vitro. The name "somatocrinin" is proposed to replace the acronym GRF.

Peptides with high intrinsic activity to release growth hormone from pituitary cells in tissue cultures were isolated from two different human pancreatic tumors that had caused acromegaly. Homogeneous peptides were obtained after gel filtration and two steps of reverse-phase high-performance liquid chromatography. From one tumor a 44-residue peptide (human pancreas growth hormone releasing factor, hpGRF-44) was isolated, together with two shorter fragments of reduced bioactivity having 40 and 37 amino acid residues (hpGRF-40, hpGRF-37). In contrast, the other tumor contained only one form of GRF which proved to be identical to hpGRF-40. These hpGRFs are indistinguishable from partially purified preparations of hypothalamic growth hormone releasing factor of human, porcine and murine origins with respect to biological activity and are very similar in their physicochemical properties (molecular weight, retention behavior on reverse-phase HPLC, absence of sulfhydryl groups). One of the pancreatic tumors also contained two forms of immunoreactive somatostatin. One form, after isolation and partial microsequencing, was identified as somatostatin-14 with a structure identical to that of the peptide found in other species. The second form has tentatively been identified as somatostatin-28 on the basis of chromatographic behavior.

The release of GH induced by purified hypothalamic GRF or native or synthetic tumor-derived GRF is antagonized by the presence of CoCl2; it is simulated by 8Br .cAMP, IBMX, cholera toxin, forskolin, with identical maximal effects (Emax). Somatocrinin (GRF) stimulates the efflux of cAMP by the pituitary cells in parallel to the release of GH. Addition of either 8Br .cAMP, IBMX, cholera toxin or forskolin to a maximally stimulating dose of GRF does not increase the response which remains GRF-Emax. In contradistinction with these results PGE2 releases GH with a dose-response curve different from that of GRF, and the combination of PGE2 + GRF produces an Emax far greater than that due to either agonist alone; showing a true additivity. The name somatocrinin is proposed to replace the acronym GRF.

In view of striking similarities between TGF-beta and inhibin, we investigated the possibility that TGF-beta might modulate pituitary hormone release in vitro. Long term incubations of beta transforming growth factor (TGF-beta) with rat anterior pituitary cells for 48 hr stimulates the basal secretion of FSH in a dose-dependent manner. The secretion of LH, TSH, GH, ACTH and PRL is not modified by TGF-beta. The minimal effective concentration of TGF-beta is 10 pg/ml (less than 500 attomolar) and is dose dependent over a range from 1 pg to 10 ng/ml. Treatment of cells with TGF-beta for short incubation times (4 hr) in assays similar to that used for hypophysial releasing factors is not effective, indicating that TGF-beta acts through a cellular mechanism distinct from that of LRF. Inhibin-A, recently characterized on the basis of its capacity to specifically inhibit the secretion of FSH in the 48 hr bioassay system inhibits the stimulatory effect of TGF-beta on FSH-release. Analyses of the dose response curves indicate that the interaction occurs in a typical non-competitive manner. The results suggest that a TGF-beta-like molecule, present in follicular fluid, may be responsible for the FSH-releasing activity ("anti-inhibin" activity) observed by us and others during the process of isolating inhibin from follicular fluids. They also suggest an important role for inhibin and the TGF-beta related molecules in modulating pituitary gonadotropin release.

The synthesis by solid-phase methodology of two glycosylated analogs of somatostatin [Glc-Asn 5 ]-SS and [NAcGlc-Asn 5 ]-SS is described. These two analogs have been biologically tested on the secretion of pituitary growth hormone, pancreatic glucagon and insulin. The results show that glycosylation of somatostatin on the Asn 5 residue decreases by a hundred fold the inhibition activity on GH release when tested in vitro . In vivo , since the activity is similar to somatostatin the carbohydrates are probably removed by some enzymatic reaction and thus liberate the full activity of somatostatin.

Immunoreactive-calcitonin was observed in all cells of the intermediate lobe of the rat pituitary gland. It may thus be a fragment of the ≥31, 000 daltons precursor molecule of adrenocorticotropin and β-lipotropin.

A radioimmunoassay (RIA) was developed to measure fibroblast growth factor (FGF) using antiserum generated against a synthetic replicate of [Tyr 10 ]FGF(1–10). The antisera, previously shown to be capable of inhibiting the biological action of FGF on bovine aortic arch endothelial cells in vitro [1], are highly specific for the amino-terminus of FGF. In the RIA, the antisera recognize the decapeptide antigen [Tyr 10 ]FGF(1–10) and the intact mitogen on an equimolar basis and show less than 0.01% cross-reactivity with N -acetyl-[Tyr 10 ]FGF(1–10). Bovine adenohypophysial cells maintained in primary monolayer culture release and ir-FGF which is indistinguishable from the intact mitogen in as much as it is retained on heparin-Sepharose affinity columns and shows a dose-dependent and parallel displacement in RIA. The release of ir-FGF by the bovine adenohypophysis can be increased with forskolin (10 −5 M) or KCl (50 mM). Preincubation of pituitary cells with 17β-estradiol has no measurable effects on basal ir-FGF, but increases the release after KCl treatment 2–3-fold. These results show that ir-FGF can be released by the bovine adenohypophysis in vitro and lend credence to the hypothesis that FGF plays a physiological role in the homeostatic mechanisms regulating mesoderm-derived cell growth.

The action of cysteamine on anterior pituitary hormone secretion was studied in vivo using conscious, freely moving male rats and in vitro using anterior pituitary cells in monolayer culture. Administration of 500 μg cysteamine into the lateral cerebral ventricles of normal rats caused the complete inhibition of pulsatile GH secretion for a minimum of 6 h. This treatment also significantly decreased plasma concentrations of LH for at least 6 h in orchiectomized rats, TSH in short-term (0.5 month) thyroidectomized rats, and PRL in long-term (6 months) thyroidectomized rats. The in vivo stimulation of GH, LH, TSH and PRL with their respective releasing hormones 60 min after administration of cysteamine was not different from the response observed in rats pretreated with saline except for PRL where cysteamine pretreatment significantly inhibited the expected PRL increase. In vitro, 1 mM cysteamine decreased basal and TRH stimulated PRL release while not affecting basal or stimulated GH, LH, TSH and ACTH secretion. These data demonstrate the dramatic and wide-ranging effects of cysteamine on anterior pituitary hormone secretion. This action appears to be mediated through hypothalamic pathways for GH, LH and TSH and through a pituitary pathway for PRL.

beta-Endorphin and enkephalin in extracts of whole brain, various brain regions, adenohypophysis, and combined pars intermedia and neurohypophysis of the rat were measured by radioimmunoassay. In brain extracts, the immunoreactive substances were further separated according to molecular size by gel filtration. beta-Endorphin was found in the diencephalon but not in the hippocampus, cerebral cortex, cerebellum, and striatum. Enkephalin was found predominantly in the striatum and diencephalon. Attention is called to possible artifactual interference by myelin basic protein in the immunoassays for beta-endorphin in some regions of the brain. In the pituitary, enkephalin was mainly restricted to the pars intermedia-neurohypophysis. Neither adrenalectomy nor hypophysectomy significantly altered levels of beta-endorphin in brain extracts. Adrenalectomy increased the levels of beta-endorphin in adenohypophysis and pars intermedia-neurohypophysis; after adrenalectomy, enkephalin was also increased in the adenohypophysis but less so in the pars intermedia-neurohypophysis. These results show that brain endorphin levels are independent of pituitary endorphin levels; they suggest that beta-endorphin-containing neurons and those containing enkephalin constitute two separate groups of brain cells.

(1) α-, β-, γ-endorphins have primary structures respectively identical to those of β-lipotropin subunits [61–76], [61–91], [61–77]. (2) These peptides exhibit opiate-like activity in several assay systems. (3) A series of analogs of the naturally occurring peptides has been prepared and their biologic activities compared. (4) Each of the endorphins produces a discrete set of profound behavioral changes when injected into the ventricular system of the brain of rats. (5) The hypothesis is advanced that alterations in the homeostasis of the β-lipotropinendorphin system may be causally involved in the mental diseases of man.

Somatostatin, a tetradecapeptide isolated from ovine hypothalamic extracts on the basis of its ability to inhibit the spontaneous secretion of growth hormone (GH) by pituitary cell cultures, has been found to inhibit the stimulated secretion of thyrotropin (TSH) mediated by TRF(pGlu-His- Pro-NH2), 10 × [K†], or theophylline in vitro, while having no effect on the secretion of luteinizing hormone (LH) due to LRF. The spontaneous release of PRL in vitro is also inhibited by somatostatin but to a lesser extent than is the spontaneous secretion of GH. In vivo, the TRF-triggered secretion of TSH but not of PRL is suppressed by somatostatin in the estrogen-progesteronepretreated male rat. The injection of TRF leads to a greater rise in both plasma TSH and PRL in estrogen-progesterone-pretreated male rats than in untreated male rats. Somatostatin acts rapidly but reversibly to inhibit the secretion of TSH due to TRF in a dose-dependent manner. Thyroid hormones and somatostatin exhibit summation in their inhibiti...

Evidence that the endorphins, metabolites of the pituitary hormone beta-lipotropin, profoundly affect mood and behavior, are secreted with ACTH in response to stress, and may act as neurotransmitters in the GI tract, suggests that they play an important role in CNS homeostasis. The fact that these peptides are synthesized in the GI tract as well as the brain casts new light on CNS-endocrine-environmental interrelationships.

Somatocrinin, a 44 amino acid peptide with potent growth hormone (GH) releasing activity in anesthetized rats, was tested in conscious freely-moving rats. When high doses of 1 to 10 μg were administered (iv) at random times between spontaneous GH pulses, the responses were inconsistent. When similar doses were tested under identical conditions but in rats pretreated with antibodies against somatostatin, all animals demonstrated a marked and immediate increase in plasma GH of 5 to 10 fold. Similarly, a 1 μg dose of somatocrinin was also ineffective in increasing plasma GH when administered to rats subjected to a 72 h fast, a paradigm known to enhance endogenous somatostatin secretion. However, plasma GH increased over 20 fold if rats were pretreated with antibodies against somatostatin. These results demonstrate the dynamic and opposite roles exerted by somatocrinin and somatostatin in regulating GH secretion.

Fractionation of monkey pituitary glands gave an oxytocin fraction in low yield which showed a counter-current distribution coefficient equivalent to that obtained with oxytocin from other species. Fractionation and chromatography of monkey vasopressin on carboxymethyl cellulose gave arginine-vasopressin of 60% purity, based on amino acid analysis and specific activity. Counter-current distribution on a small scale gave arginine-vasopressin of 89% purity. Reports by others that monkey pituitary glands contain arginine-vasopressin, based on pharmacological activities, are substantiated by the chemical data presented here.

The primary structure of ovine hypothalamic hypophysiotropic luteinizing hormone-releasing factor, LRF, has been established as pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH 2 by hydrolysis of the peptide with chymotrypsin or pyrrolidone-carboxylylpeptidase and by analysis of the products by an Edman-dansylation sequencing technique, as well as by mass spectrometry of the derived phenylthiohydantoins. A decapeptide with the proposed primary structure, prepared by total synthesis, gave the same result on sequencing. The synthetic decapeptide possesses the same biological activities as the native ovine LRF. The amino-acid sequence of ovine LRF is identical to that already published for porcine LRF.

Synthetic LRF (single IV 150 μg dose) elicited a prompt increase in circulating LH in all 7 male subjects tested. The mean maximal rise to greater than 8-fold basal levels was reached within 20–30 min. A concomitant but quantitatively smaller FSH release was observed. Variability in response between individuals was not related to body surface area or age.

The ovarian ascorbic acid depletion method for measuring LH activity has been studied on large series of rats from several strains. In agreement with the reports by Parlow and others, the method was found to give acceptable results using animals of Holtzman strain. The method, however, gives erratic results when using Wistar rats, apparently due to inadequate response of the ovaries of these animals when using Parlow's priming method. In Wistar rats, changing the priming procedure to 2 injections of 50 IU PMS each, 48 hr apart, followed 50 hr later by one injection of 25 IU HCG, yields, 6–9 days later, animals with ovarian weights and ascorbic acid contents of the order of those observed when using Sprague-Dawley or Holtzman rats. The sensitivity to LH (0.2 μg, LH, NIH-S1) of Wistar animals so prepared is still considerably below that reported by Parlow using Holtzman rats (0.04 μg, LH, NIH-S1). In all cases, but particularly so when one uses animals of the Wistar strain, adjustment of the ascorbic acid c...

An α-MSH-like peptide has been isolated from hog pituitary glands. This peptide, designated α2-CRF, has CRF activity, identical melanophoretic activity with α-MSH, but a lower inherent adrenocorticotropic activity than α-MSH. Natural or synthetic α-MSH is inactive CRF-wise at equivalent dry weight doses. α2-CRF has an identical amino acid sequence with that of α-MSH, but a discrete structural change at the N-terminus, as compared with α-MSH, may be responsible for the appearance of corticotropin-releasing activity.

The in vitro pituitary incubation system used previously for the characterization of CRF activity has been reinvestigated. Under the conditions as used to assay CRF, there is no detectable destruction of ACTH activity. The effect of CRF cannot therefore be explained on the basis of inhibition of ACTH inactivation. CRF does not act either by potentiating ACTH at the adrenal level or by an inherent adrenocorticotrophic activity. When considering the minimal active dose of purified substances, this test system appears to be highly specific for CRF. There is complete agreement as to characterization of CRF between data obtained in vitro and with recent in vivo assay systems.

The current concepts of hypothalamo-pituitary relationships are based on the results of numerous in vivo experiments (Harris, 1951; Fortier, 1951; McCann, 1953; Hume, 1949; Benoit and Assenmacher, 1953). The possibility of investigating this problem by simple in vitro techniques was particularly challenging in view of a number of findings which suggested the necessity of some hypothalamic factor, so far overlooked, for the release of pituitary hormones in vitro. Indeed, conflicting results are to be found in the literature regarding possible secretion of hormones by the various endocrines in tissue cultures. If we consider the anterior lobe of the pituitary, it has been reported as able to produce gonadotrophins (Rosenberg, 1954), or growth hormone (Gaillard, 1948; Martinovitch, 1953) whereas other investigators concluded that it could not secrete gonadotrophins (Cutting and Lewis, 1938) or any hormone whatever (Anderson and Haymaker, 1935). It appears from a study of this literature that, whenever hormon...

Somatostatin, a peptide isolated from ovine hypothalamic tissue that inhibits the release of radioimmunoassayable growth hormone in vitro from rat or human pituitary cells or in vivo in rats, has the primary structure [Formula: see text]. The structure was established by submitting the carboxymethylated peptide, the carboxymethylated tryptic digest, and the chymotryptic digest of the peptide to Edman degradation. Degradation products were analyzed by amino-acid analysis, as well as in some cases by determination of N-termini by dansylation or by determination of phenylthiohydantoins by mass spectrometry.

Intravenous administration of synthetic TRF in doses of 250–750 μg stimulates a rise in plasma HTSH levels in normal individuals. No rise in plasma HTSH occurs following 500–750 μg of TRF in subjects on 0.3 mg of Lthyroxine or in patients with panhypopituitarism. One of two euthyroid patients with a sella tumor appeared to respond suboptimally to TRF. It is condluded that synthetic TRF is a convenient means for testing the integrity of the human pituitary for TSH secretion.

The effect of pGlu-His-Pro-NH2 (TRF) on the secretion of prolactin (PRL) from rat anterior pituitary glands and cell cultures derived from normal and propylthiouracil (PTU)-fed rats is reported. TRF only slightly increased (less than 50% over control levels) the rate of secretion of radioimmunoassayable PRL or immune precipitated biosynthesized [3H]-PRL secreted by normal anterior pituitary cells, but had a greater effect on the rate of secretion of PRL by pituitary cell cultures obtained from PTU-treated rats. Furthermore, PTU-treated rats’ hemi-pituitary glands incubated in vitro also respond to TRF, demonstrating that the response to TRF by rat tissue is not a result of the cell dispersion or culture procedure. The administration of thyroid hormones which inhibit the TRF mediated secretion of TSH in vivo and in vitro suppress the secretion of PRL by hemi-pituitaries and dispersed cell cultures of anterior pituitaries from PTU-fed rats. Comparative studies of the [3H]-TRF binding to (mouse) TSH secretin...

Summary A purification sequence for the ovine hypothalamic LH-releasing factor is reported. From 300,000 sheep brains, ca. 200 μg have been obtained of a material containing ⪯ 56% peptide, of which 94% can be accounted for by the amino acids His 1, Arg 1, Ser 1, Glu 1, Pro 1, Gly 2, Leu 1, Tyr 1, the N-terminal being pyroGlu (2-pyrrolidone-5-carboxylic acid, PCA). This material stimulates release of LH in vivo and in vitro (≥0.5 ng/ml); it also stimulates release of FSH concomitantly with LH.

Intravenous administration of 0.25-2 μg/kg. body weight of highly purified β-MSH increases for long periods of time the amplitude of evoked monosynaptic potentials in the spinal cord of the cat. This is observed in midcollicular decerebrate or barbiturate anesthetized animals. These observations may be a clue to a possible role of β-MSH in the Mammalia as a neurohumor acting in the central nervous system.