Search

WOS: 000415310800007 PubMed ID: 29144972 Science is ideally suited to connect people from different cultures and thereby foster mutual understanding. To promote international life science collaboration, we have launched "The Science Bridge'' initiative. Our current project focuses on partnership between Western and Middle Eastern neuroscience communities. Medical Research Council [MC_UP_1202/5]

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.

A short historical review from the early days of neuroendocrinology is presented recognizing hormones secreted by the pituitary gland; control of its functions by nuclei of the hypothalamus through the release of unknown substances in special capillary vessels; characterization of these releasing factors as peptides; studies of their mode of action; their use in clinical medicine; new and still ongoing demonstration of their ubiquity though not random in the brain and peripheral organs; and recent implications in control of behavior in animals and humans.

The author describes his experiences as first a scientist and later an early digital artist, which led him to recognize both similarities and contrasts in the thinking and practice of art and science.

A novel method for the separation of peptides from amino acids and salts has been developed. Separation is achieved by passage of the sample through a column of octadecasilyl-silica (ODS). Peptides, but not amino acids and salts, are strongly retained by ODS in an aqueous mobile phase and can be recovered by elution with an aqueous-alcoholic mobile phase. The method is applicable to most peptides and is fast and simple. Moreover, it is characterized by good recovery yields and high sample capacity.

This paper is a short review of the traditionally obvious diseases of neuroendocrine origin (diabetes insipidus, Kallman syndrome, etc.), but also of the newly recognized participation of several peptides originally characterized in the hypothalamus and of their receptors, in a series of diseases, both in internal medicine and in psychiatry (rheumatoid arthritis, inflammation, carcinoids, anxiety, depression, etc.). The concept of neuropeptides is now vastly expanded, as these molecules and their several receptors are now known to be widely distributed throughout the brain and the periphery with increasing evidence of paracrine and autocrine modes of action.

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.

In “Scientists versus Whaling” (BioScience 52:1137–1140), Aron, Burke, and Freeman defend Japan's controversial “scientific” whaling program against a series of criticisms we made in an open letter to the Government of Japan last May in the New York Times. Our letter, signed by 21 eminent scientists, including three Nobel laureates and several pioneers of conservation biology, called on Japan to suspend its whaling program. Aron and his coauthors claim that our letter contains numerous errors of fact and law, and they cite it as an example of “science advocacy” wherein scientists, driven by passion or politics, lower their professional standards in support of popular causes. To the contrary, our overriding concern is for sound science uncorrupted by a political agenda, a standard that Japan's whaling program fails to meet. Aron and colleagues also attribute nonscientific motives to the signatories of the letter, suggesting—without supporting evidence—that politics, emotion, or sentiment have undermined our professional responsibility. Such challenges to a scientist's motivation and scientific trustworthiness should not be made lightly. Yet so far as we are aware, Aron and coauthors made no effort to determine the validity of their charges.

Last month, 70 members of the U.S. Congress, including Henry Hyde, Chairman of the House Judiciary Committee, and J. C. Watts Jr. Republican Conference Chairman, signed a letter urging the federal government to ban all research on stem cells obtained from human embryos and fetuses. The letter calls upon the U.S. Department of Health and Human Services (DHHS) to reverse National Institutes of Health (NIH) Director Harold Varmus's decision to allow funding of pluripotent stem cell research. The lawmakers object “in the strongest possible terms” to Varmus's decision, as well as to the memorandum issued in January by DHHS General Counsel Harriet Rabb, which served as the legal basis for Varmus's position. In their letter, the members of Congress state, “Any NIH action to initiate funding of such research would violate both the letter and spirit of the federal law banning federal support for research in which human embryos are harmed or destroyed.” Federal laws and regulations, they claim, have protected human embryos and fetuses “from harmful experimentation at the hands of the Federal government” for more than two decades. “This area of law has provided a bulwark against government's misuse and exploitation of human beings in the name of medical progress. It would he a travesty for this Administration to attempt to unravel this accepted ethical standard.”

The major anionic mitogenic polypeptide for endothelial cells, acidic fibroblast growth factor (FGF), has been purified to homogeneity from bovine brain and its complete primary structure established by gas-phase sequence analysis. The 140 amino acid (Mr 16,000) protein has been previously shown to be a potent growth factor for many diverse cell types of mesodermal origin, in vitro, and an angiogenic agent, in vivo. The amino acid sequence of bovine brain acidic FGF has a 53% absolute homology with that of bovine pituitary basic FGF suggesting that these endothelial cell mitogens are derived from a single ancestral gene.

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.

A peptide with somatostatin-like immuno- and bioactivity has been isolated from an aqueous extract of 96 800 rat hypothalami by means of immunoaffinity chromatography, gel filtration and reverse-phase high-performance liquid chromatography (HPLC). Chemical characterization by amino acid analysis, tryptic peptide mapping and retention behavior in two HPLC systems showed that the peptide was indistinguishable from somatostatin-28 previously characterized from several species. This evidence suggests that rat hypothalamic somatostatin-28 is identical in structure to porcine and ovine somatostatin-28.

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.

We have isolated from extracts of ovine hypothalami two molecules characterized as somatostatin-28 and somatostatin-4–28 (referred to as somatostatin-25). They were reproduced by solid phase synthesis. In equimolar ratio and depending upon the experimental conditions, synthetic somatostatin-28 and somatostatin-25 are 3–14 times more potent than somatostatin-14 to inhibit the basal in vitro secretion of growth hormone or as stimulated by prostaglandin (PGE 2 ). In early studies in vivo, somatostatin-28 and somatostatin-25 are also more potent than somatostatin-14 in inhibiting the secretion of growth hormone acutely stimulated in the rat by injection of morphine; somatostatin-28 is also longer-acting than somatostatin-14. These results suggest that somatostatin-14, as originally isolated, is a biologically active fragment of a larger molecule of greater specific activity; it should be considered as another form of somatostatin with high biological activity present in some tissues and likely secreted by the tissues along with somatostatin-14 and possibly other somatostatin-peptides of diverse sizes.

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.

Two mitogenic peptides in bovine liver extract were purified to apparent homogeneity by monitoring the purification steps with two in vitro bioassays; one based on stimulation of adult bovine aortic arch endothelial cell proliferation and the other incorporation of [ 3 H]thymidine to mouse fibroblast 3T3 cells. The purification procedure involved cation-exchange chromatography followed by affinity chromatography on heparin-Sepharose and two steps of reversed-phase HPLC. The purified material showed the same biological activity as pituitary basic fibroblast growth factor (FGF). Amino acid analyses of the purified mitogen yielded a similar, but not identical composition to that of bovine pituitary basic FGF(1–146) reported previously. Gas-phase microsequencing identified two sequences in equal amounts in the purified preparation. Furthermore, the sequencing results are in accord with the theoretical data obtained when two truncated forms of basic FGF, corresponding to FGF(12–16) and (16–146), are being sequenced simultaneously. Basic FGF(12–146) is a novel truncated form of basic FGF which has not been isolated before although the (16–146) fragment has been found previously in kidney, corpus luteum, and adrenal. SDS-PAGE analysis could not separate the two forms and showed that both migrated as a protein of about 15 100 daltons, which is slightly smaller than intact basic FGF(1–146) (16 200 daltons). These results, taken together, indicate that at least some of the mitogenic activity in liver may be derived from basic FGF-related polypeptides.

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 two major mitogenic polypeptides for endothelial cells have been purified to homogeneity. The complete primary structure of bovine pituitary basic fibroblast growth factor (FGF) and the amino-terminal amino acid sequence of bovine brain acidic FGF have been established by gas-phase sequence analyses. Homogeneous preparations of these polypeptides are potent mitogens (basic FGF, ED50 approximately equal to 60 pg/ml; acidic FGF ED50 approximately equal to 6000 pg/ml) for many diverse cell types including capillary endothelial cells, vascular smooth muscle cells, and adrenocortical and granulosa cells; in vivo, basic FGF is a powerful angiogenic agent in the chick chorioallantoic membrane assay. The available protein sequence data demonstrate the existence of significant structural homology between the two polypeptides.

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.

A 44 amino acid peptide with high intrinsic growth hormone releasing activity was isolated from 500 bovine hypothalami by means of acid extraction, immunoaffinity chromatography, gel filtration, and two steps of reverse phase HPLC. The growth hormone releasing factor was structurally characterized by gas phase sequence analysis. Reverse phase liquid chromatography of the native peptide and synthetic replicates showed that the molecule possesses an amide rather than a free acid at its carboxyl terminus. The structure of the peptide was established as: 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-Asn-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln -Gly-Ala-Lys-Val-Arg-Leu-NH2 using approximately 2 nmol of material.

The primary structures of three polypeptides, possessing high intrinsic growth hormone-releasing activity and derived from a human pancreatic carcinoma which had caused acromegaly, were established by sequence analyses of the intact peptides and their cyanogen bromide digestion fragments with a gas-phase sequenator. The three human pancreas growth hormone-releasing factors contain 44 (hpGRF-44), 40 (hpGRF-40), and 37 (hpGRF-37) amino acids in identical sequences from their NH2 termini. High pressure liquid chromatography of the native peptides and their synthetic replicates showed that hpGRF-37 and hpGRF-40 possess free carboxyl termini while that of hpGRF-44 is amidated. The structure of hpGRF-44 was established as: 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-Arg-Ala-Arg-Leu-NH2.

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.

The hypophysiotropic peptide, corticotropin-releasing factor (CRF) was isolated from caprine hypothalamic median eminence tissue by means of acid extraction, immunoaffinity chromatography, gel filtration and two steps of reverse-phase high-performance liquid chromatography (RPLC). Amino acid sequence determination using a gas-phase sequencer established the primary structure of the first 39 residues from the NH 2 -terminus. The nature of the COOH-terminal dipeptide was elucidated by Staphylococcus aureus V8 protease digestion of the native peptide, dansylation of the digest and comparative RPLC studies with the dansylated dipeptides Ile-Ala-NH 2 , Ile-Ala-OH, Ala-Ile-NH 2 and Ala-Ile-OH. The complete structure of the peptide was established as: H-Ser-Gln-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val- Leu-Glu-Met-Thr-Lys-Ala-Asp-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu- Leu-Asp-Ile-Ala-NH 2 , which is identical to that of ovine CRF.

A third melanotropin coding fragment named γ-MSH was discovered by Nakanishi et al (Nature 278 , 423–427 (1979)) in the cryptic region outside the portion coding for ACTH and β-LPH in the ACTH/β-LPH precursor mRNA isolated from the intermediate lobe of bovine pituitary. Four possible γ-MSH peptides derived from this coding fragment were synthesized by solid-phase methodology and their bioactivity determined in an in vitro MSH assay as well as the anterior pituitary primary culture assay. Relative to α-MSH, the melanotropic activities of Ac-γ1-MSH, γ1-MSH, γ2-MSH and γ3-MSH are 7.3 × 10−4, 3.3 × 10−5, 1.4 × 10−4 and 4.6 × 10−7 respectively. None of these γ-MSH peptides releases LH, FSH, PRL, GH and TSH in the pituitary culture medium at a dose as high as 100 ng per dish.

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.

The primary structure of the NH2-terminally extended somatostatins isolated from ovine hypothalamic extracts, one containing 28 residues and the other 25, has been determined. The structure of somatostatin-28 is Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH; the shorter one, somatostatin-25, has the same sequence as somatostatin-28 except that the first three NH2-terminal residues are deleted. The two peptides as isolated were found to be oxidized at the methionine residue to the methionine sulfoxide. Their structures were established by subjecting the native peptides to direct sequence analysis in a Beckman 890C sequencer and identifying the released phenylthiohydantoin derivatives by high-performance liquid chromatography. Their structures were confirmed by trypsin digestion and isolation of all the tryptic peptides, followed by amino acid analysis of the tryptic fragments. Moreover, some of the tryptic peptides were matched with their respective synthetic replicates on high-performance liquid chromatography.

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.

Summary This note describes the technical details of double antibody radio-immunoassays for the two peptides α- and β-endorphin and the specificity characteristics of the antisera. Antisera raised in rabbits to synthetic α-endorphin measure quantitatively α-endorphin; usable range is 50 pg to 5 ng. Their specific recognition site is toward the C-terminal region (Ser10-Thr16) of α-endorphin. Other antisera, raised in rabbits to synthetic β-endorphin measure quantitatively β-endorphin; usable range is 50 pg to 5 ng β-endorphin. Their specific recognition site is toward the C-terminal region (Asn20-His27) of β-endorphin; these antibodies recognize β-endorphin i.e. β-LPH-(61–91), β-lipotropin i.e. β-LPH-(1–91), and the precursor molecule to LPH-ACTH with molecular weight ca. 31 × 103 (31K-precursor). Neither antiserum binds enkephalins.

A specific radioimmunoassay for ..gamma..-melanocyte stimulating hormone-like peptides was developed. An antiserum raised in rabbit to synthetic bovine ..gamma../sub 3/-MSH, one of the possible ..gamma..-MSH peptides, specifically recognizes the portion between His/sup 5/ and Arg/sup 14/ of ..gamma../sub 3/-MSH without significant cross-reaction with other synthetic ..gamma..-MSH-like peptides, ..cap alpha..-, ..beta..-MSH, adrenocorticotropin, and ..beta..-endorphin. The usable range of this RIA is 10 pg to 600 pg of synthetic ..gamma../sub 3/-MSH. Three immunoreactive ..gamma..-MSH peaks were thus found in gel permeation chromatography of the whole bovine pituitary extract.