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This paper illustrates the role that mass spectrometry played in the first elucidation of the primary structure of a hypothalamic releasing factor. It has been demonstrated that ovine TRF has the structure 2-pyrrolidone-5-carboxylyl-histidyl-proline amide.

A study was made of the effect of a single intravenous injection of a synthetic analog of vasopressin (CRA-41) on corticotropin release during various times of the circadian rhythm of the circulating plasma 17-hydroxycorticosteroids. The material was administered at 8:00 am, 5:00 pm and 12:00 midnight, and the plasma 17-hydroxycorticosteroids were determined at intervals within 1 hr of administration. Corticotropin release, as indicated by an increase in 17-hydroxycorticosteroid levels, was greater at 5:00 pm and 12:00 midnight than at 8:00 am, and the data suggest that the amount of ACTH released at 12:00 midnight was in excess of that released during the other 2 test periods. It is proposed that release of corticotropin in response to stress varies inversely with the levels of plasma cortisol and that this may be related to variations in pituitary ACTH content. The data further indicate that there was no difference between the corticotropin-releasing activity of equipressor doses of CRA-41 and synthetic...

A purified preparation of TSH-releasing factor (TRF), specific activity 30 U/mg, of sheep hypothalamus origin, elevates the levels of circulating plasma TSH from 21 to 197 mU/100 ml in 3 min following intravenous injection in normal animals; even though less marked in amplitude, the same effect is seen in thyroidectomized animals. The material has no effect in hypophysectomized animals. (Endocrinology 77: 792, 1965)

The adrenocorticotropic activity of the purified corticotropins α1, α2, α3, β γ1 γ2, δ1 has been determined vs. the U.S.P. Reference Standard for corticotropin in an in vitro assay based on corticoidogenesis. The potencies of these various preparations in U.S.P. u./mg. were: α1 = 11.2±2.1; α3 = 34.0±2.8; α4 = 25.8±5.1; β = 94.5±10.6; γ1 = 11.6±2.0;γ2= 21.7 + 4.1; 5,δ=27.6±6.9. The pepsin degradation products of/3-corticotropin, P2,3,4 had activities respectively of 27.5±7.9; 31.4±4.7; 21.5 ±10.0, in U.S.P. U./mg. All these preparations had been reported previously to have equal and maximal activity (80–100 U./mg.) in the adrenal ascorbic acid depletion test.

The original observations of hypothalamic releasing factors for the pituitary thyroid stimulating and luteinizing hormones have now been expanded in the direction of fulfillment of the prophecy that many more factors, both releasing and inhibiting, exist. It is now possible to advance a unified concept of C N S-endocrine relationships with broad implications for many aspects of clinical medicine.

When the pituitary is induced to secrete TSH by TRF it concomitantly secretes less ACTH in response to stress; conversely, when the secretion of ACTH is inhibited as by pretreatment with dexamethasone and Nembutal, the pituitary secretes higher quantities of TSH in response to TRF. (Endocrinology 77: 797, 1965)

THE use of specific pharmacologic antagonists to study the participation of the various autonomic mediators in the activation of the anterior lobe of the pituitary during stress has been found impractical by many investigators owing to the strong ACTH-releasing effects of these substances when administered alone or concurrently with the mediator investigated. Thus, no conclusive results have been reported on the role of endogenous epinephrine in the ACTH-release mechanism (August and Gubner, 1949; Seifter et al., 1949; Paschkis et al., 1950; Ronzoni and Reichlin, 1950; Tepperman and Tepperman, 1950; Gershberg et al., 1950; Fortier, 1951a; Sawyer, 1952) nor of acetylcholine (Dordoni and Fortier, 1950) or histamine (Fortier and Guillemin, 1950; Tepperman et al., 1951). A new approach to this problem was made possible when it was shown that by “adapting” (Serve, 1946) the animals to a pharmacological agent by repeated injections it became possible to dissociate its specific pharmacodynamic effects from its n...

The bioassay based on “pituitary depletion of growth hormone content” in the rat as described by Pecile et al. (Endocrinology 77: 241, 1965) has been used to assess “growth hormone releasing activity” in crude extracts of ovine, porcine, bovine hypothalamus and purified fractions of ovine hypothalamus extracts following gel filtration on Sephadex G-25. In all experiments, control pituitary glands were assayed for growth hormone (tibia test) at 2 dose levels; experimental pituitaries were assayed for growth hormone at 2 dose levels, or at one single dose corresponding by weight of pituitary tissue to the highest dose of the control or bracketed between the 2 doses of the control glands. The results of all experiments were studied by analysis of variance, followed by the multiple comparison test of Dunnett, the multiple range test of Duncan to assess significant differences between experimental and control groups and bioassays (3-point or 4-point). With these strict mathematical criteria for validity, contr...

Inhibin is a gonadal protein that specifically inhibits the secretion of pituitary follicle-stimulating hormone (FSH). Two forms of inhibin (A and B) have been purified from porcine follicular fluid and characterized as heterodimers of relative molecular mass (Mr) 32,000 (ref. 2). Each inhibin is comprised of an identical alpha-subunit of Mr 18,000 and a distinct but related beta-subunit of Mr 13,800-14,700 linked by interchain disulphide bond(s). Throughout the purification of inhibins, we consistently observed two fractions which stimulated the secretion of pituitary FSH. We report here the isolation of one of the FSH-releasing proteins; it has a Mr of 24,000 and its N-terminal sequences up to residue 32 are identical to those of each beta-subunit of inhibins A and B. In the presence of reducing agents, SDS-polyacrylamide gel electrophoresis resolves the FSH-releasing substance into two subunits which are identical in their migration behaviour to the reduced beta-subunits of inhibins A and B. Based on the N-terminal sequence data and Mr of the intact and reduced molecules, we propose that the FSH-releasing substance, which is active in picomolar concentrations, is a heterodimeric protein composed of the two beta-subunits of inhibins A and B linked by interchain disulphide bond(s). The structural organization of the FSH-releasing substance is homologous to that of transforming growth factor-beta (TGF-beta), which also possesses FSH-releasing activity in the same bioassay. We suggest that the substance be called activin to signify the fact that it has opposite biological effects to inhibin.

Discrete cells containing neurotensin, as shown by immunofluorescence, have been observed in the lower portion of the dog ileum. This implies that neurotensin may be synthesized in the small intestine and may be involved in local regulation of intestinal functions. Neurotensin is a peptide characterized originally in the hypothalamus.

A highly specific radioimmunoassay for one of the putative adrenomedullary [Met]enkephalin precursors, BAM-12P (Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-OH), has been developed. The BAM-12P antibodies recognize the COOH-terminal fragment of the peptide from Arg7 to Glu12 and do not crossreact with [Met5]- or [Leu5]enkephalin or any of their COOH-terminal lysine or arginine extended analogs. Specificity for the COOH-terminal Glu-OH is suggested by the 100% crossreactivity with BAM-12P5-12 and 0.3% crossreactivity with BAM-12P5-12 amide. Using these antibodies, we have measured three forms of BAM-12P-like immunoreactivity in extracts of bovine adrenal medulla, of which the major form (greater than 90%) corresponds to BAM-12P by molecular weight. Extracts of bovine adrenal cortex contain 1% the amount of a BAM-12P-like material (Mr approximately 1400; 20 ng per gland), possibly due to crosscontamination with adrenomedullary tissue. The major form of BAM-12P-like material in extracts of bovine neurointermediate pituitaries is of higher molecular weight than authentic BAM-12P (Mr approximately 4000); the remaining material (10%) corresponds to BAM-12P by molecular weight. There is no detectable BAM-12P-like immunoreactivity in crude or purified extracts of bovine anterior pituitaries. Extracts of bovine hypothalamic tissues contain small amounts of BAM-12P immunoreactivity (approximately 2 ng per fragment) which can be detected as one molecular form corresponding to a 1400-dalton molecule. The results indicate that the enkephalin precursor found in the adrenal medulla also may be present in the pituitary and hypothalamus. Furthermore, the processing of this molecule appears to be tissue-specific.

A 44 amino acid peptide with growth hormone-releasing activity has been isolated from a human tumor of the pancreas that had caused acromegaly. The primary structure of the tumor-derived peptide is H-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-Ar g-Gly -Ala-Arg-Ala-Arg-Leu-NH2. The synthetic replicate has full biological activity in vitro and in vivo specifically to stimulate the secretion of immunoreactive growth hormone. The tumor-derived peptide is identical in biological activity and similar in physiochemical properties to the still uncharacterized growth hormone-releasing factor present in extracts of hypothalamic tissues.

The opiate-like peptide beta-endorphin and adrenocorticotropin are concomitantly secreted in increased amounts by the adenohypophysis in response to acute stress or long-term adrenalectomy as well as in vitro in response to purified corticotropin releasing factor and other secretagogues. Conversely, administration of the synthetic glucocorticoid dexamethasone inhibits the secretion of both adrenocorticotropin and beta-endorphin. Thus, both hormones possess common and identical regulatory mechanisms and there may be a functional role for circulating beta-endorphin.

Immunoreactive beta-endorphin was measured in the ventricular fluid of six patients with chronic pain. Stimulation of the periaqueductal gray matter in three patients with pain of peripheral origin resulted in significant increases (50 to 300 percent) in the concentration of ventricular immunoreactive beta-endorphin. In three other patients suffering deafferentation dysesthesia, stimulation of the posterior limb of the internal capsule did not alter the concentration of this peptide. These results provide evidence of the release of human immunoreactive beta-endorphin in vivo and suggest that naloxone-reversible pain relief achieved by stimulation of the periaqueductal gray matter may be in part mediated by the activation of beta-endorphin-rich diencephalic areas.

On the basis of the hypothesis that the opiate-like neuropeptides, such as beta-endorphin, may be involved in the etiology of schizophrenic symptoms, naloxone 1,2 mg and placebo were administered intravenously to 8 schizophrenic patients, using a double-blind, crossover design. Naloxone was not found to be different from placebo in effecting schizophrenic symptoms, including hallucinations and delusions.

In the myenteric plexus-longitudinal muscle bioassay, beta-endorphin, i.e., beta-lipotropin (beta-LPH)-[61-91], has a potency of 450 with confidence limits of 281-966 when Met5-enkephalin is used as a reference standard with a potency of 100. The primary amide and the ethylamide of Met5-enkephalin have potencies statistically overlapping with that of beta-endorphin. The primary amide of alpha-endorphin has twice the potency of the free acid form of alpha-endorphin. An intact NH2-terminal tyrosine is not necessary for full intrinsic activity. The shortest fragment of beta-LPH with morphinomimetic activity is beta-LPH-[61-64].

The distribution of the newly characterized peptide somatostatin-28(1-12) was determined in rat tissues using a radioimmunoassay in which the immune plasma is directed against the C-terminus of the dodecapeptide. In the central nervous system, somatostatin-28(1-12)-like immunoreactivity is highly concentrated in the hypothalamus and the amygdala. In the digestive system, the highest levels of immunoreactivity are found in the stomach, the pancreas and the colon. The immunoreactive material measured in different tissues is the heterogenous: in addition to the dodecapeptide, two N-terminally extended somatostatin-28(1-12) peptides of 4,400 and 8,000 mol wt are also detected by the immune plasma. However, the dodecapeptide itself usually accounts for the majority (66 to 75%) of the total somatostatin-28(1-12)-like immunoreactivity present in those tissues.

Rats were passively immunized with an antiserum against somatostatin and a monoclonal antibody against rat hypothalamic growth hormone-releasing factor (rGRF-mAb) which does not recognize the biologically active 1-40 amino acid fragment of hpGRF-44 (hpGRF-40). Using this paradigm we have observed that the pituitary possesses a resilient capacity to release growth hormone (GH) following repeated injections of hpGRF-40 and that this response follows a dose-dependent relationship.

Profound and long-lasting analgesia (mean duration of pain relief 33.4 h, range 22.5--73.5 h) was produced by intrathecal administration of 3 mg synthetic beta-endorphin in all of 14 patients with intractable pain due to disseminated cancer. No respiratory depression, hypotension, hypothermia, or catatonia was observed.

FOOT-SHOCK induced stress promotes a five to sixfold increase in β-endorphin plasma levels. Similar increases were also found for ACTH plasma levels. In the hypothalamus, foot-shock induced stress promotes a decrease of β-endorphin assayed by radioimmunoassays. These data suggest that physiological increases in plasma β-endorphin levels induced by stress do not result in elevated levels of brain β-endorphin.

A few years ago it was established that opiates bind to the cell membrane of some of the neurons of the central nervous system. These synaptosomal opiate receptors were shown, in all the vertebrates studied, to be distributed in discrete localizations of the brain. These areas, perhaps not too surprisingly, correspond to well characterized anatomic pathways and structures long recognized as involved in pain perception. The next obvious question was, "Why should there be in the brain of man receptors for the alkaloids of the poppy?" The logical answer was that these receptors were probably there not to accept the . . .

A peptide representing the major form of somatostatin-like immunoreactivity was isolated from 600 rat pancreata by using anti-somatostatin affinity chromatography, gel permeation chromatography and reverse-phase high-performance liquid chromatography (HPLC). The isolated peptide elutes with the same retention time as synthetic somatostatin-14 in isocratic HPLC and its amino acid composition is in agreement with that of the tetradecapeptide. We propose that the structure of the major rat pancreatic somatostatin is identical to that of somatostatin-14 characterized in other species.

Synthetic TRF (pyroglutamylhistidyl-proline amide) was infused (1 ng/min) for 20 min (1) into the anterior pituitary of 10 rats by way of a cannulated hypophysial stalk portal vessel or (2) into a femoral vein of 10 rats. At the end of the 20-min infusion period, the plasma concentration of TSH in 8 of 10 rats given TRF via a cannulated portal vessel was significantly greater than the pre-infusion level. In contrast, in only 3 of 10 rats given TRF via a femoral vein was the plasma concentration of TSH significantly greater than the pre-infusion level of TSH (Endocrinology 89: 1054, 1971)

SummaryThe adrenocortical response to injected ACTH USP Standard has been studied as a function of time in rats after hypophysectomy or placement of a lesion in median eminence of the hypothalamus. In either case, there is an exponential decrease in sensitivity to ACTH of the adrenal gland, as a function of time. This can be observed in the presence of large adrenal glands, in animals bearing a hypothalamic lesion. The progressive decrease of adrenal sensitivity to ACTH in the lesioned animal makes it desirable to use only an acute preparation as a test for CRF.

SummaryBoth arginine and lysine vasopressin have been obtained in a high degree of purity by means of chromatography on carboxymethyl cellulose using ammonium acetate as a volatile buffer and differential elution gradients. Conditions for the resolution of LVP and AVP by chromatography on this same exchanger are described. By combining efficient recovery of activity, high flow rates for the chromatography, and rapid removal of buffers by lyophilization, a simple means of purifying vasopressin is proposed.

SummaryPurification of the TRF concentrate prepared by gel filtration of acetic acid extracts of hypothalamic tissues on Sephadex G-25 can be achieved following gel chromatography on carboxymethyl-Sephadex C-50 equilibrated in 0.001M pyridine acetate. The TRF activity is quantitatively eluted upon stepwise application of 0.09M pyridine acetate buffer. The method described can be used with small columns (4 × 15 cm) with charges ≤ 1 g TRF concentrate and also with large size preparative columns (15 × 33 cm) with charges up to 25 g of the TRF concentrate.

SummaryMild nonspecific exteroceptive stimuli such as handling, transferring from one room to another, and I.P. injections, lead to a rapid decrease of plasma TSH concentrations which become undetectable within 15 minutes following the beginning of the stressing procedure. There is evidence for a circadian rhythm of plasma TSH levels in opposite phases to those of the variations of plasma ACTH or corticosteroid variations. These observations are considered to be of importance to define resting levels of plasma TSH concentrations. For rats kept at 22°C ± 0.5, rapidly decapitated at 7:00 a.m., resting levels of circulating TSH are between 25–30 mU/100 ml plasma in this laboratory.

It has long been considered that one of the drawbacks to the routine clinical use of the hypothalamic releasing factors for diagnostic or therapeutic purposes (when they would become available) would be that they could not be administered per os, being polypeptides – which usually are susceptible to proteolytic enzymes to be found in the gastrointestinal tract. The observation that the half life of the hypothalamic releasing factors is of only a few minutes, a fact probably related to the existence of enzymatic plasma inactivation, indeed demonstrated in the case of hypothalamic TRF (1,2,3,4) is another somewhat unfortunate circumstance when considering the possible clinical application and uses of these substances. Furthermore, it has long been recognized that availability of synthetic products would be a requisite for any clinical studies and/or use in view of the extremely small quantities of the natural substances of hypothalamic origin available from extraction methods.

Synthetic (linear) somatostatin (SRIF, somatotropin-release inhibiting factor) abolished the elevation of serum GH concentration induced in normal human subjects by the administration of L-dopa and arginine. Within the dose used (250 μg, iv and 500 μg constant infusion) somatostatin. did not inhibit the rise in circulating PRL normally observed with arginine infusion nor did it modify basal levels of serum GH, TSH, PRL, FSH and LH.

Tremendous excitement has been generated by the isolation, purification, and subsequent synthesis of the opioid peptides. Our collaborative efforts have been directed at the questions of where β-endorphin is stored in brain and pituitary, how such structures are related to those storing the enkephalins and possibly other biogenic substances, and the functions of these peptides expressed through their electrophysiological properties. Although none of these questions is yet completely answered, sufficient data have been accumulated (Bloom et al., 1976, 1977a, b; Guillemin et al., 1977a, b, c; Rossier et al., 1977a, b, c; Nicoll et al., 1977; Henriksen et al., 1977; French et al., 1977) to enable us to give this overview and progress report.

Basal levels of immunoreactive (ir) beta-endorphin, corticotropin (ACTH), and prolactin (PRL) in plasma of male rats decrease after dexamethasone pretreatment (400 microgram/kg at 24 hr and 200 microgram/kg at 2 hr before). Inescapable electric footshocks increase ir-beta-endorphin, ACTH, and PRL plasma levels and this effect is blocked by dexamethasone pretreatment. Morphine (20 mg/kg) also increases ir-beta-endorphin, ACTH, and PRL levels. Dexamethasone pretreatment blocks the morphine-induced release of ir-beta-endorphin but does not prevent the morphine-induced release of PRL. Naloxone, the opiate antagonist, decreases basal plasma levels of PRL and partially blocks the stress-induced increase of PRL, but it has no effect on the basal or stress-induced release of ir-beta-endorphin. These results are consistent with the proposal that beta-endorphin may interact with an opiate receptor involved in the regulation of PRL secretion.

Immunocytofluorescence techniques with well characterized anti-sera to α-endorphin and β-endorphin show presence of these two peptides in all cellular elements of the pars intermedia of the rat hypophysis, and in discrete cells of the pars distalis (adenohypophysis) at the complete exclusion of the neurohypophysis (pars nervosa, posterior lobe).

Summary Substances with β-endorphin-like and adrenocorticotropin-like immunoreactivity were observed in extracts of both differentiated and anaplastic rat medullary thyroid carcinoma. In the tumor extracts, molar ratios of immunoreactive β-endorphin to immunoreactive-adrenocorticotropin were statistically equal to 1.0 while no consistent relationship was observed in the ratios of the concentrations of these two substances to that of immunoreactive-calcitonin.

The isolation and primary structure of two peptides with morphinomimetic activity, obtained from an extract of porcine hypothalamus-neurohypophysis, are described. The amino acid sequence of the two peptides, named α-endorphin and γ-endorphin, was determined by mass spectrometry and densyl-Edman methods to be H-Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-OH and H-Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-OH, respectively. These correspond to the amino acid sequences present between residues 61 and 76 and residues 61 and 77 of the various β-lipotropins. A third peptide also obtained in pure from in these studies was found to be an unstable salt of α-endorphin.

A synthetic replicate of human pancreas growth hormone-releasing factor, hpGRF1-44-NH2, (hpGRF44), a 44-amino acid amidated peptide, was administered to seven normal men. In addition to placebo, 4 subjects received 3 doses of hpGRF44 (0.5 microgram/kg, 5 micrograms/kg, 10 micrograms/kg), while one subject received 2 doses (5 micrograms/kg, 10 micrograms/kg) and 2 subjects received one dose (0.5 microgram/kg or 5 micrograms/kg), each as a rapid intravenous injection. A significant increase in circulating growth hormone (GH) was observed at each dose (0.5 microgram/kg, p = 0.05; 5 micrograms/kg, p less than 0.005; 10 micrograms/kg, p = 0.01) when compared to placebo, with equivalent maximal responses. At all doses studied a rise in plasma GH occurred within 5 min, with a peak response in most patients at 30-45 min. There were no associated changes in any of the other anterior pituitary hormones or insulin. Other than transient flushing and minimal changes in vital signs, no side effects were noted. The results of these studies support the potential usefulness of hpGRF44 for the assessment of GH secretion and reserve, and of this peptide or an analog as a therapeutic agent to stimulate GH release.

3- O -(2-Acetamido-3-4,6-tri- O -acetyl-2-deoxy-β- d - glucopyranosyl)- N -( tert -butyloxycarbonyl)- l -serine was synthesized and condensed by the solid-phase procedure to give the sequence Gly-[β- d -Glc p NAc-(1→3)-Ser]-Ala-OH and β- d -Glc p )NAc-(1→3)-Ser-13-somatostatin. The synthetic glycopeptides appeared homogeneous on t.l.c and l.c. examination and showed the correct amino acid composition and 2-amino-2-deoxy- d -glucose content. The structure of Gly-[β- d -Glc p NAc-(1→3)-Ser]-Ala-OH was further confirmed by mass spectrometry of the N -acetyl per methyl derivative, and by n.m.r. spectroscopy.

The endogenous opioid peptide, beta-endorphin, induces nonconvulsive limbic epileptiform activity when administered intraventricularly to rats. Epileptiform activity is elicited by beta-endorphin in doses that are devoid of analgesic or behavioral signs. Equimolar intraventricular doses of morphine or of the enkephalin analog [DAIa2,Met5]enkephalin-NH2 fail to elicit this limbic epileptiform activity. These observations, together with the recent immunohistochemical localization of beta-endorphin to midline limbic structures, suggest that beta-endorphin may have an important role in the regulation of limbic excitability.

The synthesis of four enkephalinamide analogs is described in which the peptide bond between residues 4 and 5 is reversed with or without simultaneous reversal of the carboxyl-terminal amide bond. These so-called partially modified retro-inverso-isomers are new, potent, topochemical analogs of the enkephalins. Tests, both in vitro and in vivo, have shown that these analogs are considerably longer acting than any previously studied enkephalins. Thus, partial reversal of the peptide bonds of the backbone can result in peptides with enhanced activity compared to a parent compound, provide that the structural complementarity of both the side chains and end groups are conserved.

Radioimmunoassay and microdissection procedures were used to study the perinatal development of the endorphin- and enkephalin-containing systems in the rat brain. In contrast to values reported on adult rat, endorphin levels are much higher than enkephalin levels on embryonic day 16. The highest endorphin values are found in the diencephalon, midline telencephalon and medulla-midbrain regions. Perinatally, enkephalin content increases at a faster rate than endorphin in all brain regions, producing a marked drop of the endorphin/enkephalin ratios. Between postnatal days 6 and 25, both endorphin and enkephalin levels increase, approaching their adult distribution pattern. No correlation was found between regional distributions or rates of increase of endorphin and enkephalin in any of these developmental stages, suggesting that the two peptide systems develop independently from each other.

Specific radioimmunoassays for γ1-melanotropin (γ1-MSH) and γ2-melanotropin (γ2-MSH) have been developed. The γ1-MSH antibody recognizes the portion between His5 and Phe11-NH2 of γ1-MSH and shows no significant cross-reactivities with other related peptides. The γ2-MSH antibody cross-reacts with γ1-MSH and γ3-MSH to the extent of 0.004% and 0.04%, respectively, on a weight basis. Using these two different antisera on bovine pituitary extracts, two γ1-MSH-like peptides were detected only in the intermediate lobe, whereas γ2-MSH-like peptides were not detectable. Furthermore, it is likely that the smallest γ-MSH produced in the bovine intermediate pituitary is a γ1-MSH-like peptide with the COOH-terminus amidated.

Somatostatin, a recently synthesized hypothalamic peptide thought to represent growth hormone-release-inhibiting factor, has been previously shown to inhibit pancreatic glucagon secretion in species other than man. To determine whether somatostatin also inhibits human glucagon secretion, plasma glucagon responses during intravenous infusion of arginine alone (250 mg. per kilogram over twenty-five minutes) and in combination with synthetic cyclic somatostatin (20,μg. per minute) were compared in six normal and six insulin-dependent diabetic subjects. Somatostatin abolished glucagon responses to arginine in both groups, with plasma glucagon declining transiently below basal levels; this occurred despite the fact that the diabetic subjects had fasting hyperglucagonemia and exaggerated glucagon responses during control arginine infusions. In both groups plasma glucose rises seen after arginine were diminished during somatostatin infusions, and, in the normal subjects, insulin responses were also inhibited. These results demonstrate that somatostatin is a potent inhibitor of glucagon secretion in man. Accordingly, it may prove useful as an adjunct to insulin therapy in treatment of insulin-requiring diabetes mellitus.

Substantial quantities of immunoreactive (IR)-β-endorphin have been demonstrated in the retina of Rana pipiens in amounts not affected by exposure to constant light or constant darkness. IR-β-endorphin has also been measured in blood, pituitary, hypothalamus, and extrahypothalamic brain of this species, while lower concentrations of IR-Met-enkephalin and IR-Leu-enkephalin have also been found in these tissues. The significance of the widespread distribution of opioid peptide-like material in amphibian tissues remains to be determined.

The synthesis of ovine hypothalamic somatostatin-28 (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) has been accomplished by solid phase methodology. The structure of the synthetic material was verified by: (1) direct sequence analysis with a Beckman 89°C sequencer, (2) correlation of the amino acid analyses of the isolated tryptic peptide fragments with their theoretical compositions, and (3) comparison, using high performance liquid chromatography, of the synthetic methionine-sulfoxide and methionine-sulfone modified NH2-terminal peptides (residues 1–11) with the corresponding tryptic fragment from somatostatin-28.

The addition of fibroblast growth factor (FGF) to primary cultures of rat anterior pituitary cells modifies their response to thyrotropin-releasing factor in a dose-dependent manner. While the pituitary response to the other releasing factors (corticotropin-releasing factor, growth hormone-releasing factor, and gonadotropin-releasing factor) is not altered, FGF increases both the sensitivity of the cells to thyrotropin-releasing factor and the amounts of prolactin and thyrotropin released. A minimum of 24 hr of preincubation with FGF is required to modify the pituitary response, and maximal effects were observed with 48 and 72 hr of preincubation. The effective doses of FGF are similar to those described for its mitogenic activity (i.e., 1-100 pM), but inhibition of cell growth with 5-fluorodeoxyuridine does not modify the effect of FGF on thyrotropin and prolactin release. These results suggest a novel paracrine, if not autocrine, role of pituitary FGF in the homeostatic mechanisms that regulate the secretion of prolactin and thyrotropin. They also suggest that the biological significance of the presence of FGF in various tissues may not be directly related to its in vitro mitogenic activity.

Antisera were raised against synthetic replicates of the carboxyl-terminal (C-terminal) fragment of the precursor to human GH-releasing factor (GRF) (pre-proGRF) whose structure was predicted from the complementary DNA cloned from a pancreatic tumor. These antisera were used along with antisera to human GRF itself to search for the presence of related molecules in the human hypothalamus, with an immunohistochemical approach. The antisera to pre-proGRF that recognize specifically the C-terminal amidated form of pre-proGRF stain GRF neurons in their cell bodies, fibers, and nerve endings that are in contact with portal capillaries of the median eminence. Antisera against the nonamidated form of the molecule did not give any staining in the hypothalamus. These results strongly suggest that human hypothalamic GRF derives from a precursor immunologically related (and probably identical) to the tumorous one and that this precursor is cleaved inside GRF cell bodies to give, in addition to the GRF-44-NH2 a second amidated peptide, the C-terminal pre-proGRF that is transported distally to nerve endings and most probably coreleased with GRF into portal capillaries.