In vitro pituitary hormone releasing activity of 40 residue human pancreatic tumor growth hormone releasing factor

The hypophysiotropic activities of a synthetic human pancreatic growth hormone releasing factor (hpGRF) with 40 residues was examined in vitro using rat pituitary halves. At concentrations from 10(-10) M to 10(-7) M the peptide stimulated GH release in a dose-dependent manner with the ED50 being 1.2 x 10(-9) M. The concentration of 10(-10) M hpGRF is comparable to the basal hypophyseal portal blood levels of other known hypothalamic hypophysiotropic hormones. However, GH release was enhanced three-fold by concentration as low as 10(-12) M, though no dose-response relationship was observed up to 10(-10) M. Thus, this peptide not only stimulates the release of GH in a dose-dependent manner, but at lower concentrations also maintains elevated GH levels. The release of ACTH, beta-endorphin, LH, and FSH was not affected by hpGRF at any of the concentrations tested. At hpGRF concentrations less than 10(-7) M, the release of TSH and PRL were unaffected. However, at 10(-6) M, TSH release was enhanced about 2.5 fold and prolactin release was elevated slightly.     

Effects of administration of somatostatin-14 and immunoneutralization of somatostatin on endocrine and growth responses in rainbow trout

Injection of somatostatin‐14 (SS‐14) at 5 ng g^?1 body mass (BM) into rainbow trout Oncorhynchus mykiss decreased (P < 0·05, cubic, r² = 0·54) levels of growth hormone (GH) (1·5 ± 0·9 ng ml^?1v. 6·6 ± 0·6 ng ml^?1) over time when compared to controls. Somatostatin‐14 at 50 ng g^?1 BM also decreased (P = 0·064, quadratic; r² = 0·30) levels of GH (3·6 ± 2·1 ng ml^?1v. 6·6 ± 0·6 ng ml^?1) over time compared to controls. In a second study, passive immunization against SS‐14 (1 : 25 dose) increased (P = 0·10, cubic, r² = 0·12) levels of GH (11·0 ± 4·8 ng ml^?1v. 5·2 ± 1·4 ng ml^?1) over time. Passively immunizing against SS‐14 (1 : 50 dose) increased (P < 0·05, cubic, r² = 0·10) levels of GH (8·2 ± 2·3 ng ml^?1v. 5·2 ± 1·4 ng ml^?1) over time compared to controls. Overall, in the active immunization study there was no difference (P > 0·10) in specific growth rate (G) or feed conversion ratio (FCR) between the three treatment groups during the 9 weeks of the study. Only four of the fish immunized against SS‐14, however, developed antibody titres against SS. Compared to controls, these fish exhibited a G of 0·89 ± 0·09 v. 0·56 ± 0·09% per 3 weeks and FCR of 0·80 ± 0·04 v. 1·20 ± 0·05 g g^?1. In SS‐14 immunized fish, levels of GH decreased (P < 0·05) by day 63 while levels of insulin like growth factor‐I (IGF‐I) increased (P < 0·05) by day 42 and 63. These results indicate the hypothalamic hormone SS‐14 regulates GH secretion similarly in rainbow trout as it does in mammals. Active immunization against SS‐14 could improve growth performance in rainbow trout but enhanced G and FCR is dependent upon generation of antibody titres.     

Growth hormone releasing peptide-6 acts as a survival factor in glutamate-induced excitotoxicity

Chronic systemic treatment given to adult male rats with growth hormone releasing peptide-6, an agonist of the ghrelin receptor, increases insulin-like growth factor I levels in various brain regions, including the hypothalamus and cerebellum. Furthermore, intracellular signalling cascades normally associated with anti-apoptotic actions are activated in the same areas and are coincident with decreased basal cell death. Because abnormally high concentrations of glutamate can lead to overexcitation of neurones leading to cell damage and/or death, we investigated whether administration of growth hormone releasing peptide-6 attenuates monosodium glutamate-induced apoptosis in the rat hypothalamus and cerebellum. Glutamate increased activation of caspase 9 followed by cleavage of caspase 7, which in turn fragmented poly(ADP-ribose) polymerase, terminating in cell death in both the hypothalamus and cerebellum. Growth hormone releasing peptide-6 reversed glutamate-induced cell death by decreasing activation of caspases 9 and 7 and poly(ADP-ribose) polymerase fragmentation. These results provide a better understanding of the neuroprotective role of growth hormone secretagogues and the mechanisms involved.     

Cortistatin modulates the expression and release of corticotrophin releasing hormone in rat brain. Comparison with somatostatin and octreotide

Cortistatin (CST) is an endogenous neuropeptide characterized by remarkable structural and functional resemblance to somatostatin (SST), both peptides sharing the ability to bind and activate all five SST receptor subtypes. Evidence is also available showing that CST exerts biological activities independently from SST, perhaps via the activation of specific receptors that remain to be fully characterized at present. Here we have investigated the effects of CST on the gene expression and release of corticotrophin releasing hormone (CRH) from rat hypothalamic and hippocampal explants; moreover, we compared the effects of CST with those of SST and octreotide (OCT) in these models. We found that: (i) CST inhibits the expression and release of CRH from rat hypothalamic and hippocampal explants under basal conditions as well as after CRH stimulation by well known secretagogues; (ii) SST does not modify basal CRH secretion from the hypothalamus or the hippocampus, while it is able to reduce KCl-stimulated CRH release from both brain areas; (iii) OCT inhibits both basal and KCl-induced CRH secretion from rat hypothalamic explants, while it has no effect on CRH release from the hippocampus, either under basal conditions or after stimulation by high K(+) concentrations; (iv) at variance with CST; SST and OCT have not effect whatsoever on veratridine-induced CRH release from the hypothalamus. In conclusion the present findings provide in vitro evidence in support of the hypothesis that CST plays a role in the regulation of endocrine adaptive responses to stress.     

Effects of growth hormone-releasing factor, somatostatin and dopamine on growth hormone and prolactin secretion from cultured ovine pituitary cells

A synthetic form of human pancreatic growth hormone releasing factor (GRF-44-NH2) was shown to be a potent stimulator of growth hormone (GH) secretion and cellular cyclic AMP levels in cultured sheep pituitary cells. A small dose-dependent stimulation of prolactin secretion was also observed. Somatostatin (0.5 microM) completely blocked the maximal GRF (1 nM)-stimulated secretion without a significant effect on cyclic AMP levels. Dopamine (0.1 microM) inhibited the GRF-elevated GH secretion by 50% and lowered cyclic AMP levels by 30%. Dopamine (0.1 microM) inhibition of basal prolactin secretion was not affected by GRF (1 nM). The data support the hypothesis that cyclic AMP is involved in the action of GRF but suggest that somatostatin can inhibit GRF-induced secretion of GH independently of cyclic AMP.     

Isolation and characterization of the porcine hypothalamic growth hormone releasing factor

A 44 amino acid peptide with high intrinsic growth hormone releasing activity was isolated from 2500 porcine hypothalami by means of acid extraction, immunoaffinity chromatography, gel filtration, and 2 steps of reverse phase HPLC. The growth hormone releasing factor was structurally characterized by gas phase sequence analyses of the intact peptide and its carboxyl terminal cyanogen bromide digestion fragment. 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-Ser-Arg-Gln-Gln-Gly-Glu-Arg-Asn-Gln-Glu-Gln-Gly-Ala-Arg-Val-Arg-Leu-NH2 using approximately 6 nmol of material.     

The localization of oxytocin,vasopressin, somatostatin and luteinizing hormone releasing hormone in the rat neurohypophysis

Summary The hypothalamic hormones arginine-vasopressin (AVP), oxytocin (OXT), somatostatin (SOM), and luteinizing hormone-releasing hormone (LHRH) were localized in the rat neurohypophysis by the use of semithin serial sections and the unlabeled antibody enzyme method. Clusters of AVP fibres are present within the central region of the neural lobe, clusters of OXT fibres mainly in the peripheral part. The AVP fibres enter bilaterally into the neural lobe.The results call into question previous reports on the presence of AVP on receptors in the pars intermedia cells, since incubation with anti-AVP resulted in similar staining in the pars intermedia of the Wistar and homozygous Brattleboro rat, a mutant strain deficient in AVP. The same intermediate lobe cells are stained after incubation of serial sections with anti-AVP and anti--melanocyte-stimulating hormone (-MSH). This staining of anti-AVP could be removed by solid phase absorption to -MSH and is thus most probably due to cross reaction with -MSH. SOM fibres appear to be present in the peripheral parts of the proximal neurohypophysial stalk and mainly lateral in its more distal parts. In the neural lobe they rapidly decrease in number, although some fibres continue into the distal part of the neural lobe, running bilaterally and situated adjacent to the pars intermedia. The SOM staining within magnocellular elements, which has been reported in the literature, can most probably be explained by cross reaction of anti-SOM with neurophysins. LHRH fibres are very scarce in the neurohypophysial stalk and absent in the neural lobe.Supported by the Foundation for Medical Research FUNGOThe authors wish to thank Drs. J. De Mey (Beerse, Belgium), A. Arimura (New Orleans, U.S.A.), M.P. Dubois (Nouzilly, France), B.L. Baker (Ann Arbor, U.S.A.) and A.G.E. Pearse (London, U.K.) for their gifts of anti-somatostatin serum, Dr. B. Kerdelhué (Gif-sur-Yvette, France) for anti-LHRH serum, and Dr. F. Vandesande (Ghent, Belgium) for anti-neurophysin I and II serum and bovine neurophysin I and II. Dr. J.G. Streefkerk (Free University, Amsterdam) is acknowledged for critical comments and Mr. A.T. Potjer and Miss J. van der Velden for their skilled assistance     

Somatocrinin,growth hormone releasing factor,stimulates secretion of growth hormone in anesthetized rats

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, $\text{in}\text{vivo}$, contrary to $\text{in}\text{vitro}$ 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 $\text{in}\text{vivo}$ results, the three peptides with serve as powerful tools with which to investigate the mechanisms of GH secretion.     

Altered release of growth hormone from dispersed adenohypophysial cells of streptozotocin diabetic rats. I. Effects of growth hormone releasing factor and somatostatin

As growth hormone has been implicated in the "dawn phenomenon," an early morning rise in serum glucose, we have studied the control of growth hormone release in diabetes using an acutely dispersed system of adenohypophysial cells from normal or diabetic rats (65 mg/kg streptozotocin, 8 days before sacrifice; serum glucose, 490 +/- 17 mg/dL). Growth hormone release is normally controlled by the two hypothalamic hormones, growth hormone releasing factor and somatostatin. We have found cells of the diabetic rats exhibit changes in sensitivity that result in increased growth hormone release in static incubation. In normal cells, rat growth hormone releasing factor increases growth hormone release three- to four-fold with an EC50 of 151 +/- 27 pM (n = 7). In contrast, in cells from diabetic rats, there was a significant (twofold) increase in sensitivity to growth hormone releasing factor (EC50 = 75 +/- 15 pM, n = 7) which resulted in increased growth hormone release with lower but not maximal (10 nM) growth hormone releasing factor. Basal nonstimulated release was unchanged. Somatostatin inhibition of stimulated growth hormone release was reduced (n = 7); half-maximal inhibition occurred with 0.21 +/- 0.03 nM (normal) and 0.76 +/- 0.17 nM somatostatin (diabetic). In perifusion the peak secretion rate was significantly lower for diabetic cells stimulated by a maximal dose of growth hormone releasing factor. These studies suggest somatotrophs of diabetic rats have altered sensitivity in vitro to the controlling hormones growth hormone releasing factor and somatostatin.     

Pituitary response to growth hormone-releasing factor in rats with functional or anatomical lesions of the central nervous system that inhibit endogenous growth hormone secretion

The pituitary growth hormone (GH) response to the growth hormone-releasing factor, hpGRF-44, was evaluated in male rats with various lesions of the central nervous system. These included an electrical lesion of the ventromedial hypothalamus, a chemical lesion of the arcuate nucleus induced by neonatal treatment with monosodium glutamate, a functional lesion of catecholamine synthesis with alpha-methyl-p-tyrosine or a functional lesion of catecholamine storage with reserpine. The first three lesions appear to partially inhibit normal somatostatin secretion since in every instance hpGRF-44 administration induced a significant increase in plasma GH concentrations. In contrast, reserpine blocked the GH response to hpGRF-44, presumably by stimulating somatostatin secretion. The pituitary GH response to hpGRF-44 in the above described models was enhanced by pretreatment of the rats with antibodies against somatostatin. The pituitary GH response to repeated injections of hpGRF-44 was also evaluated in rats with an anatomical lesion of the arcuate nucleus or a functional lesion of catecholamine synthesis. The maximum GH response did not vary over time to the repeated injections of hpGRF-44 in rats with lesions of the arcuate nucleus; however, interruption of catecholamine synthesis resulted in a significant decrease in the GH response to hpGRF-44 over time.     

Effects of growth hormone releasing factor on pancreatic secretion in vivo and in vitro

Growth hormone releasing factor (GRF), a 44-residue peptide originally isolated from human pancreatic tumors, shows structural similarities to the members of the secretin-vasoactive intestinal peptide (VIP) peptides. This study was designed to determine the effects of human GRF (hGRF-(1-44] on pancreatic secretion in vivo in conscious dogs and in vitro in dispersed rat pancreatic acini. GRF given i.v. in graded doses in dogs caused a small but significant stimulation of pancreatic HCO3- and protein outputs and potentiated secretin- and cholecystokinin (CCK)-induced pancreatic HCO3- but not protein secretion. When given together with somatostatin, GRF failed to reverse the inhibitory action of this peptide on HCO3- and protein responses to secretin plus CCK in dogs. Studies in vitro dispersed rat pancreatic acini showed that GRF added to the incubation medium of these acini caused an increase in basal amylase release and shifted to the left the amylase dose-response curve to caerulein and urecholine but failed to affect the amylase response to VIP. This study indicates that GRF in vivo stimulates basal and augments secretin- or CCK-induced pancreatic HCO3- secretion and that this is probably due to direct stimulatory action of the peptide on pancreatic secretory cells.     

Characteristics of phorbol ester stimulated growth hormone release: inhibition by insulin-like growth factor I, somatostatin, and low calcium medium and comparison with growth hormone releasing factor

TPA (12-O-tetradecanoylphorbol 13-acetate) is one of a class of compounds known as tumor promoters which perturb the inositol phosphate pathway in a number of cells. We have used TPA in a dispersed rat adenohypophysial cell system to probe the characteristics of growth hormone (GH) release. In this system we have found that the cells release GH in response to low concentrations of TPA: the EC50 was 0.23 +/- 0.05 nM (n = 6) and the maximal concentration was 5 nM. However, the maximal TPA-induced GH release was only 34 +/- 5% (n = 7) of the GH released by maximal growth hormone releasing factor (GRF) suggesting TPA releases a subpool of stored GH. Both somatostatin and insulin-like growth factor I inhibit GH release stimulated by TPA to the same extent as that stimulated by GRF, showing that the normal inhibitory control mechanism of release is not altered. Incubation in a low calcium medium that totally blocks GRF-stimulated GH release also inhibits TPA-stimulated GH release. The calcium channel blockers nifedipine and diltiazem both partly inhibit GRF- and TPA-stimulated GH release, showing some component of the calcium necessary for GH release arises from influx across the cell membrane.     

Interaction of human pancreatic growth hormone-releasing factor, thyrotropin-releasing hormone, and somatostatin on growth hormone release in chickens

The effects of synthetic somatostatin (SRIF) on serum growth hormone (GH) concentrations stimulated by exogenous administration of synthetic thyrotropin-releasing hormone (TRH) and/or human pancreatic GH-releasing factor (hpGRF) were investigated in 4-week-old cockerels. In addition, the additive effects of TRH and hpGRF on serum GH were examined. TRH and hpGRF, when given in combination intravenously, produced an additive effect on serum GH concentration that peaked 10 min after the injection. The somatostatin did not significantly affect basal GH concentrations when given alone, but did significantly decrease the magnitude of the GH response to hpGRF. In contrast, SRIF did not significantly decrease the stimulatory effects of TRH on GH release. These results suggest that TRH and hpGRF are potent GH releasers in vivo and that their stimulating effects on GH release are additive, suggesting different mechanisms for their stimulation. The results obtained from the combination studies suggest that the main site of the stimulatory action of hpGRF is at the pituitary, and that SRIF significantly inhibited the rise in serum GH induced by a synthetic hpGRF, but not that induced by TRH.     

Developmental correlation between hypothalamic somatostatin and hypophysial growth hormone

Summary The objective of the present study was to determine, by means of immunocytochemistry, the age in fetal development at which GH is first detectable in the pituitary gland and somatostatin in the median eminence, and to correlate temporally the development of these two hormones throughout the remainder of pregnancy. Mice were studied at 15–19 days of gestation with the peroxidase-antiperoxidase (PAP) technique of Sternberger. Somatotropes in the pars distalis were initially detected at 16 days of gestation and by 17 days they were a prominent component of the parenchymal cell population of the hypophysis. These cells were ovoid and distributed uniformly throughout the pars distalis; many were located adjacent to sinusoidal capillaries. Their number and staining intensity increased by 19 days. Somatostatin was not consistently observed in the median eminence until 19 days of gestation. Reaction product indicative of the presence of somatostatin in presumptive nerve endings was located on the ventral surface of the median eminence and in the external lamina of the infundibulum in proximity to the superficial portal capillaries. Results of the present investigation support the concept that the potential for neuroendocrine control of GH secretion exists in the mouse by the end of fetal development. Several hypotheses concerning the temporal relationship between the appearance of somatostatin in the hypothalamus and of GH in the anterior pituitary gland are discussed.Supported by a Biomedical Research Support Grant (NIH RR 5417). Appreciation is extended to the National Pituitary Agency, NIAMDD for the following radioiodination-grade hormones: hGH, rPRL, rTSH, rFSH and hCG     

Stimulation of growth hormone release in dwarf and normal chickens by thyrotrophin releasing hormone (TRH) or human pancreatic growth hormone releasing factor (hpGRF)

The effect of thyrotrophin releasing hormone (TRH) or human pancreatic growth hormone releasing factor (hpGRF) on growth hormone (GH) release was studied in both dwarf and normal Rhode Island Red chickens with a similar genotype except for a sex-linked dw gene. Both TRH (10 micrograms/kg) and hpGRF (20 micrograms/kg) injections stimulated plasma GH release within 15 min in young and adult chickens. The increase in GH release was higher in young cockerels than that in adult chickens. The age-related decline in the response to TRH stimulation was observed in both strains, while hpGRF was a still potent GH-releaser in adult chickens. The maximal and long acting response was observed in young dwarf chickens, suggesting differences in GH pools releasable by TRH and GRF in the anterior pituitary gland. The pituitary gland was stimulated directly by perifusion with hpGRF (1 microgram/ml and 10 micrograms/ml) or TRH (1 microgram/ml). Repeated perifusion of GRF at 40 min intervals blunted further increase in GH release, but successive perifusion with TRH stimulated GH release. The results suggest the possibility that desensitization to the effects of hpGRF occurs in vitro and that the extent of response depends on the number of receptors for hpGRF or TRH and/or the amount of GH stored in the pituitary gland.     

Growth hormone releasing factor: comparison of two analogues and demonstration of hypothalamic defect in growth hormone release after radiotherapy.

Human pancreatic growth hormone releasing factor (hpGHRF(1-40] stimulates the release of growth hormone in normal subjects and some patients with growth hormone deficiency. A study comparing the shorter chain amidated analogue hpGHRF(1-29) with an equivalent dose of hpGHRF(1-40) in seven normal subjects showed no significant difference in growth hormone response between the two preparations. Six patients with prolactinomas were also tested; these patients had received megavoltage radiotherapy previously but had developed growth hormone deficiency as shown by insulin induced hypoglycaemia. In all six patients 200 micrograms hpGHRF(1-40) or hpGHRF(1-29)NH2 produced an increase in the serum growth hormone concentration. These data suggest that hpGHRF(1-29)NH2 may be useful for testing the readily releasable pool of growth hormone in the pituitary and that cases of hypothalamo-pituitary irradiation resulting in growth hormone deficiency may be due to failure of synthesis or delivery of endogenous GHRF from the hypothalamus to pituitary cells.