Growth Hormone-releasing Peptide Receptor Research Articles

Dexamethasone stimulates the expression of ghrelin and its receptor in rat hypothalamic 4B cells

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides that strongly induce GH release. GHRPs act via a specific receptor, the GHRP receptor (GHSR), of which ghrelin is a natural ligand. GHRPs also induce adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRPs or ghrelin stimulate ACTH release via corticotropin-releasing factor (CRF) and arginin vasopressin in the hypothalamus. Stress-activated CRF neurons are suppressed by glucocorticoids in the hypothalamic paraventricular nucleus (PVN), while CRF gene is up-regulated by glucocorticoids in the PVN cells without the influence of input neurons. However, little is known about the regulation of ghrelin and GHSR type 1a (GHSR1a) genes by glucocorticoids in PVN cells. To elucidate the regulation of ghrelin and GHSR gene expression by glucocorticoids in PVN cells, here we used a homologous PVN neuronal cell line, hypothalamic 4B, because these cells show characteristics of the parvocellular neurons of the PVN. These cells also express ghrelin and GHSR1a mRNA. Dexamethasone increased ghrelin mRNA levels. A potent glucocorticoid receptor antagonist, RU-486, significantly blocked dexamethasone-induced increases in ghrelin mRNA levels. Dexamethasone also significantly stimulated GHSR1a mRNA and protein levels. Finally, ghrelin increased CRF mRNA levels, as did dexamethasone. Incubation with both dexamethasone and ghrelin had an additive effect on CRF and ghrelin mRNA levels. The ghrelin–GHSR1a system is activated by glucocorticoids in the hypothalamic cells.

Growth hormone-releasing peptide-2 stimulates secretion and synthesis of adrenocorticotropic hormone in mouse pituitary

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides which induce strong GH release in both animals and humans. Among them, GHRP-2 is known to stimulate GH release by acting at both hypothalamic and pituitary sites, but also induces adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRP-2 may stimulate ACTH release directly via GHRP receptor type 1a in ACTH-producing tumors. GHRP-2 increases ACTH secretion in rat in vivo, but not ACTH release from rat primary pituitary cells. In the present study, in order to elucidate the mechanism underlying ACTH secretion by GHRPs, mouse pituitary cells were stimulated by GHRP-2. GHRP receptor mRNA was expressed in the mouse pituitary, and GHRP-2 directly stimulated secretion and synthesis of ACTH in the mouse anterior pituitary cells. GHRP-2 increased intracellular cyclic AMP production. H89, a potent protein kinase A (PKA) inhibitor, and bisindolylmaleimide I, a selective protein kinase C (PKC) inhibitor, inhibited the GHRP-2-induced ACTH release, and that H89, but not bisindolylmaleimide I, inhibited the GHRP-2-induced proopiomelanocortin mRNA levels. Together, the GHRP-2-induced ACTH release was regulated via both PKA and PKC pathways in the mouse pituitary cells, while ACTH was synthesized by GHRP-2 only via the PKA pathway.

Exaggerated Response of Adrenocorticotropic Hormone to Growth Hormone-Releasing Peptide-2 Test in Cushing's Disease -Case Report-

A 47-year-old woman presented with a pituitary microadenoma manifesting as typical Cushing's syndrome. The diagnosis was Cushing's disease based on the endocrinological findings. Plasma adrenocorticotropic hormone (ACTH) levels were greatly increased from 66 pg/ml to 2490 pg/ml (about 38-fold) in response to the administration of 100 microg human growth hormone-releasing peptide (GHRP)-2. GHRP receptor type 1a messenger ribonucleic acid was detected in the tumor. Therefore, GHRP-2 may stimulate ACTH via the GHRP receptor type 1a in pituitary ACTH-producing tumor. The GHRP-2 test, currently clinically available in Japan, may be a useful diagnostic tool for Cushing's disease.

The neuroendocrine response to stress is a dynamic process.

The initial neuroendocrine response to critical illness illness consists primarily of activated anterior pituitary function, the peripheral anabolic pathways being inactivated. This response presumably provides metabolic substrates, establishes the host's defences and is thus considered to be adaptive and beneficial. It was previously assumed that the acute stress response persisted throughout the course of critical illness, but this assumption has now been disproved. Indeed, a uniformly reduced pulsatile secretion of growth hormone, thyroid-stimulating hormone, prolactin and luteinizing hormone has been observed in protracted critical illness, impairing the function of target organs. A reduced availability of thyrotropin-releasing hormone, gonadotropin-releasing hormone, the endogenous ligand of the growth hormone-releasing peptide receptor (possibly ghrelin) and, in very long-stay critically ill men, also growth hormone-releasing hormone seems to be involved. The pulsatile secretion of growth hormone, thyroid-stimulating hormone, prolactin and luteinizing hormone can be re-established by relevant combinations of releasing factors, which also substantially increase the circulating levels of insulin-like growth factor-1, growth hormone dependent binding proteins, thyroxine, tri-iodothyronine and testosterone. Active feedback inhibition loops prevent the target organs being overstimulated. The metabolism is altered in a beneficial way when growth hormone-secretagogues, thyrotropin-releasing hormone and gonadotropin-releasing hormone are administered together, whereas the effect of single-hormone treatment is minor and accompanied by side-effects. This new concept of a selectively reduced stimulation of pituitary function in the chronic phase of critical illness unveils new therapeutic perspectives to reverse the paradoxical wasting syndrome' and intensive care dependency.

Somatostatin Octapeptides (Lanreotide, Octreotide, Vapreotide, and Their Analogs) Share the Growth Hormone–Releasing Peptide Receptor in the Human Pituitary Gland

The binding affinity of somatostatin-14 (SRIF), various SRIF derivatives, and some peptides belonging to the growth hormone-releasing peptide (GHRP) family to specific receptors for SRIF and GHRP in the human pituitary gland has been measured. GHRP receptors have been identified using [125I]Tyr-Ala-hexarelin, a peptide that thas been demonstrated to be a potent growth hormone (GH) releaser in humans. Tyr-Ala-hexarelin binding was displaced in a dose-dependent manner by different GHRPs (hexarelin, GHRP-2, and EP-51216). Surprisingly, some SRIF octapeptide derivatives such as vapreotide, lanreotide, octreotide, and their analogs were also able to displace the GHRP ligand. By contrast, no inhibition of Tyr-Ala-hexarelin binding was observed in the presence of SRIF or SRIF derivatives (SRIF H-2186, H-2485, and H-3382) that are known to have a weak SRIF-like activity. When [125I]Tyr1-SRIF-14 was used as a ligand, we observed displacement with SRIF and the octapeptide SRIF analogs but not with GHRPs and other SRIF derivatives. The results point to a sharing of the GHRP receptor with the octapeptide SRIF analogs, but not SRIF. Our data are consistent with the hypothesis that the putative natural GH secretagogue ligand may be a growth hormone release inhibiting factor that is different from SRIF and that is antagonized by GHRP.

The hexapeptide KP-102 (D-ala-D-beta-Nal-ala-trp-D-phe-lys-NH(2)) stimulates growth hormone release in a cichlid fish (Ooreochromis mossambicus).

Abstract Studies in mammals have shown that synthetic Met-enkephalin derivatives, called growth hormone-releasing peptides (GHRPs), stimulate growth hormone (GH) release. The present study was conducted to determine whether the GHRP, KP-102, specifically stimulates GH release in a teleost. Tilapia (Oreochromis mossambicus) were given a single intraperitoneal injection of KP-102 (D-Ala-D-beta;-Nal-Ala-Trp-D-Phe-Lys-NH(2)) or bovine GHRH(1-29)-amide or vehicle and blood was sampled at 1, 6 and 12 h after injection. KP-102 was administered at two doses of 1 ng/g and 10 ng/g body weight, whereas GHRH (positive control) was administered at a single dose of 10 ng/g body weight. Plasma levels of tilapia GH and prolactins (tPRL(177) and tPRL(188)) were determined by radioimmunoassay. As expected, GHRH injection significantly (P<0.001) elevated plasma GH levels (ng/ml) in tilapia at 6 h post-injection. KP-102 also significantly elevated GH levels (at the low dose) at 6 (P<0.05) and 12 (P<0.01) hours post-injection. There were no significant effects on plasma PRL(s) levels, although mean levels of both PRLs were elevated at 6 h post-injection. These results show for the first time that GHRPs stimulate GH release in teleosts and suggest that the GHRP receptor and possibly a "Ghrelin-like" ligand are also present in lower vertebrates.

Differential Orexigenic Effects of Hexarelin and Its Analogs in the Rat Hypothalamus: Indication for Multiple Growth Hormone Secretagogue Receptor Subtypes

We have previously reported that hexarelin and some of its analogs, including EP 50885, stimulated GH secretion and feeding after systemic administration in the rat, whereas EP 40904 selectively stimulated food intake and EP 40737 only GH release. The precise mechanism of growth hormone-releasing peptides (GHRPs) actions is still unclear, but the integrity of the arcuate nucleus of the hypothalamus (ARC) appears crucial for their endocrine effects. To better characterize the site(s) and mechanisms(s) of the orexigenic action of GHRPs, we have investigated their effects after infusion into the arcuate, paraventricular, ventromedial and medial preoptic areas of the hypothalamus. Food intake was measured for 60 min following injection of the test compound (2 µg/rat). Hexarelin, EP 40904 and EP 50885 had significant orexigenic effects after injection into the ARC. A specific NPY antagonist significantly inhibited the effect of hexarelin, whereas a GHRH antagonist was ineffective. In the paraventricular nucleus, only EP 50885 stimulated feeding, whereas all peptides were ineffective in the ventromedial nucleus and medial preoptic area. Taken altogether, these results demonstrate that GHRPs are endowed with site-specific orexigenic actions and that endogenous NPY, but not GHRH, mediates these effects. The additional orexigenic action of EP 50885 in the paraventricular nucleus suggests the existence of a GHRP receptor subtype different from the already cloned one.

Identification and characterization of a new growth hormone-releasing peptide receptor in the heart.

Hexarelin, a synthetic hexapeptide of the growth hormone-releasing peptide (GHRP) family with strong growth hormone (GH)-releasing activity, features protecting activity against postischemic ventricular dysfunction in hearts from GH-deficient and senescent rats. To document whether hexarelin action is mediated through specific cardiac receptors, perfusion of Langendorff rat hearts with hexarelin and binding studies were carried out. In the Langendorff rat heart system, hexarelin induced a dose-dependent increase in coronary perfusion pressure. Nifedipine, chelerythrine, and bisindolylmaleimide partially inhibited the vasoconstriction induced by hexarelin, suggesting that this effect was mediated at least in part by L-type Ca(2+) channels and protein kinase C. In contrast, diclofenac and 1-(7-carboxyheptyl)imidazole were without effect, suggesting that prostaglandins and thromboxanes were not involved in the coronary vasoconstriction induced by hexarelin. To characterize the hexarelin binding sites in the rat heart, [(125)I]Tyr-Bpa-Ala-hexarelin was used as photoactivatable radioligand in saturation and competitive binding studies. We specifically labeled a hexarelin receptor with an M(r) of 84 000 in rat cardiac membranes. Saturation binding curves revealed a single class of binding sites with a K(d) of 14.5 nmol/L and a density of 91 fmol/mg of protein. Competition binding studies gave an IC(50) of 2.9 micromol/L for hexarelin; MK-0677 and EP51389, both potent GH secretagogues, did not displace the binding of the photoactivatable derivative from rat cardiac membranes. Interestingly, both compounds were devoid of any vasoconstrictive activity. These results suggest the existence of a new class of hexarelin receptor in the heart, whose role in the regulation of the coronary vascular tone is yet to be determined.

Binding sites for growth hormone-releasing peptide

Growth hormone-releasing peptides (GHRPs) are known to release growth hormone (GH) in vivo and in vitro by a direct action on receptors in anterior pituitary cells. Measurement of second messengers released following somatotroph stimulation suggests the existence of more than one GHRP receptor subtype in the hypothalamic-pituitary system. Furthermore, hexarelin, a hexapeptide of the GHRP family and a potent GH secretagogue, is reported to increase left ventricular ejection fraction, suggesting the expression of specific myocardial GHRP binding sites. In order to confirm such a hypothesis, a photoactivatable derivative of hexarelin, Tyr-p-benzoyl phenylalanine-Ala-hexarelin, was developed. A putative GHRP receptor with an apparent relative molecular mass of 57,000 was specifically labelled and characterized in human, bovine and porcine anterior pituitary membranes using this hexarelin derivative. The existence of myocardial binding sites was also demonstrated using the same approach. The differential binding affinity of GHRP analogues to cardiac tissue raises the possibility of the existence of distinct GHRP receptor subtypes in the pituitary and the cardiovascular system, for which physiological roles have yet to be determined.

Hypothalamic and hypophyseal regulation of growth hormone secretion.

1. Regulation of pulsatile secretion of growth hormone (GH) relies on hypothalamic neuronal loops, major transmitters involved in their operation are growth hormone releasing hormone (GHRH) synthetized mostly in arcuate nucleus (ARC) neurons, and somatostatin (SRIH), synthetized both in hypothalamus periventricular (PVe) and ARC neurons. 2. Neurons synthetizing both peptides can inhibit each other in a reciprocal manner. Other neuropeptides synthetized in ARC neurons, such as galanin, or in ARC interneurons, such as neuropeptide Y (NPY), are able to modulate synthesis and release of GHRH and SRIH into the hypothalamohypophyseal portal system. 3. In addition, the hitherto uncharacterized endogenous ligand of the recently cloned growth hormone releasing peptide receptor, expressed mostly in the ARC, triggers GH release, presumably by actions on ARC interneurons. 4. Thyroid, gonadal, and adrenal steroid hormones also affect the GHRH-SRIH balance; a differential distribution of sex steroid receptors in the ARC and the PVe is likely to account for the different pattern of GH secretion in male and female animals. 5. Growth hormone itself is able to inhibit the amplitude of GH secretory episodes and to increase their frequency, by entering the brain (presumably by receptor-mediated internalization at the level of the choroid plexus) and acting subsequently on ARC neurons. 6. At the pituitary level, major neurotransmitters regulating GH cells act on receptors of the VIP/PACAP/GHRH family and of the somatostatin family, in particular, sst2 and sst3. Those are coupled to accumulation of cAMP as a second messenger. 7. In addition, patch-clamp experiments and measurement of intracellular Ca2+ indicate that GH cells present characteristic, GHRH-dependent, but self-maintained Ca2+ spikes and [Ca2+]i transients, which reflect adaptive mechanisms to constraints of episodic release. 8. Recent data on transcription factors affecting GH gene expression and somatotrope differentiation are also summarized. 9. Regulation and differentiation of somatotropes also depend upon paracrine processes within the pituitary itself and involve growth factors and several neuropeptides, for instance, vasoactive intestinal peptide, angiotensin 2, endothelin, and activin. 10. Finally, characteristic changes occur in the GH secretory pattern under discrete, pathological conditions, such as abnormal growth and dwarfism, diabetes, and acromegaly, as well as during inflammatory processes.

Identification of a Pituitary Growth Hormone-Releasing Peptide (GHRP) Receptor Subtype by Photoaffinity Labeling

Hexarelin, an analogue of GHRP-6, in which D-Tryptophan has been replaced by its 2-methyl derivative, is known to release growth hormone (GH) in vivo and in vitro by direct action on receptors present in anterior pituitary cells. Measurement of second messengers (c-AMP, Ca++, IP3) upon somatotrophs stimulation, suggests the existence of more than one GHRP receptor subtype. In order to document such an hypothesis, we have used a new photoactivatable derivative of Hexarelin, Tyr-Bpa-Ala-Hexarelin. This derivative was shown to be fully active in the release of GH in vivo with neonate rats. Using this photoactivatable ligand, we have specifically labeled a protein with an apparent Mr of 57 000 in human, bovine and porcine anterior pituitary membranes. Hexarelin and the spiroindoline sulfonamide MK-0677 displaced the Mr −57 000 photolabeled band with an apparent ED50 of 6×10−7 M and 2×10−5 M respectively. Taking into account the high efficiency (>60%) of covalent incorporation of the Bpa residue, this photoactivatable Hexarelin derivative has allowed the identification of a pituitary GHRP receptor subtype, which is apparently distinct from the recently cloned GH secretagogue receptor.

Identification of a pituitary growth hormone-releasing peptide (GHRP) receptor subtype by photoaffinity labeling.

Hexarelin, an analogue of GHRP-6, in which D-Tryptophan has been replaced by its 2-methyl derivative, is known to release growth hormone (GH) in vivo and in vitro by direct action on receptors present in anterior pituitary cells. Measurement of second messengers (c-AMP, Ca++, IP3) upon somatotrophs stimulation, suggests the existence of more than one GHRP receptor subtype. In order to document such an hypothesis, we have used a new photoactivatable derivative of Hexarelin, Tyr-Bpa-Ala-Hexarelin. This derivative was shown to be fully active in the release of GH in vivo with neonate rats. Using this photoactivatable ligand, we have specifically labeled a protein with an apparent Mr of 57,000 in human, bovine and porcine anterior pituitary membranes. Hexarelin and the spiroindoline sulfonamide MK-0677 displaced the Mr-57,000 photolabeled band with an apparent ED50 of 6x10(-7) M and 2x10(-5) M respectively. Taking into account the high efficiency (>60%) of covalent incorporation of the Bpa residue, this photoactivatable Hexarelin derivative has allowed the identification of a pituitary GHRP receptor subtype, which is apparently distinct from the recently cloned GH secretagogue receptor.

Growth Hormone-Releasing Peptides and Their Analogs

Growth hormone-releasing peptides (GHRPs) are a series of hepta (GHRP-1)- and hexapeptides (GHRP-2, GHRP-6, Hexarelin) that have been shown to be effective releasers of GH in animals and humans. More recently, a series of nonpeptidyl GH secretagogues (L-692,429, L-692,585, MK-0677) were discovered using GHRP-6 as a template. Some cyclic peptides as well as penta-, tetra-, and pseudotripeptides have also been described. This review summarizes recent developments in our understanding of the GHRPs, as well as the current nonpeptide pharmacologic analogs. GHRPs and their analogs have no structural homology with GHRH and act via specific receptors present at either the pituitary or the hypothalamic level. The GHRP receptor has recently been cloned and it does not show sequence homology with other G-protein-coupled receptors known so far. This evidence strongly suggests the existence of a natural GHRP-like ligand which, however, has not yet been found. Although the exact mechanism of action of GHRPs has not been fully established, there is probably a dual site of action on both the pituitary and the hypothalamus, possibly involving regulatory factors in addition to GHRH and somatostatin. Moreover, the possibility that GHRPs act via an unknown hypothalamic factor (U factor) is still open. The marked GH-releasing activity of GHRPs is reproducible and dose-related after intravenous, subcutaneous, intranasal, and even oral administration. The GH-releasing effect of GHRPs is the same in both sexes, but undergoes age-related variations. It increases from birth to puberty and decreases in aging. The GH-releasing activity of GHRPs is synergistic with that of GHRH and not affected by opioid receptor antagonists, while it is only blunted by inhibitory influences that are known to nearly abolish the effect of GHRH, such as neurotransmitters, glucose, free fatty acids, glucocorticoids, rhGH, and even exogenous somatostatin. GHRPs maintain their GH-releasing effect in somatotrope hypersecretory states, such as acromegaly, anorexia nervosa, and hyperthyroidism. On the other hand, GHRPs and their analogs have been reported to be effective in idiopathic short stature, in some situations of GH deficiency, in obesity, and in hypothyroidism, while in patients with pituitary stalk disconnection and in Cushing's syndrome the somatotrope responsiveness to GHRPs is almost absent. A potential role in the treatment of short stature, aging, catabolic states, and dilated cardiomyopathy has been envisaged.

Molecular Cloning and Gene Expression of Growth Hormone-Releasing Peptide Receptor in Rat Tissues

Yokote, R., M. Sato, S. Matsubara, H. Ohye, M. Niimi, K. Murao and J. Takahara. Molecular cloning and gene expression of growth hormone-releasing peptide receptor in rat tissues. Peptides 19(1) 15–20, 1998.—We cloned a fragment of the rat GH-releasing peptide (GHRP) receptor homologue and examined the tissue distribution of GHRP receptor mRNA in rats. Sequence analysis showed that the open reading frame is well conserved between rat and human with 96% identity in a 364-amino acid overlap. By reverse transcription-polymerase chain reaction we detected GHRP receptor mRNAs in the rat brain including the hypothalamus, anterior pituitary, and renal pelvis in twenty-eight tissues tested. Microdissection revealed that GHRP receptor mRNAs were localized predominantly in the arcuate nucleus and ventromedial hypothalamus.

Widespread transcription of the growth hormone-releasing peptide receptor gene in neuroendocrine human tumors.

GH-releasing peptides are a new class of potent GH secretagogs (GHS) in vivo and in vitro. In normal man GHS also elicit low but definite ACTH and prolactin secretion. Recently it was shown that patients with pituitary corticotrope adenomas respond to GHS with a dramatic rise in ACTH secretion, and it has been suggested that GHS may provide a diagnostic tool to differentiate Cushing's disease from the ectopic ACTH syndrome. GHS actions are mediated by a G protein-coupled receptor recently characterized and cloned in man and rat. In this study we analyzed GHS receptor (GHS-R) expression in various types of pituitary adenoma and in endocrine and non-endocrine lung tumors by RT-PCR. GHS-R transcription was detected in all normal pituitaries and GH-secreting adenomas as expected. The receptor was also transcribed in some prolactin-secreting adenomas and non-functioning adenomas, and, more strikingly, in all 18 ACTH-secreting pituitary adenomas studied. Furthermore, it was frequently expressed in endocrine bronchial tumors, especially carcinoids, whereas it was not found or barely detectable in non-endocrine bronchial tumors. Again ACTH-secreting carcinoids of the lung were all positive for GHS-R expression. These results show that GHS-R transcription is a common feature of endocrine tumors independent of their type and origin.