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Previous in vitro and in vivo studies in rat demonstrated that continuous exposure to GHRPs results in progressive attenuation of GH response. During GHRP-6 infusion in rats, GH remained elevated above spontaneous baseline and the normal GH pulsatile secretory pattern was disrupted; at the end of GHRP-6 infusion, plasma GH levels fell without resumption of normal pulsatile GH. Similar results were obtained in human. Number, duration and height of GH pulses, incremental pulse amplitude, interpeak valley concentration and individual pulse areas were significantly greater during GHRP-6 infusion than during saUne administration; as a consequence, plasma IGF-1 increased significantly; at the end of the infusion, GH response to a subsequent GHRP-6 bolus injection was significantly reduced; this attenuation of GH response was not caused by depletion of pituitary GH stores, since the response to GHRH bolus was enhanced by prior infusion of GHRP-6 (8). Daily oral administration of MK-0677 for 1 week increased circulating IGF-1 levels together with an enhancement of GH pulse frequency, but without detectably increased GH secretion. Effects of chronic administration of GHRP on hypothalamic structures received little attention.
In dwarf (dw/dw) female rats treated with GHRP-6 (1 mg/kg per 24 h) continuously for 14 days, a significant selective increase of GHRH mRNA in the posterior arcuate nucleus was seen, but no significant effect was observed in neurons of the anterior or ventromedial parts of the same nucleus. In addition, SRIH mRNA levels in the posterior periventricular nucleus were decreased. It was early recognized in clinical studies that GHRP-6 also stimulates both prolactin and Cortisol release. Bowers et al. observed a 2-foId rise in serum Cortisol and prolactin levels after i.v. injection of GHRP-6, using a dose of Ijng/kg; this increase was moderate as compared with the 120-fold increase in serum GH levels, but significant and unlikely related to stress effects; no change in LH and TSH was observed. Massoud et al. reported in human the dose-response curves for GH, prolactin and Cortisol following i.v. injection of increasing doses (0.125-1 }ig/kg) of hexarelin; GH dose-response curve reached a plateau with a dose of 1 jag/kg of hexarelin; GH maximal increase was approximately 100-fold; prolactin dose-response curve paralleled that of GH but prolactin increase was much smaller (approximately 80% increase); serum Cortisol concentrations also increased moderately, but significantly; this increase of Cortisol was not observed in all subjects; a maximal increase of approximately 40% in Cortisol levels occurred with a dose of 0.5 ^g/kg; no change in TSH, insulin or blood sugar levels was observed in this study. In monkey, no change in prolactin and TSH plasma levels was observed after GHRP-6 injection. Small, but consistent elevations in ACTH and Cortisol secretion were seen in other human studies as well as in animal models.
Acute administration of L-692,585 stimulated ACTH and Cortisol secretion in beagles; however, increment of both hormones levels was far less than that of GH level; indeed, increase in Cortisol was 2- to 3-fold as compared to 10- to 20-fold for GH; therefore, ACTH and Cortisol stimulation following administration of L-682,585 did not induce a maximal adrenal response but rather approximated an endogenous pulse (46); increase in Cortisol secretion was similar in magnitude using L-692,429, another non-peptidic GHS. GHS stimulatory effect on the hypothalamicpituitary- adrenal axis seems however transient and may not constitute an important side-effect during chronic treatment with these molecules. GHRP-6 mechanisms of action on the pituitary-adrenal axis have been investigated in animal studies. Several lines of evidence suggest a hypothalamic site of action for GHRPs. GHRPs do not directly stimulate glucocorticoid release from the adrenal glands and ACTH secretion from the pituitary gland. They do not synergize with GHRH to release more ACTH in vivo as they do to release GH. Furthermore, ACTH response to GHRP-6 injection was abolished in rats with transected pituitary stalk. Therefore, GHRPs and their analogues probably interact with the hypothalamic peptidergic systems controlling ACTH release such as corticotropin releasing hormone (CRH) and arginine vasopressin (AVP). Thomas et al. indirectly tested this hypothesis in rat and measured plasma ACTH levels after GHRP-6, CRH or AVP, alone or combined; GHRP-6 given together with CRH did not increase ACTH levels beyond its response to CRH alone whereas association of GHRP-6 and AVP markedly increased ACTH levels as compared with the effects of AVP alone (x 2.4); these data suggest that GHRP-6 acts on the hypothalamus to stimulate ACTH release; this effect is probably mediated at least partly by release of CRH.
GHS effect on ACTH is regulated by glucocorticoids. Indeed, Thomas et al. also found that GHRP-6 induced ACTH release was higher in animals with the lowest basal ACTH and corticosterone output; these findings are probably related to decreased glucocorticoid feedback on hypothalamic CRH neurons. However, in human, hexarelin showed no synergistic effect with either AVP or CRH, suggesting that the ACTH-releasing activity of GHS may be, at least partly independent of both CRH and AVP. In our laboratory, using the sheep model, a rise in CRH levels in HPB after GHRP-6 i.v. injection (2 mg/animal) was recently confirmed; in four animals, we observed a 2-fold increase in CRH levels; AVP release into HPB showed a 1.6-fold increase (G. Thomas, V. Guillaume, I. Robinson, C. Oliver, manuscript in preparation), suggesting AVP involvement in sheep, in contrast to what is assumed in rat. CRH and AVP are both synthesized in neurons of the paraventricular nucleus (PVN), where GHS receptors have recently been identified using in situ hybridization.
However, expression of GHS receptor is much higher in the arcuate nucleus; many of the arcuate neurons are NPY positive and it is known that PVN receives major projections from arcuate NPY neurons; it is therefore conceivable that GHRPs action on CRH and AVP neurons is indirectly driven, through NPY arcuate neurons. In mammals, GH secretion is pulsatile and influenced by various conditions, including stress, feeding and pharmacological manipulations, which involve central neurotransmitters. Based on experiments performed in male rat, it is generally believed that both GH secretion pulsatile pattern and GH response to physiological or pharmacological stimuU depend upon the exquisite interrelationship between GHRH and SRIH secretion and pituitary action. However, inter-species differences in GH secretion and neuroregulation have been demonstrated. Indeed, in sheep no obvious correlation was found between most GH peaks and simultaneous increase in GHRH release and decrease in SRIH release into HPB. Besides, in several species, a supramaximal dose of GHRH and several GH stimulating factors (e.g. cholinergic drugs, clonidine) have synergistic effects on GH stimulation. It is assumed that a reduction of SRIH release mediates cholinergic drugs and clonidine effects on GH secretion; however, no change in SRIH levels was detected in sheep HPB, following administration of these substances. It is tempting to speculate that a natural ligand for GHS receptors is involved in GH regulation together with both GHRH and SRIH.
The presence of such an endogenous ligand was recently reported in portal plasma of ovariectomized ewes using an in vitro assay (based on intracellular calcium responses by HEK-293 ABO cells expressing the recombinant porcine GHS receptor); biological activity of this putative natural ligand in portal plasma was found to be correlated with GH peaks. GHS may potentiate GH release and contribute to GH neuroregulation through another mechanism; indeed, Kamegai suggested that GHS-induced GHRH secretion may stimulate GHS receptor expression. GHS receptor expression in the rat arcuate and ventromedial nucleus is highly sensitive to GH, being markedly increased in the dw/dw dwarf strain and decreased after chronic (6 days) treatment with bovine GH. Expression of GHS receptor was unaltered by continuous s.c. infusion of GHRP-6. These data suggest that hypothalamic GHS receptor is involved in feedback regulation of GH, adding some evidence for the participation of an endogenous GHS receptor ligand in the regulation of GH secretion. There is now clear evidence that GHS stimulate GH release through a dual action on the pituitary and the hypothalamus. GHRH neurons are presumably the main targets of GHS. However, participation of SRIH and of a putative endogenous GHS receptor ligand cannot be excluded. The relative contribution of the pituitary and the hypothalamus in the GHSinduced GH release is still unknown.