In vitro studies have been performed to demonstrate and characterize specific binding sites for synthetic GH secretagogues (sGHS) on membranes from pituitary gland and different human brain regions. A binding assay for sGHS was established using a peptidyl sGHS (Tyr-Ala-hexarelin) which had been radioiodinated to high specific activity at the Tyr residue. Specific binding sites for 125 I-labelled Tyr-Ala-hexarelin were detected mainly in membranes isolated from pituitary gland and hypothalamus, but they were also present in other brain areas such as choroid plexus, cerebral cortex, hippocampus and medulla oblongata with no sex-related differences. In contrast, negligible binding was found in the thalamus, striatum, substantia nigra, cerebellum and corpus callosum. The binding of 125 I-labelled Tyr-Ala-hexarelin to membrane-binding sites is a saturable and reversible process, depending on incubation time and pH of the buffer. Scatchard analysis of the binding revealed a finite number of binding sites in the hypothalamus and pituitary gland with a dissociation constant (K d ) of (1·5 0·3) 10 9 and (2·1 0·4) 10 9 mol/l respectively. Receptor activity is sensitive to trypsin and phospholipase C digestion, suggesting that protein and phospholipids are essential for the binding of 125 I-labelled Tyr-Ala-hexarelin. The binding of 125 Ilabelled Tyr-Ala-hexarelin to pituitary and hypothalamic membranes was displaced in a dose-dependent manner by different unlabelled synthetic peptidyl (Tyr-Ala-hexarelin, GHRP2, hexarelin, GHRP6) and non-peptidyl (MK 0677) sGHS. An inhibition of the specific binding was also observed when binding was performed in the presence of [-Arg 1 --Phe 5 --Trp 7,9 -Leu 11 ]-substance P, a substance P antagonist that has been found to inhibit GH release in response to sGHS. In contrast, no competition was observed in the presence of other neuropeptides (GHRH, somatostatin, galanin or Met-enkephalin) which have a known influence on GH release.In conclusion, the present data demonstrate that sGHS have specific receptors in human brain and pituitary gland and reinforce the hypothesis that these compounds could be the synthetic counterpart of an endogenous GH secretagogue involved in the neuroendocrine control of GH secretion and possibly in other central activities.

At present, the mechanism(s) underlying the reduced spontaneous and stimulated GH secretion in aging is still unclear. To obtain new information on this mechanism(s), the GH responses to both single and combined administration of GH-releasing hormone (GHRH; 1 microgram/kg iv) and arginine (ARG; 30 g infused over 30 min), a well known GH secretagogue probably acting via inhibition of hypothalamic somatostatin release, were studied in seven elderly normal subjects and seven young healthy subjects. Basal GH levels were similar in both groups, while insulin-like growth factor-I levels were lower in elderly subjects (76.7 +/- 9.2 vs. 258.3 +/- 29.2 micrograms/L; P = 0.01). In aged subjects GHRH induced a GH increase (area under the curve, 314.9 +/- 91.9 micrograms/L.h) which was lower (P = 0.01) than that in young subjects (709.1 +/- 114.4 micrograms/L.h). On the other hand, the ARG-induced GH increase in the elderly was not significantly different from that in young subjects (372.8 +/- 81.8 vs. 470.6 +/- 126.5 micrograms/L.h). ARG potentiated GH responsiveness to GHRH in both elderly (1787.1 +/- 226.0 micrograms/L.h; P = 0.0001 vs. GHRH alone) and young subjects (2113.0 +/- 444.3 micrograms/L.h; P = 0.001 vs. GHRH alone). The potentiating effect of ARG on the GHRH-induced GH response was greater in elderly than in young subjects (1013.0 +/- 553.5% vs. 237.9 +/- 79.1%; P = 0.0001); thus, the GH increase induced by combined administration of ARG and GHRH overlapped in two groups. In conclusion, these results show that, differently from the GHRH-induced GH increase, the somatotroph response to combined administration of ARG and GHRH does not vary with age. Our finding suggests that an increased somatostatinergic activity may underlie the reduced GH secretion in normal aging.

The individual role played by GH and IGF-I in the regulation of hypothalamic GHRH and SRIF gene expression is still object of debate. We have investigated the effect of exogenously administered recombinant hGH (rhGH) and recombinant hIGF-I (rhIGF-I) in ad libitum fed control and starved rats, the latter an animal model which is characterized by low circulating levels of endogenous GH and IGF-I. Adult male rats were fed ad libitum (C) or food-deprived (S) for 72 hours; rats in either C or S groups were treated with systemic administration of rhGH and rhIGF-I for 3 days. GHRH, SRIF and GH mRNA levels were evaluated by Northern and slot blot hybridization. Administration of rhGH (250 micrograms/kg/twice daily, sc) induced a significant inhibition of GHRH and a significant stimulation of SRIF mRNA levels in C rats; GH treatment was, however, ineffective on both neuropeptide mRNA levels in the S group. Continuous infusion of rhIGF-I (300 micrograms/kg/day, sc) induced a significant increase of SRIF levels in both C and S rats but did not modify GHRH mRNA levels in either group. In the pituitary, GH mRNA levels followed a pattern very similar to that of GHRH. These results provide evidence for a direct role of GH in the inhibition of GHRH mRNA levels; IGF-I appears more involved in the direct stimulation of SRIF mRNA levels.

We have reported Hexarelin (HEXA), an analog of growth hormone-releasing peptide 6 (GHRP-6), potently stimulates growth hormone (GH) secretion in infant and adult rats. This study was undertaken to further investigate Hexarelin's mechanisms of action. In 10-day-old pups, treatments with HEXA (80 micrograms/kg, b.i.d.) for 3-10 days significantly enhanced, in a time-related fashion, the GH response to an acute HEXA challenge. Qualitatively similar effects were elicited in pups passively immunized against growth hormone-releasing hormone (GHRH) from birth. In adult male rats, a 5-day pretreatment with HEXA (150 micrograms/kg, b.i.d.) did not enhance the effect of the acute challenge, and the same pattern was present after a 5-day pretreatment in male rats with surgical ablation of the mediobasal hypothalamus (MBH-ablated rats). In addition, in adult sham-operated rats, Hexarelin (300 micrograms/kg, i.v.) induced a GH response greater (p < 0.05) than that induced by GHRH (2 micrograms/kg, i.v.). However, in MBH-ablated rats 7 days after surgery, GHRH was significantly (p < 0.05) more effective than HEXA, and 30 days after surgery HEXA and GHRH evoked similar rises of plasma GH. Finally, the in vitro Hexarelin (10(-6) mol/l) effect was transient while GHRH (10(-8) mol/l) induced a longer lasting and greater GH release. Three different mechanisms, not mutually exclusive, are postulated for Hexarelin stimulation of GH secretion in vivo: a direct action on the pituitary, though of minor relevance; an indirect action that involves release of GHRH, of relevance only in adult rats; and an action through the release of a still unknown hypothalamic "factor", which in infant and adult rats elicits GH release acting sinergistically with GHRH.

It is generally accepted that growth hormone influences its own secretion by modulating the activity of GHRH and SRIF neurons. To investigate if GH feedback mechanisms are already operating in the early postnatal life of the rat, we have studied in 10-day-old pups the effects of rhGH and rhIGF-I administration on GHRH and somatostatin mRNA levels. The same experiment was also performed in pups passively immunized with an anti-GHRH antiserum from the day of birth. The latter animal model had been previously characterized for presenting reduced levels of circulating GH and IGF-I. In control pups, neither rhGH (250 micrograms/kg, b.i.d., sc) nor rhIGF-I (150 micrograms/kg, b.i.d., sc) administration induced significant changes of GHRH and SRIF gene expression. The passive immunization against GHRH induced per se a trend toward an increase and a reduction of GHRH and SRIF mRNA levels, respectively. Also in these rats the treatment for 3 days with rhGH and rhIGF-I did not further modify the GHRH and SRIF mRNA levels. Based on these results, we conclude that in the 10-day-old rat GH feedback mechanisms are poorly operative, though a direct ultra-short loop mechanism involving the GHRH and SRIF systems seems already operating.