|
In sheep, we were able to demonstrate GHRH release in vivo after GHRPs injection; acute i.v, injection of hexarelin (1 mg) to adult rams induced a significant 2.5-fold enhancement of GHRH release into hypophysial portal blood (HPB) which lasted 45 min; in these animals, a 2.3-foId increase in plasma GH was observed and this GH rise was still detected 60 min after hexarelin injection; SRIH levels in HPB did not change throughout the study; the magnitude of GHRH increase after acute hexarelin administration was similar to that observed after other pharmacological stimulations of GH release, suggesting that the GHRH rise after hexarelin injection maybe sufficient to account for GH stimulation.
In another study, Fletcher gave to conscious ewes a GHRP-6 bolus injection followed by a 2-hour GHRP-6 infusion and measured GHRH and SRIH secretion in HPB; a 5.3-fold increase in plasma GH levels was observed 5-10 min after the GHRP-6 bolus injection, without a significant coincident release of GHRH; during the infusion period, there was a significant 50% increase in GHRH pulse frequency without any change in GHRH pulse amplitude; mean portal SRIH concentrations, pulse frequency and amphtude were unchanged; they concluded that GHRP-6 acts at the hypothalamic level or higher centres of the brain; however, under these experimental conditions, GH secretory response to GHRP-6 injection does not appear to be the result of GHRP-6 action on GHRH or SRIH hypothalamic neurons. The difference between Guillaume's and Fletcher's studies may be explained by the sex of the animals and by the greater potency of hexarelin. Using in situ hybridization, prominent expression of GHS receptor in the rat arcuate and ventromedial nucleus was demonstrated, supporting a direct action of GHS at the level of GHRH neurons in the hypothalamic nucleus; abundance of GHS receptors was higher in the hypothalamus than in the anterior pituitary gland. From the studies mentioned, a general consensus emerged that GHRH is integrally involved in GHS mechanisms of action.
By contrast, studies questioning GHS putative influence on SRIH neurons are far less conclusive. Indeed, no change in SRIH release into HPB has been observed in both studies performed in sheep. GHS receptor expression was either barely or not detectable in neurons of the periventricular nucleus, the major source of SRIH released into HPB and no increase in Fos immunoreactivity was detected in these neurons following GHRP-6 injection. Involvement of SRIH has also been addressed in two studies performed in rats pretreated with anti-SRIH antiserum. It was assumed that if GHS release GH through inhibition of SRIH secretion or action, SRIH antiserum pretreatment would not further increase the GH response to GHS. In one study, an augmented response to i.v. GHRP-6 was demonstrated following immunoneutralization of SRIH suggesting that SRIH is not involved in GHRP stimulation of GH release. Opposite results were obtained in another study; indeed, in freely moving rats, GHRP-6 induced GH release was not further increased by previous administration of anti-SRIH antiserum.
These last data suggest that GHRP-6 suppresses the somatostatinergic tone. They proposed that the discrepancy between both results is related to the use of different paradigms: stress-free conditions in conscious rats in their study as opposed to stressful conditions known to increase somatostatinergic tone in the former study, where therefore, SRIH immunoneutralization might not have been sufficient. Altogether, these studies bring no definite proof of an effect of GHS on GH secretion through decreased SRIH secretion into HPB. Nevertheless, an antagonistic action of GHS on SRIH inhibitory influence at the hypothalamic level was demonstrated; i.c.v. pretreatment with octreotide blocked GH-stimulating effect of i.c.v. (but not i.v.) administration of GHRP-6. these findings support an action of GHS on SRIH neurons set in the intrahypothalamic neuronal network involved in GH neuroregulation. Several experimental and clinical studies suggest that GHS may influence the release into HBP of another hypothalamic hormone than GHRH or SRIH. In a study by Bowers et al. (18), effects of GHRP-6 and GHRH on rat pituitary cell cultures were additive with only a small synergistic effect.
In vivo, a 2.5~3-fold synergism was observed with GHRP plus GHRH which cannot be simply explained by an interaction of these peptides at the pituitary level; since GHRP-6 and GHRH synergistic action on GH release was not explained by inhibition of SRIH or stimulation of GHRH (indeed, synergism was observed between GHRP-6 and supramaximal doses of GHRH), they suggested that GHRP-6 releases an unknown hypothalamic factor (U-factor) which may be an endogenous GHS receptor ligand interacting with GHRH on pituitary cells to release GH synergistically. A study performed in monkey by Malozowki supports the same hypothesis; indeed, GHS produced a greater maximal response than GHRH suggesting that a factor other than GHRH may be involved in its action either at the pituitary or hypothalamic level; pretreatment with propranolol, which is assumed to inhibit SRIH release, enhanced GH response to GHRP-6 suggesting that GHRP-6 does not affect SRIH tone; this interpretation conforms with previously discussed experiments performed in sheep, showing no change in portal SRIH levels following hexareUn or GHRP-6 administration. In human, GHRP-6 and hexarelin were able to potentiate GH release in response to a maximally stimulating dose of GHRH; stimulation of hypothalamic U-factor secretion may account for this phenomenon. Other clinical findings support this hypothesis.
In human, the GH-releasing activity of hexarelin or GIIRP-6 is partly refractory to the infusion of SRIH or to pharmacological manipulations (administration of muscarinic antagonist or p-2 adrenergic agonist) which are thought to inhibit GH secretion through SRIH release. SRIH and GHS may act as mutual functional antagonists at the pituitary and/or hypothalamic level and involve the U-factor or an endogenous GHS receptor ligand.