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Administration of GHS induces a rapid increase in plasma GH levels in a broad range of animal species i.e. rat, monkey, sheep, pig, chick, steer as well as in human. In vivo activities of GHRPs and GHRH have been compared. However comparison is intricate and depends upon many variables, such as structural form of GHS used, route of administration, age, sex, experimental procedure, and animal specie. However, GHRP-6 was found more efficient in primates than in rat, dog or farm animals. Walker et al. demonstrated that GHRP-6 was 5-20 times more effective in monkey {Macaca fasicularis) than in rat or dog; in another strain of monkeys (Cynomolgus macaque) GHRP-6 potency was lesser than that of GH; GHRP-6 and GHRH dose-response curves showed no paralleHsm, but unlike in the rat, GHRP-6 was able to evoke a much higher GH peak response than GHRH. In human, GHRP-6 is more potent than in other species; an i.v. bolus injection of 1 jig/kg b.w. GHRP-6 induced a greater GH release than GHRH, using the same dose and route of administration. The initial comparative analysis of GHRH and GHRPs effects on GH secretion suggested that their mechanisms of action were both different and complementary. Evidence for different mechanisms of action derives from the ability of GHRPs and GHRH to increase GH release beyond the maximum capacity of the other. Besides, homologous but not heterologous desensitization is observed after continuous infusion of GHRH or GHRP-6, followed by acute administration of one of these peptides. Different mechanisms of action were confirmed through identification of different receptors and signaling for GHS and GHRH.
In human, complementary effects of GHS and GHRH were found more striking in vivo than in vitro. Using maximally or submaximally effective doses of GHRP-6 and GHRH, GH secretory responses in vivo were potentiated rather than additive. Results obtained in vitro with the association of both substances are conflicting, showing either merely additive effects on rats and ovine pituitary cells, or direct synergism with GHRH on rat pituitary cells for both GHRP-6 and L-682,429. As discussed later on, the hypothalamus has been involved in the synergism between GHS and GHRH. Conversely, a functional hypothalamic-pituitary GHRH system is needed for the GH stimulating release of GHRP. Indeed, passive immunization against GHRH was shown to inhibit GHRP-6 induced GH release. Functionally intact GHRH receptors are required for pituitary action of GHRP-6, as shown by the lack of GHRP-6 evoked GH stimulation in GH deficient dwarf lit/lit mouse, whose GHRH receptor bears a point mutation in the N-terminal ligand-binding domain. In nine healthy 20-30 year-old men, Pandya et al. recently showed that the administration of a specific GHRH antagonist 20 minutes before i.v. injection of GHRP-6 severely blunts GH response to GHRP-6 (area under the curve: 376 ± 113 vs 1701 ± 278 |ig/l/min when saline was injected instead of GHRH antagonist); these data show that endogenous GHRH is necessary for most of GH response to GHRP-6 in human. These results diverge from those reported earlier by the same group in human using a different paradigm; a model of complete pituitary desensitization was designed to suppress involvement of endogenous GHRH; after a short term infusion of GHRH resulting in complete pituitary desensitization to a maximally effective dose of GHRH, GH rise in response to a bolus dose of GHRP-6 was fully preserved (16).
In agreement with the findings of Pandya et al., acute GHRP administration had only limited efficiency in individuals with GHRH deficiency. Since cultured pituitary cells respond to GHRP-6, it was first considered that GHS act primarily as a direct GH secretagogue at the pituitary level. The same group later demonstrated that GH response to GHRP-6 was higher in an hypothalamic-pituitary incubate than in an isolated pituitary incubate; these data support the concept that GHS have a hypothalamic as well as a direct pituitary site of action. Furthermore, GHS show equal potency with GHRH on GH release in vitro whereas in vivo, GH secretagogues are more efficient than GHRH in elevating plasma GH; this suggests that the pituitary gland is not the sole site of action of GHS. GH response to intracerebroventricular GHRP-6 injection was higher than that observed after systemic administration of the same dose, supporting the assumption of hypothalamic action of GHS. Anaesthesia reduced the amplitude of GH response to GHS providing another evidence for extrapituitary action of GHS; indeed, GH response to GHRP-6 is much smaller in urethane-anaesthetized than in conscious rat. Nevertheless, acute i.v. injection of GHRP-6 was still able to evoke GH release in hypothalamopituitary disconnected (HPD) sheep (wethers and ewes) indicating a pituitary site of action for this peptide; as expected, GHRP-6 was less potent than GHRH; indeed, response to GHRP-6 was 5-fold smaller in intact animals and 15-fold smaller in HPD animals; this difference may be explained by a stimulating effect of GHRP-6 on GHRH neurons and suggests that a component of GHRP-6 action is mediated through the hypothalamus. Similar findings have been reported with L-692,585 in rat and pig. In a group of 12 patients with hypothalamopituitary disconnection, GHRP-6 induced GH release was 15-fold lower than in control subjects; in these patients, GH response to GHRH was similar to that obtained in controls; they concluded that the potency of GHRP-6 action at the pituitary level is minimal and that its main action is mediated by hypothalamic structures.
Similar data have been reported by Hayashi. Several studies support this hypothesis. In rats, Dickson et al. showed that systemic administration of GHRP-6 (100 fig i.v.) activates a subpopulation of neurons in the arcuate nucleus of the hypothalamus which contains most of the GHRH neurons; increased Fos-like immunoreactivity (a marker of neuronal activation) was detected 90 min after GHRP-6 injection in many cells throughout the ventrolateral regions of the arcuate nucleus; this effect is highly specific since only a slight increase in Fos-like immunoreactivity was observed in the supraoptic nucleus and no change was seen in other hypothalamic nuclei studied (ventromedial, periventricular and paraventricular nuclei); acute i.v. injection of GHRP-6 using the same dose, stimulated the firing of putative GHRH neurons in the arcuate nucleus; this excitation began one minute after GHRP administration and lasted for at least 10 min. This response does not reflect a feedback effect involving increased GH release or increased plasma level of GHRH, since administration of a high dose of GHRH had no effect on c-fos expression in the arcuate nucleus. In the lit/lit dwarf mouse which lacks functional pituitary GHRH receptors, administration of GHRP-6 evoked arcuate nucleus neurons activation, demonstrating that central actions of GHRP-6 are not mediated by GHRH, GH or IGF-1. Dense Fos nuclear immunostaining was induced throughout the ventral part of this nucleus when GHRP-6 was given i.c.v., using a much lower dose than the dose required to stimulate GH secretion when i.v. injection is used. Similar results were obtained with L-692,585 and L-682,429. Although GHRH neurons constitute a major subgroup in this area, the arcuate nucleus is heterogeneous. Indeed, it contains several groups of neurons which project either to the median eminence and portal primary plexus (neuroendocrine cells) or to other hypothalamic or extrahypothalamic brain structures.
Besides GHRH, several peptides and monoamines have been detected, neuropeptide Y (NPY), proopiomelanocortin (POMC) and dopamine being quantitatively the most important ones. Effect of intravenous GHRP-6 on electrical activity of arcuate neurons was different in two subpopulations of cells: predominantly excitatory for putative neuroendocrine cells and inhibitory for the remaining unidentified cells. Further characterization of the subpopulation of arcuate neurons stimulated by GHRP-6 has been performed using the retrograde tracer Fluorogold which identifies neurosecretory neurons. Between 68% and 82% of the arcuate neurons expressing c-fos protein following the i.v. injection of GHRP-6 are presumably neurosecretory neurons. The majority of these cells were not identified as tyrosine hydroxylase positive or p-endorphin-containing cells. In another study, neurochemically identifiable cells expressing c-fos mRNA were shown to coexpress NPY mRNA, GHRH mRNA tyrosine hydroxylase mRNA, POMC mRNA or somatostatin mRNA. I.c.v. and i.v. injection of MK-0677 induced Fos-like immunoreactivity within the ventromedial region of the arcuate nucleus in conscious male rats. Neurons activated by MK-0677 were confined close to the wall of the third ventricule whereas GHRP-6 induced Fos-like immunoreactivity in the same area as well as in more dorsal and lateral regions of this nucleus.
Therefore, GHRP-6 may activate a broader variety of hypothalamic neurons than MK-0677; this observation might explain increased food intake observed with GHRP-6, but not with MK-0677. In urethane-anaesthetized rats, systemic injection of MK-0677 increased the electrical activity of the same population of arcuate neuroendocrine cells than GHRP-6, this effect being inhibited by the administration of SRIH. In addition, it was shown that GHRP-6-induced activation of arcuate nucleus neurons is blunted by prior central administration of a SRIH analog. These data confirm GHS action on arcuate neurons involved in the regulation of GH release.