The effect of growth hormone secretagogues and neuropeptide Y on hypothalamic hormone release from acute rat hypothalamic explants.

Growth hormone (GH) secretagogues (GH-releasing peptides and their non-peptide analogues) stimulate growth hormone release via specific G-protein coupled receptors both directly from the pituitary gland and through stimulation of the hypothalamus. The exact mechanism of action in the hypothalamus is not known. The presence of endogenous GH releasing hormone (GHRH) seems to be necessary for the in-vivo actions of growth hormone secretagogues (GHSs), but data suggest that further factors must be involved as well. The effect of GHSs is not entirely specific for the GH axis; they release prolactin and stimulate the hypothalamo-pituitary-adrenal axis causing elevations in circulating ACTH and cortisol levels in both animal and human studies. Recently, it has also been suggested that GHSs stimulate hypothalamic neuropeptide Y (NPY) neurones. In the present study, we have therefore investigated the direct effect of several GHSs (GHRP-6, hexarelin and the non-peptide analogues L-692, 429 and L-692, 585) on GHRH, somatostatin (SS), corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) release in vitro in an acute rat hypothalamic incubation system. We also assessed the effect of NPY on GHRH, SS and AVP release. Freshly removed hypothalami were incubated in control media for 20 min and then in 1-4 consecutive 20-min periods in each of the test substances at different concentrations. There was no significant change in either the basal or potassium-stimulated release of GHRH or SS at low concentrations of any of the secretagogues; however, at millimolar doses a paradoxical inhibition of GHRH was observed with GHRP-6, hexarelin and L-692 585 (data are expressed as the ratio of treated to preceding basal release; at 20 min control group: 0.97+/-0.02, GHRP-6: 0.55+/-0.04, P<0.001 compared to control group; hexarelin: 0. 56+/-0.06, P<0.001, L-692,585: 0.70+/-0.03, P<0.001), while SS was stimulated after 60 or 80 min (at 80 min control: 0.80+/-0.03, hexarelin: 1.23+/-0.07, P<0.05 and L-692,585: 1.37+/-0.11, P<0.05). GHSs stimulated hypothalamic AVP release (at 20 min control: 0. 99+/-0.06 ratio to basal release, 10-4 M concentration of GHRP-6: 6. 31+/-1, P<0.001, hexarelin: 1.88+/-0.4, P<0.01, L-692,429: 1.90+/-0. 5, P<0.05 and L-692,585: 2.34+/-0.96, P<0.01), while no stimulatory effect was found on CRH release. NPY significantly stimulated SS and inhibited basal and potassium-stimulated GHRH release, while potentiating potassium-evoked AVP secretion. The Y1 receptor antagonist BIBP 3226 did not inhibit the effects of NPY on SS, GHRH or AVP release. We therefore conclude that, in this in-vitro rat hypothalamic incubation model, growth hormone secretagogues stimulate the release of AVP but have no effect on either GHRH, SS or CRH at low doses; at high doses paradoxically they inhibit the hypothalamic GH axis similar to in-vivo data in the rat. We speculate that these effects might be mediated by NPY.

The growth hormone secretagogue hexarelin stimulates the hypothalamo-pituitary-adrenal axis via arginine vasopressin.

GH secretagogues (GHSs) act via specific receptors in the hypothalamus and the pituitary gland to release GH. GHSs also stimulate the hypothalamo-pituitary-adrenal (HPA) axis via central mechanisms probably involving CRH or arginine vasopressin (AVP). We studied the effects of hexarelin, CRH, and desmopressin, an AVP analog, on the stimulation of the HPA axis in 15 healthy young male volunteers. Circulating ACTH, cortisol, GH and PRL concentrations were measured for 2 h after the injection of hexarelin, CRH, or desmopressin alone and the combination of hexarelin plus CRH or hexarelin plus desmopressin. Symptoms during the tests were assessed by visual analog scales. Hexarelin significantly increased ACTH and cortisol release (area under the curve, 3,444+/-696 ng/L x 125 min and 45,844+/-2,925 nmol/L x 125 min, respectively), and this effect was augmented by the addition of CRH in a dose that on its own produces maximal stimulation (6,580+/-1,572 ng/mL x 125 min and 63,170+/-2,616 nmol/L x 125 min; P = 0.01 and 0.001, respectively), but was not influenced by the addition of desmopressin (3,540+/-852 ng/mL x 125 min and 35,319+/-3,252 nmol/L x 125 min; not significant). CRH on its own caused similar or slightly higher ACTH and cortisol release than hexarelin alone. Desmopressin given alone elicited a rapid rise in circulating ACTH and cortisol, but its effects were less than those of any other treatment and were not augmented by hexarelin. Hexarelin also caused significant GH and PRL release, but these effects were not influenced by the coadministration of CRH or desmopressin. Visual analog scales showed an acute small increment in appetite with hexarelin. Our data suggest that the effect of GHSs on the HPA axis involve at least in part the stimulation of AVP release. In summary, we have shown that in healthy male volunteers, the effect of hexarelin on the HPA axis does not involve CRH, but may occur through the stimulation of AVP release.

Insulin-like growth factor-I and- II in combination inhibit the release of growth hormone-releasing hormone from the rat hypothalamus in vitro.

This study was designed to investigate the feedback loop between insulin-like growth factor-I (IGF-I) and IGF-II and the hypothalamic hormones growth hormone-releasing hormone (GHRH) and somatostatin (SS) using an in vitro rat hypothalamic model. IGF-I and, to lesser extent, IGF-II, both activate type 1 IGF receptors, while type 2 receptors are activated by IGF-II alone. IGF-I, IGF-II, their various specific analogues (Des[1-3]IGF-I, [Arg54/Arg55]IGF-II and [Leu27]IGF-II), insulin and the type 2 receptor antagonist beta-galactosidase were used on their own or in combination to study their effect on GHRH and SS release. Our results suggest that the simultaneous activation of type 1 and type 2 IGF receptors is needed for the negative feedback effect of IGFs on GHRH release in this in vitro system, in agreement with earlier findings in vivo. Somatostatin was not altered by any combination of peptides.

Nitric oxide modulates the release of vasopressin from rat hypothalamic explants.

The enzyme nitric oxide (NO) synthase is present in the paraventricular nucleus, while nitric oxide has recently been shown to inhibit the stimulated release of corticotrophin-releasing hormone (CRH) in vitro. Thus the possible role of NO in regulating, vasopressin (AVP), which also plays an important role in pituitary-adrenal activity, has been investigated. The effects were studied of the NO donors, L-arginine, syndnonimine-1 (SIN-1) and sodium nitroprusside, on both the basal and stimulated release of AVP, employing a previously validated system. Rat hypothalami were incubated in either medium alone or medium containing the test substances and hormone release was measured by RIA. The effect of L-arginine in the presence of the NO synthase inhibitor, L-NMMA, was also investigated. L-arginine reduced KCl-evoked AVP release; this effect was reversed by L-NMMA and reduced by the addition of ferrous human Hb. Similarly, SIN-1 and sodium nitroprusside attenuated KCl-evoked AVP release. L-arginine also reduced IL-1 beta-stimulated AVP release. NO appears to directly and specifically inhibit the stimulated release of AVP from rat hypothalamic explants in vitro, similar to its effects on CRH. These findings provide further evidence that NO may be involved in neuroendocrine regulation.

Nitric oxide modulates the release of corticotropin-releasing hormone from the rat hypothalamus in vitro.

There is now considerable evidence that nitric oxide (NO) is an important neuroregulatory agent, but there has been very little investigation of the possible role of NO in neuroendocrine mechanisms. We have previously shown that acute rat hypothalamic explants can be used to study the regulation of hypothalamic neuropeptide release, and we have now utilised this experimental approach to investigate the putative involvement of NO in the control of the principal corticotropin-releasing hormone, CRH. We studied the direct effects of the NO precursor L-arginine (L-ARG), as well as the NO donors molsidomine and sodium nitroprusside, on both the basal and stimulated release of CRH; the stimuli used were non-specific depolarisation with potassium chloride (KCl) and the specific cytokine, interleukin-1 beta (IL-1 beta; 100 U/ml). L-ARG was tested in each experimental condition with and without contemporaneous addition of its competitive antagonist NG-monomethyl-L-arginine (L-NMMA). IL-1 beta-induced CRH release was also investigated in the presence of D-arginine (D-ARG), which is not active as a precursor to NO, and ferrous hemoglobin (Hb), a substance which is a potent inactivator of NO. None of the NO precursors (L-ARG, molsidomine, sodium nitroprusside) or antagonists (L-NMMA or Hb) was able to affect basal CRH release. However, L-ARG 10 and 100 microM were found to significantly inhibit the release of CRH induced by 40 mM KCl; CRH fell to 45% of its stimulated level at the higher dose of L-ARG. This effect was attenuated in the presence of L-NMMA at a ten-fold higher dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Pyridostigmine partially reverses dexamethasone-induced inhibition of the growth hormone response to growth hormone-releasing hormone.

The GH response to insulin-induced hypoglycaemia and growth hormone-releasing hormone (GHRH) has been shown to be impaired in subjects with Cushing's syndrome and in healthy volunteers given oral glucocorticoids. Pyridostigmine is an anticholinesterase that stimulates GH secretion, probably by inhibition of hypothalamic somatostatin secretion. This work was designed to study the site of action of glucocorticoids in inhibiting the secretion of GH. Eight healthy male volunteers were studied on three occasions in random order. They took 2 mg oral dexamethasone or placebo at precisely 6-hourly intervals for 48 h before receiving 120 mg oral pyridostigmine or placebo, followed 60 min later by GHRH (100 micrograms) i.v. Samples for measuring GH were obtained at 15 min intervals for 2 h. The 'area under the curve' (AUC) for each of the treatments was significantly different: dexamethasone-pyridostigmine-GHRH (mean +/- S.E.M., 1938 +/- 631 mU/min per l), dexamethasone-placebo-GHRH (634 +/- 211) and placebo-placebo-GHRH (4267 +/- 1183) (P < 0.02, Wilcoxon test). In conclusion, dexamethasone given for 48 h significantly inhibited the AUC for GH following treatment with GHRH. However, pretreatment with pyridostigmine significantly reversed the inhibition although this was still partial. Our data suggested that this short-term suppressive effect of dexamethasone was independent of GHRH, and most probably relates to stimulation of the release of somatostatin.