Nonpeptide agonists of each of the five somatostatin receptors were identified in combinatorial libraries constructed on the basis of molecular modeling of known peptide agonists. In vitro experiments using these selective compounds demonstrated the role of the somatostatin subtype-2 receptor in inhibition of glucagon release from mouse pancreatic alpha cells and the somatostatin subtype-5 receptor as a mediator of insulin secretion from pancreatic beta cells. Both receptors regulated growth hormone release from the rat anterior pituitary gland. The availability of high-affinity, subtype-selective agonists for each of the somatostatin receptors provides a direct approach to defining their physiological functions.

No abstract

His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6) stimulated GH release from rat primary pituitary cells in a time- and dose-dependent manner. Stimulation was observed after a 15-min, but not a 4-h, incubation. The concentrations of GHRP-6 required for half-maximal and maximal stimulation were 7 x 10(-9) and 10(-7) M, respectively. GH release induced by GHRP-6 was not affected by the addition of either naloxone or the GRF antagonist [N-Ac-Tyr1,D-Arg2]GRF-(1-29)-NH2. The latter inhibited GRF-stimulated GH release by shifting the dose-response curve to the right. His-D-Trp-D-Lys-Trp-D-Phe-Lys-NH2, an analog of GHRP-6, inhibited GH release stimulated by GHRP-6 without affecting that induced by GRF. When present together at maximal concentrations, GHRP-6 and GRF produced a synergistic effect on GH release. GHRP-6 had no effect on intracellular cAMP levels, whereas GRF increased intracellular cAMP concentrations by 3-fold. Combined treatment of pituitary cells with GRF and GHRP-6 resulted in a potentiation of the GRF-induced increase in cAMP levels. Basal GH release was reduced by 30% after pretreatment with GHRP-6 (10(-7) M) for 1 h. Pretreatment with GHRP-6 also decreased the subsequent response to GHRP-6, but not GRF. In contrast, pretreatment with GRF for 1 h had no effect on the subsequent action of GHRP-6 or GRF on GH release. The desensitization induced by GHRP-6 was completely reversed within 1 h after removal of the peptide. Results from this study indicate that GHRP-6 and GRF stimulated GH release from somatotrophs via different receptors and through discrete mechanisms.

A nonpeptidyl secretagogue for growth hormone of the structure 3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-1-([2'-(1H-tetrazol-5 -yl) (1,1'-biphenyl)-4-yl]methyl)-1H-1-benzazepin-3(R)-yl)-butanamid e (L-692,429) has been identified. L-692,429 synergizes with the natural growth hormone secretagogue growth hormone-releasing hormone and acts through an alternative signal transduction pathway. The mechanism of action of L-692,429 and studies with peptidyl and nonpeptidyl antagonists suggest that this molecule is a mimic of the growth hormone-releasing hexapeptide His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6). L-692,429 is an example of a nonpeptidyl specific secretagogue for growth hormone.

We have recently reported that His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 (GHRP-6) synergizes with GH-releasing factor (GRF) to increase GH release and cAMP accumulation in rat pituitary cells in vitro. This study was undertaken to further investigate the mechanism of action of GHRP-6 on GH release, particularly the involvement of protein kinase-C. Forskolin (10(-5) M), A23187 (10(-6) M), and phorbol 12-myristate 13-acetate (PMA; 10(-7) M) all stimulated GH release. However, only PMA can mimic the synergistic effects of GHRP-6 on GRF-stimulated GH release and intracellular cAMP accumulation. 4 alpha-Phorbol 12,13-didecanoate, an inactive phorbol ester, was unable to stimulate GH release or potentiate the effect of GRF. Extracellularly added phospholipase-C not only stimulated GH release in a dose-dependent manner, but also potentiated GRF-induced GH release. Phloretin, a protein kinase-C inhibitor, in a concentration range of 10-250 microM had very little or no effect on basal and GRF-stimulated GH release, but markedly inhibited the stimulatory effects induced by either PMA or GHRP-6. Incubation of rat pituitary cells with 10(-6) M PMA for 24 h completely down-regulated protein kinase-C, since such PMA-pretreated cells did not release GH in response to a second dose of PMA. The protein kinase-C-depleted cells had an attenuated GHRP-6 response, but they responded normally to GRF. Moreover, the synergistic effects of GHRP-6 and GRF on GH release and cAMP accumulation were also greatly inhibited by protein kinase-C down-regulation. These data suggest that the effects of GHRP-6 on GH release, either alone or together with GRF, are at least partially mediated via the activation of protein kinase-C.

The discovery and synthesis of dihydrobenzoxathiins as potent, ERalpha subtype selective ligands are described. The most active analogue, 4-D, was found to be 50-fold selective in a competitive binding assay and 100-fold selective in a transactivation assay in HEK-293 cells. The alpha selectivity was postulated to lie in the interaction of the sulfur atom of the benzoxathiin ring with the two discriminating residues in the binding pocket of the receptor isoforms.

No abstract

No abstract