Hormonal Inhibition of Adenylate Cyclase

Human platelet membranes contain a2 adrenergic receptors which mediate the guanine nucleotide-dependent inhibition of adenylate cyclase induced by epinephrine. Previous investigations have provided insight into the mechanisms of adenylate cyclase stimulation by studying hormone-induced release of radiolabeled guanine nucleotides from membranes preincubated with the nucleotides. In the present studies, we demonstrate that epinephrine promotes the release of [3H]guanylylimidodiphosphate (C3H]Gpp(NH)p) from prelabeled human platelet membranes. The effect of epinephrine is a2 adrenergic in nature and requires the presence of GTP. The maximal amount of C 3 H I Gpp(NH)p released by epinephrine, 328 2 29 fmol/mg of membrane protein (mean k S.E., n = 33), is similar to the number of platelet membrane LTZ adrenergic receptors determined by radioligand binding studies. Partial a2 adrenergic agonists release a smaller fraction of [3H]Gpp(NH)p than does the full agonist epinephrine. There is a significant correlation between an adrenergic agonist’s intrinsic activity for adenylate cyclase inhibition and its intrinsic activity for stimulation of [3H]Gpp(NH)p release. Prostaglandin El (PGE,), which stimulates adenylate cyclase activity in these platelet membranes, also promotes release of [3H]Gpp(NH)p. In the presence of both PGEl and epinephrine, more C3H] Gpp(NH)p is released than when either hormone is present alone; at low GTP concentrations, the effects were virtually additive. In the same membrane preparations in which PGEl stimulates and epinephrine inhibits adenylate cyclase activity, both hormones individually and in combination were found to stimulate the release of C3HIGpp(NH)p. Low concentrations of Mn2+, previously found to preferentially uncouple hormonal inhibition of platelet adenylate cyclase, were found to preferentially attenuate epinephrine-induced [3H]Gpp(NH)p release, whereas PGE,-stimulated nucleotide release was inhibited only at relatively higher Mn2+ concentrations. These data suggest that hormonal inhibition and stimulation of adenylate cyclase are mediated by analogous regulatory mechanisms at distinct guanine nucleotide binding sites.