Agonist-induced release of splice variants of the alpha subunit of the stimulatory G-protein from rat cardiac membranes.

It was the aim of the present study to evaluate whether, in physiological cardiac tissue, long and short splice variants of G(s)alpha (the alpha subunit of the stimulatory G-protein) differ in their susceptibility to guanine nucleotide-mediated activation. As a measure of G(s)alpha activation, we determined the proportion of G(s)alpha subunits which were released from the plasma membrane upon stimulation. Membrane preparations from heart ventricles of Wistar rats were incubated with increasing concentrations of the non-hydrolyzable GTP analogue guanylyl-imidodiphosphate (GppNHp, 0-100 micromol/L) in the absence or presence of the beta-adrenoceptor agonist isoprenaline (1 micromol/L). The 45 and 52 kDa forms of G(s)alpha (G(s)alpha-S and -L, respectively) were measured in the supernatant of the incubation mixture by immunoblotting and densitometry. The increase in cyclic AMP induced by GppNHp was measured in the same supernatant. In the absence of isoprenaline, GppNHp increased cyclic AMP formation and the concentration-dependent release of G(s)alpha-L (Friedman test, P < 0.05), whereas the amount of soluble G(s)alpha-S was not affected. After addition of isoprenaline, the redistribution of G(s)alpha-S into the soluble fraction could be stimulated by GppNHp in a concentration-dependent manner (P < 0.05). Kinetic experiments revealed that activation of G(s)alpha-S by GppNHp was rather slow, but could be markedly enhanced by isoprenaline. Thus, it is likely that the different susceptibilities of G(s)alpha-S and -L towards GppNHp reflects differences in the rate of spontaneous GDP release.