Evidence against a regulation of Na+/K+-ATpase by G(i) proteins. Failure to detect an influence of G proteins on Na+/Ca2+-exchange in cardiac sarcolemmal membranes

In the myocardium the inhibitory guanine nucleotide-binding regulatory proteins (G(i) proteins) mediate negative chronotropic and negative inotropic effects by activation of K+ channels and inhibition of adenylyl cyclase. The concept of a uniform inhibitory action of G(i) proteins on myocardial cellular activity has been questioned by the recent observations of adenosine-induced activation of the Na+/Ca2+ exchange and a carbachol-induced inhibition of the Na+/K+-ATPase activity in cardiac sarcolemmal membranes. The aim of the present study, therefore, was to reinvestigate the putative regulation of Na+/Ca2+ exchange and Na+/K+-ATPase activity in purified canine sarcolemmal membranes. These membranes were enriched in adenosine A(1) (Maximum number of receptors, B-max 0.033 pmol/mg) and muscarinic M(2) (B-max 2.9 pmol/mg) receptors and contained G(i2) and G(i3), two G(i) protein isoforms, and G(o), another pertussis toxin-sensitive G protein, as detected with specific antibodies. The adenosine A(1)-selective agonist, (-)-N-6-(2-phenylisopropyl)-adenosine, and the muscarinic agonist, carbachol, both inhibited isoprenaline-stimulated adenylyl cyclase activity by 25% and 35% respectively, and the stable GTP analogue 5'-guanylylimidodiphosphate inhibited forskolin-stimulated adenylyl cyclase activity by 35% in these membranes. The characteristics of Na+/Ca2+ exchange and Na+/K+-ATPase activity as well as those of the ouabain-sensitive, K+-activated 4-nitrophenylphosphatase, an ATP-independent, partial reaction of the Na+/K+-ATPase, were in agreement with published data with regard to specific activity, time course of activity and substrate dependency. However, none of these activities were influenced by adenosine, (-)-N-6-(2-phenylisopropyl)-adenosine, carbachol, or stable GTP analogs, suggesting that Na+/Ca2+ exchange and Na+/K+-ATPase are not regulated by G(i) proteins in canine cardiac sarcolemmal membranes.