CARDIAC ALPHA(1)-ADRENOCEPTORS STIMULATE A HIGH-AFFINITY GTPASE ACTIVITY IN SARCOLEMMAL MEMBRANES FROM RABBIT ATRIAL AND VENTRICULAR MYOCYTES

The interaction between cardiac alpha1-adrenoceptors and GTP-binding regulatory proteins was characterized in isolated rabbit cardiac Myocytes (thereby avoiding interference by other cell types present in the myocardium) by examining the alpha1-adrenergic stimulation of GTPase activity in sarcolemma-enriched membrane fractions. Stimulation of membrane-associated GTPase activity in both atrial and ventricular myocyte preparations by the alpha1-adrenergic agonists 1-noradrenaline and methoxamine (in the presence of propranolol) was observed to be both linear with time and saturable. alpha1-adrenergic stimulation did not change the K(m) for GTP (0.14-0.21 muM), but increased the V(max) by 39% and 72% above basal levels in atrial and ventricular membranes, respectively. Stimulation of GTPase activity by alpha1-agonists occurred in a concentration-dependent fashion and was blocked in the presence of the alpha-adrenoceptor antagonists phentolamine and prazosin, but not yohimbine. Prior treatment of myocytes with pertussis toxin had no effect on the alpha1-adrenergic stimulation of GTPase activity, but inhibited stimulation by muscarinic-receptor activation with carbachol. Finally, photoaffinity labelling of an approximately 75-kDa membrane-bound protein with [alpha-P-32]GTP was enhanced in the presence of the alpha1-agonist methoxamine and abolished by addition of excess nonlabelled GTP, suggesting that this GTP-binding protein may interact with cardiac alpha1-adrenoceptors; a similar GTP-binding protein which may be coupled to alpha1-adrenoceptors has been reported in rat liver plasma membranes (Im, M. J. & Graham, R. M. (1990) J. Biol. Chem. 265,18944-18951).