Cardiac α1‐adrenoceptors stimulate a high‐affinity GTPase activity in sarcolemmal membranes from rabbit atrial and ventricular myocytes

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Abstract
The interaction between cardiac α1‐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 α1‐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 α1‐adrenergic agonists 1‐noradrenaline and methoxamine (in the presence of propranolol) was observed to be both linear with time and saturable. α1‐adrenergic stimulation did not change the Km for GTP (0.14–0.21 μM), but increased the Vmax by 39% and 72% above basal levels in atrial and ventricular membranes, respectively. Stimulation of GTPase activity by α1‐agonists occurred in a concentration‐dependent fashion and was blocked in the presence of the α‐adrenoceptor antagonists phentolamine and prazosin, but not yohimbine. Prior treatment of myocytes with pertussis toxin had no effect on the α1‐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 [α‐32P]GTP was enhanced in the presence of the α1‐agonist methoxamine and abolished by addition of excess nonlabelled GTP, suggesting that this GTP‐binding protein may interact with cardiac α1‐adrenoceptors; a similar GTP‐binding protein which may be coupled to α1‐adrenoceptors has been reported in rat liver plasma membranes (Im, M. J. & Graham, R. M. (1990) J. Biol. Chem. 265, 18944–18951).