Mechanism of inhibition of adenylyl cyclase by muscarinic receptor stimulation of inhibitory g-proteins in cardiac muocytes: a biochemical approach
Creators: name: Ramaswamy, Subramanian
Publisher: name: University of British Columbia
Year: 2009

description: In the mammalian myocardium, adenylyl cyclase is under dual regulation by p adrenoceptors and muscarinic receptors via stimulatory (Gs) and inhibitory (Gj) G proteins. It is well known that activation of Gs upon stimulation of p adrenoceptors cause increase in adenylyl cyclase activity. However, the mechanism of inhibition of adenylyl cyclase upon activation of Gj is not clear and two mechanisms have been proposed (Gilman, 1987) : an indirect inhibition due to decrease in the concentration of GsaGTP by Py subunits generated upon activation of Gj and a direct inhibition by Gja.GTPIn this study we investigated the mechanism of muscarinic receptor-mediated inhibition of adenylyl cyclase in sareolemmal membranes isolated from rabbit atrial and ventricular myocyte. High affinity guanosine triphosphatase and adenylyl cyclase activity in the presence of the muscarinic agonists carbachol and oxotremorine alone and in combination with the p-adrenergic agonist isoproterenol were measured, and the different inhibitory G-protein subunits were identified in these membranes by immunoblotting. In ventricular myocyte membranes, isoproterenol, carbachol and oxotremorine alone produced significant increases in guanosine triphosphatase activity above the basal value, whereas an additive effect was observed in the presence of isoproterenol and carbachol or isoproterenol and oxotremorine together. Adenylyl cyclase activity was increased by isoproterenol alone, whereas carbachol inhibited basal cyclase activity. However, the adenylyl cyclase activity measured in the presence of isoproterenol and carbachol together was very close to the value predicted if the opposing effects of the two agonists were independent of each other. These observations suggest that in ventricular myocyte membranes, there is no interaction between p adrenoceptor and muscarinic receptor-stimulated G-proteins and inhibition of adenylyl cyclase occurs by a direct mechanism. In atrial myocyte membranes, isoproterenol, carbachol and oxotremorine alone produced significant increases in guanosine triphosphatase activity above the basal value, whereas the activity measured in the presence of isoproterenol and carbachol or isoproterenol and oxotremorine together was not different from that in the presence of carbachol or oxotremorine alone. Isoproterenol increased adenylyl cyclase above the basal level, while carbachol did not affect basal cyclase activity. In the presence of both agonists together, the isoproterenol - stimulated cyclase activity was inhibited. Although statistically not significant, our results on cyclase activity are qualitatively consistent with earlier observations from our laboratory on rabbit atrial homogenates (Ray, 1992). These observations suggest that in atrial myocyte membranes, there is an interaction between (3 adrenoceptor and muscarinic receptor-stimulated G-proteins and inhibition of adenylyl cyclase occurs by an indirect mechanism. Western blotting of ventricular myocyte membranes identified Gjai and Gi u 3 but not Gi a 2 subtypes, while the presence of Goa could not be conclusively confirmed. In atrial myocyte membranes, Western blotting identified Gio2, GJO3 and Goa subtypes. In conclusion, the results from this study suggest that there are differences in the mechanism of muscarinic receptor regulation of adenylyl cyclase in atria and ventricles, which is associated with the presence of different isoforms of Grin these two tissues.