Muscarinic cholinergic-receptor stimulation of specific GTP hydrolysis related to adenylate cyclase activity in canine cardiac sarcolemma.

Abstract

One component of muscarinic receptor inhibition of the function of cardiac ventricles is mediated by the inhibition of activated adenylate cyclase activity in sarcolemma. We have shown previously that muscarinic agonists inhibit GTP-but not Gpp(NH)p-activated adenylate cyclase activity, and various studies in other tissues indicate that nonhydrolyzable GTP analogues prevent inactivation of the enzyme. These data have suggested a role for GTP hydrolysis in the mechanism of inhibition of adenylate cyclase. The present study demonstrates that purified canine cardiac sarcolemma displays high-affinity GTPase activity that is reciprocally related to adenylate cyclase activity. The high-affinity GTPase activity was stimulated by muscarinic agonists and blocked by atropine. Furthermore, the one-half maximal effects of oxotremorine for binding to muscarinic receptors, stimulation of high-affinity GTPase activity, and inhibition of adenylate cyclase activity were similar. Muscarinic stimulation of GTPase activity and inhibition of adenylate cyclase activity required functional activity of the pertussis toxin (IAP) substrates). Treatment of sarcolemmal membranes with LAP attenuated the ability of oxotremorine to both stimulate high-affinity GTPase activity and inhibit adenylate cyclase activity. These studies indicate that muscarinic receptor stimulation of high-affinity GTPase activity dependent on functional LAP substrate(s) is closely linked to the mechanism of muscarinic inhibition of adenylate cyclase activity. (Circulation Research L988;64:340-350)

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

“…5A). However, pertussis-toxin treatment considerably reduced the level of mAChR-stimulated GTPase activity, in agreement with previous data (Fleming and Watanabe, 1988). Furthermore, examination of the pertussis-toxin substrates in membranes from both control and previously pertussis-toxin-treated myocytes using [32P]NAD revealed intense labelling of a major band of 40-41 kDa in control membranes, and only very weak labelling of this same band in membranes from pertussis-toxin-treated cells (Fig.…”

“…This contrasts with the observed maximal stimulation of cardiac mAChR-linked GTPase activity in the same atrial and ventricular sarcolemmal membranes, which was 71 % and 93%, respectively, above basal. These values are comparable to those reported earlier for mAChR-coupled GTPase activities in sarcolemmal membranes from canine ventricle (Fleming and Watanabe, 1988) and porcine atrium (Hilf and Jakobs, 1989). Taken together, these findings suggest that the modest increase in the observed a, -stimulated GTPase activity observed in myocyte sarcolemmal membranes is probably characteristic of this pathway and not a result of the membrane isolation protocol.…”

“…Taken together, these findings suggest that the modest increase in the observed a, -stimulated GTPase activity observed in myocyte sarcolemmal membranes is probably characteristic of this pathway and not a result of the membrane isolation protocol. Furthermore, the observed K, (0.14-0.21 pM GTP) for the a,-stimulated GTPase activity in both atrial and ventricular sarcolemmal membranes are in line with those previously reported mAChR-stimulated GTPase activities in canine (0.14-0.23 pM; Fleming and Watanabe, 1988) and porcine myocardium (0.22pM; Hilf and Jakobs, 1989). These observations suggest that the density of the a,-adrenoceptorlG-protein complex may be lower than that of the mAChWG-protein complex.…”

“…This latter observation is similar to that described previously for mAChR in canine cardiac sarcolemmal membranes (Fleming and Watanabe, 1988) and strongly suggests that pertussis-toxin treatment of intact myocytes effectively caused the ADP-ribosylation of pertussis-toxin-sensitive G-proteins coupled to the myocyte mAChR. This conclusion was confirmed by performing pertussis-toxin-catalyzed ADP ribosylation of membranes from control and previously pertussis-toxin-treated myocytes using [32P]NAD as the substrate.…”

Signal transduction by G proteins in cardiac tissues.

“…In several systems including cardiac sarcolemma, an additional protein has been described that is required for the cholera toxin-catalyzed ADP-ribosylation of GS. 72,96,97 This protein is often called ADP-ribosylation factor (ARF) and has been identified in plasma membranes and cellular cytosol. Interest in this protein has increased recently due to several factors.…”