J. W. Fleming

Muscarinic receptor regulation of cardiac adenylate cyclase activity.

Stimulation and inhibition of adenylate cyclase activity are mediated by the guanine nucleotide regulatory proteins Gs and Gi, respectively. Two general mechanisms have been proposed for the inhibition of activated adenylate cyclase: direct inhibition of the catalyst by Gi, and indirect inhibition of the activated catalyst mediated by Gi inhibition of Gs. We have assessed direct inhibition of adenylate cyclase by evaluating the ability of Gpp(NH)p to inhibit the forskolin-stimulated enzyme in the presence of various concentrations of magnesium ions and the guanine nucleotide. Gpp(NH)p inhibition of adenylate cyclase activity was only observed in the presence of forskolin and low concentrations of MgCl2. Muscarinic agonists did not increase Gpp(NH)p inhibition of the forskolin-stimulated enzyme, even in the presence of low concentrations of MgCl2 and guanine nucleotide (near the respective Kact or Ki). Whether in the absence or presence of muscarinic agonists, no concentration of Gpp(NH)p was found to inhibit basal adenylate cyclase activity in the absence of forskolin. In addition, muscarinic agonists had no effect on the rate constant (kon) for Gpp(NH)p activation of the enzyme. In contrast to these data, the muscarinic agonist methacholine stimulated the inactivation rate constant (koff) for isoproterenol plus GTP-activated adenylate cyclase activity 15-fold, and the increase in koff was blocked by atropine. Moreover, the sarcolemma displayed specific, high affinity GTP hydrolytic activity which was stimulated by methacholine activation of muscarinic receptors. These data further support our original hypothesis, indicating that although direct inhibition of the catalyst by Gi may occur in cardiac sarcolemma, physiologically relevant attenuation of adenylate cyclase activity by muscarinic agonists occurs by a mechanism linked to GTP hydrolysis.

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

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 (TAP) substrate(s). Treatment of sarcolemmal membranes with IAP 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 IAP substrate(s) is closely linked to the mechanism of muscarinic inhibition of adenylate cyclase activity.

Pertussis toxin-treated dog: a whole animal model of impaired inhibitory regulation of adenylate cyclase.

We have shown previously that stimulation of high-affinity GTP hydrolysis and inhibition of adenylate cyclase activity by muscarinic agonists are mediated by pertussis toxin (IAP) substrates (G1 and G0in canine cardiac sarcolemma. We have now used the pertussis toxin-treated dog as a whole animal model in which G1- and G0-mediated biochemical mechanisms can be studied. Mongrel dogs were injected intravenously with IAP 48 hours prior to death and isolation of left ventricular sarcolemma. Treatment of the animal in vivo with the toxin prevented subsequent in vitro IAP-catalyzed [32P]ADP-ribosylation of substrates in cardiac, erythrocytic, and renal cortical plasma membranes, suggesting that ADP-ribosylation occurred in vivo from endogenous substrate. Consistent with our previous results obtained by treating sarcolemma in vitro with IAP, muscarinic receptor-mediated stimulation of high-affinity GTP hydrolysis and inhibition of GTP-activated adenylate cyclase activity were attenuated in sarcolemma purified from the toxin-treated animals. Proximal to adenylate cyclase, guanine nucleotide regulation of muscarinic receptor affinity for agonists was also abolished in membranes from the toxin-treated animals. In addition, the ability of oxotremorine to attenuate GTP regulation of stimulation of adenylate cyclase activity by magnesium ions was abolished in sarcolemma from the IAP-treated dogs. Thus, cardiac sarcolemma isolated from the IAP-treated animals displayed biochemical characteristics of an adenylate cyclase system in which inhibitory regulatory pathways had been attenuated. The cardiac biochemical studies and the in vivo ADP-ribosylation of noncardiac IAP substrates also suggests considerable potential use of this model in the physiological and biochemical study of regulatory mechanisms mediated by GTP-binding proteins in other systems.