cGMP-mediated inhibition of cardiac L-type Ca2+current by a monoclonal antibody against the M2 ACh receptor

Abstract

The effects of a monoclonal antibody (B8E5) directed against the second extracellular loop of the muscarinic M2receptor were studied on the L-type Ca2+ currents ( I Ca,L) of guinea pig ventricular myocytes using the whole cell patch-clamp technique. Similar to carbachol, B8E5 reduced the isoproterenol (ISO)-stimulated I Ca,L but did not significantly affect basal I Ca,L. Atropine blocked the inhibitory effect of B8E5. The electrophysiological parameters of ISO-stimulated I Ca,L were not modified in presence of B8E5. Inhibition of I Ca,L by B8E5 was still observed when intracellular cAMP was either enhanced by forskolin or maintained constant by using a hydrolysis-resistant cAMP analog (8-bromoadenosine 3′,5′-cyclic monophosphate) or by applying the phosphodiesterase inhibitor IBMX. The effect of B8E5 was mimicked by 8-bromoguanosine 3′,5′-cyclic monophosphate, a potent stimulator of cGMP-dependent protein kinase, and prevented by a selective inhibitor of nitric oxide-sensitive guanylyl cyclase {1 H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one}. These results indicate that the antibody B8E5 inhibits the β-adrenergic-stimulated I Ca,L through activation of the M2 muscarinic receptor and further suggest that the antibody acts not via the classical pathway of decreasing intracellular cAMP, but rather by increasing cGMP.