cGMP-mediated inhibition of cardiac L-type Ca2+ current by a monoclonal antibody against the M-2 ACh receptor

The effects of a monoclonal antibody (B8E5) directed against the second ext racellular loop of the muscarinic M-2 receptor were studied on the L-type C a2+ currents (I-Ca,I-L) of guinea pig ventricular myocytes using the whole cell patch-clamp technique. Similar to carbachol, B8E5 reduced the isoprote renol (ISO)-stimulated I-Ca,I-L but did not significantly affect basal I-Ca ,I-L. Atropine blocked the inhibitory effect of B8E5. The electrophysiologi cal parameters of ISO-stimulated I-Ca,I-L were not modified in presence of B8E5. Inhibition of I-Ca,I-L by B8E5 was still observed when intracellular cAMP was either enhanced by forskolin or maintained constant by using a hyd rolysis-resistant cAMP analog (8-bromoadenosine 3',5'-cyclic monophosphate) or by applying the phosphodiesterase inhibitor IBMX. The effect of B8E5 wa s mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate, a potent stimula tor of cGMP-dependent protein kinase, and prevented by a selective inhibito r of nitric oxide-sensitive guanylyl cyclase {1H-(1,2,4)oxadiazolo[4,3-a]qu inoxaline-1-one}. These results indicate that the antibody B8E5 inhibits th e beta -adrenergic-stimulated I-Ca,I-L through activation of the M-2 muscar inic receptor and further suggest that the antibody acts not via the classi cal pathway of decreasing intracellular cAMP, but rather by increasing cGMP .