The biochemical signaling pathways involved in nitric oxide (NO)-mediated cholinergic inhibition of L-type Ca2+ current (ICa[L]) were investigated in isolated primary pacemaker cells from the rabbit sinoatrial node (SAN) using the nystatin-perforated whole-cell voltage clamp technique. Carbamylcholine (CCh; 1 microM), a stable analogue of acetylcholine, significantly inhibited ICa(L) after it had been augmented by isoproterenol (ISO; 1 microM). CCh also activated an outward K+ current, IK(ACh). Both of these effects of CCh were blocked completely by atropine. Preincubation of the SAN cells with L-nitro-arginine methyl ester (L-NAME; 0.2-1 mM), which inhibits NO synthase (NOS), abolished the CCh-induced attenuation of ICa(L) but had no effect on IK(ACh). Coincubation of cells with both L-NAME and the endogenous substrate of NOS, L-arginine (1 nM), restored the CCh-induced attenuation of ICa(L), indicating that L-NAME did not directly interfere with the muscarinic action of CCh on ICa(L). In the presence of ISO the CCh-induced inhibition of ICa(L) could be mimicked by the NO donor 3-morpholino-sydnonimine (SIN-1; 0.1 mM). SIN-1 had no effect on its own or after a maximal effect of CCh had developed, indicating that it does not inhibit ICa(L) directly. SIN-1 failed to activate IK(ACh), demonstrating that it did not activate muscarinic receptors. Both CCh and NO are known to activate guanylyl cyclase and elevate intracellular cGMP. External application of methylene blue (10 microM), which interferes with the ability of NO to activate guanylyl cyclase, blocked the CCh-induced attenuation of ICa(L). However, it also blocked the activation of IK(ACh), suggesting an additional effect on muscarinic receptors or G proteins. To address this, a separate series of experiments was performed using conventional whole-cell recordings with methylene blue in the pipette. Under these conditions, the CCh-induced attenuation of ICa(L) was blocked, but the activation of IK(ACh) was still observed. Methylene blue also blocked the SIN-1-induced decrease in ICa(L). 6-anilino-5,8-quinolinedione (LY83583; 30 microM), an agent known to decrease both basal and CCh-stimulated cGMP levels, prevented the inhibitory effects of both CCh and SIN-1 on ICa(L), but had no effect on the activation of IK(ACh) by CCh. In combination, these results show that CCh- and NO-induced inhibition of ICa(L) is mediated by cGMP.(ABSTRACT TRUNCATED AT 400 WORDS)

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