A CELLULAR MECHANISM FOR NITRIC OXIDE-MEDIATED CHOLINERGIC CONTROL OF MAMMALIAN HEART-RATE

The biochemical signaling pathways involved in nitric oxide (NO)mediated cholinergic inhibition of L-type Ca2+ current (I-Ca[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 mu M), a stable analogue of acetylcholine, significantly inhibited I-Ca(L) after it had been augmented by isoproterenol (ISO; 1 mu M). CCh also activated an outward K+ current, I-K(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 I-Ca(L) but had no effect on I-K(ACh) Coincubation of cells with both L-NAME and the endogenous substrate of NOS, L-arginine (1 mM), restored the CCh-induced attenuation of I-Ca(L), indicating that L-NAME did not directly interfere with the muscarinic action of CCh on I-Ca(L) In the presence of ISO the CCh-induced inhibition of I-Ca(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 I-Ca(L) directly. SIN-1 failed to activate I-K(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 mu M), which interferes with the ability of NO to activate guanylyl cyclase, blocked the CCh-induced attenuation of I-Ca(L). However, it also blocked the activation of l(K(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 I-Ca(L) was blocked, but the activation of I-K(ACh) was still observed. Methylene blue also blocked the SIN-1-induced decrease in I-Ca(L). 6-anilino-5,8-quinolinedione (LY83583; 30 mu M), an agent known to decrease both basal and CCh-stimulated cGMP levels, prevented the inhibitory effects of both CCh and SIN-1 on I-Ca(L), but had no effect on the activation of I-K(ACh) by CCh. In combination, these results show that CCh- and NO-induced inhibition of I-Ca(L) is mediated by cCMP. CCh could not decrease I-Ca(L) after it had been increased maximally by a membrane-permeable cAMP analogue, 8-(4-chlorophe-nylthio)-adenosine-3',5'-cyclic monophosphate (8-CPT-cAMP; 0.2 mM), which is resistant to the cAMP-specific phosphodiesterase (PDE). This finding suggests that neither the cAMP-dependent protein kinase (PKA) nor the biochemical events occurring subsequent to PKA activation are involved in the CCh- or NO-induced inhibition of I-Ca(L). I-Ca(L) was not changed significantly by the membrane-permeable cGMP analogues 8-bromoguanosine-3',5'-cyclic monophosphate (8-Br-cGMP; 0.25-0.5 mM) and 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-CPT-cGMP; 0.25 mM). These compounds selectively activate cCMP-dependent protein kinase (PKG); their effects therefore rule out a role of PKG in this NO-mediated, CCh-induced inhibition of I-Ca(L). A potent but nonspecific PDE inhibitor 3-isobutyl-1-methyl-xanthine (IBMX; 20 mu M) completely prevented the CCh-induced attenuation of I-Ca(L). Taken together, these findings suggest that in mammalian primary pacemaker SAN cells (in the presence of ISO), NO-mediated cholinergic inhibition of I-Ca(L) is due to a cGMP-stimulated cAMP-specific PDE, which hydrolyzes cAMP and thus inhibits cAMP-dependent phosphorylation of I-Ca(L) channels. Since I-Ca(L) is essential for normal pacemaker activity, this biochemical pathway represents an important new mechanism for autonomic (cholinergic) control of mammalian heart rate.