AN OBLIGATORY ROLE FOR NITRIC-OXIDE IN AUTONOMIC CONTROL OF MAMMALIAN HEART-RATE

1. Cholinergic modulation of heart rate in isolated spontaneously beating single cells from the rabbit sino-atrial node was investigated by measuring transmembrane ionic currents using the nystatin-perforated patch whole-cell voltage-clamp technique. 2. Carbamylcholine (CCh), a stable analogue of acetylcholine (ACh), significantly inhibited L-type calcium currents (I-Ca(L) which had been augmented by beta-adrenergic stimulation. In addition, CCh activated a potassium outward current (I-K(ACh)). Both effects were blocked by atropine. 3. The possible involvement of nitric oxide (NO) in these responses was evaluated by inhibiting NO synthesis. In the presence of N-G-monomethyl-L-arginine (L-NMMA, 100 mu M) or nitro-L-arginine methyl ester (L-NAME, 1 mM), two specific inhibitors of nitric oxide synthase (NOS), CCh no longer inhibited I-Ca(L). I-K(ACh) could still be activated. 4. Co-incubation of cells in L-NAME or in L-NMMA with arginine (the endogenous substrate of NOS) restored the CCh-induced attenuation of I-Ca(L), indicating that L-NAME or L-NMMA did not interfere directly with the muscarinic action of CCh on I-Ca(L). 5. Effects of the NO-releasing agent molsidomine (SIN-1) on CCh-induced changes in I-Ca(L) were also investigated. After I-Ca(L) had been augmented by beta-adrenergic stimulation, SIN-1 (0.1. mM) inhibited I-Ca(L); however, SIN-1 had no further inhibitory effect after a maximal CCh concentration had been applied. 6. These findings suggest that NO generation is an obligatory process in cholinergic inhibition of I-Ca(L) in mammalian cardiac pacemaker tissue.