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

1. Cholinergic modulation of heart rate in isolated spontaneously beat ing single cells from the rabbit sino-atrial node was investigated by measuring transmembrane ionic currents using the nystatin-perforated p atch whole-cell voltage-clamp technique. 2. Carbamylcholine (CCh), a s table 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 invol vement of nitric oxide (NO) in these responses was evaluated by inhibi ting NO synthesis. In the presence of N-G-monomethyl-L-arginine (L-NMM A, 100 mu M) or nitro-L-arginine methyl ester (L-NAME, 1 mM), two spec ific inhibitors of nitric oxide synthase (NOS), CCh no longer inhibite d I-Ca(L). I-K(ACh) could still be activated. 4. Co-incubation of cell s in L-NAME or in L-NMMA with arginine (the endogenous substrate of NO S) restored the CCh-induced attenuation of I-Ca(L), indicating that L- NAME or L-NMMA did not interfere directly with the muscarinic action o f CCh on I-Ca(L). 5. Effects of the NO-releasing agent molsidomine (SI N-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 in hibition of I-Ca(L) in mammalian cardiac pacemaker tissue.