Acetylcholine reversal of isoproterenol-stimulated sodium currents in rabbit ventricular myocytes

We have recently shown that β-adrenergic agonists enhance the cardiac sodium current (I_Na) in rabbits through dual G-protein regulatory pathways. To determine if muscarinic cholinergic receptor stimulation can also modulate I_Na, we studied the effects of acetylcholine (ACh) and carbachol on I_Na in enzymatically dispersed rabbit ventricular myocytes. Whole-cell patch-clamp experiments done at room temperature using 20 mM [Na⁺]₀ showed that 100 nM isoproterenol increased I_Na and accelerated current decay as previously described. ACh (1 μM) or carbachol (1 μM) significantly reversed the stimulatory isoproterenol effects at test potentials throughout the I_Na activation range and at holding potentials negative to -80 mV. This effect was completely inhibited by atropine (1 μM) and was confirmed by studying single-channel I_Na from cell-attached patches. When I_Na was stimulated by forskolin (1 μM), carbachol (1 μM) significantly reversed the effect. The muscarinic-mediated inhibition of I_Na was inhibited by pertussis toxin (0.1 or 1.0 μg/ml) incubation (12-15 hours), suggesting that the effect was inhibitory G-protein dependent. Further investigation of the ACh inhibitory mechanism revealed that ACh alone had no effect on I_Na and that when cells were dialyzed with cAMP (5 μM), ACh failed to inhibit I_Na. Furthermore, cGMP failed to inhibit the effect of isoproterenol on I_Na. These data suggest that ACh acts at or proximal to adenylate cyclase stimulation. Thus, rabbit cardiac Na⁺ channels are regulated by muscarinic agonists in a fashion similar to cardiac Ca²⁺ channels.