Muscarinic receptor heterogeneity in neonatal rat ventricular myocytes in culture.

Carbachol increased ventricular automaticity in a concentration-dependent fashion from a control rate of 72 ± 5 (mean ± SEM) to 86 ± 4 beats per minute at 10 -4 M carbachol. Pirenzepine, an M 1 -selective antagonist, and AFDX 116, an M 2 -selective antagonist, both at 10 -7 M, did not block the carbachol-induced positive chronotropic response. In contrast, 10 -7 M HHSiD, an M 3 -selective antagonist, completely blocked the positive chronotropic effect of carbachol. Carbachol stimulated the accumulation of IP, in a concentration-dependent manner at concentrations ≥3 x 10 -6 M. AFDX 116 had no effect on carbachol-induced IP 1 accumulation. HHSiD significantly inhibited IP 1 accumulation at concentrations ≥3 x 10 -8 M, while pirenzepine inhibited IP 1 accumulation only at concentrations ≥10 -5 M. McN A343 and methacholine, two muscarinic receptor agonists with minimal M 2 activities, and carbachol did not alter basal cAMP concentration, but all three agonists significantly attenuated the increase in cAMP accumulation in response to isoproterenol. Carbachol inhibited isoproterenol-mediated cAMP accumulation at concentrations ≥10 -7 M. AFDX 116, HHSiD, and pirenzepine blocked the carbachol-induced inhibition of isoproterenol-stimulated cAMP accumulation. At equimolar concentrations, the inhibitory effects of HHSiD and AFDX-116 were similar, while that of pirenzepine was much less. Pretreatment with pertussis toxin for 24 h did not prevent the carbachol-mediated positive chronotropic response or accumulation of IP 1 but completely abolished the inhibition of isoproterenol-stimulated cAMP accumulation. These results indicate that (a) neonatal ventricular myocytes in culture have a heterogeneous population of muscarinic (M 2 and M 3 ) receptors, (b) the M 3 receptor is coupled to pertussis toxin-sensitive and pertussis toxin-insensitive G proteins, (c) M 3 receptor stimulation activates phosphoinositide hydrolysis and increases automaticity via a pertussis toxin-insensitive G protein-dependent pathway, and (d) both M 2 and M 3 receptors couple to pertussis toxin-sensitive G protein(s) to mediate the inhibition of intracellular cAMP accumulation in response to isoproterenol stimulation.