MUSCARINIC RECEPTOR HETEROGENEITY IN NEONATAL RAT VENTRICULAR MYOCYTES IN CULTURE

Carbachol increased ventricular automaticity in a concentration-depend ent 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)-selectiv e antagonist, and AFDX 116, an M(2)-selective antagonist, both at 10(- 7) M, did not block the carbachol-induced positive chronotropic respon se. In contrast, 10(-7) M HHSiD, an M(3)-selective antagonist, complet ely blocked the positive chronotropic effect of carbachol. Carbachol s timulated the accumulation of IP1 in a concentration-dependent manner at concentrations greater than or equal to 3 x 10(-6) M. AFDX 116 had no effect on carbachol-induced IP1 accumulation. HHSiD significantly i nhibited IP1 accumulation at concentrations greater than or equal to 3 x 10(-8) M, while pirenzepine inhibited IP1 accumulation only at conc entrations greater than or equal to 10(-5) M. McN A343 and methacholin e, two muscarinic receptor agonists with minimal M(2) activities, and carbachol did not alter basal cAMP concentration, but all three agonis ts significantly attenuated the increase in cAMP accumulation in respo nse to isoproterenol. Carbachol inhibited isoproterenol-mediated cAMP accumulation at concentrations greater than or equal to 10(-7) M. AFDX 116, HHSiD, and pirenzepine blocked the carbachol-induced inhibition of isoproterenol-stimulated cAMP accumulation. At equimolar concentrat ions, 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 IP1 but completely abolished the inhibitio n of isoproterenol-stimulated cAMP accumulation. These results indicat e that (a) neonatal ventricular myocytes in culture have a heterogeneo us population of muscarinic (M(2) and M(3)) receptors, (b) the M(3) re ceptor is coupled to pertussis toxin-sensitive and pertussis toxin-ins ensitive G proteins, (c) M(3) receptor stimulation activates phosphoin ositide hydrolysis and increases automaticity via a pertussis toxin-in sensitive G protein-dependent pathway, and (d) both M(2) and M(3) rece ptors couple to pertussis toxin-sensitive G protein(s) to mediate the inhibition of intracellular cAMP accumulation in response to isoproter enol stimulation.