Supersensitivity of adenylyl cyclase after exposure to inhibitory agonists is a general means of cellular adaptation. We hypothesized that such "crosstalk" between muscarinic cholinergic agonists, beta 1-adrenoceptors, and adenylyl cyclase may be an important mechanism of cardiac adaptation to interventions that enhance vagal activity. We used primary cultures of neonatal rat ventricular myocytes and measured beta-adrenoceptors by radioligand binding and adenylyl cyclase activity by a single column method. Carbachol induced a time- and dose-dependent reversible decrease in cell surface beta 1-adrenoceptors. The peak effect occurred after 20 h of exposure to 100 microM carbachol which caused a decrease in the maximum number of binding sites for the beta-adrenoceptor antagonist 3H-CGP-12177 from 42.3 +/- 3.4 to 33.0 +/- 2.6 fmol/mg protein (n = 12, P < 0.03) without a change in antagonist affinity. Loss of cell surface receptors was prevented by atropine and by the protein kinase C inhibitor H7. The decrease in cell surface receptors was not accompanied by receptor internalization as assessed by equilibrium binding experiments in a cytosolic fraction using 125I-iodocyanopindolol. In contrast to the well-known acute inhibitory effects of carbachol on adenylyl cyclase activation, prolonged carbachol exposure preserved (-)-isoprenaline-stimulated adenylyl cyclase activity and enhanced postreceptor stimulated adenylyl cyclase activity. Carbachol did not further enhance adenylyl cyclase activity after pretreatment with pertussis toxin. The protein kinase C inhibitor chelerythrine prevented the carbachol induced enhancement of forskolin-stimulated adenylyl cyclase activity. We conclude that prolonged incubation with carbachol in rat neonatal ventricular myocytes causes a reduction in cell surface beta 1-Adrenoceptor density. beta 1-Adrenoceptor-mediated adenylyl cyclase activity is preserved and postreceptor-mediated adenylyl cyclase activity is augmented. Our data suggest that carbachol-stimulated protein kinase C activity may play a key role in the prolonged muscarinic regulation of adenylyl cyclase activity.