Heterologous regulation of muscarinic and beta-adrenergic receptors in rat cardiomyocytes in culture

Previous work indicated that hyperstimulation of muscarinic receptors brings about profound changes not only in the density of the muscarinic receptors, but also of the beta-adrenoceptors in rat heart atria in vivo. We have now investigated whether a similar receptor cross-regulation occurs in cardiomyocytes in vitro. Cardiomyocytes from 3-4 day old rats were exposed to chemical agents on days 5-6 in culture. Densities of muscarinic and beta-adrenergic receptors were measured according to the binding of N-[3H]methylscopolamine and [ H]CGP 12177, respectively, to cell surface membranes and cell homogenates. Exposure of cells to the muscarinic agonist carbachol (1 mmol/l) brought about a profound decrease in the number of muscarinic receptors. The number of beta-adrenoceptors displayed biphasic changes, being augmented after 24 h (by 20-45% on the cell surface and by 29% in the homogenate) and diminished after 48 h and 72 h (after 48 h, decrease by 44-75% on the cell surface and by 36% in the homogenate). These effects of carbachol were not prevented by dimethylaminopropyl-bis-indolylmaleimide, the inhibitor of protein kinase C. Exposure of cells to the beta-adrenoceptor agonist isoprenaline (0.1 mmol/l) strongly diminished the number of beta-adrenoceptors on the cell surface and in the homogenate. The density of muscarinic receptors on the cell surface was diminished by 24-43% after 24 h exposure to isoprenaline and unchanged after 48 h, whereas the concentration of muscarinic receptors in the homogenate was unchanged after 24 h and increased by 20% after 48 h. The isoprenaline-induced decrease in the density of cell surface muscarinic receptors could not be simulated by forskolin and was not abolished by the protein kinase A inhibitors Rp-cAMPS and HA-1004. Dibutyryl cyclic AMP diminished the density of cell surface muscarinic receptors more than that of the beta-adrenergic receptors. Our data reveal a novel phenomenon of a biphasic change (an increase followed by a loss) in the density of beta-adrenoceptors during exposure of cardiocytes to carbachol. Activation of beta-adrenoceptors brings about less conspicuous changes in the density of muscarinic receptors. The observed phenomena of receptor cross-regulation cannot be explained by simple activations of protein kinases A and C.