VISUALIZATION OF MUSCARINIC CHOLINERGIC RECEPTORS ON CHICK CARDIOMYOCYTES AND THEIR INVOLVEMENT IN PHOSPHATIDYLCHOLINE HYDROLYSIS

The purpose of this study was to visualize muscarinic receptors and th eir distribution on cardiomyocytes and to examine the effects of musca rinic cholinergic receptor (mACh-R) stimulation with carbachol on phos phatidylcholine hydrolysis. Cardiomyocytes were prepared as primary cu lture from 7-day-old chick embryo hearts. Cardiomyocytes, grown on cov er slips, were labelled with BODIPY PZ, a fluorescent analog of the mu scarinic receptor antagonist pirenzepine, and examined with a laser sc anning confocal microscope. mACh-R clusters were visualized and were f airly homogeneous in size with diameters ranging from 0.5 to 1.0 mu m. The number of receptor clusters per cell was 83.5 +/- 6.8 (mean +/- S EM) and clusters were found at the periphery of the cell. Cardiomyocyt es, grown as a monolayer in dishes, were treated with the 10(-4) M car bachol, a mACh-R agonist, and the effects on phosphatidylcholine hydro lysis were ascertained in cells preincubated with [methyl-H-3]choline for 18 h. Cells were washed, lysed, and subjected to thin-layer chroma tography to separate [H-3]choline in various metabolites of phosphatid ylcholine. Carbachol significantly (p < 0.05) increased intracellular free choline and decreased cellular phospholipid consistent with phosp hatidylcholine hydrolysis. Carbachol increased the amount of [H-3]chol ine that effluxed out of the cardiomyocyte into the medium. Carbachol- induced choline efflux was not prevented by pretreatment with n-butano l, a phospholipase D inhibitor, suggesting that other lipases such as phospholipase C are the major enzyme involved in phosphatidylcholine h ydrolysis. Pertussis toxin prevented carbachol-induced choline efflux and the changes in intracellular free choline and phospholipid. An act ion of carbachol through G proteins was supported by the ability of pe rtussis toxin to antagonize the carbachol-induced reduction in cAMP ge neration from isoproterenol. In summary, mACh-Rs, visualized in living cardiomyocytes, were peripheral to the nucleus. Phosphatidylcholine h ydrolysis induced by mACh-R stimulation may be a signal transduction p athway for mACh-R in the cardiomyocyte, operating through inhibitory G proteins sensitive to pertussis toxin.