title: Visualization of muscarinic cholinergic receptors on chick cardiomyocytes and their involvement in phosphatidylcholine hydrolysis.
abstract: The purpose of this study was to visualize muscarinic receptors and their distribution on cardiomyocytes and to examine the effects of muscarinic cholinergic receptor (mACh-R) stimulation with carbachol on phosphatidylcholine hydrolysis. Cardiomyocytes were prepared as primary culture from 7-day-old chick embryo hearts. Cardiomyocytes, grown on cover slips, were labelled with BODIPY PZ, a fluorescent analog of the muscarinic receptor antagonist pirenzepine, and examined with a laser scanning confocal microscope, mACh-R clusters were visualized and were fairly homogeneous in size with diameters ranging from 0.5 to 1.0 micron. The number of receptor clusters per cell was 83.5 +/- 6.8 (mean +/- SEM) and clusters were found at the periphery of the cell. Cardiomyocytes, grown as a monolayer in dishes, were treated with the 10(-4) M carbachol, a mACh-R agonist, and the effects on phosphatidylcholine hydrolysis were ascertained in cells preincubated with [methyl-3H]choline for 18 h. Cells were washed, lysed, and subjected to thin-layer chromatography to separate [3H]choline in various metabolites of phosphatidylcholine. Carbachol significantly (p < 0.05) increased intracellular free choline and decreased cellular phospholipid consistent with phosphatidylcholine hydrolysis. Carbachol increased the amount of [3H]choline that effluxed out of the cardiomyocyte into the medium. Carbachol-induced choline efflux was not prevented by pretreatment with n-butanol, a phospholipase D inhibitor, suggesting that other lipases such as phospholipase C are the major enzyme involved in phosphatidylcholine hydrolysis. Pertussis toxin prevented carbachol-induced choline efflux and the changes in intracellular free choline and phospholipid. An action of carbachol through G proteins was supported by the ability of pertussis toxin to antagonize the carbachol-induced reduction in cAMP generation from isoproterenol. In summary, mACh-Rs, visualized in living cardiomyocytes, were peripheral to the nucleus. Phosphatidylcholine hydrolysis induced by mACh-R stimulation may be a signal transduction pathway for mACh-R in the cardiomyocyte, operating through inhibitory G proteins sensitive to pertussis toxin.
authors.affiliation: University of British Columbia, Faculty of Medicine, Vancouver Hospital, Canada.
authors.forename: V
authors.initials: V
authors.lastname: Goutsouliak
authors.forename: Y
authors.initials: Y
authors.lastname: Wang
authors.forename: M S
authors.initials: MS
authors.lastname: Cynader
authors.forename: S W
authors.initials: SW
authors.lastname: Rabkin
mesh_terms: D020001:1-Butanol / Q000494:pharmacology; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D002642:Chick Embryo; D002794:Choline / Q000378:metabolism / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D006868:Hydrolysis / Q000187:drug effects; D018613:Microscopy, Confocal; D009206:Myocardium / Q000737:chemistry*; D037342:Pertussis Toxin; D010713:Phosphatidylcholines / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000032:analysis* / Q000187:drug effects; D013997:Time Factors; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D010713:Phosphatidylcholines; D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; D020001:1-Butanol; D002217:Carbachol; D000242:Cyclic AMP; D037342:Pertussis Toxin; D002794:Choline