Goutsouliak V, Wang Y, Cynader M S, Rabkin S W
University of British Columbia, Faculty of Medicine, Vancouver Hospital, Canada.
Biochem Cell Biol. 1997;75(2):127-36.
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.
本研究的目的是可视化毒蕈碱受体及其在心肌细胞上的分布，并研究用卡巴胆碱刺激毒蕈碱胆碱能受体（mACh-R）对磷脂酰胆碱水解的影响。从7日龄鸡胚心脏制备原代培养的心肌细胞。将生长在盖玻片上的心肌细胞用BODIPY PZ标记，BODIPY PZ是毒蕈碱受体拮抗剂哌仑西平的荧光类似物，并用激光扫描共聚焦显微镜进行检查，观察到mACh-R簇，其大小相当均匀，直径范围为0.5至1.0微米。每个细胞的受体簇数量为83.5±6.8（平均值±标准误），且簇位于细胞周边。将生长在培养皿中单层的心肌细胞用10⁻⁴M卡巴胆碱（一种mACh-R激动剂）处理，并在预先用[甲基-³H]胆碱孵育18小时的细胞中确定其对磷脂酰胆碱水解的影响。细胞经洗涤、裂解后，进行薄层层析以分离磷脂酰胆碱各种代谢产物中的[³H]胆碱。卡巴胆碱显著（p<0.05）增加细胞内游离胆碱并减少细胞磷脂，这与磷脂酰胆碱水解一致。卡巴胆碱增加了从心肌细胞流出到培养基中的[³H]胆碱量。用磷脂酶D抑制剂正丁醇预处理不能阻止卡巴胆碱诱导的胆碱流出，这表明其他脂酶如磷脂酶C是参与磷脂酰胆碱水解的主要酶。百日咳毒素可阻止卡巴胆碱诱导的胆碱流出以及细胞内游离胆碱和磷脂的变化。百日咳毒素能够拮抗卡巴胆碱诱导的异丙肾上腺素cAMP生成减少，这支持了卡巴胆碱通过G蛋白发挥作用。总之，在活心肌细胞中可视化的mACh-R位于细胞核周边。mACh-R刺激诱导的磷脂酰胆碱水解可能是心肌细胞中mACh-R的信号转导途径，通过对百日咳毒素敏感的抑制性G蛋白起作用。
