Chang Z L, Puhl H L, May L G, Williams C L, Aronstam R S
Guthrie Research Institute, Sayre, PA 18840, USA.
Biochem Pharmacol. 1997 Oct 1;54(7):833-9. doi: 10.1016/s0006-2952(97)00250-5.
The influence of ethanol on the muscarinic receptor-mediated release of inositol phosphate from Chinese hamster ovary (CHO) cells stably transfected with one of the five subtypes of muscarinic acetylcholine receptor was determined. In CHO cells expressing M3 muscarinic receptors (CHO-M3), carbamylcholine increased muscarinic receptor-induced release of inositol phosphate by 150-350% following a 15-min incubation with an EC50 of approximately 30 microM. Maximal responses were obtained with 1 mM carbamylcholine, while responses to 10 mM carbamylcholine were somewhat less than maximal. Preincubation with atropine for 10 min inhibited the response with an IC50 of approximately 30 nM. CHO cells transfected with M1, M3, and M5 receptors displayed a similar pattern of activity; CHO cells transfected with M2 and M4, as well as untransfected cells, were unresponsive to carbamylcholine. Ethanol acutely inhibited the response of CHO-M3 cells to carbamylcholine by 15% at 18 mM and by 47% at 180 mM (the highest concentration examined). CHO-M3 cells were incubated with 50 mM ethanol for 48 hr. This treatment did not affect the number of cells or their protein content (113 pg/cell). The expression of M3 muscarinic receptors (determined using [3H]N-methylscopolamine) increased from 1.34 +/- 0.23 to 1.75 +/- 0.16 pmol/mg protein (P < 0.05). In contrast, carbamylcholine-stimulated release of inositol phosphate was depressed by 40-70% in four experiments. Concentration-response analyses indicated a non-competitive inhibitory mechanism. This dissociation of muscarinic receptor expression and muscarinic signaling suggests a compensatory increase in receptor expression in response to chronic inhibition of muscarinic signaling by ethanol.
研究了乙醇对稳定转染毒蕈碱型乙酰胆碱受体五种亚型之一的中国仓鼠卵巢（CHO）细胞中，毒蕈碱受体介导的肌醇磷酸释放的影响。在表达M3毒蕈碱受体的CHO细胞（CHO-M3）中，与卡巴胆碱孵育15分钟后，卡巴胆碱使毒蕈碱受体诱导的肌醇磷酸释放增加了150%-350%，半数有效浓度（EC50）约为30微摩尔。1毫摩尔卡巴胆碱可获得最大反应，而10毫摩尔卡巴胆碱的反应略小于最大反应。用阿托品预孵育10分钟可抑制反应，半数抑制浓度（IC50）约为30纳摩尔。转染M1、M3和M5受体的CHO细胞表现出类似的活性模式；转染M2和M4的CHO细胞以及未转染的细胞对卡巴胆碱无反应。乙醇在18毫摩尔时可急性抑制CHO-M3细胞对卡巴胆碱的反应15%，在180毫摩尔时（检测的最高浓度）抑制47%。将CHO-M3细胞与50毫摩尔乙醇孵育48小时。这种处理不影响细胞数量或其蛋白质含量（113皮克/细胞）。M3毒蕈碱受体的表达（使用[3H]N-甲基东莨菪碱测定）从1.34±0.23皮摩尔/毫克蛋白质增加到1.75±0.16皮摩尔/毫克蛋白质（P<0.05）。相比之下，在四项实验中，卡巴胆碱刺激的肌醇磷酸释放降低了40%-70%。浓度-反应分析表明存在非竞争性抑制机制。毒蕈碱受体表达与毒蕈碱信号传导的这种分离表明，乙醇对毒蕈碱信号传导的慢性抑制导致受体表达出现代偿性增加。
