乙酰胆碱调控缺氧诱导Connexin43磷酸化表达

Acetylcholine-Regulated Expression of Connexin43 Phosphorylation in Hypoxia

目的研究缺氧环境下乙酰胆碱(acetylcholine,ACh)调控心肌细胞缝隙连接蛋白43(Connexin 43,Cx43)磷酸化变化,探讨其在缺血性心脏疾病中的保护机制。方法使用乙酰胆碱预处理H9c2细胞培养1小时,随后缺氧培养1小时。应用免疫印迹(Western blot)分析Cx43磷酸化的动态变化,同时使用免疫组化观察H9c2细胞Cx43表达。结果在缺氧环境下,Cx43磷酸化随着缺氧时间的延长而呈进行性降低,其分布也发生明显改变。乙酰胆碱可抑制Cx43磷酸化的降低并恢复Cx43表达。一氧化氮合成酶抑制剂N-硝基-L-精氨酸甲酯(N-nitro-L-arginine meth-yl ester,L-NAME)和K-ATP激动剂二氮嗪(Diazoxide)抑制缺氧环境下ACh上调Cx43表达。PKC抑制剂白屈菜赤碱(Chelerythrine)和ATP敏感钾通道阻滞剂格列本脲(Glybenclamide)没有显著抑制在缺氧环境下ACh上调Cx43表达。结论 Cx43磷酸化是心肌缺血的早期信号,ACh通过心肌细胞缝隙连接蛋白的心肌保护作用不是单一因素作用而是与其它信号通路联合作用的综合结果。

Objective Myocardial ischemia（MI） is usually characterized as a myocytes disease which leads to cell uncoupling and arrhythmias.It has been known that alterations in the phosphorylation state of connexins affect cell-cell communication through gap junctions.To investigate the mechanisms of signal pathway of connexins in ischemia-induced heart disease,we focused on connexin43（Cx43）,a major cardiac gap junction protein.Methods H9c2 cells were subjected to hypoxia for time-course.H9c2 cells were pretreated with acetylcholine（ACh） for 1 hour and followed by 1 hour hypoxia.The amounts of phosphorylated and non phosphorylated isoforms of Cx43 in H9c2 were detected by Western blot.The distribution of Cx43 in H9c2 was conformed by confocal microscopy.Results In response to hypoxia,phosphorylated isoform of Cx43 underwent progressive reduction with a time course.These observations suggest that hypoxia changes the phosphorylation of Cx43 and translocation of Cx43 from gap junctions into intracellular pools.However,ACh inhibited hypoxia-induced degradation of phosphorylated Cx43 and restored the function of Cx43 suppressed by hypoxia.L-NAME and Diazoxide inhibited the increase of ACh-regulation Cx43 in hypoxia.Chelerythrine and Glybenclamide did not show a remarkable inhibition effect compared to NO inhibitor and ATP agonist.Conclusion This study indicates that the change of phosphorylated Cx43 is an early sign of cardiac injury after ischemia/hypoxia.ACh cardioprotection via gap junction is not only mediated by a single factor,but also comprehensive strategy.