乙酰胆碱调控缺氧诱导Connexin43磷酸化表达

摘要

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 methyl 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 Mynrardial ischemia (MI) is usually characterized as a myocytcs disease which lends to cell un coupling and arrhythmias. It has been known that alterations in the phosphorylation statc of eonncxins affcet cell cell eommunication through gap junctions. To invcstigaitc. the mechanisms of signal pathway of conncxins in isrhcmia in duced heart disease,we focused on conncxin43 (Cx43) ,a major cardiac gap junction protein. Methods H9c2 cells were subjected to hypoxia for time course: H9c2 cells were prctreitcd with acctylcholinc (ACh) for 1 hour and followed by 1 hour hypoxii. The amounts of phosphorylitcd and non phosphorylitcd isoforms of Cx43 in H9c2 were dctectee by Western blot. The distribution of Cx43 in H9c2 was conformed by confoal microscopy. Results In response to hypoxi a, phosphorylitcd isoform of Tx43 undcrwem progressive reduction with a time course. These obscrvitions suggest that hypoxia chmges the phosphorylition of Tx43 md tnnslorition of Tx43 from gap junctions into intraccllular pools. Howcyer,ACh inhibited hypoxii induced degradation of phosphorylitcd Tx13 and restored the function of Tx43 sup pressed by hypoxia. 1. NAME and Diazoxidc inhibited the increase of ACh regulation Tx43 in hypoxia. Chclcrythrinc and Glybcnclimidc did not show a rcmirkiblc inhibition effert rompared to NO inhibitor and ATP agonist. Conclusion This study indicates that the change of phosphorylitcd Xx43 is an enrly sign of cirdiac injury after ischemia/hypoxia. ACh cardioprotoction via gap junction is not only mediatcd by a single factor, but also comprchcnsivc strategy.