乌头碱对培养新生大鼠心室肌细胞Connexin43蛋白磷酸化的影响

目的建立细胞水平的染毒模型,观察乌头碱对心肌细胞缝隙连接蛋白Connexin43(Cx43)的影响。方法实验分0.25、0.5、0.75、1.0、1.5和2.0μmol/L等6个不同浓度乌头碱染毒组,运用蛋白印迹法和免疫荧光技术,检测各组及对照组心肌细胞Cx43蛋白磷酸化状态的改变。结果蛋白印迹检测显示,不同浓度染毒组心肌细胞中Cx43蛋白总量与正常对照组无显著差异;0.5μmol/L乌头碱染毒后,Cx43开始出现脱磷酸化;当染毒浓度达到1.0μmol/L后,Cx43脱磷酸化显著,且1.5及2.0μmol/L染毒效果和1.0μmol/L染毒组相当。免疫荧光分析结果提示乌头碱作用后,心肌细胞Cx43蛋白在羧基端第368位点丝氨酸残基(Ser368)发生明显的脱磷酸化。结论一定浓度的乌头碱能诱导心肌细胞缝隙连接蛋白Cx43发生显著脱磷酸化。

Effects of aconitine on connexin43 phosphorylation status in the cultured neonatal ventricular myocytes of rat

Objective To set up an experiment model on cellular level in order to investigate the effects of aconitine on Connexin43(Cx43)in cardiomyocytes of rat.Methods The cultured cardiomyocytes of rat were incubated with aconitine at 6 different concentrations of 0.25,0.5,0.75,1.0,1.5 and 2.0 μmol/L.The changes of Cx43 phosphorylation status of each group were investigated by Western blot analysis and immunofluorescent microscopy techniques.Results The total amount of Cx43 had not changed in cardiomyocytes after aconitine incubation by western blot analysis,but it began to induce Cx43 dephosphorylation after incubation of the cultures with 0.5 μmol/L aconitine and Cx43 underwent significant dephosphorylation when the concentration of aconitine elevated to 1.0 μmol/L,and the dephosphorylation effects of 1.5 and 2.0 μmol/L aconitine on Cx43 were similar to that of 1.0 μmol/L aconitine.Quantitative immunofluorescent microscopy revealed that Ser368,one of the serine amino acid phosphorylation sites in the C-terminal domain of Cx43,underwent significant dephosphorylation when incubation of the cardiomyocytes with 1.0 μmol/L aconitine.Conclusion Under certain concentration conditions,aconitine can induce significant dephosphorylation of Cx43 in the cardiomyocytes of rat.