目的评价CX43蛋白磷酸化在大鼠逼尿肌过度活动症(detrusor overactivity,DO)中的作用,并初步探讨PP1和PP2A对CX43蛋白磷酸化修饰状态的影响。方法建立大鼠DO模型作为实验组,假手术组作为对照组,Western blot检测CX43蛋白磷酸化水平、PP1及PP2A的表达变化,免疫荧光双染对CX43与PP1、PP2A的定位及表达变化进行检测,荧光漂白恢复实验(FRAP)比较正常组、DO组、DO+碱性磷酸酶(AP)组的细胞间通讯功能(gap junction intercellular communication,GJIC)的差异。结果与正常组相比,DO组膀胱中的CX43蛋白的不同磷酸化修饰状态(P0、P1、P2)的表达水平均明显升高(P〈0.05),PP2A的表达明显降低(P〈0.05),PP1的表达未发现明显改变(P〉0.05)。DO组的GJIC较正常组明显增加(P〈0.05),DO+AP组的GJIC较正常组和DO组明显降低(P〈0.05)。结论CX43蛋白的磷酸化水平上调可能在DO的发病过程中发挥着重要的作用,这种磷酸化水平的上调可能与PP2A的表达减少相关。
Objective To evaluate the effect of Connexin 43 (CX43) phosphorylation on detrusor overactivity (DO) in rats, and preliminarily investigate the influence of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) on CX43 phosphorylation. Methods DO rat model was established as the experimental group, and sham-operated rats were selected as the control group. The CX43 protein phosphorylation levels and the PP1 and PP2A expression changes were detected by Western blotting. Double immunofluorescence staining was used for detecting the localization and expression PP2A. Fluorescence recovery after photobleaching (FRAP) was employed to intercellular communication (GJIC) of the control group, the DO group and the changes of CX43, compare the gap DO + alkaline ph PP1 and junction osphatase (AP) group. Results Compared with the control group, the expression of CX43 protein with different phosphorylation statuses ( P0, P1, and P2) in bladder was markedly increased in the DO group (P 〈 0. 05 ), while PP2A expression was decreased (P 〈0. 05 ), but PP1 expression had no significant change. The GJIC of the DO group was significantly increased as compared to the control group, and the GJIC of the DO + AP group was significantly decreased as compared to the control group and the DO group. Conclusion Up- regulation of CX43 protein phosphorylation may play an important role in the progress of DO, and may be closely related with the decrease of PP2A expression.