细胞内Ca^2+在轮状病毒感染对Caco-2细胞NHE3蛋白调控中的作用

Role of intracellular Ca^2+ in regulating NHE3 expression in Caco-2 cells during rotavirus infection

目的研究轮状病毒(rotavirus,RV)感染对Caco-2细胞表面钠-氢(Na^+-H^+)交换蛋白3(NHE3)水平和生物活性的影响及其调控机制。方法构建表达NHE3的Caco-2细胞模型,分为对照组(CTL组)、RV组、细胞内Ca^2+络合剂BAPTA-AM组和BAPTA-AM+RV组,用BCECF-AM法和细胞表面NHE3生物素化法测定Na^+-H^+交换蛋白活性和细胞表面NHE3蛋白水平,Western blot测定细胞Cdc42蛋白水平。结果与CTL组相比,RV感染可引起细胞Na^+-H^+交换蛋白活性以及细胞表面NHE3蛋白水平明显降低,此抑制作用可被BAPTA-AM拮抗,此外RV感染可引起Cdc42蛋白水平升高,这一作用也可被BATPA-AM拮抗。结论RV感染对NHE3蛋白水平及生物活性的调控可能与细胞内Ca^2+介导的Cdc42依赖性内吞途径有关。

Objective To investigate the effects of rotavirus(RV)on the expression and bioactivity of Na^+-H^+ exchanger 3(NHE3)in Caco-2 cells and the possible regulatory mechanism.Methods Caco-2 cells expressing NHE3 were constructed and divided into four groups as follows:control(CTL)group,RV group,BAPTA-AM(a Ca^2+ chelator)group and BAPTA-AM+RV group.Na^+-H^+ exchanger activity and NHE3 expression on cell surface were determined using BCECF-AM and biotinylation assay,respectively.Expression of Cdc42 at protein level was measured by Western blot.Results Compared with the control group,RV infection significantly decreased the activity of NHE3 and its expression on cell surface.BATPA-AM antagonized the inhibitory effects on NHE3.Moreover,the expression of Cdc42 at protein level was increased following RV infection,which was also antagonized by BATPA-AM.Conclusions Intracellular Ca^2+-mediated Cdc42-dependent endocytosis pathway might be involved in regulating the expression and bioactivity of NHE3 during RV infection.