Intrahepatic imaging and molecular analysis of MRP2 localization regulated by the intracellular redox status

众所周知，氧化应激是胆汁淤积症的常见特征。我们已经描述了参与胆盐非依赖性胆汁流动的多药耐药相关蛋白2(MRP2)在急性氧化应激下的内化，以及最终导致新的蛋白激酶C(NPKC)激活的一系列信号通路；然而，当细胞内的氧化还原状态从氧化应激中恢复时，内化的MRP2的定位是否重新定位到小管膜上尚不清楚。在这项研究中，我们证明了由叔丁基氢过氧化氢(t-BHP)引起的小管内MRP2表达的减少可以通过GSH乙酯(一种细胞通透性的GSH)补充GSH而恢复到小管膜上。此外，用秋水仙碱和蛋白激酶A(PKA)抑制剂预处理大鼠肝细胞不影响t-BHP诱导的MRP2内化过程，但可阻止GSH补充诱导的MRP2循环过程。此外，细胞内cAMP浓度随着细胞内GSH含量的变化而变化。综上所述，我们的数据清楚地表明，氧化还原敏感的PKA/PKC激活平衡通过两条不同的途径调节可逆的MRp2定位，即微管非依赖性内化途径和MRp2依赖的循环途径。

Oxidative stress is known to be a common feature of cholestatic syndrome. We have described the internalization of multidrug resistance-associated protein 2 (Mrp2), a biliary transporter involved in bile-salt independent bile flow, under acute oxidative stress and a series of signaling pathways finally leading to the activation of novel protein kinase C (nPKC) were involved in this mechanism; however, it has been unclear whether the internalized Mrp2 localization was re-localized to the canalicular membrane when the intracellular redox status was recovered from oxidative stress. In this research, we demonstrated that decreased canalicular expression of Mrp2 induced by tertiary-butyl hydroperoxide (t-BHP) was recovered to the canalicular membrane by the replenishment of GSH by GSH-ethyl ester, a cell-permeable form of GSH. Moreover, pretreatment of isolated rat hepatocytes with colchicine and protein kinase A (PKA) inhibitor did not affect the t-BHP induced Mrp2 internalization process, but did prevent the Mrp2 recycling process induced by GSH replenishment. Moreover, intracellular cAMP concentration similarly changed with the change of intracellular GSH content. Taken together, our data clearly indicate that the redox-sensitive balance of PKA/PKC activation regulates the reversible Mrp2 localization in two different pathways, the microtubule-independent internalization pathway and -dependent recycling pathway of Mrp2.