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

细胞内氧化还原状态调节的MRP2定位的肝内成像和分子分析

基本信息

批准号：
18390012
负责人：
HORIE Toshiharu
金额：
$ 8.97万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18390012/
关键词：
Biophysical Signal transduction Drug reactivity Protein 蛋自質

项目摘要

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.

氧化应激是胆汁淤积综合征的共同特征。我们描述了急性氧化应激下多药耐药相关蛋白2 （Mrp2）的内化，这是一种胆道转运蛋白，参与胆盐不依赖的胆汁流动，以及一系列最终导致新型蛋白激酶C （nPKC）激活的信号通路。然而，当细胞内氧化还原状态从氧化应激中恢复时，内化的Mrp2定位是否重新定位到小管膜尚不清楚。在这项研究中，我们证明了叔丁基过氧化氢（t-BHP）诱导的Mrp2小管表达的降低，通过GSH-乙酯（GSH的一种细胞渗透性形式）补充GSH，恢复到小管膜上。此外，用秋水仙碱和蛋白激酶A （PKA）抑制剂预处理离体大鼠肝细胞不会影响t-BHP诱导的Mrp2内化过程，但会阻止GSH补充诱导的Mrp2再循环过程。此外，细胞内cAMP浓度也随着细胞内GSH含量的变化而变化。综上所述，我们的数据清楚地表明，PKA/PKC激活的氧化还原敏感平衡通过两种不同的途径调节Mrp2的可逆定位，即Mrp2的微管非依赖性内化途径和依赖于Mrp2的再循环途径。