Bile acid secretion and direct targeting of mdr1-green fluorescent protein from Golgi to the canalicular membrane in polarized WIF-B cells.
Bile acid secretion and direct targeting of mdr1-green fluorescent protein from Golgi to the canalicular membrane in polarized WIF-B cells.
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发表时间：
1999-12
影响因子：
4
通讯作者：
Yoshimichi Sai;A. Nies;Irwin M. Arias
中科院分区：
文献类型：
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作者：
Yoshimichi Sai;A. Nies;Irwin M. Arias
胆小管膜含有几种atp依赖性转运蛋白，这些转运蛋白参与胆汁分泌。小管转运蛋白在内质网中合成，在高尔基体中修饰，然后转运到根尖质膜。然而，atp依赖性转运蛋白在细胞内运输的途径和调控尚未阐明。在本研究中，我们构建了mdr1和绿色荧光蛋白的翻译融合，并研究了WIF-B细胞中胆汁酸分泌和mdr1的细胞内运输。与肝细胞相似，wi - b细胞分泌胆汁酸和有机阳离子（如罗丹明-123）进入胆管。牛磺酸胆酸盐和磷酸肌肽3-激酶十肽激活剂可刺激小管荧光素异硫氰酸-糖胆酸盐的分泌，渥曼宁可降低小管荧光素的分泌。用mdr1-GFP构建物短暂而稳定地转染wi - b9细胞。荧光在管膜、管周点状结构和高尔基区可见。延时显微镜显示mdr1-GFP以管状囊泡结构从高尔基体转移，其中大部分直接转移到小管膜。在管膜和根尖下区域之间也观察到循环。基底外侧质膜未检测到mdr1-GFP。15℃时，mdr1-GFP在高尔基体中积累；转移到37℃后，荧光直接移动到管膜上。牛磺胆酸盐增强了这一过程，wortmannin阻断了这一过程。在这些研究中，也没有观察到mdr1-GFP荧光在任何时候的基底外膜或其他胞内细胞器。总之，在wi - b细胞中，存在从高尔基体到管膜运输mdr1的直接途径，mdr1是一种胆管atp依赖的有机阳离子转运体。与正常肝细胞一样，磷酸肌肽3激酶调节wi - b细胞胆汁酸分泌和mdr1的细胞内转运。稳定转染mdr1-GFP的wi - b细胞为研究小管转运蛋白的运输和调控提供了一个重要的模型。在线影片：http://www.healthsci.tufts.edu/LABS/IMArias++ + /Sai_F9.html
The bile canalicular membrane contains several ATP-dependent transporters that are involved in biliary secretion. Canalicular transporters are synthesized in ER, modified in Golgi and transported to the apical plasma membrane. However, the route and regulation of intracellular trafficking of ATP-dependent transporters have not been elucidated. In the present study, we generated a translational fusion of mdr1 and green fluorescent protein and investigated bile acid secretion and intracellular trafficking of mdr1 in WIF-B cells, a polarized liver derived cell line. Similar to hepatocytes, WIF-B cells secrete bile acids and organic cations (i.e. rhodamine-123) into the bile canaliculi. Canalicular secretion of fluorescein isothiocyanate-glycocholate was stimulated by taurocholate and a decapeptide activator of phosphoinositide 3-kinase and was decreased by wortmannin. WIF-B9 cells were transiently and stably transfected with a mdr1-GFP construct. Fluorescence was observed in the canalicular membrane, pericanalicular punctate structures and Golgi region. Time lapse microscopy revealed that mdr1-GFP is transferred from Golgi as tubular vesicular structures the majority of which traveled directly to the canalicular membrane. Recycling between the canalicular membrane and subapical region was also observed. At no time was mdr1-GFP detected in the basolateral plasma membrane. At 15 degrees C, mdr1-GFP accumulated in Golgi; after a shift to 37 degrees C, fluorescence moved directly to the canalicular membrane. This process was enhanced by taurocholate and blocked by wortmannin. In these studies as well, no mdr1-GFP fluorescence was observed at any time in basolateral membranes or other intracellular organelles. In conclusion, in WIF-B cells, there is a direct route from Golgi to the canalicular membrane for trafficking of mdr1, a bile canalicular ATP-dependent transporter of organic cations. As in normal hepatocytes, phosphoinositide 3-kinase regulates bile acid secretion and intracellular trafficking of mdr1 in WIF-B cells. WIF-B cells stably transfected with mdr1-GFP provide an important model in which to study trafficking and regulation of canalicular transporters. Movies available on-line: http://www.healthsci.tufts.edu/LABS/IMArias++ + /Sai_F9.html