Expression of the apical conjugate export pump, Mrp2, in the polarized hepatoma cell line, WIF‐B
Expression of the apical conjugate export pump, Mrp2, in the polarized hepatoma cell line, WIF‐B
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顶端缀合物输出泵 Mrp2 在极化肝癌细胞系 WIF-B 中的表达
DOI：
10.1002/hep.510280523
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发表时间：
1998
期刊：
影响因子：
13.5
通讯作者：
D. Keppler
中科院分区：
文献类型：
--
作者：
A. Nies;T. Cantz;M. Brom;I. Leier;D. Keppler
极化的大鼠肝癌/人成纤维细胞杂交细胞系WIF-B形成顶端空泡，亲胆汁物质分泌到其中。我们研究了顶端共轭输出泵MRP2在WIF-B细胞中的表达、定位和功能。MRP2是多药耐药蛋白的顶端亚型，也被称为管状MRP或管状多特异性有机阴离子转运体，是一种190kd的膜糖蛋白，介导依赖于三磷酸腺苷的葡萄糖醛酸类化合物、谷胱甘肽S结合物和其他两亲性阴离子通过肝细胞管膜进入胆汁。用逆转录聚合酶链式反应(RT-PCR)检测大鼠mrp2基因在WIF-B细胞中的表达，并对扩增出的789个碱基的片段进行测序。免疫印迹法利用与大鼠MRP2氨基末端或羧基末端序列反应的抗体，在WIF-B细胞匀浆中检测到190kd的糖蛋白。免疫荧光显微镜下可见MRP2定位于根尖膜区。用钙依赖的荧光指示剂Fluo-3的膜透性酯预负载WIF-B细胞，然后由MRp2介导的两亲性阴离子Fluo-3分泌到顶端空泡。这种转运被添加到培养基中的环孢菌素A有效地抑制。直接测量含mRp2的质膜小泡与缺乏mrp2的质膜小泡的依赖三磷酸腺苷转运的结果表明，Fluo-3是由mrp2转运的，Km值为3.7Mol/L。我们的结果表明，极化的WIF-B细胞表达大鼠顶端结合转运型ATPase的同源基因，mRp2。因此，可以使用荧光两亲性阴离子Fluo-3来分析MRP2的功能以及抑制剂的作用。
The polarized rat hepatoma/human fibroblast hybrid cell line, WIF‐B, forms apical vacuoles into which cholephilic substances are secreted. We studied expression, localization, and function of the apical conjugate export pump, Mrp2, in WIF‐B cells. Mrp2, the apical isoform of the multidrug resistance protein, alternatively termed canalicular Mrp (cMrp) or canalicular multispecific organic anion transporter (cMoat), is a 190‐kd membrane glycoprotein mediating adenosine triphosphate (ATP)‐dependent transport of glucuronides, glutathione S‐conjugates, and other amphiphilic anions across the hepatocyte canalicular membrane into bile. Expression of the rat mrp2 gene in WIF‐B cells was shown by reverse‐transcription polymerase chain reaction (PCR), followed by sequencing of the amplified 789‐bp fragment. Immunoblotting, using antibodies reacting with the amino‐terminal or with the carboxyl‐terminal sequence of rat Mrp2, detected the 190‐kd glycoprotein in WIF‐B cell homogenates. Immunofluorescence microscopy localized Mrp2 to the apical membrane domain. Preloading of WIF‐B cells with a membrane‐permeable ester of the calcium‐dependent fluorescent indicator, Fluo‐3, was followed by Mrp2‐mediated secretion of the amphiphilic anion, Fluo‐3, into the apical vacuoles. This transport was potently inhibited by cyclosporin A added to the culture medium. Direct measurements of ATP‐dependent transport into Mrp2‐containing plasma membrane vesicles in comparison with Mrp2‐deficient vesicles established that Fluo‐3 is transported by Mrp2 with a Km value of 3.7 μmol/L. Our results indicate that the polarized WIF‐B cells express the rat ortholog of the apical conjugate–transporting ATPase, Mrp2. The function of Mrp2 as well as the action of inhibitors can thus be analyzed by use of the fluorescent amphiphilic anion, Fluo‐3.
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影响因子：
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作者：
Shanks,MR;Cassio,D;Lecoq,O;Hubbard,AL
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DOI：
10.1016/s0021-9258(18)83166-0
发表时间：
1989-05
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10.1016/s0021-9258(18)83165-9
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1989-05
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The Journal of biological chemistry
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DOI：
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发表时间：
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The Journal of biological chemistry
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影响因子：
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作者：
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