Vectorial transport by double-transfected cells expressing the human uptake transporter SLC21A8 and the apical export pump ABCC2
Vectorial transport by double-transfected cells expressing the human uptake transporter SLC21A8 and the apical export pump ABCC2
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DOI：
10.1124/mol.60.5.934
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发表时间：
2001-11-01
影响因子：
3.6
通讯作者：
Keppler, D
中科院分区：
文献类型：
--
作者：
Cui, YH;König, J;Keppler, D
内源性物质、药物和毒素的载体运输是极化细胞的重要功能。我们构建了一种双转染的Madin-Darby犬肾（MDCK）细胞系，永久表达基侧膜中有机阴离子的重组摄取转运蛋白和顶膜中阴离子偶联物的atp依赖输出泵。基底外侧摄取由人体有机阴离子转运蛋白8 （OATP8，符号SLC21A8）介导，随后由多药耐药蛋白2 （MRP2，符号ABCC2）向顶端输出。在生理条件下，这两种转运蛋白都在肝细胞中强烈表达，并有助于肝胆清除有机阴离子。通过免疫印迹和共聚焦激光扫描显微镜证实了在Transwell膜插入上生长的MDCK细胞中OATP8和MRP2的表达和定位。h -3标记的次溴代眼蛋白（BSP）是这两种转运蛋白的底物，双转染MDCK-MRP2/OATP8细胞从基底外侧转移到根尖室的速度比单转染MDCK-OATP8或MDCK-MRP2细胞快至少6倍。对于h -3标记的底物白三烯C-4、17 β -葡萄糖醛酸雌二醇和硫酸脱氢表雄酮，荧光阴离子底物氟-3和抗生素利福平，双转染细胞的载体转运率也比单转染细胞高得多。抑制研究表明，2,4-氯二硝基苯在细胞内形成S-（2,4-二硝基苯）-谷胱甘肽选择性地抑制mrp2介导的[H-3]BSP的跨细胞转运。双转染细胞为鉴定转运底物和转运抑制剂（包括候选药物）提供了一个有用的系统。
Vectorial transport of endogenous substances, drugs, and toxins is an important function of polarized cells. We have constructed a double-transfected Madin-Darby canine kidney (MDCK) cell line permanently expressing a recombinant uptake transporter for organic anions in the basolateral membrane and an ATP-dependent export pump for anionic conjugates in the apical membrane. Basolateral uptake was mediated by the human organic anion transporter 8 (OATP8; symbol SLC21A8) and subsequent apical export by the multidrug resistance protein 2 (MRP2; symbol ABCC2). Under physiological conditions, both transport proteins are strongly expressed in hepatocytes and contribute to the hepatobiliary elimination of organic anions. Expression and localization of OATP8 and MRP2 in MDCK cells growing on Transwell membrane inserts was demonstrated by immunoblotting and confocal laser scanning microscopy. H-3-Labeled sulfobromophthalein (BSP) was a substrate for both transport proteins and was transferred from the basolateral to the apical compartment at a rate at least six times faster by double-transfected MDCK-MRP2/OATP8 cells than by single-transfected MDCK-OATP8 or MDCK-MRP2 cells. Vectorial transport at a much higher rate by double-transfected than by sing le-transfected cells was also observed for the H-3-labeled substrates leukotriene C-4, 17 beta -glucuronosyl estradiol, and dehydroepiandrosterone sulfate, for the fluorescent anionic substrate fluo-3, and for the antibiotic rifampicin. Inhibition studies indicated that intracellular formation of S-(2,4-dinitrophenyl)-glutathione from 2,4-chlorodinitrobenzene selectively inhibits the transcellular transport of [H-3]BSP at the site of MRP2-mediated export. The double-transfected cells provide a useful system for the identification of transport substrates and transport inhibitors including drug candidates.