Human hepatobiliary transport of organic anions analyzed by quadruple-transfected cells
Human hepatobiliary transport of organic anions analyzed by quadruple-transfected cells
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DOI：
10.1124/mol.105.014605
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发表时间：
2005-10-01
影响因子：
3.6
通讯作者：
Keppler, D
中科院分区：
文献类型：
--
作者：
Kopplow, K;Letschert, K;Keppler, D
许多有机阴离子的肝胆消除由OATP 1B 1（OATP 2、LST-1、OATP-C）、OATP 1B 3（OATP 8）和OATP 2B 1（OATP-B）启动，它们是人肝细胞的主要摄取转运蛋白。此后，单向外排泵ABCC 2（多药耐药蛋白2）介导有机阴离子（包括谷胱甘肽结合物和葡萄糖醛酸苷）转运到胆汁中。在本研究中，我们生成了在基底外侧膜中稳定表达重组OATP 1B 1、OATP 1B 3和OATP 2B 1并在顶膜中稳定表达ABCC 2的Madin-Darby犬肾（MDCKII）细胞系。将稳定表达ABCC 2以及OATP 1B 1、OATP 1B 3或OATP 2B 1的双转染MDCK II细胞作为对照细胞。四重转染的细胞表现出高速率的有机阴离子，包括溴磺酞，胆囊收缩素肽（CCK-8），和雌酮3-硫酸的载体运输。四重转染的细胞能够鉴定在双转染细胞系研究中可能遗漏的摄取或载体转运底物，如CCK-8所示，其是OATP 1B 3的底物，但不是OATP 1B 1或OATP 2B 1的底物。三种肝细胞OATP转运蛋白覆盖的广泛底物谱使得能够代表性地分析许多有机阴离子在人肝细胞中的摄取。由四重转染细胞载体运输的广谱有机阴离子也提供了ABCC 2的底物选择性的有价值的信息，而不需要在含有ABCC 2蛋白的膜囊泡内进行研究。因此，四重转染的MDCKII-ABCC 2/OATP 1B 1/1B 3/2B 1细胞可用于鉴定底物和抑制剂，包括候选药物，在比人肝细胞原代培养物更好定义的条件下，这些底物和抑制剂被人肝细胞摄取和分泌。
Hepatobiliary elimination of many organic anions is initiated by OATP1B1 (OATP2, LST-1, OATP-C), OATP1B3 (OATP8), and OATP2B1 (OATP-B), which are the predominant uptake transporters of human hepatocytes. Thereafter, the unidirectional efflux pump ABCC2 (multidrug resistance protein 2) mediates the transport of organic anions, including glutathione conjugates and glucuronosides, into bile. In this study, we generated a Madin-Darby canine kidney (MDCKII) cell line stably expressing recombinant OATP1B1, OATP1B3, and OATP2B1 in the basolateral membrane and ABCC2 in the apical membrane. Double-transfected MDCKII cells stably expressing ABCC2 together with OATP1B1, OATP1B3, or OATP2B1 served as control cells. The quadruple-transfected cells exhibited high rates of vectorial transport of organic anions, including bromosulfophthalein, cholecystokinin peptide (CCK-8), and estrone 3-sulfate. The quadruple-transfected cells enabled the identification of substrates for uptake or vectorial transport that may be missed in studies with a double-transfected cell line, as exemplified by CCK-8, which is a substrate for OATP1B3 but not for OATP1B1 or OATP2B1. The broad substrate spectrum covered by the three hepatocellular OATP transporters enables representative analyses of the uptake of many organic anions into human hepatocytes. The broad spectrum of organic anions transported vectorially by the quadruple-transfected cells also provides valuable information on the substrate selectivity of ABCC2, without the need for studies in inside-out membrane vesicles containing the ABCC2 protein. The quadruple-transfected MDCKII-ABCC2/OATP1B1/1B3/2B1 cells may thus be useful for the identification of substrates and inhibitors, including drug candidates, undergoing uptake and secretion by human hepatocytes, under conditions that may be better defined than in primary cultures of human hepatocytes.