Vectorial transport of the plant alkaloid berberine by double-transfected cells expressing the human organic cation transporter 1 (OCT1, SLC22A1) and the efflux pump MDR1 P-glycoprotein (ABCB1)
Vectorial transport of the plant alkaloid berberine by double-transfected cells expressing the human organic cation transporter 1 (OCT1, SLC22A1) and the efflux pump MDR1 P-glycoprotein (ABCB1)
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DOI：
10.1007/s00210-007-0219-x
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发表时间：
2008-02-01
影响因子：
3.6
通讯作者：
Keppler, Dietrich
中科院分区：
文献类型：
--
作者：
Nies, Anne T.;Herrmann, Elke;Keppler, Dietrich
肝细胞的一项重要功能是通过胆汁消除内源性和外源性小分子，其中许多是有机阳离子。为了研究有机阳离子的这种矢量转运，我们构建了双转染的 Madin-Darby 犬肾品系 II (MDCKII) 细胞系，该细胞系在基底外侧膜中永久表达人类有机阳离子转运蛋白 1 (OCT1, SLC22A1)，在顶端表达 MDR1 P-糖蛋白 (MDR1 P-gp, ABCB1)，这是一种依赖于三磷酸腺苷 (ATP) 的有机阳离子外排泵。膜。此外，还生成了稳定表达 OCT1、MDR1 P-gp 或人有机阳离子转运蛋白 2（OCT2、SLC22A2）的 MDCKII 单转染子。针对OCT1或OCT2的抗血清特异性地检测到人肝细胞基底外侧膜中的OCT1、人肾肾小管上皮细胞中的OCT2以及MDCKII转染子基底外侧膜中各自的重组转运蛋白。我们鉴定出亲脂性有机阳离子小檗碱（一种具有广泛生物活性的荧光植物生物碱）作为 OCT1 和 OCT2 的底物，米氏常数分别为 14.8 μM 和 4.4 μM。小檗碱还抑制 MDCK-OCT1 和 MDCK-OCT2 转染子对原型阳离子四乙铵和 1-甲基-4-苯基吡啶鎓的摄取。当转染细胞在渗透性过滤器支持物上极化生长时，MDCK-OCT1/MDR1 P-gp 双转染子将小檗碱从基底外侧转移到顶室的速度比 MDCK-OCT1 或 MDCK-MDR1 P-gp 单转染子快很多倍。特异性 MDR1 P-gp 抑制剂 zosuquidar 三盐酸盐 (LY335979) 强烈抑制小檗碱流入心尖室。 MDCK-OCT1/MDR1 P-gp 双转染子可用于鉴定 OCT1 和 MDR1 P-gp 的其他阳离子底物和抑制剂，包括候选药物。
An important function of hepatocytes is the biliary elimination of endogenous and xenobiotic small molecules, many of which are organic cations. To study this vectorial transport of organic cations, we constructed a double-transfected Madin-Darby canine kidney strain II (MDCKII) cell line permanently expressing the human organic cation transporter 1 (OCT1, SLC22A1) in the basolateral membrane and MDR1 P-glycoprotein (MDR1 P-gp, ABCB1), an adenosine triphosphate (ATP)-dependent efflux pump for organic cations, in the apical membrane. Additionally, MDCKII single transfectants stably expressing OCT1, MDR1 P-gp, or human organic cation transporter 2 (OCT2, SLC22A2) were generated. Antisera directed against OCT1 or OCT2 specifically detected OCT1 in the basolateral membrane of human hepatocytes, OCT2 in tubular epithelial cells of human kidney, and the respective recombinant transporter in the basolateral membrane of MDCKII transfectants. We identified the lipophilic organic cation berberine, a fluorescent plant alkaloid exhibiting a broad range of biological activities, as substrate of OCT1 and OCT2 with Michaelis-Menten constants of 14.8 mu M and 4.4 mu M, respectively. Berberine also inhibited the uptake of the prototypic cations tetraethylammonium and 1-methyl-4-phenylpyridinium by MDCK-OCT1 and MDCK-OCT2 transfectants. When transfected cells were grown polarized on permeable filter supports, berberine was transferred from the basolateral to the apical compartments many times faster by MDCK-OCT1/MDR1 P-gp double transfectants than by MDCK-OCT1 or MDCK-MDR1 P-gp single transfectants. The specific MDR1 P-gp inhibitor, zosuquidar trihydrochloride (LY335979), strongly inhibited berberine efflux into the apical compartment. The MDCK-OCT1/MDR1 P-gp double transfectants may be useful to identify additional cationic substrates and inhibitors of OCT1 and MDR1 P-gp, including drug candidates.