von Dippe P, Amoui M, Stellwagen R H, Levy D
University of Southern California, School of Medicine, Department of Biochemistry and Molecular Biology, Los Angeles, California 90033, USA.
J Biol Chem. 1996 Jul 26;271(30):18176-80. doi: 10.1074/jbc.271.30.18176.
Previous studies have suggested that the enzyme microsomal epoxide hydrolase (mEH) is able to mediate sodium-dependent transport of bile acids such as taurocholate into hepatocytes (von Dippe, P., Amoui, M., Alves, C., and Levy, D.(1993) Am. J. Physiol. 264, G528-G534). In order to characterize directly the putative transport properties of the enzyme, a pCB6 vector containing the cDNA for this protein (pCB6-mEH) was transfected into Madin-Darby canine kidney (MDCK) cells, and stable transformants were isolated that could express mEH at levels comparable with the levels expressed in hepatocytes. Sodium-dependent transport of taurocholate was shown to be dependent on the expression of mEH and to be inhibited by the bile acid transport inhibitor 4,4'-diisothiocyanostilbene-2,2'disulfonic acid (DIDS), as well as by other bile acids. Kinetic analysis of this system indicated a Km of 26.3 microM and a Vmax of 117 pmol/mg protein/min. The Km value is essentially the same as that observed in intact hepatocytes. The transfected MDCK cells also exhibited sodium-dependent transport of cholate at levels 150% of taurocholate in contrast to hepatocytes where cholate transport is only 30% of taurocholate levels, suggesting that total hepatocyte bile acid transport is a function of multiple transport systems with different substrate specificities, where mEH preferentially transports cholate. This hypothesis is further supported by the observation that a monoclonal antibody that partially protects (26%) taurocholate transport from inhibition by DIDS in hepatocytes provides almost complete protection (88%) from DIDS inhibition of hepatocyte cholate transport, suggesting that taurocholate is also taken up by an alternative system not recognized by this antibody. Additional support for this concept is provided by the observation that the taurocholate transport system is almost completely protected (92%) from DIDS inhibition by this antibody in MDCK cells that express mEH as the only bile acid transporter. These results demonstrate that mEH is expressed on the surface of hepatocytes as well as on transfected MDCK cells and is able to mediate sodium-dependent transport of taurocholate and cholate.
以往的研究表明，微粒体环氧化物水解酶（mEH）能够介导牛磺胆酸盐等胆汁酸依赖钠的转运进入肝细胞（冯·迪普，P.，阿莫伊，M.，阿尔维斯，C.，和利维，D.（1993年）《美国生理学杂志》264卷，G528 - G534页）。为了直接表征该酶假定的转运特性，将含有此蛋白cDNA的pCB6载体（pCB6 - mEH）转染到犬肾Madin - Darby（MDCK）细胞中，并分离出稳定的转化体，其mEH表达水平与肝细胞中的表达水平相当。结果表明，牛磺胆酸盐依赖钠的转运依赖于mEH的表达，并受到胆汁酸转运抑制剂4,4'-二异硫氰基芪-2,2'-二磺酸（DIDS）以及其他胆汁酸的抑制。对该系统的动力学分析表明，Km为26.3微摩尔，Vmax为117皮摩尔/毫克蛋白/分钟。Km值与在完整肝细胞中观察到的值基本相同。与肝细胞中胆酸盐转运仅为牛磺胆酸盐水平的30%相比，转染的MDCK细胞中胆酸盐的钠依赖性转运水平为牛磺胆酸盐的150%，这表明总肝细胞胆汁酸转运是具有不同底物特异性的多种转运系统的功能，其中mEH优先转运胆酸盐。这一假设得到了进一步支持，即一种单克隆抗体在肝细胞中能部分保护（26%）牛磺胆酸盐转运免受DIDS抑制，而对肝细胞胆酸盐转运免受DIDS抑制则提供几乎完全的保护（88%），这表明牛磺胆酸盐也通过该抗体未识别的另一种系统摄取。该概念的额外支持来自以下观察结果：在将mEH作为唯一胆汁酸转运蛋白表达的MDCK细胞中，该抗体对牛磺胆酸盐转运系统几乎完全保护（92%）免受DIDS抑制。这些结果表明，mEH在肝细胞表面以及转染的MDCK细胞表面表达，并且能够介导牛磺胆酸盐和胆酸盐依赖钠的转运。
