Abstract
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.
Highlights
Sodium-dependent transport of bile acids such as tauro- These compounds play a critical role in numerous physiological cholate into hepatocytes (von Dippe, P., Amoui, M., Alves, C., and Levy, D. (1993) Am
Transfection of Madin-Darby canine kidney (MDCK) Cells and Expression of microsomal epoxide hydrolase (mEH)—The expression vector pCB6-mEH was transfected into MDCK cells, and stable transformants were selected based on G-418 resistance
In addition to the close correspondence of mEH levels and molecular weight in MDCK cells compared with hepatocytes, the specific enzymatic activity of mEH was shown to be 85% of that observed in hepatocytes
Summary
Construction of Expression Vector pCB6-mEH—The cDNA for mEH was prepared from rat liver poly(A)ϩ mRNA using reverse transcriptase and the polymerase chain reaction amplification procedure [27] and subsequently ligated into the BglII/KpnI sites of the pCB6 vector under the control of the butyrate-inducible cytomegalovirus promoter for expression in MDCK cells. Transport of Taurocholate and Cholate by MDCK Cells and Hepatocytes—MDCK cells were removed from tissue culture dishes (100 mm) by initial treatment with 4.5 ml of a 0.002% trypsin solution in phosphate-buffered saline (PBS), pH 7.4, for 5 min at 37 °C, followed by washing with 2 ml of medium/fetal bovine serum followed by 10 ml of PBS. Uptake was measured by adding 125 l of buffer containing sodium or choline chloride with various amounts of [3H]taurocholate or [3H]cholate as well as unlabeled bile acid to 125 l of buffer containing approximately 2 ϫ 106 MDCK cells. The effect of mAb 25A-3 (1 mg/ml) on DIDS inhibition of bile acid transport was evaluated by preincubating the cells with the antibody for 30 min at 37 °C prior to the addition of DIDS.
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