The functional expression of sodium-dependent bile acid transport in Madin-Darby canine kidney cells transfected with the cDNA for microsomal epoxide hydrolase.
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.
von Dippe
P
P
University of Southern California, School of Medicine, Department of Biochemistry and Molecular Biology, Los Angeles, California 90033, USA.
Amoui
M
M
Stellwagen
R H
RH
Levy
D
D
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D001647:Bile Acids and Salts / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D019826:Cholic Acid; D002793:Cholic Acids / Q000378:metabolism; D018076:DNA, Complementary; D004285:Dogs; D004851:Epoxide Hydrolases / Q000235:genetics / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D013656:Taurocholic Acid / Q000378:metabolism; D014162:Transfection
D001647:Bile Acids and Salts; D002352:Carrier Proteins; D002793:Cholic Acids; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013656:Taurocholic Acid; D012964:Sodium; D004851:Epoxide Hydrolases; D019826:Cholic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid