Document Detail


The functional expression of sodium-dependent bile acid transport in Madin-Darby canine kidney cells transfected with the cDNA for microsomal epoxide hydrolase.
MedLine Citation:
PMID:  8663355     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
P von Dippe; M Amoui; R H Stellwagen; D Levy
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  271     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1996 Jul 
Date Detail:
Created Date:  1996-09-03     Completed Date:  1996-09-03     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  18176-80     Citation Subset:  IM    
Affiliation:
University of Southern California, School of Medicine, Department of Biochemistry and Molecular Biology, Los Angeles, California 90033, USA.
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MeSH Terms
Descriptor/Qualifier:
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / pharmacology
Animals
Bile Acids and Salts / metabolism*
Biological Transport / drug effects
Carrier Proteins / genetics,  metabolism*
Cells, Cultured
Cholic Acid
Cholic Acids / metabolism
DNA, Complementary
Dogs
Epoxide Hydrolases / genetics,  metabolism*
Kidney / cytology,  metabolism
Kinetics
Microsomes, Liver / enzymology*
Recombinant Proteins / metabolism
Sodium / metabolism*
Taurocholic Acid / metabolism
Transfection
Grant Support
ID/Acronym/Agency:
DK 25836/DK/NIDDK NIH HHS
Chemical
Reg. No./Substance:
0/Bile Acids and Salts; 0/Carrier Proteins; 0/Cholic Acids; 0/DNA, Complementary; 0/Recombinant Proteins; 53005-05-3/4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; 7440-23-5/Sodium; 81-24-3/Taurocholic Acid; 81-25-4/Cholic Acid; EC 3.3.2.-/Epoxide Hydrolases

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