title: Expression and characterization of a functional rat liver Na+ bile acid cotransport system in COS-7 cells.
abstract: A cDNA for the rat liver sodium-dependent bile acid cotransporter was expressed in COS-7 cells to study the functional properties of the translated protein in a mammalian cell line. A 1.2-kb insert was ligated into a pMAMneo vector and transiently transfected using electroporation. After optimal conditions were established, the transiently transfected COS cells were screened with fluorescent-conjugated labeled bile acids for evidence of expression of the cotransporter after 48 h. The uptake of [3H]taurocholate ([3H]TC) was then determined in cells transfected with or without the bile acid insert. Progressive uptake of [3H]TC (0.45 microM) was observed for 30 min in the presence of sodium. In contrast, no uptake of [3H]TC was observed in the absence of sodium, in nontransfected COS cells, or in COS cells transfected with the empty plasmid. Kinetic studies revealed a Michaelis constant (Km) of 29 microM, essentially identical to the Km of this cotransporter described in intact rat hepatocytes and membrane vesicles. Uptake of [3H]TC (5.0 microM) at 5 min (n = 3-6) was inhibited by 100 microM taurochenodeoxycholic acid (81%), tauroursodeoxycholic acid (77%), cholic acid (55%), chenodeoxycholic acid (74%), and ursodeoxycholic acid (56%) but not by 100 microM taurodehydrocholate, 1 mM probenecid, or 100 microM bilirubin. In contrast, bumetanide (500 microM) inhibited [3H]TC uptake by 52%. These studies indicate that the isolated cDNA codes for a physiological bile acid transporter present in rat hepatocytes and that posttranslational factors present in mammalian cells may not be as important in defining properties of this cotransport system.
authors.affiliation: Liver Center, Yale University School of Medicine, New Haven, Connecticut 06510-8050.
authors.forename: J L
authors.initials: JL
authors.lastname: Boyer
authors.forename: O C
authors.initials: OC
authors.lastname: Ng
authors.forename: M
authors.initials: M
authors.lastname: Ananthanarayanan
authors.forename: A F
authors.initials: AF
authors.lastname: Hofmann
authors.forename: C D
authors.initials: CD
authors.lastname: Schteingart
authors.forename: B
authors.initials: B
authors.lastname: Hagenbuch
authors.forename: B
authors.initials: B
authors.lastname: Stieger
authors.forename: P J
authors.initials: PJ
authors.lastname: Meier
mesh_terms: D000818:Animals; D001647:Bile Acids and Salts / Q000493:pharmacokinetics; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D005453:Fluorescence; D008099:Liver / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D029363:Organic Anion Transporters, Sodium-Dependent; D051381:Rats; D027981:Symporters; D013656:Taurocholic Acid / Q000493:pharmacokinetics; D014162:Transfection
chemical_list: D001647:Bile Acids and Salts; D002352:Carrier Proteins; D018076:DNA, Complementary; D029363:Organic Anion Transporters, Sodium-Dependent; D027981:Symporters; C071908:sodium-bile acid cotransporter; D013656:Taurocholic Acid