Functional characterization of the hepatic sodium-dependent taurocholate transporter stably transfected into an immortalized liver-derived cell line and V79 fibroblasts.
Bile acids are taken up into liver parenchymal cells by active, carrier-mediated transport. This transport is lost during cell transformation in permanent growing liver tumor cell lines. In order to establish bile acid uptake in a permanent mammalian cell culture system, we transfected the cDNA from the cloned rat liver Na(+)-taurocholate cotransporting polypeptide (Ntcp) in Chinese hamster lung fibroblasts (V79 cells) and in a "hepatocyte-like" cell line HPCT-1F3 with three different gene transfer methods (calcium phosphate precipitation, lipofection, electroporation). A stable integration of the cDNA in both cell genomes was observed. However, in V79 fibroblasts, a permanent functional expression of taurocholate transport was not achieved. The sodium-dependent uptake of taurocholate was expressed permanently only in HPCT-1E3 cells, if the Ntcp was transfected by electroporation. In this cell line (HPCT-1E3-TC-6/2), substrate specificity, sodium- and energy dependence, as well as the kinetic parameters of the transfected single transporter were measured. The sodium-dependent taurocholate uptake was inhibited by addition of non-labeled bile acids, bumetanide, sulfobromophthalein and oligomycin. Pretreatment with 10 mM Na(+)-butyrate of this cell culture for 22 h stimulated taurocholate uptake twofold. Neither butyrate-stimulated cells nor unstimulated cells transport glycocholate or cholate. Besides taurocholate a fluorescence-labeled taurocholate derivative, NBD-taurocholate, was taken up by the HPCT-1E3-TC cells. In conclusion, the specific gene transfer with the electroporation technique in combination with the "right" cell line, HPCT-1E3, has been successful for the permanent and functional expression of the Ntcp. This allowed direct monitoring of the solitary sodium-dependent taurocholate transport system in a "liver cell-like" environment.
Platte
H D
HD
Institut für Pharmakologie und Toxikologie, Justus-Liebig-Universităt, Giessen/Germany.
Honscha
W
W
Schuh
K
K
Petzinger
E
E
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology*; D005455:Fluorescent Antibody Technique; D051381:Rats; D012964:Sodium / Q000494:pharmacology; D013656:Taurocholic Acid / Q000378:metabolism; D014162:Transfection
D002352:Carrier Proteins; D013656:Taurocholic Acid; D012964:Sodium