PURPOSE: The purpose of the present study is to investigate the expression of canalicular multispecific organic anion transporter (cMOAT) by its cDNA transfection in polarized Madin-Darby canine kidney cells (MDCK). METHODS: MDCK cells were transfected with an expression vector (pCXN2) containing the rat cMOAT cDNA with lipofectamine to obtain the stable transfectant under G418. Cells from a single colony whose cMOAT expression was the highest were seeded to form a tight epithelial monolayer on microporous membrane filters. Export of glutathione S-bimane (GS-B) from monolayers was determined after preloading its precursor, monochloro bimane (MCB). RESULTS: A comparable amount of GS-B was excreted to the apical and basal compartments in the vector-transfected cells. In contrast, in cMOAT-transfected cells, the amount apically excreted was approximately twice that excreted into the basal compartment. Cyclosporin A (CsA) (30 microM), an inhibitor of cMOAT at higher concentrations, inhibited the preferential apical export of GS-B from cMOAT-transfected cells. CONCLUSIONS: Rat cMOAT is functionally expressed on the apical membrane of MDCK cells after transfection.
PURPOSE: The purpose of the present study is to investigate the expression of canalicular multispecific organic anion transporter (cMOAT) by its cDNA transfection in polarized Madin-Darby canine kidney cells (MDCK). METHODS: MDCK cells were transfected with an expression vector (pCXN2) containing the ratcMOAT cDNA with lipofectamine to obtain the stable transfectant under G418. Cells from a single colony whose cMOAT expression was the highest were seeded to form a tight epithelial monolayer on microporous membrane filters. Export of glutathione S-bimane (GS-B) from monolayers was determined after preloading its precursor, monochloro bimane (MCB). RESULTS: A comparable amount of GS-B was excreted to the apical and basal compartments in the vector-transfected cells. In contrast, in cMOAT-transfected cells, the amount apically excreted was approximately twice that excreted into the basal compartment. Cyclosporin A (CsA) (30 microM), an inhibitor of cMOAT at higher concentrations, inhibited the preferential apical export of GS-B from cMOAT-transfected cells. CONCLUSIONS:RatcMOAT is functionally expressed on the apical membrane of MDCK cells after transfection.
Authors: R P Oude Elferink; C T Bakker; H Roelofsen; E Middelkoop; R Ottenhoff; M Heijn; P L Jansen Journal: Hepatology Date: 1993-03 Impact factor: 17.425
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