Vectorial transport by double-transfected cells expressing the human uptake transporter SLC21A8 and the apical export pump ABCC2

Vectorial transport of endogenous substances, drugs, and toxins is an impor tant function of polarized cells. We have constructed a double-transfected Madin-Darby canine kidney (MDCK) cell line permanently expressing a recombi nant uptake transporter for organic anions in the basolateral membrane and an ATP-dependent export pump for anionic conjugates in the apical membrane. Basolateral uptake was mediated by the human organic anion transporter 8 ( OATP8; symbol SLC21A8) and subsequent apical export by the multidrug resist ance protein 2 (MRP2; symbol ABCC2). Under physiological conditions, both t ransport proteins are strongly expressed in hepatocytes and contribute to t he hepatobiliary elimination of organic anions. Expression and localization of OATP8 and MRP2 in MDCK cells growing on Transwell membrane inserts was demonstrated by immunoblotting and confocal laser scanning microscopy. H-3- Labeled sulfobromophthalein (BSP) was a substrate for both transport protei ns and was transferred from the basolateral to the apical compartment at a rate at least six times faster by double-transfected MDCK-MRP2/OATP8 cells than by single-transfected MDCK-OATP8 or MDCK-MRP2 cells. Vectorial transpo rt at a much higher rate by double-transfected than by sing le-transfected cells was also observed for the H-3-labeled substrates leukotriene C-4, 17 beta -glucuronosyl estradiol, and dehydroepiandrosterone sulfate, for the f luorescent anionic substrate fluo-3, and for the antibiotic rifampicin. Inh ibition studies indicated that intracellular formation of S-(2,4-dinitrophe nyl)-glutathione from 2,4-chlorodinitrobenzene selectively inhibits the tra nscellular transport of [H-3]BSP at the site of MRP2-mediated export. The d ouble-transfected cells provide a useful system for the identification of t ransport substrates and transport inhibitors including drug candidates.