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Vol. 60, Issue 5, 934-943, November 2001
Division of Tumor Biochemistry, Deutsches Krebsforschungszentrum,
Heidelberg, Germany
Vectorial transport of endogenous substances, drugs, and toxins is an
important function of polarized cells. We have constructed a
double-transfected Madin-Darby canine kidney (MDCK) cell line permanently expressing a recombinant 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 resistance
protein 2 (MRP2; symbol ABCC2). Under physiological conditions, both
transport proteins are strongly expressed in hepatocytes and contribute
to the 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. 3H-Labeled sulfobromophthalein (BSP)
was a substrate for both transport proteins 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 transport
at a much higher rate by double-transfected than by single-transfected
cells was also observed for the 3H-labeled substrates
leukotriene C4, 17
-glucuronosyl estradiol, and
dehydroepiandrosterone sulfate, for the fluorescent anionic substrate
fluo-3, and for the antibiotic rifampicin. Inhibition studies indicated
that intracellular formation of
S-(2,4-dinitrophenyl)-glutathione from
2,4-chlorodinitrobenzene selectively inhibits the transcellular transport of [3H]BSP at the site of MRP2-mediated export.
The double-transfected cells provide a useful system for the
identification of transport substrates and transport inhibitors
including drug candidates.
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