Yh. Cui et al., Vectorial transport by double-transfected cells expressing the human uptake transporter SLC21A8 and the apical export pump ABCC2, MOLEC PHARM, 60(5), 2001, pp. 934-943
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.