Transcellular Transport of Organic Anions Across a Double- transfected Madin-Darby Canine Kidney II Cell Monolayer Expressing Both Human Organic Anion-transporting Polypeptide (OATP2/SLC21A6) and Multidrug Resistance-associated Protein 2 (MRP2/ABCC2)*

From the ‡Department of Biopharmaceutics, School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, the §Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, 1-33, Yayoi-chou, Inage-ku, Chiba 263-8522, Japan, and the ¶Division of Nephrology, Endocrinology, and Vascular Medicine, Department of Medicine, Tohoku University Graduate School of Medicine, Sendai, 1-1, Seriyo-cho, Aoba-ku 980-8575, Japan

These anionic compounds taken up into hepatocytes are then excreted into the bile via apically located ATP-dependent transporters, such as bile salt export pump (symbol ABCB11) (13) and multidrug resistance-associated protein 2 (MRP2, ABCC2) (4,14,15). Bile salt export pump is responsible for the biliary excretion of monovalent bile acids (13), whereas MRP2 mediates the excretion of a variety of anionic compounds, including conjugated metabolites (4,14). The substrate specificity of MRP2 has been determined by comparing the transport across the bile canalicular membrane between normal and MRP2-deficient mutant rats and by examining the ATP-dependent uptake into membrane vesicles expressing MRP2 (4,14). Because of the similar substrate specificity between OATP2 and MRP2, it is considered that these transporters participate jointly in the transport of their substrates from blood into bile. However, no in vitro model to reflect the vectorial transport of organic anions has been reported. Evers et al. (16) and we ourselves (17) have established MDCK cell lines that stably express human and rat MRP2, respectively, and demonstrated the preferential apical excretion of intracellularly formed glutathione conjugates. These results are consistent with the apical localization of MRP2 in transfected MDCK cells (16). Although Evers et al. (16) have also shown that some hydrophobic cations (such as vinblastine), which may be cotransported with reduced glutathione by MRP2, are transported from the basal to apical compartments in an MRP2expressing MDCK monolayer, the vectorial transport of organic anions has not been demonstrated due to their poor penetration into cell monolayers across the basolateral membrane. We focused on the fact that OATP2 is expressed on the basolateral membrane of transfected MDCK cells (9) and, in the present article, we report the establishment of a double-transfected MDCK monolayer, which stably expresses OATP2 and MRP2 on basolateral and apical membranes, respectively, as an in vitro model to determine the transcellular transport of a series of organic anions. As model compounds, we used E 2 17␤G, pravastatin, and leukotriene C 4 (LTC 4 ), which are transported by OATP2 and MRP2 (5,6), along with E 1 S and DHEAS, which are transported by OATP2 but not by MRP2. We also examined the transcellular transport of taurolithocholate sulfate (TLC-S), which is transported by rat oatp1 and Mrp2 (18 (16), kindly provided by Dr. Piet Borst, were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum at 37°C under 5% CO 2 . The transfection of plasmids into MDCK II cells was performed using Lipo-fectAMINE (Invitrogen, Gaithersburg, MD). At 30% confluency, cells in 150-mm dishes were exposed to serum-free DMEM containing plasmid (1 g/ml) and LipofectAMINE (1 g/ml). At 5 h after the initiation of transfection, the plasmid-LipofectAMINE solution was removed, and the medium, consisting of DMEM supplemented with 10% fetal bovine serum, was added. After 2 weeks of Zeocin selection (700 g/ml), single colonies were screened for the expression of OATP2 by Northern blot analysis.
Western Blot Analysis-For the Western blot analysis, crude membrane was prepared from MDCK II cells according to the method of Gant et al. (20). Cells were homogenized in five volumes of 0.1 M Tris-HCl buffer, pH 7.4, containing 1 g/ml leupeptin and pepstatin A and 50 g/ml phenylmethylsulfonyl fluoride with 20 strokes of a Dounce homogenizer. After centrifugation (1500 ϫ g for 10 min) of the homogenate, the supernatant was recentrifuged (100,000 ϫ g for 30 min). The precipitate was suspended in Tris-HCl buffer and recentrifuged (100,000 ϫ g for 30 min). The crude membrane fraction was resuspended in the 0.1 M Tris-HCl buffer containing the proteinase inhibitors with five strokes of a Dounce homogenizer and stored at Ϫ80°C before being used for Western blot analysis. All procedures were performed at 0 -4°C. The membrane protein concentrations were determined by the method of Lowry et al. (21) with bovine serum albumin (BSA) as a standard.
For the Western blot analysis, 20 g of crude membrane was dissolved in 10 l of 0.25 M Tris-HCl buffer containing 2% SDS, 30% glycerol, and 0.01% bromphenol blue, pH 6.8, and subjected onto a 7.5% SDS-polyacrylamide gel electrophoresis with a 4.4% stacking gel. The molecular weight was determined using a prestained protein marker (New England BioLabs, Beverly, MA). Proteins were transferred electrophoretically to a nitrocellulose membrane (Millipore, Bedford, MA) using a blotter (Bio-Rad Laboratories, Richmond, CA) at 15 V for 1 h. The membrane was blocked with Tris-buffered saline containing 0.05% Tween 20 (TBS-T) and 5% BSA overnight at 4°C. After washing with TBS-T, the membrane was incubated for 1 h at room temperature in TBS-T containing 5% BSA and 500-fold diluted anti-OATP2 rabbit serum, which was raised in rabbits against the 21 amino acids at the carboxyl terminus of the deduced OATP2 sequence (ESLNKNKHFVP-SAGADSETHC) (9), or with 500-fold diluted anti-MRP2 rabbit serum, which was raised against 12 amino acids at the carboxyl terminus of the deduced MRP2 sequence (EAGIENVNSTKF). For the detection of OATP2, the membrane was allowed to bind to 125 I-labeled sheep antirabbit IgG in TBS-T containing 5% BSA for 1 h at room temperature. Then, the membrane was exposed to imaging plates (Fuji Film, Tokyo, Japan) overnight at room temperature. The intensity of the specific band was quantified using a Bio-Image Analyzer (Bas 1800, Fuji Film). For the detection of MRP2, the membrane was allowed to bind to donkey anti-rabbit IgG conjugated with the horseradish peroxidase (Amersham Biosciences, Inc., Buckinghamshire, UK). The enzyme activity was assessed by using ECL Plus Western blotting Starter Kit (Amersham Biosciences, Inc.) with luminescent image analyzer (LAS-1000 plus, Fuji Film).
Immunofluorescence Microscopy of Transfected Cells-Transfected MDCK cells were grown on Transwell membrane inserts (pore size of 3 m; Falcon, Bedford, MA). Sodium butyrate was added to the culture medium 24 h before the experiment. After fixation with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min and permeabilization in 1% Triton X-100 in PBS for 10 min, cells were incubated with the previously described polyclonal antibody against OATP2 (diluted 50-fold in PBS) or the monoclonal antibody against MRP2 (Alexis Biochemicals, M2II-12; diluted 40-fold in PBS) for 30 min at room temperature. Cells were then washed three times with PBS and incubated with Goat anti-rabbit IgG (Alexa 488) or Goat anti-mouse IgG (Alexa 546) (Molecular Probes, Inc., Eugene, OR, diluted 250-fold in PBS) for 30 min at room temperature. Nuclei were stained with SYTO61 (Molecular Probes, diluted 1000-fold in PBS). Membranes were cut from the inserts and mounted onto slides with 50% glycerol in PBS. Confocal laser-scanning immunofluorescence microcopy was performed using an LSM-410 apparatus from Carl Zeiss.
Transcellular Transport Study-Transfected MDCK II cells were seeded in 24-well plates at a density of 1.4 ϫ 10 5 cells per well and cultured with 10 mM sodium butyrate for 24 h (8). For uptake studies, cells were washed three times and preincubated with Krebs-Henseleit buffer or Na ϩ -free buffer, prepared by substituting Na ϩ by choline, at 37°C for 5 min. Krebs-Henseleit buffer consisted of 142 mM NaCl, 23. Data Analysis-For the kinetic analysis, the transcellular transport of ligands determined over 2 h was used. The kinetic parameters for transcellular transport of [ 3 H]E 2 17␤G and [ 3 H]pravastatin were estimated from the following Michaelis-Menten equation where v 0 is the initial uptake rate of substrates (picomoles/min/mg of protein), S is the substrate concentration in medium (M), K m is the Michaelis constant (M), V max is the maximum uptake rate (pmol/min/mg of protein), and P diff is the non-saturable transport clearance (microliters/min/mg of protein). The uptake data were fitted to this equation by a nonlinear least-squares method with a MULTI program (22) to obtain estimates of the kinetic parameters. The input data were weighted as the reciprocals of the squares of the observed values.
The permeability-surface area product across the apical membrane (PS apical ) was calculated by dividing the rate for the transcellular transport of [ 3 H]E 2 17␤G determined over 2 h by the cellular concentration of [ 3 H]E 2 17␤G determined at the end of the experiments (2 h).

RESULTS
Expression of OATP2 and MRP2 in MDCK II Cells-The expression of OATP2 and MRP2 in MDCK II cells was determined by confocal immunofluorescence laser scanning microscopy. As shown in Fig. 1, OATP2 and MRP2 were localized on the lateral and apical membranes of transfected MDCK II cells, respectively, and their pattern of expression was not superimposable in the double transfectant. The expression level of these transporters was also confirmed by Western blot analysis (Fig. 2). Semiquantitative analysis of the results of Western blot analysis showed that the expression levels of OATP2 and MRP2 in the single-transfected MDCK II cells were about the same as those in the double-transfected MDCK II cells; that is, the expression levels of OATP2 and MRP2 in the single-transfected cells were ϳ1.2 and 0.9 times those in the double-transfected cells.
Transcellular Transport of E 2 17␤G Across MDCK II Monolayers-Transcellular transport of E 2 17␤G across MDCK II monolayers expressing OATP2 and MRP2, along with that across the double-transfected monolayer, was compared with that across the control monolayer. As shown in Fig. 3, a symmetrical flux of E 2 17␤G was observed across the control and MRP2-expressing MDCK II monolayer. The basal-to-apical flux of E 2 17␤G across the OATP2-expressing monolayer was approximately two times higher than that in the opposite direction (Fig. 3), whereas the basal-to-apical flux of E 2 17␤G was ϳ9 times higher than that in the opposite direction in the double-transfected cells (Fig. 3). The cellular accumulation of E 2 17␤G, determined at 2 min, in the double-transfected cells was significantly lower than that in OATP2 expressing cells (17.8 Ϯ 0.8 pmol/mg of protein versus 34.0 Ϯ 4.9 pmol/mg of protein, n ϭ 3, p Ͻ .05).
The basal-to-apical flux of E 2 17␤G across the MDCK II monolayer expressing OATP2 and the double transfectant was saturable (Fig. 4). Kinetic analysis revealed that the saturation can be best described by assuming the presence of one saturable and one non-saturable components. The analysis gave K m , V max and P diff values of 23.8 Ϯ 6.1 M, 249 Ϯ 60 (pmol/min/mg of protein) and 0.81 ϩ 0.41 (l/min/mg of protein), respectively, for the OATP2 expressing MDCK II monolayer. This P diff value was comparable to that determined in the parental MDCK II monolayer (1.10 Ϯ 0.03 l/min/mg of protein). On the other hand, K m , V max and P diff values of 27.9 Ϯ 4.1 M, 560 Ϯ 89 (pmol/min/mg of protein), and 6.01 Ϯ 0.61 (l/min/mg of protein), respectively, were obtained also for the double-transfected cells (Fig. 4).
Furthermore, to quantitatively evaluate the transport activity across the apical membrane, the PS apical for E 2 17␤G was also determined. As shown in Fig. 5, the PS apical in the double transfectant was significantly higher than that in the control monolayer. Moreover, the PS apical for E 2 17␤G in the double-transfected cells was not saturated even if the concentration of E 2 17␤G in the basal compartment was increased to 150 M (Fig. 5).
Transcellular Transport of Pravastatin, LTC 4 , TLC-S, E 1 S, and DHEAS Across an MDCK II Monolayer-Transcellular transport of pravastatin, LTC 4 , TLC-S, E 1 S, and DHEAS across an MDCK II monolayer was also determined. As shown in Fig. 6, the basal-to-apical transport of pravastatin was 2.5 times higher than that in the opposite direction in the doubletransfected cells. The transcellular transport of pravastatin across the monolayer was saturable, with K m , V max , and P diff values of 24.3 Ϯ 10.4 M, 149 Ϯ 56 pmol/min/mg of protein, and 0.99 Ϯ 0.32 l/min/mg of protein, respectively (Fig. 7). The cellular accumulation of pravastatin, determined at 2 min, in the double-transfected cells was significantly lower than that in OATP2-expressing cells (4.7 Ϯ 0.6 pmol/mg of protein versus 12.0 Ϯ 2.9 pmol/mg of protein, n ϭ 3, p Ͻ 0.05).
For LTC 4 , the basal-to-apical flux was two and four times higher than that in the opposite direction in an MDCK II monolayer expressing OATP2 and the double transfectant, respectively (Fig. 8). In the same manner, the basal-to-apical flux of TLC-S was 3.1 and 11.5 times higher than that in the opposite direction in an MDCK II monolayer expressing OATP2 and the double transfectant, respectively (Fig. 9).
Transcellular transport of E 1 S and DHEAS was also determined across the monolayers. As shown in Figs. 10 and 11, the basal-to-apical flux of E 1 S and DHEAS in OATP2-expressing MDCK II monolayer was about 2 and 1.5 times higher than that in the opposite direction, respectively. This vectorial flux of E 1 S and DHEAS was not further stimulated by expressing MRP2 (Figs. 10 and 11). The cellular accumulation of E 1 S, determined at 2 min, was not different between the OATP2expressing monolayer and the double transfectant (37.7 Ϯ 0.7 pmol/mg of protein versus 42.0 Ϯ 3.4 pmol/mg of protein, n ϭ 3, p Ͼ 0.05). DISCUSSION In the present study, we have established the doubletransfected MDCK II cells, which express both OATP2 and MRP2. Immunohistochemical analysis with confocal laser microscopy suggested the basal and apical expression of OATP2 and MRP2, respectively (Fig. 1), which is consistent with the previously reported localization of these transporters (9,16,17). Because the antibodies against OATP2 and MRP2 were raised against their intracellular domains and the MDCK II cells were permeabilized for the immunohistochemical studies, it is possible that these transporters are also expressed in the intracellular vesicles near to the plasma membrane. However, the fact that Evers et al. (16) could demonstrate the apical preferential efflux of 2,4-dinitrophenyl-S-glutathione, an MRP2 substrate, from MRP2 expressing MDCK II monolayer after preloading the cells with its precursor (1-chloro-2,4-dinitrobenzene), along with the fact that we ourselves could find the vectorial transport of several ligands across the cell monolayers in the present study, suggests that these transporters may also be located on the  Transport Across MDCK II Cells Co-expressing OATP2 and MRP2 plasma membrane to a significant level. The Western blot analysis revealed that the expression levels of OATP2 and MRP2 in the single-transfected MDCK II cells were about the same as those in the double-transfected cells (Fig. 2). We have examined the transcellular transport of several kinds of ligands using this characterized MDCK II monolayer.
The basal-to-apical flux of E 2 17␤G across OATP2 expressing an MDCK II monolayer was approximately two times higher than that in the opposite direction (Fig. 3). This result may be accounted for by assuming the presence of one or more endogenous transporters on the apical membrane, which is able to extrude E 2 17␤G from the cells; E 2 17␤G molecules in the basal compartment are taken up into the monolayer via OATP2 and then excreted into the apical compartment with the aid of such an endogenous transporter. If OATP2 is not expressed on the basolateral membrane, E 2 17␤G molecules are not significantly taken up by MDCK II monolayers, resulting in the observed symmetrical transport in control and MRP2-expressing MDCK II cells (Fig. 3). The basal-to-apical flux of E 2 17␤G was stimulated to a greater extent by co-expressing MRP2 with OATP2 (Fig. 3), suggesting that E 2 17␤G molecules taken up into the monolayer are much more efficiently extruded into the apical compartment with the aid of MRP2. This suggestion is further supported by the fact that the cellular accumulation of E 2 17␤G was significantly lower in the double-transfected cells compared with OATP2 expressing cells (see "Results").
Kinetic analysis was also performed for the transcellular transport of E 2 17␤G. The K m value for the transcellular transport of E 2 17␤G across MDCK II cells expressing OATP2 (single transfectant) and those expressing both OATP2 and MRP2 (double transfectant) was 23.8 Ϯ 6.1 and 27.9 Ϯ 4.1 M, respectively (Fig. 4). These K m values are comparable with those reported for OATP2-mediated transport of E 2 17␤G in cRNA-injected Xenopus laevis oocytes (9.7 M) (23) and in cDNA-transfected MDCK II cells (8.2 M) (9). Moreover, the PS apical was not saturated even if the concentration of E 2 17␤G in the basal compartment was increased up to 150 M (Fig. 5). Taking all these observations into consideration, the saturation observed in the transcellular transport of E 2 17␤G may be ascribed to the saturation of OATP2mediated uptake, suggesting that the uptake may be the rate-determining step in the transcellular transport of E 2 17␤G. In other words, E 2 17␤G molecules taken up into the MDCK II monolayer may be efficiently extruded into the apical compartment with the aid of MRP2. The reason for the high P diff value for E 2 17␤G across the double transfectant (Fig. 4) remains unknown.
In the same manner, it was found that the vectorial transport of TLC-S, which is transported by rat oatp1 and Mrp2 (18,24), was also stimulated by OATP2 and MRP2 expression ( Fig. 9), suggesting that this sulfated bile acid is transported by human OATP2 and MRP2. Moreover, we have examined the transcellular transport of pravastatin and LTC 4 , which are substrates of both OATP2 (5, 6) and MRP2 (4), to perform kinetic analysis. As shown in Fig. 6, the vectorial transport of pravastatin from the basal to apical compartment was stimulated by expressing both OATP2 and MRP2. The K m value for the transcellular transport of pravastatin (24.3 Ϯ 10.4 M; Fig. 7) also agrees with the OATP2-mediated uptake of this drug determined in cRNA-injected oocytes (33.7 M) (6). Because pravastatin is a low affinity substrate of MRP2 (220 M) (25), the rate-determining step in the transcellular transport of this compound may also be the uptake mediated by OATP2. This hypothesis is also consistent with our previous observations that uptake is the rate-determining step for the biliary excretion of pravastatin in rats (25).
Moreover, transcellular transport of LTC 4 was examined for the purpose of comparing its transport properties with those of E 2 17␤G. An analysis of the data shown in Fig. 8 indicated that the transport activity for the basal to apical flux of LTC 4 was ϳ4 l/min/mg of protein, which is significantly lower than that for E 2 17␤G (ϳ12 l/min/mg of protein; Fig. 3). These results should be discussed in the light of the transport properties of these compounds mediated by OATP2 and MRP2. In OATP2expressing oocytes, Abe et al. (5) and Kullak-Ublick et al. (11) demonstrated that the transport activity for LTC 4 is ϳ4.8 and 2.6 times lower than that for E 2 17␤G, respectively. In contrast, using membrane vesicles isolated from MRP2-transfected HEK-293 cells, Cui et al. (26) demonstrated that the transport activity for LTC 4 is much higher than that for E 2 17␤G (351 versus 20 l/min/mg of protein). Collectively, our finding that E 2 17␤G, rather than LTC 4 , is efficiently transported across the double-transfected MDCK II monolayer expressing both OATP2 and MRP2 may also be accounted for by the fact that the transcellular transport rate of ligands is governed by the cellular uptake rate.
In addition, transcellular transport of E 1 S and DHEAS was also examined. As shown in Figs. 10 and 11, basal-to-apical flux of E 1 S and DHEAS was stimulated by OATP2 transfection, which is consistent with the previous finding that E 1 S and DHEAS are substrates of OATP2 (5). In contrast, additional expression of MRP2 did not further stimulate the transcellular transport of these sulfated conjugates (Figs. 10 and 11). Moreover, the cellular accumulation of E 1 S was not significantly different between OATP2-expressing cells and the double transfectant (see "Results"). These results are consistent with the previous finding that E 1 S may not be a good substrate of MRP2. Indeed, no ATP-dependent transport of E 1 S or DHEAS has been observed in isolated bile canalicular membrane vesicles, which expresses MRP2, and E 1 S actually stimulated MRP2-mediated transport of substrates such as 2,4-dinitrophenyl-S-glutathione (27). Molecular cloning of a transporter, which is responsible for the canalicular efflux of E 1 S, is required to establish an in vitro model to predict the hepatobiliary excretion of sulfated conjugates.
In conclusion, we have been able to establish an MDCK II cell line that expresses both OATP2 and MRP2 on the basal and apical membranes, respectively. Kinetic analysis revealed the rate-determining step in the transcellular transport of OATP2 and MRP2 bisubstrates across this cell monolayer. By normalizing the level of expression of these transporters in human liver, it may be possible to quantitatively predict the in vivo human biliary excretion of substrates of these transporters.