Characterization of the Transport Properties of Cloned Rat Multidrug Resistance-associated Protein 3 (MRP3)*

Tomoko Hirohashi, Hiroshi SuzukiDagger , and Yuichi Sugiyama

From the Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have previously cloned rat MRP3 as an inducible transporter in the liver (Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T., and Sugiyama, Y. (1998) Mol. Pharmacol. 53, 1068-1075). In the present study, the function of rat MRP3 was investigated using membrane vesicles isolated from LLC-PK1 and HeLa cell population transfected with corresponding cDNA. The ATP-dependent uptake of both 17beta estradiol 17-beta -D-glucuronide ([3H]E217beta G) and glucuronide of [14C] 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole (E3040), but not that of [3H]leukotriene C4 and [3H]2,4-dinitrophenyl-S-glutathione, was markedly stimulated by MRP3 transfection in both cell lines. The Km and Vmax values for the uptake of [3H]E217beta G were 67 ± 14 µM and 415 ± 73 pmol/min/mg of protein, respectively, for MRP3-expressing membrane vesicles and 3.0 ± 0.7 µM and 3.4 ± 0.4 pmol/min/mg of protein, respectively, for the endogenous transporter expressed on HeLa cells. [3H]E217beta G had also a similar Km value for MRP3 when LLC-PK1 cells were used as the host. All glucuronide conjugates examined (E3040 glucuronide, 4-methylumbelliferone glucuronide, and naphthyl glucuronide) and methotrexate inhibited MRP3-mediated [3H]E217beta G transport in LLC-PK1 cells. Moreover, [3H]methotrexate was transported via MRP3. The inhibitory effect of estrone sulfate, [3H]2,4-dinitrophenyl-S-glutathione, and [3H]leukotriene C4 was moderate or minimal, whereas N-acetyl-2,4-dinitrophenylcysteine had no effect on the uptake of [3H]E217beta G. The uptake of [3H]E217beta G was enhanced by E3040 sulfate and 4-methylumbelliferone sulfate. Thus we were able to demonstrate that several kinds of organic anions are transported via MRP3, although the substrate specificity of MRP3 differs from that of MRP1 and cMOAT/MRP2 in that glutathione conjugates are poor substrates for MRP3.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The membrane proteins belonging to the ATP binding cassette (ABC)1 transporter family play an important role in the extrusion of many substrates from cells. Among them, the GS-X pump plays an important role in cell survival (1). Many electrophiles entering cells are conjugated with glutathione and then extruded from the cells with the aid of GS-X pumps (1). Multidrug resistance-associated protein (MRP1), initially cloned from a multidrug resistant tumor cell line, was the first molecule to be associated with the GS-X pump activity (1-3). The substrate specificity of MRP1 was determined in a series of transport studies using MRP1-transfected or MRP1-overexpressing cells (1-3). MRP1 accepts many conjugates as substrates such as glutathione conjugates (e.g. 2,4-dinitrophenyl-S-glutathione (DNP-SG), glutathione disulfide, and leukotriene C4 (LTC4)), glucuronide conjugates (e.g. 17beta estradiol-17beta -D-glucuronide (E217beta G)), and sulfated conjugates of certain bile acids (e.g. 3alpha -sulfatolithocholyltaurine) (1-3).

Although MRP1 is widely expressed in many somatic cells, its hepatic expression is not marked. In the liver, canalicular multispecific organic anion transporter (cMOAT/MRP2), another member of the GS-X pump family, is expressed on the bile canalicular membrane, mediating the efficient biliary excretion of many organic anions (3-5). The similar substrate specificity of cMOAT/MRP2 and MRP1 has been established by comparing the transport properties across the bile canalicular membrane between normal rats and mutant rats whose cMOAT/MRP2 activity is hereditarily defective (e.g. transport-deficient (TR-) rats and Eisai hyperbilirubinemic rats (EHBR)) (3-5). These mutant rats have been used as an animal model for Dubin-Johnson syndrome found in humans (6-14). cDNA cloning and functional analysis of its product, along with a mutation analysis (15, 16), have been performed in this and other laboratories.

It is possible that transporters other than cMOAT/MRP2 may be also involved in the hepatic transport of organic anions. Indeed, we were able to amplify two kinds of novel transporters, which were initially referred to as MRP-like protein (MLP) 1 and 2 from EHBR liver using RT-PCR with the degenerated primers designed for the highly conserved carboxyl-terminal ABC region of human MRP1 (17). The sequence alignment of the full-length of cDNA indicated that MLP-1 and 2 correspond to MRP6 and 3, respectively (17, 18). Northern blot analysis showed that the hepatic expression of MRP3 was significantly enhanced in EHBR compared with Sprague-Dawley (SD) rats, although the extent of MRP6 expression is comparable in the two rat strains (17). It is possible that MRP3 may compensate for the defective expression of cMOAT/MRP2 (17, 19). In addition, because Northern blot analysis indicated that there was extensive expression of MRP3 in intestinal tissues of rats and human, it is possible that MRP3 is also responsible for the intestinal excretion of organic anions (17, 20).

In the present study, we examined the function of rat MRP3 using membrane vesicles from LLC-PK1 and HeLa cells transfected with an expression vector containing the cloned MRP3 cDNA. MRP3 shows different substrate specificity to MRP1 and cMOAT/MRP2 in that glutathione conjugates are poor substrates for the former.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Materials-- [3H]LTC4 (111 µCi/nmol) and [3H]E217beta G (55.0 µCi/nmol) were purchased from NEN Life Science Products. [3H]Methotrexate (30 µCi/nmol) was purchased from American Radiolabeled Chemicals, Inc. Unlabeled and 3H-labeled DNP-SG (22.5 µCi/nmol) were synthesized enzymatically using [glycine-2-3H]glutathione (NEN Life Science Products) and 1-chloro-2,4-dinitrobenzene and glutathione S-transferase (Sigma) as described previously (21). [14C]E3040 glucuronide (84.5 µCi/µmol) and unlabeled E3040 glucuronide and sulfate were prepared from E3040 (supplied by Eisai Co., Ltd, Tsukuba, Japan) as described previously (22). NAc-DNP-Cys was synthesized as described previously (23). 4-Methylumbelliferone glucuronide, 4-methylumbelliferone sulfate, LTC4, methotrexate, alpha -naphthyl beta -D-glucuronide, estrone 3-sulfate, and acivicin were purchased from Sigma.

Preparation of Transfected Cells-- - Rat MRP3 cDNA, excised with EcoRI from pBluescript II SK(-) vector (17), was then inserted into the EcoRI site in a mammalian expression vector (pCXN2; supplied by Dr. J. Miyazaki, Osaka University, School of Medicine) (24). After transfection with LipofectAMINE (Life Technologies, Inc.), the LLC-PK1 and HeLa cells were maintained in the presence of 800 µg/ml G418 (Geneticin, Life Technologies) to obtain colonies. Three weeks later, the expression level of MRP3 was determined in several independently transfected cell populations using Northern blot analysis. Membrane vesicles were prepared from the cell population exhibiting the highest MRP3 expression.

The cDNA fragment containing the carboxyl-terminal ABC region of rat MRP3 was prepared to undergo Northern hybridization according to the method described previously (9, 17). Filters were exposed to Fuji imaging plates (Fuji Photo Film Co., Ltd., Kanagawa, Japan) for 3 h at room temperature and analyzed with an imaging analyzer (BAS 2000, Fuji Photo Film Co., Ltd.).

Transport Studies with HeLa Membrane Vesicles-- Membrane vesicles were prepared from 1 × 108 of the MRP3- and vector-transfected HeLa and LLC-PK1 cell populations as described previously (25). The membrane vesicles were frozen in liquid nitrogen and then transferred to a freezer (-100 °C) until required.

The transport studies were performed using the rapid filtration technique (26). Briefly, 16 µl of transport medium (10 mM Tris-HCl, 250 mM sucrose, 10 mM MgCl2, pH 7.4) containing radiolabeled compounds with or without unlabeled substrate was preincubated at 37 °C for 3 min and then rapidly mixed with 4 µl of membrane vesicle suspension (8~10 µg of protein). The reaction mixture contained 5 mM ATP or 5 mM AMP and ATP-regenerating system (10 mM creatine phosphate, 100 µg/ml creatine phosphokinase). In some instances, the membrane vesicles were treated with acivicin (final concentration of 6 mM) at 25 °C for 15 min before initiation of the transport studies to avoid possible degradation of glutathione-S-conjugates by gamma -glutamyltranspeptidase. Acivicin did not affect MRP3-mediated transport of E217beta G. The transport reaction was stopped by the addition of 1 ml of ice-cold buffer containing 250 mM sucrose, 0.1 M NaCl, 10 mM Tris-HCl (pH 7.4). The stopped reaction mixture was filtered through a 0.45-µm membrane filter (GVWP; Millipore Corp., Bedford, MA) and then washed twice with 5 ml of stop solution. Radioactivity retained on the filter was determined using a liquid scintillation counter (LSC-3500, Aloka Co., Tokyo, Japan). The ATP-dependent uptake of ligands was calculated by subtracting the ligand uptake in the absence of ATP from that in the presence of ATP.

    RESULTS
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INTRODUCTION
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Uptake of Glucuronides and Glutathione Conjugates into Membrane Vesicles-- The expression of rat MRP3 in the transfected cells was confirmed by Northern blot analysis (Fig. 1). The length of the transcript in MRP3-transfected LLC-PK1 and HeLa cells was comparable with the band observed in Sprague-Dawley rat colon (17) (Fig. 1).


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Fig. 1.   Northern blot analysis of the expression of MRP3. The membrane hybridized with 32P-labeled cDNA probes encoding the carboxyl-terminal ABC region of rat MRP3 were rehybridized with 32P-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA. Lane 1, SD rat colon (3 µg of poly(A)+ RNA); lane 2, MRP3-transfected LLC-PK1 cells; lane 3, control vector-transfected LLC-PK1 cells; lane 4, MRP3-transfected HeLa cells; lane 5, control vector-transfected HeLa cells. Lanes 2~5, 50 µg of total RNA.

The time profiles for the uptake of [3H]E217beta G, [14C]E3040 glucuronide, [3H]DNP-SG, and [3H]LTC4 by the membrane vesicles from LLC-PK1 cells are shown in Fig. 2. The ATP-dependent uptake of [3H]E217beta G and [14C]E3040 glucuronide at 10 min was 4.1-fold and 6.9-fold higher in MRP3-transfected LLC-PK1, respectively, compared with the control vector-transfected LLC-PK1 (Fig. 2, a and b). In contrast, the ATP-dependent uptake of [3H]DNP-SG and [3H]LTC4 was not stimulated by MRP3 transfection (Fig. 2, c and d). The same results were obtained in membrane vesicles from MRP3-transfected HeLa cells (Fig. 3). These results indicate that MRP3 preferentially accepts these two glucuronides as substrates, whereas these two glutathione conjugates are poor substrates of MRP3.


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Fig. 2.   Time profiles for the uptake of [3H]E217beta G (a), [14C]E3040 glucuronide (b), [3H]DNP-SG (c), and [3H]LTC4 (d) into membrane vesicles. Membrane vesicles (10 µg of protein) from MRP3 (circles)- or vector (squares)- transfected LLC-PK1 cells were incubated at 37 °C in medium containing 100 nM [3H]E217beta G (panel a), 10 µM [14C]E3040 glucuronide (panel b), 100 nM [3H]DNP-SG (panel c), and 5 nM [3H]LTC4 (panel d) in the presence (closed symbols) and absence (open symbols) of ATP. Each point and vertical bar represents the mean ±S.E. of triplicate determinations (closed symbols) or the mean value from two determinations (open symbols).


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Fig. 3.   Uptake of glucuronide and glutathione conjugates into membrane vesicles. Membrane vesicles from MRP3- or vector-transfected HeLa cells were incubated at 37 °C for 10 min in medium containing 100 nM [3H]E217beta G (panel a), 10 µM [14C]E3040 glucuronide (panel b), 100 nM [3H]DNP-SG (panel c), and 5 nM [3H]LTC4 (panel d) in the presence (closed columns) and absence of ATP (open columns). Each column and vertical bar represents the mean ±S.E. of triplicate determinations (closed columns) or the mean value from two determinations (open columns).

Transport Kinetics of [3H]E217beta G-- -The ATP-dependent uptake of [3H]E217beta G into membrane vesicles was saturable (Fig. 4). Nonlinear regression analysis of the uptake by MRP3- and control vector-transfected LLC-PK1 cells revealed that the saturable uptake can be described by a Km of 110 ± 20 µM and Vmax of 1570 ± 300 pmol/min/mg of protein and a Km of 13 ± 6 µM and Vmax of 35 ± 11 pmol/min/mg of protein, respectively (Fig. 4a). In MRP3- and control vector-transfected HeLa cells, kinetic parameters for the saturable uptake of [3H]E217beta G were determined to be Km = 67 ± 14 µM and Vmax = 415 ± 73 pmol/min/mg of protein and Km = 3.0 ± 0.7 µM and Vmax = 3.4 ± 0.4 pmol/min/mg of protein, respectively (Fig. 4b).


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Fig. 4.   Concentration dependence of [3H]E217beta G uptake by membrane vesicles. Membrane vesicles from MRP3- or vector-transfected LLC-PK1 (10 µg of protein, panel a) or HeLa (8 µg of protein, panel b) cells were incubated at 37 °C for 5 min (panel a) or 10 min (panel b) in medium containing 3H-labeled (100 nM) and unlabeled E217beta G. The circles and squares represents the ATP-dependent uptake by the membrane vesicles from rat MRP3- and control vector-transfected cells, respectively. Each point and vertical/horizontal bar represents the mean ±S.E. of triplicate determinations. Eadie-Hofstee plots were generated, and the kinetic parameters (Km and Vmax) were calculated.

Characterization of MRP3-mediated Transport of [3H] E2 17beta G-- Table I shows the effect of anionic compounds on the ATP-dependent uptake of [3H]E217beta G in MRP3-expressing LLC-PK1 and HeLa cells. The uptake was reduced by E3040 glucuronide, DNP-SG, and MTX in a concentration-dependent manner, whereas the inhibitory effect of LTC4 was only minimal in both cell lines up to 1 µM. 4-Methylumbelliferone glucuronide, alpha -naphthyl beta -D-glucuronide, and estrone 3-sulfate had inhibitory effects on the uptake of [3H]E217beta G, whereas NAc-DNP-Cys had no inhibitory effect in LLC-PK1 at a concentration of 500 µM (Table I). In contrast, E3040 sulfate and 4-methylumbelliferone sulfate enhanced the uptake of [3H]E217beta G in LLC-PK1 cells (Table I).

                              
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Table I
Effects of various compounds on the ATP-dependent uptake of [3H]E217beta G by membrane vesicles
Membrane vesicles from MRP3-transfected LLC-PK1 or HeLa cells were incubated at 37 °C for 5 min LLC-PK1 cells or 10 min HeLa cells in medium containing 100 nM [3H]E2 17G with or without (control) the inhibitors. ATP-dependent uptake was calculated by subtracting values in the presence of 5 mM AMP from those in the presence of 5 mM ATP. Transport was expressed as percent of the control uptake. Data represent mean ± S.E. triplicate determinations.

Because MTX inhibited MRP3-mediated transport of [3H]E217beta G, the transport of [3H]MTX was examined in membrane vesicles isolated from LLC-PK1 cells. The uptake of MTX was significantly enhanced by transfection of MRP3 (Fig. 5), indicating that MTX is a substrate for MRP3.


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Fig. 5.   Time profiles for the uptake of [3H]MTX into membrane vesicles. Membrane vesicles (10 µg of protein) from MRP3 (circles)- or vector (squares)-transfected LLC-PK1 cells were incubated at 37 °C in medium containing 120 nM [3H]MTX in the presence (closed symbols) and absence (open symbols) of ATP. Each point and vertical bar represents the mean ±S.E. of triplicate determinations (closed symbols) or the mean value from two determinations (open symbols).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, we examined the function of the recently cloned rat MRP3 (17) by using membrane vesicles prepared from LLC-PK1 and HeLa cell populations transfected with the corresponding cDNA. The ATP-dependent uptake of glucuronide conjugates (E217beta G and E3040 glucuronide), but not that of glutathione conjugates (DNP-SG and LTC4), was markedly stimulated by MRP3 transfection (Figs. 2 and 3), indicating that MRP3 accepts the glucuronide conjugates much more readily as substrates than the glutathione conjugates. In addition, the transport characteristics of MRP3 are the same if this transporter is expressed on HeLa or LLC-PK1 cells (Figs. 2 and 3, Table I).

The transport characteristics of MRP3 differ from those of MRP1 and cMOAT/MRP2 in that LTC4 is a much better substrate than E217beta G for the latter two transporters. In MRP1-transfected HeLa cells, the Vmax/Km for the ATP-dependent uptake was 1030, 110, and 42 µl/min/mg of protein for LTC4, DNP-SG, and E217beta G, respectively (27, 28). This result is consistent with the finding that the Vmax/Km value for the ATP-dependent uptake of LTC4 was 30 times higher than that of E217beta G in MRP1-transfected HEK cells (29). For murine mrp1, this value for LTC4 was 200 times higher than that of E217beta G in the transfected HEK cells (29). In rat CMVs, the Vmax/Km for the ATP-dependent uptake of LTC4, DNP-SG, and E217beta G was 268, 58, and 34 ml/min/mg of protein, respectively, indicating that the clearance for uptake mediated by cMOAT/MRP2 is 8 times higher for LTC4 than E217beta G.2 Kinetic analysis revealed that the Km value for MRP3 was 110 µM and 67 µM in MRP3-transfected LLC-PK1 and HeLa cells, respectively (Fig. 4), which is much higher than that reported for MRP1-transfected HeLa and HEK cells (1.5-4.8 µM) (28-30) and in CMVs from SD rats (6.3 µM)3.

We also examined the inhibitory effect of several anionic compounds on the MRP3-mediated transport of E217beta G (Table I). LTC4 inhibited the transport of E217beta G only minimally even at 1 µM, which is much higher than its Km value for MRP1 (0.1 µM) (27, 29, 31) and cMOAT/MRP2 (0.25 µM) (26). Taking this together with the results of the transport studies shown in Figs. 2 and 3, LTC4 has, therefore, been shown to be a poor substrate for MRP3. alpha -Naphthyl beta -D-glucuronide reduced the MRP3-mediated transport of E217beta G with an IC50 of 20- 50 µM (Table I), consistent with the hypothesis that this compound is also recognized by MRP3. The inhibitory effect of E3040 glucuronide (IC50 < 5 µM) and 4-methylumbelliferone glucuronide (IC50 ~ 50 µM) was in marked contrast to the stimulatory effect of the corresponding sulfates (Table I). Although the mechanism for stimulation still remains unclear, such a stimulatory effect by E3040 sulfate and 4MU sulfate has been demonstrated in uptake of DNP-SG into CMVs from SD rats (32). Moreover, the low affinity of MRP3 toward NAc-DNP-Cys suggests that MRP3 gene may not encode MOAT4, whose transport properties had previously been characterized in mouse L1210 cells (33). It has been shown that MOAT4 mediates the low affinity transport of DNP-SG (Km = 450 µM) and exhibits high sensitivity toward NAc-DNP-Cys (Ki = 5.0 µM) and alpha -naphthyl beta -D-glucuronide (Ki = 8.5 µM) (33). Because NAc-DNP-Cys did not affect MRP3-mediated transport, even at a concentration of 500 µM, irrespective of the fact that DNP-SG can act as an inhibitor with an IC50 of 50~100 µM (Table I), suggests that MOAT4 differs from MRP3.

Previously, we found that the expression of MRP3 is induced in EHBR liver (17). It is also induced in SD rat liver by phenobarbital treatment and by treatment which increases plasma bilirubin and/or its glucuronide (e.g. the cholestasis induced by common bile duct ligation and by alpha -naphthylisothiocyanate treatment) (17, 19). It is plausible that, in EHBR, MRP3 is induced to compensate for the physiological function of cMOAT/MRP2 to excrete bilirubin glucuronides from hepatocytes (17, 19). This hypothesis has been proposed from the previous finding that, in mdr 1a knock-out mice, the hepatic function of mdr 1a is compensated for by the increased expression of mdr 1b, whose substrate specificity resembles that of mdr 1a (34). Although we reported that E3040 glucuronide is taken up by CMVs from EHBR in an ATP-dependent manner (32), the comparison of the transport properties between CMVs and MRP3-expressing membrane vesicles suggested that the uptake into EHBR CMVs may not be mediated by MRP3. This suggestion was proposed based on the finding that neither the uptake of E217beta G3 nor MTX (35) was stimulated by the addition of ATP if CMVs were isolated from EHBR. Together with the finding that all of the E217beta G, E3040 glucuronide, and MTX are transported via MRP3 (Figs. 2, 3, and 5) suggests that the previously described ATP-dependent transport of E3040 glucuronide in CMVs from EHBR should be attributed to another transporter.

The extensive expression of MRP3 in rat and human intestinal tissues may be related to the intestinal excretion of glucuronides. Indeed, intestinal excretion of ethinylestradiol glucuronide (36) and 1-naphthol glucuronide (37) has been demonstrated in rats in in situ experiments. In addition, the extent of excretion of 1-naphthol glucuronide was comparable in normal and cMOAT/MRP2-deficient rats, suggesting the presence of another transporter responsible for the excretion of this glucuronide (38). It is possible that MRP3 is responsible for the intestinal excretion of this conjugated metabolite.

In conclusion, although MRP3 mediates the transport of several kinds of organic anions, the substrate specificity of MRP3 is in marked contrast to that of MRP1 and cMOAT/MRP2 in that glutathione conjugates are poor substrates for MRP3. It is possible that MRP3 acts as an inducible transporter compensating for the cMOAT/MRP2 function. In addition, MRP3 may be responsible for the cellular extrusion of glucuronide conjugates in the small intestine.

    FOOTNOTES

* This work was supported by Grant-in-Aid for Scientific Research on Priority Areas ABC proteins 10044243 from the Ministry of Education, Science, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 81-3-5841-4774; Fax: 81-3-5800-6949; E-mail: suzuki{at}seizai.f.u-tokyo.ac.jp.

2 K. Niinuma, Y. Kato, H. Suzuki, C. A. Tyson, V. Weizer, J. E. Dabbs, R. Froelich, C. E. Green, and Y. Sugiyama, Y., submitted for publication.

3 A. Morikawa, H. Suzuki, T. Hirohashi, and Y. Sugiyama, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: ABC, ATP binding cassette; CMVs, canalicular membrane vesicles; cMOAT, canalicular multispecific organic anion transporter; DNP-SG, 2,4-dinitrophenyl-S-glutathione; EHBR, Eisai hyperbilirubinemic rat; E3040, 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole; LTC4, leukotriene C4; MTX, methotrexate; MRP, human multidrug resistance-associated protein; NAc-DNP-Cys, N-acetyl-2,4-dinitrophenylcysteine; E217beta G, 17beta estradiol 17beta -D-glucuronide; SD, Sprague-Dawley rats.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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