Functional Analysis of a Canalicular Multispecific Organic Anion Transporter Cloned from Rat Liver*

Kousei ItoDagger , Hiroshi SuzukiDagger , Tomoko HirohashiDagger , Kazuhiko Kume§, Takao Shimizu§, and Yuichi SugiyamaDagger

From the Dagger  Faculty of Pharmaceutical Sciences and the § Faculty of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Transport of many organic anions across the bile canalicular membrane is mediated by the canalicular multispecific organic anion transporter (cMOAT). Previously, we cloned cDNA that may encode cMOAT from Sprague-Dawley rat liver (Ito, K., Suzuki, H., Hirohashi, T., Kume, K., Shimizu, T., and Sugiyama, Y. (1997) Am. J. Physiol. 272, G16-G22). In the present study, the function of this cloned cDNA was investigated by examining the ATP-dependent uptake of S-(2,4-dinitrophenyl)-glutathione (DNP-SG) into membrane vesicles isolated from an NIH/3T3 cell line transfected with an expression vector containing the cloned cDNA. Although the membrane vesicles from the control NIH/3T3 cells exhibited endogenous activity in transporting DNP-SG and leukotriene C4 in an ATP-dependent manner, the transfection of cMOAT cDNA resulted in a significant increase in the transport activity for these ligands. The uptake of DNP-SG into membrane vesicles was osmotically sensitive and was stimulated to some extent by other nucleotide triphosphates (GTP, UTP, and CTP) but not by AMP or ADP. The Km and Vmax values for the uptake of DNP-SG by the membrane vesicles were 0.175 ± 0.031 µM and 11.0 ± 0.73 pmol/min/mg protein, respectively, for the transfected rat cMOAT and 0.141 ± 0.036 µM and 3.51 ± 0.39 pmol/min/mg protein, respectively, for the endogenous transporter expressed on control NIH/3T3 cells. These results suggest that the product of the previously cloned cDNA has cMOAT activity being able to transport organic anions in an ATP-dependent manner. Alternatively, it is possible that the cDNA product encodes an activator of endogenous transporter since the Km value for DNP-SG was comparable between the vector- and cMOAT-transfected cells. The transport activity found in the control NIH/3T3 cells may be ascribed to mouse cMOAT since Northern blot analysis indicated the presence of a transcript that hybridyzed to the carboxyl-terminal ATP-binding cassette sequence of the murine protein.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

It is well established that the biliary excretion of organic anions is mediated by the canalicular multispecific organic anion transporter (cMOAT)1 (1-4). We and others clarified that the substrates for cMOAT include the glutathione conjugates (such as leukotriene C4 (LTC4) (5, 6), glutathione disulfide (GSSG) (7), and S-(2,4-dinitrophenyl)-glutathione (DNP-SG) (8)), glucuronide conjugates (such as glucuronides of bilirubin (9) and xenobiotics (10-13)), and anionic xenobiotics without further metabolism (such as dibromosulfophthalein (6, 14), pravastatin (15), and temocaprilat (16)). Furthermore, the mutant rat strains whose cMOAT function is hereditarily defective have been used as an excellent animal model for the Dubin-Johnson syndrome found in humans (17). These include TR- (18) and Eisai hyperbilirubinemic rats (EHBR) (19) established from Wistar and SD rats, respectively. The reduced excretion of bilirubin glucuronide has been identified as the cause of jaundice in both TR- and EHBR (18, 19).

Recently, the molecular features of cMOAT have been clarified (20-22). Focusing on the fact that 1) the substrate specificity of cMOAT is similar to that of human multidrug resistance-associated protein (hMRP) (23-25) and 2) that the highly conserved ATP-binding cassette (ABC) region is observed among a series of ABC transporters (23), we and others recently cloned cDNA (4,623 base pairs) that may encode cMOAT from SD and Wistar rat liver based on the homology with ABC region of hMRP, respectively (20-22, 26). Northern blot analysis revealed that the expression of cMOAT is defective both in TR- and EHBR (20-22). In addition, studies with antibodies indicated selective loss of the expression of cMOAT from the canalicular membrane in TR- and EHBR (20, 21). Further analysis by Paulusma et al. (20) revealed that a 1-base pair deletion at amino acid 393 resulted in the introduction of the stop codon at amino acid 401 in TR- rats. We also found that a 1-base pair replacement (G right-arrow A) at amino acid 855 resulted in the introduction of the premature stop codon in EHBR (22). Since EHBR and TR- are allelic mutants (27) and both strains exhibit an autosomal recessive inheritance in the biliary excretion of organic anions (18), it was suggested that the impaired expression of this particular protein is related to the pathogenesis of hyperbilirubinemia in the mutant animals.

The impaired expression of cMOAT in a patient suffering from Dubin-Johnson syndrome was also demonstrated immunohistochemically (4, 28). Recently, the human homologue of rat cMOAT was cloned from several human tumor cell lines (29). Northern blot analysis suggested that the expression of a transcript which can hybridize with the human cMOAT probe is enhanced in cisplatin-resistant cell lines (29). If we consider the fact that 1) cisplatin is metabolized within the cells to form the glutathione conjugate (30) and 2) that cMOAT can accept many glutathione conjugates as a substrate (1-4), it is possible that this human homologue of rat cMOAT has the function of reducing the intracellular concentration of this antitumor drug, thus conferring drug resistance.

Functional analysis, however, remains to be performed to finally show that the previously cloned cDNA actually encodes a protein with cMOAT activity. In the present study, we established an NIH/3T3 cell line transfected with an expression vector containing cMOAT cDNA and examined the transport activity using membrane vesicles isolated from these transfected cells.

    EXPERIMENTAL PROCEDURES
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Results
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References

Materials-- [14,15,19,20-3H]LTC4 (128 Ci/mmol) was purchased from Amersham International Ltd (Buckinghamshire, UK). Unlabeled and 3H-labeled DNP-SG (50.0 µCi/nmol) were synthesized enzymatically using [2-glycine-3H]glutathione (NEN Life Science Products), 1-chloro-2,4-dinitrobenzene, and glutathione S-transferase (Sigma) as described previously (8), and the purity (>90%) was checked by thin layer chromotography. pCXN2 mammalian expression vector (31) was supplied by Dr. J. Miyazaki, Osaka University.

Preparation of the Transfected Cell Line-- cMOAT cDNA with the shortest 3'-UTR length in pBluescript II SK(-) vector described previously (22) was excised with EcoRI and then inserted into the EcoRI site in the pCXN2 vector. The NIH/3T3 cells, transfected with pCXN2 vector by Lipofectin (Life Technologies), was maintained in the presence of 800 µg/ml G418 (Geneticin, Life Technologies) to obtain the colonies. We identified five colonies and determined the expression of cMOAT in each of these using Northern blot analysis. We prepared membrane vesicles from one clone whose cMOAT expression was highest.

Transport Studies-- Membrane vesicles were prepared from 2 × 108 of the control and transfected NIH/3T3 cells as described previously (32) and were frozen in liquid nitrogen and stored at -100 °C until use. Protein concentrations were determined by the Lowry method. In addition, the orientation of membrane vesicles was determined by examining the nucleotide pyrophosphatase accessibility (33).

The transport study was performed using the rapid filtration technique (5). Briefly, transport medium (10 mM Tris, 250 mM sucrose, 10 mM MgCl2, pH 7.4), containing radiolabeled compounds (16 µl) with or without unlabeled substrate was preincubated at 37 °C for 3 min, and then rapidly mixed with 4 µl of membrane vesicle suspension (10 µg of protein), with or without 5 mM ATP and ATP-regenerating system (10 mM creatine phosphate, 100 µg/ml creatine phosphokinase). In some instances, ATP was replaced by AMP, ADP, GTP, UTP, or CTP. 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 GVWP filter (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).

Northern Blot Analysis-- The cDNA fragment containing the amino-terminal ABC region of rat cMOAT (nucleotides 2122-3154) was prepared as described previously (22). The cDNA fragment encoding the carboxyl-terminal ABC region of mouse cMOAT and MRP was amplified from BALB/c mouse liver and lung RNA, respectively, by RT-PCR using degenerated primers as described previously (26). The amplified PCR product was subcloned into the EcoRV site of pBluescript II SK(-), and then the sequence was determined. This cDNA fragment was excised by digestion with EcoRI and HindIII for use as the probe. Northern hybridization was performed as described previously (26). Filters were exposed to Fuji imaging plates (Fuji Photo Film Co., Ltd., Kanagawa, Japan) for 3 h at room temperature and analyzed by a BAS imaging analyzer (Fuji Photo Film Co., Ltd.).

    RESULTS
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Procedures
Results
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References

Northern Blot Analysis of the Expression of Rat cMOAT-- The expression of rat cMOAT in the cells cultured for 10 weeks after transfection was confirmed by Northern blot analysis. As shown in Fig. 1A, rat cMOAT probe hybridized to the NIH/3T3 cells transfected with a vector containing rat cMOAT cDNA, but not to those transfected with the vector. The length of the transcript in cMOAT-transfected NIH/3T3 cells was comparable with the shortest band observed in SD rat liver (22).


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Fig. 1.   Northern blot analysis of the expression of cMOAT. A, expression of rat cMOAT; lane 1, SD rat liver (2 µg of poly(A)+ RNA); lane 2, vector-transfected control NIH/3T3 cells (3 µg of poly(A)+ RNA); lane 3, cMOAT-transfected NIH/3T3 cells (3 µg of poly(A)+ RNA). B, expression of endogenous transporters; lane 1, BALB/c mouse liver (50 µg total RNA); lane 2, untransfected NIH/3T3 cells (3 µg poly(A)+ RNA); lane 3, vector-transfected control NIH/3T3 cells (3 µg poly(A)+ RNA). The autoradiographs were probed with 32P-labeled cDNA, including the amino-terminal ABC region of rat cMOAT (nucleotide 2122-3154) (panel A) and 32P-labeled cDNA encoding the carboxyl-terminal ABC region of mouse cMOAT (panel B) had a 3-h exposure at room temperature to a Fujix 2000 BAS imaging plate.

Uptake of [3H]DNP-SG and [3H]LTC4 into Membrane Vesicles-- The enrichment of leucine amino peptidase was 8.1- and 6.6-fold in plasma membrane vesicles from cMOAT-transfected and vector-transfected cells relative to the cell homogenate, respectively. The sideness of the membrane vesicles was also comparable between these cells; 36 and 34% of the membrane vesicles were inside-out for cMOAT-transfected and vector-transfected NIH/3T3 cells, respectively. Fig. 2 shows the time profiles for the uptake of [3H]DNP-SG and [3H]LTC4 by membrane vesicles. Although the membrane vesicles from the control cells exhibited the ability to transport [3H]DNP-SG and [3H]LTC4 in an ATP-dependent manner, the stimulating effect of ATP was greater in the cMOAT-transfected NIH/3T3 cells (Fig. 2, A and B). The clearance for the initial uptake of DNP-SG into cMOAT-transfected cells was 9.85 ± 0.42 µl/min/mg of protein (mean ± S.E.; n = 3), which is significantly (p < 0.05) higher than that observed in NIH/3T3 cells without any plasmid (3.15 ± 0.075 µl/min/mg of protein; n = 3) or vector-transfected NIH/3T3 cells (2.66 ± 0.22 µl/min/mg of protein; n = 3). The clearance for the uptake of [3H]DNP-SG into membrane vesicles isolated from NIH/3T3 cells not transfected with any plasmid (3.15 ± 0.075 µl/min/mg of protein; n = 3) was not significantly different from that observed for the vector-transfected NIH/3T3 cells (2.66 ± 0.22 µl/min/mg of protein; n = 3). Moreover, we found that the expression of rat cMOAT in the transfected cells was reduced during storage in liquid N2; after thawing, expression of the transcript in the transfected cells fell below the limit of detection. In accordance with the reduced expression of transfected cMOAT, the uptake of DNP-SG into membrane vesicles from thawed cells was significantly (p < 0.05) reduced to 3.13 ± 0.22 µl/min/mg of protein (n = 3), a figure not significantly different from that obtained in parental and vector-transfected NIH/3T3 cells.


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Fig. 2.   Time profiles for the uptake of [3H]DNP-SG and [3H]LTC4 into membrane vesicles. Membrane vesicles (10 µg) prepared from cMOAT-transfected (bullet ) or vector-transfected NIH/3T3 cell lines (black-square) were incubated at 37 °C in 20 µl of medium (10 mM Tris-HCl, 250 mM sucrose, 10 mM MgCl2, pH 7.4) containing 1 µM [3H]DNP-SG (panel A) or 10 nM [3H]LTC4 (panel B) with (closed symbols) or without (open symbols) ATP regenerating system (5 mM ATP, 10 mM creatine phosphate, 100 µg/ml creatine phosphokinase). Each point and vertical bar represent the mean ± S.E. of triplicate determinations in a typical experiment.

In addition, the uptake of [3H]DNP-SG into membrane vesicles from cMOAT-transfected and vector-transfected cells was reduced by increasing the sucrose concentration in the medium. The y-intercept for relationship between the amount of DNP-SG associated with the vesicles versus the reciprocal of the sucrose concentration in the medium was almost 0. GTP, CTP, and UTP stimulated the uptake of [3H]DNP-SG into membrane vesicles isolated from cMOAT-transfected cells to 38.3, 41.2, and 44.4% of that observed in the presence of ATP. In the same manner, the uptake of [3H]DNP-SG into membrane vesicles from vector-transfected cells in the presence of GTP, CTP, and UTP was 55.6, 54.4, and 56.6% of that observed in the presence of ATP. In contrast, no effect of ADP or AMP was observed for both membrane vesicle preparations. Vanadate (100 µM) reduced the ATP-dependent uptake of [3H]DNP-SG to 38.4% of the control value.

The ATP-dependent uptake of [3H]DNP-SG into membrane vesicles was saturable (Fig. 3). The nonlinear regression analysis of the vector-transfected cells revealed that the uptake can be described by a saturable (Km = 0.141 ± 0.036 µM, Vmax = 3.51 ± 0.39 pmol/min/mg of protein) and a non-saturable component with a clearance of 0.228 ± 0.030 µl/min/mg of protein. The ability of rat cMOAT to transport [3H]DNP-SG was determined by subtracting the uptake in the vector-transfected cells from that in the cMOAT-transfected cells (Fig. 3). The Km and Vmax values for the transport of [3H]DNP-SG by rat cMOAT were 0.175 ± 0.031 µM and 11.0 ± 0.73 pmol/min/mg of protein, respectively.


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Fig. 3.   Concentration-dependence of [3H]DNP-SG uptake by membrane vesicles. Membrane vesicles were incubated at 37 °C with 50 nM [3H]DNP-SG in 20 µl of medium (10 mM Tris-HCl, 250 mM sucrose, 10 mM MgCl2, pH 7.4) containing varying concentrations of DNP-SG for 10 min, during which linearity is observed. The closed triangle (black-triangle) represents the uptake mediated by the transfected rat cMOAT calculated by subtracting the endogenous uptake by vector-transfected NIH/3T3 vesicles (black-square) from that of cMOAT-transfected NIH/3T3 vesicles (bullet ). Each point and vertical/horizontal bar represents the mean ± S.E. of triplicate determinations in a typical experiment.

Northern Blot Analysis of the Expression of Endogenous Transporters-- Expression of MRP/cMOAT related proteins in the control NIH/3T3 cells was examined by Northern blot analysis. The amplified cDNA fragment encoding carboxyl-terminal ABC region of mouse cMOAT (367 base pairs) (Fig. 4) exhibited 92.6 and 94.3% homology at the cDNA and deduced amino acid level with rat cMOAT (20-22), respectively, and hybridyzed with the poly(A)+ RNA from NIH/3T3 cells to produce the ~6-kilobase band (Fig. 1B). The deduced amino acid sequence of the amplified cDNA fragment of mouse MRP was the same as that reported previously (34). The homology in the carboxyl-terminal ABC region between mouse MRP and mouse cMOAT was 68.0 and 78.0% at the cDNA and deduced amino acid level, respectively. Northern blot indicated that the expression of mouse MRP in NIH/3T3 cells was below the detection limit (data not shown).


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Fig. 4.   Alignment of the nucleotide and deduced amino acid sequence of the cDNA probe for mouse cMOAT and mouse MRP. Carboxyl-terminal ABC region of the mouse cMOAT and mouse MRP were amplified from BALB/c mouse liver and lung, respectively, using degenerated PCR primers described previously (26). The sequence between forward and reverse primers is listed in this figure. Asterisks represent the consensus sequence. Dots represent the amino acids homologous to each other.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

In the present study, we examined the function of the product of the recently cloned rat cDNA, whose expression is defective in EHBR and TR- (20-22), by examining the transport of typical substrates for cMOAT in the cDNA transfected cells. Since 1) the uptake of [3H]DNP-SG into membrane vesicles from the cMOAT-transfected cells was stimulated to a greater extent by ATP compared with that from the vector-transfected cells (Fig. 2A) and 2) since the expression of rat cMOAT in the cMOAT-transfected cells was confirmed by Northern blot analysis, it was concluded that rat cMOAT activity is associated with its expression at an mRNA level. Uptake of DNP-SG and LTC4 into membrane vesicles isolated from the control NIH/3T3 cells was stimulated by ATP, suggesting the presence of endogenous ABC transporters for these glutathione conjugates (Fig. 2). To estimate the function of rat cMOAT, therefore, the uptake in control cells should be subtracted from that in cMOAT-transfected NIH/3T3 cells. Since the uptake of DNP-SG into membrane vesicles was comparable between the parent and vector-transfected NIH/3T3 cells (see "Results"), the vector introduction may not affect the expression of endogenous transporters.

The uptake of [3H]DNP-SG was osmotically sensitive (see "Results"), suggesting that a large part of the accumulation by vesicles can be accounted for by transport into the intravesicular space, but not by binding to the vesicle surface. GTP, CTP, and UTP could also enhance the uptake of DNP-SG by cMOAT to some extent (see "Results"), which was consistent with hMRP (35). Vanadate was effective in reducing the ATP-stimulated uptake of DNP-SG, which was consistent with the observations in CMVs (8) and in hMRP (32).

Kinetic analysis revealed that the Km of rat cMOAT was 0.175 µM (see "Results"), which was more than 10-fold lower than that reported for the uptake of DNP-SG by CMVs; using rat CMVs, Kobayashi et al. (8) reported a Km of 4 µM for DNP-SG although the Km reported by Akerboom was 70 µM (36). The Km value determined in the present study is similar to that found for ATP-dependent uptake of DNP-SG into plasma membrane vesicles from murine leukemia cells (L1210; Km = 0.63 µM) (37). Comparison of the previous reports revealed the presence of a 10-fold difference in the Km value for DNP-SG between hMRP-overexpressing tumor cells (GLC4/ADR, Km = 30 µM) (32) and hMRP-transfected HeLa cells (3.6 µM) (38). Although we do not have a good explanation to account for the discrepancy in the Km values between the cMOAT-transfected mouse (NIH/3T3) cells and rat hepatocytes, one hypothesis involves considering the difference in the "atmosphere" of the protein molecule due to the difference in lipid composition between the two animal species. Alternatively, it is also plausible that the cDNA product encodes an activator of endogenous transporter since the Km value of DNP-SG was comparable between the vector- and cMOAT-transfected NIH/3T3 cells. Such protein-protein interaction has been demonstrated on the plasma membrane. For example, Inagaki et al. (39) reported that the co-expression of ATP-dependent K+-channel activity (beta  cell inward rectifier (BIR)) and sulfonylurea receptor (SUR) is required for BIR activity in COS cells. They found that COS-1 cells transfected with BIR alone or SUR alone did not exhibit this function (39).

In the present study, we found the presence of ATP-dependent transport of DNP-SG and LTC4 in control NIH/3T3 cells. The presence of such endogenous activity on mouse plasma membrane has been reported previously; Saxena and Henderson (37) found that DNP-SG is taken up into membrane vesicles from L1210 cells via high (Km = 0.63 µM) and low (Km = 450 µM) affinity systems, the former being inhibited by LTC4 with a Ki value of 0.20 µM. Northern blot analysis using a mouse MRP probe suggested that the expression of MRP in NIH/3T3 cells was minimal (see "Results"). In contrast, a transcript which hybridizes to the carboxyl-terminal ABC region of mouse cMOAT was observed at the position which was same as that in mouse liver (Fig. 1), suggesting that the mouse cMOAT rather than MRP may be responsible for the endogenous activity in transporting DNP-SG in NIH/3T3 cells.

In conclusion, we have shown that the product of the previously cloned cDNA (20-22) has the ability to transport glutathione conjugates in an ATP-dependent manner, which is the most important characteristic of cMOAT. Together with the previous finding that the expression of the cloned cDNA 1) is predominantly observed in the liver among all the tissues examined (20, 22), 2) is almost exclusively observed on the bile canalicular membrane (20, 21), and 3) is hereditarily defective in both allelic mutant rat strains (EHBR and TR-) (20-22) and humans (28), this leads us to conclude that the defective expression of this transporter is the pathogenesis for the jaundice in the Dubin-Johnson syndrome found in humans.

    Note Added in Proof

After submission of this manuscript, Madon et al. (1997) FEBS Lett. 406, 75-78, communicated the accelerated efflux of DNP-SG from oocytes and COS-7 cells expressing rat cMOAT.

    FOOTNOTES

* 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB008832.

To whom correspondence should be addressed. Tel.: 81-3-5802-2045; Fax: 81-3-5800-6949.

1 The abbreviations used are: cMOAT, canalicular multispecific organic anion transporter; CMVs, canalicular membrane vesicles; LTC4, leukotriene C4; GSSG, glutathione disulfide; DNP-SG, S-(2,4-dinitrophenyl)-glutathione; EHBR, Eisai hyperbilirubinemic rats; hMRP, human multidrug resistance-associated protein; ABC, ATP-binding cassette.

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Abstract
Introduction
Procedures
Results
Discussion
References

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