From the Faculty of Pharmaceutical Sciences and the
§ Faculty of Medicine, The University of Tokyo, Hongo,
Bunkyo-ku, Tokyo 113, Japan
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ABSTRACT |
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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.
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INTRODUCTION |
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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
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.
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EXPERIMENTAL PROCEDURES |
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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).
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.).
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RESULTS |
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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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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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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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DISCUSSION |
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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 ( 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.
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Note Added in Proof |
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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.
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FOOTNOTES |
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* 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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REFERENCES |
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