From the Graduate School of Pharmaceutical Sciences, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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ABSTRACT |
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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 17 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. 17 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 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.
Materials--
[3H]LTC4 (111 µCi/nmol) and [3H]E217 Preparation of Transfected Cells--
- Rat MRP3 cDNA,
excised with EcoRI from pBluescript II SK(
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 (
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 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).
The time profiles for the uptake of
[3H]E217 Transport Kinetics of
[3H]E217 Characterization of MRP3-mediated Transport of
[3H] E2 17
Because MTX inhibited MRP3-mediated transport of
[3H]E217 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
(E217 The transport characteristics of MRP3 differ from those of MRP1 and
cMOAT/MRP2 in that LTC4 is a much better substrate than E217 We also examined the inhibitory effect of several anionic compounds on
the MRP3-mediated transport of E217 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 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.
estradiol
17-
-D-glucuronide ([3H]E217
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]E217
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]E217
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]E217
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]E217
G. The
uptake of [3H]E217
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
estradiol-17
-D-glucuronide (E217
G)), and sulfated conjugates of certain bile acids (e.g.
3
-sulfatolithocholyltaurine) (1-3).
) 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.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
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,
-naphthyl
-D-glucuronide, estrone 3-sulfate, and acivicin were purchased from Sigma.
) 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.
100 °C) until required.
-glutamyltranspeptidase.
Acivicin did not affect MRP3-mediated transport of
E217
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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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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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.
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]E217
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]E217 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]E217
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]E217 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).
G--
-The
ATP-dependent uptake of
[3H]E217
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]E217
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]E217 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 E217
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.
G--
Table
I shows the effect of anionic compounds
on the ATP-dependent uptake of
[3H]E217
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,
-naphthyl
-D-glucuronide, and estrone 3-sulfate had
inhibitory effects on the uptake of
[3H]E217
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]E217
G in LLC-PK1 cells (Table I).
Effects of various compounds on the ATP-dependent uptake of
[3H]E217G by membrane vesicles
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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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).
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 E217
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 E217
G in MRP1-transfected HEK cells
(29). For murine mrp1, this value for LTC4 was 200 times
higher than that of E217
G in the transfected HEK cells (29). In rat CMVs, the
Vmax/Km for the
ATP-dependent uptake of LTC4, DNP-SG, and
E217
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
E217
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.
G (Table I).
LTC4 inhibited the transport of E217
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.
-Naphthyl
-D-glucuronide reduced the
MRP3-mediated transport of E217
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
-naphthyl
-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.
-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
E217
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 E217
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.
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FOOTNOTES |
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* 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.
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.
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ABBREVIATIONS |
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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;
E217G, 17
estradiol 17
-D-glucuronide;
SD, Sprague-Dawley rats.
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