From the Cancer Research Laboratories, Queen's
University, Kingston, Ontario, Canada K7L 3N6 and the
§ Center for Experimental Therapeutics and Department of
Pharmacology, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104
Received for publication, September 8, 2000, and in revised form, November 30, 2000
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ABSTRACT |
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Multidrug resistance protein 1 (MRP1) is an
ATP-binding cassette (ABC) transporter that transports a range of
hydrophobic xenobiotics, as well as relatively hydrophilic organic
anion conjugates. The protein is present at high levels in testicular
Leydig and Sertoli cells. Studies with knockout mice suggest that MRP1
may protect germ cells from exposure to some cytotoxic xenobiotics, but
potential endobiotic substrates in this organ have not been identified.
Previously, we have shown certain D-ring, but not A-ring, estrogen
glucuronides can act as competitive inhibitors of MRP1 mediated
transport, suggesting that they are potential substrates for the
protein. In the case of
17 Human multidrug resistance protein
(MRP)1 1 is a member of the
ATP-binding cassette superfamily of transmembrane transporters, which
was originally discovered by virtue of its association with drug
resistance in tumor cells (1). It is now also known to be a primary
active transporter of many conjugated organic anions (2). The first
substrate shown to be actively transported by MRP1 using inside-out
membrane vesicles was the glutathione-conjugated leukotriene,
LTC4 (3, 4). Since then, the spectrum of molecules transported by MRP1 has been extended to include many other GSH conjugates, as well as several glucuronate and sulfate conjugates (5,
6). A number of unconjugated amphiphilic anions have also been
demonstrated to be substrates for MRP1 (7). However, using an in
vitro membrane vesicle system unmodified forms of the natural
product drugs to which MRP1 confers resistance are not directly
transported by this protein, although primary active transport of some
of them has been observed in the presence of reduced glutathione (GSH)
(8-11). Potential endogenous substrates of MRP1 that show a similar
dependence on the presence of GSH have not been identified, although it
has been observed that the intracellular levels of GSH are reduced in
some drug selected and transfected cells overexpressing MRP1 in the
absence of an exogenous substrate (12-14). In addition, GSH levels are
elevated in some tissues in
mrp1 MRP1/mrp1 is highly expressed in the sex hormone-producing Leydig cells
of the human and mouse testis (16, 17), as well as in Sertoli cells of
the mouse (17). Both of these cell types have high levels of GSH
S-transferase activity (18, 19) and are thought to
contribute to detoxification in the testis by the formation of GS
conjugates, which may be substrates for MRP1 or related transporters.
Studies of mrp1 In addition to a protective role with respect to xenobiotic exposure,
the relatively high levels of MRP1 in Leydig cells may also serve to
protect the testis from the potential feminizing effects of
endogenously produced estrogen conjugates. Estrogen is synthesized in
the testis and is required for normal testicular function, as revealed
by studies of mice in which the estrogen receptor Materials--
[6,7-3H]Estrone sulfate (53 Ci
mmol Cell Culture--
The HeLa T5 cells transfected with the pRcCMV
vector containing the MRP1 coding sequence and the HeLa C1 cells
transfected with empty pRcCMV vector have been described previously
(27). Both T5 and C1 were cultured in RPMI 1640 medium with 5% defined bovine calf serum and maintained in 400 µg
ml Membrane Vesicle Preparation and Transport Studies--
Plasma
membrane vesicles were prepared as described (8). Briefly, cell pellets
were covered with buffer containing 50 mM Tris-HCl, 250 mM sucrose, 0.25 mM CaCl2, and
protease inhibitors and frozen at
ATP-dependent transport of
[3H]LTC4 into the inside-out membrane
vesicles was measured by a rapid filtration technique (8). In standard
transport assays, 2.5 µg of membrane vesicles were used in a 25-µl
reaction volume and incubated at 23 °C in the presence of 50 nM [3H]LTC4, 10 mM
MgCl2, and 4 mM ATP or AMP in transport buffer. Where indicated, GSH was added to 1 mM unless otherwise
stated. Uptake was stopped by rapid dilution in ice-cold transport
buffer and followed by filtration through glass fiber (type A/E)
filters (Gelman Sciences, Dorval, Quebec, Canada) that had been
presoaked overnight at 4 °C in transport buffer. All of the data had
been corrected for the amount of [3H]LTC4
that remained bound to the filter in the absence of the membrane
vesicles, which was usually less than 5-10% of the total radioactivity. For kinetic analysis of LTC4 transport in
the presence of estrogen sulfates and/or GSH, LTC4 was
included at concentrations ranging from 16 nM to 1 µM and ATP-dependent
[3H]LTC4 uptake was determined as above.
ATP-dependent uptake of [3H]estrone 3-sulfate
was measured by rapid filtration as above, except that the incubation
temperature was 37 °C and substrate concentration was 300 nM unless otherwise indicated. Uptake was stopped after
60 s or at the time indicated by rapid dilution in ice-cold
buffer, and the reaction mixture was filtered through glass fiber
filters. Where indicated, MRP1-specific mAbs were added to 10 µg
ml
Transport of [3H] GSH into the membrane vesicles was also
measured by rapid filtration as above. In a 50-µl reaction volume, 22 µg of membrane vesicle protein were incubated at 37 °C for 20 min
in the presence of 100 µM [3H]GSH (80 or
288 nCi/reaction), 10 mM DTT, 10 mM
MgCl2, and 4 mM ATP or AMP in transport buffer.
Estrone 3-sulfate or estradiol 3-sulfate was added to several
concentrations ranging from 0.2 to 20 µM. Verapamil was
used a positive control for stimulation of [3H]GSH
transport (28) and was added to 100 µM. All data were corrected by subtracting the amount of [3H]GSH that
remained bound to the filter in the presence of 4 mM AMP,
which was usually less than 5% of the total radioactivity.
Photoaffinity Labeling of MRP1 with
[3H]LTC4 and Inhibition of Labeling by
Estrogen Sulfates--
Membrane vesicles (75 µg of protein in 35 µl) were incubated with [3H]LTC4 (0.25 µCi, 200 nM) in the absence or presence of various concentrations of estrone 3-sulfate or estradiol 3-sulfate at room
temperature for 10 min and frozen in liquid nitrogen. Samples were
alternately irradiated for 30 s at 312 nm in a Stratalinker, followed by snap-freezing in liquid nitrogen, for a total of 10 min.
Radiolabeled vesicles were solubilized in Laemmli's buffer and
analyzed on a 7.5% gel by SDS-polyacrylamide gel electrophoresis. The
gel was fixed in isopropanol:water:acetic acid (25:65:10) for 30 min
and then soaked in Amplify for 15-20 min. After drying under vacuum at
80 °C, the gel was placed in close contact with x-ray film at
[3H]Estradiol Sulfate Accumulation in Intact HEK
Cells--
The expression vectors pCDNA3 containing mouse
estrogen-sulfotransferase cDNA (pCDNA3-est) and pCEBV7
containing human MRP1 cDNA (pCEBV7-MRP1) were described previously
(29, 30), and transfection was performed with FuGENETM 6 transfection
reagent (Roche Molecular Biochemicals). HEK293 cells (2 × 106) were seeded into each T75 flask and transfected on the
following day with a mixture of 40 µl of FuGENETM reagent and 10 µg of pCDNA3-est or together with 10 µg of pCEBV7-MRP1. After
66 h, the cells were harvested. Half of the cells were then used
for steroid accumulation experiments and the other half for an EST
activity assay. [3H]Estradiol sulfate accumulation was
initiated by addition of [3H]estradiol to a final
concentration of 500 nM to a suspension of untransfected or
transfected cells (5 × 105 cells in 100 µl of
serum-free DMEM) at 37 °C and stopped at 20 min by adding 1 ml of
ice-cold phosphate-buffered saline. The cells were washed twice with
ice-cold phosphate-buffered saline., solubilized in 200 µl of 0.5%
SDS, and extracted with 400 µl of ethyl acetate. The aqueous phase
was taken for liquid scintillation counting, and 3H-labeled
aqueous soluble steroid accumulation was expressed as dpm/106 cells. The procedures for preparation of cytosolic
proteins and EST activity assay were the same as described previously
(29, 31).
Inhibition of MRP1-mediated LTC4 Transport by Estrogen
Sulfates--
To determine whether estrogen sulfates were potential
substrates for MRP1, we initially tested the ability of estrone and estradiol 3-sulfate to compete for transport of the high affinity MRP1
substrate, LTC4. Neither estrogen sulfate alone was a
potent inhibitor of [3H]LTC4 uptake by
vesicles prepared from MRP1-transfected HeLa T5 (HeLa-MRP) cells (Fig.
1, A and B). At
lower concentrations of the conjugated estrogens (20-200
nM), a reproducible 25-50% increase in the rate of
LTC4 transport was observed with significant inhibition of
transport being observed when concentrations of the estrogen sulfates
reached more than 20 µM. Previously, GSH has been shown
to increase the inhibitory potency of some hydrophobic xenobiotics,
which are themselves poor competitive inhibitors of LTC4
transport (8). However, no effect on the transport of conjugated
endobiotics has been reported. We found that addition of 1 mM GSH markedly increased the inhibitory potency of the
sulfated estrogens, resulting in a decrease in IC50 values
from 31 and 50 µM to 0.2 and 0.3 µM, for
estrone sulfate and estradiol sulfate, respectively (Fig. 1,
A and B).
To determine whether inhibition of LTC4 transport by the
estrogen sulfates was competitive, Eadie-Hofstee plots of
LTC4 transport in the absence or presence of 2 µM estrogen sulfate were constructed. At this
concentration, estrone sulfate alone inhibited
[3H]LTC4 transport by ~10% while estradiol
sulfate stimulated transport by ~25% (Fig. 1, A and
B). However, in the presence of 1 mM GSH, both
estrone and estradiol sulfate behaved as competitive inhibitors of
[3H]LTC4 transport, with apparent
Ki values of 0.45 and 0.38 µM,
respectively (Fig. 1, C and D).
Attenuation of [3H]LTC4 Photolabeling of
MRP1 by Estrogen Sulfates--
To determine whether inhibition of
LTC4 transport was the result of direct competition for
binding, we examined the ability of the conjugated estrogens to inhibit
[3H]LTC4 photolabeling of MRP1 in T5 membrane
vesicles in the absence or presence of GSH. GSH alone weakly inhibited
photolabeling with [3H]LTC4 (Fig.
2A), decreasing labeling by
20-25% at a concentration of 1 mM. Low concentrations of
estrone or estradiol sulfate alone (0.2 µM) resulted in
small but reproducible increases in [3H]LTC4
photolabeling of MRP1 (Fig. 2B), while higher concentrations either decreased (20 µM) or abolished (200 µM) labeling. The enhanced photolabeling of MRP1 observed
with low concentrations (0.2-2 µM) of sulfated estrogens
alone was not apparent in the presence of 1 mM GSH (Fig.
2B), and [3H]LTC4 labeling was
completely abolished in the presence of 1 mM GSH and 20 µM estrone sulfate or estradiol sulfate (Fig.
2B). Furthermore, the effect of GSH could be fully mimicked
by a nonreducing GSH derivative, S-methyl GSH (Fig.
2C), indicating that GSH-enhanced inhibition of
[3H]LTC4 labeling of MRP1 was not caused by a
change in the redox state of the protein.
[3H]Estrone 3-Sulfate Transport in T5 Membrane
Vesicles: Osmotic Sensitivity and Inhibition by MRP1-specific
mAbs--
To determine whether it was possible to detect
ATP-dependent MRP1-mediated transport of the estrogen
sulfates, we determined the rate of [3H]estrone 3-sulfate
(300 nM) uptake using T5 membrane vesicles. The rate of
ATP-dependent uptake of estrone 3-sulfate was very low
(5-6 pmol mg
To confirm that vesicle-associated estrone sulfate was indicative of
transport into the vesicle lumen, rather than surface or intramembrane
binding, we determined the effect of increasing extravesicular
osmolarity. As expected for true transport, the amount of
vesicle-associated [3H]estrone sulfate decreased with
increasing concentrations of sucrose extrapolating to zero at infinite
osmolarity (Fig. 4A).
To confirm that transport was MRP1-mediated, we used previously
characterized conformation-dependent MRP1-specific mAbs,
which have been shown to inhibit MRP1-mediated transport of
LTC4 and other MRP1 substrates (8-10, 26, 28, 32). As
observed for other MRP1 substrates, three mAbs that recognize distinct
conformation-dependent epitopes in the first (mAbs QCRL-2
and QCRL-3) and second (mAb QCRL-4) nucleotide-binding domains of MRP1
(26) completely inhibited ATP-dependent and GSH-stimulated
estrone sulfate transport by T5 vesicles (Fig. 4B). In
contrast, mAb QCRL-1, which recognizes a linear epitope in the linker
region of MRP1(33), had no inhibitory effect.
To determine the kinetic parameters of estrone sulfate transport and
the effect of GSH, rates of ATP-dependent uptake in the presence and absence of 1 mM GSH were determined at several
concentrations of estrone 3-sulfate (125 nM to 16 µM) to obtain Km and
Vmax values (Fig. 3C). A nonlinear
regression analysis of the data yielded an apparent
Km of 0.73 ± 0.17 µM for
estrone 3-sulfate and a Vmax of 440 ± 27 pmol mg
When the nonreducing S-methyl GSH was used in place of GSH,
1 mM S-methyl GSH stimulated estrone 3-sulfate
uptake by T5 membrane vesicles, somewhat more effectively than the same
concentration of GSH (Fig.
5A). In contrast, the
sulfhydryl reducing agents DTT (10 mM) and
2-mercaptoethanol (5 mM) and oxidized glutathione GSSG
(0.05 mM) had no effect on estrone 3-sulfate transport.
Since S-methyl GSH appeared to be a more potent stimulator
of estrone sulfate transport than GSH, rates of transport were
determined as a function of GSH or S-methyl GSH
concentration (Fig. 5B). Both compounds exhibited a similar
concentration dependence with 50% of maximal stimulation being
observed at a concentration of ~0.5 mM. However, the
maximal rate of estrone sulfate transport obtained with
S-methyl GSH was ~2.5-fold greater than with GSH itself.
Inhibition of Estrone 3-Sulfate Transport by
LTC4--
Since we found that estrogen sulfates
competitively inhibited MRP1-mediated
[3H]LTC4 transport in the presence of GSH, we
determined whether the reverse was also true. Thus, the ability of
LTC4 to inhibit [3H]estrone 3-sulfate
transport by MRP1 was examined in T5 vesicles. GSH-enhanced estrone
3-sulfate uptake was inhibited by LTC4 in a
dose-dependent manner (Fig.
6A). An Eadie-Hofstee plot of
estrone 3-sulfate uptake in the presence of 0.2 µM
LTC4 indicated that the inhibition was competitive, with an
apparent Ki (LTC4) of 0.2 µM (Fig. 6B).
Effect of Sulfated Estrogens on MRP1-mediated [3H]GSH
Transport--
Previous studies of the GSH stimulated transport of the
unconjugated xenobiotic vincristine revealed a reciprocal stimulation of GSH transport by the drug, suggesting a cotransport mechanism (10).
In addition, verapamil was shown to markedly stimulate GSH transport by
MRP1, but in this case no transport of verapamil could be detected
(28). Consequently, we examined the ability of the estrogen sulfates to
stimulate GSH transport using verapamil as a positive control. As
expected, verapamil at 100 µM significantly enhanced GSH
transport by about 3-fold. In the first set of experiments, 80 nCi of
[3H]GSH was used in each reaction and no stimulation of
GSH transport was observed with either estrogen sulfate at 0.2 and 2 µM. A modest stimulation (25%) was observed at 20 µM but only with estrone sulfate (Fig.
7A). In a second set of
experiments, the amount of [3H]GSH was increased to 288 nCi/reaction to obtain a higher sensitivity of the transport assay. The
modest stimulation of GSH transport by estrone sulfate was also
detected at the concentration of 100 µM. However,
addition of 100 µM estradiol sulfate inhibited rather than enhanced the low basal level of ATP-dependent
[3H]GSH transport, which was ~25-30 pmol
mg [3H]Estradiol Sulfate Accumulation in Intact HEK
Cells Transiently Expressing est or est plus MRP1--
To determine
whether the MRP1-mediated estrogen sulfate transport observed in
vitro with membrane vesicles could also be detected in intact
cells. HEK cells were transfected with pCDNA3-est alone and with
pCEBV7-MRP1 together. The production and accumulation of
[3H]estradiol sulfate in the est-transfected cells was
then determined and compared with untransfected control cells, and with
cells coexpressing est and MRP1. EST specifically catalyzes estrogen sulfation, and the identities of the products have been confirmed previously by high performance liquid chromatography to be estrogen 3-sulfates (31). Thus, an increased accumulation of tritium in the
est-transfected cells incubated with [3H]estradiol
reflects the accumulation of [3H]estradiol 3-sulfate,
which unlike estradiol does not readily diffuse across the plasma
membrane. Cytosolic extracts were also prepared from control and
transfected cells and assayed directly for est activity as described
previously (29, 31). The level of est activity obtained with extracts
from cells transfected with either the est vector alone or
cotransfected with the MRP1 vector were ~10-fold higher than in
control cells. Cotransfection with the MRP1 vector had no detectable
effect on the levels of EST activity (data not shown). As shown in Fig.
8, over a 20-min period, cells
transfected with the EST vector accumulated ~2.5-fold more tritium
than control cells and cotransfection with the MRP1 vector decreased
this accumulation to a level that was only 50% higher than the
controls. This decrease was statistically significant (p < 0.01, unpaired t test) (Fig. 8),
consistent with an MRP1-mediated efflux of sulfated estrogen from the
intact cells.
Our data derived from studies with MRP1-enriched membrane vesicles
and intact cells demonstrate that estrone and estradiol 3-sulfates are
potential endogenous substrates for MRP1. The in vitro
experiments, in which ATP-dependent transport of
[3H]estrone sulfate was examined directly using plasma
membrane vesicles from MRP1-transfected cells, confirmed that the
conjugated estrogen is an MRP1 substrate and also revealed that a
physiological concentration of GSH is required for its efficient
transport. Direct demonstration of MRP1-mediated estrogen sulfate
efflux from intact cells was precluded by the fact that the hydrophilic conjugates enter intact cells very poorly. Consequently, it was necessary to produce the estradiol 3-sulfate intracellularly by conjugation of [3H]estradiol. This was accomplished by
using short term transfection to express est in HEK cells in the
presence and absence of MRP1. The results demonstrated that the
presence of MRP1 significantly decreased accumulation of the
water-soluble estrogen conjugate produced by est.
The Km value (0.73 µM) for
MRP1-mediated and GSH-enhanced estrone sulfate transport is comparable
with that for organic anion transporter 3 (OAT3)- or organic anion
transporter 4 (OAT4)-mediated transport of estrone sulfate (34, 35).
However, these latter transporters have a more restricted tissue
distribution than MRP1/mrp1 and are involved in the uptake rather than
efflux of organic anions. The relatively high coexpression of MRP1/mrp1
with EST in Leydig cells suggests that MRP1 is likely to be involved in
estrogen sulfate efflux from the testis. Since estrone sulfate can be
converted back into estrone by estrogen sulfatase, efficient removal of estrone sulfate by an export pump is expected to be important for
maintenance of low estrogen levels in organs such as the testis. Studies of mrp1 The requirement of GSH for efficient transport of the sulfated estrogen
provides the first example of GSH-enhanced transport of a conjugated
endogenous substrate by MRP1. Previous studies have shown that GSH is
required for the transport of some xenobiotics including unmodified
chemotherapeutic agents such as vincristine (8-10), daunorubicin (9,
11) and aflatoxin B1 (32), and enhances transport of
etoposide conjugated with glucuronate (37). GSH has been reported
recently to stimulate transport of [3H]luteolin
7-O-diglucuronyl-4'-O-glucuronide in plant
leaves, but the transporter involved has not been characterized
structurally. However, it appears functionally to be a
membrane-potential sensitive member of the ABC superfamily (38).
We have previously proposed that MRP1 contains a bipartite site to
which the hydrophobic and anionic moieties of its conjugated substrates
bind (8). Studies of the GSH-stimulated transport of vincristine, and
the influence of GSH on the ability of vincristine to compete for
LTC4 transport by MRP1, indicate that GSH not only increases the Vmax for the drug but also the
affinity with which it interacts with the protein, as reflected by an
approximate 20-fold increase in its inhibitory potency. These studies
also indicated that vincristine reciprocally increases the affinity of
MRP1 for GSH (10). The data suggest that initial low affinity binding
of either GSH or drug by MRP1 induces a conformational change in the
protein such that high affinity binding of the cotransported substrate
can occur. In this respect, the model differs from the two-site model
proposed by Borst et al. (39) in which the protein is
envisaged to have two binding sites: one that has a relatively high
affinity for drug and low affinity for GSH and another with high
affinity for GSH and low affinity for drug (39). Although this model
may explain the ability of drug and GSH to reciprocally stimulate
cotransport, it is difficult to explain why GSH markedly increases the
ability of drug to competitively inhibit transport of high affinity,
conjugated substrates such as LTC4. Consistent with the
existence of a low affinity site for GSH alone, our photoaffinity experiments with [3H]LTC4 indicate that GSH
and S-methyl GSH decrease [3H]LTC4
labeling of MRP1 in a concentration-dependent manner in the
millimolar range, suggesting that the binding sites for
LTC4 encompass a low affinity site to which GSH (or
S-methyl GSH) can bind.
Recent studies of drug binding and transport by the bacterial,
homodimeric ABC multidrug resistance protein, Lmra, suggest positive
cooperativity between allosterically linked low and high affinity
substrate binding sites (40). A "two-cylinder engine" model, which
embodies the alternating catalytic sites model proposed by Senior
et al. (41), has been proposed in which the transport of
drug from a high affinity intracellular site to a low affinity extracellular site on the same subunit of the homodimer is driven by
the alternating hydrolysis of ATP at one or the other nucleotide binding domain. Thus, the two subunits cycle with respect to exposure of high or low affinity sites (40).
The evidence for positive cooperativity is based in part on the
observation that two substrates that compete for transport at high
concentrations reciprocally stimulate transport of each other at lower
concentrations. Unlike the bacterial homodimeric transporters and
transporters such as P-glycoprotein, the nucleotide binding domains of
the MRP-related proteins are relatively divergent. Evidence to date
suggests that they are also not functionally equivalent and may not
alternate catalytically (42). Similarly, there is no evidence of
primary structure conservation between the NH2 and
COOH-proximal membrane-spanning domains of MRP1. However, we have
consistently observed that low concentrations of the estrogen sulfates
in the absence of GSH stimulate LTC4 transport and binding, as evidenced by photocross-linking studies, while they compete at
higher concentrations. Thus, the data are also consistent with the
existence of interacting binding sites being present on MRP1. However,
since both the estrogen sulfates and LTC4 are relatively hydrophilic and the duration of transport assays is extremely short,
the stimulation observed suggests that the sites are accessible from
the cytoplasmic face of the membrane. In addition, the stimulation of
binding occurs in the complete absence of nucleotide, indicating that
if allosteric changes in structure occur following initial interaction
with substrate, they do not require the binding and/or hydrolysis of ATP.
In some respects, the results we have obtained in the present study
with estrone sulfate are similar to previous observations on the effect
of GSH on rate of transport and apparent binding affinity of unmodified
hydrophobic substrates such as vincristine, despite the fact that the
compound is conjugated. However, in the case of vincristine, aflatoxin
B1, and daunorubicin, it has not been possible to determine
kinetic parameters of transport in the absence of GSH. Our data
demonstrate that GSH decreases the Km for estrone
sulfate from 4.2 to 0.73 µM and increases Vmax from 107 to 440 pmol
min Earlier studies with conjugated estrogens indicated that
E217 It has been observed that GSH levels are increased in some tissues of
mrp knockout mice (15) and decreased in drug-selected or
transfected cells that overexpress MRP1 (12, 13), suggesting that MRP1
might efflux GSH either alone or via cotransport with currently
unidentified, endogenous substrates. The stimulation of estrogen
sulfate transport by GSH suggested that these compounds might be
candidates for such substrates. Thus far, vesicle transport studies
have provided strong evidence for cotransport of GSH with some
xenobiotics (15, 10). However, in other cases, it has not been possible
to detect a xenobiotic-dependent stimulation of GSH
transport (10, 11). In addition, we have shown that some compounds,
such as verapamil, can markedly stimulate MRP1-mediated GSH transport
with no detectable net transport of the compound itself (28).
Despite the readily demonstrable GSH stimulation of estrone sulfate
transport, we have not been able to detect reciprocal stimulation of
GSH transport by the conjugated estrogen. No significant increase in
GSH uptake by membrane vesicles could be detected in the presence of
either estradiol or estrone sulfate over a 100-fold range of conjugated
estrogen concentrations (0.2-20 µM). However, because of
practical limitations, the highest concentration of
[3H]GSH used in these studies is ~100 µM,
and at this concentration the rate of estrone sulfate transport is
~25-30 pmol min-estradiol-17
-D-glucuronide, this has been
confirmed by direct transport studies. The Leydig cell is the major
site of estrogen conjugation in the testis. However, the principal
products of conjugation are A-ring estrogen sulfates, which are then
effluxed from the cell by an unknown transporter. To determine whether
MRP1/mrp1 could fulfill this function, we used membrane vesicles from
MRP1-transfected HeLa cells to assess this possibility. We found that
estradiol and estrone 3-sulfate alone were poor competitors of
MRP1-mediated transport of the cysteinyl leukotriene, leukotriene
C4. However, in the presence of reduced glutathione (GSH),
their inhibitory potency was markedly increased. Direct transport
studies using [3H]estrone 3-sulfate confirmed that the
conjugated estrogen could be efficiently transported
(Km = 0.73 µM,
Vmax = 440 pmol mg
1
protein min
1), but only in the presence of
either GSH or the nonreducing alkyl derivative, S-methyl
GSH. In contrast to previous studies using vincristine as a substrate,
we detected no reciprocal increase in MRP1-mediated GSH transport.
These results provide the first example of GSH-stimulated,
MRP1-mediated transport of a potential endogenous substrate and expand
the range of MRP1 substrates whose transport is stimulated by GSH to
include certain hydrophilic conjugated endobiotics, in addition to
previously identified hydrophobic xenobiotics.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice (15). Whether this is
attributable to the efflux of GSH in association with transport of
endogenous compounds has not been established.
/
mice have
already provided evidence that testicular mrp1 protects the local
tissue against drug-induced damage (17). Thus, the testes of
mrp1-deficient mice treated with the anticancer drug, etoposide
phosphate, showed aberrant spermatogenesis with no sign of meiotic
divisions and an increased number of prematurely released round germ
cells. In contrast, the same treatment of the wild-type mice produced
only a partially distorted spermatogenesis and meiotic divisions still occurred.
or P450 aromatase
genes have been disrupted (20-22). MRP1 has been shown to transport
certain estrogen glucuronides but the major metabolite in Leydig cells
is estrogen sulfate produced by estrogen sulfotransferase (EST) (5,
23). Using 3'-phosphoadenosine 5'-phosphosulfate as a sulfate donor,
this sulfation takes place at the 3-hydroxyl group of the parent
molecule, generating the more hydrophilic estrogen 3-sulfate. The
relative hydrophilicity of estrogen 3-sulfate prevents its ready
diffusion across the plasma membrane. Thus, it has been assumed that an
export pump is involved in its efflux from the cell. Testicular
expression of estrogen sulfotransferase is mainly localized in Leydig
cells and is regulated by luteinizing hormone via modulation of cAMP levels (23, 24). Interestingly, the testes of 9-12-month-old est
/
mice displayed Leydig cell
hypertrophy/hyperplasia, as well as seminiferous tubule
disruption.2 The
colocalization of MRP1 and estrogen sulfotransferase in the testis
prompted us to investigate whether estrogen sulfates were also
substrates of MRP1. We found that estrogen sulfates alone were very
poor substrates and competed poorly for transport and binding of the
high affinity MRP1 substrate LTC4. However, in the presence
of GSH, their inhibitory potency was significantly increased and
GSH-enhanced direct transport of [3H]estrone sulfate
could be easily detected. These results provide the first examples of
potential endogenous MRP1 substrates that depend on the presence of GSH
for efficient transport and the first examples of conjugated
endobiotics that display this type of dependence.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1), [2,4,6,7-3H]estradiol
(87.6 Ci mmol
1), and
[glycine-2-3H]GSH (42 Ci
mmol
1) were purchased from PerkinElmer Life
Sciences; [14,15-3H]-LTC4 (38 Ci
mmol
1) and fluorographic reagent
Amplify® were from Amersham Pharmacia Biotech (Oakville,
Ontario, Canada). Estrogen 3-sulfates, nucleotides, GSH, verapamil,
S-methyl GSH, glutathione disulfide (GSSG),
2-mercaptoethanol, and DTT were purchased from Sigma. Estrogen
3-sulfates were dissolved with H2O to prepare stock
solutions at 10 mM and diluted with transport buffer. The
MRP1-specific murine monoclonal antibodies (mAbs) QCRL-1, QCRL-2,
QCRL-3, and QCRL-4 have been described previously (25, 26).
1 Geneticin (G418). HEK293 cells were grown
in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal bovine serum.
70 °C overnight. The cells were
then thawed and disrupted by N2 cavitation (10-min
equilibrium at 200 p.s.i.). EDTA was added to 1 mM
before centrifugation at 500 × g for 15 min to remove
cell debris. To increase the yield of membrane vesicles, the resulting
pellet was washed once with 5 ml of transport buffer (50 mM
Tris-HCl and 250 mM sucrose, pH 7.5) and centrifuged again. The supernatants were pooled, layered over 35% (w/w) sucrose in 10 mM Tris-HCl (pH 7.5), and centrifuged at 100,000 × g for 1 h. The interface was collected in washing
buffer (10 mM Tris-HCl and 25 mM sucrose) and
followed by centrifugation at 100,000 × g for 30 min.
The membrane pellet resuspended in transport buffer and passed 10 times
through a 27.5-gauge needle for vesicle formation. The membrane
vesicles were then aliquoted and stored at
70 °C until use.
1 and preincubated with membrane vesicles
on ice for 1 h. All data were corrected by subtracting nonspecific
binding of [3H]estrone 3-sulfate to the filter, which was
usually less than 5% of the total radioactivity. For kinetic analysis
of GSH-enhanced estrone 3-sulfate transport in the absence or presence
of LTC4, estrone 3-sulfate was included at concentrations
ranging from 125 nM to 16 µM and
ATP-dependent [3H]estrone 3-sulfate uptake
was determined as above.
70 °C for 2 weeks (8).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of estrogen sulfates on
ATP-dependent [3H]LTC4 transport
by membrane vesicles prepared from MRP1-transfected HeLa T5 cells.
Panels A and B, the rates of
[3H]LTC4 uptake by vesicles were determined
at a fixed substrate concentration (50 nM) but at various
concentrations of estrone 3-sulfate (A) or estradiol
3-sulfate (B) (20 nM to 200 µM)
for 30 s at 23 °C. The transport assays were performed in the
presence of 4 mM ATP alone ( ) or in combination with 1 mM GSH (
). Results are expressed as a percentage of the
control value obtained in the absence of competitors. Data points are
means (± S.E.) of triplicate determinations in a single experiment.
Panels C and D, ATP-dependent
[3H]LTC4 uptake by membrane vesicles was
determined at various substrate concentrations (18 nM to 1 µM) in the absence (open symbols)
or presence (closed symbols) of 1 mM
GSH and 2 µM estrone 3-sulfate (panel C) and
estradiol 3-sulfate (panel D) for 30 s at 23 °C. The
inhibitions were competitive according to the Eadie-Hofstee plots since
the kinetic parameters were significantly altered for only
Km but not Vmax. In the
absence and presence of 2 µM estrone sulfate and 1 mM GSH, the values of Km for
LTC4 were 167 ± 15 nM and 910 ± 190 nM, whereas those of Vmax were
330 ± 19 pmol mg
1
min
1 and 366 ± 62 pmol
mg
1 min
1,
respectively. The estimated Ki value for estrone
sulfate was 0.45 µM. Similarly, in the absence and
presence of 2 µM estradiol sulfate and 1 mM
GSH, the values of Km for LTC4 were
136 ± 10 nM and 860 ± 266 nM,
whereas those of Vmax were 361 ± 16 pmol
mg
1 min
1 and
276 ± 68 pmol mg
1
min
1, respectively. The estimated
Ki value for estradiol sulfate was 0.38 µM. For clarity the data for the uptakes in the presence
of 1 mM GSH alone or 2 µM estrogen sulfates
alone were not shown, and no significant affect was observed under
these conditions. Data points are the means (± S.E.) of triplicate
determinations in a single experiment.
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Fig. 2.
Photoaffinity labeling of MRP1-enriched
membrane vesicles by [3H]LTC4 and inhibition
of labeling by estrone 3-sulfate and estradiol 3-sulfate in the absence
and presence of GSH. MRP1-enriched T5 membrane vesicles (75 µg)
were incubated with [3H]LTC4 (200 nM, 0.25 µCi) alone or in the presence of various
concentrations of competitors at 22 °C for 10 min. Samples were
irradiated at 312 nm prior to being subjected to SDS-polyacrylamide gel
electrophoresis and fluorography (see "Experimental
Procedures"). Panel A, effect of GSH (0.3-3
mM) and S-methyl GSH (0.3-3 mM) alone on
[3H]LTC4 labeling of MRP1. Panel
B, inhibition of [3H]LTC4 labeling by
estrone sulfate or estradiol sulfate (0.2-200 µM) alone
and by the sulfates (0.2-20 µM) in the presence of 1 mM GSH. Panel C, effect of a combination of 1 mM S-methyl GSH with several concentrations of estrone
sulfate (0.2-20 µM) on
[3H]LTC4 labeling of MRP1.
S-MeGSH, S-methyl GSH.
1 protein
min
1). However, an approximate 10-fold
increase in the rate of transport was observed when 1 mM
GSH was present (Fig. 3A).
GSH-stimulated transport was linear for up to 60 s at a rate of
~60 pmol mg
1 protein
min
1 with an initial concentration of 300 nM estrone 3-sulfate. In the presence of 4 mM
AMP and 1 mM GSH, estrone 3-sulfate uptake by the vesicles
was 2-4 pmol mg
1 protein
min
1. ATP-dependent and
GSH-stimulated estrone 3-sulfate transport was not detected with
membrane vesicles prepared from vector-transfected HeLa C1 control
cells (Fig. 3B).
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Fig. 3.
Time course and kinetic parameters of
[3H]estrone 3-sulfate uptake by membrane vesicles from
MRP1-transfected HeLa cells. [3H]Estrone 3-sulfate
uptake by membrane vesicles prepared from MRP1-transfected HeLa cells
(T5) (panel A) or vector-transfected HeLa control cells (C1)
(panel B) was determined at 37 °C in the presence ( ,
) or absence (
,
) of 1 mM GSH for the times
indicated. The initial concentration of [3H]estrone
3-sulfate was 300 nM with a specific activity of 17 Ci
mmol
1. Closed symbols
(
,
) indicate uptake in the presence of 4 mM ATP, and
open symbols (
,
) represent uptake in the
presence of 4 mM AMP. Panel C,
[3H]estrone sulfate uptake by T5 vesicles was measured at
estrone sulfate concentrations ranging from 125 nM to 16 µM for 1 min at 37 °C in the presence of 4 mM ATP and 1 mM GSH. Kinetic parameters were
determined from nonlinear regression analysis. The calculated
Km and Vmax for
GSH-stimulated ATP-dependent [3H]estrone
sulfate uptake were 0.73 ± 0.17 µM and 440 ± 27 pmol mg
1 min
1,
respectively. The inset shows Eadie-Hofstee transformation
of the data. Data points are the means (± S.E.) of triplicate
determinations in a typical experiment.
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Fig. 4.
Osmotic sensitivity and MRP1-specific mAbs
inhibition of GSH-stimulated [3H]estrone sulfate
transport by T5 membrane vesicles. Panel A, T5 membrane
vesicles were preincubated on ice for 10 min in transport buffer
containing sucrose (0.25-1 M). [3H]Estrone
sulfate uptake was measured at 37 °C for 1 min in the presence of a
combination of 1 mM GSH and 4 mM ATP ( ) or 4 mM AMP (
). Panel B, T5 membrane vesicles were
preincubated on ice for 1 h with the indicated mAbs (10 µg
ml
1). [3H]Estrone sulfate
uptake was then measured at 37 °C for 1 min in the presence of 4 mM ATP and 1 mM GSH. Data points in both panels
are means (± S.E.) of triplicate determinations in a typical
experiment.
1 protein
min
1 in the presence of GSH, and an apparent
Km of 4.2 ± 1.1 µM for estrone 3-sulfate and a Vmax of 107 ± 10 pmol mg
1 protein
min
1 in its absence. The inset
shows an Eadie-Hofstee transformation of the data (Fig. 3C).
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Fig. 5.
Effect of S-methyl GSH,
sulfhydryl reducing agents, and GSSG on [3H]estrone
3-sulfate transport by MRP1-enriched T5 membrane vesicles.
Panel A, ATP-dependent [3H]estrone
3-sulfate uptake was determined at 37 °C for 1 min in the presence
of reducing agents (1 mM GSH, 5 mM
2-mercaptoethanol, or 10 mM DTT) or GSH derivatives (1 mM S-methyl GSH, 0.05 mM GSSG). The
initial concentration of [3H]estrone 3-sulfate was 300 nM. Control (Con) shows uptake in the presence
of 4 mM ATP alone, whereas other bars represent
uptake in the presence of 4 mM ATP and one of the indicated
agents. Bars represent the means (± S.E.) of triplicate
determinations in a typical experiment. S-MeGSH,
S-methyl GSH; 2-ME, 2-mercaptoethanol;
GSSG, glutathione disulfide. Panel B,
ATP-dependent [3H]estrone 3-sulfate uptake
was determined at various concentrations of GSH ( ) and
S-methyl GSH (
) ranging from 31 µM to 4 mM for 1 min at 37 °C in the presence of a fixed initial
concentration of [3H]estrone 3-sulfate (300 nM). Data points are means (± S.E.) of triplicate
determinations in a typical experiment.
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Fig. 6.
Inhibition by LTC4 of
ATP-dependent and GSH-enhanced [3H]estrone
3-sulfate transport. Panel A, [3H]estrone
3-sulfate uptake by T5 membrane vesicles was determined at a fixed
substrate concentration (300 nM) and various concentrations
of LTC4 (5-1000 nM) for 1 min at 37 °C in
the presence of 4 mM ATP and 1 mM GSH. Results
are expressed as a percentage of the control value obtained in the
absence of LTC4. The control uptake rate was 76 pmol
mg 1 min
1, and each
data point is the mean (± S.E.) of triplicate determinations in a
single experiment. Panel B, ATP-dependent and
GSH-enhanced [3H]estrone 3-sulfate uptake by T5 membrane
vesicles was determined at various substrate concentrations (0.125-16
µM) in the absence (
) or presence (
) of 200 nM LTC4 for 1 min at 37 °C. ATP (4 mM) and GSH (1 mM) were present in all the
assays. Eadie-Hofstee plots were generated to show that
LTC4 competitively inhibited GSH-enhanced estrone sulfate
transport. In the absence or presence of 200 nM
LTC4, the values of Km for estrone
sulfate were 2.1 ± 0.2 µM and 4.8 ± 0.6 µM, whereas those of Vmax were
959 ± 60 pmol mg
1
min
1 and 823 ± 76 pmol
mg
1 min
1. The
apparent Ki for LTC4 was 160 nM. Data points are the means (± S.E.) of triplicate
determinations in a single experiment.
1 min
1 at an
initial concentration of 100 µM (Fig. 7B).
This rate is approximately equivalent to the rate of
[3H]estrone sulfate uptake in the presence of 100 µM GSH. To determine whether the basal level of
ATP-dependent GSH transport observed with vesicles from
MRP1 transfected cells was MRP1-mediated, we also examined the rate of
ATP-dependent GSH transport by vesicles from HeLa C1 cells
transfected with an empty vector and the ability of the MRP1 mAb QCRL-3
to inhibit transport. The rate of [3H]GSH transport by C1
vesicles was less than 5 pmol mg
1
min
1, and basal GSH transport by T5 vesicles
was completely inhibited by QCRL-3 at 10 µg
ml
1.
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Fig. 7.
Effect of estrogen sulfates on
[3H]GSH uptake by T5 membrane vesicles. Panel
A, membrane vesicles (22 µg of protein) were incubated with 100 µM [3H]GSH (80 nCi/reaction) for 20 min at
37 °C in the presence of 5 mM DTT, 4 mM ATP,
and the indicated concentrations (0.2-20 µM) of estrone
3-sulfate (E1S) or estradiol 3-sulfate (E2S).
Verapamil (VRP, 100 µM) was used as a positive
control stimulating MRP1-mediated GSH transport. Control
(CON) represents ATP-dependent GSH transport in
the absence of a second substrate. All data were corrected by
subtracting [3H]GSH uptake in the presence of 4 mM AMP, and bars represent the means (± S.E.)
of triplicate determinations in a single experiment. Panel
B, experimental conditions were the same as above except that the
amount of [3H] GSH was increased to 288 nCi/reaction.
Control (CON) represents ATP-dependent GSH
transport in the absence of a second substrate. Estrone 3-sulfate
(E1S) and estradiol 3-sulfate (E2S) were added to
100 µM. All data were corrected by subtracting
[3H] GSH uptake in the presence of 4 mM AMP,
and bars represent the means (± S.E.) of triplicate
determinations in a single experiment.
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Fig. 8.
[3H]Estradiol sulfate
accumulation by intact HEK cells transiently expressing est or est and
MRP1. Untransfected control HEK cells and those transfected with
the expression vector pCDNA3 containing est cDNA alone or in
combination with pCEBV7-MRP1 for 66 h were harvested and incubated
with 500 nM [3H]estradiol in serum-free DMEM
at 37 °C for 20 min. [3H]Estradiol sulfate
accumulation was then determined as described under "Experimental
Procedures." Bars represent means (± S.E.) of triplicate
determinations in a single experiment. *, p < 0.01 when compared with untransfected control cells; **, p < 0.01 when compared with the cells transfected with pCDNA3-est
alone. The other half of aliquots of suspended cells were sonicated to
prepare cytosolic proteins for est activity assay. An ~10-fold
increase in the activity was found in the est-transfected cells, and
MRP1 expression did not affect the activity.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice treated
with etoposide phosphate strongly suggest that mrp1 may also protect
the testis from exposure to cytotoxic xenobiotics (17). In addition,
the recent report that EST is able to sulfate environmental estrogens,
such as bis-, 4-octyl-, and p-nonylphenols, raises the
possibility that MRP1 or related proteins could play a role in the
cellular elimination of the conjugates of these estrogenic compounds
(36).
1 mg protein
1.
In addition, GSH also markedly enhanced the ability of estrone sulfate
to inhibit photolabeling of MRP1 with LTC4. In these
experiments, the GSH-enhanced inhibitory potency of estrone sulfate was
obtained in the absence of ATP, indicating that any induced change in
affinity for the conjugated estrogen occurs without a requirement for
either nucleotide binding or hydrolysis.
G could compete for LTC4 binding and
transport by MRP1 while estrogens conjugated with glucuronide at the 3 position of the A-ring are very poor inhibitors (5). These studies also
revealed that a change in site of glucuronidation from the 17
to
16
position on the D-ring markedly decreased the affinity for the
protein, as judged by the difference between the Ki
values for E317
G (1.4 µM) and
E316
G (45 µM) as inhibitors of
E217
G transport. Taken together, the data indicate that
strict structural requirements with respect to the site of
glucuronidation of the steroid nucleus must be met to interact with
MRP1 as a substrate or competitive inhibitor. Sulfation at the
3-position of the A-ring of E217
G had little or no
effect on its ability to compete for transport, implying that the
presence of the sulfate group neither precluded nor enhanced
interaction with the protein. In contrast, the conjugated bile salt
glycolithocholate 3-sulfate was an effective inhibitor of
E217
G transport when compared with bile salts that were
not conjugated at this position of the A-ring. This is consistent with
the possibility that sulfation of the A-ring, in the absence of
additional anionic conjugation, might enhance interaction with MRP1. A
low level of transport of estrone sulfate could be detected in the
absence of GSH, suggesting that sulfation resulted in the formation of
a relatively low affinity, low capacity substrate in which the sulfate
presumably does not prevent GSH from interacting with the protein,
either because it binds to a different site or because it interacts
only weakly with the GSH binding site. This latter possibility would be
consistent with the inhibition of basal MRP1-mediated GSH transport by
HeLa T5 vesicles observed at high concentrations of estradiol sulfate.
However, the GSH has no effect on the inhibitory potency of other
A-ring conjugates, such as E23
G or on the transport of
E217
G, or estradiol itself (data not shown). Thus with
respect to stimulation of transport by MRP1, it remains difficult to
predict which conjugated or nonconjugated compounds might be affected
by the presence of GSH.
1 mg
protein
1. Using [3H]GSH with a
7-fold higher specific activity than used in previous studies, we were
able to detect a low but significant rate of ATP-dependent,
MRP1-mediated GSH transport in the absence of a second substrate. This
rate of basal GSH transport was approximately equivalent to the rate of
estrone sulfate transport under the experimental conditions used. This
leaves open the possibility that the "basal" rate of GSH transport
by the protein is adequate under the experimental conditions to support
cotransport of some substrates. However, this would imply that the
interaction of substrates such as the estrogen sulfates, in contrast to
compounds like verapamil, does not significantly affect the binding and transport of GSH. These studies expand the range of potential MRP1
substrates that require GSH for efficient transport to include certain
hydrophilic anionic as well as previously identified hydrophobic, unconjugated compounds.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank our colleagues Drs. Q. Mao and W. Qiu for providing membrane vesicles for initial experiments.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from Eli Lilly and Co. and the National Cancer Institute of Canada with funds from the Terry Fox Run.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.
¶ Senior scientist of Cancer Care Ontario.
Stauffer research professor of Queen's University. To
whom correspondence should be addressed: Cancer Research Laboratories, Botterell Hall, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Tel.: 613-533-2981; Fax: 613-533-6830; E-mail:
deeleyr@post.queensu.ca.
Published, JBC Papers in Press, December 1, 2000, DOI 10.1074/jbc.M008251200
2 Y. Qian, X. Sun, X. Li, and W. Song, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MRP, multidrug
resistance protein;
ABC, ATP-binding cassette;
LTC4, leukotriene C4;
E217G, 17
-estradiol-17
-D-glucuronide;
E23
G, 17
-estradiol-3
-D-glucuronide;
E316
G, 16
,17
-estriol 16
-D-glucuronide;
GSH, reduced
glutathione;
GSSG, glutathione disulfide;
DTT, dithiothreitol;
mAb, monoclonal antibody;
EST, estrogen sulfotransferase;
DMEM, Dulbecco's
modified Eagle's medium.
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