(Received for publication, November 30, 1995; and in revised form, January 29, 1996)
From the
In addition to its ability to confer resistance to a range of
natural product type chemotherapeutic agents, multidrug resistance
protein (MRP) has been shown to transport the cysteinyl leukotriene,
LTC, and several other glutathione (GSH) S-conjugates. We now demonstrate that its range of potential
physiological substrates also includes cholestatic glucuronidated
steroids. ATP dependent, osmotically sensitive transport of the
naturally occurring conjugated estrogen, 17
-estradiol
17-(
-D-glucuronide) (E
17
G), was readily
demonstrable in plasma membrane vesicles from populations of
MRP-transfected HeLa cells (V
1.4 nmol
mg
min
, K
2.5 µM). The involvement of MRP was confirmed
by demonstrating that transport was completely inhibited by a
monoclonal antibody specific for an intracellular conformational
epitope of the protein. MRP-mediated transport of LTC
was
competitively inhibited by E
17
G (K
22 µM),
despite the lack of structural similarity between these two substrates.
Competitive inhibition of
[
H]E
17
G transport was also
observed with a number of other cholestatic conjugated steroids. All of
these compounds prevented photolabeling of MRP with
[
H]LTC
, demonstrating that the
cholestatic steroid and leukotriene conjugates compete either for the
same or possibly overlapping sites on the protein. Consistent with the
presence of overlapping but non-identical sites, studies using
chemotherapeutic drugs to inhibit MRP-mediated E
17
G
transport indicated that daunorubicin had the highest relative potency
of the drugs tested, whereas it was the least potent inhibitor of
LTC
transport. Non-cholestatic steroids glucuronidated at
the 3 position of the steroid nucleus, such as 17
-estradiol
3-(
-D-glucuronide), did not compete for transport of
E
17
G by MRP, nor did they inhibit photolabeling of the
protein with [
H]LTC
. These data
identify MRP as a potential transporter of cholestatic conjugated
estrogens and demonstrate site-specific requirements for
glucuronidation of the steroid nucleus.
Increased expression of multidrug resistance protein (MRP) ()or its cognate mRNA has been found in drug selected cell
lines from a wide range of tumor types (1, 2, 3, 4, 5, 6, 7, 8) .
These cell lines have a phenotype similar in many respects to that
conferred by P-glycoproteins. Transfection of MRP expression vectors
into drug-sensitive cells has confirmed that MRP confers resistance to
a spectrum of natural product type chemotherapeutic
agents(9, 10) . MRP, like the P-glycoproteins, is a
member of the ATP-binding cassette superfamily of transmembrane
transporters. However, unlike the P-glycoproteins, it has not been
possible to demonstrate that MRP binds and actively transports
unmodified forms of the drugs to which it confers
resistance(11, 12, 55) .
The ability of
MRP to function as an ATP-dependent transporter has been investigated
using plasma membrane vesicles prepared from drug-selected and MRP
transfected cells(13, 14, 15, 55) .
These studies have shown that vesicles enriched in MRP display elevated
levels of ATP-dependent, high affinity transport of cysteinyl
leukotrienes. Evidence has also been presented that the protein can
transport other organic glutathione
conjugates(13, 14) , as well as oxidized glutathione
itself(16) . In the accompanying article (55) , we
provide immunological confirmation that MRP is a primary active
transporter of LTC and demonstrate for the first time that
in the presence of GSH, MRP can actively transport the Vinca alkaloid, vincristine.
ATP-dependent transport systems for
organic GSH conjugates (17) , GSSG(18) , and other
organic anions (19, 20) have been characterized
functionally in a number of tissues. Studies with intact cells or
membrane vesicle preparations indicate that at least one of the
transporters involved, frequently referred to as the multispecific
organic anion transporter (MOAT), has a broad substrate specificity
that includes other organic anions in addition to glutathione
conjugates(21) . MOAT has been studied most extensively in bile
canalicular membranes where it is believed to contribute to the hepatic
clearance of a wide range of xenobiotic and endogenous organic anions,
notably the glucuronidated conjugates of bile acids and
bilirubin(22) . The transporter is functionally defective in
hepatocanalicular membranes of the TR mutant Wistar
rat, the phenotype of which is similar to that observed in the human
Dubin-Johnson syndrome (23, 24) and the mutant
Corriedale sheep(21, 25) . All of these congenital
conditions are associated with chronic conjugated hyperbilirubinemia.
In the TR
rat model, although hepatocanalicular MOAT
activity is markedly reduced, organic anion transport in other tissues
appears to be normal(26) . Whether this indicates the existence
of structurally distinct hepatic and non-hepatic forms of the
transporter, or is attributable to a liver-specific defect in its
expression has not been established.
Recent immunohistological data
indicate that subcellular localization of MRP, or an MRP-related
protein, may be abnormal in the livers of TR rats(27) . This suggests that MRP could be the
transporter functionally described as MOAT. However, although MRP mRNA
can be detected in both rodent and human liver, its levels are low when
compared with other tissues such as muscle, testes, heart, and
lung(1, 28, 29, 30) . To further
define the substrate specificity of MRP, we have used plasma membrane
vesicles from drug-selected and MRP-transfected cells, to examine ATP
dependent transport of [
H]17
-estradiol
17-(
-D-glucuronide) (E
17
G). This
estradiol metabolite is formed in the liver and subsequently excreted
into bile(31, 32) . Increases in the levels of
E
17
G and some other conjugated estrogens have been
implicated in the development of cholestasis during the later stages of
pregnancy, a condition which occurs with exceptional frequency in the
Dubin-Johnson syndrome(33) . Consequently, the potential for
other known cholestatic and non-cholestatic compounds to inhibit
MRP-mediated E
17
G transport has also been determined.
Finally, we have examined the relative abilities of several natural
product drugs to which MRP confers resistance to inhibit transport of
the conjugated steroid and determined the influence of GSH and
glucuronate on their potencies.
Figure 1:
Time course of
[H]E
17
G uptake by membrane
vesicles from MRP-transfected HeLa cells (T14), control HeLa (C6)
cells, drug-sensitive H69 cells, multidrug-resistant H69AR cells,
revertant H69PR cells, and P-glycoprotein overexpressing 8226/Dox40
cells. Membrane vesicles were incubated at 37 °C in transport
buffer containing [
H]E
17
G (50
nM, specific activity 13 Ci mmol
) and ATP
(4 mM) (closed symbols) or AMP (4 mM) (open symbols) for the times indicated. Vesicles were derived
from the following cells, as described under ``Experimental
Procedures:'' Panel A, HeLa C6 (
,
) and T14
(
,
); Panel B, H69 (
,
) and H69AR
(
,
); Panel C, H69PR (
,
); Panel
D, 8226/Dox40 (
,
). The broken curves in Panels A and B indicate ATP-dependent uptake for T14
and H69AR cells, respectively. Data points represent the means
(± S.E.) of triplicate determinations in a typical
experiment.
Correlation
between ATP-dependent [H]E
17
G
transport rates and levels of MRP expression was examined using
vesicles isolated from drug-sensitive H69, drug-resistant H69AR, and
revertant H69PR cells. The rate of ATP-dependent
[
H]E
17
G uptake in H69AR vesicles
(60 pmol mg
min
) was
approximately 3-fold higher than for T14 vesicles (Fig. 1B), consistent with the 3-4-fold higher
levels of MRP in H69AR cells (12) and vesicle preparations
(data not shown). No ATP dependence of
[
H]E
17
G uptake could be
demonstrated with H69 vesicles. The rate of ATP-dependent
[
H]E
17
G uptake in H69PR membrane
vesicles was approximately 1% that of H69AR cells (Fig. 1C). This is consistent with the low, but
detectable levels of MRP mRNA (1) and protein (34) in
these cells. To examine the ability of P-glycoprotein to transport
E
17
G, we used vesicles from the multidrug-resistant
myeloma cell line, 8226/Dox40, which overexpresses P-glycoprotein (37) and has MRP levels comparable to those in H69PR cells
(data not shown). Vesicles from these cells exhibited levels of
[
H]E
17
G transport no higher than
those of H69PR-derived vesicles (Fig. 1D). Thus the
data are consistent with E
17
G transport by 8226/Dox40
vesicles being attributable to their low levels of MRP. They also
indicate that there is little if any transport of E
17
G
by P-glycoprotein.
Figure 2:
Effect of
[H]E
17
G and ATP concentration on
[
H]E
17
G uptake by T14 vesicles. Panel A, the rate of ATP dependent
[
H]E
17
G uptake by T14 membrane
vesicles was measured at various E
17
G concentrations
(12.5 nM to 25 µM) for up to 60 s at 37 °C in
the presence of a fixed concentration of nucleotide (4 mM), as
described under ``Experimental Procedures.'' Data were
plotted as V
versus [S] to
confirm that the concentration range selected was appropriate to
observe both zero-order and first-order rate kinetics. Kinetic
parameters (K
2.5 µM and V
1.4 nmol mg
min
) were determined from regression analysis
of the Lineweaver-Burk transformation of the data (inset). Panel B, ATP-dependent uptake of
[
H]E
17
G was measured as
described for Panel A at various concentrations of nucleotide
(1 to 4000 µM) in the presence of a fixed concentration of
[
H]E
17
G (50 nM). An
apparent K
of 390 µM for ATP
was determined from regression analysis of the Lineweaver-Burk data
transformation (inset).
Figure 3:
Osmotic sensitivity and nucleotide
specificity of [H]E
17
G transport
by T14 membrane vesicles. Panel A, T14 membrane vesicles were
preincubated for 10 min in transport buffer containing sucrose at
concentrations ranging from 250 to 1000 mM. Rates of
[
H]E
17
G uptake at 37 °C
under various conditions of osmolarity were measured at a substrate
concentration of 50 nM in the presence of 4 mM AMP
(
) or ATP (
), as described under ``Experimental
Procedures.'' Panel B, rates of
[
H]E
17
G uptake were determined
at 37 °C, in the presence of various hydrolyzable and
non-hydrolyzable nucleotides (4 mM), as described under
``Experimental Procedures'' except that the nucleoside
triphosphate regenerating system was omitted. The rate observed in the
presence of 4 mM ATP was not affected by omission of the
regenerating system (data not shown). The rates of uptake supported by
various nucleotides have been expressed as a percentage of that
obtained with ATP. The results shown are the means (± S.E.) of
triplicate determinations in a single experiment. The rate of
ATP-dependent uptake in the control was 31.7 pmol mg
min
.
Nucleotide dependent
transport was not detectable when AMP-PNP, AMP-PCP, or ATPS (4
mM) were substituted for ATP, thus indicating a requirement
for ATP hydrolysis (Fig. 3B). As expected when compared
with other nucleotide triphosphates, ATP supported the highest rate of
[
H]E
17
G transport. However, the
rate of transport in the presence of GTP was substantial and approached
approximately 70% of that achievable in the presence of ATP.
Figure 4:
Effect of glucuronidated estradiols on
[H]LTC
uptake by T14 membrane
vesicles. The rates of [
H]LTC
uptake
by T14 vesicles were determined at 23 °C, at various substrate
concentrations (25 to 1000 nM) in the absence (
) or
presence (
, 20 µM;
, 40 µM;
, 100 µM) of E
17
G (upper
panel) or E
3
G (lower panel).
Double-reciprocal plots were generated for each concentration of
potential inhibitor and used to determine K
. The results shown are the means
(±S.E.) of triplicate determinations at each substrate and
inhibitor concentration.
Lineweaver-Burk plots of
[H]E
17
G uptake by T14 membrane
vesicles indicated that LTC
behaved as a competitive
inhibitor of [
H]E
17
G transport,
with a K
of 0.53 µM (Fig. 5). Inhibition by E
17
G (K
1.4 µM) and the
cholestatic bile salt glycolithocholate-3-sulfate (K
1.4 µM) (data not
shown) was also competitive. E
16
G, which is of
intermediate cholestatic potency (40) , was a less effective
competitive inhibitor of transport, with a K
of 45 µM. Although
non-cholestatic in rodents, E
3SO
17
G, was
also an effective competitive inhibitor of E
17
G
transport by MRP (K
1.7
µM).
Figure 5:
Effect of LTC, steroid
glucuronides, and bile salt derivatives on transport of
[
H]E
17
G by T14 membranes. The
rates of uptake of [
H]E
17
G by
T14 vesicles were determined at 37 °C, at various substrate
concentrations (0.05-25 µM) in the absence or
presence of the indicated concentration of LTC
(1
µM) (upper panel), E
17
G (1
µM) or E
16
G (100 µM) (middle panel), and E
3SO
17
G (1
µM) (lower panel). Double reciprocal plots were
generated for each inhibitor and used to determine a K
. Results shown are the means
(±S.E.) of triplicates determinations at each substrate and
inhibitor concentration.
Figure 6:
Photoaffinity labeling of T14 membrane
vesicles by [H]LTC
and inhibition of
labeling by E
17
G and other steroid glucuronides. Panel A, T14 vesicles (150 µg of membrane protein) were
incubated with [
H]LTC
(50
nM) alone or in the presence of various concentrations of
E
17
G (5-100 µM) or 100 µM E
3
G, as indicated. Samples were irradiated at 312
nm prior to being subjected to SDS-PAGE and fluorography, as described (55) . Panel B, photoaffinity labeling of T14 membrane
vesicles with [
H]LTC
was carried out
as above in the presence of various glucuronides and bile salt
derivatives (100 µM), as indicated. Photolabeled membranes
were analyzed by SDS-PAGE and fluorography. DMSO, dimethyl
sulfoxide.
Figure 7:
Effects of chemotherapeutic agents on
[H]E
17
G uptake by T14 vesicles
in the absence or presence of GSH or glucuronic acid. Upper
panel, the rate of uptake of
[
H]E
17
G (50 nM) by T14
vesicles was determined at 37 °C in the presence of various natural
product drugs (100 µM) alone (open bars) and in
combination with GSH (1 mM) (hatched bars) or
glucuronic acid (5 mM) (solid bars). Results are
expressed as a percentage of the control value obtained in the absence
of drug and anion. The drug vehicle (dimethyl sulfoxide) was present at
a concentration of 0.5% in control incubations lacking drug and had no
effect on uptake under the conditions of the assay. The results are the
means (±S.E.) of triplicate determinations in a single
experiment. The control uptake rate in this experiment was 19.2 pmol
mg
min
. DOX,
doxorubicin; DNR, daunorubicin; VBL, vinblastine; VCR, vincristine. Lower panel, inhibition of
[
H]E
17
G uptake by T14 membrane
vesicles by various concentrations of DNR (
), DOX (
), VCR
(
), or VBL (
). Data points represent means of duplicate
determinations in a single experiment and are plotted as a percentage
of control values obtained in the absence of drug, but in the presence
of 0.5% (v/v) dimethyl sulfoxide. The control uptake rate in the
experiment shown was 25.8 pmol mg
min
.
MRP is expressed in a wide range of normal tissues (1, 28, 29, 30) and is overexpressed in many multidrug-resistant cell lines(1, 3, 5, 6, 41) . Elevated levels of MRP in membrane vesicles from drug-selected cell lines and MRP transfectants have been correlated with increased ATP-dependent transport of cysteinyl leukotrienes and other glutathione S-conjugates(13, 14, 15) . It has been suggested on the basis of these data, that one of the physiological roles of MRP is that of a GSH conjugate or GS-X pump(42, 43) . However, the ability to transport certain glutathione conjugates does not explain the drug cross-resistance profile of MRP-transfected or selected cells(1, 5, 12) . Current data indicate that the major classes of drugs to which MRP confers resistance are not metabolized via GSH conjugation(44) , although a GSH-dependent transport mechanism may exist for some compounds(55) . Other conjugation pathways, such as glucuronidation, appear more important for the detoxification of natural product drugs such as VP-16(45) , at least in the liver, where glucuronidation is a major biotransformation pathway for steroid hormones and bile salts. Our data demonstrate that some glucuronide conjugates are potential substrates for MRP.
The rates of ATP-dependent transport of
E17
G by membrane vesicles from MRP-transfected HeLa
T14 cells were more than 20-fold higher than vesicles from cells
transfected with vector alone. In addition, the rates of
E
17
G transport obtained with vesicles from previously
characterized H69, H69AR, and H69PR small cell lung cancer cell lines
correlate well with their levels of MRP(1, 34) .
Several other lines of evidence confirm that MRP is the primary active
transporter involved. 1) A conformation dependent, MRP-specific mAb
that inhibits LTC
transport by MRP(55) , also
inhibits E
17
G transport; 2) E
17
G and
LTC
compete for ATP-dependent transport by MRP enriched
vesicles; and 3) E
17
G blocks the photolabeling of MRP
by [
H]LTC
in a
concentration-dependent manner. Thus LTC
and
E
17
G appear to interact with similar or at least
mutually exclusive sites on MRP.
Despite the ability of MRP to
transport anionic compounds with no apparent structural similarity,
studies with steroid conjugates reported here indicate that substrate
affinity can be markedly influenced by the site of anionic conjugation.
In human liver, E17
G and E
3
G are
formed in approximately equal amounts(46, 47) .
However, only the former is cholestatic. It has been suggested that one
mechanism by which some estrogen D-ring conjugates may diminish bile
flow is by competing for transport by one or more of the ATP-dependent
hepatocanalicular transport proteins. Whether or not MRP is the
transporter involved remains to be firmly established. However, the
substrate specificity of MRP correlates well with the cholestatic
potential of A-ring and D-ring steroid glucuronides. Non-cholestatic
E
3
G does not inhibit
[
H]LTC
transport by MRP nor does it
inhibit photolabeling of the protein. Similarly, E
3
G
and E
3
G do not compete for
[
H]E
17
G transport. Thus A-ring
glucuronidation is insufficient to result in detectable interaction
with MRP. Alternative forms of anionic modification of the A-ring, such
as sulfation, also have little effect on the inhibitory potency of
17-(
-D-glucuronides) (e.g. E
3SO
17
G has a K
of 1.7 µM with respect to E
17
G
transport). In contrast, sulfation of the A-ring of bile salts may
enhance inhibitory potency, since glycocholic acid itself is a
relatively weak inhibitor of E
17
G transport (30%
inhibition at 10 µM) compared with the cholestatic bile
salt glycolithocholate-3-sulfate (approximately 95% inhibition at 10
µM). More surprising, given the major structural
differences between some MRP substrates, is the finding that inhibitory
potency is sensitive to the position of glucuronidation within the
D-ring itself. The K
of the moderately cholestatic
glucuronide, E
16
G, is more than 20-fold higher than
that of E
17
G. These studies demonstrate that the
protein can be highly selective with respect to the site of anionic
conjugation of some substrates and provide information of potential use
in designing agents capable of blocking MRP function. The behavior of
the synthetic estrogen, 17
-ethinyl-17
-estradiol is an
exception to the correlation between cholestatic potential and the
ability to inhibit MRP-dependent transport. This compound does not
inhibit transport of E
17
G in vitro but is
cholestatic. However, it is thought that the glucuronide of this
synthetic estrogen, produced in the liver, rather than the parent
compound is responsible for the cholestasis(33, 48) .
It has been suggested previously that ATP-dependent
hepatocanalicular transport of E17
G is attributable to
P-glycoprotein(32) . This suggestion stems from studies with
two drug-selected cell lines known to overexpress P-glycoprotein, which
displayed approximately 5-fold increased resistance to cytotoxic
concentrations of E
17
G and a 2-3-fold decrease
in accumulation of the compound. We have found that
E
17
G transport in vesicles from the P-glycoprotein
overexpressing myeloma cell line, 8226/Dox40, is approximately 20- and
100-fold lower than in vesicles from MRP transfected T14 and
drug-selected H69AR cells, respectively. Moreover, the drug-resistant
myeloma cells also express low but sufficient levels of MRP to account
for the low level of E
17
G transport observed. These
data combined with recent reports of multidrug-resistant cell lines
that express elevated levels of both P-glycoprotein and MRP (8, 49, 50) suggest that MRP rather than
P-glycoprotein is responsible for the previously observed transport of
E
17
G(32) .
Direct binding and transport of
chemotherapeutic drugs by MRP has not been demonstrated, but
30-60% decreases in MRP-dependent LTC transport have
been observed in high concentrations (approximately 100
µM) of certain chemotherapeutic agents (15, 55) . With some drugs (e.g. vincristine
and vinblastine), we have shown that inhibition is potentiated by
physiological concentrations of GSH and have demonstrated
ATP/GSH-dependent transport of vincristine using T14
vesicles(55) . Data presented here demonstrate similar
inhibition of E
17
G transport with several
chemotherapeutic agents and potentiation of inhibition by vincristine
in the presence of GSH. A possible explanation of this behavior is that
inhibition and/or transport is enhanced as a result of independent
interactions of GSH and the Vinca alkaloids with distinct
regions of a composite binding site on MRP. Consequently, we determined
whether the anionic substituents of known MRP substrates, other than
GSH, might act in a similar fashion. However, no potentiation of
inhibition was observed in the presence of glucuronate with any of the
drugs tested and the anion also failed to enhance inhibition
E
17
G transport by 17
-estradiol. Inorganic sulfate
was also without effect (data not shown). Thus the ability to enhance
inhibition and/or transport appears to be GSH-specific, rather than a
general property of the known anionic substituents of MRP substrates.
In addition, although all drugs tested inhibited E
17
G
and LTC
transport, their relative potencies differ. For
example, daunorubicin is the most potent inhibitor of
E
17
G transport while it is the least potent inhibitor
of LTC
transport(51, 55) . Similar
differences in the relative potency of various inhibitors have been
observed previously in the transport and/or binding of different
substrates by P-glycoprotein(52, 53) . One
interpretation is that interaction of potential substrates or
inhibitors with both proteins may occur via multiple, overlapping but
not identical sites.
Data presented here combined with those of previous studies demonstrate that the substrate specificities of MRP and MOAT overlap extensively (27, 21, 40, 54) . Our studies on the transport of steroid glucuronides are also consistent with a potential role for MRP in bile formation and in the development of cholestasis. However, it remains to be established whether MOAT is a single protein or whether functionally similar but structurally distinct tissue-specific forms exist. With detailed knowledge of the substrate specificities of MRP and the availability of mAbs capable of specifically inhibiting its function, it should soon be possible to determine whether MRP and MOAT are the same protein or different functionally related transporters.