JBC GenomeOne product landing page

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M104833200 on July 10, 2001

J. Biol. Chem., Vol. 276, Issue 36, 33747-33754, September 7, 2001
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
276/36/33747    most recent
M104833200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Chen, Z.-S.
Right arrow Articles by Kruh, G. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, Z.-S.
Right arrow Articles by Kruh, G. D.

Transport of Cyclic Nucleotides and Estradiol 17-beta -D-Glucuronide by Multidrug Resistance Protein 4

RESISTANCE TO 6-MERCAPTOPURINE AND 6-THIOGUANINE*

Zhe-Sheng ChenDagger§, Kun LeeDagger, and Gary D. Kruh||

From the Medical Science Division, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

Received for publication, May 26, 2001, and in revised form, June 26, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Human multidrug resistance protein 4 (MRP4) has recently been determined to confer resistance to the antiviral purine analog 9-(2-phosphonylmethoxyethyl)adenine and methotrexate. However, neither its substrate selectivity nor physiological functions have been determined. Here we report the results of investigations of the in vitro transport properties of MRP4 using membrane vesicles prepared from insect cells infected with MRP4 baculovirus. It is shown that expression of MRP4 is specifically associated with the MgATP-dependent transport of cGMP, cAMP, and estradiol 17-beta -D-glucuronide (E217beta G). cGMP, cAMP, and E217beta G are transported with Km and Vmax values of 9.7 ± 2.3 µM and 2.0 ± 0.3 pmol/mg/min, 44.5 ± 5.8 µM and 4.1 ± 0.4 pmol/mg/min, and 30.3 ± 6.2 µM and 102 ± 16 pmol/mg/min, respectively. Consistent with its ability to transport cyclic nucleotides, it is demonstrated that the MRP4 drug resistance profile extends to 6-mercaptopurine and 6-thioguanine, two anticancer purine analogs that are converted in the cell to nucleotide analogs. On the basis of its capacity to transport cyclic nucleotides and E217beta G, it is concluded that MRP4 may influence diverse cellular processes regulated by cAMP and cGMP and that its substrate range is distinct from that of any other characterized MRP family member.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The multidrug resistance protein (MRP)1 family of ATP-binding cassette transporters first came to light as a result of the identification in drug-resistant cell lines of the Mr 190,000 protein product and cDNA of its founding member, MRP1 (1-3). Based upon the determination of complete coding sequences and putative topologies, this family is now known to consist of at least seven members (4). The substrate selectivities and drug resistance profiles of several of these pumps have been determined. MRP1, MRP2 (cMOAT), and MRP3 (MOAT-D), which confer resistance to certain natural product agents and methotrexate (5-14), are the best characterized family members. These three transporters are lipophilic anion pumps whose substrates include glutathione S-conjugates, such as leukotriene C4 (LTC4) and S-(2,4-dinitrophenyl)glutathione (DNP-SG), and glucuronate conjugates such as estradiol 17-beta -D-glucuronide (E217beta G) (10, 11, 15-22). However, whereas MRPs 1-3 have similar substrate ranges, they subserve distinct physiological functions. MRP1 is distinguished from MRP2 and MRP3 by its higher affinity for LTC4, a feature that is reflected in the specific role it plays in mediating immune responses involving cellular export of this cysteinyl leukotriene (23, 24). By contrast with MRP1, which is ubiquitously expressed and localized at basolateral surfaces of polarized cells (25-27), MRP2 is primarily expressed in the hepatocyte canaliculus where it functions as an apical efflux pump for organic anions such as bilirubin glucuronide and in provision of the biliary fluid constituent glutathione (28). MRP3 is also a glutathione and glucuronate conjugate pump but has the additional capability of mediating the transport of monoanionic bile acids (22, 29). This substrate selectivity, together with its induction at basolateral surfaces of hepatocytes under cholestatic conditions (30-32), has led to the notion that it functions as a compensatory backup mechanism to eliminate from hepatocytes potentially toxic compounds that are ordinarily excreted into the bile.

Two members of the MRP family, MRP4 (MOAT-B) and MRP5 (MOAT-C), are no more related to each other than they are to MRPs 1-3 in terms of degree of amino acid identity, but they are structurally distinct from the latter proteins in that MRPs 4 and 5 do not possess a third (N-terminal) membrane spanning domain (4, 33, 34). The topological dissimilarity of MRP5 is reflected in its distinct drug resistance capabilities and substrate selectivity. By contrast with MRPs 1-3, MRP5 is not known to confer resistance to natural product anticancer agents or methotrexate, but instead it has the facility for conferring resistance to purine analogs (35, 36). Similarly, membrane vesicle transport assays suggest that glutathione and glucuronate conjugates are not substrates of MRP5. Instead it is able to transport cyclic nucleotides (37).

The functional characteristics of MRP4, the other MRP family member that lacks an N-terminal membrane spanning domain, have yet to be defined in any detail. The drug resistance capabilities of MRP4 have been assessed to some degree in transfected NIH3T3 cells and in a drug-selected cell line in which MRP4 is overexpressed (38, 39). These studies indicate that MRP4 has the facility for conferring resistance to the antimetabolite methotrexate and the antiviral purine analog 9-(2-phosphonylmethoxyethyl)adenine (PMEA). However, almost nothing is known about its in vitro transport properties or physiological functions. In the present report we begin to address these questions by the analysis of MRP4-mediated transport in membrane vesicles prepared from MRP4-enriched insect cells. In so doing it is demonstrated that MRP4, like MRP5, catalyzes the MgATP-energized transport of cGMP and cAMP. However, by contrast with MRP5, MRP4 is also able to transport the glucuronide E217beta G and is a higher affinity transporter of cAMP. In addition, it is shown that the resistance profile of MRP4 extends to include anticancer purine analogs. These findings indicate that the substrate range of MRP4 is distinct from all other characterized MRPs and have important implications regarding the cellular physiology of cyclic nucleotides and cellular resistance mechanisms associated with commonly used anticancer purine analogs.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and Cell Lines-- [3H]cGMP (6.8 Ci/mmol), [3H]cAMP (21.9 Ci/mmol), and [14C]6-mercaptopurine (6-MP) (54 mCi/mmol) were purchased from Moravek Biochemicals (Brea, CA). 2-Deoxycoformycin (DCF) was kindly provided by Supergen Pharmaceutical Research Institute (Pleasanton, CA). [3H]E217beta G (44 Ci/mmol), [Gly-2-3H]glutathione (44.8 Ci/mmol), and [3H]LTC4 (130 Ci/mmol) were purchased from PerkinElmer Life Sciences. Creatine phosphokinase, creatine phosphate, ATP, AMP, 6-MP, 2-mercaptopurine (2-MP), 6-thioguanine (6-TG), 2-chlorodeoxyadenosine (CDA), E217beta G, LTC4, cGMP, and cAMP were purchased from Sigma. DNP-SG and [3H]DNP-SG were synthesized from 1-chloro-2,4-dinitrobenzene and unlabeled or labeled [3H]glutathione, respectively, as described previously (40). The MRP4-transfected NIH3T3 cell line (3T3/MRP4-3) and NIH3T3 cells transfected with parental vector (3T3/pSRalpha ) were described previously (38). NIH3T3 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% calf serum, penicillin/streptomycin, and glutamine. Insect cells (Sf9) were cultured and infected with MRP4 baculovirus as described previously (38). Monoclonal antibody directed against MRP4 was described previously (38).

Immunoblot Analysis-- Membrane vesicles preparations were analyzed by 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as described previously (41). Proteins were transferred to nitrocellulose filters using a wet transfer system as described previously (42). MRP4 was detected using monoclonal MRP4 antibody (1:2000) and alkaline phosphatase-conjugated secondary antibody.

Analysis of Drug Sensitivity-- Drug sensitivity was analyzed by use of a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt/phenazine methosulfate (MTS/PMS) microtiter plate assay (CellTiter 96 Cell Proliferation Assay; Promega Corp., Madison, WI). Control and MRP4-transfected cells were seeded in triplicate at 5,000 cells/well in 96-well dishes in DMEM supplemented with 10% calf serum. The next day drugs at various concentrations were added to the growth medium. Growth assays were performed after 72 h of growth in the presence of drug.

Preparation of Membrane Vesicles and Transport Experiments-- Membrane vesicles were prepared by the nitrogen cavitation method as described previously (43). Transport experiments were performed using the rapid filtration method essentially as described previously (16). Transport experiments were carried out in medium containing membrane vesicles (10 µg), 0.25 M sucrose, 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 4 mM ATP, 10 mM phosphocreatine, 100 µg/ml creatine phosphokinase, and radiolabeled substrate ± unlabeled substrate, in a total volume of 50 µl. Reactions were carried out at 37 °C and stopped by the addition of 3 ml of ice-cold stop solution (0.25 M sucrose, 100 mM NaCl, 10 mM Tris-HCl, pH 7.4). Samples were passed through 0.22-µm Durapore membrane filters (Millipore, Bedford, MA) under vacuum. The filters were washed 3 times with 3 ml of ice-cold stop solution and dried at room temperature for 30 min. Radioactivity was measured by the use of a liquid scintillation counter. Rates of net ATP-dependent transport were determined by subtracting the values obtained in the presence of 4 mM AMP from those obtained in the presence of 4 mM ATP. Uptake rates were linear for up to 5 min, and rates for concentration dependence experiments were measured at 5 min.

Drug Accumulation and Efflux-- For accumulation experiments subconfluent 3T3/pSRalpha and 3T3/MRP4-3 cells seeded in triplicate in 100-mm plastic dishes were incubated overnight in DMEM growth medium. After growth overnight the cells were incubated at 37 °C with 10 µM [14C]6-MP for 10, 30, and 60 min. Cells were washed 3 times with cold PBS and immediately harvested by trypsinization. The cells were washed 2 times with cold PBS, and an aliquot was used to count cell number. Radioactivity was measured by the use of a liquid scintillation counter. For efflux experiments, subconfluent 3T3/pSRalpha and 3T3/MRP4-3 cells seeded in triplicate in 100-mm plastic dishes were incubated overnight in DMEM growth medium. The next day the cells were washed and incubated for 2 h in energy depletion medium (glucose-free, pyruvate-free DMEM containing 10% dialyzed calf serum, 10 mM deoxyglucose, and 10 mM sodium azide) containing 10 µM [14C]6-MP. The cells were then washed 3 times with a total volume of 20 ml of PBS, and the medium was replaced with ordinary growth medium without radiolabeled drug. The cells were incubated at 37 °C, and at various time points medium was collected for measuring radioactivity. Cell-associated radioactivity was counted at the end of the 2-h incubation in energy depletion medium containing radiolabeled drug and after 2 h of efflux.

Data Analysis-- Kinetic parameters were computed by nonlinear least squares analysis (44) using the Ultrafit computer software (BioSoft, Ferguson, MO). For drug sensitivity experiments the nonparametric two-tailed Wilcoxon test was used to make inferences about the significance of differences in the IC50 values of MRP4-transfected and control cells.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Transport of cGMP, cAMP, and E217beta G by MRP4-- MRP4-dependent transport activity was assayed on density-fractionated membrane vesicles prepared from insect (Sf9) cells infected with MRP4 baculovirus. As determined by immunoblot analysis, these membranes are a rich source of MRP4 protein, which migrates as an Mr 150,000 electrophoretic species (Fig. 1). As observed previously (38) MRP4 expressed in insect cells, which are unable to synthesize complex N-linked oligosaccharide chains, migrates with a lower apparent molecular weight than the same recombinant protein expressed in NIH3T3 cells (Mr 170,000).


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 1.   Immunoblot detection of MRP4 in membrane vesicle preparations. Membrane vesicles were prepared from insect cells (Sf9) infected with MRP4 baculovirus (lane 1) or from uninfected insect cells (lane 2). Protein (5 µg/lane) was resolved by SDS-polyacrylamide gel electrophoresis on 7.5% gels, electrotransferred to nitrocellulose membranes, and incubated with monoclonal MRP4 antibody. The sizes of molecular weight standards (in kilodaltons) are indicated. The arrow indicates MRP4 protein.

Previous studies employing MRP4-transfected NIH3T3 cells and PMEA-selected cells indicate that the pump confers resistance to the antiviral purine analog PMEA (38, 39). The topological resemblance of MRP4 to MRP5 (4, 34), and the observation that MRP5, which also confers resistance to purine analogs including PMEA (36), is competent in the transport of cyclic nucleotides (37), suggested that cyclic nucleotides might also be transport substrates of MRP4. To explore this possibility, uptake of cGMP and cAMP into inside-out membrane vesicles prepared from insect cells infected with MRP4 baculovirus was examined. To assess the relative contribution of MRP4 to overall uptake, parallel experiments were also performed on membrane vesicles purified from uninfected insect cells.

Both cGMP and cAMP were indeed subject to MRP4-mediated, MgATP-dependent transport (Fig. 2, A and B). When measured at initial concentrations of 1.0 µM and at the 5-min time point of the assay, [3H]cGMP and [3H]cAMP were taken up by MRP4-enriched vesicles at rates of 1.42 and 0.49 pmol/mg/min, respectively, from media containing MgATP and at rates of less than 0.39 and 0.17 pmol/mg/min, respectively, from media containing MgAMP. By contrast, the rates of uptake of cGMP and cAMP, respectively, for membranes prepared from uninfected insect cells were consistently less than 0.39 and 0.17 pmol/mg/min, under either energized or nonenergized conditions.


View larger version (15K):
[in this window]
[in a new window]
  		Fig. 2.   Time course of ATP-dependent uptake of [3H]cGMP, [3H]cAMP, and [3H]E217beta G into membrane vesicles. Membrane vesicles (10 µg) prepared from insect cells infected with MRP4 baculovirus (circles) or uninfected insect cells (squares) were incubated at 37 °C in uptake media containing 1.0 µM [3H]cGMP (A), 1.0 µM [3H]cAMP (B), or 1.0 µM [3H]E217beta G (C). Closed symbols, uptake from media containing 4 mM MgATP; open symbols, uptake from media containing 4 mM MgAMP. Values shown are means ± S.E.

Glucuronate and glutathione conjugates are established substrates of MRPs 1-3, but not of MRP5 (37). To determine whether conjugates are substrates of MRP4, transport of E217beta G, DNP-SG, and LTC4, prototypical glucuronate and glutathione conjugates, were selected as model test compounds. Of these three compounds, robust uptake was observed only for E217beta G (Fig. 2C). When measured at initial concentrations of 1.0 µM and at the 5-min time point of the assay, [3H]E217beta G was taken up by MRP4-enriched membranes at a rate of 4.4 pmol/mg/min from media containing MgATP and at a rate of only 1.0 pmol/mg/min from media containing MgAMP. Uptake rates of less than 1.1 pmol/mg/min from media containing either MgATP or MgAMP were observed for membranes prepared from uninfected insect cells.

Transport of [3H]LTC4 and [3H]DNP-SG uptake was not consistently observed in that low levels of uptake of these compounds were detected in some but not all membrane vesicle preparations (data not shown).

Osmotic Sensitivity of [3H]cGMP Transport by MRP4-- The osmotic sensitivity of [3H]cGMP uptake was examined to confirm that radiolabel retained by MRP4-enriched membrane vesicles was largely attributable to transport of the substrate into the intravesicular compartment as opposed to nonspecific binding to the vesicles and/or filters. MgATP-dependent uptake of 1.0 µM cGMP increased as a linear function of the reciprocal of the sucrose concentration of the uptake medium, indicating that the transport substrate was delivered into an osmotically active compartment (Fig. 3). By contrast, the sucrose concentration exerted a moderate effect on substrate retention measured in medium containing MgAMP, suggesting that under nonenergized conditions an appreciable fraction of the apparent uptake measured represented binding to the membranes and/or filters. The magnitude of the ordinate intercepts indicated that nonspecific substrate binding constituted 18% of the radiolabel retained by MRP4-enriched membranes in media containing MgATP, but as much as 62% of the radioactivity retained in media containing MgAMP. MgATP-dependent uptake of [3H]cAMP and [3H]E217beta G was also osmotically sensitive (data not shown).


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 3.   Osmotic sensitivity of [3H]cGMP uptake by MRP4. Membrane vesicles (10 µg) prepared from insect cells infected with MRP4 baculovirus were preincubated in uptake medium containing 0.25-1.0 M sucrose for 5 min before measuring uptake of 1.0 µM [3H]cGMP at 37 °C in media containing 4 mM MgATP (closed symbols) or 4 mM MgAMP (open symbols). Uptake was measured at 5 min. Values shown are means ± S.E.

Kinetics of cGMP, cAMP, and E217beta G Uptake by MRP4-- The substrate concentration dependence of MgATP-energized [3H]cGMP, [3H]cAMP, and [3H]E217beta G uptake by membrane vesicles prepared from insect cells infected with MRP4 baculovirus approximated Michaelis-Menten kinetics. When measured over a broad range of substrate concentrations, the initial rates of MgATP-dependent uptake of both compounds, enumerated as the difference between uptake rates in media containing MgATP and uptake in media containing MgAMP, exhibited saturation kinetics (Fig. 4). Nonlinear least squares fitting of the data to the Michaelis-Menten equation for transport of cGMP, cAMP, and E217beta G yielded Km and Vmax values of 9.69 ± 2.3 µM and 2.01 ± 0.34 pmol/mg/min, 44.5 ± 5.8 µM and 4.14 ± 0.40 pmol/mg/min, and 30.3 ± 6.2 µM and 102 ± 16 pmol/mg/min, respectively (Table I). The efficiencies of transport fell in the rank order E217beta G (Vmax/Km = 3.4) > cGMP (0.21) > cAMP (0.09) (Table I).


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 4.   Concentration dependence of [3H]cGMP, [3H]cAMP, and [3H]E217beta G uptake by MRP4. The rates of MgATP-dependent uptake of [3H]cGMP (A), [3H]cAMP (B), and [3H]E217beta G (C) into membrane vesicles (10 µg) prepared from insect cells infected with MRP4 baculovirus were measured at 37 °C. Values shown (means ± S.E.) are rates measured in the presence of MgATP minus rates measured in the presence of MgAMP for triplicate determinations. Uptake rates were measured at 5 min. The lines of best fit and kinetic parameters were computed by nonlinear least squares analysis (44). Representative experiments are shown.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Summary of kinetic parameters for MRP4-mediated transport
Kinetic parameters were computed as described in the legend to Fig. 4 and under "Experimental Procedures." Values shown are means ± S.E. of at least three measurements performed in triplicate.

Inhibition of MRP4-mediated Transport of E217beta G-- These membrane vesicle experiments indicated that MRP4 is able to transport cGMP, cAMP, and E217beta G, and in a previous report (38) we inferred from drug sensitivity studies that MRP4 can transport methotrexate. Taken together these studies suggest that MRP4 is able to transport diverse amphipathic anions. To gain further insight into the substrate selectivity of MRP4, the ability of cGMP, cAMP, and methotrexate to inhibit transport was examined in experiments in which E217beta G was employed as the test substrate. As would be expected if these substrates were transported by a common mechanism, all three were capable of inhibiting E217beta G transport (Table II). cGMP was the most potent inhibitor (83.8% inhibition at 300 µM), consistent with its higher affinity by comparison with cAMP. The degree of inhibition exerted by methotrexate (59.7%) was comparable to that of cAMP (60.4%) at 300 µM concentrations but slightly higher at concentrations of 30 and 100 µM.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Inhibition of MRP4-mediated transport of E217beta G
Membrane vesicles prepared from MRP4-enriched Sf9 cells were incubated at 37 °C for 5 min in medium containing 1 µM [3H]E217beta G in the presence or absence of the indicated compounds. ATP-dependent uptake was calculated by subtracting values obtained in the presence of 4 mM MgATP from those in the presence of 4 mM MgAMP. Transport is expressed as percent of uptake in the absence of inhibitor. Values shown are means ± S.E. of at least three measurements performed in duplicate.

Sensitivity of MRP4-transfected NIH3T3 Cells to Anticancer Purine Analogs-- By having determined that the pump has the facility for MgATP-dependent transport of cyclic purine nucleotides, and knowing from previous studies (38, 39) that MRP4 confers resistance to the antiviral purine analog PMEA, an acyclic phosphonate that cannot be converted in the cell to a nucleoside phosphate (45), the involvement of MRP4 in resistance to anticancer purine analogs that are known to be metabolized in the cell to nucleotides was examined. For this purpose growth assays were performed on MRP4-transfected NIH3T3 cells (3T3/MRP4-3) and parental vector-transfected control cells (3T3/pSRalpha ) in the presence and absence of several agents. From these experiments it was determined that MRP4 is not only able to confer resistance to antiviral agents but also to anticancer purine analogs.

Three purine analogs, 6-MP, 2-MP, and 6-TG, and two purine nucleoside analogs, CDA and DCF, were selected for analysis. Of the agents examined, 3T3/MRP4-3 exhibited significantly higher levels of resistance by comparison with 3T3/pSRalpha for two of the three purine analogs (Fig. 5 and Table III). The MRP4-transfected cells exhibited 4.6-fold resistance for 6-MP and 2.7-fold resistance for 6-TG. A difference in sensitivity was also observed for 2-MP, but this value did not reach statistical significance. By contrast, MRP4 did not confer resistance to the two anticancer nucleoside analogs tested.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5.   Sensitivity of MRP4-transfected and parental vector-transfected NIH3T3 cells to 6-MP and 6-TG. The drug sensitivity of parental vector-transfected cells (3T3/pSRalpha , open symbols) or NIH3T3/MRP4-3 cells (closed symbols) were analyzed using the MTS/PMS assay as described under "Experimental Procedures." Values are means ± S.E. Representative experiments are shown.

                              
View this table:
[in this window]
[in a new window]
 
Table III
Drug sensitivity of MRP4-transfected NIH3T3 cell to purine analogs
The sensitivities of 3T3/pSRalpha and 3T3/MRP4-3 cells were measured as described in the legend to Fig. 5 and under "Experimental Procedures." The IC50 is the concentration at which growth is inhibited by 50%. Fold resistance is obtained by dividing the IC50 of 3T3/MRP4-3 by the IC50 of 3T3/pSRalpha .

Analysis of [14C]6-MP Accumulation and Efflux in MRP4-transfected NIH3T3 Cells-- To gain insight into the mechanism by which MRP4 confers resistance to purine analogs, accumulation and efflux of [14C]6-MP were analyzed in 3T3/MRP4-3 and 3T3/pSRalpha cells. From these experiments it was determined that expression of MRP4 is associated with reduced drug accumulation and enhanced drug efflux, as would be expected if resistance were based upon the operation of a plasma membrane efflux pump.

When incubated in media containing 10 µM [14C]6-MP, 3T3/MRP4-3 exhibited markedly reduced intracellular drug accumulation compared with 3T3/pSRalpha over the 1-h time course of the assay (Fig. 6A). By comparison with 3T3/pSRalpha , 3T3/MRP4-3 cells accumulated 64, 61, and 51% drug at 10, 30, and 60 min, respectively.


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 6.   Analysis of the cellular kinetics of [14C]6-MP in MRP4-transfected and parental vector-transfected NIH3T3 cells. A, accumulation of [14C]6-MP in MRP4-transfected (3T3/MRP4-3) and parental vector transfected (3T3/pSRalpha ) NIH3T3 cells. Cells were incubated in 10 µM [14C]6-MP and intracellular radioactivity measured at various time points. B, efflux of drug into medium. 3T3/MRP4 and 3T3/pSRalpha cells were incubated for 2 h in the presence of 10 µM [14C]6-MP under energy-depletion conditions as described under "Experimental Procedures," and the medium was changed to complete medium lacking drug. Efflux of radioactivity into the medium was then measured at various time points. C, intracellular radioactivity at the beginning and ending of the efflux experiment shown in B. Open columns and open circles, 3T3/pSRalpha ; striped bars and closed circles, 3T3/MRP4 cells. Values are means ± S.E.

To determine whether reduced accumulation was consequent upon enhanced drug efflux, extrusion of radiolabeled drug into the growth medium was measured over a 2-h time course. In order to perform this experiment under conditions in which intracellular drug levels were comparable in the two cell lines at the beginning of the assay, 3T3/MRP4-3 and 3T3/pSRalpha cells were first incubated in the presence of 10 µM [14C]6-MP under energy-depletion conditions for 2 h. Following this incubation period, the growth medium was replaced with complete medium lacking drug, and efflux of radiolabeled drug into the medium was measured (Fig. 6B). As anticipated, 3T3/MRP4-3 cells exhibited markedly enhanced drug efflux by comparison with the control cells. After 30 min the MRP4-transfected cells effluxed 1.8-fold more drug than the control cells, and this ratio was maintained throughout the subsequent course of the assay (1.8-fold at 2 h).

Examination of intracellular drug content at the beginning and ending of the efflux assay indicated that by contrast with the ability of MRP4 to diminish accumulation under ordinary growth conditions (Fig. 6A), under energy-depletion conditions drug accumulation in the two cell lines differed by no more than 6% (Fig. 6C). However, after 2 h of efflux in complete media intracellular drug in 3T3/MRP4-3 cells was 0.48-fold less than the 3T3/pSRalpha control cells (Fig. 6C). This value is in reasonably good agreement with the 1.8-fold increased level of drug effluxed into the medium by 3T3/MRP4-3 cells (Fig. 6B).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study the in vitro transport properties of human MRP4 were investigated to gain insight into its substrate selectivity and potential physiological functions. Cyclic nucleotides were selected as one class of target compounds because MRP4 has been determined previously to confer resistance to the antiviral nucleotide analog PMEA (38, 39) and because cyclic nucleotides have been established recently as transport substrates of an MRP family member (MRP5) whose protein topology resembles that of MRP4 (37). In agreement with these structural and drug resistance features, it was determined that MRP4 can indeed transport cGMP and cAMP. In addition, it is shown that cGMP is a higher affinity substrate of MRP4 than is cAMP, as is also the case for MRP5. However, whereas both transporters are able to mediate transport of cyclic nucleotides there are significant differences in their kinetic parameters. The affinity of MRP4 for cGMP (Km = 9.7 µM) is ~5-fold lower than that of MRP5 (Km = 2.1 µM) (37). By contrast, the affinity of MRP4 for cAMP (Km = 44.5 µM) is ~9-fold higher than that reported for MRP5 (Km = 379 µM). The markedly higher affinity of MRP4 for cAMP may be of considerable significance in view of the involvement of this signaling molecule in diverse regulatory processes.

Cellular efflux of cyclic nucleotides has been described in both prokaryotes and eukaryotes (46-49). Analyses employing a variety of cultured cells and membrane vesicle preparations have established that cyclic nucleotide efflux in mammals is energy-dependent and mediated by amphipathic anion transporters in that it can be blocked by inhibitors of organic anion pumps (46, 47, 50-63). The present study and that by Jedlitschky et al. (37) indicate that MRP4 and MRP5, respectively, are components of the previously described membrane efflux systems for these critical signaling molecules (Fig. 7). However, while these studies have identified molecular components of the export systems, the precise physiological roles of cyclic nucleotide efflux are not completely understood. A well defined role for cellular export of cyclic nucleotides is best established for the slime mold Dictyostelium discoideum, for which cAMP effluxed by solitary amoebae under low nutrient conditions acts both as a chemoattractant that mediates formation of multicellular aggregates and as a differentiation agent (64). In mammals it is thought that efflux of cyclic nucleotides subserves two functions. One proposed function is that it contributes to the modulation of cyclic nucleotide signaling by reducing intracellular levels of these second messengers. In support of this notion is the consistent observation that triggered elevations in intracellular cyclic nucleotide levels are associated with enhanced cellular efflux (52-55, 57). A second proposed function for efflux is in provision of extracellular cAMP involved in intercellular signaling. This idea is consistent with the detection of cAMP in a variety of extracellular fluids (65-67) and is also supported by characterizations of cellular activities attributed to extracellular cAMP and presumably mediated by proteins located in the plasma membranes of target cells (see for example Refs. 68-71). It might be expected that MRP4, as a result of its higher affinity for cAMP by comparison with MRP5, plays a more prominent role in modulating intracellular cAMP levels and in efflux of cAMP involved in intercellular signaling. On the other hand MRP5 might be a more potent factor in the modulation of intracellular cGMP levels. Detailed studies concerning the tissue-specific expression patterns of MRP4 and MRP5, which are currently understood primarily at the transcript level (33, 34, 38, 72), should provide further insights as to which of these pumps are deployed in specific situations.


View larger version (13K):
[in this window]
[in a new window]
  		Fig. 7.   Schematic diagram depicting the role played by MRPs in the cellular physiology of cyclic nucleotides. cGMP is synthesized by guanylyl cyclases located in the cytoplasm (sGC) and plasma membrane (GC) when triggered by nitric oxide (NO) or peptide ligands (L), respectively. cAMP is synthesized by adenylyl cyclases (AC) located in the cytoplasm (sAC) or associated with G protein-coupled receptors (GPCR) in the plasma membrane, when trigged by bicarbonate or peptide ligands (L), respectively. Cyclic nucleotides are enzymatically degraded by specific phosphodiesterases (PDEs) or extruded from the cell by an efflux system that includes MRP4 and MRP5.

Whereas the substrate selectivity of MRP4 is similar to that of MRP5 with regard to transport of cyclic nucleotides, our experiments indicate that there are also significant differences. By contrast with MRP5 (37), MRP4 is able to transport the glucuronide E217beta G. In this regard MRP4 is similar to MRPs 1-3, for which this compound is an established substrate. The affinity of E217beta G transport by MRP4 (Km = 30.3 µM) is comparable to the Km value we previously reported for human MRP3 (25.6 µM) (22). However, both MRP1 (Km = 1.5-2.5 µM) and MRP2 (Km = 7.2 µM) are higher affinity transporters of this substrate (10, 15, 17). In addition to the transport of E217beta G, the substrate range of MRP4 is distinct from MRP5 with regard to at least one other compound, namely methotrexate. Transport of this anionic antimetabolite was inferred previously from studies demonstrating that MRP4-transfected cells are resistant to and accumulate reduced amounts of this agent (38). In further support of the notion that methotrexate is an MRP4 substrate, it is demonstrated here that this agent can inhibit MRP4-mediated transport of E217beta G. As with E217beta G transport, MRP4-mediated transport of methotrexate is similar to MRPs 1-3 which are also able to confer resistance to and transport methotrexate (13, 14, 22).

In the present study we demonstrate that the drug resistance profile of MRP4 extends beyond the antiviral purine analog PMEA, an acyclic phosphonate, to include the commonly used anticancer purine analog 6-MP. It is further demonstrated that the pump functions to reduce intracellular levels of this agent by an energy-dependent efflux mechanism. However, we have not detected transport of 6-MP in membrane vesicle assays.2 It is therefore unlikely that 6-MP or 6-TG, both of which are uncharged purine base analogs, are direct substrates of MRP4. Rather, the facility of MRP4 for transporting cyclic nucleotides and E217beta G, both of which are amphipathic anions, suggests that the nucleotide metabolites of 6-MP and 6-TG, which are the toxic forms of these agents, are likely to be the anionic species effluxed by the pump. By contrast with 6-MP and 6-TG, PMEA is an amphipathic anion (45). Hence, in this case it is likely that either PMEA and/or its di- or triphosphorylated metabolites are direct substrates of MRP4. These notions concerning how MRP4 confers resistance to antiviral and anticancer purine analogs are supported by analyses of species effluxed from MRP4-overexpressing CEMr-1 cells treated with PMEA and MRP5-transduced cells treated with PMEA and 6-MP (13, 73).

In view of the fact that 6-MP and methotrexate are significant components of chemotherapeutic regimens used in the treatment of childhood leukemias, the ability of MRP4 to confer resistance to both of these antimetabolites is noteworthy. In this regard MRP4 is unique among characterized MRP family members that confer resistance to either methotrexate (MRPs 1-3) or 6-MP (MRP5) but not to both agents. Whether MRP4 or MRP5 contributes to clinical resistance associated with either of these agents remains to be determined.

    ACKNOWLEDGEMENTS

We thank Susan Walther for assistance with generation of MRP4 baculovirus and infection of insect cells, Hao Wang for assistance with statistical analyses, and Hongxie Shen for assistance with DNP-SG synthesis.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant CA73728 (to G. D. K.) and by an appropriation from the Commonwealth of Pennsylvania.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.

Dagger Contributed equally to this work.

§ Recipient of a Japan Research Foundation for Clinical Pharmacology award.

Recipient of National Institutes of Health Fellowship CA74518.

|| To whom correspondence should be addressed: Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. Tel.: 215-728-5317; Fax: 215-728-3603; E-mail: GD_Kruh@fccc.edu.

Published, JBC Papers in Press, July 10, 2001, DOI 10.1074/jbc.M104833200

2 Z.-S. Chen and G. D. Kruh, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: MRP, multidrug resistance protein (MRP1-MRP5, gene symbols ABCC1-ABCC5); LTC4, leukotriene C4; DNP-SG, S-(2, 4-dinitrophenyl)glutathione; E217beta G, estradiol 17-beta -D-glucuronide; MOAT, multispecific organic anion transporter (MOAT-B, MOAT-C, and MOAT-D are alternative names for MRP4, MRP5, and MRP3, respectively, and cMOAT is an alternative name for MRP2); 6-MP, 6-mercaptopurine; DCF, 2-deoxycorfomycin; 2-MP, 2-mercaptopurine; 6-TG, 6-thioguanine; CDA, 2-chlorodeoxyadenosine; DMEM, Dulbecco's modified Eagle's medium; MTS/PMS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt/phenazine methosulfate; PMEA, 9-(2-phosphonylmethoxyethyl)adenine; PBS, phosphate-buffered saline.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Marquardt, D., McCrone, S., and Center, M. S. (1990) Cancer Res. 50, 1426-1430
2. McGrath, T., Latoud, C., Arnold, S. T., Safa, A. R., Felsted, R. L., and Center, M. S. (1989) Biochem. Pharmacol. 38, 3611-3619
3. Cole, S. P., Bhardwaj, G., Gerlach, J. H., Mackie, J. E., Grant, C. E., Almquist, K. C., Stewart, A. J., Kurz, E. U., Duncan, A. M., and Deeley, R. G. (1992) Science 258, 1650-1654
4. Hopper, E., Belinsky, M. G., Zeng, H., Tosolini, A., Testa, J. R., and Kruh, G. D. (2001) Cancer Lett. 162, 181-191
5. Grant, C. E., Valdimarsson, G., Hipfner, D. R., Almquist, K. C., Cole, S. P., and Deeley, R. G. (1994) Cancer Res. 54, 357-361
6. Kruh, G. D., Chan, A., Myers, K., Gaughan, K., Miki, T., and Aaronson, S. A. (1994) Cancer Res. 54, 1649-1652
7. Breuninger, L. M., Paul, S., Gaughan, K., Miki, T., Chan, A., Aaronson, S. A., and Kruh, G. D. (1995) Cancer Res. 55, 5342-5347
8. Zaman, G. J., Flens, M. J., van Leusden, M. R., de Haas, M., Mulder, H. S., Lankelma, J., Pinedo, H. M., Scheper, R. J., Baas, F., Broxterman, H. J., and Borst, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8822-8826
9. Cole, S. P., Sparks, K. E., Fraser, K., Loe, D. W., Grant, C. E., Wilson, G. M., and Deeley, R. G. (1994) Cancer Res. 54, 5902-5910
10. Cui, Y., Konig, J., Buchholz, J. K., Spring, H., Leier, I., and Keppler, D. (1999) Mol. Pharmacol. 55, 929-937
11. Kawabe, T., Chen, Z. S., Wada, M., Uchiumi, T., Ono, M., Akiyama, S., and Kuwano, M. (1999) FEBS Lett. 456, 327-331
12. Zeng, H., Bain, L. J., Belinsky, M. G., and Kruh, G. D. (1999) Cancer Res. 59, 5964-5967
13. Kool, M., van der Linden, M., de Haas, M., Scheffer, G. L., de Vree, J. M., Smith, A. J., Jansen, G., Peters, G. J., Ponne, N., Scheper, R. J., Elferink, R. P., Baas, F., and Borst, P. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6914-6919
14. Hooijberg, J. H., Broxterman, H. J., Kool, M., Assaraf, Y. G., Peters, G. J., Noordhuis, P., Scheper, R. J., Borst, P., Pinedo, H. M., and Jansen, G. (1999) Cancer Res. 59, 2532-2535
15. Jedlitschky, G., Leier, I., Buchholz, U., Barnouin, K., Kurz, G., and Keppler, D. (1996) Cancer Res. 56, 988-994
16. Leier, I., Jedlitschky, G., Buchholz, U., Cole, S. P., Deeley, R. G., and Keppler, D. (1994) J. Biol. Chem. 269, 27807-27810
17. Loe, D. W., Almquist, K. C., Cole, S. P., and Deeley, R. G. (1996) J. Biol. Chem. 271, 9683-9689
18. Ito, K., Suzuki, H., Hirohashi, T., Kume, K., Shimizu, T., and Sugiyama, Y. (1998) J. Biol. Chem. 273, 1684-1688
19. Madon, J., Eckhardt, U., Gerloff, T., Stieger, B., and Meier, P. J. (1997) FEBS Lett. 406, 75-78
20. van Aubel, R. A., van Kuijck, M. A., Koenderink, J. B., Deen, P. M., van Os, C. H., and Russel, F. G. (1998) Mol. Pharmacol. 53, 1062-1067
21. Hirohashi, T., Suzuki, H., and Sugiyama, Y. (1999) J. Biol. Chem. 274, 15181-15185
22. Zeng, H., Liu, G., Rea, P. A., and Kruh, G. D. (2000) Cancer Res. 60, 4779-4784
23. Wijnholds, J., Evers, R., van Leusden, M. R., Mol, C. A., Zaman, G. J., Mayer, U., Beijnen, J. H., van der Valk, M., Krimpenfort, P., and Borst, P. (1997) Nat. Med. 3, 1275-1279
24. Robbiani, D. F., Finch, R. A., Jager, D., Muller, W. A., Sartorelli, A. C., and Randolph, G. J. (2000) Cell 103, 757-768
25. Kruh, G. D., Gaughan, K. T., Godwin, A., and Chan, A. (1995) J. Natl. Cancer Inst. 87, 1256-1258
26. Flens, M. J., Zaman, G. J., van der Valk, P., Izquierdo, M. A., Schroeijers, A. B., Scheffer, G. L., van der Groep, P., de Haas, M., Meijer, C. J., and Scheper, R. J. (1996) Am. J. Pathol. 148, 1237-1247
27. Evers, R., de Haas, M., Sparidans, R., Beijnen, J., Wielinga, P. R., Lankelma, J., and Borst, P. (2000) Br. J. Cancer 83, 375-383
28. Keppler, D., and Kartenbeck, J. (1996) Prog. Liver Dis. 14, 55-67
29. Hirohashi, T., Suzuki, H., Takikawa, H., and Sugiyama, Y. (2000) J. Biol. Chem. 275, 2905-2910
30. Hirohashi, T., Suzuki, H., Ito, K., Ogawa, K., Kume, K., Shimizu, T., and Sugiyama, Y. (1998) Mol. Pharmacol. 53, 1068-1075
31. Konig, J., Rost, D., Cui, Y., and Keppler, D. (1999) Hepatology 29, 1156-1163
32. Ortiz, D. F., Li, S., Iyer, R., Zhang, X., Novikoff, P., and Arias, I. M. (1999) Am. J. Physiol. 276, G1493-G1500
33. Lee, K., Belinsky, M. G., Bell, D. W., Testa, J. R., and Kruh, G. D. (1998) Cancer Res. 58, 2741-2747
34. Belinsky, M. G., Bain, L. J., Balsara, B. B., Testa, J. R., and Kruh, G. D. (1998) J. Natl. Cancer Inst. 90, 1735-1741
35. McAleer, M. A., Breen, M. A., White, N. L., and Matthews, N. (1999) J. Biol. Chem. 274, 23541-23548
36. Wijnholds, J., Mol, C. A., van Deemter, L., de Haas, M., Scheffer, G. L., Baas, F., Beijnen, J. H., Scheper, R. J., Hatse, S., De Clercq, E., Balzarini, J., and Borst, P. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 7476-7481
37. Jedlitschky, G., Burchell, B., and Keppler, D. (2000) J. Biol. Chem. 275, 30069-30074
38. Lee, K., Klein-Szanto, A. J., and Kruh, G. D. (2000) J. Natl. Cancer Inst. 92, 1934-1940
39. Schuetz, J. D., Connelly, M. C., Sun, D., Paibir, S. G., Flynn, P. M., Srinivas, R. V., Kumar, A., and Fridland, A. (1999) Nat. Med. 5, 1048-1051
40. Awasthi, Y. C., Garg, H. S., Dao, D. D., Partridge, C. A., and Srivastava, S. K. (1981) Blood 58, 733-738
41. Laemmli, U. K. (1970) Nature 227, 680-685
42. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354
43. Cornwell, M. M., Gottesman, M. M., and Pastan, I. H. (1986) J. Biol. Chem. 261, 7921-7928
44. Marquardt, D. W. (1963) J. Soc. Ind. Appl. Math. 11, 431-441
45. Pisarev, V. M., Lee, S. H., Connelly, M. C., and Fridland, A. (1997) Mol. Pharmacol. 52, 63-68
46. Brunton, L. L., and Mayer, S. E. (1979) J. Biol. Chem. 254, 9714-9720
47. Brunton, L. L., and Heasley, L. E. (1988) Methods Enzymol. 159, 83-93
48. Goldenbaum, P. E., and Hall, G. A. (1979) J. Bacteriol. 140, 459-467
49. Saier, M. H., Jr., Feucht, B. U., and McCaman, M. T. (1975) J. Biol. Chem. 250, 7593-7601
50. Heasley, L. E., Azari, J., and Brunton, L. L. (1985) Mol. Pharmacol. 27, 60-65
51. Steinberg, R. A., Steinberg, M. G., and van Daalen Wetters, T. (1979) J. Cell. Physiol. 100, 579-588
52. Billiar, T. R., Curran, R. D., Harbrecht, B. G., Stadler, J., Williams, D. L., Ochoa, J. B., Di Silvio, M., Simmons, R. L., and Murray, S. A. (1992) Am. J. Physiol. 262, C1077-C1082
53. Patel, M. J., Wypij, D. M., Rose, D. A., Rimele, T. J., and Wiseman, J. S. (1995) J. Pharmacol. Exp. Ther. 273, 16-25
54. Woods, M., and Houslay, M. D. (1991) Biochem. Pharmacol. 41, 385-394
55. Hamet, P., Pang, S. C., and Tremblay, J. (1989) J. Biol. Chem. 264, 12364-12369
56. Podevin, R. A., Boumendil-Podevin, E. F., Bujoli-Roche, J., and Priol, C. (1980) Biochim. Biophys. Acta 629, 135-142
57. Fehr, T. F., Dickinson, E. S., Goldman, S. J., and Slakey, L. L. (1990) J. Biol. Chem. 265, 10974-10980
58. Strewler, G. J. (1984) Am. J. Physiol. 246, C224-C230
59. Millul, V., Prie, D., Geniteau-Legendre, M., Verpont, M. C., Baudouin, B., and Ronco, P. M. (1996) Am. J. Physiol. 270, C1051-C1060
60. Schultz, C., Vaskinn, S., Kildalsen, H., and Sager, G. (1998) Biochemistry 37, 1161-1166
61. Sundkvist, E., Jaeger, R., and Sager, G. (2000) Biochim. Biophys. Acta 1463, 121-130
62. Vaskinn, S., Sundkvist, E., Jaeger, R., and Sager, G. (1999) Mol. Membr. Biol. 16, 181-188
63. Sager, G., Orbo, A., Pettersen, R. H., and Kjorstad, K. E. (1996) Scand. J. Clin. Lab. Invest. 56, 289-293
64. Janssens, P. M., and Van Haastert, P. J. (1987) Microbiol. Rev. 51, 396-418
65. Ashman, D. F., Lipton, R., Melicow, M. M., and Price, T. D. (1963) Biochem. Biophys. Res. Commun. 11, 330-334
66. Huang, C. L., Ives, H. E., and Cogan, M. G. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8015-8018
67. Broadus, A. E., Kaminsky, N. I., Hardman, J. G., Sutherland, E. W., and Liddle, G. W. (1970) J. Clin. Invest. 49, 2222-2236
68. Friedlander, G., Couette, S., Coureau, C., and Amiel, C. (1992) J. Clin. Invest. 90, 848-858
69. Kubler, D., Pyerin, W., Bill, O., Hotz, A., Sonka, J., and Kinzel, V. (1989) J. Biol. Chem. 264, 14549-14555
70. Wen, S. C., Chang, C. M., Reitherman, R. W., and Harding, B. W. (1985) Endocrinology 116, 935-944
71. Sorbera, L. A., and Morad, M. (1991) Science 253, 1286-1289
72. Kool, M., de Haas, M., Scheffer, G. L., Scheper, R. J., van Eijk, M. J., Juijn, J. A., Baas, F., and Borst, P. (1997) Cancer Res. 57, 3537-3547
73. Robbins, B. L., Connelly, M. C., Marshall, D. R., Srinivas, R. V., and Fridland, A. (1995) Mol. Pharmacol. 47, 391-397

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


This article has been cited by other articles:


Home page
 Mol. Pharmacol.Home page
C. D. Cropp, T. Komori, J. E. Shima, T. J. Urban, S. W. Yee, S. S. More, and K. M. Giacomini
Organic Anion Transporter 2 (SLC22A7) Is a Facilitative Transporter of cGMP
Mol. Pharmacol., April 1, 2008; 73(4): 1151 - 1158.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
Z. P. Lin, Y.-L. Zhu, D. R. Johnson, K. P. Rice, T. Nottoli, B. C. Hains, J. McGrath, S. G. Waxman, and A. C. Sartorelli
Disruption of cAMP and Prostaglandin E2 Transport by Multidrug Resistance Protein 4 Deficiency Alters cAMP-Mediated Signaling and Nociceptive Response
Mol. Pharmacol., January 1, 2008; 73(1): 243 - 251.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
V. Reichel, R. Masereeuw, J. J. M. W. van den Heuvel, D. S. Miller, and G. Fricker
Transport of a fluorescent cAMP analog in teleost proximal tubules
Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2007; 293(6): R2382 - R2389.
[Abstract] [Full Text] [PDF]

Home page
 Drug Metab. Dispos.Home page
A. J. Lickteig, C. D. Fisher, L. M. Augustine, L. M. Aleksunes, D. G. Besselsen, A. L. Slitt, J. E. Manautou, and N. J. Cherrington
Efflux Transporter Expression and Acetaminophen Metabolite Excretion Are Altered in Rodent Models of Nonalcoholic Fatty Liver Disease
Drug Metab. Dispos., October 1, 2007; 35(10): 1970 - 1978.
[Abstract] [Full Text] [PDF]

Home page
 Nephrol Dial TransplantHome page
N. A. Wolff, B. C. Burckhardt, G. Burckhardt, M. Oellerich, and V. W. Armstrong
Mycophenolic acid (MPA) and its glucuronide metabolites interact with transport systems responsible for excretion of organic anions in the basolateral membrane of the human kidney
Nephrol. Dial. Transplant., September 1, 2007; 22(9): 2497 - 2503.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
K. Takenaka, J. A. Morgan, G. L. Scheffer, M. Adachi, C. F. Stewart, D. Sun, M. Leggas, K. F.K. Ejendal, C. A. Hrycyna, and J. D. Schuetz
Substrate Overlap between Mrp4 and Abcg2/Bcrp Affects Purine Analogue Drug Cytotoxicity and Tissue Distribution
Cancer Res., July 15, 2007; 67(14): 6965 - 6972.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
D. Willoughby and D. M. F. Cooper
Organization and Ca2+ Regulation of Adenylyl Cyclases in cAMP Microdomains
Physiol Rev, July 1, 2007; 87(3): 965 - 1010.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. G. Belinsky, P. Guo, K. Lee, F. Zhou, E. Kotova, A. Grinberg, H. Westphal, I. Shchaveleva, A. Klein-Szanto, J. M. Gallo, et al.
Multidrug Resistance Protein 4 Protects Bone Marrow, Thymus, Spleen, and Intestine from Nucleotide Analogue-Induced Damage
Cancer Res., January 1, 2007; 67(1): 262 - 268.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
M. J. Zamek-Gliszczynski, K.-i. Nezasa, X. Tian, A. S. Bridges, K. Lee, M. G. Belinsky, G. D. Kruh, and K. L. R. Brouwer
Evaluation of the Role of Multidrug Resistance-Associated Protein (Mrp) 3 and Mrp4 in Hepatic Basolateral Excretion of Sulfate and Glucuronide Metabolites of Acetaminophen, 4-Methylumbelliferone, and Harmol in Abcc3-/- and Abcc4-/- Mice
J. Pharmacol. Exp. Ther., December 1, 2006; 319(3): 1485 - 1491.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
H. Sun, L. Liu, and K. S. Pang
Increased Estrogen Sulfation of Estradiol 17beta-D-Glucuronide in Metastatic Tumor Rat Livers
J. Pharmacol. Exp. Ther., November 1, 2006; 319(2): 818 - 831.
[Abstract] [Full Text] [PDF]

Home page