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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Functional Expression of the Multidrug Resistance Protein 1 in Microglia

Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada (S.D., R.B.); Division of Cellular and Molecular Biology, Toronto Western Research Institute, University Health Network, and Department of Physiology, University of Toronto, Toronto, Ontario, Canada (X.Z., L.S.); and New Agent and Innovative Therapy Program, Division of Hematology/Oncology, The Hospital for Sick Children, Toronto, Ontario, Canada (S.B.)

Received May 13, 2003; accepted June 11, 2003.

	Abstract

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Brain expression of the multidrug resistance proteins (MRPs), a collection of membrane-associated ATP-dependent efflux transporters, is poorly understood. Although several studies have examined the expression of these proteins within the brain barriers (i.e., the blood-brain barrier and choroid plexus), little information is available with respect to brain parenchyma cells such as microglia and astrocytes. Because microglia are the primary brain cells infected by the human immunodeficiency virus type 1 (HIV-1), MRP1 expression within microglia may contribute to lower brain accumulation of anti-HIV drugs. To examine the expression pattern of MRP1 within microglia, we performed reverse transcriptase-polymerase chain reaction analysis and Western blotting on a rat brain microglia cell line MLS-9, and in primary cultures of rat microglia. Both rat MRP1 (rMPR1) mRNA and protein were expressed in the cell line, as well as the primary cultures. We then characterized rMRP1-mediated transport properties in MLS-9 cells using [³H]vincristine, a known MRP1 substrate. Vincristine accumulation by monolayers of MLS-9 cells increased significantly in the presence of several well established MRP1 inhibitors (MK571, genistein, sulfinpyrazone, probenecid, and indomethacin), protease inhibitors, or the ATPase inhibitor sodium azide. In addition, vincristine accumulation was significantly modulated by altering the intracellular concentration of the reduced form of glutathione, further suggesting the involvement of rMRP1-mediated transport. These results provide strong evidence that the MRP1 protein is both expressed and functional in microglia cells. They also suggest that brain parenchyma can act as a "second" barrier to drug permeability and regulate brain distribution/accumulation of various xenobiotics, including protease inhibitors.

At present, nine MRP proteins ranging in size from 1,325 to 1,545 amino acids have been identified in humans (hMRP1–9). Amino acid comparisons indicate 30 to 56% homology between the various isozymes. Complementary DNAs for rat homologs of MRP1–6 have now been characterized (designated rMRP1–6) and exhibit 66 to 94% homology with their human counterparts. Most mammalian cells seem to express multiple forms of MRP, generally with one predominant isozyme (Borst et al., 1999).

Although MRP1 expression is most often associated with multidrug resistance to cancer chemotherapy agents, antiretroviral agents used in the treatment of human immunodeficiency virus-1 (HIV-1) infection may also interact with these proteins. Studies in MRP1-overexpressing cell lines (e.g., hMRP1-transfected LLC-PK1 kidney cell line; hMRP1-overexpressing CEM lymphocyte cell lines) and isolated capillaries from rat and pig brain indicate that the protease inhibitors saquinavir, ritonavir, nelfinavir, and indinavir can interact with MRP1 (Srinivas et al., 1998; Miller et al., 2000; Jones et al., 2001). Recently, Williams et al. (2002) provided direct evidence that saquinavir is a substrate for MRP1 and MRP2 in Madin-Darby canine kidney II-overexpressing hMRP1 and hMRP2 cell lines, respectively. Furthermore, the ability of MRP1 to limit antiretroviral drug exposure in the clinic has been confirmed in HIV-infected patients using flow cytometry (Meaden et al., 2002). Significantly lower accumulation of saquinavir and ritonavir in peripheral blood mononuclear cells was seen in patients whose cells had greater MRP1 expression (Meaden et al., 2002).

HIV-1 infection within the central nervous system causes a number of neurological and psychiatric disorders in a large proportion of infected patients (Koutsilieri et al., 2001). Symptoms range from mild cognitive impairment to frank acquired immunodeficiency syndrome dementia, a progressive neurological disorder characterized by motor and behavioral abnormalities. HIV-1 infection within the brain seems to be limited primarily to microglia, the immune cells of the brain (Bagasra et al., 1996; Epstein, 1998). Microglia act as a cellular reservoir for the virus and upon activation release a number of factors that lead to neurotoxicity (Epstein, 1998). Because suboptimal levels of anti-HIV-1 drugs likely contribute to the emergence of drug-resistant viral strains, effective eradication of the virus from the brain requires sufficient penetration of antiretroviral drugs into microglia. The presence of efflux transporters within the therapeutic target of HIV-1 in the brain (i.e., microglia) may be one mechanism by which antiretroviral resistance emerges in patients.

MRP1 was previously identified in multiple brain compartments of several animal species, including bovine blood-brain barrier endothelial cells, mouse blood-cerebrospinal barrier endothelial cells and rat astrocytes (Declèves et al., 2000; Wijnholds et al., 2000; Zhang et al., 2000). We previously characterized the cellular/subcellular localization and functional expression of P-gp in microglia and microvessel endothelial cells from rat brain (Lee et al., 2001b; Bendayan et al., 2002). Recently, rMRP1 transcripts were identified in cultured rat microglia cells (Ballerini et al., 2002; Hirrlinger et al., 2002); however, levels of rMRP1 protein were not determined and complete functional studies specific to the rMRP1 isoform were not performed. Thus, the objective of the present study was to confirm the expression of the rMRP1 protein and extensively characterize its transport properties in microglia.

	Methods and Materials

Top Abstract Methods and Materials Results Discussion References

 
Chemicals. [³H]Vincristine (2.5 Ci/mmol), [³H]digoxin (55 mCi/mmol), and ritonavir were purchased from Moravek Biochemicals (Brea, CA). Anti-actin (clone AC-40), L-buthionine-[S,R]-sulfoximine (BSO), digoxin, EDTA, indomethacin, genistein, probenecid, and sulfinpyrazone were purchased from Sigma-Aldrich (Oakville, ON, Canada). MK571 and indinavir were a kind gift from Merck Research Labs (West Point, PA). Saquinavir was kindly provided from Roche Diagnostics (Hertfordshire, UK). The monoclonal antibodies MRPr1 and M2III-6 were purchased from Kamiya Biomedical (Seattle, WA).

Cell Culture. The microglia cell line (MLS-9) was derived from neopallia of 2- or 3-day-old Wistar rat pups, as described previously (Zhou et al., 1998; Cayabyab and Schlichter, 2002). Briefly, microglia were isolated by enzymatic dissociation of tissue, and cultured in endotoxin-free medium (Invitrogen, Burlington, ON, Canada) for 10 to 12 days without feeding. Essentially pure microglia cultures (>98% as determined by isolectin B4 labeling) obtained from shaking the 10- to 12-day-old flasks were treated with colony stimulating factor-1 to increase proliferation. From these cultures microglia colonies arose after 2 to 6 weeks that continued to grow in the absence of added growth factors. The MLS-9 cell line was established from one of these colonies. The cell line displays morphological and phenotypical attributes characteristic of microglia, including pinocytosis of dyes (diI-acetylated low-density lipoprotein or lucifer yellow) and staining for several microglia cell markers: isolectin B4 (100% of cells), OX-42 antibody (98%), and ED-1 antibody (99%). The cells were negative for characteristic markers of astrocytes (glial fibrillary acidic protein) and fibroblasts (fibronectin) (Zhou et al., 1998).

The MLS-9 cells (passages 4–32) were grown as monolayers on 75-cm² polystyrene tissue culture flasks or multiwell dishes (Sarstedt, St. Leonard, QC, Canada) at 37°C, in 5% CO₂, 95% air and 95% humidity. Cells were grown to confluence (4–6 days after seeding) in minimal essential medium (pH 7.2) containing 2 mM L-glutamine, 5.55 mM D-glucose, 5% fetal bovine serum, 5% horse serum, and 0.5% penicillin/streptomycin. For passaging, subconfluent MLS-9 cells (75% confluent) were released from the flask with a 15 mM sodium citrate solution containing 130 mM NaCl, 10 mM D-glucose, and 10 mM HEPES, pH 7.4.

The human MRP1-overexpressing cell line (WT-MRP1) and parent cell line (VF-HeLa) were kindly provided by Dr. S. P. C. Cole (Queen's University, Kingston, ON, Canada). The WT-MRP1 is a HeLa cell line, which is stably transfected with a pRc/CMV vector containing the complete coding sequence of hMRP1. The cells were maintained in Dulbecco's modified Eagle's medium containing 4 mM L-glutamine, 25 mM glucose, 400 µg/ml G418 with 10% fetal bovine serum, (pH 7.1) and subcultured weekly using 0.25% trypsin.

A human P-gp-overexpressing cell line (CH^RC5), originally derived from a colchicine-resistant Chinese hamster ovary cell line, was a kind gift of Dr. V. Ling (University of British Columbia, Vancouver, BC, Canada). The cells were maintained in minimal essential medium- containing 2 mM L-glutamine, 5.55 mM D-glucose, 10% fetal bovine serum, with 0.5% penicillin/streptomycin, pH 7.2, and subcultured weekly using 0.25% trypsin. We have previously confirmed through Western blotting analysis overexpression of P-gp in this cell line, compared with MLS-9 (Lee et al., 2001b).

RT-PCR Analysis. RT-PCR was performed as described previously by our group (Lee et al., 2001b). Total RNA from MLS-9 cells was isolated using TRIzol reagent (Invitrogen, Burlington, ON, Canada). We used a cDNA reaction mixture containing 0.5 µg of pd(N)₆ and 1 µg of RNA, 50 mM Tris-HCl buffer, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mM dithiothreitol, 0.5 mM dNTPs, and 200 units of Superscript II reverse transcriptase. Then 2 µl of cDNA was amplified by PCR using a GeneAmp PCR 2400 system (PerkinElmer Life Sciences, Mississauga, ON, Canada) in 20 µl of reaction mixture containing 1 unit of Platinum Taq polymerase, 1x PCR buffer, 1.5 mM MgCl₂, 0.2 mM dNTPs, and 0.8 µM forward and reverse primers. The sequences of rMRP1 primers were: forward primer 5'-AGAAGCAGCGTGTGAGCCT-3' and reverse primer 5'-GCCAATAGTTAGAAGCC AGTGC-3'. The RT-PCR program consisted of 35 cycles of three phases: denaturing (20 s at 94°C), annealing (30 s at 60°C), and extension (30 s at 72°C). Amplified DNA products were resolved on a 1.5% agarose gel with 0.5 mg/ml ethidium bromide. The RT-PCR product was purified and sequenced (ACGT Corp., Toronto, ON, Canada). All reagents were from Invitrogen.

Western Blotting. Crude membrane proteins were obtained as follows: primary cultures of microglia, MLS-9, and WT-MRP1 cells were harvested by centrifugation (400g), the pellet collected, and cells lysed for 30 min at 4°C in a 250 mM sucrose buffer containing 1 mM EDTA and protease inhibitor cocktail (P8340; Sigma-Aldrich). Adult rat kidney tissue was cut into small pieces and washed several times in phosphate-buffered saline and homogenized using a polytron homogenizer (Kinematica, Basel, Switzerland). The resulting lysate was centrifuged at 400g to remove any blood cells and debris and then incubated on ice with lysis buffer (as described above) for 30 min. Lysates from cells and tissues were then homogenized in a Dounce homogenizer at 10,000 rpm for three cycles of 20 s each. Homogenates were centrifuged at 3,000g to remove cellular debris, and the supernatant was further centrifuged at 100,000g for 1 h at 4°C. The resulting membrane pellet was resuspended in a 10 mM Tris-HCl suspension buffer, pH 8.8, and frozen at –20°C until use.

Crude membrane samples (12.5–200 µg) were separated on a 10% polyacrylamide gel using a minigel system (Bio-Rad, Hercules, CA) and transferred electrophoretically to polyvinylidene difluoride membranes. Protein transfer was verified using Ponceau S staining. The membranes were blocked overnight at 4°C in Tris-buffered saline (15 mM Tris-HCl, and 150 mM NaCl, pH 7.6) containing 0.05% Tween 20 (TBS-T) and 5% dry skim milk powder. After three washes (5 min each) with TBS-T, the membranes were incubated at room temperature with either the monoclonal MRP1 antibody MRPr1 at a dilution of 1:1,000 in 5% milk in TBS-T for 4 h for MRP1 detection, or the actin antibody AC-40 (1:500; 5% milk) for actin detection. After a second wash, the membranes were then incubated for 2 h in the presence of either anti-mouse (1:5,000) or anti-rat (1:5000) horse-radish peroxidase-linked secondary antibodies (Sigma-Aldrich) in 5% milk in TBS-T. Proteins were visualized using enhanced chemiluminescence according to the manufacturer's instructions (Pierce Chemical, Rockford, IL). Densitometric analysis was performed on the immunoreactive bands using Kodak ID image analysis software version 2.0.1 (Eastman Kodak, Rochester, NY). Expression of MRP2 was determined in a similar manner using the M2III-6 (1:2,000) antibody and an anti-mouse horseradish peroxidase-linked secondary antibody (1:5,000).

[³H]Vincristine and [³H]Digoxin Transport Studies. MLS-9, WT-MRP1, VF-HeLa, and CH^RC5 cells were plated onto multiwell plates and uptake/accumulation of radiolabeled vincristine (30 nM) or digoxin (100 nM) was measured as described previously by our laboratory (Lee et al., 2001b). Briefly, cells were washed once and preconditioned for 30 min at 37°C with Earle's Balanced Salt Solution (EBSS), containing 1.8 mM CaCl₂, 5.4 mM KCl, 0.8 mM MgSO₄, 138 mM NaCl, 1.0 mM Na₂HPO₄, 5.5 mM D-glucose, and 20 mM HEPES, pH 7.4. Cells were then incubated for the desired time with transport medium containing [³H]vincristine (30 nM) or digoxin (100 nM) with or without an inhibitor. At completion of the uptake/accumulation period, the transport medium was removed by aspiration and uptake was terminated by adding ice-cold 0.16 M NaCl. Cells were solubilized for 30 min with 1 M NaOH and then transferred to scintillation vials containing 2 N HCl and scintillation cocktail (Beckman Coulter, Inc., Fullerton, CA). Cellular incorporation of the radiolabeled probe was measured using liquid scintillation counting. The sample counts were corrected for "zero time" uptake in each individual experiment, and data were standardized to the amount of cell protein (milligrams per milliliter) in each culture plate determined by the Bradford colorimetric method (Bio-Rad) with bovine serum albumin (Sigma-Aldrich) as the standard.

Efflux of [³H]vincristine was assessed as described previously, but with a few modifications (Lee et al., 2001b). After preincubation with EBSS-transport buffer, cells were incubated with 30 nM [³H]vincristine for 1 h. After this initial incubation, the reaction was stopped by addition of 1 ml of ice-cold 0.16 M NaCl. The amount of intracellular vincristine accumulated at this time was designated as the "initial value" at time 0. Cells were then incubated for the specified time using either transport buffer, or transport buffer with added inhibitor. At the appropriate time, the reaction was stopped with ice-cold 0.16 M NaCl and the cells were solubilized. Intracellular radioactivity remaining in the cells was quantified as described above.

The energetics of rMRP1-mediated transport of vincristine was investigated in MLS-9 cells using the ATPase inhibitor sodium azide. Accumulation studies were carried out as described previously, with a few modifications. The MLS-9 cells were preincubated in either EBSS containing 5.5 mM D-glucose or EBSS containing equimolar amounts of mannitol. Accumulation of [³H]vincristine was then assessed in EBSS (control cells) or in transport buffer containing 5.5 mM mannitol and 10 mM sodium azide.

The modulatory effects of the reduced form of glutathione (GSH) on vincristine or digoxin transport were studied by either depleting or augmenting intracellular GSH concentrations. GSH was depleted using the GSH-synthesis inhibitor BSO, which is a standard method for depleting cellular GSH (Anderson, 1998). Cells were incubated with 25 µM BSO for 24 h at 37°C, 5% CO₂, 95% air and 95% humidity. Accumulation studies were then conducted as described above.

Several approaches have been used in whole cell transport studies to increase intracellular GSH concentrations, including using GSH pro-drugs (i.e., N-acetylcysteine, GSH ethylester) or GSH precursors, such as cysteine or methionine (Anderson, 1998). Since the extent of de novo synthesis of GSH in MLS-9 cells is not known, direct use of GSH precursors was not considered. Furthermore, in macrophages, cells that are derived from the same precursors as microglia, adding GSH itself increases intracellular GSH slightly more than does GSH ethyl ester (Minhas and Thornalley, 1995), possibly because GSH is imported intact through a bidirectional GSH transporter. Therefore, for transport experiments involving augmentation of GSH, MLS-9 cells were incubated with EBSS medium containing [³H]vincristine (30 nM) and 5 mM GSH.

HPLC Analysis. Intracellular concentrations of GSH after GSH depletion (BSO-treated) or augmentation (GSH-treated) were determined using a slightly modified HPLC method previously described and validated by our group (Stempak et al., 2001). MLS-9 and CH^RC5 cells were cultured to 90% confluence in 75-cm² tissue culture flasks as described above. After a 24-h incubation with either fresh medium (control) or medium containing 25 µM BSO (GSH depletion), cells were washed and incubated with EBSS transport buffer. Similarly, augmentation of intracellular GSH was determined in MLS-9 in the presence and absence of 5 mM GSH. At the conclusion of the incubation periods, cells were washed twice with Hanks' balanced salt solution (Invitrogen, Carlsbad, CA) and removed from the flask surface using 15 mM sodium citrate. Cells were counted using a hemocytometer, solubilized with 15% perchloric acid containing 2 mM EDTA, and immediately assayed by HPLC.

Intracellular GSH was measured using a Shimatzu HPLC system that consisted of a SIL-6B autoinjector, LCAT pump, and SCL-6B system controller (Mantech, Guelph, ON, Canada). Isocratic elution was performed on a Prodigy C-18 ODS column (5 µm particles; 150 x 4.6 mm) using a mobile phase of 10 mM NaH₂PO₄·H₂O, 0.03 mM octane sulfonic acid, and 2% acetonitrile, pH 2.7, at a flow rate of 1 ml/min. The detector was a porous graphite, dual-electrode electrochemical detector (model 5010; ESA, Bedford, MA), which was connected to a porous graphite guard cell (ESA model 5020). The potentials applied to the guard cell, E1 and E2 electrodes were 850, 300, and 800 mV, respectively. The R2 sensitivity (gain range) for E2 was set at 50 to 100 µA. GSH peak height values were corrected per 1 million cells and BSO- or GSH-treated cells were compared with control cells and expressed as a percent value. The intraday and interday variability of the assay is less than 10% (Stempak et al., 2001).

Data Analysis. [³H]Vincristine and [³H]digoxin accumulation and efflux values are expressed as picomoles per milligram of protein and are presented as mean ± S.D. from a minimum of three separate experiments. In an individual experiment, each data point represents quadruplicate trials. Intracellular GSH values, as determined by HPLC, are expressed as mean percent control ± S.D., based on comparison of GSH peak heights/10⁶ cells for three separate experiments. Statistical analysis for unpaired experimental data was carried out using the unpaired Student's t test. For multiple comparisons, the test of repeated measures analysis of variance and/or the post hoc multiple-comparison Bonferroni t test was used. A value of p < 0.05 was considered statistically significant.

	Results

Top Abstract Methods and Materials Results Discussion References

 
MRP1 mRNA and Protein Expression. RT-PCR analysis was used to assess the expression of rMRP1 mRNA in MLS-9 cells (Fig. 1A). Based on the existing partial sequence of rMRP1 available at the time, we designed RT-PCR primers to amplify a region that is conserved across mice, rats, and humans. Subsequently, the presumed full-length sequence of rMRP1 was published (Yang et al., 2002; American Association of Pharmaceutical Scientists Web site at http://www.aapspharmsci.org/scientificjournals/pharmsci/journal/ps040315.htm Accessed Nov 15, 2002), showing that the primers we used are in the middle of the rat gene. hMRP1 has an additional 400 base pairs at the C terminus. These primers worked well for RT-PCR of the MLS-9 cell line and primary cultured microglia, the cells from which the MLS-9 cell line was derived. They amplified a single band of the expected size (669 base pairs) in both cell types (lanes 3 and 4), which were confirmed to be rMRP1 by dideoxy sequencing.

View larger version (45K): [in this window] [in a new window]   Fig. 1. Expression of rMRP1 in MLS-9 cells. A, RT-PCR analysis of rMRP1. Lane 1, negative control (no cDNA); lane 2, negative control (no RNA), lane 3, MLS-9 microglia cell line; lane 4, primary microglia; lane 5, size marker (1-kb Plus DNA ladder; Invitrogen). B, Western Blot analysis of MRP1 in crude membrane fractions were prepared from cell lysates and tissue samples and then were separated on a 10% polyacrylamide gel containing 0.1% SDS. Proteins were transferred to polyvinylidene difluoride membrane electrophoretically. The blot was incubated with MRP1 monoclonal antibody MRPr1 (1:1,000) and an anti-actin antibody (1:500). Lane 1, 25 µg of primary rat microglia; lane 2, 25 µg of MLS-9 microglia cell line; lane 3, 12.5 µg of hMRP1-transfected HeLa cells (WT-MRP1); lane 4, 12.5 µg of adult rat kidney. C, Western blot analysis of rMRP2 in crude membrane fractions were prepared in a similar manner as MRP1. The blot was incubated with MRP2 monoclonal antibody M2III-6 (1:2,000) and an anti-actin antibody (1:4,000). Lane 1, 200 µg of rat liver; lane 2, 200 µg of MLS-9 microglia cell line.

Protein expression of rMRP1 and hMRP1 was detected using Western blotting (Fig. 1B). The monoclonal antibody MRPr1, which was raised against a bacterial fusion protein (amino acids 238–247) of hMRP1, recognized a band of 190 kDa in primary rat microglia (lane 1) and MLS-9 cells (lane 2). This antibody does not cross-react with P-gp or MRP isoforms 2 to 6 (Hipfner et al., 1998). The same single band was also detected in two positive control samples: WT-MRP1, a HeLa cell line that was stably transfected with hMRP1 (lane 3) and rat kidney (lane 4). Using the same amount of crude membrane protein in each lane (25 µg), primary rat microglia produced a stronger rMRP1 band than MLS-9 cells. It is possible that rMRP1 is down-regulated in the cell line (see Discussion). A single band of 43 kDa corresponding to actin was detected in each lane, which verified that an appropriate amount of protein was loaded for each sample.

Functional Studies. The chemotherapeutic agent vincristine has been used widely to characterize MRP1-mediated transport in vitro (Loe et al., 1996). Vincristine is, however, a substrate of several ATP-dependent efflux transporters, including MRP1, MRP2, and P-gp (Seetharaman et al., 1998; Borst et al., 1999). As shown in Fig. 1C, we confirmed that rMRP2 is not expressed in MLS-9 (lane 2) applying Western blotting analysis. Using the MRP2-specific antibody M2III-6, an 190-kDa band was detected in rat liver (lane 1), a tissue well known for high MRP2 expression (Borst et al., 1999). This antibody recognizes a 202 amino acid epitope in the C terminus of hMRP2 and rMRP2 proteins and does not cross-react with P-gp, MRP1, MRP3, or MRP5 (Scheffer et al., 2000). Despite loading a large amount of protein (200 µg), no rMRP2 band was observed in MLS-9 cells.

Given that the MRP1-mediated transport properties of vincristine have been examined extensively, and several properties (i.e., GSH-dependence; use of MRP-specific inhibitors) can distinguish P-gp from MRP1-mediated effects, we determined transport properties of radiolabeled vincristine as one measure of rMRP1 functional expression in MLS-9. Furthermore, we verified the specificity of the inhibitors and glutathione modulation by measuring digoxin transport (a "reference" P-gp substrate) in a P-gp-overexpressing cell line.

Accumulation and Efflux. A time-dependent increase in vincristine accumulation by MLS-9 cell monolayers was observed at 37°C, with a plateau beginning to be reached by 4 h (Fig. 2). Vincristine accumulation was significantly increased (i.e., 64% at 2 h) in the presence of the leukotriene D4 antagonist MK571, a potent inhibitor of MRP1. Increasing concentrations of MK571 over a 150-fold concentration range (0.5–75 µM) produced a dose-dependent increase in vincristine accumulation at 2 h (Fig. 3), and a plateau was observed at 50 µM MK571.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Effect of MK571 on vincristine accumulation by MLS-9 cells. Vincristine accumulation (30 nM) was measured at 37°C in the presence of the MRP1 inhibitor MK571 (5 µM) at 0.25, 0.5, 1, 2, 3, and 4 h. The results are expressed as mean ± S.D. of three separate experiments, with each data point in an individual experiment representing quadruplicate measurements. ***, p < 0.001, significantly different from control.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Dose-dependent effect of MK571 on vincristine accumulation by MLS-9 cells. Vincristine accumulation (30 nM) was measured at 2 h (37°C) in the presence of increasing concentrations of MK571 (0.5–75 µM). The results are expressed as mean ± S.D. of five separate experiments, with each data point in an individual experiment representing quadruplicate measurements. ***, p < 0.001, significantly different from control.

Because MRP1 is an efflux transporter, time-dependent vincristine efflux was directly examined in the presence or absence of MK571 (Fig. 4). After 1 h of preloading with vincristine, a time-dependent decrease was observed in the amount of vincristine remaining in the cells. Adding 5 µM MK571 at the start of the efflux studies significantly increased the intracellular drug concentration after 2 h (p < 0.001), suggesting that decrease in vincristine efflux in MLS-9 in the presence of the inhibitor was most likely mediated by rMRP1.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Effect of MK571 on vincristine efflux by MLS-9 cells. Vincristine efflux (30 nM) by MLS-9 was measured at 37°C in the presence of MK571 (5 µM) at 0.5, 1, and 2 h. Amount of radioactivity remaining intracellularly was measured and is expressed as a percentage of initial value (i.e., at time 0). The results are expressed as mean ± S.D. of three separate experiments with each data point in an individual experiment representing quadruplicate measurements. *, p < 0.05; ***, p < 0.001, significantly different from control.

Energetics. The energetic properties of vincristine transport by MRP1 are well characterized (Versantvoort et al., 1995; Loe et al., 1996). To confirm that rMRP1-mediated transport of vincristine was ATP-dependent, effects of the ATPase inhibitor, sodium azide were assessed in MLS-9 cells (Fig. 5). In the presence of 10 mM sodium azide, accumulation of vincristine was increased significantly (p < 0.001) at three time points: 30, 45, and 60 min.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Effect of sodium azide on vincristine accumulation by MLS-9 cells. Vincristine accumulation (30 nM) was measured in the presence of the metabolic inhibitor sodium azide (10 mM) at 0.5, 0.75, and 1 h. The results are expressed as mean ± S.D. of three separate experiments, with each data point in an individual experiment representing quadruplicate measurements. ***, p < 0.001, significantly different from control.

Efficient MRP1-mediated efflux of vincristine across cell membranes also requires physiological concentrations (1–5 mM) of intracellular GSH (Loe et al., 1996). That is, lowering the intracellular GSH concentration decreases vincristine efflux, whereas increasing GSH increases drug efflux. Because this property is specific to MRP1-mediated transport, modulation by GSH can discriminate between transport of vincristine by P-gp and MRP1. After depletion of MLS-9 intracellular GSH using BSO, accumulation of vincristine was significantly increased at several time points, e.g., by 52% at 2 h (Fig. 6A). Conversely, adding 5 mM exogenous GSH significantly (p < 0.001) decreased vincristine accumulation in MLS-9 at2hby 44%. No significant alteration in the accumulation of digoxin, an established P-gp substrate, was observed in the P-gp-overexpressing cell line CH^RC5 after GSH depletion (Fig. 6B). To confirm that altered vincristine accumulation in BSO- or GSH-treated cells was accompanied by parallel changes in intracellular GSH levels, GSH was measured by HPLC analysis. After 24-h incubation with 25 µM BSO, the GSH concentration in MLS-9 and CH^RC5 cells was decreased by >94%. Conversely, addition of 5 mM exogenous GSH in MLS-9 cells increased GSH by 30% within the first 10 min of the incubation period.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Glutathione modulation of vincristine and digoxin accumulation by MLS-9 or CHRC5 cells. A, vincristine accumulation (30 nM) was measured after depletion of intracellular glutathione with 25 µM BSO (24 h) at 0.5, 1, and 2 h at 37°C. B, accumulation of digoxin (100 nM) was measured at 0.5, 1, and 2 h in the P-gp-overexpressing cell line CHRC5 after intracellular glutathione depletion using 25 µM BSO (24 h). Results are expressed as mean ± S.D. of three to five separate experiments, with each data point in an individual experiment representing quadruplicate measurements. ***, p < 0.001, significantly different from control.

Specificity. Several other well established MRP1 inhibitors were tested for their effect on vincristine transport by MLS-9 cells (Fig. 7A). The flavanoid genistein (200 µM), the nonsteroidal anti-inflammatory drug indomethacin (50 µM), and the antihyperuricemic agents probenecid (100 µM) and sulfinpyrazone (2 mM) significantly increased vincristine accumulation at 2 h from the control value (2.2 ± 0.2 pmol/mg protein) to 3.9 ± 0.1, 3.07 ± 0.3, 3.1 ± 0.3, and 3.2 ± 0.5 pmol/mg protein, respectively. The inhibitors used also significantly enhanced vincristine accumulation in the hMRP1-overexpressing cell line WT-MRP1 by up to 355% compared with control (Fig. 7B), whereas no effect was observed in VF-Hela, the hMRP1-deficient parental cell line (data not shown). In addition, the inhibitors did not cross-react with P-gp when their effect was evaluated on digoxin accumulation by CH^RC5 cells (Fig. 8).

View larger version (16K): [in this window] [in a new window]   Fig. 7. Effect of MRP1 inhibitors on vincristine accumulation by MLS-9 and WT-MRP1 cell lines. A, accumulation of vincristine (30 nM) in MLS-9 at 2 h was measured in the presence of 5 µM MK571, 200 µM genistein (GEN), 2 mM sulfinpyrazone (SULF), 50 µM indomethacin (INDO), or 100 µM probenecid (PROB) at 37°C. B, accumulation of vincristine (30 nM) in WT-MRP1 at 2 h was measured in the presence of 5 µM MK571, 200 µM genistein (GEN), 2 mM sulfinpyrazone (SULF), 50 µM indomethacin (INDO), or 100 µM probenecid (PROB) at 37°C. The results are expressed as mean ± S.D. of five separate experiments, with each data point in an individual experiment representing quadruplicate measurements. ***, p < 0.001, significantly different from control.

View larger version (16K): [in this window] [in a new window]   Fig. 8. Effect of MRP1 inhibitors on digoxin accumulation by CHRC5 cells. Accumulation of digoxin (100 nM) in the presence of 5 µM MK571, 200 µM genistein (GEN), 2 mM sulfinpyrazone (SULF), 50 µM indomethacin (INDO), or 100 µM probenecid (PROB) at 1 h (37°C). The results are expressed as mean ± S.D. of three separate experiments, with each data point in an individual experiment representing quadruplicate measurements.

The antiretroviral protease inhibitor drugs indinavir, ritonavir, and saquinavir are known to interact with MRP1 (Srinivas et al., 1998; Miller et al., 2000; Jones et al., 2001; Williams et al., 2002). In this study, indinavir (50 µM), ritonavir (10 µM), and saquinavir (10 µM) increased vincristine accumulation by up to 76 and 400% in MLS-9 (Fig. 9A) and in the hMRP1-overexpressing cell line WT-MRP1 (Fig. 9B), respectively.

View larger version (13K): [in this window] [in a new window]   Fig. 9. Effect of protease inhibitors on vincristine accumulation by MLS-9 and WT-MRP1 cell lines. A, accumulation of vincristine (30 nM) in MLS-9 at 2 h was determined at 37°C in the presence of 10 µM saquinavir (SAQ), 10 µM ritonavir (RIT), or 50 µM indinavir (IND). B, accumulation of vincristine (30 nM) in WT-MRP1 at 2 h was determined at 37°C in the presence of 10 µM saquinavir (SAQ), 10 µM ritonavir (RIT), or 50 µM indinavir (IND). The results are expressed as mean ± S.D. of three separate experiments, with each data point in an individual experiment representing quadruplicate measurements. ***, p < 0.001, significantly different from control.

	Discussion

Top Abstract Methods and Materials Results Discussion References

 
The MRP family of efflux transport proteins is known to confer multidrug resistance to a variety of structurally unrelated compounds (Borst et al., 1999). The expression of these proteins in the brain has recently been examined within the blood-brain and blood-cerebrospinal fluid barriers (Wijnholds et al., 2000; Zhang et al., 2000). Multiple MRP homologs are expressed within these barriers and seem to contribute to the low brain penetration of a variety of therapeutic compounds, including antiretroviral drugs used in the treatment of HIV-1 (Miller et al., 2000). The primary cells infected by HIV-1 in the brain are microglia (brain macrophages) (Bagasra et al., 1996; Epstein, 1998) but little is known about expression of MRP family members in microglia. The presence of rMRP1 transcripts in cultured rat microglia was recently reported (Ballerini et al., 2002; Hirrlinger et al., 2002); however, protein levels and functional activity of rMRP1 were not extensively examined. The present study investigates the expression of rMRP1 in a rat microglia cell line (MLS-9) at both the protein and functional levels.

We had previously found rMRP1 transcripts in primary rat microglia but did not detect them in MLS-9 cells using primers that recognize a region in the 3' end of the rat gene (Lee et al., 2001b). In the present study, we amplified a region in the middle of the gene from primary microglia and from MLS-9 cells and obtained a single band for each cell type. Each product was the expected size, and sequencing confirmed they were both rMRP1. Western blotting using the monoclonal antibody MRPr1 confirmed the expression of rMRP1 protein in primary cultures of rat microglia and the MLS-9 microglia cell line. This antibody recognizes hMRP1 and rMRP1 (Rao et al., 1999) and does not cross-react with MRP2–6 (Hipfner et al., 1998). A more robust rMRP1 band was detected in primary cultures of rat microglia than in MLS-9 cells, possibly due to differences in protein processing or down-regulation of rMRP1 in the cell line. A similar discrepancy in rMRP1 expression between primary cultures and their immortalized counterparts has been reported, with less rMRP1 protein levels in an immortalized rat brain endothelial cell line (RBE4) than in primary cultures of rat brain endothelial cells (Regina et al., 1998).

To characterize rMRP1 function in MLS-9 cells, we investigated the transport properties of vincristine, a well established MRP1 substrate (Loe et al., 1996; Renes et al., 1999). However, vincristine is also a substrate for P-gp and MRP2. Lack of rMRP2 expression in MLS-9 was confirmed by Western blotting using M2III6, an MRP2-specific antibody that does not cross-react with either MRP1, MRP3, MRP5, or P-gp (Scheffer et al., 2000). However, because MLS-9 cells do express both rMRP1 and P-gp, it was important to characterize separately the transport mediated by P-gp versus rMRP1 (Lee et al., 2001b). To do this, we used inhibitors specific to the MRP family and studied a selective modulator of MRP1 transport energetics. Several potent MRP1 inhibitors have been identified, including MK571, a leukotriene D4 receptor antagonist that competitively inhibits the transport of various MRP1 substrates (K_i = 0.6 µM), including leukotriene C4 and glutathione disulfide (Leier et al., 1996). Accumulation of vincristine by MLS-9 cells was significantly increased and its efflux reduced by 5 µM MK571. Four other well established MRP1 inhibitors (indomethacin, genistein, probenecid, and sulfinpyrazone) also significantly increased the accumulation of vincristine by 42% or greater in MLS-9 cells. These results agree with recent findings by Ballerini et al. (2002) where 2 mM sulfinpyrazone and 1 mM probenecid increased the accumulation of 10 nM vincristine by greater than 40% in primary cultures of microglia. Genistein (400 µM) was the strongest inhibitor of vincristine transport (10 nM) in primary cultures of brain endothelial cells and an immortalized cell line derived from these cells (RBE4), followed by 2 mM sulfinpyrazone and 1 mM probenecid (Regina et al., 1998). A similar pattern of inhibition emerged in MLS-9. In WT-MRP1, an hMRP1 transfected cell line that expresses much higher levels of MRP1 than MLS-9, these same inhibitors significantly increased vincristine accumulation up to 355%.

To verify the specificity of the inhibitors for MRP1, we examined the ability of the compounds to alter the accumulation of digoxin in vitro. Digoxin is a prototypical P-gp substrate that has been shown to interact with the transporter at both the in vitro and in vivo level and is often used as a "reference" compound for P-gp transport studies (Fromm et al., 1999; Stephens et al., 2002). At the concentrations used in the present study, none of the MRP1 inhibitors tested significantly altered the transport of digoxin in a P-gp-overexpressing cell line (CH^RC5). It is therefore unlikely that these inhibitors modulate P-gp-mediated vincristine transport in MLS-9.

Although P-gp and MRP1 have several functional similarities, the effect of GSH on MRP1 transport remains an identifiable difference between the two proteins. Efficient vincristine transport by MRP1 requires physiological intracellular concentrations (1–5 mM) of GSH (Loe et al., 1996). It is well established that vincristine and GSH are cotransported by MRP1 (Zaman et al., 1995; Loe et al., 1998). While the exact nature of this cotransport process remains unresolved, proposed models include bipartite and multipartite binding, since neither GSH nor vincristine can be transported alone (Leslie et al., 2001). Therefore, we depleted intracellular GSH concentrations in MLS-9 cells using BSO, an irreversible inhibitor of -glutamylcysteine synthetase, the first cellular enzyme used in the biosynthesis of GSH. Overnight incubation of MLS-9 cells with 25 µM BSO decreased intracellular GSH levels by >94%, as determined by HPLC analysis. This was a greater decrease than previously reported for MRP1-over-expressing cells treated in a similar manner, where intracellular GSH was depleted by 80–87% (Feller et al., 1995; Zaman et al., 1995). We used a highly sensitive and specific HPLC assay coupled to electrochemical detection, which we have recently shown simultaneously detects GSH and other nonprotein thiols (Stempak et al., 2001). In BSO-treated MLS-9 cells, vincristine accumulation consistently increased at several time intervals, suggesting that GSH-dependent drug efflux was decreased. However, BSO did not alter digoxin accumulation by CH^RC5 cells, further confirming the specificity of GSH dependence for MRP1.

Adding 5 mM GSH to the MLS-9 cells increased intracellular GSH by 30%, and decreased vincristine accumulation by 44%, presumably due to faster efflux. These results concur with studies by Loe et al. (1996) and Renes et al. (1999) where increased vincristine accumulation in the presence of 5 mM GSH was observed in membrane vesicles from hMRP1-transfected and hMRP1-overexpressing cells. Together, our results demonstrate functional activity of rMRP1 in MLS-9 cells.

Microglia cells are the primary targets of HIV-1 infection in vivo (Epstein, 1998). We were therefore interested in examining the ability of these cells to interact with a number of antiretroviral drugs (i.e., protease inhibitors). Numerous in vitro studies suggest that protease inhibitors interact with MRP1 (Srinivas et al., 1998; Jones et al., 2001). In hMRP1 overexpressing lymphocytes, protease inhibitors significantly increase the efflux of calcein, a known MRP1 substrate, at concentrations of 50 µM (Srinivas et al., 1998). Recently, direct evidence that saquinavir is a substrate for MRP1 has been shown in MDCKII overexpressing hMRP1 cells (Williams et al., 2002). In the present study, the protease inhibitors saquinavir, indinavir, and ritonavir significantly increased the accumulation of vincristine in both the microglia cell line, and hMRP1-overexpressing cells. However, the concentration of indinavir required to achieve similar levels of vincristine inhibition was 5 times that of saquinavir and ritonavir. The ability of these three compounds to also alter P-gp mediated transport (Srinivas et al., 1998; Jones et al., 2001; Lee et al., 2001b), precludes any comparison of the relative contribution of rMRP1 versus P-gp-mediated transport of vincristine in MLS-9 cells. Future studies examining the direct transport properties of protease inhibitors in cultured microglia should help us understand the relative contribution of P-gp versus MRP1 in this brain compartment.

In summary, we have shown that rMRP1, which is expressed in cultured microglia, is functionally active in a cell line derived from these microglia. Combined with our previous results showing functional expression of P-gp in this cell line (Lee et al., 2001b), our present data show that microglia express multiple subfamilies of ATP-binding cassette transporters. The novel expression of drug-efflux transporters in the brain parenchyma warrants a reconsideration of brain barriers and drug transport. It seems that membranes of microglia can act as a second "barrier" to drug permeability (Lee et al., 2001a). Because microglia are the primary targets of HIV-1 infection in the brain, the expression of transporters in these cells may play a significant role in the antiretroviral drug response and in the development of drug resistance.

	Acknowledgements

 
We thank Mahendra Kumar Pallapothu, Meenal Joshi, and Diana Stempak for excellent technical support.

	Footnotes

 
This work is supported by grants to R.B. from the Canadian Institutes for Health Research (HOP-56976) and the Ontario HIV Treatment Network, and to L.C.S. by Canadian Institutes for Health Research (MT-13657). S.D. has received a graduate studentship from the Ontario HIV Treatment Network. This work was presented in a preliminary format at The American Association of Pharmaceutical Scientists Annual Meeting, Denver, CO, October 2001, and the 10th Annual Canadian Association for HIV Research Conference, Toronto, ON, June 2001.

ABBREVIATIONS. P-gp, P-glycoprotein; MRP, multidrug resistance protein; hMRP1, human multidrug resistant protein 1; rMRP1, rat multidrug resistance protein 1; HIV-1, human immunodeficiency virus type 1; BSO, L-buthionine-[S,R]-sulfoximine; RT-PCR, reverse transcriptase-polymerase chain reaction; TBS-T, Tris-buffered saline/Tween 20; EBSS, Earle's balanced salt solution; GSH, glutathione.

DOI: 10.1124/jpet.103.054304.

Address correspondence to: Dr. Reina Bendayan, Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Toronto, 19 Russell St., Toronto, ON M5S 2S2, Canada. E-mail: r.bendayan{at}utoronto.ca

	References

Top Abstract Methods and Materials Results Discussion References

 

Anderson ME (1998) Glutathione: an overview of biosynthesis and modulation. Chem Biol Interact 111–112: 1–14.

Bagasra O, Lavi E, Bobroski L, Khalili K, Pestaner JP, Tawadros R, and Pomerantz RJ (1996) Cellular reservoirs of HIV-1 in the central nervous system of infected individuals: identification by combination of in situ polymerase chain reaction and immunocytochemistry. AIDS 10: 573–585.[Medline]

Ballerini P, Di Iorio P, Ciccarelli R, Nargi E, D'Alimonte L, Traversa U, Rathbone MP, and Caciagli F (2002) Glial cells express multiple ATP binding cassette proteins which are involved in ATP release. Neuroreport 13: 1789–1792.[CrossRef][Medline]

Bendayan R, Lee G, and Bendayan M (2002) Functional expression and localization of P-glycoprotein at the blood-brain barrier. Microsc Res Tech 57: 365–380.[CrossRef][Medline]

Borst P, Evers R, Kool M, and Wijnholds J (1999) The multidrug resistance protein family. Biochim Biophys Acta 1461: 347–357.[Medline]

Cayabyab FS and Schlichter LC (2002) Regulation of an ERG K⁺ current by Src tyrosine kinase. J Biol Chem 277: 13673–13681.[Abstract/Free Full Text]

Cole SPC, Bhardwaj G, Gerlach JH, Mackie JE, Grant CE, Almquist KC, Stewart AJ, Kurz EU, Duncan AMV, and Deeley RG (1992) Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science (Wash DC) 258: 1650–1654.[Abstract/Free Full Text]

Cole SPC and Deeley RG (1998) Multidrug resistance mediated by the ATP-binding cassette transporter protein MRP. Bioessays 20: 931–940.[CrossRef][Medline]

Declèves X, Regina A, Laplanche JL, Roux F, Boval B, Launay JM, and Scherrmann JM (2000) Functional expression of P-glycoprotein and multidrug resistance-associated protein (Mrp1) in primary cultures of rat astrocytes. J Neurosci Res 60: 594–601.[CrossRef][Medline]

Epstein LG (1998) HIV neuropathogenesis and therapeutic strategies. Acta Paediatr Jpn 40: 107–111.[Medline]

Feller N, Broxterman HJ, Währer DC, and Pinedo HM (1995) ATP-dependent efflux of calcein by the multidrug resistance protein (MRP): no inhibition by intracellular glutathione depletion. FEBS Lett 368: 385–388.[CrossRef][Medline]

Fromm MF, Kim RB, Stein CM, Wilkinson GR, and Roden DM (1999) Inhibition of P-glycoprotein-mediated drug transport – a unifying mechanism to explain the interaction between digoxin and quinidine. Circulation 99: 552–557.[Abstract/Free Full Text]

Hipfner DR, Gao M, Scheffer G, Scheper RJ, and Cole SPC (1998) Epitope mapping of monoclonal antibodies specific for the 190-kDa multidrug resistance protein (MRP). Br J Cancer 78: 1134–1140.[Medline]

Hirrlinger J, König J, and Dringen R (2002) Expression of mRNAs of multidrug resistance proteins (Mrps) in cultured rat astrocytes, oligodendrocytes, microglial cells and neurones. J Neurochem 82: 716–719.[CrossRef][Medline]

Jones K, Bray PG, Khoo SH, Davey RA, Meaden ER, Ward SA, and Back DJ (2001) P-glycoprotein and transporter MRP1 reduce HIV protease inhibitor uptake in CD4 cells: potential for accelerated viral drug resistance? AIDS 15: 1353–1358.[CrossRef][Medline]

Koutsilieri E, ter Meulen V, and Riederer P (2001) Neurotransmission in HIV associated dementia: a short review. J Neural Transm 108: 767–775.[CrossRef]

Lee G, Dallas S, Hong M, and Bendayan R (2001a) Drug transporters in the central nervous system: brain barriers and brain parenchyma considerations. Pharmacol Rev 53: 569–596.[Abstract/Free Full Text]

Lee G, Schlichter L, Bendayan M, and Bendayan R (2001b) Functional expression of P-glycoprotein in rat brain microglia. J Pharmacol Exp Ther 299: 204–212.[Abstract/Free Full Text]

Leier I, Jedlitschky G, Buchholz U, Center M, Cole SPC, Deeley RG, and Keppler D (1996) ATP-dependent glutathione disulphide transport mediated by the MRP gene-encoded conjugate export pump. Biochem J 314: 433–437.

Leslie EM, Deeley RG, and Cole SPC (2001) Toxicological relevance of the multidrug resistance protein 1, MRP1 (ABCC1) and related transporters. Toxicology 167: 3–23.[CrossRef][Medline]

Loe DW, Almquist KC, Deeley RG, and Cole SPC (1996) Multidrug resistance protein (MRP)-mediated transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles. Demonstration of glutathione-dependent vincristine transport. J Biol Chem 271: 9675–9682.[Abstract/Free Full Text]

Loe DW, Deeley RG, and Cole SPC (1998) Characterization of vincristine transport by the M_r 190, 000 multidrug resistance protein (MRP): evidence for cotransport with reduced glutathione. Cancer Res 58: 5130–5136.[Abstract/Free Full Text]

Meaden ER, Hoggard PG, Newton P, Tjia JF, Aldam D, Cornforth D, Lloyd J, Williams I, Back DJ, and Khoo SH (2002) P-glycoprotein and MRP1 expression and reduced ritonavir and saquinavir accumulation in HIV-infected individuals. J Antimicrob Chemother 50: 583–588.[Abstract/Free Full Text]

Miller DS, Nobmann SN, Gutmann H, Toeroek M, Drewe J, and Fricker G (2000) Xenobiotic transport across isolated brain microvessels studied by confocal microscopy. Mol Pharmacol 58: 1357–1367.

Minhas HS and Thornalley PJ (1995) Comparison of the delivery of reduced glutathione into P388D₁ cells by reduced glutathione and its mono- and diethyl ester derivatives. Biochem Pharmacol 49: 1475–1482.[CrossRef][Medline]

Rao VV, Dahlheimer JL, Bardgett ME, Snyder AZ, Finch RA, Sartorelli AC, and Piwnica-Worms D (1999) Choroid plexus epithelial expression of MDR1 Pglycoprotein and multidrug resistance-associated protein contribute to the blood-cerebrospinal-fluid drug-permeability barrier. Proc Natl Acad Sci USA 96: 3900–3905.[Abstract/Free Full Text]

Regina A, Koman A, Piciotti M, El Hafny B, Center MS, Bergmann R, Couraud PO, and Roux F (1998) Mrp1 multidrug resistance-associated protein and P-glycoprotein expression in rat brain microvessel endothelial cells. J Neurochem 71: 705–715.[Medline]

Renes J, de Vries EG, Nienhuis EF, Jansen PL, and Müller M (1999) ATP- and glutathione-dependent transport of chemotherapeutic drugs by the multidrug resistance protein MRP1. Br J Pharmacol 126: 681–688.[CrossRef][Medline]

Scheffer GL, Kool M, Heijn M, de Haas M, Pijnenborg AC, Wijnholds J, van Helvoort A, de Jong MC, Hooijberg JH, Mol CA, et al. (2000) Specific detection of multidrug resistance proteins MRP1, MRP2, MRP3, MRP5 and MDR3 P-glycoprotein with a panel of monoclonal antibodies. Cancer Res 60: 5269–5277.[Abstract/Free Full Text]

Seetharaman S, Barrand MA, Maskell L, and Scheper RJ (1998) Multidrug resistance-related transport proteins in isolated human brain microvessels and in cells cultured from these isolates. J Neurochem 70: 1151–1159.[Medline]

Srinivas RA, Middlemas D, Flynn P, and Fridland A (1998) Human immunodeficiency virus protease inhibitors serve as substrates for multidrug transporter proteins MDR1 and MRP1 but retain antiviral efficacy in cell lines expressing these transporters. Antimicrob Agents Chemother 42: 3157–3162.[Abstract/Free Full Text]

Stempak D, Dallas S, Klein J, Bendayan R, Koren G, and Baruchel S (2001) Glutathione stability in whole blood: effects of various deproteinizing agents. Ther Drug Monit 23: 542–549.[CrossRef][Medline]

Stephens RH, O'Neill CA, Bennett J, Humphrey M, Henry B, Rowland M, and Warhurst G (2002) Resolution of P-glycoprotein and non-P-glycoprotein effects on permeability using intestinal tissues from mdr1a (–/–) mice. Br J Pharmacol 135: 2038–2046.[CrossRef][Medline]

Versantvoort CH, Broxterman HJ, Bagrij T, Scheper RJ, and Twentyman PR (1995) Regulation by glutathione of drug transport in multidrug resistant human lung tumour cell lines overexpressing multidrug resistance-associated protein. Br J Cancer 72: 82–89.[Medline]

Wijnholds J, de Lange ECM, Scheffer GL, van der Berg DJ, Mol CAA, van der Valk M, Schinkel AH, Scheper RJ, Breimer DD, and Borst P (2000) Multidrug resistance protein 1 protects the choroid plexus epithelium and contributes to the blood-cerebrospinal fluid barrier. J Clin Investig 105: 279–285.[Medline]

Williams GC, Liu A, Knipp G, and Sinko PJ (2002) Direct evidence that saquinavir is transported by multidrug resistance-associated protein (MRP1) and canalicular multispecific organic anion transporter (MRP2). Antimicrob Agents Chemother 46: 3456–3462.[Abstract/Free Full Text]

Zaman GJR, Lankelma J, van Tellingen O, Beijnen J, Dekker H, Paulusma C, Oude Elferink RPJ, Baas F, and Borst P (1995) Role of glutathione in the export of compounds from cells by the multidrug resistance-associated protein. Proc Natl Acad Sci USA 92: 7690–7694.[Abstract/Free Full Text]

Zhang Y, Han H, Elmquist WF, and Miller DW (2000) Expression of various multidrug resistance-associated protein (MRP) homologues in brain microvessel endothelial cells. Brain Res 876: 148–153.[CrossRef][Medline]

Zhou W, Cayabyab FS, Pennefather PS, Schlichter LC, and DeCoursey TE (1998) HERG-like K⁺ channels in microglia. J Gen Physiol 111: 781–794.[Abstract/Free Full Text]

Yang Z, Li CS, Shen DD and Ho RJ (2002) Cloning and characterization of the rat multidrug resistance-associated protein 1. AAPS Pharm Sci 4: 1–7.

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