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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Plasma Membrane Localization of Multidrug Resistance-Associated Protein Homologs in Brain Capillary Endothelial Cells

Pfizer Global Research and Development, PDM Department, Ann Arbor, Michigan (Y.Z.); Department of Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee (J.D.S.); Department of Pharmaceutics, University of Minnesota, Minneapolis, Minnesota (W.F.E.); and Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska (D.W.M.)

Received March 16, 2004; accepted June 21, 2004.

	Abstract

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Several multidrug resistance-associated protein (MRP) homologs are expressed in brain microvessel endothelial cells forming the blood-brain barrier (BBB). The influence of these MRP transporters on BBB permeability will be dependent on their localization within the brain microvessel endothelial cells. Using two different and complementary approaches, the localization of various MPR homologs (MRP1, MRP4, and MRP5) was examined in primary cultured bovine brain microvessel endothelial cells (BBMECs). The first approach involved centrifugal separation of apical and basolateral plasma membranes of cultured BBMECs. The membrane fractions were then subjected to Western blot analysis for MRPs. The second approach used confocal laser scanning microscopy to determine membrane localization of MRPs in BBMECs. Results show a predominantly apical plasma membrane distribution for MRP1 and MRP5, and an almost equal distribution of MRP4 on the apical and basolateral plasma membrane of BBMECs. These studies provide the first demonstration of the localization of MRP1, MRP4, and MRP5 homologs in brain microvessel endothelial cells. The present studies also indicate that the localization of MRPs in the endothelial cells forming the BBB is different from that observed in polarized epithelial cells and thus may contribute to the reduced entry and enhanced elimination of organic anions and nucleotides in the brain.

Although much attention has been focused on inward transport systems in the BBB, the presence of outwardly directed (i.e., efflux) transporters has both physiological and pathological implications. P-glycoprotein (P-gp) is an example of a drug efflux transport system in the BBB. Originally found in drug-resistant tumor cells, Pgp actively transports a wide range of compounds out of the cell. In brain microvessel endothelial cells, P-gp is found on the luminal (apical) plasma membrane, where it has an important role in limiting the accumulation of drugs and xenobiotics in the brain (Schinkel et al., 1994, 1996). The multidrug resistance-associated proteins (MRPs) are also drug efflux transporters belonging to the ATP-binding cassette superfamily of membrane transporter proteins. In contrast to P-gp, which transports lipophilic and cationic compounds, MRPs transport organic anions, glutathione, or glucuronide-conjugated compounds, and various nucleoside analogs (Jedlitschky et al., 1996; Borst et al., 2000). Human MRP1, along with eight other homologs, including MRP2/canalicular multispecific organic anion transporter, and MRP3–MRP9, have been identified in cancer cells and in normal human tissues (Kool et al., 1997, 1999; Bera et al., 2001; Hopper et al., 2001; Tammur et al., 2001). Although the expression of several MRP homologs in the brain microvessel endothelial cells has been reported previously (Huai-Yun et al., 1998; Regina et al., 1998; Seetharaman et al., 1998; Gutmann et al., 1999; Zhang et al., 2000), the localization of these homologs in the BBB remains unknown.

The purpose of the current study was to determine the localization of various MRP homologs expressed in brain microvessel endothelial cells forming the BBB. Using primary cultured bovine brain microvessel endothelial cells (BBMECs) as an in vitro model of the BBB, localization of MRP1, MRP4, and MRP5 was examined using isolated membrane fractions and confocal microscopy. Western blot analysis of isolated apical and basolateral membrane fractions from BBMEC monolayers indicated a higher expression of MRP1 and MRP5 in the apical membrane compared with the basolateral fraction. In contrast, immunoblots indicated MRP4 was equally distributed in both apical and basolateral fractions. A similar pattern of distribution was observed using confocal microscopy. This is the first report concerning the localization of MRP1, MRP4, and MRP5 homologs in brain microvessel endothelial cells. These studies suggest that the plasma membrane localization of MRP transporters are different from that observed in polarized epithelial cells. The distribution of MRP transporters in the plasma membrane of brain microvessel endothelial cells indicate these transporters may have an important role in determining BBB permeability.

	Materials and Methods

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Cell Isolation and Culturing
The BBMECs were isolated from fresh cow brains using a combination of enzyme digestion and centrifugal separation techniques (Miller et al., 1992). The cells were cultured to confluence (typically 12–14 days) on either collagen-coated, fibronectin-treated culture flasks or polycarbonate Transwell membranes (24 mm in diameter; 0.4-µm pore size; Costar, Corning, NY) using minimal essential medium:F-12 media supplemented with 10% horse serum, 50 µg/ml gentamicin, 2.5 µg/ml amphotericin B, and 100 µg/ml heparin. Two transfected cell lines were also used in the present study. Madin-Darby canine kidney II (MDCKII) cells transfected with human MRP1 (MDCKII-MRP1) or MRP2 (MDCKII-MRP2) were kindly provided by Dr. Piet Borst (Netherlands Cancer Institute, Amsterdam, The Netherlands) and cultured in Dulbecco's modified Eagle's media (Cellgro) with 10% fetal bovine serum. Cells were seeded on polycarbonate Transwell membrane filters at a density of 2 x 10⁶ cells/well and were used on the 3rd day after seeding.

Isolation of Apical and Basolateral Plasma Membranes
Total Plasma Membrane Isolation. Total plasma membrane vesicles were prepared using the method of Peterson and Hawkins (1998), with modifications described below. Primary cultured BBMECs were grown to confluence on collagen-coated, fibronectin-treated 175-cm² culture flasks (seeding density approximately 5 x 10⁶ cells/flask) and harvested using a rubber scraper. The following experimental procedures were carried out at 4°C. The cell suspension was centrifuged at 1000g for 15 min. The resulting cell pellet was resuspended in 40-fold hypotonic lysis buffer (1 mM sodium bicarbonate with a cocktail of protease inhibitors, pH 7.0) (Roche Diagnostics, Indianapolis, IN) and incubated for 14 to 16 h. The cell lysate was then centrifuged at 100,000g for 30 min, and the resulting pellet was resuspended in 10-fold of TSEM buffer (10 mM Tris-HCl, pH 7.4, 250 mM sucrose, 0.1 mM EGTA, and 0.5 mM MgCl₂) and homogenized using a glass homogenizer (25 strokes). The homogenate was centrifuged at 5500g for 10 min, and the supernatant was collected. The pellet was resuspended in 9-fold of TSEM buffer, rehomogenized, and centrifuged at 5500g for 10 min. The resulting supernatant was pooled with the supernatant from the previous step, and MgSO₄ was added to reach a final concentration of 10 mM. The mixture was stirred for 30 min and centrifuged at 4000g for 10 min. The supernatant was further centrifuged at 100,000g for 1 h, and the resulting pellet containing total cell membrane was collected.

Isolation of Apical and Basolateral Plasma Membranes. Total cell membrane from BBMECs was suspended in TSE buffer (TSEM without MgCl₂) and dissociated by passing sequentially through a 20-gauge syringe needle and then a 27-gauge syringe needle. The membrane suspension was then layered onto a discontinuous Ficoll gradient (5, 10, 15, and 20% Ficoll) and centrifuged at 163,000g for 2.5 h. Four fractions were collected at the interphases between the different Ficoll layers. Membrane fractions corresponding to the 0/5% (fraction 1) and 10/15% (fraction 3) Ficoll interphases were reported previously to correspond to the apical and basolateral plasma membranes of brain capillary endothelial cells, respectively (Peterson and Hawkins, 1998). The membranes were kept in storage buffer (290 mM mannitol and 10 mM Tris-HCl, pH 7.4) and frozen at –80°C.

Western Blot Studies
Western blot studies were performed on the various plasma membrane fractions using techniques described previously (Miller et al., 1996). The proteins examined included P-gp, MRP1, MRP4, and MRP5. The antibodies used for MRP1, MRP5, and P-gp were MRPm6 (Kamiya Biomedical, Seattle, WA), M₅II-54 (Alexis Corporation, San Diego, CA), and C219 (DakoCytomation California Inc., Carpinteria, CA), respectively. A monoclonal antibody to human MRP4 developed in the laboratory of Dr. John Schuetz (Schuetz et al., 1999) was used to identify MRP4 in the BBMEC membranes. Antibodies to MRP1 and P-gp were used at 1:100 dilution, whereas antibodies to MRP4 and MRP5 were used at 1:200 and 1:50 dilution, respectively. Secondary IgG horseradish peroxidase-conjugated antibodies were used at a 1:1500 dilution and were purchased from Amersham Biosciences Inc. (Cleveland, OH) and Stressgen (Victoria, BC, Canada). Immunoreactivity was visualized with a chemiluminescence kit (Pierce Chemical, Rockford, IL). The intensity of the protein bands was measured using a densitometer. The relative localization of the MRP homologs was expressed as the ratio of protein band intensity in the apical and basolateral membrane fractions (band intensity_apical/band intensity_basolateral).

Immunocytochemistry
BBMEC, MDCKII-MRP1, or MDCKII-MRP2 were grown on microporous polystyrene membrane filters until confluent. Cells were fixed for 10 min in 3% (v/v) formaldehyde in 1% bovine serum albumin/phosphate-buffered saline followed by permeabilization with 0.5% Triton X-100 for 5 min at room temperature. Cells were incubated with 14% goat serum in phosphate-buffered saline for 30 min to reduce background staining. Cells were then incubated with the various primary monoclonal antubodies for the drug efflux transporters overnight at 4°C. The dilutions for anti-MRP1, MRP2 (M₂III-6; Alexis Corporation), MRP4, and MRP5 were 1:10, 1:50, 1:25, and 1:25, respectively. After incubation with primary antibodies, cells were washed three times, and secondary antibody biotin-tagged goat anti-mouse (for MRP1 and MRP2) or goat anti-rabbit (for MRP4) was added (13 µg/ml; Molecular Probes, Eugene, OR). After incubation with the secondary antibody for 45 min at 4°C, the cells were washed and exposed to FITC-labeled streptavidin (10 µg/ml; Molecular Probes). To aid in the localization of the antibody staining, nuclei were counterstained with 0.5 µg/ml propidium iodide (Sigma-Aldrich). The localization of MRP5 was examined using Texas Redtagged goat anti-rat IgG secondary antibody (10 µg/ml; Molecular Probes), and nuclei were stained using a 1:5000 dilution of Toto-3 (Molecular Probes). The negative controls were treated as described above with the exception of the omission of primary antibody. Cells were examined using a confocal microscope (Carl Zeiss, Thornwood, NY).

	Results

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Membrane Vesicle Isolation Studies. Preliminary data showed the membrane enrichment of various efflux transporters compared with the cell lysates from BBMECs (Table 1). The expression of P-gp, MRP1, MRP4, and MRP5 was 38-, 9.3-, 18-, and 5.4-fold enriched in the total membrane fraction compared with whole cell lysates (Table 1). The present study further examined the localization of the transporters in the plasma membrane of brain microvessel endothelial cells. Four turbid layers were obtained after centrifugation of the BBMEC membranes in a discontinuous Ficoll gradient. According to Peterson and Hawkins (1998), the first and the third layer of the Ficoll separation correspond to the apical and basolateral membrane fractions, respectively. Because P-gp has been localized to the apical membrane of brain endothelial cells (Cordon-Cardo et al., 1989), it was used as a marker for the apical membrane of BBMECs. Whereas the P-gp antibody detected an approximately 170-kDa band of protein in all four BBMEC membrane fractions collected from the Ficoll gradient centrifugation, the highest level of expression was detected in the apical membrane fraction (F1) (Fig. 1). Quantitative comparison of the apical-to-basolateral distribution of P-gp indicated an approximately 4-fold enrichment of P-gp immunoreactivity in the apical plasma membrane (Fig. 3). A similar expression pattern was also observed for both MRP1 and MRP5, with the highest expression levels found in the apical membrane fraction (F1) compared with the basolateral membrane fraction (F3) (Fig. 2, A and C, respectively). Densitometry analysis of the apical and basolateral distribution of MRP1 and MRP5 showed a 3- and 5-fold apical enrichment, respectively (Fig. 3). In contrast, the expression of MRP4 was similar in the first three BBMEC membrane fractions (Fig. 2B), resulting in an apical-to-basolateral ratio near 1 (Fig. 3).

View larger version (12K): [in this window] [in a new window]   Fig. 1. Representative Western blot of P-gp in plasma membrane fractions separated from BBMEC monolayers. The positive control used for P-gp was MDCK-MDR1 (1 µg; lane P). Membrane fractions loaded were shown as lanes F1–F4, with F1 being the apical membrane fraction and F3 containing the basolateral fraction. The amount of protein used in each lane was 0.7 µg.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Distribution of the MRP homologs in apical and basolateral membrane fractions separated from the BBMEC monolayers. Relative localization of each protein on the plasma membrane was expressed as the ratio of band density from the apical fraction to that of the basolateral fraction using a densitometer. P-gp was used as an apical marker. Values represent the mean ± S.E.M. of five different membrane isolations.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Representative Western blot of MRP1 (A), MRP4 (B), and MRP5 (C) in plasma membrane fractions separated from BBMEC monolayers. The positive controls used for MRP1 and MRP4 were cell lysates of COR-L23/R (1 µg; lane COR) and MRP4-transfected MCF-7 (5 µg; lane MCF), respectively. Membrane fractions loaded were shown as lanes F1–F4, with F1 being the apical and F3 being the basolateral fraction, respectively. The amount of protein loaded in lanes F1–F4 was 0.7 µg.

Immunocytochemistry and Confocal Microscopy. The localization of MRP1 and MRP4 in polarized BBMEC was also determined by immunocytochemistry using appropriate antibodies and confocal laser scanning microscopy. A top view of the polarized BBMEC monolayers showed a similar staining pattern with the MRP1 or MRP4 antibody, although staining was more intense with MRP4 (Fig. 4, A and B). In contrast, cross-sectional views of the BBMEC monolayers indicated distinct differences in the staining pattern, with MRP1 immunostaining confined to the apical plasma membrane (Fig. 4C) and MRP4 staining both apical and basolateral membranes of the BBMECs (Fig. 4D). There was only weak staining in the negative controls where primary antibodies were omitted (Fig. 4, E and F). Staining for MRP5 was predominantly oriented toward the apical membrane of the BBMEC cells (Fig. 5). Consistent with previous findings (Zhang et al., 2000), there was no MRP2 staining observed (data not shown).

View larger version (103K): [in this window] [in a new window]   Fig. 4. Immunolocalization of MRP1 (A, C, and E) and MRP4 (B, D, and F) in BBMECs by confocal microscopy. MRP1 and MRP4 were detected by biotin-secondary antibody with streptavidin-FITC (green). Cell nuclei were counterstained by propidium iodide (red). Negative controls for MRP1 and MRP4, are shown in E and F, respectively. Cross-sections of the cells for MRP1 and MRP4 are shown in C and D, respectively. The lines indicate the position where the sections were made. Scale bars, 25 µm.

View larger version (73K): [in this window] [in a new window]   Fig. 5. Immunolocalization of MRP5 in BBMEC monolayers by confocal microscopy. Localization of MRP5 was detected with a Texas Red-labeled secondary antibody (red staining). Cell nuclei were stained using Toto-3 (blue staining). Pictured are a top view of MRP5 staining in BBMEC monolayers (A), cross-sectional staining of the cells for MRP5 (B), cell nuclei (C), a computerized overlay of MRP5 and nuclei staining (D), and negative controls exposed to the MRP5 secondary antibody only (E). Scale bars, 25 µm.

Similar confocal studies were performed in MRP1- and MRP2-transfected MDCKII cells (Fig. 6). In contrast to the pattern of distribution observed in BBMEC, MRP1 staining was predominantly localized to the (baso)lateral membrane of the MDCKII-MRP1 cells (Fig. 6C). Examination of MRP2 localization in the MDCKII-MRP2 cells showed a distinct apical plasma membrane distribution (Fig. 6D).

View larger version (89K): [in this window] [in a new window]   Fig. 6. Immunolocalization of MRP1 and MRP2 in MDCKII-MRP1 (A, C, and E) and MDCKII-MRP2 (B, D, and F) using confocal microscopy. MRP1 and MRP2 were detected by biotin-secondary antibody with streptavidin-FITC (green). Cell nuclei were counterstained by propidium iodide (red). Cross sections of cell monolayer staining for MRP1 (C) and MRP2 (D) are shown. The lines indicate the position where the sections were made. The negative controls of MRP1 and MRP2 are shown in E and F, respectively. Scale bars, 25 µm.

	Discussion

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In the present study, the localization of MRP1, MRP4, and MRP5 was examined using primary cultured BBMEC as an in vitro model of the BBB. The rationale for selecting these particular homologs is based on previous reverse transcription-polymerase chain reaction studies, demonstrating the expression of MRP1, MRP4, and MRP5 in both cultured BBMECs and the capillary-enriched fraction from fresh bovine brain homogenates (Zhang et al., 2000). Whereas relatively high expression levels of MRP6 were also found in BBMEC monolayers (Zhang et al., 2000), the lack of availability of an MRP6 antibody prevented the localization of this particular homolog in the bovine brain endothelial cells at the present time.

The localization of MRP homologs in BBMEC monolayers was determined using plasma membrane isolation techniques and confocal microscopy. Based on immunoblots, all the MRP homologs examined were enriched in the plasma membrane fraction compared with cell lysates. The degree of plasma membrane enrichment varied from 5-fold with MRP5 to approximately 18-fold for MRP4. This enrichment in the plasma membrane fraction compared with whole cell lysates suggests a potential role for the MRPs in controlling brain capillary permeability.

Further analysis of the plasma membrane fractions from BBMEC in the current study demonstrated a 3- and 5-fold enrichment of MRP1 and MRP5, respectively, in the apical membrane. The magnitude of enrichment in the apical membrane fraction of the BBMECs was similar to that observed with P-gp (4-fold apical enhancement), a drug efflux transporter that is highly localized to the apical (luminal) plasma membrane of brain microvessel endothelial cells (Cordon-Cardo et al., 1989). In contrast, MRP4 seemed to be equally distributed in both the apical and basolateral plasma membrane of BBMECs with an apical-to-basolateral ratio of approximately 1. Confocal microscopy studies confirmed the localization of MRP1 and MRP5 to the apical plasma membrane and MRP4 distribution to both sides of the BBMEC monolayer. The fact that these two independent methods were in agreement as to the localization of the various MRP transporters in BBMEC provides compelling evidence, supporting the apical (luminal) distribution of MRP1 and MRP5, and a mixed distribution of MRP4 to the apical and basolateral (abluminal) sides of the BBB.

Although the present study is the first to apply discontinuous Ficoll gradient separation techniques to primary cultured BBMECs, the technique has been used previously in isolated brain capillaries to identify glucose transporter distribution (Simpson et al., 2001). In these studies, a polarized apical membrane distribution of the GLUT1 transporter was demonstrated from both binding and Western blot analysis. Likewise, confocal microscopy has been applied to the examination of drug efflux transporters in freshly isolated brain capillaries (Miller et al., 2000), but it has not been reported for cultured brain microvessel endothelial cells. Because the current study was performed with cultured brain endothelial cells, there is a possibility that the gradient densities of the apical and basolateral plasma membrane may be different from the ones reported previously in freshly isolated brain capillaries. However, the fact that P-gp expression was greatest in the 0/5% Ficoll interface (fraction 1), which has been previously reported to be the apical membrane fraction in freshly isolated brain capillaries (Peterson and Hawkins, 1998), suggests the density fractions used in the present study are representative of the apical and basolateral plasma membranes.

It should be noted that the membrane localization for MRP1, and MRP5 in the BBMEC monolayers, is in direct contrast to the localization of these MRP homologs in epithelial cells. Both functional studies and confocal immunohistochemical studies in various epithelial preparations have demonstrated a basolateral distribution for MRP1 (Evers et al., 1996; Wright et al., 1998). Indeed, a basolateral localization of MRP1 was also observed in MDCK-MRP1-transfected cells in the present study. Whereas considerably less is known about the localization of MRP5, it also seems to have a predominantly basolateral localization in polarized epithelial cells based on confocal microscopy studies in a transfected cell line (Wijnholds et al., 2000).

The subcellular localization pattern of MRP4 seems to show variability in localization even within epithelial cells of various origins. Immunohistochemical studies by Lai and Tan (2002) indicated a basolateral localization of MRP4 in MDCKII-MRP4-transfected cells. Basolateral localization was also found in tubuloacinar cells of human prostate (Lee et al., 2000). In contrast, studies by van Aubel et al. (2002) indicated that MRP4 was localized in the apical (brush-border) membrane of proximal tubular cells of human kidney.

The reasons for the different localization patterns for MRP in the brain microvessel endothelial cells compared with epithelial cells are not clear. It is not likely to be due to artifacts in the methods, because two different procedures provided confirmatory results regarding the localization patterns for the transporters in the BBMECs. Furthermore, confocal studies performed on MRP1- and MRP2-transfected MDCKII cells, under identical conditions as those used for the BBMEC, found localization patterns consistent with previous studies in MDCK transfectants. Incomplete polarization of the BBMEC monolayers is also an unlikely explanation. If the BBMEC were not sufficiently polarized, one would anticipate a roughly equal distribution of the various transporters in both the apical and basolateral membrane fractions. Although this did occur with MRP4, it was clearly not the case with the other transporters examined, including P-gp, which displayed a predominantly apical localization in the BBMEC plasma membrane.

There is precedent for differences in localization of membrane proteins in the brain microvessel endothelial cells compared with epithelial cells. An example of this is the transferrin receptor that has a predominantly basolateral plasma membrane distribution in polarized epithelial cells (Fuller and Simons, 1986) and a predominantly apical plasma membrane distribution in the brain microvessel endothelial cells of the BBB (Fishman et al., 1987).

The various MRP homologs show a great deal of overlap in both substrates and inhibitors. However, in general, the MRP1-MRP3 homologs seem to favor anionic- and/or glutathione- or glucuronide-conjugated substrates, whereas the MRP4 and MRP5 homologs tend to favor nucleoside-based substrates (Borst et al., 1999; Schuetz et al., 1999; Lee et al., 2000; Schinkel and Jonker, 2003). Given the general substrate characteristics for the MRP homologs, the apical localization of MRP1 and MRP5 determined in the current study is consistent with previous reports of anion and nucleoside efflux transporters in the BBB. In vivo studies by Adkinson et al. (1994) and Wong et al. (1993) have reported the presence of probenecid-sensitive efflux transporters for valproate and 3'-azido-3'-deoxythymidine (AZT), respectively. In addition, MRP1-mediated efflux transport of grepafloxacin in the BBB has been reported in both mice and rats (Tamai et al., 2000). Together, these findings support the apical (luminal) expression of efflux transporters, such as MRP1 and MRP5, in brain microvessel endothelial cells.

It should be noted that the localization and potential contribution of MRP1 in the BBB is somewhat controversial at this time. Recent studies by Cisternino et al. (2003) using the in situ brain perfusion method reported no differences in the blood-brain transport kinetics for a number of MRP substrates in mrp1 (–/–) knockout mice compared with wild-type controls. However, these studies are in direct opposition to the recent studies by Sugiyama et al. (2003), which examined MRP1 function in the BBB in both wild-type and mrp1 (–/–) knockout mice using the brain efflux index. The results of these studies demonstrated a significantly lower efflux of 17-estradiol-D-17-glucuronide, an MRP1 substrate, from the brains of the mrp1 knockout mice compared with wild-type controls. Based on these results, the authors concluded that MRP1 is located on the apical side of brain microvessel endothelial cells and is functionally important in the elimination of selected anions and conjugated metabolites from the brain. Both Cisternino et al. (2003) and Sugiyama et al. (2003) used the same probe to examine MRP1 function in the BBB (17-estradiol-D-17-glucuronide), thus the differences in the results are most likely attributed to the different techniques for assessing BBB efflux transport processes.

The findings of the present study do not preclude the expression of additional MRP-like transporters in the apical membrane of brain microvessel endothelial cells. Previous studies have shown MRP6 expression at the mRNA level (Zhang et al., 2000), although the localization of the protein in the brain endothelial cells is not currently known. An apical localization of MRP2 has also been reported in rat brain capillaries, suggesting that this particular MRP homolog may contribute to the brain efflux of organic anions and glutathione conjugates (Miller et al., 2000). However, no MRP2 expression was observed in the present study or in previous studies using primary cultured bovine brain microvessel endothelial cells and freshly isolated bovine brain capillaries (Zhang et al., 2000). Others have also failed to demonstrate MRP2 expression or activity in both the primary cultured mouse brain capillary endothelial cells (MBEC4) and isolated rat brain capillaries (Kusuhara et al., 1998; Sugiyama et al., 2003). The reason for the discrepancy in MRP2 expression in the various BBB preparations remains unknown. It could be related to differences in the reverse transcription-polymerase chain reaction primers and/or antibodies used to identify the transporter in the various studies. Alternatively, the expression of MRP2 in the BBB could be species-dependent.

Although unexpected, the presence of MRP4 on both the apical and basolateral plasma membrane of brain microvessel endothelial cells is not unique. A similar distribution pattern is observed with selected amino acid transporters in the BBB. Neutral amino acids enter the brain and exchange readily between the blood and the brain through the non-Na⁺-dependent facilitative transport system L1, which is localized on both the luminal (apical) and the abluminal (basolateral) membrane of the cerebral endothelial cells (Sanchez del Pino et al., 1995). Another example is the nonpolarized localization of organic anion transporter protein 2. Studies in rat brain capillary endothelial cells using confocal microscopy suggest that this transporter is localized along both the luminal and abluminal membranes of the BBB (Gao et al., 1999).

In summary, the present study is the first to identify the localization of several MRP homologs (MRP1, MRP4, and MRP5) in brain microvessel endothelial cells. The apical (luminal) distribution of MRP1 and MRP5 suggests these transporters may play an important role in restricting the passage of selected therapeutic agents into the brain. The observation that MRP4 is expressed on both the apical and basolateral plasma membranes of cultured BBMECs suggests that MRP4 may influence nucleoside transport both into and out of the brain. For those compounds that are substrates of MRP4 and MRP5, the directionality of transport will be dependent on the level of expression of transporters in both the apical and basolateral membrane of the brain endothelial cells and whether the compound of interest is present in the blood or extracellular fluid of the brain.

	Footnotes

 
This research was supported in part by U.S. Public Health Service grants from the National Institutes of Health, R01-CA93558 (to D.W.M.) and R29-CA75466 (to W.F.E.), and a Nebraska Department of Health, Cancer and Smoking Related Diseases research grant (to D.W.M.).

doi:10.1124/jpet.104.068528.

ABBREVIATIONS: BBB, blood-brain barrier; P-gp, P-glycoprotein; MRP, multidrug resistance-associated protein; BBMEC, bovine brain microvessel endothelial cell; MDCK, Madin-Darby canine kidney; FITC, fluorescein isothiocyanate.

Address correspondence to: Dr. Donald W. Miller, Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025. E-mail: dwmiller{at}unmc.edu

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