Expression of various multidrug resistance-associated protein (MRP) homologues in brain microvessel endothelial cells

Brain Research 876 (2000) 148–153 www.elsevier.com / locate / bres

Research report

Expression of various multidrug resistance-associated protein (MRP) homologues in brain microvessel endothelial cells Yan Zhang, Huaiyun Han, William F. Elmquist, Donald W. Miller* Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska, 986025 Nebraska Medical Center, Omaha, NE 68198 -6025, USA Accepted 27 June 2000

Abstract Multidrug resistance-associated protein (MRP) actively transports a broad range of anionic compounds out of the cell. To date, six different homologues of MRP (i.e. MRP1–MRP6) have been identified. The current study examines the expression of the various MRP homologues in both primary cultured bovine brain microvessel endothelial cells (BBMEC) and the capillary-enriched fraction from bovine brain homogenates. RT–PCR analysis demonstrated the presence of MRP1, MRP4, MRP5 and MRP6 in both BBMEC and the capillary-enriched fractions of brain homogenates. While low levels of MRP3 were detected in the BBMEC, it was not observed in the capillary-enriched fraction. In addition, RT–PCR and Western blot studies indicated an absence of MRP2 expression in both blood–brain barrier preparations. The presence of several different MRP homologues in the brain microvessel endothelial cells may be important in controlling the permeability of the blood–brain barrier to organic anions.  2000 Elsevier Science B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Blood–brain barrier Keywords: Blood–brain barrier; Multidrug resistance-associated protein; Brain microvessel; Reverse transcriptase–polymerase chain reaction; Western blot

1. Introduction The brain microvessel endothelial cells that form the blood–brain barrier (BBB) have an important role in controlling the composition of the extracellular fluid environment of the brain. While much attention has been focused on BBB transport systems directed into the brain, transport directed out of the brain also has important implications in drug delivery and CNS toxicity. An example of such a drug efflux transporter in the BBB is P-glycoprotein (P-gp). A member of the ATP-binding cassette (ABC) family of transport proteins, P-gp was first identified in cancer cells, where its presence was associated with multidrug resistance through active transport of drugs out of the cell [29]. However, the expression of P-gp is not limited to malignant cells, as it is also found in normal cells such as the intestine, liver and brain microvessel endothelial cells forming the BBB [6,25]. Both in vitro and *Corresponding author. Fax: 11-402-559-9543. E-mail address: [email protected] (D.W. Miller).

in vivo studies have demonstrated the importance of P-gp in limiting drug permeability in the BBB and CNS toxicity [3,22,27]. Multidrug resistance-associated protein (MRP) is another member of the ABC superfamily of transport proteins. It has approximately 15% amino acid sequence homology to P-gp, and the characteristic ATP binding sites that allow for the active transport of a diverse array of compounds out of the cell. In contrast to P-gp, which in general transports lipophilic and cationic compounds [8], MRP transports organic anions, and glucuronide or glutathione conjugated compounds [2,10,12]. The ability of MRP to transport glucuronide and glutathione conjugates most likely accounts for the apparent overlap observed with some P-gp substrates. While MRP is also found in many multidrug resistant cancer cells and in various normal tissues, determination of the functional significance of this particular drug efflux transporter is more complex due to the existence of several different homologues of MRP. Currently, there are six different genes that have been identified encoding six homologues of the protein,

0006-8993 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 00 )02628-7

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MRP1–MRP6 [5,14,15]. Previous studies reported the expression of MRP1 in cultured brain microvessel endothelial cells [9,11], and to a lesser extent, in freshly isolated brain microvessels [20,24]. However, the expression of the remaining MRP homologues in the BBB remains unknown. The purpose of the present study was to determine the extent to which the various MRP homologues are expressed in the brain endothelial cells that form the BBB. The level of expression of the various MRP homologues in the in vivo BBB, as determined by capillary depletion studies, was compared with the expression levels for MRP homologues in primary cultured brain microvessel endothelial cells. Although there were quantitative differences in the level of expression of the MRP homologues in the capillary depletion and primary cultured BBMEC preparations, the results of the present study indicate that MRP1, MRP4, MRP5, and MRP6 are present in both BBB preparations. Also of note was the variable expression of MRP3 in the BBMEC monolayers and the lack of detectable expression of MRP2 in either the capillary depletion or primary cultured BBMEC preparations. These studies demonstrate that the expression of MRP-related drug efflux transporters in the BBB is complex and involves multiple homologues of the transporter.

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ture was increased to 60 o C for the MRP3 and MRP6 primers. The primers used for MRP1 were first described in Huai-Yun et al. [11], while the primers for MRP2– MRP6 were reported by Kool et al. [14,15]. The primers used for MRP1–MRP6 yield PCR products of 361, 241, 262, 239, 381, and 237 bp, respectively. Samples of each single-strand cDNA were also treated with primers that amplify a 661 bp fragment of the b-actin gene (Stratagene, La Jolla, CA) to allow for quantitative comparison of the expression levels of the MRP homologues. The products from the PCR amplification were separated on 2% agarose gel and stained with ethidium bromide.

2.3. Isolation of bovine liver canalicular membranes Canalicular membranes were obtained from fresh bovine liver lobes using the method described by Rosario et al. [21]. Briefly, the liver was cut into thin slices and added to chilled buffer solution (mannitol 300 mM; EGTA 5 mM; Tris–HCl 18 mM; and PMSF 0.1 mM; pH 7.4). The slices were homogenized and the homogenate was centrifuged at 48 0003g for 30 min. The resulting pellet was resuspended in buffer containing MgCl 2 (15 mM), and centrifuged for 15 min at 24453g. The supernatant was then centrifuged a final time for 30 min at 48 0003g to obtain the canalicular fraction.

2. Materials and methods

2.4. Western blot

2.1. Cell isolation and culturing

Identification of MRP1, MRP2 and P-gp was done using immunoblot techniques described previously [18]. The monoclonal antibodies used for MRP1, MRP2 and P-gp were MRPm6 (Kamiya Biomedical, Seattle, WA), M 2 III-6 (Alexis Corp., San Diego, CA), and C219 (Dako Corp., Carpinteria, CA), respectively. They were all used at 1:100 dilution. The secondary horseradish peroxidase anti-mouse Ig antibody (1:1500 dilution) was purchased from Amersham Life Sciences (Cleveland, OH). The specific protein bands were visualized using a chemiluminescence kit (Pierce, Rockford, IL).

The BBMEC were isolated from fresh cow brains using a combination of enzyme digestion and centrifugal separation techniques [17]. The BBMEC were seeded onto collagen-coated, fibronectin-treated six-well culture plates as previously described [17], and used at confluency (12– 14 days). The hepatoma-derived rat / human fibroblast hybrid cell line, WIF-B, was used as an MRP2-positive cell line [19]. The cells were kindly provided by Dr Ann Hubbard (Johns Hopkins University, Baltimore, MD) and cultured as previously described [19].

2.2. Reverse transcriptase–polymerase chain reaction ( RT–PCR) studies Total RNA from five different preparations of confluent BBMEC monolayers and from five capillary-enriched bovine brain homogenates was isolated as previously described [11]. Single-strand cDNA was synthesized from 1 mg of RNA by reverse transcription using a random hexamer (Perkin Elmer, Branchburg, NJ). Fragments specific to MRP1, MRP2, MRP4, and MRP5 genes were amplified through 45 cycles of 958C for 15 s, 558C for 30 s, and 728C for 15 s. To reduce the nonspecific bands resulting from the GC-rich primers, the annealing tempera-

2.5. Capillary depletion An enriched capillary fraction was collected from brain homogenates using the capillary depletion technique described by Triguero et al. [26]. Following removal of the meninges, the gray matter was collected and homogenized with a glass homogenizer in a pH 7.4 buffer solution consisting of: HEPES (10 mM), NaCl (141 mM), KCl (4 mM), CaCl 2 (2.8 mM), MgSO 4 (1 mM), NaH 2 PO 4 (1 mM) and D-glucose (10 mM). The homogenate was centrifuged in a 13% dextran solution at 54003g for 15 min at 48C. The resulting pellet consisted of brain microvessel segments, red blood cells and brain nuclei. The capillary enrichment of the resulting pellet is demonstrated by Western blots examining P-gp expression

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while MRP1 has a more ubiquitous distribution in the brain [20].

3. Results and discussion

Fig. 1. Western blot analysis of P-gp and MRP1 in brain microvessel endothelial cells. The expression of P-gp (top blot) and MRP1 (bottom blot) was examined in both the capillary depletion and BBMEC monolayer preparations. Samples consisted of the capillary-depleted fraction (lane 1), the capillary-enriched fraction (lane 2), BBMEC lysates (lane 3) and whole brain homogenate (lane 4). A total of 25 mg of protein was loaded onto 7.5% polyacrylamide gels. Primary antibodies for P-gp (C-219) and MRP1 (MRPm6) were used at dilutions of 1:100, secondary antibody was used at a 1:1500 dilution. The protein bands were detected using chemiluminescence.

(Fig. 1). In the brain homogenate, there is a slight but readily apparent band detected with the C219 antibody for P-gp. However, there is no visible staining for P-gp in the non-vascular supernatant and enriched staining for P-gp observed in the capillary pellet (Fig. 1). In contrast, MRP1, is detected in the capillary-enriched pellet, but has greater apparent expression in non-vascular cells in the CNS. These results are consistent with previous studies that indicate P-gp is predominantly localized in the BBB [6],

Of the six MRP homologues examined, MRP1, MRP4, MRP5, and MRP6 were consistently expressed in both the capillary-enriched fraction of the brain homogenate and the confluent BBMEC monolayers (Fig. 2). The expression of MRP3 was variable in BBMEC, being present in only two out of the five samples, and was absent in the capillaryenriched fraction of the brain homogenates. Furthermore, the initial RT–PCR analysis indicated no evidence of MRP2 expression in either the capillary depletion or BBMEC preparations (Fig. 2). To determine if the absence of expression of MRP2 in the BBMEC and capillary depletion preparations were related to species differences in the primer sequence used in the RT–PCR studies, western blot experiments were also performed. Using the specific MRP2 antibody (M 2 III6), an approximately 190 kDa protein was observed in WIFB cell lysates and in canalicular membrane enriched samples from bovine liver homogenates (Fig. 3). In contrast, no MRP2 was detected in the immunoblots of the

Fig. 2. Detection of various MRP homologues in brain microvessel endothelial cells using RT–PCR. Samples prepared from either confluent BBMEC monolayers (lane B) or the capillary-enriched fraction of bovine brain homogenates (lane C) were examined using specific primers for MRP1, MRP4, and MRP5 (A), and MRP2, MRP3, and MRP6 (B). A 100 bp DNA ladder is shown in the far left lane in both panels. In addition, the expression of b-actin was determined for each sample and is shown directly below the respective MRP homologues. RT–PCR analysis for the MRP1, MRP2, MRP4, and MRP5 were performed under 558C annealing temperature, while the MRP3 and MRP6 were performed under 608C annealing temperature.

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Fig. 3. Absence of MRP2 expression in brain microvessel endothelial cells. Western blot analysis of MRP2 was performed using M 2 III-6 monoclonal antibody (1:100 dilution). Protein samples from BBMEC lysates (12 mg; lane 1), bovine brain homogenate (6 mg; lane 4), capillary-enriched fraction of bovine brain homogenate (6 mg; lane 5) and capillary-depleted fraction of bovine brain homogenate (6 mg; lane 6) were examined. Samples from bovine liver canalicular membrane (1 mg; lane 2) and WIF-B cells (0.5 mg; lane 3) were also included as positive controls for MRP2 protein expression.

BBMEC cell lysates, the enriched capillary fraction, or whole brain homogenate (Fig. 3). The detection of MRP2 in the WIFB cell lysates and liver canalicular membranes was anticipated based on previous studies and the high expression of MRP2 in the liver [13,19]. Furthermore, the presence of MRP2 protein band in the bovine canalicular membrane sample indicates that the MRP2 antibody does cross-react with bovine MRP2. Based on the RT–PCR and Western blot studies there appears to be little, if any, expression of MRP2 in the brain microvessel endothelial cells that form the BBB. Comparison of the expression levels of the various MRP homologues indicates a relatively high expression of MRP5 in both the capillary depletion and primary cultured BBMEC preparations (Fig. 4). This finding is in good agreement with the previous studies by Kool et al. [14], demonstrating a high level of MRP5 expression in brain tissue. To insure that the RT–PCR primer, which is based on the sequence of human MRP5, is recognizing bovine MRP5, the 381 bp nucleotide band amplified by the MRP5

Fig. 4. Comparison of the expression levels of the MRP homologues in brain microvessel endothelial cells using RT–PCR. The levels of MRP1– MRP6 were examined in capillary-enriched fractions of bovine brain homogenates (capillary depletion method; dark bars) and BBMEC monolayers (light bars). Values represent the mean6S.E.M. of five separate preparations, except for MRP3 expression in the BBMEC which was only observed in two separate preparations. Quantitative expression levels of the MRP homologues were expressed as the ratio of the MRP band intensity to that of the b-actin band intensity using a densitometer. *P,0.05 compared with the same MRP homologue in capillary depletion group.

primers was subjected to sequence analysis. The results indicated a greater than 92% homology with the predicted base pair sequence for the MRP5 fragment. Based on the current studies, a portion of the MRP5 expression observed previously in the whole brain homogenate [14] appears to be localized in the BBB. While MRP5 is known to function as an anion transporter [16], its localization within the brain microvessel endothelial cells and its contribution in the permeability of anionic compounds and drugs in the BBB remains to be determined. The results of the present study indicate an absence of MRP2 expression and low to undetectable levels of MRP3 expression in BBMEC and capillary depletion preparations (Fig. 4). These findings are also consistent with previous studies reporting little if any MRP2 and MRP3 in whole brain [4,14]. While MRP3 expression was observed in two of the five BBMEC preparations examined, even when present, the MRP3 expression levels were low in comparison to other MRP homologues. This variability in MRP3 detection in the BBMEC may be due to the possibility that the level of MRP3 expression is at or near the sensitivity limits of the RT–PCR. The absence of significant MRP2 and MRP3 expression in the capillary depletion and BBMEC monolayer preparations suggest that the contribution of these particular MRP homologues in organic anion and drug transport in the BBB is minimal. In contrast to the studies by Kool et al. [14] examining MRP homologue expression in whole brain extracts, there were significant levels of MRP4 expression observed in both the capillary depletion and BBMEC preparations (Fig. 4). This discrepancy may reflect an enrichment of MRP4 in the BBB fraction, which may not be apparent when examining expression in whole brain tissue where the brain endothelial cells account for only a small portion of the tissue sampled. Sequence analysis of the 239 bp fragment amplified by the MRP4 primers indicated approximately 94% sequence homology to the predicted sequence for the MRP4 fragment (data not shown). The high level of sequence homology indicated the human based MRP4 primers were interacting with the MRP4 homologue in the bovine preparations. While the function of MRP4 in the brain microvessels remains to be determined, recent studies demonstrating MRP4-mediated resistance to antiviral nucleosides [23] suggest that MRP4 may be involved in nucleoside transport in the BBB. In this regard, it

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should be noted that a probenecid-sensitive efflux transport system for the antiviral nucleoside, AZT, has been demonstrated in the BBB [28]. The present study is the first to examine the expression of the various MRP homologues in the BBB. Previous studies have demonstrated the expression of MRP1 in cultured brain microvessel endothelial cells [9,11,20,24]. However, studies by Seetharaman et al. [24] and Regina et al. [20] have suggested the expression of MRP1 is greater in cultured brain microvessel endothelial cells than that observed in vivo. In the current study, no statistical difference in MRP1 expression was found in the cultured BBMEC compared with the capillary depletion preparation. Since the capillary-enriched fraction isolated from the brain homogenate is likely to contain red blood cells trapped within the capillary segments and closely associated astrocytes, some of the MRP1 expression observed in the capillary depletion preparation may be from either erythrocyte or astrocyte sources. Alternatively, the upregulation of MRP1 expression reported previously in cultured rat brain endothelial cells may be attributable to differences in the culturing conditions used in the current study. In this regard, it should be noted that both the studies by Regina et al. [20] and Seetharaman et al. [24] supplemented the brain microvessel endothelial cell culture media with growth factors that may have had an effect on MRP expression. The current studies were in good agreement with previous reports indicating the expression of MRP1 in the BBB was much less than that found at other sites in the brain [20]. Western blot analysis of MRP1 in brain homogenate, and the capillary-enriched and capillary-depleted fractions of brain homogenate show a stronger MRP1 protein band in both the brain homogenate and capillary-depleted fractions than that observed in the capillary-enriched pellet. The current studies indicate the expression of MRP1 is not confined to the BBB, and even within the brain microvessel endothelial cells, expression of MRP1 is less than that observed with other homologues such as MRP5. This suggests that other MRP homologues in addition to MRP1 may play an important role in the BBB permeability of organic anions. In general, there was good qualitative agreement in the expression of the MRP homologues in the enriched brain capillary fraction and the primary cultured BBMEC. In essence, those homologues expressed in the capillaryenriched fraction of the brain homogenates (MRP1, MRP4, MRP5, MRP6) were also detected in the BBMEC monolayers. However, quantitative differences were observed in the expression levels of MRP5 and MRP6 homologues in the two different BBB preparations, with significantly higher levels of MRP5 and significantly lower levels of MRP6 in the capillary depletion preparations compared with cultured BBMEC monolayers (Fig. 4). The functional significance of these quantitative differences in MRP homologue expression in the BBMEC and capillary depletion preparations remains to be determined.

The present study demonstrates the expression of MRP1, MRP4, MRP5, and to a much lesser extent, MRP6, in capillary endothelial cells forming the BBB. These same transporters were also detected in primary cultured BBMEC, an in vitro model of the BBB. Previous studies using fluorescein as a marker of organic anion efflux transport have shown enhanced accumulation following treatment with MRP inhibitors in BBMEC monolayers [11]. There is also ample evidence of organic anion efflux transport systems in the in vivo BBB [1,7,28]. These transporters have been described mostly by the compounds which inhibit efflux (i.e. probenecid-sensitive). While the results of the present study suggest that organic anion transport at the BBB may be influenced by several different MRP homologues, it is difficult at this time to identify which particular transporter(s) is involved in this process. Furthermore, the present study does not exclude the involvement of other anionic transporters in addition to the MRP homologues discussed, in the BBB transport of organic anions. Depending on the localization of the various MRP isoforms in the brain microvessel endothelial cells, these transporters could function to either restrict the permeability of organic anions and selected therapeutic agents into the central nervous system or remove organic anions and metabolic products from the brain.

Acknowledgements The current study was supported by NIH Grants R15NSOD35364 (D.W.M.) and R29-CA75466 (W.F.E.). The authors wish to thank Dr Ann Hubbard for the WIF-B cells used in the present study.

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