Introduction

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The brain uptake of compounds is restricted by the BBB that acts as a shield to protect the brain. The BBB is formed by the tight junction, an anatomical feature of brain capillary endothelial cells, which connects them to each other (Bradbury, 1979; Pardridge, 1991; Rapoport, 1976). Because the molecules in the circulating blood have to be transported across the cerebral endothelial cells transcellularly to enter the brain due to the presence of the tight junction and the paucity of fenestra or pinocytotic vesicles, the brain penetration of compounds particularly those with high hydrophilicity and/or high molecular weight is restricted (Bradbury, 1979; Pardridge, 1991; Rapoport, 1976). Although several cationic or neutral compounds are lipophilic, their penetration into the brain is restricted by P-gp, a primary active transporter on the luminal membrane of the cerebral endothelial cells that extrudes cationic or neutral compounds from the brain into the circulating blood (Tamai and Tsuji, 1996; Tatsuta et al., 1992; Schinkel et al., 1994).

In addition, cumulative evidence suggests that several organic anions are transported from the brain to the blood across the BBB (Suzuki et al., 1997); Leininger et al. (1991) indicated that the elimination of 1-naphthyl--D-glucuronide after microinjection into the cerebral cortex is mediated by a saturable process. We also analyzed the time profiles of the brain concentrations of an anionic -lactam antibiotic, cefodizime after i.v. administration and found that this compound is actively transported from the brain to the blood across the BBB (Matsushita et al., 1991). The fact that both 1-naphthyl--D-glucuronide and cefodizime are substrates for the primary active transporter for organic anions located on the bile canalicular membrane (cMOAT) (Kobayashi et al., 1990; Kusuhara et al., in press; Sathirakul et al., 1994; Yamazaki et al., 1993) prompts us to hypothesize that cMOAT and/or its related transporter(s) may be expressed on the BBB.

Our purpose is to examine this hypothesis in an in vitro model for studying ligand transport across the BBB. As a cell line, we used MBEC4 cells, established by infecting isolated mouse cerebral endothelial cells with SV40 (Tatsuta et al., 1992). Because 1) MBEC4 cells retain properties specific to cerebral endothelial cells in the brain such as the expression of acetyl low-density lipoprotein receptors, -glutamyl transpeptidase and alkaline phosphatase, 2) a monolayer of MBEC4 cells with little paracellular leakage can be formed with the localized luminal expression of P-gp, this cell line has been used to characterize the transport properties of P-gp located on the BBB (Tatsuta et al., 1992). In our study, we examined the ATP-dependent uptake of DNP-SG and LTC₄, typical substrates for primary active transporters (such as cMOAT and MRP) (Lautier et al., 1996; Loe et al., 1996a; Keppler and Arias, 1997; Kusuhara et al., in press; Oude-Elferink et al., 1995; Yamazaki et al., 1993) into membrane vesicles prepared from MBEC4 cells. In addition, we examined the expression of MRP along with that of cMOAT by Northern and Western blot analyses.

	Materials and Methods

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Chemicals. Unlabeled and [³H]DNP-SG (50.0 µCi/nmol) were synthesized enzymatically using [glycine-2-³H]glutathione (New England Nuclear, Boston, MA), 1-chloro-2,4-dinitrobenzene and glutathione S-transferase (Sigma Chemical Co., St. Louis, MO) as described previously (Kobayasi et al., 1990), and the purity was checked by thin-layer chromatography. [14,15,19,20-³H]LTC₄ (128 µCi/nmol), [¹²⁵I]sheep anti-mouse immunoglobulin and [¹²⁵I]sheep anti-rat immunoglobulin antibodies were purchased from Amersham International (Buckinghamshire, UK). Unlabeled LTC₄ was purchased from Sigma. Rat anti-hMRP antibody, MRPr1 and mouse anti-human P-gp antibody, C219 were purchased from Kamiya Biomedical (Tukwila, WA) and Centocor (Malvern, PA), respectively. All other chemicals were commercially available, of reagent grade and used without further purification.

Cell lines. MBEC4 cells, established by immortalizing the isolated mouse brain capillary endothelial cells by SV40 infection (Tatsuta et al., 1992), were used in our study. The cells were maintained in Dulbecco's modified Eagle's medium (low glucose) supplemented with 10% fetal bovine serum in 5% CO₂-95% air at 37°C.

Membrane vesicle preparation. All steps were performed at 0 to 4°C. Membrane vesicles were prepared by nitrogen cavitation from MBEC4 cells according to the method described previously (Fujii et al., 1994) with minor modification. Cell monolayers were washed and scraped into phosphate-buffered saline. The cells were washed by centrifugation (4000 × g for 10 min) in phosphate-buffered saline and then in buffer A (10 mM Tris-HCl, 250 mM sucrose and 2 mM CaCl₂, pH 7.5). The pellet was stored at -100°C until required. The defrosted cells were equilibrated at 4°C under a nitrogen pressure of 63 kg/cm² for 30 min, and then depressurized rapidly. EDTA (final concentration 1 mM) and 3 volumes of buffer B (10 mM Tris-HCl and 250 mM sucrose, pH 7.5) were added to the lysed cell suspension, then centrifuged at 1000 × g for 10 min at 4°C to remove nuclei and unlysed cells. The supernatant was layered onto a 35% sucrose cushion (10 mM Tris-HCl, 35% sucrose and 1 mM EDTA, pH 7.5) and centrifuged for 30 min at 16,000 × g at 4°C. The interface was collected, diluted with 4 volumes of buffer B and centrifuged at 100,000 × g for 45 min at 4°C. The vesicle pellet was resuspended in buffer B using a 25-gauge needle. Vesicles were stored at -100°C until required. The orientation of membrane vesicles was determined by examining the nucleotide pyrophosphatase in the presence and absence of 1% Triton X with p-nitrophenyl-thymidine 5'-monophosphate as the substrate (Böhme et al., 1994).

Isolation of brain capillaries. Capillaries were isolated from Wistar rats (Male, 240-270 g; Nihon Ikagaku, Tokyo, Japan) using the method described previously (Pardridge et al., 1985) with minor modification. After decapitation, the cerebrum was removed quickly, rinsed with ice-cold buffer containing 122 mM NaCl, 25 mM NaHCO₃, 10 mM D-glucose, 3 mM KCl, 1.4 mM CaCl₂, 1.2 mM MgSO₄, 0.4 mM K₂HPO₄ and 10 mM HEPES (pH 7.4). The minced cerebrum was homogenized with 2 volumes of buffer using a glass/Teflon Potter homogenizer for five periods of 1 min each at 200 rpm. The homogenate was filtered through a 32-µm nylon mesh. Trapped brain capillaries were stored at -80°C until assayed.

Northern blot analysis. Northern hybridization was performed as described previously (Ito et al., 1996-1998). The cDNA fragment encoding the carboxy-terminal ABC region of murine MRP was amplified from MBEC4 cell RNA by RT-PCR using degenerated PCR primers as described previously (Ito et al., 1998). The cDNA fragments containing the linker region of murine mdr1a and mdr1b (nucleotide 1343-1476 and 1921-2098, respectively; Croop et al., 1989) were also prepared by RT-PCR, using RNA from ddy mouse liver as a template. The amplified PCR products were subcloned into the EcoR V site of pBluescript II SK(-) and then the sequence was determined. The cDNA fragments were excised by digestion with EcoRI and HindIII as the probe.

Western blot analysis. A total of 25 µg of the proteins of MBEC4 membrane vesicles and rat brain capillary was fractionated on a 7.5% (w/v) polyacrylamide slab gel containing 0.1% (w/v) SDS and then transferred onto a nitrocellulose filter by electroblotting. The filter was incubated for at least 1 hr in 10 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, 0.05% Tween-20 and 5% (w/v) bovine serum albumin for MRPr1 and in 10 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, 0.05% Tween-20 and 5% (w/v) milk powder for C219 to prevent nonspecific binding of antibodies. Then, it was incubated with MAbs (MRPr1, 1:50; C219, 1:100) for 12 hr, and with [¹²⁵I] sheep anti-rat immunoglobulin and [¹²⁵I]sheep anti-mouse immunoglobulin antibodies for 1 hr at room temperature in the same buffer. Filters were exposed to Fuji imaging plates for 3 h at room temperature and analyzed by a BAS imaging analyzer.

	Results

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Uptake of [³H]DNP-SG and [³H]LTC₄ into membrane vesicles. Determination of nucleotide pyrophosphatase in the presence and absence of 1% Triton X revealed that 65% of the membrane vesicles were inside out. Figure 1 shows the time-profiles for the uptake of [³H]DNP-SG (1 µM) and [³H]LTC₄ (2 nM) into membrane vesicles prepared from MBEC4 cells in the presence or absence of 5 mM ATP. The uptake of both ligands into membrane vesicles was stimulated by ATP (fig. 1).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Time-profiles for the uptake of [3H]DNP-SG (a) and [3H]LTC4 (b) into membrane vesicles. Membrane vesicles (10 µg protein) prepared from MBEC4 cells were incubated at 37°C in 20 µl medium (10 mM Tris-HCl, 250 mM sucrose and 10 mM MgCl2, pH 7.4) containing 1 µM [3H]DNP-SG (a) or 2 nM [3H]LTC4 (b) in the presence (closed symbols) or absence (open symbols) of ATP and its regenerating system (5 mM ATP, 10 mM creatine phosphate and 100 µg/ml creatine phosphokinase). For closed symbols, each point and vertical bar represent the mean ± S.E. from one preparation performed in triplicate. The S.E. is included within the symbol used to designate the mean where the bar is not represented. For open symbols, each point represents a single experiment for the uptake in the absence of ATP.

Osmotic sensitivity of the uptake of [³H]DNP-SG into membrane vesicles. Osmotic sensitivity was studied by examining the uptake of [³H]DNP-SG into membrane vesicles in the presence of several concentrations of sucrose in the medium to confirm that a major part of the accumulation can be accounted for by transport into the intravesicular space, but not by binding to the vesicle surface. As shown in figure 2, the uptake of [³H]DNP-SG at steady-state was reduced as the sucrose concentration in the medium increased. The y-intercept for the relationship between the amount of DNP-SG associated with the vesicles versus the reciprocal of the sucrose concentration in the medium was 5.5 µl/mg protein (fig. 2). The amount of DNP-SG bound to the vesicle surface was less than 10% of the total vesicle uptake, if the transport experiment was performed in isotonic medium.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Osmotic sensitivity of [3H]DNP-SG uptake into membrane vesicles. Membrane vesicles (10 µg protein) prepared from MBEC4 cells were incubated at 37°C in increasing concentrations of sucrose (0.25 M, 0.33 M, 0.50 M, 0.66 M and 1.0 M) in 20 µl incubation medium containing 10 mM Tris-HCl (pH 7.4), 10 mM MgCl2 and 1 µM [3H]DNP-SG in the presence (closed symbols) or absence (open symbols) of ATP and its regenerating system (5 mM ATP, 10 mM creatine phosphate and 100 µg/ml creatine phosphokinase). Accumulation of [3H]DNP-SG was measured at 60 min by rapid filtration. For closed symbols, each point and vertical bar represent the mean ± S.E. from one preparation performed in quadruplicate. The S.E. is included within the symbol used to designate the mean where the bar is not represented. For open symbols, each point represents a single experiment for the uptake in the absence of ATP.

Nucleotide specificity of [³H]DNP-SG uptake into membrane vesicles. Nucleotide specificity of [³H]DNP-SG uptake was examined by replacing ATP by other nucleotides. As shown in table 1, DNP-SG uptake was most efficient with ATP, but not ADP or AMP. GTP was to some extent also able to stimulate the uptake of [³H]DNP-SG into membrane vesicles prepared from MBEC4 cells. One hundred micromolar vanadate, an inhibitor of ATPases, reduced the ATP-dependent uptake of [³H]DNP-SG to 60% of the control value.

Kinetics of the uptake of [³H]DNP-SG and [³H]LTC₄ into membrane vesicles. To obtain the kinetic parameters (K_m and V_max), the saturation of the ATP-dependent uptake of [³H]DNP-SG and [³H]LTC₄ into membrane vesicles was examined (fig. 3). Nonlinear regression analysis yielded K_m values of 0.557 ± 0.066 µM and 0.221 ± 0.047 µM, and V_max values of 5.47 ± 2.66 pmol/min/mg protein and 27.5 ± 3.9 pmol/min/mg protein for DNP-SG and LTC₄, respectively. The fit in figure 3b seems poor. Due to the saturation, the transport in the presence and absence of ATP was similar at higher concentrations of LTC₄ and this fact resulted in the poor fit along with the large deviations in V_max.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Concentration-dependence of [3H]DNP-SG (a) and [3H]LTC4 (b) uptake by membrane vesicles. Membrane vesicles (10 µg protein) were incubated at 37°C with 0.1 µM [3H]DNP-SG or 2 nM [3H]LTC4 in 20 µl medium (10 mM Tris-HCl, 250 mM sucrose and 10 mM MgCl2, pH 7.4) containing different concentrations of DNP-SG or LTC4 for 5 or 2 min, during which linearity is observed in the presence (closed symbols) or absence (open symbols) of ATP and its regenerating system (5 mM ATP, 10 mM creatine phosphate and 100 µg/ml creatine phosphokinase). ATP-dependent uptake was obtained by subtracting the uptake in the absence of ATP from that in its presence. For closed symbols, each point and vertical/horizontal bar represent the mean ± S.E. from one preparation performed in quadruplicate. The S.E. is included within the symbol used to designate the mean where the bar is not represented. For open symbols, each point represents a single experiment for the uptake in the absence of ATP.

Northern and Western blot analyses. The expression of cMOAT and MRP in MBEC4 cells was examined by Northern blot analysis. As shown in figure 4, murine MRP probe hybridized with poly A⁺ RNA from MBEC4 cells at the same location (6.0 kb) as that reported previously (Stride et al., 1996). In contrast, no band was observed if a cMOAT probe was used, although two bands (6.0 and 8.2 kb) were detected in BALB/C mouse liver (data not shown). Furthermore, RT-PCR failed to amplify the cMOAT cDNA fragment with MBEC4 cDNA.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Northern (a) and Western blot (b, c) analyses for MRP in MBEC4 and rat cerebral endothelial cells. a, Northern blot analysis. Five micrograms poly(A)+ RNA from MBEC4 cells was subjected to analysis. The autoradiograph of the blots, probed with [32P]labeled cDNA encoding the carboxy terminal ABC region of murine MRP (lane 1) and the linker region of the transmembrane domain of mdr1b (lane 2) had a 3-day exposure at -80°C to x-ray film. b and c, Western blot analysis. A total of 25 µg protein of rat cerebral endothelial cells (lane 1) and membrane vesicles from MBEC4 cells (lane 2c) was subjected to analysis. The autoradiograph of the blots, probed with anti-MRP antibody, MRPr1 (b) and anti-P-gp antibody, C219 (c) had a 3-day exposure at -80°C to x-ray film.

	Discussion

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We examined the presence of an efflux transporter on the BBB for organic anions by examining the transport of DNP-SG and LTC₄, typical substrates of cMOAT and MRP, into membrane vesicles prepared from MBEC4 cells. ATP stimulated the uptake of [³H]DNP-SG and [³H] LTC₄ into membrane vesicles (fig. 1). This ATP-dependent uptake was osmotically sensitive (fig. 2), suggesting that the major part of the uptake of [³H]DNP-SG is actually due to uptake, and not adsorption to membrane vesicles. Because 1) ATP-dependent uptake of [³H]DNP-SG was inhibited by vanadate and 2) ADP or AMP did not stimulate the uptake of [³H]DNP-SG (table 1), the uptake of [³H]DNP-SG requires hydrolysis of ATP. Not only ATP but also GTP could stimulate the uptake of [³H]DNP-SG into membrane vesicles (table 1), which is consistent with a previous observation in rat bile canalicular membrane vesicles (Ballatori and Truong, 1995; Kobayashi et al., 1990) and in membrane vesicles prepared from hMRP-transfected HeLa cells (Loe et al., 1996b). These results indicate that MRP/cMOAT activity is associated with the plasma membrane of MBEC4 cells. Because Northern and Western blot analyses suggest the expression of MRP in MBEC4 cells, it is plausible that the transport activity is mediated, at least in part, by MRP. In principle, the ATP-dependent uptake of ligands can be ascribed to uptake into inside out membrane vesicles that account for 65% of the prepared vesicles, and therefore, the transport observed in vitro represents efflux of ligands from cells under physiological conditions.

Kinetic parameters for the uptake of DNP-SG and LTC₄ should be compared in several cell lines. Because 1) the ATP-dependent uptake of these glutathione conjugates into membrane vesicles from MBEC4 cells consisted of one saturable component (fig. 3), and 2) Northern and Western blot analyses indicated the expression of MRP but not cMOAT in MBEC4 cells (fig. 4), the Michaelis constants determined in our study may represent the K_m value of the respective ligands for MRP. Kinetic analysis revealed that the K_m value for DNP-SG uptake into MBEC4 membrane vesicles was 0.56 µM (fig. 3), which was consistent with that determined in membrane vesicles prepared from murine leukemia cells, L1210 (K_m = 0.6 µM) (Saxena and Henderson, 1995). In addition, Saxena and Henderson (1995) found that LTC₄ inhibits the ATP-dependent uptake of [³H]DNP-SG into membrane vesicles from L1210 with a K_i of .20 µM, which is also consistent with the K_m of the ATP-dependent uptake of LTC₄ into MBEC4 membrane vesicles (K_m = 0.22 µM) (fig. 3). Collectively, the transport properties of primary active transporter(s) expressed on L1210 and MBEC4 cells resemble each other kinetically. Kinetic parameters determined in these mouse cell lines, however, were different from those reported for hMRP. Jedlitschky et al. (1996) determined the kinetic parameters for the uptake of DNP-SG and LTC₄ in hMRP-transfected HeLa cells and found that the affinity of LTC₄ (0.097 µM) for hMRP is approximately 40-fold higher than that of DNP-SG (3.6 µM). Because the V_max values of LTC₄ and DNP-SG for hMRP are 100 and 409 pmol/min/mg protein in hMRP-transfected HeLa cells, respectively, the clearance for the uptake at tracer concentrations (CL_uptake) defined as the V_max/K_m of LTC₄ (1031 µl/min/mg protein) is much higher than that of DNP-SG (114 µl/min/mg protein) (Jedlitschky et al., 1996). Although the CL_uptake of LTC₄ (124 µl/min/mg protein) was approximately 13-fold higher than that of DNP-SG (9.82 µl/min/mg protein) in MBEC4 vesicles, this difference is ascribed predominantly to the difference in V_max values between the two ligands (27.5 pmol/min/mg protein and 5.47 pmol/min/mg protein for LTC₄ and DNP-SG, respectively). Collectively, these results suggest that the transport characteristics of MRP may be different in mice and humans. Such a difference in the transport properties of MRP between mice and humans has been reported previously. Stride et al. (1997) examined the resistance of murine and human MRP-transfected HEK293 cells to anti-tumor drugs and found that murine and human MRP conferred similar resistance profiles with the exception that only hMRP conferred resistance to anthracyclines. In addition, the accumulation of [³H]vincristine and [³H]VP-16 was reduced and efflux of [³H]vincristine was increased in both murine and human MRP-transfectants, although only hMRP-transfectants displayed reduced accumulation and increased the efflux of [³H]daunomycin (Stride et al., 1997).

Although expression of MRP in the brain was reported using RNase protection assay/Northern blot analysis (Flens et al., 1996; Stride et al., 1996), localization in the brain has not been examined. Western blot analysis revealed that MRP or closely related protein is expressed on the rat brain capillary (fig. 4). Although the localization of this protein on brain capillary endothelial cells (luminal or antiluminal) has not been clarified yet, the results of the present study are consistent with the hypothesis that MRP and/or its related protein, along with P-gp, mediates the efflux of xenobiotics from the CNS. Because 1) glucuronide and glutathione conjugates are substrates for MRP (Lautier et al., 1996; Loe et al., 1996a) and 2) UDP-glucuronosyl transferase and glutathione-S-transferase are expressed in brain parenchyma and cerebral endothelial cells (Ghersi-Egea et al., 1994), it is plausible that the conjugated metabolites formed in the CNS are transported to the blood across the BBB. The presence of such a sequential detoxification of xenobiotics by metabolic enzymes and efflux transporter(s) in the liver has been suggested (Ishikawa, 1992). Based on the results of the transport studies in normal rats (such as Sprague-Dawley and Wistar strains) and mutant rats whose cMOAT function is hereditarily defective (such as Eisai hyperbilirubinemic and TR strains), we and others have demonstrated that the substrate for cMOAT includes glutathione conjugates (such as DNP-SG, LTC₄ and glutathione disulfide) and glucuronide conjugates (such as glycyrrhizin, glucuronides of bilirubin and 6-hydroxy 5,7-dimethyl-2-methylamino-4-(3-pyridylmethyl) benzothiazole dihydrochloride and liquiritigenin) (Lautier et al., 1996; Leier et al., 1994; Loe et al., 1996a; Keppler and Arias, 1997; Kusuhara et al., in press; Oude-Elferink et al., 1995; Yamazaki et al., 1993). In addition, we demonstrated that the nonconjugated compounds such as pravastatin, temocaprilat, cefodizime and the carboxylate form of CPT-11 and SN-38 are extruded via cMOAT (Kusuhara et al., in press; Yamazaki et al., 1993). Because the substrate specificity of cMOAT and MRP is similar (Lautier et al., 1996; Loe et al., 1996a; Keppler et al., 1997; Kusuhara et al., in press; Oude-Elferink et al., 1995; Yamazaki et al., 1993), it is plausible that the penetration of these compounds into the brain is restricted by MRP expressed on cerebral capillary endothelial cells. Although Cornford et al. (1985) and Masereeuw et al. (1994) along with Takasawa et al. (1997) reported active efflux of valproic acid and 3'-azide-3'-deoxythymidine across the BBB by examining the time-dependent change in the brain uptake index and by examining the elimination after administration into the cerebral cortex, respectively, it remains to be established whether these ligands are actively transported across the BBB via MRP.

In conclusion, our study demonstrated the expression of MRP on MBEC4 cells, retaining BBB properties. It is possible that the expression of such efflux transporter(s) along with the presence of metabolic enzymes endows cerebral endothelial cells with the ability to detoxify xenobiotics thereby providing a blood-brain barrier function.