Introduction

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The blood-brain barrier (BBB) is formed by the tight junction, which connects each cerebral endothelial cell to its neighbors (Rapoport, 1976; Bradbury, 1979; Pardridge, 1991, 1995). Along with the characteristics of these cerebral endothelial cells, i.e., the paucity of fenestra or pinocytotic vesicles, the tight junction acts as a barrier to restrict penetration of compounds that are hydrophilic and/or have a high molecular weight (Rapoport, 1976; Bradbury, 1979; Pardridge, 1991, 1995). In terms of a defense system against xenobiotics, therefore, the BBB was previously believed to function as a static wall. However, recent studies suggest the presence of P-glycoprotein on the luminal membrane of the cerebral endothelial cells, which is responsible for the active efflux of some lipophilic ligands from the central nervous system (CNS) into the blood (Tatsuta et al.,1992; Schinkel et al.,1994; Tamai and Tsuji, 1996; Kusuhara et al., 1997). Thus, the BBB is now also considered to be involved in the detoxification of xenobiotics from the brain.

In addition to P-glycoprotein, many kinds of somatic cells have the ability to export organic anions, including glutathione conjugates (Kusuhara et al., 1998a). Based on this transport property, such efflux transporters are referred to as the glutathione S-conjugates (GS-X) pump (Ishikawa, 1992). The GS-X pump consists of many kinds of superfamily members, some of which have been cloned previously (Kool et al., 1997; Hirohashi et al., 1998). These include multidrug resistance-associated protein (MRP) overexpressed on some tumor cells that have acquired the multidrug resistance (Lautier et al., 1997; Loe et al., 1997) and canalicular multispecific organic anion transporter, which is responsible for the biliary excretion of anionic compounds (Oude-Elferink et al., 1995; Yamazaki et al., 1996; Keppler and König, 1997; Kusuhara et al., 1998a). Considering this fact, we can assume the presence of a similar transport system on the BBB (Suzuki et al., 1997), i.e., the hypothesis should be examined that the BBB has a function enabling it to export conjugated metabolites of drugs into the circulating blood and protect the brain from invasion by xenobiotics.

The purpose of the present study was to examine this hypothesis in MBEC4, a cell line established by infecting mouse cerebral endothelial cells with simian virus 40. Because several proteins specific to the cerebral endothelial cells are expressed on MBEC4, and P-glycoprotein is expressed on the luminal membrane, MBEC4 has been used as an in vitro experimental model to characterize the transport properties across the BBB (Tatsuta et al., 1992). Although we previously suggested the expression of MRP and/or its related protein(s) in MBEC4 by Western blot analysis and by demonstrating the ATP-dependent uptake of 2,4-dinitrophenyl-S-glutathione (DNP-SG) and leukotriene C₄ into membrane vesicles isolated from MBEC4 cells (Kusuhara et al., 1998b), the transport properties in the intact cells remain to be clarified. Moreover, it is essential to determine whether the transporter(s) for the glutathione conjugates is/are expressed on the luminal or abluminal membrane of MBEC4 cells. In the present study, we performed a kinetic investigation of the export of glutathione bimane (GS-B) after preloading MBEC4 with its precursor, monochlorobimane (MCB) (Oude-Elferink et al., 1993). In addition, the mechanism for the transport of [³H]GS-B was studied using membrane vesicles isolated from MBEC4 cells.

	Materials and Methods

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Materials. Twenty-four-well multiwell plates and Falcon Cell Culture Inserts (0.31-cm² and 3.0-µm pore size) and Companion TC plates were purchased from Becton Dickinson Labware (Franklin Lakes, NJ). MCB was purchased from Molecular Probes (Eugene, OR). Glycine-2-[³H]glutathione was purchased from New England Nuclear (Boston, MA). Glutathione-S-transferase was purchased from Sigma Chemical (St. Louis, MO). All other chemicals were commercially available, of reagent grade, and were used without further purification.

Cell Culture. MBEC4 cells, established by immortalizing the mouse cerebral endothelial cells by the infection of simian virus 40 (Tatsuta et al.,1992), were used in the present study. Cells were maintained at 37°C in Dulbecco's modified Eagle's medium (low glucose) containing 10% fetal bovine serum and antibiotics under an atmosphere of 5% CO₂/95% air.

Transport Study. Cells were seeded on 24-well multiwell plates at a density of 1.0 × 10⁵ cells per well and cultured for 16 h. The medium used in the transport study was composed of 122 mM NaCl, 3 mM KCl, 0.4 mM K₂HPO₄, 25 mM NaHCO₃, 1.4 mM CaCl₂, 1.2 mM MgSO₄, 10 mM HEPES, and 10 mM glucose, pH 7.4. Five hundred microliters of this medium was added to each well. The cells were preincubated for 20 min with the medium containing 20 µM MCB at 37°C. At time 0, the medium was replaced with fresh medium and incubated at 37°C. At designated times, 100-µl aliquots of medium were collected and the cells were solubilized by adding 500 µl of 10 mM phosphate buffer (pH 6.5) containing 0.1% SDS. To determine the effect of ATP depletion, the medium was replaced at time 0 with fresh medium containing 10 mM NaF, 10 mM NaN₃, and 10 mM 3-O-methyl-D-glucose (3-OMG) (instead of glucose). Also, to determine the effect of 1-chloro-2,4-dinitrobenzene (CDNB), the medium was replaced at time 0 with fresh medium containing CDNB at the concentration indicated. In each instance, after a 20-min incubation, medium was collected and cells were solubilized as described previously. All specimens were diluted with 2 ml of 10 mM phosphate buffer (pH 6.5) to determine the concentration of GS-B by measuring the fluorescence intensity (Ex 386 nm; Em 476 nm) in a fluorospectrophotometer (F-2000, Hitachi Ltd., Tokyo, Japan). To analyze the effect of CDNB kinetically, the efflux rate constant of GS-B was calculated by dividing the GS-B efflux velocity by the intracellular amount of GS-B, estimated by taking the mean value of the intracellular content at 0 and 20 min.

Transwell Study. Cells were seeded on cell culture inserts at a density of 1.0 × 10⁴ cells per well and cultured for 3 days to allow them to form a polarized monolayer. The medium used in the transwell study had the same composition as that described above, and 250 µl and 950 µl of medium was added to the luminal and abluminal compartments of the monolayer, respectively. To determine the time-dependent efflux of GS-B, the cells were preincubated for 20 min with medium containing 20 or 10 µM MCB. At time 0, the medium was replaced by fresh medium and incubated at 37°C. At designated times, 100-µl aliquots of medium were collected from the luminal and abluminal compartments and replaced by equivalent volumes of fresh medium. To determine the dependence on the amount of GS-B preloaded, cells were preincubated with MCB at the indicated concentration and at time 0 the medium was replaced by fresh medium and incubated at 37°C. After 20 min incubation, 100-µl aliquots of medium were collected from the luminal and abluminal compartments. All specimens were diluted with 2 ml of 10 mM phosphate buffer (pH 6.5) to measure the fluorescence intensity.

Preparation of Labeled or Unlabeled GS-B. [³H]GS-B was synthesized enzymatically from glycine-2-[³H]glutathione and MCB using glutathione S-transferase from equine liver. Dithiothreitol was removed from the glycine-2-[³H]glutathione solution by extraction with a 10-fold excess of ethyl acetate in samples acidified to pH 2 with 2 M HCl. Extracted glycine-2-[³H]glutathione was reacted with excess MCB in 50 mM potassium phosphate buffer, pH 6.5, at 37°C in the presence of 30 µg/ml of glutathione S-transferase for 60 min. [³H]GS-B was purified from residues using high-performance liquid chromatography with an ODS2 column (LiChrosorb RP-18, ø 4.6 × 250 mm; GL Science, Tokyo, Japan) equipped with UV detector. The mobile phase consisted of CH₃CN, and H₂O containing 0.1% CF₃COOH (13:87) was applied to the column. [³H]GS-B elution was detected by UV detection at 386 nm (purity >99.5%). Unlabeled GS-B was synthesized based on the same principle and also purified using high-performance liquid chromatography.

Membrane Vesicle Preparation. All steps were performed at 0-4°C. MBEC4 cell monolayers were washed and scraped into phosphate-buffered saline and washed by centrifugation (4000g for 10 min). The pellet was stored at 100°C until required. Membrane vesicles were prepared from defrosted MBEC4 cells according to the method described previously with minor modification (Müller et al., 1994). Vesicles were frozen in liquid nitrogen and stored at 100°C until required. Protein concentrations were determined by the Lowry method using bovine serum albumin as a standard (Lowry et al., 1951). The orientation of membrane vesicles was determined by examining the nucleotide pyrophosphatase in the presence and absence of 1% Triton X-100 with p-nitrophenyl-thymidine 5-monophosphate as the substrate (Böhme et al., 1994).

Transport Study with Membrane Vesicles. The transport study was performed using the rapid filtration technique described in a previous report (Ishikawa et al., 1990). Transport medium (10 mM Tris, 250 mM sucrose, 10 mM MgCl₂, pH 7.4) containing radiolabeled compounds (15 µl), with or without unlabeled substrate, was preincubated at 37°C for 3 min and then was rapidly mixed with 5 µl of membrane vesicle suspension (10 µg of protein) with or without 5 mM ATP and ATP-regenerating system (10 mM creatine phosphate and 100 µg/ml creatine phosphokinase). To check the ATP dependence, ATP was replaced by AMP. The transport reaction was stopped by adding 1 ml of ice-cold buffer containing 250 mM sucrose, 0.1 M NaCl, and 10 mM Tris-HCl (pH 7.4). The stopped reaction mixture was filtered through a 0.45-µm HA filter (Millipore Corp., Bedford, MA) and washed twice with 5 ml of stop solution. Radioactivity retained on the filter was determined using a liquid scintillation counter (LSC-3500, Aloka Co., Tokyo, Japan). The kinetic parameters for the uptake of GS-B into membrane vesicles prepared from MBEC4 were estimated from the following equation: where v₀ is the initial uptake rate of the substrate (in picomoles per minute per milligram of protein), S is the substrate concentration in the medium (in micromolar concentrations), K_m,1 and K_m,2 is the Michaelis-Menten constant (in micromolar concentrations), V_max,1 and V_max,2 is the maximum uptake rate (in picomoles per minute per milligram of protein), and P_dif is the clearance for the nonspecific uptake (in microliters per minute per milligram of protein). The equation was fitted to the ATP-dependent transport velocity, which was defined as the difference in the transport velocity in between the presence and absence of ATP, by an iterative nonlinear least-squares method using a MULTI program (Yamaoka et al., 1981) to obtain estimates of the kinetic parameters. The input data were weighted as the reciprocal of the observed values, and the Damping Gauss Newton algorithm was used for fitting. The fitted line was converted to the v₀/S versus v₀ form (Eadie-Hofstee plot).

	Results

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Export of GS-B from MBEC4. Figure 1 shows the time profiles for the export of GS-B from MBEC4 after preincubating the cells with MCB 20 µM for 20 min. The amount of GS-B excreted into medium increased and the amount remaining in the cells decreased in a time-dependent manner (Fig. 1). A time-dependent increase was observed in the total amount of GS-B (Fig. 1), indicating that MCB continued to be conjugated to GS-B during this period (0-60 min).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Export of GS-B from MBEC4. MBEC4 was preincubated with 20 µM MCB for 20 min. At time 0 the medium was replaced with fresh medium without MCB. The amount of GS-B excreted into the medium (), the amount remaining in the cells (), and the total amount synthesized within the cells () are shown as a function of time. Each point and vertical bar represent the mean ± S.E. (n = 3).

View larger version (59K): [in this window] [in a new window]   Fig. 2.   Effect of ATP depletion on GS-B export. MBEC4 was preincubated with 20 µM MCB for 20 min. At time 0 the medium was replaced with fresh medium containing glucose 10 mM (ATP+) or 10 mM 3-OMG, 10 mM NaF, 10 mM NaN3 (ATP). The amount of GS-B excreted into the medium (dotted column), the amount remaining in the cells (wavy lined column), and the total amount synthesized within the cells (slashed column) up to 20 min are shown. Each point and vertical bar represent the mean ± S.E. (n = 3) in a typical experiment.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Concentration-dependent effect of CDNB on GS-B export. MBEC4 was preincubated with 20 µM MCB for 20 min. At time 0 the medium was replaced with fresh medium containing the indicated concentration of CDNB. The amount of GS-B excreted into the medium (), the amount remaining in the cells (), and the total amount synthesized within the cells () up to 20 min are shown in A. The mean efflux rate constant of GS-B over 20 min is shown in B. Each point and vertical bar represent the mean ± S.E. (n = 3).

Export of GS-B from MBEC4 Monolayer. To clarify the distribution of this export activity, the export of GS-B into the luminal and abluminal compartments from the MBEC4 monolayer was examined. Figure 4 shows the time profiles for the export of GS-B after preincubating the cells with 10 and 20 µM MCB, respectively. After preloading the cells with 10 µM MCB, the amount of GS-B excreted into the luminal compartment was approximately twice that excreted into the abluminal compartment (Fig. 4A), whereas the amount of GS-B excreted into both sides were almost identical at 20 µM (Fig. 4B).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Export of GS-B from MBEC4 monolayer. MBEC4 monolayer was preincubated with 10 µM (A) or 20 µM (B) MCB for 20 min. At time 0 the medium was replaced with fresh medium without MCB. The amount of GS-B excreted into the luminal compartment () and abluminal compartment () at the designated times is shown in each figure. Each point and vertical bar represent the mean ± S.E. (n = 3).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Concentration dependence of GS-B export from MBEC4 monolayer. MBEC4 monolayer was preincubated with the indicated concentrations of MCB for 20 min. At time 0 the medium was replaced with fresh medium without MCB. The amount of GS-B excreted into the luminal compartment () and abluminal compartment () up to 20 min is shown. Each point and vertical bar represent the mean ± S.E. (n = 3).

Transport Study with MBEC4 Membrane Vesicles. The enrichment of leucine amino peptidase was 7.4-fold in plasma membrane vesicles relative to the cell homogenate. In addition, 65% of the membrane vesicles were inside out. Figure 6 shows the time profiles for the uptake of [³H]GS-B (20 nM) into the membrane vesicles prepared from MBEC4 cells in the presence of ATP or 5 mM AMP. The uptake into the membrane vesicles was stimulated by ATP (Fig. 6). The ATP-dependent uptake was linear up to 10 min (Fig. 6).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Time profiles for the uptake of [3H]GS-B into membrane vesicles. Membrane vesicles (10 µg of protein) prepared from MBEC4 cells were incubated at 37°C in 20 µl of medium (10 mM Tris-HCl, 250 mM sucrose, and 10 mM MgCl2, pH 7.4) containing [3H]GS-B (20 nM) in the presence of 5 mM ATP () or 5 mM AMP () and ATP-regenerating system (10 mM creatine phosphate and 100 µg/ml creatine phosphokinase). Each point and vertical bar represent the mean ± S.E. (n = 3).

View larger version (11K): [in this window] [in a new window]   Fig. 7.   Concentration dependence of [3H]GS-B uptake by membrane vesicles. Membrane vesicles (10 µg of protein) were incubated at 37°C with [3H]GS-B in 20 µl of medium (10 mM Tris-HCl, 250 mM sucrose, and 10 mM MgCl2, pH 7.4) containing different concentrations of unlabeled GS-B for 5 min, during which time linearity was observed in the presence of ATP 5 mM () or AMP 5 mM () and ATP-regenerating system (10 mM creatine phosphate and 100 µg/ml creatine phosphokinase) (A). B, active uptake of GS-B into vesicles obtained as the difference in the uptake between the presence of ATP and the presence of AMP. Each point and vertical/horizontal bar represent the mean ± S.E. (n = 3).

	Discussion

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In the present study it was suggested that MBEC4 is able to form glutathione conjugates of compounds (such as MCB) that have invaded the cells and excrete these conjugates from them (Fig. 1). From the following evidence, it has been confirmed that this efflux is mediated by a specific primary active transporter. First, the addition of metabolic inhibitors such as 3-OMG, NaF, and NaN₃ to the medium resulted in a reduction in the amount of GS-B excreted into the medium and an increase in the amount remaining in the cells (Fig. 2). This result suggests that the efflux of GS-B requires energy. Second, CDNB inhibited the efflux of GS-B in a concentration-dependent manner. Because we previously indicated that DNP-SG is taken up into the membrane vesicles isolated from MBEC4 cells in an ATP-dependent manner (Kusuhara et al., 1998b), this result may be accounted for by assuming that CDNB is conjugated with glutathione intracellularly to form DNP-SG, and that the latter competitively inhibits GS-B efflux (Fig. 3A). Third, the uptake of [³H]GS-B into plasma membrane vesicles prepared from MBEC4 showed ATP dependence (Fig. 6).

To investigate the localization of these transport activities, we studied the export of GS-B into the luminal and abluminal compartments from MBEC4 monolayers. After preloading the monolayers with MCB 10 µM, the amount of GS-B excreted into the luminal compartment was approximately twice that excreted into the abluminal compartment (Fig. 4A). In contrast, almost identical amounts of GS-B were excreted into both luminal and abluminal compartments after preloading with 20 µM (Fig. 4B). To characterize this phenomenon from a kinetic point of view, we investigated the concentration dependence of the efflux of GS-B from MBEC4 monolayers. The amount of GS-B excreted into the abluminal compartment was markedly affected by the preloading MCB concentration, whereas that excreted into the luminal compartment was less affected (Fig. 5). These results suggest that the efflux of GS-B into the luminal compartment is almost saturated, even at the lowest concentration (5 µM precursor). In contrast, between 5 and 25 µM MCB, the abluminal efflux of GS-B is under linear conditions (Fig. 5). The contribution of passive diffusion to the efflux of GS-B into the abluminal compartment may be minimal at the higher concentration of MCB, because CDNB markedly reduced the efflux of GS-B after preloading the cells with 20 µM MCB (Fig. 3). Collectively, the affinity of the efflux transport system on the luminal membrane is considered to be higher than that on the abluminal membrane. Under conditions in which the ligand concentration is low enough, preferential luminal efflux should be observed in the BBB (Fig. 5).

To analyze the mechanism for the transport of GS-B, we investigated the uptake of [³H]GS-B into membrane vesicles prepared from MBEC4. The uptake of [³H]GS-B was stimulated in the presence of ATP (Fig. 6), suggesting the existence of primary active transport for the cellular efflux of GS-B. Kinetic analyses showed that two differential systems were involved in the ATP-dependent uptake of GS-B; a high-affinity-low-capacity system (K_m = 35 nM, V_max = 1.12 pmol/min/mg protein) and a low-affinity-high-capacity system (K_m = 14 µM, V_max = 8.83 pmol/min/mg protein) (Fig. 7B). Together with the results from the efflux studies in the monolayers, it seems that the luminal preferential efflux is mediated by the high-affinity transporter.

In our previous report we found that MRP and/or its related protein(s) are expressed in both MBEC4 and rat cerebral microvessel endothelial cells (BBB) using Northern and Western blot analyses (Kusuhara et al., 1998b). Taken together, the export of glutathione conjugates such as GS-B, DNP-SG, and leukotriene C₄ may be mediated by these transporters. In particular it is suggested that the high-affinity efflux system located on the luminal side of the BBB can serve as one of the detoxification systems of the CNS by excreting conjugated metabolites into the circulating blood. Because it has been shown that cerebral endothelial cells are endowed with high enzyme activity, including UDP-glucuronosyl transferase and glutathione-S-transferase (Ghersi-Egea et al., 1994), and that the substrate specificity of MRP and its related proteins is very similar because glutathione and glucuronide conjugates are transported (Lautier et al., 1996; Loe et al., 1996a,b), it is suggested that lipophilic compounds invading the BBB are metabolized by these enzymes and converted into more hydrophilic anions and sequentially excreted into the circulating blood with the aid of MRP and/or its related proteins. Indeed, Ishikawa and collaborators (1992) proposed that this kind of sequential biochemical system of metabolism (molecular conversion) and excretion (molecular transport) acts as a defense system to detoxify xenobiotics in the liver and some tumor cells. In addition, if we consider that several clinically important anionic drugs [such as methotrexate (Masuda et al., 1997), pravastatin (Yamazaki et al., 1996), temocaprilat (Ishizuka et al., 1997), and CPT-11 (Chu et al., 1997a,b)] are substrates for canalicular multispecific organic anion transporter, an MRP-related protein, along with cumulative in vivo evidence suggesting the presence of an efflux transport system(s) for organic anions from CNS to circulating blood across the BBB (Suzuki et al., 1997), it is possible that the brain entry of these drugs is restricted by the presence of an efflux transporter located on the luminal membrane of the BBB.

In conclusion, the results of the present study suggest the expression of a high-affinity transport system for the primary active export of glutathione conjugates on the luminal membrane of MBEC4 cells. This transport system may play an important role in restricting the brain entry of xenobiotics, including clinically important anionic drugs. In addition, monolayers of MBEC4 are an excellent tool for studying the transport of anionic compounds across the BBB.