Introduction

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L-Cystine and L-glutamic acid (L-Glu) exchange transporter, referred to as system x_c, is composed of the heavy chain of 4F2 cell surface antigen (4F2hc/CD98) and xCT protein (Sato et al., 1999). The physiological flux via system x_c involves entry of L-cystine and exit of L-Glu. Therefore, it plays a key role in the synthesis of glutathione because L-cysteine (L-Cys), which is reduced from L-cystine in the cells, is one of the rate-limiting precursor amino acids for glutathione synthesis (Bannai and Tateishi, 1986). Under oxidative stress in the brain, L-cystine and/or L-Cys need to undergo influx transport from the circulating blood to the brain across the blood-brain barrier (BBB) to synthesize glutathione as a protection against free radicals, peroxides, and other toxic compounds in the central nervous system (CNS) (Meister and Anderson, 1983). Glutathione is present in a relatively high concentration (2 µmol/g brain) in brain parenchymal cells (Folbergrova et al., 1979), and its depletion causes a serious disorder in the CNS (Skullerud et al., 1980; Herrera et al., 2001). L-Cys is usually transported by a neutral amino acid transporter, such as system L, which is present at the BBB (Smith et al., 1987; Boado et al., 1999). However, the concentration of L-Cys in the plasma (10-20 µM) is 10 times lower than that of L-cystine (100-200 µM) (Dröge et al., 1991).

Benrabh and Lefauconnier (1996) suggested that system x_c is not present at the BBB under normal conditions since [¹⁴C]L-Glu transport from blood to the brain, studied using the brain perfusion technique, was not reduced by L-cystine. Nevertheless, system x_c is an inducible transporter under oxidative stress conditions since xCT mRNA is induced by treatment with diethyl maleate (DEM), lipopolysaccharide, and nitric oxide donor (Sato et al., 1999; Bridges et al., 2001; Tomi et al., 2002). DEM is often used as a reagent to reduce intracellular glutathione, i.e., oxidative stress condition, because it is relatively less toxic than some other electrophilic agents (Bannai, 1984; Sato et al., 1999; Kim et al., 2001). Using in vivo integration plot analysis, we recently reported that L-cystine uptake by brain and eye is activated following a 12-h DEM infusion from the external carotid artery, and this enhancement is inhibited in the presence of L-Glu and L--aminoadipic acid (L-AAA), substrates of system x_c. This suggests that L-cystine influx transport via system x_c is activated by DEM at the BBB and blood-retinal barrier in vivo (Hosoya et al., 2001).

The purpose of this study was to elucidate the mechanism of L-cystine uptake enhancement, as well as the expression and regulation of system x_c under oxidative stress, following DEM treatment using an immortalized mouse brain capillary endothelial cell line, MBEC4, as an in vitro model of the BBB (Tatsuta et al., 1992).

	Materials and Methods

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Animals. Male ddY mice, weighing 25 to 30 g, were purchased from Charles River (Yokohama, Japan) and used as positive controls for reverse transcription-polymerase chain reaction (RT-PCR). The investigations using mice described in this report conformed to the guidelines of the Animal Care Committee, Graduate School of Pharmaceutical Sciences, Tohoku University.

Cell Culture. MBEC4 cells (passages 10-40) were maintained in Dulbecco's modified Eagle's medium (Nissui Co., Tokyo, Japan) supplemented with 10% fetal bovine serum (Moregate, Bulimba, Australia) (Tatsuta et al., 1992) in the presence or absence of DEM (Wako Pure Chemicals, Osaka, Japan), a sulfhydryl-reactive agent, at 37°C in a humidified atmosphere of 5% CO₂/95% air.

RT-PCR Analysis. Total RNA was prepared from phosphate-buffered saline (PBS)-washed cells using Trizol reagent (Invitrogen, Carlsbad, CA). Single-strand cDNA was made from 1 µg of total RNA by RT using oligo(dT) primer. PCR was performed using GeneAmp (PCR system 9700; Applied Biosystems, Foster City, CA) with xCT- or 4F2hc-specific primers through 40 cycles of 94°C for 30 sec, 60°C for 1 min, and 72°C for 1 min. The sequences of the specific primers were as follows: the sense sequence was 5'-CCTGGCATTTGGACGCTACAT-3' and antisense sequence was 5'-TCAGAATTGCTGTGAGCTTGCA-3' for mouse xCT, and the sense sequence was 5'-CTCCCAGGAAGATTTTAAAGACCTTCT-3' and antisense sequence was 5'-TTCATTTTGGTGGCTACAATGTCAG-3' for mouse 4F2hc. The PCR products were separated by electrophoresis on an agarose gel in the presence of ethidium bromide and visualized using an imager (EPIPRO 7000; Aisin, Aichi, Japan). The PCR products of the expected length were then cloned into a plasmid vector using p-GEM-T Easy Vector system I (Promega, Madison, WI) and amplified in Escherichia coli. Several clones were then sequenced from both directions using a DNA sequencer (model 4200; LI-COR Biosciences, Lincoln, NE).

Quantitative Real-Time RT-PCR. Quantitative real-time RT-PCR analysis was performed using an ABI PRISM 7700 sequence detector system (Applied Biosystems) with 2× SYBR green PCR master mix (Applied Biosystems) according to manufacturer's protocol. To quantify the amount of specific mRNA in the samples, a standard curve was generated for each run using the plasmid (pGEM-T Easy Vector; Promega) containing the gene of interest. This enabled standardization of the initial mRNA content of MBEC4 cells relative to the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PCR was performed using xCT, 4F2hc, or GAPDH-specific primers, and the cycling parameters are those given above. The specific primers for xCT and 4F2hc are those listed above and for GAPDH as follows: the sense sequence was 5'-TGATGACATCAAGAAGGTGGTGAAG-3', and antisense sequence was 5'-TCCTTGGAGGCCATGTAGGCCAT-3'.

Determination of Intracellular Glutathione. Measurement of the total glutathione of PBS-washed cells using a BIOXYTECH GSH-420 kit was performed according to manufacturer's protocol (Oxis Research International, Portland, OR). The method is based on the formation of a chromophoric thione (Griffith, 1980). Protein assay was performed with a DC protein assay kit (Bio-Rad, Hercules, CA) with bovine serum albumin as a standard.

[¹⁴C]L-Cystine and [³H]L-Glutamic Acid Uptake. L-[U-¹⁴C]Cystine ([¹⁴C]L-cystine, 303 mCi/mmol; PerkinElmer Life Sciences, Boston, MA) or L-[2,3-³H]glutamic acid ([³H]L-Glu, 24 Ci/mmol; PerkinElmer Life Sciences) uptake was measured according to a previous report (Terasaki et al., 1991). Cells (5 × 10 ⁴ cells/cm²) were cultured at 37°C for 2 days on a 24-well plate (BD Biosciences, San Jose, CA) and washed with 1 ml of extracellular fluid (ECF) buffer consisting of 122 mM NaCl, 25 mM NaHCO₃, 3 mM KCl, 1.4 mM CaCl₂, 1.2 mM MgSO₄, 0.4 mM K₂HPO₄, 10 mM D-glucose, and 10 mM Hepes (pH 7.4) at 37°C. Uptake was initiated by applying 200 µl of ECF buffer containing 0.1 µCi of [¹⁴C]L-cystine (1.7 µM) or 0.5 µCi of [³H]L-Glu (104 nM) at 37°C in the presence or absence of inhibitors. Na⁺-free ECF buffer was prepared by equimolar replacement of NaCl and NaHCO₃ with choline chloride and choline bicarbonate, respectively. After a predetermined time period, uptake was terminated by removing the solution and then immersing cells in ice-cold ECF buffer. The cells were then solubilized in 750 µl of 1% Triton X-100/PBS. An aliquot (15 µl) was taken for protein assay using a DC protein assay kit with bovine serum albumin as a standard. The remaining solution (500 µl) was mixed with 5 ml of scintillation cocktail (Hionic-Fluor; Packard BioScience, Meriden, CT) for measurement of radioactivity in a liquid scintillation counter (LS6500; Beckman Coulter Inc., Fullerton, CA).

Data Analysis. For kinetic studies, the Michaelis-Menten constant (K_m) and maximum uptake rate (V_max) of L-cystine or L-Glu uptake were calculated from eq. 1 using the nonlinear least-squares regression analysis program, MULTI (Yamaoka et al., 1981).

(1)

where V and C are the uptake rate of L-cystine or L-Glu at 5 min and the concentration of L-cystine or L-Glu, respectively. To analyze the mechanism of inhibition of [¹⁴C]L-cystine uptake by L-Glu and [³H]L-Glu uptake by L-cystine, the inhibitory constant (K_i) was calculated from eq. 2 using MULTI (Yamaoka et al., 1981).

(2)

where I is the concentration of L-Glu or L-cystine.

	Results

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Carrier-Mediated Uptake of L-Cystine by MBEC4 Cells. The time courses of [¹⁴C]L-cystine uptake by MBEC4 cells are shown in Fig. 1. [¹⁴C]L-Cystine uptake was increased linearly for at least 30 min at 37°C. The [¹⁴C]L-cystine uptake (cell-to-medium ratio) was 239 ± 23 µl/mg of protein and 644 ± 60 µl/mg of protein at 10 and 30 min, respectively. At 4°C and 30 min, [¹⁴C]L-cystine uptake was reduced by 97.6% (15.4 ± 1.8 µl/mg of protein) (Fig. 1). Under Na⁺-free conditions, L-cystine uptake remained unchanged compared with that in the presence of Na⁺ (Table 1). L-Cystine uptake was saturable with a K_m of 63.7 ± 13.9 µM (mean ± S.D.) (Fig. 2A). Moreover, the Lineweaver-Burk plot showed that the two lines of the L-cystine uptake in the presence or absence of 160 µM L-Glu intersected the ordinate axis. This indicates that L-Glu competitively inhibited L-cystine uptake with a K_i of 83.5 ± 10.3 µM (mean ± S.D.) (Fig. 2B). These results suggest that L-cystine uptake by MBEC4 cells is temperature- and concentration-dependent as well as Na⁺-independent. The inhibition study was performed to characterize the [¹⁴C]L-cystine uptake by MBEC4 cells (Table 1). [¹⁴C]L-Cystine uptake was inhibited by more than 90% by L-cystine and L-Glu in the presence or absence of Na⁺ and by L-AAA, L-homocysteic acid (L-HCA), and L-quisqualic acid (L-QQA) in the presence of Na⁺, all of which are substrates for system x_c (Sato et al., 1999). Partial inhibition was observed by DL-diaminopimelic acid (DL-DPA) and L-aspartic acid (L-Asp) by up to 42%, whereas L-leucine (L-Leu), L-lysine (L-Lys), L-arginine (L-Arg), -aminobutyric acid, and p-aminohippuric acid had no effect on [¹⁴C]L-cystine uptake.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Time courses of [14C]L-cystine uptake by MBEC4 cells. [14C]L-Cystine (1.7 µM) uptake was performed at 37°C () and 4°C (). Each point represents the mean ± S.E.M. (n = 4).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Concentration dependence of L-cystine uptake by MBEC4 cells (A) and Lineweaver-Burk plot of L-cystine uptake by MBEC4 cells showing competitive inhibition by L-Glu (B). [14C]L-Cystine (1.7 µM) uptake was performed in the absence () or presence of 160 µM L-Glu () at 37°C for 5 min. Each point represents the mean ± S.E.M. (n = 3-4). The Km for L-cystine uptake is 63.7 ± 13.9 µM (mean ± S.D.) according to eq. 1. The Ki for L-glutamic acid is 83.5 ± 10.3 µM (mean ± S.D.) according to eq. 2.

Carrier-Mediated Uptake of L-Glutamic Acid by MBEC4 Cells. Although L-Glu exhibited competitive inhibition of L-cystine uptake in Fig. 2B, [³H]L-Glu uptake was performed to characterize the L-Glu transport of L-cystine and L-Glu exchange transporter, system x_c, in MBEC4 cells. Figure 3 shows the time courses of [³H]L-Glu uptake by MBEC4 cells in the presence or absence of Na⁺ at 37 and 4°C. [³H]L-Glu uptake was increased linearly for at least 30 min in the presence or absence of Na⁺ at 37°C. The cell-to-medium ratio of [³H]L-Glu at 30 min was 1110 ± 140 µl/mg of protein and 123 ± 13 µl/mg of protein in the presence and absence of Na⁺, respectively. In contrast, at 4°C, the cell-to-medium ratio of [³H]L-Glu at 30 min was 4.66 ± 0.59 µl/mg of protein. This was reduced by 99.6 and 96.2% compared with that in the presence and absence of Na⁺ at 37°C, respectively. Although this suggests that L-Glu uptake by MBEC4 cells exhibits both Na⁺-dependent and -independent processes, a further study was performed under Na⁺-free conditions because system x_c has been reported by others to be Na⁺-independent (Sato et al., 1999), and this is shown in Table 1. [³H]L-Glu uptake in the absence of Na⁺ was saturable with a K_m of 48.1 ± 14.2 µM (mean ± S.D.) (Fig. 4A). A Lineweaver-Burk plot showed that the two lines of the L-Glu uptake in the presence or absence of 100 µM L-cystine intersected the ordinate axis. This indicates that L-cystine competitively inhibited L-Glu uptake with a K_i of 24.9 ± 3.5 µM (mean ± S.D.) (Fig. 4B). Table 2 shows the inhibitory effect of several amino acids on [³H]L-Glu uptake at 5 min. [³H]L-Glu uptake by MBEC4 cells was inhibited by more than 90% by L-Glu, L-cystine, L-AAA, L-HCA, and L-QQA. It was partially inhibited (50%) by L-Asp, whereas L-Leu and L-Arg had no effect on [³H]L-Glu uptake.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Time courses of L-Glu uptake by MBEC4 cells. [3H]L-Glu (104 nM) uptake was performed in the presence of Na+ at 37°C () and 4°C () or absence of Na+ at 37°C (). Each point represents the mean ± S.E.M. (n = 4).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Concentration dependence of L-Glu uptake by MBEC4 cells (A) and Lineweaver-Burk plot of L-Glu uptake by MBEC4 cells showing competitive inhibition by L-cystine (B). [3H]L-Glu (104 nM) uptake was performed in the absence () or presence of 100 µM L-cystine () at 37°C and 5 min. Each point represents the mean ± S.E.M. (n = 3-4). The Km for L-Glu uptake is 48.1 ± 14.2 µM (mean ± S.D.) according to eq. 1. The Ki for L-cystine is 24.9 ± 3.5 µM (mean ± S.D.) according to eq. 2.

Expression of xCT and 4F2hc mRNA in MBEC4 Cells. The expression of xCT and 4F2hc mRNA in MBEC4 cells was analyzed by RT-PCR. The bands corresponding to the expected 182 and 141 base pairs for xCT and 4F2hc, respectively, were amplified from MBEC4 cells and mouse brain as a positive control (lane 1) (Fig. 5). The DNA sequence of the bands of MBEC4 cells was identical to mouse xCT (Sato et al., 1999) and 4F2hc (Parmacek et al., 1989) with a homology of 100%.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   RT-PCR analysis of xCT (A) and 4F2hc (B) in MBEC4 cells. Lane 1, mouse brain as a positive control in both xCT (A) and 4F2hc (B); lane 2, MBEC4 cells; , in the absence of reverse transcriptase for MBEC4 cells.

Effect of DEM on System x_c Expression in MBEC4 Cells. The effect of DEM treatment for 12 h on mRNA expression, [¹⁴C]L-cystine uptake, and glutathione concentration was examined in MBEC4 cells. The expression of xCT mRNA was significantly increased up to 500 µM in a concentration-dependent manner for DEM treatment, whereas 4F2hc was unchanged (Fig. 6A). Corresponding to xCT mRNA expression, [¹⁴C]L-cystine uptake was enhanced up to 500 µM in a concentration-dependent manner for DEM treatment (Fig. 6B). The intracellular glutathione concentration correlated with the L-cystine uptake rate (r ² = 0.80) (Fig. 7). The xCT mRNA level, [¹⁴C]L-cystine uptake activity, and intracellular glutathione concentration following 12 h of 500 µM DEM treatment were 2.88-, 2.20-, and 1.44-fold greater than that of the control (no treatment), respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effect of DEM concentration on mRNA expression of xCT and 4F2hc (A) and [14C]L-cystine uptake activity (B) in MBEC4 cells. A, , xCT; , 4F2hc; mRNA expression level. Each mRNA expression level was normalized by the GAPDH mRNA expression and plotted as a relative ratio to the control (no treatment). B, DEM treatment was performed for 12 h. Each point represents the mean ± S.E.M. (n = 3-8). , p < 0.05; , p < 0.001, significantly different from respective controls (no treatment).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Relationship between L-cystine uptake rate and the intracellular glutathione concentration in MBEC4 cells. DEM treatment was performed for 12 h. The solid line represents the correlation between [14C]L-cystine uptake rate (results from Fig. 6B) and intracellular glutathione concentration, r 2 = 0.80. Each point represents the mean ± S.E.M. (n = 3-4).

	Discussion

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The present study demonstrates that MBEC4 cells used as an in vitro model of the BBB express xCT and 4F2hc mRNA (Fig. 5), and L-cystine and L-Glu uptakes via system x_c are Na⁺-independent and concentration-dependent (Figs. 1-4). The K_m value for L-cystine uptake is 63.7 µM (Fig. 2), which is very similar to those of 70 and 81 µM reported in cultured human umbilical vein endothelial cells (Miura et al., 1992) and mouse xCT and 4F2hc cRNA-coinjected Xenopus laevis oocytes, respectively (Sato et al., 1999). The K_m value for L-Glu is 48.1 µM (Fig. 4), which is 4.2-fold smaller than that of 200 µM in cultured human fibroblasts (Bannai and Kitamura, 1980). Nevertheless, mutual inhibition was observed for L-cystine and L-Glu uptake by MBEC4 cells. The [¹⁴C]L-cystine uptake was competitively inhibited by 160 µM L-Glu with a K_i of 83.5 µM (Fig. 2B), which is comparable with the K_m value of L-Glu uptake in this study. The [³H]L-Glu uptake was also competitively inhibited by 100 µM L-cystine with a K_i of 24.9 µM (Fig. 4B), which is comparable with the K_m value of L-cystine uptake in this study. These results support the hypothesis that both L-cystine and L-Glu uptake is mediated via system x_c in MBEC4 cells. Moreover, the uptake of both [¹⁴C]L-cystine and [³H]L-Glu is strongly inhibited by system x_c substrates, such as L-AAA, L-HCA, and L-QQA (Tables 1 and 2). This manner of inhibition is consistent with system x_c characteristics as reported elsewhere (Miura et al., 1992; Sato et al., 1999). System b^0,+, which is also an Na⁺-independent transporter, mediates the transport of L-cystine, L-Leu, and basic amino acids (Pfeiffer et al., 1999). [¹⁴C]L-Cystine uptake by MBEC4 cells excludes the involvement of system b^0,+ since L-Leu, L-Lys, and L-Arg produced no marked inhibition (Table 1). Na⁺-dependent L-Glu uptake is present in MBEC4 cells (Fig. 3), and this suggests that an Na⁺-dependent L-Glu and L-Asp transporter, system X_AG, is involved in L-Glu transport in the presence of Na⁺ since excitatory amino acid transporter subtypes 1~3 are expressed at the BBB (O'Kane et al., 1999). Although L-Asp and DL-DPA are dicarboxylic amino acids like L-Glu and L-cystine, the inhibition ratio was lower than that of L-Glu and L-cystine. It seems that these amino acids have a lower affinity for system x_c than L-Glu and L-cystine since L-Asp and DL-DPA have, respectively, shorter and longer carbon chains than L-Glu (Tables 1 and 2).

Our previous in vivo study indicated that [¹⁴C]L-cystine uptake by the brain following a 12-h DEM infusion (7.5 µM) via the external carotid artery was significantly increased (1.6-fold), compared with saline infusion (Hosoya et al., 2001). In the presence of L-Glu and L-AAA, [¹⁴C]L-cystine uptake by the brain was inhibited by 80% of the amount of enhanced uptake produced by DEM treatment in the brain, suggesting that system x_c-mediated L-cystine transport is activated under oxidative stress conditions at the BBB following DEM treatment. The present study demonstrates the induction and function of the L-cystine transporter in MBEC4 cells used as an in vitro model for the BBB. DEM induced xCT mRNA as well as L-cystine uptake in a concentration-dependent manner following DEM treatment (Fig. 6). This induction under oxidative stress conditions following DEM treatment is in good agreement with data in mouse macrophages (Sato et al., 1999), human glioma cells (Bannai, 1984), and a rat retinal capillary endothelial cell line (Tomi et al., 2002). However, the 4F2hc mRNA level remained unchanged (Fig. 6A), and this is probably due to the fact that the amount of 4F2hc mRNA was 46-fold greater than xCT mRNA, according to quantitative real-time RT-PCR analysis under normal conditions (data not shown). Therefore, the 4F2hc protein is large enough to bind to xCT protein, although xCT mRNA increased 2.9-fold following treatment with 500 µM DEM for 12 h. The intercellular glutathione concentration was also enhanced with increasing L-cystine uptake by MBEC4 cells (Fig. 7), supporting the hypothesis that activation of L-cystine uptake via system x_c in MBEC4 cells stimulates glutathione synthesis in the cells under oxidative stress conditions. Since [¹⁴C]L-cystine uptake by the brain is activated following DEM infusion in vivo (Hosoya et al., 2001), glutathione could be synthesized in brain parenchymal cells (Sagara et al., 1993) as well as brain endothelial cells. Although L-Glu transport from blood to brain can be enhanced due to induction of system x_c at the BBB under the oxidative stress conditions, it may not affect L-Glu levels in the brain. The brain efflux index method has demonstrated that L-Glu undergoes efflux from brain to blood (Hosoya et al., 1999). Moreover, O'Kane et al. (1999) suggested that excitatory amino acid transporter subtypes 1~3 are present on the abluminal (brain) side of the BBB and mediate brain-to-blood efflux transport of L-Glu.

A possible physiological role for the induction of system x_c includes action as a detoxifying system in the brain and brain capillary endothelial cells by supplying L-cystine/L-Cys for the synthesis of glutathione. Maintaining the glutathione concentration in the brain is essential to support normal CNS functions (Skullerud et al., 1980). Therefore, under oxidative stress conditions, system x_c is likely to be induced at the BBB to supply L-cystine/L-Cys to the brain as well as the brain endothelial cells. Parkinson's disease, Alzheimer's disease, dementia, and Huntington's chorea appear to be associated with oxidative stress in the brain (Karelson et al., 2001; Maksimovic et al., 2001; Serra et al., 2001) due to a fall in glutathione levels in neuronal and glial cells (Herrera et al., 2001). Therefore, system x_c at the BBB may play a number of important roles in supplying L-cystine to the brain under oxidative stress conditions and maintaining the glutathione concentration in the brain to protect it against CNS disorders. Although Benrabh and Lefauconnier (1996) have suggested that system x_c is not present at the BBB under normal conditions, system x_c is likely to be induced under oxidative stress conditions and to stimulate the supply of L-cystine to the brain. MBEC4 cells express xCT and 4F2hc mRNA and play a role in L-cystine uptake even under normal conditions. However, our previous in vivo study indicated that the apparent influx clearance of [¹⁴C]L-cystine was 3.63 µl/(min · g brain) following a saline infusion (control) (Hosoya et al., 2001). This value is 12-fold greater than that of inulin [0.308 µl/(min · g brain)] (Kakee et al., 1996), suggesting that the L-cystine transport system may operate at the BBB and supply L-cystine to the brain under normal conditions. There are two possible hypotheses to explain this: 1) the BBB expresses system x_c even under normal conditions, and 2) MBEC4 cells acquire system x_c during development of this cell line. Further studies are needed to see whether system x_c is present at the BBB under normal conditions in vivo. Taking all these current and previous in vivo results into consideration, we have shown that L-cystine undergoes influx transport from the circulating blood to the brain via system x_c at the BBB, under oxidative stress conditions, following DEM treatment to protect the brain from oxidative damage.

In conclusion, the xCT mRNA level, L-cystine transport activity, and intracellular glutathione level are all enhanced under oxidative stress conditions following DEM treatment of MBEC4 cells used as an in vitro model for the BBB. Our current and previous findings represent an important contribution to a better understanding of the supply of L-cystine to the brain to combat oxidative stress and of the detoxifying and neuroprotective role of the BBB.