Abstract
The purpose of the present study was to elucidate the mechanism of enhancement of l-cystine uptake at the blood-brain barrier (BBB). The uptake of [14C]l-cystine and [3H]l-glutamic acid (l-Glu) was determined using a mouse brain endothelial cell line (MBEC4) as an in vitro BBB model. The mRNA levels ofl-cystine/l-Glu exchanger, system xc−, which consists of xCT and 4F2hc, were determined by quantitative real-time reverse transcription-polymerase chain reaction analysis. The [14C]l-cystine uptake by MBEC4 cells appeared to be mediated via an Na+-independent saturable process. The corresponding Michaelis-Menten constant (Km) was 63.7 μM. In the presence ofl-Glu, there was competitive inhibition with an inhibition constant (Ki) of 83.5 μM. [3H]l-Glu uptake in the absence of Na+ was saturable with a Km of 48.1 μM, and it exhibited competitive inhibition with aKi of 24.9 μM in the presence ofl-cystine. The mutual inhibition betweenl-cystine and l-Glu and the type of inhibition suggest that system xc− operates in MBEC4 cells. The xCT and 4F2hc mRNAs were expressed in MBEC4 cells and, following diethyl maleate (DEM) treatment, the xCT mRNA level andl-cystine uptake in MBEC4 cells were enhanced in parallel with an increase in DEM concentration (up to 500 μM). Concomitantly, the glutathione concentration in MBEC4 cells was increased. In conclusion, system xc−-mediatedl-cystine uptake takes place in MBEC4 cells.l-Cystine transport via system xc−at the BBB is likely to be induced under oxidative stress conditions following DEM treatment due to enhanced transcription of the xCT gene.
l-Cystine and l-glutamic acid (l-Glu) exchange transporter, referred to as system xc−, 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 xc− involves entry ofl-cystine and exit of l-Glu. Therefore, it plays a key role in the synthesis of glutathione becausel-cysteine (l-Cys), which is reduced froml-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 xc− 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 xc− 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 ofl-Glu and l-α-aminoadipic acid (l-AAA), substrates of system xc−. This suggests thatl-cystine influx transport via system xc− 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 ofl-cystine uptake enhancement, as well as the expression and regulation of system xc− 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
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) orl-[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 4cells/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 mMd-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 (Km) and maximum uptake rate (Vmax) ofl-cystine or l-Glu uptake were calculated from eq. 1 using the nonlinear least-squares regression analysis program, MULTI (Yamaoka et al., 1981).
Unless otherwise indicated, all data represent means ± S.E.M. Statistical significance of differences among means of several groups was determined by one-way analysis of variance followed by modified Fisher's least-squares difference method.
Results
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+ (Table1). l-Cystine uptake was saturable with a Km of 63.7 ± 13.9 μM (mean ± S.D.) (Fig. 2A). Moreover, the Lineweaver-Burk plot showed that the two lines of thel-cystine uptake in the presence or absence of 160 μM l-Glu intersected the ordinate axis. This indicates that l-Glu competitively inhibitedl-cystine uptake with aKi of 83.5 ± 10.3 μM (mean ± S.D.) (Fig. 2B). These results suggest thatl-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 andl-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 xc− (Sato et al., 1999). Partial inhibition was observed bydl-diaminopimelic acid (dl-DPA) and l-aspartic acid (l-Asp) by up to 42%, whereasl-leucine (l-Leu),l-lysine (l-Lys),l-arginine (l-Arg), γ-aminobutyric acid, and p-aminohippuric acid had no effect on [14C]l-cystine uptake.
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 xc−, in MBEC4 cells. Figure3 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 xc−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 aKm of 48.1 ± 14.2 μM (mean ± S.D.) (Fig. 4A). A Lineweaver-Burk plot showed that the two lines of thel-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 aKi 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% byl-Glu, l-cystine,l-AAA, l-HCA, andl-QQA. It was partially inhibited (50%) byl-Asp, whereas l-Leu andl-Arg had no effect on [3H]l-Glu uptake.
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%.
Effect of DEM on System xc− 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.
Discussion
The present study demonstrates that MBEC4 cells used as an in vitro model of the BBB express xCT and 4F2hc mRNA (Fig. 5), andl-cystine and l-Glu uptakes via system xc− are Na+-independent and concentration-dependent (Figs. 1-4). The Km value forl-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 Km value forl-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 forl-cystine and l-Glu uptake by MBEC4 cells. The [14C]l-cystine uptake was competitively inhibited by 160 μM l-Glu with aKi of 83.5 μM (Fig. 2B), which is comparable with the Km value ofl-Glu uptake in this study. The [3H]l-Glu uptake was also competitively inhibited by 100 μM l-cystine with a Ki of 24.9 μM (Fig. 4B), which is comparable with the Km value of l-cystine uptake in this study. These results support the hypothesis that both l-cystine andl-Glu uptake is mediated via system xc− in MBEC4 cells. Moreover, the uptake of both [14C]l-cystine and [3H]l-Glu is strongly inhibited by system xc−substrates, such as l-AAA,l-HCA, and l-QQA (Tables 1and 2). This manner of inhibition is consistent with system xc− characteristics as reported elsewhere (Miura et al., 1992; Sato et al., 1999). System b0,+, 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 b0,+ since l-Leu,l-Lys, and l-Arg produced no marked inhibition (Table 1). Na+-dependentl-Glu uptake is present in MBEC4 cells (Fig. 3), and this suggests that an Na+-dependentl-Glu and l-Asp transporter, system XAG−, 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 anddl-DPA are dicarboxylic amino acids likel-Glu and l-cystine, the inhibition ratio was lower than that of l-Glu andl-cystine. It seems that these amino acids have a lower affinity for system xc−than l-Glu and l-cystine since l-Asp and dl-DPA have, respectively, shorter and longer carbon chains thanl-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 xc−-mediatedl-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 increasingl-cystine uptake by MBEC4 cells (Fig. 7), supporting the hypothesis that activation of l-cystine uptake via system xc− 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. Althoughl-Glu transport from blood to brain can be enhanced due to induction of system xc− at the BBB under the oxidative stress conditions, it may not affectl-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 xc− 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 xc− 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 xc− at the BBB may play a number of important roles in supplyingl-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 xc− is not present at the BBB under normal conditions, system xc− is likely to be induced under oxidative stress conditions and to stimulate the supply ofl-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 xc− even under normal conditions, and 2) MBEC4 cells acquire system xc− during development of this cell line. Further studies are needed to see whether system xc− is present at the BBB under normal conditions in vivo. Taking all these current and previous in vivo results into consideration, we have shown thatl-cystine undergoes influx transport from the circulating blood to the brain via system xc− 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.
Acknowledgments
We thank Dr. T. Masuko for valuable discussions and N. Funayama for secretarial assistance.
Footnotes
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This study was supported, in part, by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture, Japan. It was also supported, in part, by The Suzuken Memorial Foundation, The Nakatomi Foundation, The Uehara Memorial Foundation, and The Mochida Memorial Foundation for Medical and Pharmaceutical Research.
- Abbreviations:
- BBB
- blood-brain barrier
- CNS
- central nervous system
- DEM
- diethyl maleate
- l-AAA
- l-α-aminoadipic acid
- MBEC4
- mouse brain endothelial cell line
- RT-PCR
- reverse transcription-polymerase chain reaction
- PBS
- phosphate-buffered saline
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- [14C]l-cystine
- [U-14C]l-cystine
- [3H]l-Glu
- l-[2,3-3H]glutamic acid
- ECF
- extracellular fluid
- l-HCA
- l-homocysteic acid
- l-QQA
- l-quisqualic acid
- dl-DPA
- dl-diaminopimelic acid
- Received January 31, 2002.
- Accepted March 15, 2002.
- The American Society for Pharmacology and Experimental Therapeutics