INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

GLUTATHIONE (GSH), a major ubiquitous antioxidant, is a tripeptide (-glutamylcysteinylglycine) synthesized from cysteine, glutamate, and glycine. The availability of cysteine in body fluids is low. The cysteine for GSH synthesis is therefore provided by the intracellular reduction of cystine, which is taken up from the extracellular space via a cystine/glutamate antiporter (x transport system) (2). In the presence of high extracellular concentrations of glutamate, the cystine/glutamate antiporter functions in reverse, taking up glutamate in exchange for intracellular cystine. This heterodimeric transporter includes the CD98 heavy chain, which is present in various transporters, and the xCT light chain, which confers substrate specificity (22). The rate of cystine uptake by the cystine/glutamate antiporter is the determining factor in the regulation of GSH concentration. Indeed, stimulation of mouse peritoneal macrophages by lipopolysaccharide (LPS), or acute exposure of endothelial cells to nitric oxide, increases cystine uptake and intracellular GSH levels (14, 21). In contrast, blocking the cystine/glutamate antiporter by competitive inhibition, with high extracellular concentrations of glutamate, for example, leads to GSH depletion and possibly cell death in C6 glioma cells and peritoneal macrophages (11, 20). Thus intracellular GSH depletion by competitive inhibition of cystine uptake is a nonexcitotoxic mechanism by which glutamate may induce cell death.

In 1992, a new family of glutamate transporters was cloned from mammalian tissues (1, 8, 10, 17, 23). These transporters, Na⁺-dependent high-affinity glutamate transporters (excitatory amino acid transporters, EAATs), are essentially present in the central nervous system on astrocytes and neurons, and they protect against excitotoxicity by clearing extracellular glutamate. EAATs transport L-glutamate (L-Glu) and D- (D-Asp) and L-aspartate (L-Asp) and couple the electrochemical gradient of three cotransported sodium ions and one countertransported potassium ion with that of the amino acids (X system) (27). A third glutamate transport system has also been described in rat alveolar type 2 cells and in astrocytes. This system is Na⁺ dependent, and it transports cystine, glutamate, and aspartate (X_AG system ) (Refs. 5, 6, 12; see Table 1). Because both cystine and glutamate are substrates of the X_AG system, they compete for entry. This competition is also seen for the x system but not for the EAATs.

We demonstrated previously (19) that human macrophages derived from monocytes (MDMs) have both a Na⁺-independent glutamate transport system and EAATs. We found that EAAT activity in MDMs was similar to that of neonatal astrocytes and embryonic neurons and that MDMs overcame glutamate toxicity in neuron cultures by clearing glutamate from the medium. We also showed that extracellular glutamate increased intracellular GSH levels in MDMs, suggesting that EAATs may be involved in the regulation of GSH synthesis (19). In this study, we aimed to determine the mechanisms by which glutamate and cystine transporters interact in the regulation of intracellular levels of GSH in MDMs. X_AG system expression by MDMs has not yet been reported, and this transport system would interfere if active in our X- and x-positive MDMs. Accordingly, we first determined whether or not MDMs express this transport system. We then studied how the expressed transport systems interact.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Human monocyte isolation and differentiation. Human peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy human immunodeficiency virus (HIV)-, hepatitis B virus-, and hepatitis C virus-seronegative donors by Ficoll-Hypaque density gradient centrifugation. Monocytes were separated from PBMCs by countercurrent centrifugal elutriation. Monocytes (2 × 10⁶ cells/well) were seeded in 48-well plates in DMEM (Roche Diagnostics, Meylan, France) supplemented with 10% heat-inactivated (+56°C for 30 min) fetal calf serum (FCS; Roche Diagnostics), 2 mM L-glutamine (Roche Diagnostics), and 1% antibiotic mixture (penicillin, streptomycin, and neomycin; Life Technologies, Grand Island, NY). Cells were maintained at +37°C in a humidified atmosphere containing 5% CO₂. In our hands, blood monocytes (95% pure after elutriation) began to adhere after 1 h of culture, spontaneously detached from the plastic after 24 h, and retained a monocyte-like appearance for 5 days. Monocytes were then washed with phosphate-buffered saline (PBS) and dispensed into 48-well plates (0.5 × 10⁶ cells/well) in 10% FCS culture medium supplemented with 15% human PBMC-conditioned medium (7 days of culture). After 7-8 days in culture, the cells adhered tightly to the plastic and morphological differentiation occurred such that the monocytes-macrophages became fibroblast-like. Between days 9 and 12, the cells became large, well-dispersed, rounded macrophages, and they retained this appearance for ~25 days. All experiments were performed using 9- to 12-day-old MDMs, at which age EAAT activity is maximal (19).

Glutamate and cystine uptake. Glutamate uptake was determined for MDMs seeded in 48-well plates. The uptake medium contained (in mM) 137 NaCl, 0.7 K₂HPO₄, 1 CaCl₂, 1 MgCl₂, 5 glucose, and 10 HEPES, pH 7.4. We assessed Na⁺ dependence by replacing 137 mM NaCl with 137 mM choline chloride (Sigma). Cells were washed with 1 ml of PBS and incubated for 20 min at 37°C in 200 µl of uptake medium with changes in ion concentration or inhibitors if necessary. The medium was removed by vacuum aspiration and replaced with 100 µl of uptake medium (with changes in ion concentration or inhibitors if necessary) containing 1 µM L-[2,3-³H]glutamic acid for the glutamate uptake assay (30-60 Ci/mmol; ICN, Irvine, CA) or 10 µM L-[³⁵S]cystine (0.1 Ci/mmol) for the cystine uptake assay. Uptake was stopped after 5 min by removing the medium and washing twice with 1 ml of cold PBS. Cells were then lysed with 130 µl of 100 mM NaOH. The radioactivity of 60 µl of cell lysate was determined by liquid scintillation counting. The protein content of 60 µl of cell lysate was determined by the Bradford method. All experiments were performed in triplicate. Uptake is expressed as picomoles of glutamate or cystine taken up per milligram of protein per minute.

Glutamate transporter competitive inhibitors. DL-threo--hydroxyaspartic acid (THA), L-trans-pyrrolidine-2,4-dicarboxylic acid (trans-PDC), dihydrokainate (DHK), L-Asp and D-Asp, quisqualic acid (Quis), and L-homocysteate were purchased from Sigma (see Table 1).

Intracellular glutathione content. MDMs were cultured overnight in DMEM without cystine, glutamine, and glutamate (DMEM; Life Technologies) but supplemented with 0.1% FCS. Cells were then washed with PBS and incubated with 300 µl of DMEM-0.1% FCS in the presence or absence of glutamine (Life Technologies), cystine, glutamate, L- or D-Asp, L-buthionine-[S,R]-sulfoximine (BSO), or THA for 4.5 h. Glutamine was stored as single-use frozen aliquots and thawed just before use to avoid degradation before the experiment. A pH of 7.4 was maintained by adding KOH as necessary. MDMs were washed with PBS and lysed in 150 µl of PBS-0.1% Tween 20 for 1 h. GSH content was determined by enzyme assay (Cayman Chemical, Ann Arbor, MI) as specified by the manufacturer. The protein content of cell lysates was determined by the Bradford method. All experiments were performed in triplicate. GSH content is expressed as nanomoles per milligram of protein.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Characterization of glutamate transporters in MDMs. We demonstrated previously that MDMs possess a Na⁺-dependent, THA- and trans-PDC-sensitive glutamate transport system (EAATs) and a Na⁺-independent glutamate transport system (19). We investigated whether the Na⁺-independent glutamate transport system involved the cystine/glutamate transporter or the X_AG transporter by performing [³H]glutamate and [³⁵S]cystine uptake experiments. Na⁺-dependent glutamate transport was inhibited by THA, trans-PDC, and L- and D-Asp by 91%, 83%, 88%, and 83.5%, respectively, but was insensitive to L-homocysteate and Quis (Fig. 1A). Na⁺-independent glutamate transport was inhibited only by L-homocysteate (85%) and Quis (63%) (Fig. 1A). Cystine transport was Na⁺ independent, insensitive to THA and L- or D-Asp, and inhibited by L-homocysteate, Quis, and L-Glu (by 84.2%, 63%, and 59% respectively; Fig. 1B). Thus there are two distinct glutamate transport systems in MDMs, the first involving EAATs (X system) and the second involving the cystine/glutamate antiporter (x system). The X_AG transport system is not expressed at detectable levels in MDMs (see Table 1).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Effect of transport inhibitors on the uptake of glutamate and cystine by macrophages derived from monocytes (MDMs). Uptake of 1 µM [3H]glutamate (A) or 10 µM [35S]cystine (B) by MDMs was measured over 5 min in uptake buffer containing 137 mM NaCl or 137 mM choline chloride in the presence or absence of 1 mM transport inhibitors. Data are from 1 of 3 independent experiments and are expressed as means ± SD of triplicate determinations. THA, DL-threo--hydroxyaspartic acid; TPDC, L-trans-pyrrolidine-2,4-dicarboxylic acid; L-Asp and D-Asp, L- and D-aspartate; Quis, quisqualic acid; L-Glu, L-glutamate.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Inhibition of cystine transport by L-glutamate. MDMs were incubated for 5 min in uptake buffer containing 10 µM [35S]cystine and various concentrations of glutamate. The results are expressed as % of inhibition of cystine uptake. Data are means ± SE obtained with MDMs from 4 donors.

Effect of glutamate on GSH synthesis. We tested the ability of cystine, alone or with L-Glu, to support GSH synthesis by MDMs. Cystine increased the concentration of GSH in a dose-dependent manner (Fig. 3). This observation is consistent with cystine being limiting for cysteine availability and cysteine being the limiting precursor for GSH synthesis. The addition of 100 µM glutamate to the culture medium increased intracellular GSH levels by 37%. BSO inhibits -glutamyl-cysteine synthetase, an enzyme catalyzing the synthesis of -glutamylcysteine, an intermediate in GSH synthesis. When 5 mM BSO was added to culture medium containing cystine and glutamate, the intracellular GSH levels recorded were not significantly different from those measured in cystine-free medium. When MDMs were cultured in the presence of 100 µM cystine, glutamate increased GSH concentration in a dose-dependent manner, the maximal effect (79% of stimulation) being obtained with a concentration of 1,000 µM glutamate (Fig. 4). A similar increase in GSH synthesis also occurred when there was a 50-fold excess of glutamate over cystine in the culture medium. However, a 100-fold excess of glutamate over cystine inhibited cystine uptake by x (Fig. 1B).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Effect of cystine and glutamate on glutathione (GSH) concentration. MDMs were cultured for 4.5 h in DMEM without cystine, glutamine, and glutamate (DMEM) supplemented with various concentrations of cystine in the presence or absence of 100 µM glutamate or 5 mM L-buthionine-[S,R]-sulfoximine (BSO). Intracellular GSH levels were determined as described in MATERIALS AND METHODS. Data are from 1 of 2 independent experiments and are expressed as means ± SD of triplicate determinations.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Dose-dependent effect of glutamate on GSH synthesis. MDMs were cultured for 4.5 h in DMEM supplemented with 100 µM cystine (DMEM/cyst+) in the presence of various concentrations of glutamate, and intracellular GSH levels were determined. Data are from 1 of 3 independent experiments and are expressed as means ± SD of triplicate determinations.

Effect of amino acids on GSH synthesis. We compared the effects of various amino acids on cystine-induced GSH synthesis by MDMs. Glutamine increased cystine-induced GSH synthesis in a dose-dependent manner (79% for 1,000 µM glutamine; Fig. 5). When MDMs were cultured in the presence of 300 µM glutamine, the addition of 1,000 µM L-glutamate did not potentiate the effect of glutamine on GSH synthesis (data not shown), suggesting that MDMs may use either glutamine or glutamate for GSH synthesis. L-Asp, another amino acid that can be transported by EAATs, also increased cystine-induced GSH synthesis to an extent similar to that observed with L-Glu (45% and 42% for 1,000 µM L-Asp and L-Glu, respectively, in this experiment). D-Asp, the nonmetabolizable analog of L-Asp, which is also transported by EAATs, had no effect on cystine-induced GSH synthesis (<5%; Fig. 5). In the absence of cystine, none of these amino acids induced GSH synthesis (data not shown). Thus cystine is the limiting factor, but MDMs may also use extracellular glutamine, glutamate, or L-Asp to increase GSH synthesis.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Effect of various amino acids on GSH synthesis. MDM were cultured for 4.5 h in DMEM/cyst+ in the presence or absence of 100, 1,000, or 5,000 µM L-glutamine (Gln), L-Glu, L-Asp, or D-Asp. Data are from 1 of 2 experiments and are expressed as means ± SD of triplicate determinations.

Effect on intracellular GSH content of inhibiting EAAT-mediated uptake. D-Asp is a competitive inhibitor of glutamate uptake by EAATs, but, unlike L-Asp, it is not metabolized. We therefore used this amino acid to block L-Glu uptake by EAATs. D-Asp, at a concentration of 10 mM, abolished the increase in GSH synthesis induced by 100 µM glutamate (Fig. 6). This demonstrates that glutamate-induced increase in GSH concentration is indeed dependent on EAAT-mediated uptake of glutamate.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Effect of D-Asp, a nonmetabolizable excitatory amino acid transporter (EAAT) competitive inhibitor, on glutamate-induced GSH synthesis. MDMs were cultured for 4.5 h in DMEM/cyst+ in the presence or absence of 100 µM L-Glu and/or 10 mM D-Asp. Results are expressed as % of control (i.e., GSH level in DMEM/cyst+). Data are means ± SE obtained with MDMs from 4 donors.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

We have demonstrated that MDMs possess two transport systems for glutamate, differing in Na⁺ dependence. We had shown previously (19) that the Na⁺-dependent transport system involves EAAT1 and EAAT2 activity (system X) (Fig. 1). We show here that cystine transport is 100% Na⁺ independent (Fig. 1B) and that the Na⁺-independent uptake of both cystine and glutamate is inhibited by excess L-homocysteate, Quis, and L-Glu but not by L-Asp, D-Asp, or THA (Fig. 1). Thus the Na⁺-independent glutamate transport system in MDMs does indeed make use of the cystine/glutamate antiporter (x system). The third known glutamate transporter system, X_AG, is not detectable in our MDMs, as no Na⁺-dependent cystine uptake was observed (see Table 1). MDMs may, as a consequence, take up cystine and/or glutamate only through the X and x systems, which may interact for GSH synthesis independent of other known systems.

Previous reports have demonstrated, with different cell types, that extracellular glutamate decreases intracellular levels of GSH by competing with cystine for use of the cystine/glutamate antiporter, reducing intracellular cystine availability (11, 16, 20). Because MDMs may take up glutamate by the cystine/glutamate antiporter as well as by EAATs, we investigated the effects of glutamate on GSH synthesis. The incubation of MDMs for 4.5 h in the presence of a 50-fold excess of glutamate over cystine did not inhibit GSH synthesis and even increased it (Fig. 4). During this 4.5-h culture period, competition between cystine and glutamate for x transport was likely to occur. Indeed, we had already shown (19) that MDMs clear <20% of 1 mM added glutamate over a 6-h culture period. Moreover, as shown in Fig. 2, excess extracellular glutamate does compete with cystine uptake in a 5-min assay under our conditions. We therefore cannot explain GSH synthesis enhancement by glutamate clearance from the medium. Nevertheless, enhancement of GSH intracellular level by excess extracellular glutamate shows that cystine entry over a 4.5-h culture period was sufficient to support increased GSH synthesis. This strongly suggests that in the presence of extracellular glutamate EAATs efficiently fuel the intracellular glutamate pool, permitting an increase in x transport velocity that leads to enhanced cystine uptake, albeit under competition conditions (trans-stimulation). Indeed, in human fibroblasts that transport glutamate through the x system, Bannai and Ishii (3) showed that the intracellular pool of glutamate is a limiting factor for cystine uptake and is rapidly depleted in cystine-containing medium. To assess whether our observation was dependent on EAAT activity, we used L- and D-Asp instead of glutamate and measured GSH levels (Fig. 5). These amino acids are two other substrates for EAATs that efficiently compete with glutamate uptake. As expected, L-Asp increased GSH synthesis, because it can be converted to L-Glu by aspartate aminotransferase and subsequently contribute to the glutamate pool. In contrast, D-Asp is not a substrate for aspartate aminotransferase and did not modify GSH levels. Moreover, excess D-Asp over glutamate abrogated the effect of glutamate on GSH synthesis, showing that EAAT-mediated uptake of glutamate is necessary for GSH synthesis enhancement (Fig. 6).

We showed that, in the presence of cystine, MDMs may also use L-glutamine for GSH synthesis (Fig. 5). Tritsch and Moore (24) showed that spontaneous glutamine decomposition in FCS-containing culture media (PBS, Eagle's medium, and medium 213) was 10%/day at 37°C and pH 7.2. The enhancement of GSH synthesis by exogenous glutamine may thus have two mechanisms. First, glutamine decomposition produces pyrrolidonecarboxylic acid (pyroglutamate) that might be processed to glutamate if decyclization occurs. Glutamate would then enhance GSH synthesis as already shown. On the other hand, glutamine may also act as described by Bannai and Ishii (3) in fibroblasts. According to this model, glutamine would be taken up by MDMs by one or more of the known glutamine transport systems (systems A, L, and ASC; Refs. 4, 7, 15, and 25), be processed to glutamate by cellular glutaminase, and fuel the limiting intracellular pool of glutamate for cystine uptake. Glutamine decomposition kinetics in culture medium at 37°C and neutral pH indicate that <10% of added glutamine would be converted to pyroglutamate (24), and the second mechanism thus seems the most likely.

Our data strongly suggest that intracellular GSH level is controlled by the activity of transporters for cystine, glutamate, and glutamine. To maintain their intracellular pool of glutamate MDMs may thus use either glutamine, as fibroblasts do (3), or glutamate itself, as previously described in astrocytes (13). Indeed, Kranich et al. (13) reported that astrocytes, unlike neurons and fibroblasts, preferentially use extracellular glutamate rather than glutamine for GSH synthesis, probably because of their high level of EAAT activity. In contrast, extracellular glutamate inhibits GSH synthesis in C6 glioma cells (11). However, these authors also demonstrated that extracellular cystine inhibits glutamate uptake in these cells, suggesting that C6 cells mainly use the cystine/glutamate antiporter rather than EAATs (11). Moreover, glioma cell lines derived from human tumors exhibit a lower level of Na⁺-dependent glutamate uptake and a higher level of cystine/glutamate antiporter activity than astrocytes (26). As a consequence of the reduction of EAAT activity in these cells, competition occurs between cystine and glutamate, leading to an increase in extracellular glutamate concentration if an excess of extracellular cystine is present. The presence of the X_AG transport system, which transports glutamate, cystine, and aspartate, on cultured astrocytes derived from neonatal rats also suggests that competition between these amino acids may occur and may have profound effects on the redox status and structural and functional integrity of the central nervous system (5). Consistent with our observations, Reichelt et al. (18) reported that extracellular glutamate increases intracellular GSH level in retinal Müller glial cells dependent on EAATs. This was confirmed by Igo and Ash (9), who demonstrated, using somatic cell mutants (CHO-K1 Xag-null), that X provides a significant proportion of the glutamate used in the uptake of cystine for GSH synthesis. From these numerous observations in different cell types, it appears that cells that express the cystine/glutamate antiporter are sensitive to GSH depletion and subsequent oxidative stress induced by glutamate unless they also express another glutamate transporter that fuels their intracellular pool of glutamate, then increasing their antioxidant potential. The presence of both the cystine/glutamate transporter and EAATs on macrophages, and the absence of the X_AG transport system, may accordingly increase the antioxidant activity of macrophages by increasing intracellular GSH concentration when extracellular glutamate concentration increases (see Fig. 7).

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Role of glutamate transporters in the regulation of intracellular GSH levels in MDMs. MDMs possess the X and x transport systems, but not the XAG system, under physiological conditions. Extracellular cystine is transported into MDMs by the x transporter in exchange for glutamate. The X transporters take up extracellular glutamate, thereby fueling the intracellular pool of glutamate for trans-stimulation of the x transporter. Thus both glutamate transporters participate in the regulation of intracellular GSH levels, even if extracellular glutamate concentration increases. Aspartate and glutamine also fuel the intracellular pool of glutamate. Glutamate might also be produced through decyclization of pyroglutamate. 1: -Glutamyl-cysteine synthetase. 2: Glutathione synthetase. 3: Aspartate transpeptidase. 4: Glutaminase. 5: Spontaneous degradation of glutamine. CD98/xCT, CD98 heavy chain and xCT light chain.

We demonstrated previously that freshly sorted tissue macrophages and blood monocytes do not possess functional EAATs and that X transport by differentiating MDMs may be under the control of inflammatory molecules such as TNF- (19). In addition, TNF- and LPS increase cystine uptake by macrophages (19, 20). This suggests that during inflammatory processes, the acquisition of functional EAATs on monocytes-macrophages and the increase in cystine uptake via x may increase antioxidant activity. The modulation of the activity of both glutamate transporters on macrophages should therefore be evaluated in pathological conditions, such as neurological diseases or HIV infection, in which an increase in extracellular glutamate concentration associated with a decrease in EAAT activity on astrocytes and a deficit in GSH concentration have been reported.

In conclusion, we demonstrated previously that EAATs on macrophages protect neurons from excitotoxicity and we show in this study that they are also involved, along with the cystine/glutamate antiporter, in the regulation of GSH synthesis in two ways: first, by stimulating cystine uptake through the cystine/glutamate antiporter and second, by providing intracellular glutamate for direct insertion into GSH (see Fig. 7). The activity of the two glutamate transporters on macrophages is therefore determinant for limiting excitotoxicity and protection against oxygen free radicals in pathological conditions.