Role of glutamate transporters in the regulation of
glutathione levels in human macrophages
Anne-Cécile
Rimaniol1,
Patricia
Mialocq1,
Pascal
Clayette1,2,
Dominique
Dormont1, and
Gabriel
Gras1
1 Service de Neurovirologie, CEA, DSV/DRM, Centre de
Recherches du Service de Santé des Armées, Ecole
Pratique des Hautes Etudes, Institut Paris Sud sur les Cytokines,
92265 Fontenay-aux-Roses cedex; and 2 Société de
Pharmacologie et d'Immunologie-BIO, 91741 Massy, France
 |
ABSTRACT |
Cysteine is the limiting precursor for
glutathione synthesis. Because of its low bioavailability, cysteine is
generally produced from cystine, which may be taken up through two
different transporters. The cystine/glutamate antiporter
(x
system) transports extracellular cystine in
exchange for intracellular glutamate. The XAG transport
system takes up extracellular cystine, glutamate, and aspartate. Both
are sensitive to competition between cystine and glutamate, and excess
extracellular glutamate thus inhibits glutathione synthesis, a
nonexcitotoxic mechanism for glutamate toxicity. We demonstrated
previously that human macrophages express the glutamate transporters
excitatory amino acid transporter (EAAT)1 and EAAT2 (which do not
transport cystine, X
system) and overcome
competition for the use of cystine transporters. We now show
that macrophages take up cystine through the x
and
not the XAG system. We also found that glutamate, although competing with cystine uptake, dose-dependently increases glutathione synthesis. We used inhibitors to demonstrate that this increase is
mediated by EAATs. EAAT expression in macrophages thus leads to
glutamate-dependent enhancement of glutathione synthesis by providing
intracellular glutamate for direct insertion in glutathione and also
for fueling the intracellular pool of glutamate and
trans-stimulating the cystine/glutamate antiporter.
cystine; glutamate/cystine antiporter; oxidative stress; glutamine
 |
INTRODUCTION |
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 (XAG system ) (Refs. 5,
6, 12; see Table
1). Because both cystine and glutamate
are substrates of the XAG 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. XAG 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 |
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).
All culture medium contained <1 ng/ml endotoxin as assessed by the
Limulus test and did not induce tumor necrosis factor-
(TNF-
) production by differentiated macrophages (data not shown). Moreover, we verified that our MDMs are not sensitive to LPS doses of
<1 ng/ml (TNF-
production). This rules out the possible artifacts induced by undetectable endotoxin contamination. We also tested the
ability of 1 µg/ml LPS to modulate either X
or
x
transport systems. This dose of LPS applied to 9- to 12-day-old MDMs did not modulate the X
system
and enhanced the x
system by ~80% (data not
shown). The latter result is concordant with the data of Sato et al.
(20) on x
transport.
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 |
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 XAG 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
XAG transport system is not expressed at detectable levels
in MDMs (see Table 1).

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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.
|
|
We generated dose-response curves for the inhibition of cystine uptake
(10 µM) by L-Glu and obtained an inhibition constant of
650 ± 143 µM (Fig. 2). This value
and the observation that 75% of the extracellular glutamate was
transported by EAATs and 25% by the cystine/glutamate antiporter
(Na+ dependence; Fig. 1A) suggest that
extracellular glutamate is taken up with higher affinity by EAATs than
by the cystine/glutamate antiporter.

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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).

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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.
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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.

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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.

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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.
|
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 |
DISCUSSION |
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, XAG, 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
XAG 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 XAG transport system,
may accordingly increase the antioxidant activity of macrophages by increasing intracellular GSH concentration when extracellular glutamate
concentration increases (see Fig. 7).

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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.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by grants from the Agence Nationale
de Recherches sur le SIDA (ANRS) and SIDACTION/Ensemble Contre le
SIDA. A. C. Rimaniol is a recipient of a fellowship from the ANRS.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: A.-C.
Rimaniol, Service de Neurovirologie, DSV/DRM, CEA, BP 6, 18 route du
Panorama, 92265 Fontenay-aux-Roses, France (E-mail:
rimaniol{at}dsvidf.cea.fr).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 4 December 2000; accepted in final form 8 August 2001.
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