Glutamate induces release of glutathione from cultured rat astrocytes--a possible neuroprotective mechanism?

Glutamate induces release of glutathione from cultured rat
astrocytes – a possible neuroprotective mechanism?
Joa˜o Frade,* Simon Pope,� Maike Schmidt,� Ralf Dringen,� Rui Barbosa,*,§ Jennifer Pocock,¶
Joa˜o Laranjinha*,** and Simon Heales�,��
*Centre for Neurosciences and Cell Biology, University of Coimbra, Coimbra, Portugal
�Department of Clinical Biochemistry (Neurometabolic Unit), National Hospital of Neurology and Neurosurgery, London, UK
�Center of Biomolecular Interactions, University of Bremen, Faculty 2 (Biology/Chemistry), Bremen, Germany
§Biochemistry Laboratory, Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal
¶Department of Neuroinflammation, Institute of Neurology, University College London, London, UK
**Laboratory of Instrumental Analysis, University of Coimbra, Coimbra, Portugal
��Department of Molecular Neuroscience, Institute of Neurology, University College London, London, UK
Glutamate is the major excitatory amino acid of the
mammalian brain (Danbolt 2001). It acts through a variety
of ionotropic and metabotropic receptors: the first exert their
effects via ligand-gated ion channels, whereas the second act
through coupling to G proteins and activation of intracellular
secondary messengers (Greenamyre and Porter 1994; Mel-
drum 2000). Although glutamate is an important excitatory
neurotransmitter it can be toxic if its extracellular levels are
not tightly controlled. In conditions where release and/or
uptake of glutamate are altered, extracellular glutamate can
accumulate causing a persistent or excessive activation of
glutamate-gated ion channels (excitotoxicity) (Mark et al.
2001; Coyle and Puttfarcken 1993). A number of pathways
have been implicated in glutamate excitotoxicity, namely
calcium deregulation, loss of membrane potential, mito-
chondrial impairment and production of reactive nitrogen/
oxygen species (RNOS), which can lead to oxidative/
nitrosative stress and ultimately cell death (Coyle and
Received September 14, 2007; revised manuscript received December 1,
2007; accepted December 19, 2007.
Address correspondence and reprint requests to Simon Pope,
Department of Clinical Biochemistry (Neurometabolic Unit), National
Hospital of Neurology and Neurosurgery, London WC1N 3BG, UK.
E-mail: spope@ion.ucl.ac.uk
Abbreviations used: cGT, c-glutamyl transferase; AMPA, a-amino-3-
hydroxy-5-methyl-4-isoxazole propionic acid; BSO, buthionine sul-
phoxime; DIV, day in vitro; GSH, glutathione; GSx, total glutathione
(amount of GSH + twice the amount of GSSG); LDH, lactate dehy-
drogenase; MM, minimal medium; NO, nitric oxide; RNOS, reactive
nitrogen/oxygen species.
Abstract
Glutamate is the major excitatory amino acid of the mamma-
lian brain but can be toxic to neurones if its extracellular levels
are not tightly controlled. Astrocytes have a key role in the
protection of neurones from glutamate toxicity, through regu-
lation of extracellular glutamate levels via glutamate trans-
porters and metabolic and antioxidant support. In this study,
we report that cultures of rat astrocytes incubated with high
extracellular glutamate (5 mM) exhibit a twofold increase in
the extracellular concentration of the tripeptide antioxidant
glutathione (GSH) over 4 h. Incubation with glutamate did not
result in an increased release of lactate dehydrogenase,
indicating that the rise in GSH was not because of membrane
damage and leakage of intracellular pools. Glutamate-induced
increase in extracellular GSH was also independent of de
novo GSH synthesis, activation of NMDA and non-NMDA
glutamate receptors or inhibition of extracellular GSH break-
down. Dose–response curves indicate that GSH release from
rat astrocytes is significantly stimulated even at 0.1 mM glu-
tamate. The ability of astrocytes to increase GSH release in
the presence of extracellular glutamate could be an important
neuroprotective mechanism enabling neurones to maintain
levels of the key antioxidant, GSH, under conditions of glu-
tamate toxicity.
Keywords: antioxidant, astrocytes, cell culture, glutamate,
glutathione, oxidative stress.
J. Neurochem. (2008) 105, 1144–1152.
d JOURNAL OF NEUROCHEMISTRY | 2008 | 105 | 1144–1152 doi: 10.1111/j.1471-4159.2008.05216.x
1144 Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1144–1152
� 2008 The Authors

Puttfarcken 1993; Massieu and Garcia 1998; Pitt et al.
2000).
The extracellular levels of glutamate have been measured
in various in vivo disease models by microdialysis and have
been shown to reach concentrations of > 500 lM following
spinal cord injury (McAdoo et al. 1999) and be maintained
at concentrations of > 50 lM for 1–2 h during and
following ischaemic insult (Orwar et al. 1994; Ritz et al.
2004; Homola et al. 2006). As extracellular glutamate
derives from intracellular vesicles (whose glutamate con-
centrations are between 0.24 and 11 mM; Harris and Sultan
1995), the local concentration of glutamate in these
conditions is likely to be even higher. Prolonged exposure
to such concentrations of glutamate is likely to result in
significant neurotoxicity (Liu et al. 1999). Astrocytes have
a fundamental role in the regulation of extracellular
glutamate levels and in the protection of neurones from
glutamate toxicity (Hertz and Zielke 2004). In normal
synaptic transmission, glutamate released into the synaptic
cleft by neurones is accumulated in astrocytes (Hertz et al.
1978) by means of glutamate transporters such as glutamate
transporter 1 and glutamate aspartate transporter (Gadea and
Lopez-Colome 2001), after which it is returned to neurones
in the form of glutamine.
Astrocytes also protect neurones in other ways such as
through metabolic and antioxidant support. One of the
most important molecules in this respect is the antioxidant
glutathione (GSH) (Schulz et al. 2000). The trafficking of
GSH between astrocytes and neurones is particularly
important in conditions of oxidative stress (Dringen
2000). Astrocytes are able to increase neuronal GSH
levels by secreting GSH into the extracellular environment
(Sagara et al. 1996; Dringen et al. 1999; Stewart et al.
2002). Neurones are unable to take up GSH directly but
can make use of cysteinyl glycine and cysteine, which are
produced from GSH by the consecutive action of c-
glutamyl transferase (cGT) and aminopeptidase N, two
enzymes expressed on the surface of astrocytes and
neurones respectively (Dringen et al. 1997, 2001). Cyste-
ine is the rate-limiting substrate for GSH synthesis in
neurones, so the supply of this substrate by astrocytes is
essential for the maintenance of GSH levels in neurones
(Dringen et al. 1999). Previous studies have shown that
astrocytes increase GSH release in response to increases in
RNOS, such as nitric oxide (NO) (Gegg et al. 2003) and
hydrogen peroxide (Sagara et al. 1996). This increase in
GSH release is hypothesised to be a neuroprotective
mechanism which maintains and/or increases neuronal
GSH levels to counteract the damaging effects of RNOS.
As oxidative stress is considered to be a key component of
glutamate toxicity it was the aim of this study to
investigate whether high concentrations of extracellular
glutamate also had an effect on GSH release from
astrocytes.
Materials and methods
Reagents
Minimum essential medium (L-valine based) and foetal bovine
serum were purchased from Gibco-Invitrogen (Paisley, UK). Cell
culture flasks were purchased from Nalgene Nunc International
(Naperville, IL, USA). Six-well plates were purchased from
Corning Costar (High Wycombe, UK). All other chemical reagents
were purchased from Sigma Chemical Company (Poole, UK). For
the experiments performed on cell cultures on 24-well dishes the
following reagents were used: Dulbecco’s modified Eagle’s
medium was from Gibco-Invitrogen (Karlsruhe, Germany). Foetal
calf serum and penicillin/streptomycin stock solution were from
Biochrom (Berlin, Germany). Sulphosalycilic acid and NADPH
were from AppliChem (Darmstadt, Germany). Glutathione reduc-
tase and GSSG were obtained from Roche Diagnostics (Mannheim,
Germany). All other chemicals were obtained from Sigma
(Steinheim, Germany), Fluka (Neu-Ulm, Germany) or Merck
(Darmstadt, Germany). Sterile 24-well dishes were from
Sarstedt (Nu¨mbrecht, Germany).
Primary cultures of astrocytes
Primary cortical astrocytes cultures were prepared from Wistar rat
neonates (0–2 days). The cerebral hemispheres were removed from
the skull under the dissecting microscope, and cortex and
hippocampus were isolated and manipulated separately. Cortical
and hippocampal astrocytes were prepared as described previously
(Griffin et al. 2005). Astrocytes on day in vitro (DIV) 13 were
removed from the flasks with 0.01% trypsin, and seeded on to poly-
D-lysine-coated six-well plates at a density of 1 · 106 cells/well.
Experiments on these secondary astrocyte cultures were conducted
at DIV 14. The experiments shown in Fig. 1b and Table 1 were
performed on primary astrocyte cultures that were prepared
according to the method described by Hamprecht and Loeffler
(1985) by seeding 3 · 105 cells per well of 24-well dishes. These
cultures were used at DIV 15–23.
GSH release from astrocytes
The media of six-well plates containing secondary astrocyte
cultures at DIV 14 was removed and the cells were washed twice
in 1 mL Hank’s buffered saline solution; 1 mL minimal medium
(MM) (44 mM NaHCO3, 110 mM NaCl, 1.8 mM CaCl2, 5.4 mM
MgSO4, 0.92 mM NaH2PO4 and 5 mM glucose, adjusted with
CO2 to pH 7.4) was added to each well, supplemented with 5 mM
sodium glutamate, 5 mM buthionine sulphoxime (BSO) or both.
For BSO experiments, cells were incubated in MM containing
5 mM BSO for 2 h before and during supplementation with
glutamate. After stimulation for 15, 45, 120 and 240 min, 500 lL
of medium was removed and centrifuged at 3000 g for 5 min to
remove cell debris (NB: Different wells were used for each time
point). A total of 250 lL of supernatant was added to the same
volume of 30 mM o-phosphoric acid and kept at )80�C for up to
3 weeks until HPLC determination of GSH. For experiments on
primary cultures on 24-well dishes, cells were washed with 0.5 mL
of pre-warmed (37�C) MM, pre-incubated for 2 h in 0.5 mL MM
with 100 lM of the c-glutamyl transpeptidase (cGT)-inhibitor
acivicin (Dringen et al. 1997) in the absence or the presence of
BSO (5 mM), and incubated in the cell incubator with 0.5 mL
� 2008 The Authors
Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1144–1152
Glutamate induces release of glutathione from cultured rat astrocytes | 1145

incubation medium (MM with 100 lM acivicin) in the absence or
presence of glutamate (5 mM) and/or BSO (5 mM). Extracts of
cells and media in 1% (w/v) of sulphosalicylic acid were used to
determine the total glutathione content (GSx = amount of GSH
plus twice the amount of GSSG). For determination of the content
of GSSG in lysates or media the GSH present was derivatised with
2-vinylpyridine as described previously (Minich et al. 2006). For
all conditions investigated the GSSG values were in the range of
the detection limit of the assay used (< 5% of GSx). Therefore, the
GSx amounts determined are considered and addressed here as
GSH amounts.
GSH quantification
GSH levels were determined electrochemically following extraction
of GSH into 15 mM o-phosphoric acid (final concentration) and
separation by reverse-phase HPLC (Riederer et al. 1989). The levels
of GSx and of GSSG in cells and media of primary astrocyte
cultures in wells of 24-well dishes were determined as previously
described (Minich et al. 2006) by a modification of the colorimetric
Tietze assay.
Lactate dehydrogenase release
Lactate dehydrogenase (LSH) activity was determined by measure-
ment of NADH oxidation at 340 nm in the presence of pyruvate.
The assay was performed in 96-well plates as described (Dringen
et al. 1998). The percentage of LDH released into medium was
calculated for three separate preparations (mean ± SEM) by the
following: (LDH activity in medium/Total LDH in medium after cell
lysis with Triton X-100) · 100.
Statistical analysis
Results are expressed as a mean ± SEM values for the number of
preparations indicated. Statistical significance for the comparison of
two groups was assessed using Student’s t-test. Multiple compar-
isons were made by one-way ANOVA followed by the Bonferroni test
unless otherwise stated. A value of p < 0.05 was considered
significant. Data expressed as ratios were transformed as previously
described (Gegg et al. 2003), prior to statistical analysis.
Table 1 Cellular and extracellular GSH contents (nmol/well) of primary astrocyte cultures that were treated with glutamate and/or BSO
0 min Cells 240 min Cells 240 min Media 240 min Cells + Media
Cont 1.9 ± 0.2 1.0 ± 0.1 (53 ± 5%) 0.9 ± 0.1 (45 ± 4%) 1.9 ± 0.1 (98 ± 3%)
Glu 1.9 ± 0.2 1.1 ± 0.1 (57 ± 5%) 1.4 ± 0.1* (70 ± 7%)* 2.5 ± 0.2 (127 ± 10%)
Cont + BSO 1.7 ± 0.2 0.7 ± 0.0* (41 ± 3%) 0.9 ± 0.1 (51 ± 5%) 1.6 ± 0.1 (92 ± 7%)
Glu + BSO 1.7 ± 0.2 0.8 ± 0.1 (48 ± 5%) 1.2 ± 0.1 (70 ± 7%)* 2.0 ± 0.2 (118 ± 11%)
Primary astrocyte cultures in wells of 24-well dishes were pre-incubated for 2 h in MM without or with BSO (5 mM) before they were incubated for
4 h in 0.5 mL MM in the presence or absence of glutamate (5 mM) and/or BSO (5 mM). The basal cellular GSH content of untreated primary
astrocyte cultures was 23.0 ± 1.8 nmol/mg protein. The 2 h pre-incubation of these cultures without and with BSO (5 mM) lowered the GSH
content to 19.7 ± 1.0 nmol/mg and 17.5 ± 0.5 nmol/mg respectively. The data presented are mean ± SEM of experiments performed on 4
independently prepared cultures. The significance of differences to the data obtained for the control condition (no glutamate and no BSO) are
indicated as *p < 0.05, analysed by ANOVA followed by the Tukey post hoc test. For all conditions, the extracellular activity of LDH was less than
10% of initial cellular LDH and the values did not differ significantly between the individual groups. GSH release rates from cultured astrocytes have
previously been reported to be between 2 and 4 nmol/mg/h (Sagara et al. 1996; Hirrlinger et al. 2002; Gegg et al. 2003). In the current study, the
GSH release rate was 2.25 nmol/mg/h under control conditions and 3.5 nmol/mg/h after addition of glutamate. LDH, lactate dehydrogenase; BSO,
buthionine sulphoxime.
Fig. 1 Glutamate induces an increase in extracellular GSH in
astrocyte cultures. (a) Cortical astrocytes on six-well plates (a) and
primary astrocytes on 24-well plates (b) were incubated with (h,
dashed line) or without ( , full line) 5 mM glutamate in MM and
extracellular GSH quantified at the indicated time points. Glutamate
induced a marked increase in extracellular GSH when compared
with control astrocytes. Astrocytes were also incubated with 5 mM
of the GSH synthesis inhibitor BSO for 2 h prior to and throughout
experiments with (s, dashed line) or without (d, full line) 5 mM
glutamate. No significant differences were observed in extracellular
GSH between BSO treated and untreated cells. (n = 4–6 different
cell preparations, *p < 0.05 and **p < 0.01 control vs. glutamate
conditions).
Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1144–1152
� 2008 The Authors
1146 | J. Frade et al.

Results
Glutamate induces an increase in extracellular GSH in
cultures of rat cortical astrocytes
To assess the effect of extracellular glutamate on GSH
release, rat cortical astrocytes were treated with glutamate
and extracellular GSH was measured at various time points
by HPLC (Fig. 1a). In these initial experiments 5 mM
glutamate was used. Although this could be thought of as a
comparatively high glutamate concentration to use, similar
glutamate concentrations are thought to be reached in the
synaptic cleft following release of a single synaptic vesicle
(hypothesised to be between 0.24 and 11 mM) (Harris and
Sultan 1995) and millimolar glutamate has been used before
to model glutamate excitotoxicity in astrocytes (Chen et al.
2000).
In the absence of glutamate, extracellular GSH increased
to 0.52 ± 0.05 lM after 120 min and 1.23 ± 0.18 lM after
240 min (Fig. 1a ). In the presence of 5 mM glutamate, the
concentration of extracellular GSH was significantly higher
after 120 and 240 min compared with control astrocytes,
reaching 1.22 ± 0.08 and 2.33 ± 0.17 lM respectively
(Fig. 1ah, p < 0.05). Similar results were obtained for
primary astrocyte cultures on 24-well dishes using a different
assay to determine GSH (Fig. 1b). These results indicate that
glutamate, at this concentration, induces a strong increase in
extracellular GSH in rat astrocyte cultures. However, an
apparent increase in extracellular GSH could be the result of
increased GSH synthesis following incubation with gluta-
mate, increased leakage of intracellular contents because of
glutamate toxicity or inhibition of extracellular GSH break-
down. These were investigated in turn.
Increased extracellular GSH is not a result of de novo
synthesis
Glutamate can be used by cells for GSH synthesis, provided
other precursors are not limited (Dringen and Hamprecht
1998), and constitutive GSH release from astrocytes corre-
lates with intracellular GSH concentration (Sagara et al.
1993). The increase in extracellular GSH observed in the
presence of high extracellular glutamate could therefore
result from increased GSH synthesis. To determine whether
this was the case, glutamate-induced GSH release was
measured in the presence and absence of the GSH synthesis
inhibitor BSO (Fig. 1). Astrocytes were incubated with or
without 5 mM BSO (a concentration that has previously
been shown to inhibit de novo GSH synthesis (Gegg et al.
2002)) in MM for 2 h prior to and throughout experiments.
In the absence of glutamate, extracellular GSH levels for
BSO-treated astrocytes were not significantly different from
control astrocytes, reaching 0.99 ± 0.09 lM after 240 min
(Fig. 1ad). When glutamate was added to BSO-treated
astrocytes a significant increase in extracellular GSH was
detected, reaching 2.28 ± 0.17 lM after 240 min (Fig. 1as,
Cont + BSO vs. Glu + BSO, p < 0.05), similar to what was
observed in glutamate-treated astrocytes in the absence of
BSO. These results were confirmed for primary astrocyte
cultures on 24-well dishes (Fig. 1b).
Glutamate does not induce LDH release from cortical
astrocytes
In order to determine if the increase in extracellular GSH was
because of glutamate-induced cellular damage, LDH levels
were measured in media and cells as an indicator of
membrane disruption. As determined for the 240 min time
point, LDH levels were not significantly different between
control (1.6 ± 0.3%) and glutamate-treated astrocytes
(2.0 ± 1.2%), suggesting that the increase in extracellular
GSH was not a consequence of leakage of intracellular
content. LDH release levels were also not significantly
different between glutamate-treated and control astrocytes in
the presence of BSO (2.2 ± 0.3% vs. 2.4 ± 0.6% respec-
tively).
Increased extracellular GSH is not a result of inhibition
of cGT by glutamate
c-Glutamyl transferase, expressed on the surface of astro-
cytes, breaks down extracellular GSH by catalysing the
transfer of the glutamyl residue of GSH to a variety of amino
acid and dipeptide acceptors (Dringen et al. 1997). Inhibition
of cGT by acivicin has been shown to result in an increase in
extracellular GSH (Dringen et al. 1997). To investigate the
possibility that glutamate was increasing extracellular GSH
levels by inhibiting cGT, the effect of acivicin with or
without glutamate on the release of extracellular GSH by
primary rat astrocytes was tested (Fig. 2). Treatment with
100 lM acivicin resulted in a slight but not significant
increase in extracellular GSH in control astrocytes after
240 min (1.42 ± 0.01 lM for Cont vs. 1.70 ± 0.03 lM for
Cont + Aciv), suggesting that cGT was not particularly
active in our cultures to metabolise the GSH released from
the cells. However, a combination of acivicin and glutamate
did result in a significant increase in extracellular GSH after
240 min compared with astrocytes treated with glutamate
alone (3.17 ± 0.13 lM for Glu alone vs. 3.79 ± 0.13 lM for
Glu + Aciv, p < 0.05). As acivicin was used at a concentra-
tion which has previously been reported to maximally inhibit
cGT (Dringen et al. 1997) and glutamate increased extra-
cellular GSH even in the presence of acivicin, this data
suggests that glutamate does not act by inhibiting cGT.
Determination of cellular GSH
In order to better understand the effects of glutamate and
BSO on GSH metabolism and GSH release in astrocytes,
intra- and extracellular GSH was measured before and after
glutamate stimulation of primary astrocytes on 24-well
� 2008 The Authors
Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1144–1152
Glutamate induces release of glutathione from cultured rat astrocytes | 1147

dishes. In the absence of glutamate, approximately 50% of
the initial cellular GSH was found in the medium after
240 min incubation. This amount was increased to approx-
imately 70%, if glutamate was present during the incubation.
In contrast, the presence of BSO did not alter the extracel-
lular GSH content compared with the respective controls
without BSO. The differences found for the sum of cellular
plus extracellular GSH after 240 min of incubation were not
significant (p > 0.05). For all conditions shown in Table 1,
GSSG accounted for less than 5% of the GSx contents in
cells or media (data not shown), indicating that GSH and not
GSSG is released from astrocytes and that the presence of
glutamate does not significantly affect the extracellular GSH/
GSSG ratio.
Effect of glutamate receptor agonists on GSH release
To study whether GSH release was dependent on activation
of glutamate receptors, agonists were added to astrocyte
cultures. Incubation of astrocytes with agonists to the NMDA
(50 lM) or non-NMDA ionotropic glutamate receptors
[50 lM a-amino-3-hydroxy-5-methyl-4-isoxazole propionic
acid (AMPA)] had no significant effect on GSH release at the
240 min time point compared with control astrocytes
(Table 2).
Glutamate-induced GSH release from hippocampal
astrocytes
In order to investigate whether glutamate-induced increase in
extracellular GSH could be observed in astrocytes from other
brain regions, hippocampal astrocytes at DIV 14 were
compared with cortical astrocytes. In the absence of gluta-
mate, extracellular GSH increased to 1.14 ± 0.23 lM after
240 min in hippocampal cultures compared with 1.22 ±
0.08 lM for cortical cultures (Fig. 3). In the presence of
5 mM glutamate, the concentration of extracellular GSH in
hippocampal cultures was significantly increased compared
with controls (2.73 ± 0.42 lM vs. 1.14 ± 0.23 lM, respec-
tively, at 240 min; p < 0.05). This increase in extracellular
GSH is of the same order of magnitude to that observed in
cortical astrocyte cultures. As observed for cortical astro-
cytes, no significant difference could be observed between
Fig. 3 Glutamate induces release of GSH in hippocampal and cortical
astrocytes. Incubation with 5 mM glutamate for 15, 45, 120 and
240 min induced a significant increase in extracellular GSH in hippo-
campal astrocytes when compared with control without glutamate
(n = 3 separate cell preparations, *p < 0.05 control vs. glutamate
conditions), similar to the increase observed for glutamate-treated
cortical astrocytes.
Fig. 2 The effect of acivicin on glutamate-induced GSH release from
rat astrocytes. Cortical astrocytes were incubated with (Glu) or without
(Cont) 5 mM glutamate for 240 min and extracellular GSH levels were
determined. The same experiment was repeated in the presence of
100 lM acivicin (Glu + Aciv and Cont + Aciv columns). Acivicin did
not have a significant effect on extracellular GSH in control astrocytes
(Cont vs. Cont + Aciv; 1.42 ± 0.01 lM vs. 1.70 ± 0.03 lM; p > 0.05)
but did significantly increase extracellular GSH in the presence of
glutamate (Glu vs. Glu + Aciv; 3.17 ± 0.13 lM vs. 3.79 ± 0.13 lM;
**p < 0.01), suggesting that glutamate does not act by inhibition of
cGT (n = 3 separate wells from the same astrocyte preparation).
Table 2 Effect of glutamate receptor agonists on GSH release from
astrocytes
Extracellular
GSH (lM,
mean ± SEM)
Percentage
control (%) n
Control 1.4 ± 0.2 100 ± 13.9 6
5 mM glutamate 2.8 ± 0.3 208.8 ± 24.1 6
50 lM NMDA 1.3 ± 0.3 104.1 ± 14.8 3
50 lM AMPA 1.2 ± 0.2 93 ± 7.1 3
Cortical astrocyte cultures were incubated for 4 h with or without
glutamate or glutamate receptor agonists, as indicated. Only gluta-
mate had a significant effect on extracellular GSH compared with
controls (p < 0.01). Percentage control is the extracellular GSH con-
centration after 4 h compared with the control for that experiment.
n numbers are as indicated. AMPA, a-amino-3-hydroxy-5-methyl-
4-isoxazole propionic acid.
Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1144–1152
� 2008 The Authors
1148 | J. Frade et al.