Differential effects of iodoacetamide and iodoacetate on glycolysis and glutathione metabolism of cultured astrocytes.

Frontiers in Neuroenergetics www.frontiersin.org March 2009 | Volume 1 | Article 1 | 1
NEUROENERGETICS
ORIGINAL RESEARCH ARTICLE
published: 24 March 2009
doi: 10.3389/neuro.14.001.2009
Differential effects of iodoacetamide and iodoacetate on
glycolysis and glutathione metabolism of cultured astrocytes
Maike M. Schmidt1,2 and Ralf Dringen1,2,3*
1 Center for Biomolecular Interactions Bremen, University of Bremen, Bremen, Germany
2
Center for Environmental Research and Sustainable Technology, University of Bremen, Bremen, Germany
3
School of Psychology, Psychiatry, and Psychological Medicine, Monash University, Clayton, Australia
Iodoacetamide (IAA) and iodoacetate (IA) have frequently been used to inhibit glycolysis,
since these compounds are known for their ability to irreversibly inhibit the glycolytic enzyme
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). However, the consequences of a
treatment with such thiol reagents on the glutathione (GSH) metabolism of brain cells have not
been explored. Exposure of astroglia-rich primary cultures to IAA or IA in concentrations of up
to 1 mM deprived the cells of GSH, inhibited cellular GAPDH activity, lowered cellular lactate
production and caused a delayed cell death that was detectable after 90 min of incubation.
However, the two thiol reagents differed substantially in their potential to deprive cellular GSH
and to inhibit astrocytic glycolysis. IAA depleted the cellular GSH content more effi ciently than
IA as demonstrated by half-maximal effects for IAA and IA that were observed at concentrations
of about 10 and 100 µM, respectively. In contrast, IA was highly effi cient in inactivating GAPDH
and lactate production with half-maximal effects observed already at a concentration below
100 µM, whereas IAA had to be applied in 10 times higher concentration to inhibit lactate
production by 50%. These substantial differences of IAA and IA to affect GSH content and
glycolysis of cultured astrocytes suggest that in order to inhibit astrocytic glycolysis without
substantially compromising the cellular GSH metabolism, IA – and not IAA – should be used
in low concentrations and/or for short incubation periods.
Keywords: alkylation, astrocytes, carboxymethylation, GAPDH, glycolysis, GSH, lactate, thiol reagents
excluded. In this context, especially the reactions of IAA and IA
with low molecular weight cellular thiols such as GSH (Chen and
Stevens, 1991; Liu et al., 1996) have to be considered.
The tripeptide GSH (γ-L-glutamyl-L-cysteinylglycine) is a cel-
lular thiol that is present in millimolar concentrations in mam-
malian cells (Cooper and Kristal, 1997). GSH has many important
functions in cells. Among those, the antioxidative and detoxifying
functions of GSH are most likely the most important ones for
many cell types and tissues. GSH serves as electron donor for the
reduction of peroxides by glutathione peroxidases (Margis et al.,
2008) and is substrate for the detoxifi cation of xenobiotics in the
reactions that are catalyzed by glutathione-S-transferases (Hayes
et al., 2005).
In the brain, astrocytes play a very important role in the anti-
oxidative defense and in the detoxifi cation of xenobiotics (Aoyama
et al., 2008; Ballatori et al., 2008; Cooper and Kristal, 1997; Dringen,
2000, 2009). Cultured astrocytes contain GSH in a cytosolic concen-
tration of 8 mM (Dringen and Hamprecht, 1998). These cells rely
on a high GSH concentration for the rapid clearance of peroxides
(Dringen et al., 2005; Liddell et al., 2006a,b) for the resistance
against oxidative stress (Bi et al., 2008; Bishop et al., 2007; Gegg
et al., 2003, 2005; Giordano et al., 2008) and for the GSH- dependent
detoxifi cation of xenobiotics (Kubatova et al., 2006; Sagara and
Sugita, 2001; Waak and Dringen, 2006).
Although IAA and IA have often been used to modulate the
glucose metabolism of cultured astrocytes, the consequences of a
INTRODUCTION
The thiol reagents iodoacetamide (IAA) and iodoacetate (IA)
(Figure 1) are frequently used as alkylating reagents to modify
thiol groups in proteins by S-carboxyamidomethylation and
S-carboxymethylation, respectively. IAA and IA have been used to
study the importance of cysteine residues in catalytic reactions of
enzymes and in transport processes of brain cells (Albrecht et al.,
1993; Aschner et al., 1994; Gali and Board, 1997) and to derivatise
cysteine residues in proteins for proteomic approaches (Adachi
et al., 2005; Sun et al., 2008; Williams et al., 2008). The inactivation
of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by IAA
and IA (Sabri and Ochs, 1971; Williamson, 1967) is often used
as text book example for an irreversible enzyme inhibition. The
essential cysteine residue in the active center of GAPDH forms a
thioether bond with IAA or IA and can therefore not react anymore
with the physiological substrate glyceraldehyde-3-phosphate. As a
consequence, GAPDH is inactivated after exposure to IAA or IA
and glycolysis is inhibited.
On cultured astrocytes and glial cell lines, IAA and IA have been
used to inhibit glycolysis (Bakken et al., 1998; Bickler and Kelleher,
1992; Gemba et al., 1994; Loreck et al., 1987; Ogata et al., 1995)
and to study the consequences of ATP depletion and hypoglycemia
(Kauppinen et al., 1988; Nodin et al., 2005; Parkinson et al., 2002;
Sun et al., 1993; Taylor et al., 1996). However, during treatment
of cells or tissue with IAA or IA, side effects that are caused by
the unspecifi c reaction of IAA and IA as thiol reagents cannot be
Edited by:
Luc Pellerin, University of Lausanne,
Switzerland
Reviewed by:
Carole Escartin, CEA, France
Suzie Lavoie, CHUV-UNIL, Lausanne
*Correspondence:
Ralf Dringen, Center for Biomolecular
Interactions Bremen, University of
Bremen, P.O. Box 330440, D-28334
Bremen, Germany.
e-mail: ralf.dringen@uni-bremen.de

Frontiers in Neuroenergetics www.frontiersin.org March 2009 | Volume 1 | Article 1 | 2
Schmidt and Dringen Iodoacetamide and iodoacetate
treatment of astrocytes with such thiol reagents on the GSH content
of the cells have not been reported. Here we describe that exposure
of cultured astrocytes to IAA or IA lowers the cellular GSH con-
tent and inhibits glycolysis, but that the two thiol reagents differ
strongly in their potential to do so. IA is highly effi cient in low
concentrations to inhibit GAPDH activity and to lower cellular
lactate production of cultured astrocytes, whereas IAA is much
more potent to deprive cells of GSH than to inactivate glycolysis.
These results suggest that for experiments that require inhibition
of glycolysis in astrocytes IA – but not IAA – should be used in
micromolar concentrations or only for short incubation periods to
avoid a severe compromise of cellular GSH metabolism.
MATERIALS AND METHODS
MATERIALS
IAA, IA and 5,5′-dithio-bis(2-nitrobenzoic acid) were obtained from
Sigma-Aldrich (Steinheim, Germany). Glucose-6-phosphate, gluta-
mate pyruvate transaminase, GSH, glutathione disulfi de (GSSG),
glutathione reductase (GR), glyceraldehyde-3-phosphate, lactate
dehydrogenase (LDH), maleimide and potassium arsenate were
from Roche Diagnostics (Mannheim, Germany). Bovine serum
albumin, NAD+, NADH, NADP+, NADPH and sulfosalicylic acid
were purchased from Applichem (Darmstadt, Germany). Fetal calf
serum, streptomycin sulfate and penicillin G were from Biochrom
(Berlin, Germany). Dulbecco’s modifi ed Eagle’s medium (DMEM)
was purchased from Invitrogen (Karlsruhe, Germany). All other
chemicals were obtained from Fluka (Buchs, Switzerland) or Merck
(Darmstadt, Germany) at analytical grade. Sterile cell culture
material and unsterile 96-well plates were from Nunc (Roskilde,
Denmark) and Sarstedt (Karlsruhe, Germany).
CELL CULTURES
Astroglia-rich primary cultures derived from the whole brains
of neonatal Wistar rats were prepared as previously described
(Hamprecht and Löffl er, 1985). Three hundred thousand viable
cells were seeded per well of a 24-well dish in 1 mL culture medium
(90% DMEM, 10% fetal calf serum, 20 U/mL of penicillin G and
20 µg/mL of streptomycin sulfate) and cultured in a cell incubator
(Sanyo, Osaka, Japan) that contained a humidifi ed atmosphere of
10% CO
2
/90% air. The culture medium was renewed every seventh
day. The results described here were obtained on 14- to 23-day-old
cultures.
EXPERIMENTAL INCUBATION OF CELLS
To study the consequences of a treatment of astrocytes with IAA or
IA, the cells were washed with 1 mL of prewarmed (37°C) incuba-
tion buffer (IB; 1.8 mM CaCl
2
, 1 mM MgCl
2
, 5.4 mM KCl, 145 mM
NaCl, 0.8 mM Na
2
HPO
4
, 20 mM HEPES, 5 mM d-Glucose, pH 7.4)
and incubated in the cell incubator with 0.5 mL IB containing IAA
or IA in the concentrations given in the fi gures. For analysis of the
total cellular glutathione content (GSx = amount of GSH plus twice
the amount of GSSG), the cells were washed with 1 mL phosphate-
buffered saline (PBS; 10 mM potassium phosphate buffer, 150 mM
NaCl, pH 7.4) and lysed with 0.5 mL 1% (w/v) sulfosalicylic acid.
Ten microliters of aliquote parts of the lysates were used to quantify
the cellular GSx content.
CHEMICAL REACTION OF GSH WITH IAA OR IA
GSH (10 µM) was incubated with the indicated concentrations
of IAA or IA in a total volume of 1 mL at room temperature in
IB for up to 60 min. At the time points indicated, 10 µL sample of
the reaction mixture were mixed with 10 µL 1% sulfosalicylic acid
before the GSx content was determined as described below.
DETERMINATION OF GLUTATHIONE
The contents of GSx and GSSG in cell lysates and incubation media
were determined as described previously (Dringen and Hamprecht,
1996; Dringen et al., 1997) in microtiter plates according to the
colorimetric method originally described by Tietze (Tietze, 1969).
The detection limit of this assay was about 0.2 nmol GSx per 500 µL
lysate or medium.
DETERMINATION OF LACTATE IN CULTURE MEDIUM
Extracellular lactate concentration in culture media was determined
using a modifi cation of an established assay (Dringen et al., 1993).
Briefl y, 20 µL media sample were diluted with 160 µL purifi ed
water in wells of a microtiter plate and mixed with 180 µL reac-
tion mixture (5.6 mM NAD+, 19.9 U/mL LDH, 1.94 U/mL gluta-
mate pyruvate transaminase in 250 mM glutamate/NaOH buffer,
pH 8.9). After incubation for 90 min in a humidifi ed atmosphere
at 37°C the absorbance of the NADH generated from lactate was
determined at 340 nm using a Sunrise microtiter plate photometer
(Tecan, Austria). Media samples containing no lactate were used
as blanks.
DETERMINATION OF ENZYME ACTIVITIES
The cells were washed with 1 mL ice-cold PBS and subsequently
lysed by incubation with 200 µL 20 mM potassium phosphate
buffer (pH 7.0) for 10 min on ice. The lysates were scrapped of
the wells and transferred into Eppendorf cups. After centrifugation
(1 min, 12,000g, room temperature) 20 µL volumes of the superna-
tant were used to determine enzyme activities in wells of microtiter
plates at room temperature. GAPDH activity was determined using
the method described by Bisswanger (2004). The reaction mixture
contained in a total volume of 360 µL 0.9 mM glyceraldehyde-3-
phosphate, 3 mM potassium dihydrogen arsenate, 2 mM NAD+
and 93 mM triethylamine hydrochloride/NaOH buffer, pH 7.6. The
increase of absorbance at 340 nm due to the reduction of NAD+
to NADH was followed over 10 min time. Glucose-6-phosphate
dehydrogenase (G6PDH) activity was determined according to
Deutsch (1984). The reaction mixture contained in a total volume
of 200 µL 6.3 mM MgCl
2
, 5 mM maleimide, 3.3 mM glucose-6-
phosphate, 0.4 mM NADP+ and 75 mM Tris/HCl buffer, pH 7.5.
The increase of absorbance at 340 nm due to the reduction of
NADP+ to NADPH was followed over 5 min. LDH activity was
FIGURE 1 | Structural formulas of iodoacetamide (IAA) and iodoacetate (IA).

Frontiers in Neuroenergetics www.frontiersin.org March 2009 | Volume 1 | Article 1 | 3
Schmidt and Dringen Iodoacetamide and iodoacetate
determined using the method described previously (Dringen et al.,
1998). The reaction mixture contained in a total volume of 360 µL
1.8 mM pyruvate, 0.2 mM NADH and 80 mM Tris/HCl buffer
containing 200 mM NaCl, pH 7.2. The decrease of absorbance
at 340 nm due to the oxidation of NADH to NAD+ was followed
over 5 min. GR activity was determined using a method described
previously (Gutterer et al., 1999). The reaction mixture contained
in a total volume of 300 µL 1 mM GSSG, 0.2 mM NADPH and
1 mM EDTA in 100 mM potassium phosphate buffer, pH 7.0. The
decrease of absorbance at 340 nm due to the oxidation of NADPH
was followed over 5 min.
DETERMINATION OF CELL VIABILITY AND PROTEIN CONTENT
Loss of cell viability was analyzed by comparing the activity of
LDH in the incubation medium with that of the cells using the
microtiter plate assay described previously (Dringen et al., 1998).
The presence of IAA or IA in a concentration of 1 mM did not affect
LDH activity in lysates of cultured astrocytes (data not shown).
The protein content per well of a 24-well dish was quantifi ed after
solubilization of the cells in 200 µL of 0.5 M NaOH according to
the Lowry method (Lowry et al., 1951) using bovine serum albumin
as a standard. Total protein content per well and cytosolic protein
in the supernatants of cell lysates (cytosolic protein) were used
to calculate specifi c GSx or GSSG contents and specifi c enzyme
activities, respectively.
PRESENTATION OF THE DATA
If not stated otherwise, the data are presented as mean ± SD of
values obtained in experiments on three independently prepared
cultures. In the fi gures the bars have been omitted if they were
smaller than the symbols representing the mean values. Statistical
analysis of the signifi cance of differences between multiple sets
of data were performed by ANOVA followed by Bonferroni post
hoc test. If not stated otherwise, statistical analysis of the signifi -
cance of differences between two sets of data was performed using
the paired t-test. The data shown in Figure 2 for IAA and IA were
obtained separately and were therefore analyzed for signifi cance by
the unpaired t-test. p > 0.05 was considered as not signifi cant.
RESULTS
CHEMICAL REACTION OF IAA OR IA WITH GSH
To test whether IAA and IA react chemically with GSH, IAA or IA
were mixed with GSH and the amount of detectable GSx was deter-
mined during an incubation period of up to 60 min. In the presence
of IAA (Figure 2A) or IA (Figure 2B) the amount of detectable
GSx was lowered in a time- and concentration-dependent manner.
For all concentrations of IAA or IA applied, the amount of GSx
determined after 60 min of incubation was signifi cantly lower after
treatment with IAA than after exposure to IA (Figure 2C). Half-
maximal effects on the GSx content after 60 min of incubation
with IAA and IA were observed for concentrations of about 0.2
and 1 mM, respectively (Figure 2C), demonstrating that IAA was
more potent to react with GSH than IA.
INACTIVATION OF ASTROCYTIC GAPDH BY IAA OR IA
IAA and IA are well known to inhibit GAPDH activity (Sabri and
Ochs, 1971; Williamson, 1967). To test for the potential of these
compounds to inactivate astrocytic GAPDH, cultured astrocytes
were lysed and the GAPDH activity in the lysates was determined
in the absence or presence of IAA or IA. In the absence of IAA and
IA, the increase in absorbance that was caused by the GAPDH-
dependent formation of NADH was almost linear for up to 30 min
2345
G
S
x
(%
o
f
in
iti
a
l)
0
25
50
75
100
1 mM
10 mM
A
time of incubation (min)
0 5 15 30 60
G
S
x
(%
o
f
in
iti
a
l)
0
25
50
75
100
1 mM
10 mM
B
- log ([IAA] or [IA] (M))
G
S
x
(%
o
f
in
iti
a
l)
0
25
50
75
100
IA
IAA
C
0 mM
0.1 mM
0 mM
0.1 mM
## ## ###
#
FIGURE 2 | Disappearance of GSH after exposure to IAA or IA. GSH in a
concentration of 10 µM was incubated with the indicated concentrations of
IAA (A) or IA (B) for up to 60 min. (C) shows the GSx content that was
detected after 60 min incubation with the given concentrations of IAA or IA.
The data shown represent mean ± SD of 6 values that were obtained in two
independent experiments, each performed in triplicates with individually
prepared solutions. The signifi cance of the differences between the values
obtained for IAA and IA was calculated by the unpaired t-test and is indicated
by #p < 0.05, ##p < 0.01 or ###p < 0.001).

Frontiers in Neuroenergetics www.frontiersin.org March 2009 | Volume 1 | Article 1 | 4
Schmidt and Dringen Iodoacetamide and iodoacetate
(Figure 3). If the GAPDH reaction was monitored in the presence
of IAA, the increase in NADH absorbance during the fi rst 20 min of
incubation was not signifi cantly different (p > 0.05) from that of the
control condition (absence of inhibitor) and it took about 60 min
before the enzyme was completely inhibited (Figure 3). In contrast,
in the presence of IA the increase in NADH absorbance was com-
pletely prevented within a few minutes (Figure 3).
CONSEQUENCES OF A TREATMENT OF ASTROCYTE CULTURES WITH
IAA OR IA
To test for the consequences of a treatment of cultured astrocytes
with IAA or IA, the cells were incubated with various concentrations
of these compounds for up to 2 h. After the indicated incubation
times the extracellular activity of LDH as indicator for the loss of
cell viability, the specifi c cellular GSx content as indicator for cel-
lular GSH, and the lactate concentration in the medium as indicator
for glycolytic activity were monitored.
During the fi rst 60 min of incubation with IAA or IA, the cells
remained viable as indicated by the lack of any increase (p > 0.05)
in extracellular LDH activity compared to control (absence of IAA
and IA) (Figures 4A,B). However, incubation of astrocytes with
IAA or IA for longer than 60 min caused a signifi cant increase in
extracellular LDH activity that was moderate for 0.1 mM IAA or
IA but more severe for higher concentrations of both compounds
(Figures 4A,B).
Exposure of cultured astrocytes to IAA caused a rapid decline of
the cellular specifi c GSx content (p < 0.001 already after 5 min) that
resulted after 30 min incubation in cellular GSx levels below 20%
of control (absence of IAA) (Figure 4C). Also IA caused a decline
time of incubation (min)
0 20 40 60 80 100 120
a
b
so
rb
a
n
ce
(
3
4
0
n
m
)
0.0
0.2
0.4
0.6
0.8
1.0
no inhibitor
1 mM IAA
1 mM IA
FIGURE 3 | GAPDH activity in cell lysates of astrocyte cultures in the
absence or the presence of IAA or IA. The absorbance at 340 nm that
indicates the formation of NADH by GAPDH was monitored. The results are
presented as mean ± SD of data that were obtained on cell lysates derived
from three independently prepared cultures.
iodoacetamide iodoacetate
0 15 30 60 90 120
ce
llu
la
r
G
S
x
(%
o
f
in
iti
a
l)
0
25
50
75
100
0 15 30 60 90 120
e
xt
ra
ce
llu
la
r
L
D
H
a
ct
iv
ity
(
%
o
f
in
iti
a
l)
0
25
50
75
100
0 mM
0.1 mM
0.3 mM
1 mM
0 mM
0.1 mM
0.3 mM
1 mM
A B
C D
time of incubation (min)
***
***
***
***
***
*
***
***
**
***
**
**
*
**
or
***
FIGURE 4 | Consequences of an application of IAA (A,C) or IA (B,D) on
cellular viability (A,B) and specifi c cellular GSx content (C,D) in astrocyte
cultures. The cells were incubated for up to 120 min without or with IAA or IA
in the concentrations indicated in (A) and (B). The results represent
mean ± SD of data that were obtained on three independently prepared
cultures. The cultures contained initial protein contents of 80 ± 8 µg protein
per well and initial specifi c GSx contents (100%) of 46.2 ± 10.2 nmol/mg
protein. The signifi cance of differences to the data obtained for the control
condition (absence of inhibitor) are indicated by *p < 0.05, **p < 0.01 or
***p < 0.001.

Frontiers in Neuroenergetics www.frontiersin.org March 2009 | Volume 1 | Article 1 | 5
Schmidt and Dringen Iodoacetamide and iodoacetate
Exposure of astrocytes to IAA hardly affected the extracellular
accumulation of lactate during the fi rst 60 min of incubation
compared to control (Figure 6A), whereas a further increase of
extracellular lactate was slowed during longer incubations, at least
for IAA concentrations of 0.3 and 1 mM (Figure 6A). The extracel-
lular lactate concentration determined for cells that were treated
with IAA for 60 min did not differ signifi cantly to that of control
cells (Figure 7A). In contrast, incubation of astrocytes with IA in
a concentration of 1 mM inhibited extracellular lactate accumu-
lation much quicker than IAA (Figure 6B). In concentrations of
0.1 or 0.3 mM IA almost completely prevented extracellular lac-
tate accumulation within 30 min of incubation (Figure 6B). After
60 min of incubation the extracellular lactate concentrations of
astrocyte cultures that were treated with 0.1, 0.3 and 1 mM IA were
signifi cantly lowered to 40 ± 7, 24 ± 16 and 6 ± 8%, respectively, of
the concentration determined for cells that were incubated without
inhibitor (Figure 7A).
To demonstrate that indeed the inhibition of GAPDH by IAA
or IA was responsible for the observed decline in the rate of glyco-
lytic lactate production in astrocyte cultures, the specifi c activity of
GAPDH was determined for cells that were treated for 60 min with
various concentrations of IAA or IA. When the cells were exposed to
0.1 mM IAA the specifi c GAPDH activity was signifi cantly lowered
to half of that of controls (Figure 7B). In contrast, already after
treatment of the cells with 0.1 mM IA for 60 min GAPDH activity
was not detectable anymore (Figure 7B). In concentrations of 0.3
and 1 mM, both IAA and IA completely inhibited GAPDH activity
of astrocyte cultures within 60 min of incubation (Figure 7B).
To analyze how quickly the cellular GAPDH activity of cultured
astrocytes was inactivated after exposure to IAA or IA, the cells
were incubated with various concentrations of IAA or IA for time
periods in the minute range (Figure 8). Half-maximal inhibition
of astrocytic GAPDH by IAA that was applied in concentrations
of 0.1, 0.3 and 1 mM was observed after about 60, 30 and 5 min of
incubation, respectively (Figure 8A). In contrast, inactivation of
GAPDH by the presence of IA was much faster that that by IAA.
GAPDH activity was not detectable anymore after exposure of the
cells for 5, 15 and 30 min to IA in concentrations of 1, 0.3 and
0.1 mM, respectively (Figure 8B).
To test whether IAA or IA are also able to inactivate other meta-
bolic enzymes in cultured astrocytes, the cells were exposed for
60 min to IAA or IA in concentrations of 1 mM. Although these
-log ([IAA] or [IA] (M))
ce
llu
la
r
G
S
x
(%
o
f
in
iti
a
l)
0
25
50
75
100
IA
IAA
**
***
6 5 4 3
* *
***
###
FIGURE 5 | Concentration dependency of the GSH depletion by IAA or IA
in cultured astrocytes. The cells were incubated for 60 min with IAA or IA in
concentrations of up to 1 mM. The results represent mean ± SD of data that
were obtained on three independently prepared cultures that contained
80 ± 8 µg protein per well. The initial specifi c GSx content (100%)
corresponded to 46.2 ± 10.2 nmol/mg protein. The signifi cance of differences
to the data obtained for the control condition (absence of inhibitor) are
indicated by *p < 0.05, **p < 0.01 or ***p < 0.001. The signifi cance of
differences between the data observed after treatment with identical
concentrations of IAA and IA was calculated by the paired t-test and is
indicated by ###p < 0.001.
in cellular GSx content that depended on the concentration of IA
applied (Figure 4D). However, this decline in cellular GSx after IA
application (Figure 4D) was much slower than that observed for
identical concentrations of IAA (Figure 4C) (p < 0.001 after 5 min
for all concentrations of IAA and IA applied). The concentrations
causing half-maximal deprivation of cellular GSx after exposure
of cultured astrocytes for 60 min to IAA and IA were about 10
and 100 µM, respectively, and differed by one order of magnitude
(Figure 5).
The decline in cellular GSx that was observed after exposure
of cultured astrocytes to IAA or IA (Figures 4C,D and 5) was not
accompanied by an increase in cellular GSSG nor by an increase
in the extracellular concentrations of GSx or GSSG (Table 1). In
contrast, the extracellular contents of GSx and GSSG were lowered
after incubation of the cells with IAA or IA (Table 1).
Table 1 | Cellular and extracellular GSx and GSSG contents and extracellular LDH activity of primary astrocyte cultures after exposure to IAA or IA.
Control IAA IA n
Cellular GSx (nmol/mg) 41.9 ± 7.6 3.1 ± 3.6*** 8.9 ± 5.6*** 4
Cellular GSSG (nmol/mg) 5.1 ± 0.6 2.0 ± 2.0 2.9 ± 1.1 3
Extracellular GSx (nmol/mg) 4.7 ± 0.5 1.4 ± 1.1* 2.4 ± 1.4 3
Extracellular GSSG (nmol/mg) 3.8 ± 0.5 1.6 ± 0.9* 2.6 ± 0.9 3
Extracellular LDH (%) 6.4 ± 1.8 12.2 ± 6.5 8.9 ± 2.2 4
Primary astrocyte cultures were incubated in the absence (control) or the presence of 1 mM IAA or IA. The cellular and extracellular contents of GSx and GSSG and
extracellular LDH activity (as % of initial) were determined after 60 min of incubation. The results represent mean ± SD of data that were obtained on n independently
prepared cultures that contained 87 ± 16 µg protein per well. The signifi cance of differences to the data obtained for the control condition (absence of inhibitor) are
indicated by *p < 0.05 or ***p < 0.001.