Differential regulation of glutathione by oxidants and
dexamethasone in alveolar epithelial cells
Irfan
Rahman1,
Agnes
Bel1,
Brigitte
Mulier1,
Kenneth
Donaldson2, and
William
MacNee1
1 The Rayne Laboratory,
Respiratory Medicine Unit, Department of Medicine, University of
Edinburgh, Edinburgh EH8 9AG; and
2 Department of Biological
Sciences, Napier University, Edinburgh EH10 5DT, United Kingdom
 |
ABSTRACT |
We studied the regulation of GSH and the enzymes
involved in GSH regulation,
-glutamylcysteine synthetase (
-GCS)
and
-glutamyl transpeptidase (
-GT), in response to the oxidants
menadione, xanthine/xanthine oxidase, hyperoxia, and cigarette smoke
condensate in human alveolar epithelial cells (A549). Menadione (100 µM), xanthine/xanthine oxidase (50 µM/10 mU), and cigarette smoke
condensate (10%) exposure produced increased GSH levels (240 ± 6, 202 ± 12, and 191 ± 2 nmol/mg protein, respectively;
P < 0.001) compared with the control
level (132 ± 8 nmol/mg protein), which were associated with a significant increase in
-GCS activity (0.18 ± 0.006, 0.16 ± 0.01, and 0.17 ± 0.008 U/mg protein, respectively;
P < 0.01) compared with the control
level (0.08 ± 0.001 U/mg protein) at 24 h. Exposure to hyperoxia
(95% O2) resulted in a
time-dependent increase in GSH levels.
-GCS activity increased
significantly at 4 h (P < 0.001),
returning to control values after 12 h of exposure. Dexamethasone (3 µM) exposure produced a significant time-dependent decrease in the
levels of GSH and
-GCS activity at 24-96 h. The activity of
-GT did not change after oxidant treatment; however, it was
decreased significantly by dexamethasone at 24-96 h. Thus oxidants
and dexamethasone modulate GSH levels and activities of
-GT and
-GCS by different mechanisms. We suggest that the increase in
-GCS activity but not in
-GT activity may be required for the
increase in intracellular GSH under oxidative stress in alveolar
epithelial cells.
-glutamylcysteine synthetase;
-glutamyl transpeptidase; A549
cells
 |
INTRODUCTION |
THE RESPIRATORY EPITHELIUM is often exposed to
oxidants, whether inhaled, such as cigarette smoke and ozone, or from
reactive oxygen intermediates released from neutrophils recruited to
the lungs during airway inflammation. GSH, a ubiquitous cellular
nonprotein sulfhydryl, is an important antioxidant in the maintenance
of intracellular redox balance and is involved in the detoxification of
oxidants, free radicals, electrophiles, and organic peroxides either
through direct thiol conjugation or by enzyme-catalyzed reactions
(26, 28).
The synthesis of intracellular GSH is controlled by two rate-limiting
factors. One is the enzyme
-glutamylcysteine synthetase (
-GCS),
which catalyzes the first reaction of de novo GSH synthesis. The other
is the availability of cysteine (18, 30), which is produced as a
breakdown product of extracellular GSH by the membrane-bound enzyme
-glutamyl transpeptidase (
-GT) (7). The activities of both of
these enzymes are important in maintaining the intracellular GSH pool
and thus preventing oxidative injury to cells. Intracellular GSH can
also be increased by cysteine transport (9, 17) in cells under
oxidative stress, which can subsequently be used for the synthesis of
GSH by
-GCS. Hence the maintenance of intracellular GSH is mediated
either by modification of cellular uptake of its precursors (cysteine)
or by an increase in the activity of
-GCS. Rahman and colleagues
(24, 27) recently showed that GSH synthesis is induced in
lung cells during oxidative stress as an adaptive response. However,
different forms of oxidant stress may have differential effects on GSH
regulation through the enzymes
-GCS and
-GT. Therefore, in this
study, we used a range of different oxidative stresses including
cigarette smoke condensate (CSC) to assess their effects on GSH
homeostasis in lung type II epithelial cells. The oxidants that were
studied were hyperoxia (>95%
O2); xanthine/xanthine oxidase
(X/XO), an intracellular generator of superoxide anion
(O
2·) (11);
2-methyl-1,4-naphthoquinone [menadione (MQ)], a quinone that generates O
2· and hydrogen
peroxide (H2O2)
by redox cycling (2); and CSC, a complex oxidant that contains
1014 to
1016 free radicals/puff and
electrophilic compounds capable of generating H2O2
(21).
Corticosteriods such as dexamethasone are widely used as for their
anti-inflammatory properties in various inflammatory lung diseases (1).
The anti-inflammatory action of dexamethasone appears to be mediated
through an effect on transcription factors such as nuclear factor-
B
and activator protein-1 (AP-1) for inflammatory mediator gene
expression (1). The AP-1 binding site also appears to be critical for
-GCS gene regulation and hence GSH synthesis (27). The effects of
corticosteroids on lung GSH metabolism have not been studied so far.
Therefore, we studied the effects of the oxidants described above and
dexamethasone on GSH and its enzymes
-GCS and
-GT, which are
critical for GSH synthesis in human alveolar epithelial cells (A549).
 |
MATERIALS AND METHODS |
Unless otherwise stated, all of the biochemical reagents used in this
study were purchased from Sigma (Poole, UK); cell culture media were purchased from GIBCO-BRL
(Paisley, UK).
A549 epithelial cells. The type II
alveolar epithelial cell line A549 (European Culture Collection no.
86012804), which was mycoplasma free, was maintained in
continuous culture at 37°C with 5%
CO2 in DMEM, sodium bicarbonate,
L-glutamate, and 10% fetal
bovine serum.
Preparation of CSC. CSC (100%) was
produced from standard cigarettes, each containing 23 mg of tar and 2.2 mg of nicotine (University of Kentucky UK2R1). CSC was produced by
blowing smoke generated by a smoking machine with a vacuum syringe
system from three cigarettes over 3 ml of PBS in a siliconized glass
tonometer (Vitalograph, Buckingham, UK). The smoke-generating machine
delivers a 37-ml puff of whole cigarette smoke, including particulates, every minute to the tonometer, which was rotated gently (13). The
solution was made up fresh on the morning of each experiment, and CSC
(100%) was filtered with a 0.22-mm filter (Millipore, Molsheim,
France) to remove large particles and bacteria (27). Ten percent CSC
was prepared from this solution by dilution with PBS.
To define a convenient surrogate marker for the amount of cigarette
smoke entering into the solution, we measured the products of nitric
oxide, nitrite, and nitrate in the filtered CSC solution by the Griess
reaction (31). We found that the concentration was 12.2 ± 1.2 µM,
which is in agreement with the earlier report (5).
Epithelial cell exposure to CSC, X/XO, MQ, and
dexamethasone. Monolayers of confluent epithelial cells
were prepared by seeding 3 × 106 cells/well in a six-well plate
and reculturing in DMEM with 10% fetal bovine serum at 37°C with
5% CO2 for 24 h. Confluent
monolayers were rinsed two times with DMEM and exposed to CSC (10%),
X/XO (50 µM/10 mU), MQ (100 and 200 µM), or dexamethasone (3 µM)
for time intervals between 1, 6, and 24 h in 2 ml of full medium
incubated at 37°C with 5%
CO2. In some experiments to study
the recovery of intracellular GSH and enzyme activities after various
exposures, monolayers were washed with fresh medium after exposure to
the oxidants for 1 h and reincubated for 24 h. Thereafter, the
monolayers were washed two times with cold PBS (pH 7.4), scraped into
PBS, and centrifuged at 250 g for 5 min at 4°C. Cell viability was determined by staining with trypan
blue.
Exposure of epithelial cells to
hyperoxia. Cell monolayers were placed in anaerobic
chambers humidified with wet tissue paper at 37°C. The chambers
were sealed, and 95% O2-5% air
was flushed through for 3 min (until the reading on the
O2 monitor was 95%). The level of
O2 was then maintained at 95% for
a subsequent 24 h and flushed at 12-h intervals.
GSH,
-GCS, and
-GT
assays. After centrifugation of the epithelial cells at
250 g for 5 min at 4°C, the cell
pellets were suspended in 1 ml of cold 0.6% sulfosalicyclic acid,
sonicated on ice, homogenized with a Teflon pestle, and vortexed
vigorously. The cells were then centrifuged at 4,000 g for 5 min at 4°C. The supernatant was immediately used in the soluble GSH assay with the
5,5'-dithio-bis(2-nitrobenzoic acid)-GSSG reductase recycling method described by Tietze (38).
The cells were suspended in 100 mM Tris · HCl buffer
and used for the
-GT activity assay. For the
-GCS activity assay,
the cell pellets were resuspended in 100 mM
Tris · HCl buffer, pH 8.0, briefly sonicated, and
homogenized with a Teflon pestle on ice with Triton X-100 to a final
concentration of 0.1% (vol/vol) followed by vortexing. The cell
extracts were spun at 4,000 g for 15 min at 4°C. The resultant cell supernatants and the whole cell
suspension were immediately analyzed for
-GT and
-GCS activity.
-GCS activity was assayed with the method described by Seelig and
Meister (32) using the coupled assay with pyruvate kinase and lactate
dehydrogenase. The rate of decrease in absorbance at 340 nm at 37°C
was followed. Enzyme specific activity was defined as micromoles of
NADH oxidized per minute per milligram of protein, which is equal to 1 IU. Buthionine sulfoximine (50 µM) was used to inhibit
-GCS
activity and was used to test the specificity of the reaction.
-GT activity was assayed with the method of Tate and Meister (37)
using 0.1 M glycyl-glycine and 5 mM
L-
-glutamyl-p-nitroanilidine. The rate of formation of
p-nitroaniline was recorded at 410 nm at 37°C for 1 min. Purified
-GT was used as a standard, and 1 IU
of
-GT was defined as the micromoles of
p-nitroanilinine released from
L-
-glutamyl-p-nitroaniline
per minute per milligram of protein. Acivicin (0.5 mM) was used as a
specific inhibitor of
-GT (36) to confirm the enzyme activity.
Protein and DNA assays. The protein
concentration was determined with the bicinchoninic acid reagent assay
(Pierce, Rockford, IL) (35). The DNA concentration was estimated with
the method of Richards (29) using the diphenylamine reagent.
Statistical analysis. The results are
expressed as means ± SE. Differences between values were compared
by Duncan's multiple range test.
 |
RESULTS |
Effects of MQ, CSC, X/XO, hyperoxia, and dexamethasone
on GSH levels in alveolar epithelial cells. Rahman and
colleagues (24, 27) previously demonstrated that MQ (100 µM) and CSC
(10%) deplete GSH levels significantly at 1 h followed by a
significant increase in GSH levels 24 h after exposure. In this study,
A549 cells were exposed to various generators of oxidant stress to
investigate whether these agents exert their effects on GSH levels in a
time-dependent manner. MQ at concentrations of 100 and 200 µM
significantly decreased GSH levels at 1 h
(P < 0.01) compared with the control
values. A return to the control levels by 6 h and a significant
increase in intracellular GSH concentration after a 24-h exposure were observed with both concentrations of MQ (Fig.
1). X/XO did not produce any change in GSH
after 1 and 6 h of exposure. However, GSH levels significantly
increased at 24 h compared with the control values (Fig.
2). Exposure to hyperoxia produced a
significant increase in GSH concentration at 2, 8, 12, and 24 h, the
level peaking at 12 h in comparison with the control value (Fig.
3). Dexamethasone (3 µM) incubation
produced a time-dependent depletion of intracellular GSH (Fig.
4). Cell viability remained >94% after all of the above treatments. The mean GSH level in the A549 cells was
132 ± 8 nmol/mg protein, which is in agreement with previous reports (8, 24).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of 100 and 200 µM menadione (MQ) at different time intervals
on GSH levels in A549 type II alveolar epithelial cells. Each symbol is
mean ± SE of 6 experiments. Significant difference compared with
control value: ** P < 0.01;
*** P < 0.001.
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of xanthine/xanthine oxidase (X/XO; 50 µM/10 mU) at 1, 6, and
24 h on GSH levels in A549 cells. Each symbol is mean ± SE of 6 experiments. ** Significant difference compared with control
value, P < 0.01.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of hyperoxic (95% O2) exposure at 2, 8, 12, and 24 h on GSH levels in A549 cells. Each symbol is mean ± SE of 4 experiments. Significant difference compared with control value:
** P < 0.01;
*** P < 0.001.
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of 3 µM dexamethasone at different time points on GSH levels
in A549 epithelial cells. Each symbol is mean ± SE of 4 experiments. Significant difference compared with control value:
* P < 0.05;
** P < 0.01.
|
|
Effects of MQ, CSC, X/XO, hyperoxia, and dexamethasone
on
-GCS activity in alveolar epithelial
cells. Previously, Rahman et al. (27) described an
increase in the activity of
-GCS after CSC exposure at 24 h. In this
study, we found that MQ (100 and 200 µM) increased
-GCS activity
at 24 h (P < 0.001) without any change at 1 and 6 h (Fig. 5). Similarly,
X/XO did not produce any change in
-GCS activity at 1 and 6 h;
however, the activity was significantly increased after a 24-h exposure
(Fig. 6). Hyperoxia exposure resulted in a
significant increase in the
-GCS activity at 4 h, but the activity
had normalized after 12 and 24 h of treatment (Fig.
7). Dexamethasone did not
produce any significant change in the
-GCS activity after 4 and 10 h
of treatment; however, the activity was significantly decreased at 24, 48, 72, and 96 h compared with the control values (Fig.
8).

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of MQ on -glutamylcysteine synthetase ( -GCS) enzyme
activity. Cells were incubated with 100, and 200 µM MQ for different
time intervals. Each bar is mean ± SE of 4 experiments.
***Significant difference compared with control value,
P < 0.001.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of X/XO (50 µM/10 mU) on -GCS activity in A549 type II
alveolar epithelial cells. Each bar is mean ± SE of 4 experiments.
** Significant difference compared with control value,
P < 0.01.
|
|

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of hyperoxic exposure at different time points on -GCS
activity in A549 cells. Each bar is mean ± SE of 4 experiments.
*** Significant difference compared with control value,
P < 0.001.
|
|

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 8.
Effect of 3 µM dexamethasone at different time points on -GCS
activity in A549 alveolar epithelial cells. Each bar is mean ± SE
of 4 experiments. Significant difference compared with control value:
** P < 0.01;
*** P < 0.001.
|
|
Effects of MQ, CSC, X/XO, hyperoxia, and dexamethasone
on
-GT activity in alveolar epithelial
cells. The activity of
-GT was not affected by any
of the oxidants after 1, 6, and 24 h of treatment (Table
1). By contrast, dexamethasone produced a
significant decrease in
-GT activity at 24, 48, 72, and 96 h (Table
2) compared with the control values. There
were no changes in enzyme activity after 4 and 10 h of treatment with
dexamethasone. The activity of
-GCS and
-GT in these cells was
0.08 ± 0.01 and 0.2 ± 0.02 U/mg protein, respectively, which is
in agreement with several earlier reports (10, 20, 24).
Effects of MQ, X/XO, hyperoxia, CSC, and dexamethasone
on protein and DNA concentrations in A549 cells. We
determined the concentration of protein per cell or per milligram of
DNA after treatment with oxidants and dexamethasone in A549 cells. The
concentrations of cellular protein after MQ (100 µM), X/XO,
hyperoxia, and CSC exposures at 24 h were not significantly different
(0.84 ± 0.1, 0.86 ± 0.07, 0.84 ± 0.1, and 0.79 ± 0.08 mg protein/106 cells,
respectively) compared with the control value (0.82 ± 0.1 mg
protein/106cells;
n = 3 experiments). Similarly, the
concentration of protein per milligram of DNA remained constant after
MQ, X/XO, hyperoxia, and CSC treatments in A549 cells at 24 h (6.7 ± 0.6, 6.8 ± 0.9, 7.2 ± 1.1, and 6.8 ± 0.4 mg
protein/mg DNA, respectively) compared with the control value (6.8 ± 0.8 mg protein/mg DNA; n = 3 experiments). By contrast, dexamethasone produced a slight but
insignificant time-dependent decrease in cellular protein concentration
at 24, 48, 72, and 96 h (0.72 ± 0.1, 0.69 ± 0.08, 0.65 ± 0.1, and 0.63 ± 0.11 mg
protein/106 cells, respectively)
compared with the control values (0.82 ± 0.1, 0.78 ± 0.07, 0.80 ± 0.1 and 0.74 ± 0.12 mg
protein/106 cells;
P > 0.05;
n = 3 experiments). However,
dexamethasone produced a time-dependent increase in the concentration
of protein per milligram of DNA (7.8 ± 0.3, 8.7 ± 0.5, 9.1 ± 0.4, and 9.5 ± 0.4 mg protein/mg DNA) compared with the
control values (6.6 ± 0.2, 6.6 ± 0.1, 7.0 ± 0.4 and 6.9 ± 0.6 mg protein/mg DNA; P < 0.05; n = 3 experiments) at 24, 48, 72, and 96 h, respectively.
 |
DISCUSSION |
The capacity of mammalian cells to maintain homeostasis of cellular
functions during oxidative stress depends on the rapid induction of
protective antioxidant enzymes (25, 34). In the present study, we
demonstrated that exposure to sublethal concentrations of MQ and CSC
caused a transient depletion of GSH followed by an elevation in
intracellular GSH levels in A549 epithelial cells. X/XO also produced
an elevation in GSH levels at 24 h without an initial depletion, at
least at the time points at which measurements were made. One mechanism
we considered for this elevation in GSH was an increase in
-GCS
activity, the rate-limiting enzyme of GSH biosynthesis. The results of
this study demonstrated that the increase in GSH levels was associated
with an increase in
-GCS activity that occurred in response to
exposure of cells to MQ and X/XO. A previous study (27) showed a
similar effect with CSC.
Several mechanisms can account for the de novo synthesis of GSH in
response to sublethal oxidative stress. It has been reported that
-GCS normally functions submaximally due to feedback inhibition by
GSH (30). A transient decrease in cellular GSH content could release
this feedback inhibition of
-GCS activity, leading to higher GSH
levels. This could partially explain the increased intracellular GSH
levels produced by MQ and CSC. As the GSH levels increase, further
feedback inhibition may result, causing a subsequent decrease in
-GCS activity. However, an initial depletion of GSH cannot explain
the later increase in GSH synthesis after X/XO or hyperoxia exposure.
The lack of an initial depletion of GSH with hyperoxia in contrast to
MQ and CSC may be explained by the fact that MQ and CSC result in GSH
depletion by a mechanism that produces GSH conjugates (26), which does
not occur with hyperoxia or X/XO, or that a transient fall in GSH was
missed because measurements were made at limited time points.
A second mechanism that can result in increased GSH synthesis is an
increase in
-GCS activity through increased synthesis of the enzyme.
Recently, Rahman and colleagues (24, 27) showed that cigarette smoke
and MQ can induce
-GCS-heavy subunit (HS) mRNA expression in human
alveolar type II cells that possess the catalytic activity of this
enzyme.
Other investigators (15, 22, 33) have also shown that oxidants can
increase GSH synthesis in various cell lines. We suggested that
reactive oxygen species, particularly
O
2· and
H2O2,
released from MQ (2) and
H2O2
generated by CSC-derived free radicals and electrophilic compounds (21)
could result in transcriptional upregulation of
-GCS-HS. Our data
showing an increase in GSH levels and
-GCS activity by X/XO
therefore suggest upregulation of GSH synthesis by the direct action of O
2· within the cell. This
observation is supported by previous studies showing increased
-GCS-HS mRNA expression after MQ (24) and CSC (27).
Hyperoxia is known to increase GSH levels by increasing cystine uptake
in endothelial cells (17). Our data showing increased levels of GSH
2-24 h after hyperoxia exposure in A549 cells suggest that an
increased synthesis of GSH occurred in response to hyperoxia. However,
the activity of
-GCS was only increased at 4 h and not at 12 and 24 h. This suggested that the increased GSH levels produced by hyperoxia
may not be related to increased
-GCS activity in alveolar epithelial
cells.
Activity of
-GT has been suggested to be important in maintaining
intracellular GSH (28). GSH levels have been shown to be twofold higher
in the epithelial lining fluid of chronic cigarette smokers (3, 19). In
this study, we hypothesized that cigarette smoke may increase the level
of epithelial lining fluid GSH, which might result from the inhibition
of
-GT in epithelial cells, which would prevent the degradation of
extracellular GSH. However, we showed that CSC had no effect on
-GT
activity in epithelial cells. Thus our data suggest that
-GT may not
be involved in the regulation of GSH synthesis in alveolar epithelial
cells in response to CSC. Indeed the previous work by Rahman and
colleagues (24, 27) suggests that the increase in GSH after CSC is
mainly due to transcriptional upregulation of
-GCS-HS mRNA. In
contrast,
-GT has been shown to be induced in rat lung epithelial L2
cells by MQ and tert-butylhydroquinone
(12, 14). However, our data failed to show any change in the
-GT
activity in human alveolar epithelial cells in response to MQ, X/XO, or
hyperoxia. The possible explanation for differential regulation of
-GT activity in response to oxidative stress may be due to
differential expression of the
-GT gene in different cell lines and
organs (4, 6, 23). A similar discrepancy was also observed recently in
a study (39) on the regulation of
-GT by hyperoxia in rat lung type
II cells. However, further studies are required on the effects of
oxidants on
-GT gene expression in human alveolar epithelial cells
to explain this difference.
Corticosteroids such as dexamethasone are thought to suppress
inflammation by interacting with various transcription factors (1). The
role of redox status, particularly GSH redox status, in the regulation
of transcription factors is of considerable interest currently (1).
However, the effect of dexamethasone on GSH synthesis has not been
extensively studied so far. We show for the first time a time-dependent
depletion of intracellular GSH by dexamethasone in A549 alveolar
epithelial cells. The decrease in GSH is associated with inhibition of
the activity of
-GCS and
-GT. Similar hormonal inhibition of GSH
synthesis has been observed in a rat hepatic cell line (16).
Dexamethasone treatment also produced a time-dependent small decrease
in the concentration of protein per cell and the milligrams of protein
per milligram of DNA. However, the extent of the decrease in protein or
DNA levels did not account for the dramatic decrease in the levels of
GSH,
-GCS, and
-GT activity (30-77%) in response to
dexamethasone in A549 cells because the decrease in the level of
milligrams of protein per cell and per milligrams of DNA was only
12-15 and 15-27%, respectively. The reason for the
inhibition of
-GCS by dexamethasone is not known; however, it is of
interest that the
-GCS gene is regulated by an AP-1 transcription
factor (27), which may be inhibited by dexamethasone. Further studies
are required to understand the molecular mechanism(s) of the action of
dexamethasone on the GSH synthesis.
In conclusion, this study has shown that sublethal concentrations of
MQ, X/XO, and hyperoxia produce elevations in intracellular GSH content
in alveolar epithelial cells. This elevation appears to be dependent on
an increase in
-GCS enzymatic activity but not on the increase in
-GT activity. Dexamethasone decreases GSH levels by inhibiting
-GT and
-GCS activity and hence GSH synthesis. We suggest that
-GT activity may not be necessary for the regulation of GSH under
oxidative stress in human alveolar epithelial cells.
 |
ACKNOWLEDGEMENTS |
This work was supported by the British Lung Foundation and the
Chest Heart and Stroke Association (Scotland).
 |
FOOTNOTES |
Address for reprint requests: W. MacNee, Respiratory Medicine Unit,
Dept. of Medicine, Univ. of Edinburgh, Royal Infirmary, Lauriston
Place, Edinburgh EH3 9YW, UK.
Received 21 November 1997; accepted in final form 3 April 1998.
 |
REFERENCES |
1.
Barnes, P. J.,
and
I. Adcock.
Anti-inflammatory actions of steroids: molecular mechanism.
Trends Pharmacol. Sci.
14:
436-441,
1993[Medline].
2.
Brunmark, A.,
and
E. Cadenas.
Redox and addition chemistry of quinoid compounds and its biological implications.
Free Radic. Biol. Med.
7:
435-477,
1989[Medline].
3.
Cantin, A. M.,
S. L. North,
R. C. Hubbard,
and
R. G. Crystal.
Normal alveolar epithelial lining fluid contains high levels of glutathione.
J. Appl. Physiol.
63:
152-157,
1987[Abstract/Free Full Text].
4.
Courtay, C.,
N. Heisterkamp,
G. Siest,
and
J. Groffen.
Expression of multiple gamma-glutamyltransferase genes in man.
Biochem. J.
297:
503-508,
1994[Medline].
5.
Cueto, R.,
and
W. A. Pryor.
Cigarette smoke chemistry: conversion of nitric oxide to nitrogen dioxide and reactions of nitrogen oxides with other smoke components as studied by Fourier transform infrared spectroscopy.
Vib. Spectrosc.
86:
6377-6381,
1989.
6.
Darbouy, M.,
M.-N. Chobert,
O. Lahuna,
T. Okamoto,
J.-P. Bonvalet,
N. Farman,
and
Y. Laperche.
Tissue-specific expression of multiple
-glutamyl transpeptidase mRNAs in rat epithelia.
Am. J. Physiol.
261 (Cell Physiol. 30):
C1130-C1137,
1991[Abstract/Free Full Text].
7.
Hanigan, M. H.,
and
W. A. Ricketts.
Extracellular glutathione is a source of cysteine for cells that express gamma-glutamyl transpeptidase.
Biochemistry
32:
6302-6306,
1993[Medline].
8.
Hatcher, E. L.,
J. M. Alexander,
and
Y. J. Kang.
Decreased sensitivity to adriamycin in cadmium-resistant human lung carcinoma A549 cells.
Biochem. Pharmacol.
53:
747-754,
1997[Medline].
9.
Jenkinson, S. G.,
R. A. Lawrence,
C. A. Zamora,
and
S. M. Deneke.
Induction of intracellular glutathione in alveolar type II pneumocytes following BCNU exposure.
Am. J. Physiol.
266 (Lung Cell. Mol. Physiol. 10):
L125-L130,
1994[Abstract/Free Full Text].
10.
Kang, Y. J.,
Y. Feng,
and
E. L. Hatcher.
Glutathione stimulates A549 cell proliferation in glutamine-deficient culture: the effect of glutamate supplementation.
J. Cell Biol.
161:
589-596,
1994.
11.
Kim, J. H.,
and
R. Hille.
Reductive half-reaction of xanthine oxidase with xanthine.
J. Biol. Chem.
268:
44-51,
1993[Abstract/Free Full Text].
12.
Kugelman, A.,
H. A. Choy,
R. Liu,
M. M. Shi,
E. Gozal,
and
H. J. Forman.
-Glutamyl transpeptidase is increased by oxidative stress in rat alveolar L2 epithelial cells.
Am. J. Respir. Cell Mol. Biol.
11:
586-592,
1994[Abstract].
13.
Lannan, S.,
K. Donaldson,
D. Brown,
and
W. MacNee.
Effects of cigarette smoke and its condensates on alveolar cell injury in vitro.
Am. J. Physiol.
266 (Lung Cell. Mol. Physiol. 10):
L92-L100,
1994[Abstract/Free Full Text].
14.
Liu, R. M.,
H. Hu,
T. W. Robinson,
and
H. J. Forman.
Differential enhancement of
-glutamyl transpeptidase and
-glutamylcysteine synthetase by tert-butylhydroquinone in rat lung epithelial L2 cells.
Am. J. Respir. Cell Mol. Biol.
14:
186-191,
1996[Abstract]. '
15.
Liu, R. M.,
H. Hu,
T. W. Robinson,
and
H. J. Forman.
Increased
-glutamylcysteine synthetase and
-glutamyl transpeptidase activities enhance resitance of rat lung epithelial L2 cells to quinone toxicity.
Am. J. Respir. Cell Mol. Biol.
14:
192-197,
1996[Abstract].
16.
Lu, S. C.,
J. Kuhlenkamp,
C. Garcia-Ruiz,
and
N. Kaplowitz.
Hormone-mediated down-regulation of hepatic glutathione synthesis in the rat.
J. Clin. Invest.
88:
260-269,
1991[Medline].
17.
Miura, K.,
T. Ishii,
Y. Sugita,
and
S. Bannai.
Cystine uptake and glutathione level in endothelial cells exposed to oxidative stress.
Am. J. Physiol.
262 (Cell Physiol. 31):
C50-C58,
1992[Abstract/Free Full Text].
18.
Morris, P. E.,
and
G. R. Bernard.
Significance of glutathione in lung disease and implications for therapy.
Am. J. Med. Sci.
307:
119-127,
1994[Medline].
19.
Morrison, D.,
S. Lannan,
A. Langridge,
I. Rahman,
and
W. MacNee.
Effect of acute cigarette smoking on epithelial permeability, inflammation and oxidant status in the airspaces of chronic smokers (Abstract).
Thorax
49:
1077,
1994.
20.
Morstyn, G.,
A. Russo,
D. N. Carney,
E. Karawya,
S. H. Wilson,
and
J. B. Mitchell.
Heterogeneity in the radiation survival curves and biochemical properties of human lung cancer cell lines.
J. Natl. Cancer Inst.
73:
801-807,
1984[Medline].
21.
Nakayama, T.,
D. F. Church,
and
W. A. Pryor.
Quantitative analysis of the hydrogen peroxide formed in aqueous cigarette tar extracts.
Free Radic. Biol. Med.
7:
9-15,
1989[Medline].
22.
Ochi, T.
Hydrogen peroxide increases the activity of
-glutamylcysteine synthetase in cultured Chinese hamster V79 cells.
Arch. Toxicol.
70:
96-103,
1995[Medline].
23.
Pawlak, A.,
O. Lahunat,
F. Bulle,
A. Suzuki,
N. Ferry,
S. Siegrist,
N. Chikhi,
M. N. Chobert,
G. Guellaent,
and
Y. Laperche.
gamma-Glutamyl transpeptidase: a single copy gene in the rat and multigene family in the human genome.
J. Biol. Chem.
263:
9913-9916,
1988[Abstract/Free Full Text].
24.
Rahman, I.,
A. Bel,
B. Mulier,
M. F. Lawson,
D. J. Harrison,
W. MacNee,
and
C. A. D. Smith.
Transcriptional regulation of
-glutamylcysteine synthetase-heavy subunit by oxidants in human alveolar epithelial cells.
Biochem. Biophys. Res. Commun.
229:
832-837,
1996[Medline].
25.
Rahman, I.,
L. Clerch,
and
D. Massaro.
Rat lung antioxidant enzyme induction by ozone.
Am. J. Physiol.
260 (Lung Cell. Mol. Physiol. 4):
L412-L418,
1991[Abstract/Free Full Text].
26.
Rahman, I.,
X. Y. Li,
K. Donaldson,
D. J. Harrison,
and
W. MacNee.
Glutathione homeostasis in alveolar epithelial cells in vitro and lung in vivo under oxidative stress.
Am. J. Physiol.
269 (Lung Cell. Mol. Physiol. 13):
L285-L292,
1995[Abstract/Free Full Text].
27.
Rahman, I.,
C. A. D. Smith,
M. F. Lawson,
D. J. Harrison,
and
W. MacNee.
Induction of
-glutamylcysteine synthetase by cigarette smoke is associated with AP-1 in human alveolar epithelial cells.
FEBS Lett.
396:
21-25,
1996[Medline].
28.
Reed, D. J.
Glutathione: toxicological implications.
Annu. Rev. Pharmacol. Toxicol.
30:
603-631,
1990[Medline].
29.
Richards, G. M.
Modifications of the diphenylamine rection giving increased sensitivity and simplicity in the estimation of DNA.
Anal. Biochem.
57:
369-376,
1974[Medline].
30.
Richman, P. G.,
and
A. Meister.
Regulation
-glutamylcysteine synthetase by nonallosteric feedback inhibition by glutathione.
J. Biol. Chem.
250:
1422-1426,
1975[Abstract].
31.
Schmidt, H. H. W.,
and
M. Kelm.
Determination of nitrite and nitrate by the Griess reaction.
In: Methods in Nitric Oxide Research, edited by M. Feelisch,
and J. S. Stamler. Chichester, UK: Wiley, 1996, p. 491-497.
32.
Seelig, G. F.,
and
A. Meister.
-Glutamylcysteine synthetase.
J. Biol. Chem.
259:
3534-3538,
1984[Abstract/Free Full Text].
33.
Shi, M. M.,
T. Iwamoto,
and
H. J. Forman.
-Glutamylcysteine synthetase and GSH increase in quinone-induced oxidative stress in BPAEC.
Am. J. Physiol.
267 (Lung Cell. Mol. Physiol. 11):
L414-L421,
1994[Abstract/Free Full Text].
34.
Shi, M. M.,
A. Kugelman,
I. Takeo,
L. Tian,
and
H. J. Forman.
Quinone-induced oxidative stress elevates glutathione and induces
-glutamylcysteine synthetase activity in rat lung epithelial L2 cells.
J. Biol. Chem.
269:
26512-26517,
1994[Abstract/Free Full Text].
35.
Smith, P. K.,
R. I. Krohn,
G. T. Hermanson,
A. K. Mallia,
F. H. Gartner,
M. D. Provenzano,
E. K. Fujimoto,
N. M. Goeke,
B. J. Olson,
and
D. C. Klenk.
Measurement of protein using bicinchoninic acid.
Anal. Biochem.
150:
76-85,
1985[Medline].
36.
Stole, E.,
T. K. Smith,
J. M. Manning,
and
A. Meister.
Interaction of
-glutamyl transpeptidase with acivicin.
J. Biol. Chem.
269:
21435-21439,
1994[Abstract/Free Full Text].
37.
Tate, S.,
and
A. Meister.
-Glutamyl transpeptidase from kidney.
Methods Enzymol.
113:
400-405,
1985[Medline].
38.
Tietze, F.
Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissue.
Anal. Biochem.
27:
502-522,
1969[Medline].
39.
Van Klaveren, R. J.,
D. Dinsdale,
J. L. Pype,
M. Demedts,
and
B. Nemery.
Changes in
-glutamyltransferase activity in rat lung tissue, BAL, and type II cells after hyperoxia.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L537-L547,
1997[Abstract/Free Full Text].
Am J Physiol Lung Cell Mol Physiol 275(1):L80-L86
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society