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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We studied the regulation of GSH and the enzymes involved in GSH regulation, gamma -glutamylcysteine synthetase (gamma -GCS) and gamma -glutamyl transpeptidase (gamma -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 gamma -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. gamma -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 gamma -GCS activity at 24-96 h. The activity of gamma -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 gamma -GT and gamma -GCS by different mechanisms. We suggest that the increase in gamma -GCS activity but not in gamma -GT activity may be required for the increase in intracellular GSH under oxidative stress in alveolar epithelial cells.

gamma -glutamylcysteine synthetase; gamma -glutamyl transpeptidase; A549 cells

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 gamma -glutamylcysteine synthetase (gamma -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 gamma -glutamyl transpeptidase (gamma -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 gamma -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 gamma -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 gamma -GCS and gamma -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-kappa B and activator protein-1 (AP-1) for inflammatory mediator gene expression (1). The AP-1 binding site also appears to be critical for gamma -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 gamma -GCS and gamma -GT, which are critical for GSH synthesis in human alveolar epithelial cells (A549).

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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, gamma -GCS, and gamma -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 gamma -GT activity assay. For the gamma -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 gamma -GT and gamma -GCS activity.

gamma -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 gamma -GCS activity and was used to test the specificity of the reaction.

gamma -GT activity was assayed with the method of Tate and Meister (37) using 0.1 M glycyl-glycine and 5 mM L-gamma -glutamyl-p-nitroanilidine. The rate of formation of p-nitroaniline was recorded at 410 nm at 37°C for 1 min. Purified gamma -GT was used as a standard, and 1 IU of gamma -GT was defined as the micromoles of p-nitroanilinine released from L-gamma -glutamyl-p-nitroaniline per minute per milligram of protein. Acivicin (0.5 mM) was used as a specific inhibitor of gamma -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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 gamma -GCS activity in alveolar epithelial cells. Previously, Rahman et al. (27) described an increase in the activity of gamma -GCS after CSC exposure at 24 h. In this study, we found that MQ (100 and 200 µM) increased gamma -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 gamma -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 gamma -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 gamma -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 gamma -glutamylcysteine synthetase (gamma -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 gamma -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 gamma -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 gamma -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 gamma -GT activity in alveolar epithelial cells. The activity of gamma -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 gamma -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 gamma -GCS and gamma -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).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effects of MQ, X/XO, CSC, and hyperoxia on gamma -GT activity in alveolar epithelial cells

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Effect of dexamethasone on gamma -GT activity in alveolar epithelial cells

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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 gamma -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 gamma -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 gamma -GCS normally functions submaximally due to feedback inhibition by GSH (30). A transient decrease in cellular GSH content could release this feedback inhibition of gamma -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 gamma -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 gamma -GCS activity through increased synthesis of the enzyme. Recently, Rahman and colleagues (24, 27) showed that cigarette smoke and MQ can induce gamma -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 gamma -GCS-HS. Our data showing an increase in GSH levels and gamma -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 gamma -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 gamma -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 gamma -GCS activity in alveolar epithelial cells.

Activity of gamma -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 gamma -GT in epithelial cells, which would prevent the degradation of extracellular GSH. However, we showed that CSC had no effect on gamma -GT activity in epithelial cells. Thus our data suggest that gamma -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 gamma -GCS-HS mRNA. In contrast, gamma -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 gamma -GT activity in human alveolar epithelial cells in response to MQ, X/XO, or hyperoxia. The possible explanation for differential regulation of gamma -GT activity in response to oxidative stress may be due to differential expression of the gamma -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 gamma -GT by hyperoxia in rat lung type II cells. However, further studies are required on the effects of oxidants on gamma -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 gamma -GCS and gamma -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, gamma -GCS, and gamma -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 gamma -GCS by dexamethasone is not known; however, it is of interest that the gamma -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 gamma -GCS enzymatic activity but not on the increase in gamma -GT activity. Dexamethasone decreases GSH levels by inhibiting gamma -GT and gamma -GCS activity and hence GSH synthesis. We suggest that gamma -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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
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 gamma -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. gamma -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 gamma -glutamyl transpeptidase and gamma -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 gamma -glutamylcysteine synthetase and gamma -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 gamma -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 gamma -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 gamma -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 gamma -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. gamma -Glutamylcysteine synthetase. J. Biol. Chem. 259: 3534-3538, 1984[Abstract/Free Full Text].

33.   Shi, M. M., T. Iwamoto, and H. J. Forman. gamma -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 gamma -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 gamma -glutamyl transpeptidase with acivicin. J. Biol. Chem. 269: 21435-21439, 1994[Abstract/Free Full Text].

37.   Tate, S., and A. Meister. gamma -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 gamma -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