Am J Physiol Cell Physiol Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 290: C262-C270, 2006. First published September 14, 2005; doi:10.1152/ajpcell.00070.2005
0363-6143/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/1/C262    most recent
00070.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (13)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cantin, A. M.
Right arrow Articles by Hanrahan, J. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cantin, A. M.
Right arrow Articles by Hanrahan, J. W.

MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Oxidant stress suppresses CFTR expression

André M. Cantin,1 Ginette Bilodeau,1 Cristine Ouellet,1 Jie Liao,2 and John W. Hanrahan2

1Pulmonary Research Unit, Faculty of Medicine, Université de Sherbrooke, Sherbrooke; and 2Department of Physiology, McGill University, Montreal, Quebec, Canada

Submitted 17 February 2005 ; accepted in final form 1 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Epithelial mucous membranes are repeatedly exposed to oxidants and xenobiotics. CFTR plays a role in glutathione transepithelial flux and in defining the hydration and viscoelasticity of protective mucus. We therefore hypothesized that CFTR expression and function may be modulated by oxidant stress. A sublethal oxidant stress (tert-butylhydroquinone, BHQ) in CFTR-expressing epithelial cells (T84) induced a significant increase in cellular glutathione that was associated with an increase in expression of the gene encoding the heavy subunit of the rate-limiting enzyme for glutathione synthesis, {gamma}-glutamylcysteine synthetase ({gamma}-GCShs). CFTR gene expression was markedly decreased according to a time course that mirrored the changes in {gamma}-GCShs. Western blot analysis confirmed that the decrease in CFTR gene expression was associated with a decrease in CFTR protein. cAMP-dependent iodide efflux was also decreased by the oxidant stress. Nuclear run-on assays indicated that the oxidant stress had no effect on CFTR gene transcription, but the mRNA stability in the oxidant-stressed cells was markedly reduced. Furthermore, BHQ increased {gamma}-GCShs mRNA while decreasing CFTR mRNA in Calu-3 cells, and taurine chloramine induced similar effects in T84 cells. We conclude that suppression of CFTR expression may represent an adaptive response of mucosal epithelium to an exogenous oxidant stress.

antioxidants; chloride channels; epithelial cells; cystic fibrosis; reactive oxygen species

THE LOSS OF FUNCTIONAL CFTR leads to cystic fibrosis, a disease characterized by the obstruction of epithelial tissues that produce mucin-rich secretions (14), probably due to volume depletion of mucus and epithelial surface fluid layers (25, 33, 42). However, the links between CFTR function and normal epithelial physiology are not fully understood (57). CFTR is an anion channel located at the apical surface of epithelial cells, and it regulates the permeability of chloride and bicarbonate through a cAMP-dependent process (2, 22, 43). CFTR also has finite permeability to large organic anions and can mediate membrane permeability of the antioxidant glutathione (26, 29).

Glutathione is an antioxidant tripeptide that plays an essential role in protecting epithelial cells against oxidant stress and xenobiotics (35). The cytoplasmic levels of glutathione rapidly increase in epithelial cells exposed to oxidative stress (46, 47). Glutathione synthesis results from reactions catalyzed by two enzymes, {gamma}-glutamylcysteine synthase ({gamma}-GCS) and glutathione synthetase (7). {gamma}-GCS represents the rate-limiting enzyme in glutathione synthesis (21). This enzyme consists of two subunits, a light and a heavy chain, each encoded by separate genes. The gene encoding the heavy subunit ({gamma}-GCShs) contains regulatory elements in the 5'-flanking region that allow a marked increase in transcription on exposure of epithelial cells to an oxidative stress (36, 51). An example of such an oxidative stress is cigarette smoke, which increases the transcription of {gamma}-GCShs in lung epithelial cells (46, 48, 49). Recently, cigarette smoke was also shown to inhibit chloride secretion in human bronchial epithelium, suggesting that oxidants may modulate CFTR function (27).

In addition to regulating glutathione synthesis, epithelial cells can modulate transmembrane glutathione transport (32). Membrane transport systems for glutathione have been identified in liver and heart cells and are likely to occur in many other tissues such as those affected by cystic fibrosis. Linsdell and Hanrahan (29) provided electrophysiological evidence that CFTR is permeable to glutathione, implicating it in glutathione transport across epithelial cell membranes. This concept is consistent with previous in vivo observations by Roum et al. (52) of decreased glutathione content in the lower respiratory tract extracellular space of patients with cystic fibrosis and subsequent in vitro studies of cell monolayers expressing either wild-type or mutant nonfunctional CFTR, which also demonstrated that CFTR plays an essential role in maintaining high levels of extracellular glutathione (15). Glutathione permeability has been observed in patch-clamp experiments, and glutathione flux through CFTR channels has been demonstrated biochemically (26). In addition, cells expressing wild-type functional CFTR have significantly less glutathione and are more susceptible to oxidant-dependent apoptosis than cells expressing no functional CFTR (24). A large number of studies have clearly indicated that high intracellular glutathione concentrations are essential to protect cells against oxidant injury. One would therefore predict that during an oxidative stress in mucosal tissues, epithelial cells might respond by using mechanisms to increase cellular glutathione such as enhanced glutathione synthesis and decreased glutathione efflux.

In addition to its role in glutathione transport, CFTR is a cAMP-dependent anion channel permeable to chloride and bicarbonate. CFTR also regulates amiloride-sensitive sodium channels and the chloride/bicarbonate exchanger. A decrease in CFTR function increases mucus concentration and decreases mucus pH, the latter of which favors interchain cross-linking of mucins and increased mucus viscosity (42). Because mucins have been shown to scavenge free radicals (12, 17), a decrease in CFTR would likely increase the antioxidant protection provided by mucus.

In the context of the above, we hypothesized that CFTR is a physiologically important regulator of the response to oxidative stress and that its expression in epithelial cells may decrease in the presence of an oxidant burden. To verify this hypothesis, cellular glutathione, expression of the {gamma}-GCShs gene, and expression of CFTR at the gene, protein, and functional levels were characterized in cultured epithelial cell lines exposed to the oxidants tert-butylhydroquinone (BHQ) and taurine chloramine (TauNHCl).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell lines and reagents. Human colon adenocarcinoma T84 cells [American Type Culture Collection (ATCC) cell line CCL248; Rockville, MD] were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (GIBCO, Invitrogen Life Technologies, Burlington, ON, Canada) supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2.5 µg/ml Fungizone, and 5% fetal bovine serum at 37°C in 5% CO2. The cells were seeded at 6 x 106 cells/well in six-well tissue culture plates (Linbro Chemical, New Haven, CT) and grown to confluence. The cells were treated with 0–1,600 µM BHQ (Sigma-Aldrich, Oakville, ON, Canada) for 0–24 h, according to the protocols described below. TauNHCl was generated by adding taurine (Sigma-Aldrich) and NaOCl at a 2-to-1 ratio to the culture medium as previously reported (6). The human airway epithelial cell line Calu-3 (ATCC HTB 55) was cultured in MEM supplemented with MEM nonessential amino acids (0.1 mM) and sodium pyruvate (1 mM), 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2.5 µg/ml Fungizone, and 15% fetal bovine serum at 37°C in 5% CO2. Calu-3 cells were seeded at 2 x 106 cells/well in six-well tissue culture plates and cultured until confluent. Forskolin, IBMX, dibutyryl cAMP, and mucin from porcine stomach were from Sigma-Aldrich. 51Cr and 125I were from PerkinElmer Life Sciences (Boston, MA). The antibody used for Western blot analysis of CFTR, MAb-450, was a generous gift from T. J. Jensen and J. R. Riordan (S. C. Johnson Medical Research Center, Mayo Clinic, Scottsdale, AZ). This antibody recognizes the R domain of CFTR.

Cytotoxicity and glutathione assays. To determine the effect of BHQ on cell viability, T84 cells were grown to confluence in 24-well plates and labeled with 5 µCi/well 51Cr for 18 h at 37°C. The cells were then washed three times with Earle’s balanced salt solution (EBSS; GIBCO) and incubated for 7 h at 37°C in 5% CO2 in the presence of 0–1,600 µM BHQ in EBSS. At the end of the incubation period, 100 µl of supernatant was collected from each well to determine radioactivity in a gamma counter (model Wizard 1470; PerkinElmer Life Sciences). An index of cytotoxicity was calculated for each sample by subtracting the radioactivity of untreated cells (background) from test supernatants and dividing this value by the difference in radioactivity between supernatants from cells incubated with 1% Triton X-100 and background. Cellular glutathione was determined with a model DU7 spectrophotometer (Beckman Instruments Canada, Mississauga, ON, Canada) to determine the reduction of DTNB in the presence of glutathione reductase and NADPH as previously described (8). This assay measures both reduced glutathione and glutathione disulfide.

RNA extraction and Northern blot analysis. Cells were incubated for 0, 2, 4, 6, 18, and 24 h in the presence of 100 (T84) or 300 (Calu-3) µM BHQ before being harvested for Northern blot analysis as previously described (1). Total cell RNA was isolated with a one-step guanidinium-phenol-chloroform extraction procedure (9). RNA was separated by electrophoresis on 1% agarose and transferred onto a hybond-N+ membrane (Amersham, Oakville, ON, Canada) for analysis. Membranes were prehybridized for 4 h in a mixture containing 120 mM Tris, 600 mM NaCl, 0.1% Na4P2O7, 8 mM EDTA, 0.2% SDS, 625 µg/ml heparin, and 10% dextran sulfate at pH 7.4. Hybridization was performed overnight at 68°C in the same buffer. The human {gamma}-GCS probe was obtained from ATCC (GenBank/EMBL M90656 [GenBank] ), and the multidrug resistance protein 1 (MRP1) probe was a gift from Dr. Susan Cole of Queen’s University (Kingston, ON, Canada). The CFTR pcDNA3 was a gift from Dr. Gergely Lukacs of the Hospital for Sick Children (Toronto, ON, Canada). The probe was prepared by using a 1.1-kb fragment after EcoRI digestion. Each probe was labeled with the multiprime DNA labeling system (Amersham) using [{alpha}-32P]dCTP (specific activity >3,000 Ci/mM). The membrane was then washed once at room temperature for 20 min in 2x SSC and for 1 h at 68°C in 0.1% SDS, 0.1x SSC and was rinsed at room temperature in 0.1x SSC. The membrane was exposed to X-OMAT film (Kodak, Rochester, NY) with intensifying screens at –80°C. As a control for RNA integrity, the blot was hybridized with a 1-kb PstI cDNA probe (ATCC) of the housekeeping gene GAPDH. Signal intensity was quantitated densitometrically with a PowerLook II scanner (UMAX Technologies, Dallas, TX). Densitometric values are expressed as the ratio of {gamma}-GCShs to GAPDH, MRP1 to GAPDH, or CFTR to GAPDH densitometric quantifications.

mRNA stability and nuclear run-on assays. To determine mRNA stability in the presence and absence of BHQ, T84 cells were seeded at a density of 6 x 106 cells/well in six-well plates and cultured to confluence in Dulbecco’s modified Eagle’s medium with Ham’s F-12 medium (1:1) supplemented with 5% FBS under 5% CO2 at 37°C. The cells were then washed, and fresh medium containing 100 µM BHQ or medium alone was added to the cells for 3 h. The supernatant was then replaced by medium free of BHQ and containing 8 µg/ml actinomycin D. Northern blot analysis was performed on cell extracts 3, 6, and 9 h after the addition of actinomycin D. We also determined the transcriptional effect of BHQ on cystic fibrosis gene expression with nuclear run-on assays as previously described (1). Confluent T84 cells were incubated with 300 µM BHQ for 0, 1, or 3 h. The cells were washed in PBS and lysed in 1 ml of lysis solution [in mM: 10 Tris·HCl (pH 7.4), 5 MgCl2, and 10 KCl with 0.5% NP-40], and the nuclei were isolated. The nuclei were resuspended in nuclei storage buffer [in mM: 50 Tris·HCl (pH 7.4), 5 MgCl2, and 0.5 DTT with 40% glycerol] containing 20 U/ml RNAguard (Pharmacia). Nascent transcripts were elongated in vitro for 30 min at 30°C for run-on analysis as previously described. Nuclear [{alpha}-32P]UTP-labeled RNAs were hybridized to linearized DNA of CFTR and GAPDH.

Western blot analysis. T84 cells were cultured in 100-mm tissue culture dishes to confluence in medium alone or with 100 or 300 µM BHQ for 24 h. The cells were then washed twice with cold PBS, harvested by scraping, and resuspended in 0.5 ml of RIPA buffer composed of 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 0.08% sodium deoxycholate, 20 mM Tris·HCl (pH 8.0), and 1 protease inhibitor cocktail tablet/50 ml (Roche, Mannheim, Germany) before being placed on ice for 10 min to lyse the cells. The lysate was centrifuged at 9,000 g for 10 min at 4°C, and the supernatant was collected. The protein concentration of the lysate was estimated with the Bio-Rad Protein Assay Kit. Samples containing 50 µg of protein were electrophoresed on a 8% SDS-polyacrylamide gel. The separated proteins were electrotransferred to a nitrocellulose membrane (Bio-Rad Laboratories, Mississauga, ON, Canada) for Western blot analysis.

cAMP-dependent iodide efflux assay. cAMP-dependent anion efflux was determined in T84 cells. Cells were cultured to 90% confluence in six-well plates and labeled with 15 µCi/well 125I for 1 h at room temperature. The cells were then washed six times in efflux buffer (in mM: 119 Na gluconate, 1.2 K2HPO4, 0.6 KH2PO4, 25 NaHCO3) and incubated in efflux buffer. Supernatants were collected every minute for 4 min and replaced with fresh efflux buffer. After 4 min, efflux buffer containing 0.5 mM 2'-O-dibutyryl cAMP, 10 µM IBMX, and 10 µM forskolin (cAMP buffer) was added, and supernatants were collected every minute and replaced with fresh cAMP-containing medium. The radioactivity of the supernatants was determined using a gamma counter. Results are expressed as the percentage of counts per minute measured before the addition of IBMX, forskolin, and dibutyryl cAMP.

Mucin and BHQ cytotoxicity. Secretory mucins are the major glycoproteins present in mucosal secretions at epithelial surfaces. One of the major roles of CFTR is to regulate the hydration and viscosity of mucins by modulating transepithelial chloride, sodium, bicarbonate, and glutathione flux (15, 45). It has been suggested that mucins have antioxidant properties (13, 17). A potential advantage of CFTR suppression during sustained oxidant exposure may be to increase the concentration and viscosity of mucins at epithelial surfaces. We therefore hypothesized that mucins protect epithelial T84 cells against cytotoxicity induced by high concentrations of BHQ. To test this hypothesis, T84 cells were labeled with 51Cr as described above and washed, and 0–10 mg/ml mucin (Sigma-Aldrich) was added to 500 µl of EBSS. At these concentrations, mucin was freely soluble and did not form layers in the medium. Subsequently, 1 mM BHQ was added to each well, and supernatants were collected after 7 h to calculate the cytotoxicity.

Statistics. Statistical analysis was performed with Prism software (GraphPad, San Diego, CA) version 4.0 for Macintosh. The results are presented as means ± SE. The data from experiments with only one variable were analyzed using one-way ANOVA, and two-way ANOVA was applied to data from experiments with more than one variable. A Bonferroni multiple comparison test was applied after ANOVA. A value of P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
HBQ oxidative stress. Exposure of the T84 cell line to BHQ resulted in a significant oxidative stress that at high concentrations induced measurable cytotoxicity, as determined by the release of chromium into the cell supernatants. Exposure of cells to ≤400 µM BHQ did not induce significant cytotoxicity, whereas a concentration of 800 µM induced a cytotoxicity index of 10.3 ± 0.8% (n = 12; P < 0.05 compared with 0 µM BHQ). Exposure of T84 and Calu-3 cells to 300 µM BHQ for 24 h did not decrease the cell number [T84 controls = 37.0 ± 3.0 x 106 cells, 300 µM BHQ = 38.0 ± 3.6 x 106 cells (P > 0.05); Calu-3 controls = 13.8 ± 1.0 x 106 cells, 300 BHQ = 12.2 ± 0.9 x 106 cells (P > 0.05)], cell protein content [T84 controls = 46.8 ± 3.7 mg/ml, 300 µM BHQ = 43.3 ± 1.8 mg/ml (P > 0.05); Calu-3 controls = 20.5 ± 3.4 mg/ml, 300 BHQ = 20.1 ± 1.0 mg/ml (P > 0.05)], or viability as determined by Trypan blue dye exclusion. Increasing concentrations of BHQ resulted in progressively higher cytotoxicity (Fig. 1).



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 1. Induction of an oxidant stress in T84 cells by exposure to tert-butylhydroquinone (BHQ). Cells were incubated with 51Cr overnight, followed by extensive washing and the addition of 0–1,600 µM BHQ for 7 h, and the radioactivity released into the supernatants was measured to determine the cytotoxicity index. Results are expressed as means ± SE (n = 12). *P < 0.05, **P < 0.001 vs. 0 BHQ.

 
BHQ exposure and cellular glutathione. Exposure of confluent monolayers of T84 cells to BHQ for 6–24 h resulted in time-dependent increases in cellular glutathione (Fig. 2A). At 6 h, the glutathione content of cells exposed to 100 µM BHQ was significantly increased above control (44.8 ± 0.6 vs. 32.3 ± 0.3 nmol/106 cells; P < 0.001, n = 12), and the increase persisted at all time points up to 24 h (24 h = 94.9 ± 2.5 vs. 45.5 ± 0.9 nmol/106 cells; P < 0.001 for all times compared with controls, n = 12). In contrast, extracellular glutathione from the same experiments (Fig. 2B) was slightly increased only at 12 h (BHQ 1.80 ± 0.05 vs. control 1.30 ± 0.04 nmol/106 cells; P < 0.001, n = 12) and did not differ at any time subsequent to 12 h (P > 0.05 at all other times).



View larger version (31K):
[in this window]
[in a new window]
  		Fig. 2. Effect of BHQ exposure on cellular (A) and extracellular (B) glutathione content. Confluent monolayers of T84 cells were exposed to medium alone or with 100 µM BHQ for 6–24 h. The supernatants were collected, and the cells were lysed. Both preparations were then assayed for glutathione. *P < 0.001, BHQ vs. control; n = 12 per condition.

 
Effect of oxidant stress on gene expression. Exposure of T84 cells to BHQ was associated with a marked increase in {gamma}-GCShs mRNA expression (Fig. 3, A and B). At 4 and 6 h, the increase in {gamma}-GCShs mRNA reached maximal levels, returning to control levels by 18–24 h ({gamma}-GCShs-to-GAPDH ratio at 6 h: control = 5.3 ± 1.1 vs. 25.3 ± 7.2 ratio units; P < 0.01, n = 6). In striking contrast, CFTR mRNA showed changes synchronous to those of {gamma}-GCShs but in the opposite direction, with a marked decrease in CFTR mRNA detected after 6-, 18-, and 24-h exposure to 100 µM BHQ (Fig. 3, A and C; CFTR/GAPDH at 6 h: control = 8.2 ± 0.8 vs. BHQ = 1.8 ± 0.3 ratio units, P < 0.001; 18 h, P < 0.001; 24 h, P < 0.01; n = 6 independent experiments). The oxidant stress did not alter mRNA levels of the gene encoding the ATP-binding cassette (ABC) protein MRP1 (Fig. 3D; P > 0.05 for all comparisons).



View larger version (51K):
[in this window]
[in a new window]
  		Fig. 3. Northern blot analysis of mRNA expression of genes encoding the heavy subunit of {gamma}-glutamylcysteine synthase ({gamma}-GCShs), CFTR, multidrug resistance protein 1 (MRP1), and GAPDH. A: Northern blot from 1 representative experiment. B–D: mean ± SE ratios of the densitometric signals for {gamma}-GCShs/GAPDH (B), CFTR/GAPDH (C), and MRP1/GAPDH (D) of cells exposed to 100 µM BHQ vs. control (n = 6). *P < 0.01, **P < 0.001 vs. control.

 
CFTR nuclear run-on assay and mRNA stability. To determine whether oxidant stress affected CFTR expression at the level of transcriptional initiation, cells were treated with 300 µM BHQ for 0, 1, and 3 h, and the cell nuclei were prepared for nuclear run-on assays. Equal counts of radioactively labeled transcripts were hybridized to DNA fragments immobilized on nitrocellulose filters. Signals were compared with the transcriptional levels of stable GAPDH. No difference in signal was observed at any time point, suggesting that oxidant stress did not affect CFTR transcription (Fig. 4A). In contrast, the rate of CFTR mRNA degradation was clearly accelerated in cells exposed to 100 µM BHQ (Fig. 4B). The mRNA degradation curves of BHQ-treated and control cells were significantly different from each other as determined by repeated-measures two-way ANOVA (P < 0.01, n = 4).



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 4. Regulation of CFTR gene transcription and mRNA stability by oxidative stress. A: transcriptional effect of BHQ on CFTR gene expression was determined by incubating T84 cells with 300 µM BHQ for 0, 1, or 3 h. Cells were washed and lysed, and the nuclei were isolated. Nascent transcripts were elongated in vitro for 30 min at 30°C for run-on analysis. Radiolabeled RNAs were hybridized to linearized DNA of CFTR and GAPDH. Results are representative of 3 separate experiments in which densitometric analysis of CFTR gene-to-GAPDH mRNA ratios showed no significant differences between each time point. B: stability of the CFTR gene mRNA was determined after 3-h exposure of cells to 100 µM BHQ or medium (Control), followed by the addition of 8 µg/ml actinomycin D. Northern blot analysis was performed on cell extracts 3, 6, and 9 h after the addition of actinomycin D. Shown are the means ± SE of 4 separate experiments.

 
CFTR protein and oxidant stress. Exposure of T84 cells to 100 and 300 µM BHQ for 24 h resulted in decreased levels of CFTR protein as determined by Western blot analysis (Fig. 5). A concentration-dependent decrease in CFTR protein could be observed after 24 h with 100 and 300 µM BHQ (control = 101.3 ± 4.6, BHQ 100 µM = 84.7 ± 4.1, 300 µM = 61.2 ± 5.2 CFTR/actin protein density as %control; P < 0.05 and P < 0.01 for BHQ 100 and 300 µM vs. control, respectively, n = 9). The level of the reference protein actin was not affected by either concentration of BHQ, regardless of exposure time.



View larger version (23K):
[in this window]
[in a new window]
  		Fig. 5. Oxidant-mediated modulation of CFTR protein expression as determined by Western blot analysis. T84 cells were exposed to 100 or 300 µM BHQ for 24 h. The cells were then harvested, and extracts of cell lysates were electrophoresed. A: protein bands were revealed with antibodies against either CFTR (top) or actin (bottom). B: results shown represent 3 separate experiments, each performed in triplicate. *P < 0.05, **P < 0.01.

 
cAMP-dependent 125I efflux. A concentration-dependent decrease in cAMP-dependent 125I efflux was observed when cells were exposed for 24 h to 0–400 µM BHQ (Fig. 6A). The peak cAMP-stimulated 125I effluxes at 6, 7, and 8 min were decreased in cells exposed to 400 µM BHQ (125I efflux at 7 min: control = 188.5 ± 30.8% vs. 400 µM BHQ = 123.3 ± 14.2%; P < 0.001, n = 6), as well as in cells exposed to 300 and 200 µM BHQ. An analysis of the area under the curve for each concentration of BHQ showed a substantial decrease in 125I efflux that reached statistical significance at 300 and 400 µM BHQ (P < 0.05; Fig. 6B).



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 6. cAMP-dependent 125I efflux from T84 cells exposed to 0–400 µM BHQ for 24 h. Confluent cells previously loaded with 125I were washed every minute for 4 min, followed by the addition of 10 µM IBMX, 10 µM forskolin, and 500 µM dibutyryl cAMP. 125I efflux was determined by measuring the radioactivity of the culture supernatants every minute. A: representative efflux curves at each BHQ concentration are expressed as %control counts per minute measured before the addition of IBMX, forskolin, and dibutyryl cAMP at 4 min. B: data representing the means of the area under the curve (AUC) of 6 separate experiments. *P < 0.05 vs. control, {dagger}P < 0.05 vs. 50 µM BHQ.

 
Effect of oxidant stress on gene expression in Calu-3 cell line. Exposure of Calu-3 cells to BHQ was also associated with a marked increase in {gamma}-GCShs and a decrease in CFTR mRNA expression (Fig. 7). At 12, 18, and 24 h, {gamma}-GCShs mRNA expression was significantly higher than in cells not exposed to 300 µM BHQ ({gamma}-GCShs-to-GAPDH ratio at 12, 18, and 24 h: P < 0.001 compared with controls at either 6 or 24 h; n = 6). In striking contrast, CFTR mRNA showed changes synchronous to those of {gamma}-GCShs but in the opposite direction, with a marked decrease in CFTR mRNA detected after 12-, 18-, and 24-h exposure to 300 µM BHQ (CFTR-to-GAPDH ratio at 12, 18 and 24 h: P < 0.001 compared with controls at either 6 or 24 h; n = 6).



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 7. Northern blot analysis of mRNA expression of genes encoding {gamma}-GCShs (A) and CFTR (B) relative to GAPDH expression in Calu-3 cells exposed to 300 µM BHQ for the indicated times. *P < 0.001 vs. control at 6 or 24 h; n = 5.

 
TauNHCl, glutathione, {gamma}-GCShs, and CFTR mRNA in T84 cells. Glutathione levels of T84 cells exposed to 500 µM TauNHCl for 24 h were markedly increased (control: glutathione = 49.2 ± 2.0 nmol/106 cells; 500 µM TauNHCl: glutathione = 151.0 ± 3.9 nmol/106 cells; P < 0.0001, n = 16; Fig. 8A). Exposure of T84 cells to 0–500 µM TauNHCl induced a concentration-dependent increase in {gamma}-GCShs-to-GAPDH ratio (P < 0.01 for 250 and 500 µM vs. control, n = 5; Fig. 8B) and a synchronous concentration-dependent decrease in the CFTR-to-GAPDH ratio (P < 0.05 for 62.5 µM vs. control; P < 0.01 for 125, 250, and 500 µM vs. control, n = 5; Fig. 8C).



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 8. Effect of T84 cell exposure to taurine chloramine (TauNHCl) on cellular glutathione after incubation with 500 µM TauNHCl for 24 h (*P < 0.0001, n = 16; A) and on {gamma}-GCShs (B) and CFTR (C) mRNA expression as determined by Northern blot analysis at 24 h (*P < 0.05, {dagger}P < 0.01; n = 5).

 
Protection of T84 cells against BHQ-induced cytotoxicity by mucin. Mucin from porcine stomach provided concentration-dependent protection of T84 cells against cytotoxicity induced by 1 mM BHQ (Fig. 9). The cytotoxicity index decreased from 51.8 ± 3.0% in the absence of mucin to 22.0 ± 2.7% and 12.3 ± 4.8% in the presence of 5 and 10 mg/ml mucin, respectively (P < 0.01 and P < 0.001 compared with no mucin; n = 3).



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 9. Protection of T84 cells against injury induced by 1 mM BHQ. Mucin was added to the culture medium at the stated concentrations during exposure of the cells to BHQ. The chromium released in the supernatant after 7 h was assayed as an index of cytotoxicity. *P < 0.01, **P < 0.001; n = 3.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Glutathione levels of airway epithelial cells are regulated, at least in part, by CFTR expression (15, 24). In the present study, we report that CFTR is suppressed by oxidant stress at the mRNA, protein, and functional levels. The oxidant-mediated actions on CFTR are associated with a marked increase in cellular glutathione, the major soluble antioxidant present in eukaryotic cells. Although cellular glutathione was markedly increased, extracellular levels of glutathione did not increase proportionately, suggesting that suppression of CFTR function may represent an adaptive mechanism of epithelial cells that contributes to the preservation of cellular glutathione during an oxidant stress. Interestingly, the decrease in CFTR gene expression is synchronous with a marked increase in expression of the gene encoding {gamma}-GCShs, the heavy subunit of the rate-limiting enzyme in glutathione synthesis. The decrease in CFTR mRNA is specific, because the oxidant BHQ did not decrease the amount of mRNA of either GAPDH or another member of the ABC protein superfamily, MRP1. Similar effects of BHQ on {gamma}-GCS and CFTR mRNA were also observed in Calu-3 cells, although some differences in the capacity to recover normal {gamma}-GCS and CFTR mRNA expression after 24 h of oxidant exposure were observed between the cell lines, with partial recovery in T84 cells but no evidence of recovery in Calu-3 cells. In addition, higher concentrations of BHQ were needed to induce changes in Calu-3 cells similar to those observed in T84 cells. Because Calu-3 cells are derived from the human airways and T84 cells from the human intestine (37, 53), the effects of oxidant stress on CFTR expression are not cell or tissue specific. The oxidant-mediated decrease of CFTR in T84 cells was also associated with an accelerated degradation of CFTR mRNA as well as decreases in CFTR protein and function.

The effects of BHQ on glutathione, {gamma}-GCShs, and CFTR mRNA expression were not specific to BHQ. TauNHCl, a physiologically relevant oxidant produced by activated neutrophils, is thought to play a role in inflammatory bowel disease and possibly in lung disease associated with cystic fibrosis (34, 59). Chloramine concentrations of 30–100 µM have been measured in the supernatants of activated neutrophils at a density of 0.2–1.6 x 107/ml (16, 38, 41). In the present study, TauNHCl increased cellular glutathione and {gamma}-GCShs mRNA expression in T84 cells, whereas as little as 62.5 µM TauNHCl decreased CFTR mRNA expression. The TauNHCl concentrations at which CFTR mRNA suppression was observed are within the range reported to be present in the supernatants of activated neutrophils and in cystic fibrosis sputum (16, 38, 59). Further evidence supporting the concept that oxidants other than BHQ can suppress CFTR gene expression was presented by Baudouin-Legros et al. (3), who showed that ouabain, which induces the generation of reactive oxygen species (30), can suppress CFTR mRNA expression in Calu-3 cells and that this effect is blocked by the antioxidants N-acetyl-L-cysteine and diphenyleneiodonium.

Oxidative stress results in epithelial cell responses that can be characterized as either immediate or adaptive (51). The biphasic nature of such responses is illustrated in lung airway epithelium exposed to cigarette smoke. Acute exposure results in depletion of the epithelium’s surface fluid glutathione, whereas chronic smoke exposure results in an increase of glutathione (8, 28, 48). The immediate decrease in cytoplasmic glutathione during an acute oxidant stress results from the rapid scavenging of radical oxygen species as a first line of defense. The subsequent adaptive response involves a complex network of transcriptional and posttranscriptional events coordinated to maximize the effectiveness of cellular antioxidant defenses by increasing cellular glutathione (10, 46). Several genes encoding peptides and proteins related to the synthesis and metabolism of glutathione are targets of these adaptive responses. The rate-limiting step in glutathione synthesis is the formation of the peptide {gamma}-glutamylcysteine catalyzed by the enzyme complex {gamma}-GCS (21, 35). The {gamma}-GCS complex is the product of two genes, one encoding a light subunit and the other a heavy subunit (20, 21). Transcription of the {gamma}-GCShs gene has been reported to markedly increase in cells exposed to oxidative stress (48, 54). Similar results were observed in the present study, with epithelial cell {gamma}-GCShs gene expression increasing after 4 and 6 h of exposure to BHQ. The increase in {gamma}-GCShs was associated with a significant increase in cellular glutathione in both intestine- and airway-derived epithelial cell lines, suggesting a common adaptive response to the oxidant burden.

In addition to {gamma}-GCShs, another key factor that regulates epithelial glutathione is transepithelial glutathione transport (7). Recently, at least three transmembrane proteins from the ABC family have been shown to facilitate the export of glutathione S-conjugates and glutathione across epithelial cell membranes: the multidrug resistance protein MRP1 (ABCC1), CFTR (ABCC7), and MRP4 (ABCC4) (15, 50). Patients lacking functional CFTR have low levels of extracellular glutathione as determined in their lung epithelial lining fluid and in their serum (52). Glutathione efflux from CFTR-deficient epithelial cells is defective and can be restored by CFTR repletion. Cells expressing CFTR have lower glutathione levels and are more sensitive to oxidative stress-induced apoptosis than cells not expressing CFTR (24). These observations are consistent with the present study, in which the oxidative stress-mediated decrease in CFTR gene, protein, and function was associated with an increase in cellular glutathione. Interestingly, a decrease in CFTR expression occurred at the same time as expression of the {gamma}-GCShs gene was increased, suggesting that the cell has a coordinated adaptive response that increases synthesis and decreases export of cellular glutathione during oxidant stress. The mRNA encoding the structurally related MRP1 did not decrease on exposure to oxidant stress.

The effects of oxidant stress on chloride efflux and CFTR function have been studied by several investigators (11, 23, 40, 55). Most of these studies have focused on the immediate response of CFTR function within minutes of exposure to an oxidant stress. Using a wide range of oxidants, including tert-butylhydroperoxide, monochloramine, and hydrogen peroxide, these studies have consistently demonstrated that chloride efflux is rapidly increased. In contrast, we have observed that a prolonged exposure to oxidant stress over several hours has the opposite effect. Together these observations suggest that oxidant-mediated CFTR activation is an immediate host response, perhaps representing an attempt to flush the oxidant source away from the cell surface, whereas CFTR suppression represents a long-term adaptive response to favor preservation of intracellular glutathione and increase the viscosity of the mucous barrier to prevent oxidation of vital cellular components. Consistent with this concept, recent work reported by Kreindler et al. (27) indicates that cigarette smoke exposure can induce changes in human bronchial epithelial cells that mimic ion transport defects observed in cystic fibrosis epithelia.

The results of this study and the work of others suggest that oxidant-mediated CFTR suppression may occur through several mechanisms. Using nuclear run-on assays, we were unable to observe an oxidant-dependent decrease in CFTR gene transcription. However, CFTR mRNA stability was clearly decreased after oxidant exposure. Enhanced degradation of CFTR mRNA has been reported previously for cells exposed to TNF-{alpha} and {gamma}-interferon, suggesting that posttranscriptional regulation may play a key role in defining CFTR mRNA levels generally (5, 39). In addition to decreasing CFTR mRNA levels, oxidant stress also decreased steady-state CFTR protein level and channel function. A decrease in CFTR mRNA may contribute to lower CFTR protein and function, but it is unlikely to account for all of the loss of CFTR function. It was recently shown that reactive nitrogen species can directly accelerate CFTR protein degradation (4). Furthermore, oxidants could act directly through interactions with cysteine residues in the nucleotide binding domains of CFTR, which results in the slowing of channel gating (18, 19), although this would be unlikely to persist for 24 h in the absence of oxidant stress. Severe oxidant stress may lead to reversible glutathionylation of CFTR and inhibition of its channel activity (58).

BHQ is a redox cycling quinone that produces hydrogen peroxide and hydroxyl radical, reactive oxygen species that can injure epithelial cells (31, 44). Mucin, a glycoprotein concentrated at the surface of mucosal epithelia, can scavenge hydroxyl radicals and is thought to protect epithelial cells against oxidant-mediated damage (17). One of the major functions of CFTR is to regulate chloride and bicarbonate secretion, thus modulating the water content and pH of mucus, the key parameters defining the viscoelastic properties of mucin (33, 56). Our data indicate that increasing concentrations of mucin protect epithelial cells against BHQ-mediated injury. We therefore suggest that in addition to modulating glutathione transport, a potential benefit of decreased CFTR expression during an oxidant stress may be the increased antioxidant protection provided by viscous mucin at the epithelial surface.

In summary, we have shown that oxidant stress can suppress CFTR gene expression, protein expression, and function while increasing the cellular antioxidant glutathione. We propose that CFTR suppression during an oxidant stress is an antioxidant glutathione-sparing response that may also increase mucous barrier viscosity to help protect the epithelium and occurs despite the risk of impaired mucus clearance. In contrast to the sustained deficiency of CFTR observed in cystic fibrosis that leads to substantial respiratory, gastrointestinal, and reproductive disease from mucus obstruction, the oxidant-mediated effects on CFTR expression are transient. These results may be highly relevant to situations in which mucosal epithelial tissues are chronically exposed to a significant oxidant burden such as occurs in the airways of cigarette smokers and in the intestinal tract of patients with inflammatory bowel diseases.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was funded by a grant from the Canadian Cystic Fibrosis Foundation. J. W. Hanrahan was a senior scientist of the Canadian Institutes of Health Research. A. M. Cantin was a chercheur national of the Fonds de la Recherche en Santé du Québec.


   ACKNOWLEDGMENTS
 
The authors thank Dr. Susan Cole for providing the MRP1 cDNA, Dr. Gergely Lukacs for the CFTR cDNA, and Tim Jensen and Dr. John Riordan for the CFTR antibody.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. M. Cantin, Pulmonary Research Unit, Faculty of Medicine, Univ. of Sherbrooke, 3001, 12ième Ave. Nord, Sherbrooke, QC, Canada J1H 5N4 (e-mail: Andre.Cantin{at}USherbrooke.ca)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
1. Arsalane K, Dubois CM, Muanza T, Begin R, Boudreau F, Asselin C, and Cantin AM. Transforming growth factor-{beta}1 is a potent inhibitor of glutathione synthesis in the lung epithelial cell line A549: transcriptional effect on the GSH rate-limiting enzyme {gamma}-glutamylcysteine synthetase. Am J Respir Cell Mol Biol 17: 599–607, 1997.[Abstract/Free Full Text]

2. Ballard ST, Trout L, Bebok Z, Sorscher EJ, and Crews A. CFTR involvement in chloride, bicarbonate, and liquid secretion by airway submucosal glands. Am J Physiol Lung Cell Mol Physiol 277: L694–L699, 1999.[Abstract/Free Full Text]

3. Baudouin-Legros M, Brouillard F, Tondelier D, Hinzpeter A, and Edelman A. Effect of ouabain on CFTR gene expression in human Calu-3 cells. Am J Physiol Cell Physiol 284: C620–C626, 2003.[Abstract/Free Full Text]

4. Bebok Z, Varga K, Hicks JK, Venglarik CJ, Kovacs T, Chen L, Hardiman KM, Collawn JF, Sorscher EJ, and Matalon S. Reactive oxygen nitrogen species decrease cystic fibrosis transmembrane conductance regulator expression and cAMP-mediated Cl– secretion in airway epithelia. J Biol Chem 277: 43041–43049, 2002.[Abstract/Free Full Text]

5. Besancon F, Przewlocki G, Baro I, Hongre AS, Escande D, and Edelman A. Interferon-{gamma} downregulates CFTR gene expression in epithelial cells. Am J Physiol Cell Physiol 267: C1398–C1404, 1994.[Abstract/Free Full Text]

6. Cantin AM. Taurine modulation of hypochlorous acid-induced lung epithelial cell injury in vitro. Role of anion transport. J Clin Invest 93: 606–614, 1994.[ISI][Medline]

7. Cantin AM and Begin R. Glutathione and inflammatory disorders of the lung. Lung 169: 123–138, 1991.[ISI][Medline]

8. Cantin AM, North SL, Hubbard RC, and Crystal RG. Normal alveolar epithelial lining fluid contains high levels of glutathione. J Appl Physiol 63: 152–157, 1987.[Abstract/Free Full Text]

9. Chomczynski P and Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159, 1987.[ISI][Medline]

10. Comhair SA and Erzurum SC. Antioxidant responses to oxidant-mediated lung diseases. Am J Physiol Lung Cell Mol Physiol 283: L246–L255, 2002.[Abstract/Free Full Text]

11. Cowley EA and Linsdell P. Oxidant stress stimulates anion secretion from the human airway epithelial cell line Calu-3: implications for cystic fibrosis lung disease. J Physiol 543: 201–209, 2002.[Abstract/Free Full Text]

12. Cross CE, Halliwell B, and Allen A. Antioxidant protection: a function of tracheobronchial and gastrointestinal mucus. Lancet 1: 1328–1330, 1984.[CrossRef][ISI][Medline]

13. Cross CE, van der Vliet A, O’Neill CA, Louie S, and Halliwell B. Oxidants, antioxidants, and respiratory tract lining fluids. Environ Health Perspect 102, Suppl 10: 185–191, 1994.

14. Davis PB, Drumm M, and Konstan MW. Cystic fibrosis. Am J Respir Crit Care Med 154: 1229–1256, 1996.[ISI][Medline]

15. Gao L, Kim KJ, Yankaskas JR, and Forman HJ. Abnormal glutathione transport in cystic fibrosis airway epithelia. Am J Physiol Lung Cell Mol Physiol 277: L113–L118, 1999.[Abstract/Free Full Text]

16. Grisham MB, Jefferson MM, Melton DF, and Thomas EL. Chlorination of endogenous amines by isolated neutrophils. Ammonia-dependent bactericidal, cytotoxic, and cytolytic activities of the chloramines. J Biol Chem 259: 10404–10413, 1984.[Abstract/Free Full Text]

17. Grisham MB, Von Ritter C, Smith BF, Lamont JT, and Granger DN. Interaction between oxygen radicals and gastric mucin. Am J Physiol Gastrointest Liver Physiol 253: G93–G96, 1987.[Abstract/Free Full Text]

18. Harrington MA, Gunderson KL, and Kopito RR. Redox reagents and divalent cations alter the kinetics of cystic fibrosis transmembrane conductance regulator channel gating. J Biol Chem 274: 27536–27544, 1999.[Abstract/Free Full Text]

19. Harrington MA and Kopito RR. Cysteine residues in the nucleotide binding domains regulate the conductance state of CFTR channels. Biophys J 82: 1278–1292, 2002.[Abstract/Free Full Text]

20. Huang CS, Anderson ME, and Meister A. Amino acid sequence and function of the light subunit of rat kidney {gamma}-glutamylcysteine synthetase. J Biol Chem 268: 20578–20583, 1993.[Abstract/Free Full Text]

21. Huang CS, Chang LS, Anderson ME, and Meister A. Catalytic and regulatory properties of the heavy subunit of rat kidney {gamma}-glutamylcysteine synthetase. J Biol Chem 268: 19675–19680, 1993.[Abstract/Free Full Text]

22. Hug MJ, Tamada T, and Bridges RJ. CFTR and bicarbonate secretion by epithelial cells. News Physiol Sci 18: 38–42, 2003. [Corrigenda. News Physiol Sci 18: April 2003, p. 59.][Abstract/Free Full Text]

23. Jung JS, Lee JY, Oh SO, Jang PG, Bae HR, Kim YK, and Lee SH. Effect of t-butylhydroperoxide on chloride secretion in rat tracheal epithelia. Pharmacol Toxicol 82: 236–242, 1998.[ISI][Medline]

24. Jungas T, Motta I, Duffieux F, Fanen P, Stoven V, and Ojcius DM. Glutathione levels and BAX activation during apoptosis due to oxidative stress in cells expressing wild-type and mutant cystic fibrosis transmembrane conductance regulator. J Biol Chem 277: 27912–27918, 2002.[Abstract/Free Full Text]

25. Knowles MR and Boucher RC. Mucus clearance as a primary innate defense mechanism for mammalian airways. J Clin Invest 109: 571–577, 2002.[CrossRef][ISI][Medline]

26. Kogan I, Ramjeesingh M, Li C, Kidd JF, Wang Y, Leslie EM, Cole SP, and Bear CE. CFTR directly mediates nucleotide-regulated glutathione flux. EMBO J 22: 1981–1989, 2003.[CrossRef][ISI][Medline]

27. Kreindler JL, Jackson AD, Kemp PA, Bridges RJ, and Danahay H. Inhibition of chloride secretion in human bronchial epithelial cells by cigarette smoke extract. Am J Physiol Lung Cell Mol Physiol 288: L894–L902, 2005.[Abstract/Free Full Text]

28. Li XY, Rahman I, Donaldson K, and MacNee W. Mechanisms of cigarette smoke induced increased airspace permeability. Thorax 51: 465–471, 1996.[Abstract]

29. Linsdell P and Hanrahan JW. Glutathione permeability of CFTR. Am J Physiol Cell Physiol 275: C323–C326, 1998.[Abstract/Free Full Text]

30. Liu J, Tian J, Haas M, Shapiro JI, Askari A, and Xie Z. Ouabain interaction with cardiac Na+/K+-ATPase initiates signal cascades independent of changes in intracellular Na+ and Ca2+ concentrations. J Biol Chem 275: 27838–27844, 2000.[Abstract/Free Full Text]

31. Liu RM, Shi MM, Giulivi C, and Forman HJ. Quinones increase {gamma}-glutamyl transpeptidase expression by multiple mechanisms in rat lung epithelial cells. Am J Physiol Lung Cell Mol Physiol 274: L330–L336, 1998.[Abstract/Free Full Text]

32. Lorico A, Rappa G, Finch RA, Yang D, Flavell RA, and Sartorelli AC. Disruption of the murine MRP (multidrug resistance protein) gene leads to increased sensitivity to etoposide (VP-16) and increased levels of glutathione. Cancer Res 57: 5238–5242, 1997.[Abstract/Free Full Text]

33. Matsui H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis CW, and Boucher RC. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 95: 1005–1015, 1998.[CrossRef][ISI][Medline]

34. McKenzie SJ, Baker MS, Buffinton GD, and Doe WF. Evidence of oxidant-induced injury to epithelial cells during inflammatory bowel disease. J Clin Invest 98: 136–141, 1996.[ISI][Medline]

35. Meister A and Anderson ME. Glutathione. Annu Rev Biochem 52: 711–760, 1983.[CrossRef][ISI][Medline]

36. Mulcahy RT and Gipp JJ. Identification of a putative antioxidant response element in the 5'-flanking region of the human {gamma}-glutamylcysteine synthetase heavy subunit gene. Biochem Biophys Res Commun 209: 227–233, 1995.[CrossRef][ISI][Medline]

37. Murakami H and Masui H. Hormonal control of human colon carcinoma cell growth in serum-free medium. Proc Natl Acad Sci USA 77: 3464–3468, 1980.[Abstract/Free Full Text]

38. Nagl M, Hess MW, Pfaller K, Hengster P, and Gottardi W. Bactericidal activity of micromolar N-chlorotaurine: evidence for its antimicrobial function in the human defense system. Antimicrob Agents Chemother 44: 2507–2513, 2000.[Abstract/Free Full Text]

39. Nakamura H, Yoshimura K, Bajocchi G, Trapnell BC, Pavirani A, and Crystal RG. Tumor necrosis factor modulation of expression of the cystic fibrosis transmembrane conductance regulator gene. FEBS Lett 314: 366–370, 1992.[CrossRef][ISI][Medline]

40. Nguyen TD and Canada AT. Modulation of human colonic T84 cell secretion by hydrogen peroxide. Biochem Pharmacol 47: 403–410, 1994.[CrossRef][ISI][Medline]

41. Passo SA and Weiss SJ. Oxidative mechanisms utilized by human neutrophils to destroy Escherichia coli. Blood 63: 1361–1368, 1984.[Abstract/Free Full Text]

42. Perez-Vilar J and Boucher RC. Reevaluating gel-forming mucins’ roles in cystic fibrosis lung disease. Free Radic Biol Med 37: 1564–1577, 2004.[CrossRef][ISI][Medline]

43. Pilewski JM and Frizzell RA. Role of CFTR in airway disease. Physiol Rev 79: S215–S255, 1999.[Medline]

44. Pinkus R, Weiner LM, and Daniel V. Role of oxidants and antioxidants in the induction of AP-1, NF-{kappa}B, and glutathione S-transferase gene expression. J Biol Chem 271: 13422–13429, 1996.[Abstract/Free Full Text]

45. Puchelle E, Bajolet O, and Abely M. Airway mucus in cystic fibrosis. Paediatr Respir Rev 3: 115–119, 2002.[CrossRef][Medline]

46. Rahman I and MacNee W. Lung glutathione and oxidative stress: implications in cigarette smoke-induced airway disease. Am J Physiol Lung Cell Mol Physiol 277: L1067–L1088, 1999.[Abstract/Free Full Text]

47. Rahman I and MacNee W. Oxidative stress and regulation of glutathione in lung inflammation. Eur Respir J 16: 534–554, 2000.[Abstract]

48. Rahman I, Smith CA, Lawson MF, Harrison DJ, and MacNee W. Induction of {gamma}-glutamylcysteine synthetase by cigarette smoke is associated with AP-1 in human alveolar epithelial cells. FEBS Lett 396: 21–25, 1996.[CrossRef][ISI][Medline]

49. Rahman I, van Schadewijk AA, Hiemstra PS, Stolk J, van Krieken JH, MacNee W, and de Boer WI. Localization of {gamma}-glutamylcysteine synthetase messenger RNA expression in lungs of smokers and patients with chronic obstructive pulmonary disease. Free Radic Biol Med 28: 920–925, 2000.[CrossRef][ISI][Medline]

50. Rappa G, Gamcsik MP, Mitina RL, Baum C, Fodstad O, and Lorico A. Retroviral transfer of MRP1 and {gamma}-glutamyl cysteine synthetase modulates cell sensitivity to L-buthionine-S,R-sulphoximine (BSO): new rationale for the use of BSO in cancer therapy. Eur J Cancer 39: 120–128, 2003.[CrossRef][ISI][Medline]

51. Ray S, Watkins DN, Misso NL, and Thompson PJ. Oxidant stress induces {gamma}-glutamylcysteine synthetase and glutathione synthesis in human bronchial epithelial NCI-H292 cells. Clin Exp Allergy 32: 571–577, 2002.[CrossRef][ISI][Medline]

52. Roum JH, Buhl R, McElvaney NG, Borok Z, and Crystal RG. Systemic deficiency of glutathione in cystic fibrosis. J Appl Physiol 75: 2419–2424, 1993.[Abstract/Free Full Text]

53. Shen BQ, Finkbeiner WE, Wine JJ, Mrsny RJ, and Widdicombe JH. Calu-3: a human airway epithelial cell line that shows cAMP-dependent Cl– secretion. Am J Physiol Lung Cell Mol Physiol 266: L493–L501, 1994.[Abstract/Free Full Text]

54. Shi MM, Kugelman A, Iwamoto T, Tian L, and Forman HJ. 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]

55. Tamai H, Kachur JF, Baron DA, Grisham MB, and Gaginella TS. Monochloramine, a neutrophil-derived oxidant, stimulates rat colonic secretion. J Pharmacol Exp Ther 257: 887–894, 1991.[Abstract/Free Full Text]

56. Veerman EC, Valentijn-Benz M, and Nieuw Amerongen AV. Viscosity of human salivary mucins: effect of pH and ionic strength and role of sialic acid. J Biol Buccale 17: 297–306, 1989.[ISI][Medline]

57. Verkman AS, Song Y, and Thiagarajah JR. Role of airway surface liquid and submucosal glands in cystic fibrosis lung disease. Am J Physiol Cell Physiol 284: C2–C15, 2003.[Abstract/Free Full Text]

58. Wang W, Oliva C, Li G, Holmgren A, Lillig CH, and Kirk KL. Reversible silencing of CFTR chloride channels by glutathionylation. J Gen Physiol 125: 127–141, 2005.[Abstract/Free Full Text]

59. Witko-Sarsat V, Delacourt C, Rabier D, Bardet J, Nguyen AT, and Descamps-Latscha B. Neutrophil-derived long-lived oxidants in cystic fibrosis sputum. Am J Respir Crit Care Med 152: 1910–1916, 1995.[Abstract]




This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
A. Rab, R. Bartoszewski, A. Jurkuvenaite, J. Wakefield, J. F. Collawn, and Z. Bebok
Endoplasmic reticulum stress and the unfolded protein response regulate genomic cystic fibrosis transmembrane conductance regulator expression
Am J Physiol Cell Physiol, February 1, 2007; 292(2): C756 - C766.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
N. A. Bradbury
Freedom of expression. Focus on "Endoplasmic reticulum stress and the unfolded protein response regulate cystic fibrosis transmembrane conductance regulator expression"
Am J Physiol Cell Physiol, February 1, 2007; 292(2): C687 - C688.
[Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
E. F. McKone, J. Shao, D. D. Frangolias, C. L. Keener, C. A. Shephard, F. M. Farin, M. R. Tonelli, P. D. Pare, A. J. Sandford, M. L. Aitken, et al.
Variants in the Glutamate-Cysteine-Ligase Gene Are Associated with Cystic Fibrosis Lung Disease
Am. J. Respir. Crit. Care Med., August 15, 2006; 174(4): 415 - 419.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
A. M. Cantin, J. W. Hanrahan, G. Bilodeau, L. Ellis, A. Dupuis, J. Liao, J. Zielenski, and P. Durie
Cystic Fibrosis Transmembrane Conductance Regulator Function Is Suppressed in Cigarette Smokers
Am. J. Respir. Crit. Care Med., May 15, 2006; 173(10): 1139 - 1144.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/1/C262    most recent
00070.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow