Departments of 1 Molecular Pharmacology and Toxicology and 2 Medicine, Will Rogers Institute Pulmonary Research Center, University of Southern California, Los Angeles, California 90033; and 3 Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599
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ABSTRACT |
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Glutathione (GSH)
is a potentially important component of antioxidant defense in the
epithelial lung lining fluid. Cystic fibrosis (CF) patients have
chronic inflammation in which oxidative stress can be a factor. To
examine the hypothesis that the transport of GSH content was defective
in CF patients, intracellular and extracellular GSH were measured by
HPLC. Four cell lines were used: CFT1 cells [with defective CF
transmembrane conductance regulator (CFTR), F508 homozygous, two
clones] and one of the CFT1 clones transfected with either normal
CFTR (CFTR repleted) or
-galactosidase. GSH content in the apical
fluid was 55% lower in CFTR-deficient cultures than in CFTR-repleted
cells (P < 0.001). In contrast,
intracellular GSH content was similar in CFT1 cells and CFTR-repleted
cells.
-Glutamyl transpeptidase activity, which degrades
extracellular GSH, did not account for differences in apical GSH.
Rather, GSH efflux of CFTR-deficient cells was lower than that of
CFTR-repleted cells. These studies suggested that decreased GSH content
in the apical fluid in CF resulted from abnormal GSH transport
associated with a defective CFTR.
-glutamyl transpeptidase; epithelial lung lining fluid; cystic
fibrosis transmembrane conductance regulator; thiol
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INTRODUCTION |
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CYSTIC FIBROSIS (CF) is a systemic disorder primarily
manifested in the lung, pancreas, and intestinal tract. CF is caused by
mutations of the cystic fibrosis transmembrane conductance regulator
(CFTR) gene. About 70% of mutations of the CFTR gene in the Caucasian
population display deletion of phenylalanine at position 508 (F508).
CFTR is an apical membrane glycoprotein that functions as a
cAMP-dependent chloride channel. In addition to regulating chloride
transport of epithelia, CFTR also appears to modulate sodium transport
(1).
Lung dysfunction is a major cause of death in CF patients. The major clinical manifestations affecting the respiratory tract are mucus accumulation, Pseudomonas aeruginosa infection, and chronic inflammation. Replacement gene therapy may eventually provide a cure for CF (18). Improvement in delivery of the CFTR gene is the crucial step for such gene therapy. In the meantime, pharmacological remedies for correcting the defect may be attempted. For example, 4-phenylbutyrate has been found to restore CFTR function in vitro (23). More immediately, CF therapy is targeted at treating the resulting physiological and pathological manifestations, such as chronic inflammation.
Inflammation presents an oxidative challenge to the lung. Invading
neutrophils produce superoxide, hydrogen peroxide, and hypochlorous
acid. Glutathione (GSH) is a major component of cellular defense
against oxidative injury. GSH is normally present at 2-10 mM
concentrations inside cells. Most cells depend on de novo GSH synthesis
to maintain their intracellular GSH content. -Glutamylcysteine synthetase (GCS) and cysteine are the rate-limiting enzyme and substrate, respectively, for the de novo synthesis of GSH. On the other
hand,
-glutamyl transpeptidase (GGT), which is located on the outer
surface of the plasma membrane, can degrade extracellular GSH. GCS and
GGT constitute part of a system for transport of GSH between organs and
its recycling between the extracellular and intracellular compartments.
As a consequence of GSH export by epithelial cells, GSH is found in
high concentration in some extracellular fluids, such as the epithelial
lung lining fluid (ELF; see Refs. 3, 25). Normal human ELF contains a
high GSH concentration (i.e., ~400 µM) that is 140-fold higher than that in the plasma (3). Extracellular GSH can serve as a scavenger of
carbon-centered free radicals produced by lipid peroxidation and
hypochlorous acid produced by neutrophils during inflammation. Therefore, a decrease in GSH content in ELF may increase the
susceptibility of the lung epithelium to chronic inflammation such as
occurs in CF. Evidence of oxidative stress has also been found in CF patients along with pulmonary dysfunction (2, 12). GSH content is
decreased in the lung lavage fluid from adult CF patients (22). However, GSH content is either unchanged in the lavage fluid from CF
children without inflammation or tends to decrease but is not statistically significant from CF children with inflammation (12). Because of the technical difficulties in making an accurate measurement of GSH, especially in damaged lungs, there may be an advantage in
studying cells derived from CF patients and controls in vitro.
In hepatocytes and kidney, GSH transport has been found to be membrane potential dependent (9, 14, 15). In hepatocytes, GSH transport has also been shown to be carrier mediated (20). Another study has also suggested GSH transport via vesicle secretion in myeloma cells and Xenopus laevis oocytes (4). Whether similar GSH transporters exist in lung is still not clear.
Recently, CFTR has been suggested to be permeable to several organic anions and GSH (16, 17). Furthermore, mutations of CFTR cause abnormal chloride and sodium transport, which may cause changes in the fluid composition in ELF as well as in plasma membrane potential. Both changes in membrane potential and mutations of CFTR might affect GSH transport and homeostasis in CF lung epithelium. Therefore, the purposes of the present study were to examine 1) whether intracellular/extracellular GSH balance in cells with defective CFTR was abnormal; 2) whether this biochemical imbalance could be corrected by stable transfection with the normal CFTR gene; and 3) the possible mechanisms of any abnormality in GSH balance.
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MATERIALS AND METHODS |
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Chemicals. Acivicin,
2,4-dinitrofluorobenzene, and a somatic cell ATP assay kit were
purchased from Sigma (St. Louis, MO). -Glutamyl-glutamic acid was
purchased from Bachem Bioscience (Torrance, CA). All chemicals used
were at least analytical grade.
Cell culture and treatment. CFT1 clone
2 (CFT1-c2) and clone 9 (CFT1-c9) cells are immortalized human tracheal
epithelial cells from a homozygous F508 CF patient (27). CFT1-LC3
cells are CFT1-c2 cells transfected with
-galactosidase as
transfection control, and CFT1-LCFSN cells are CFT1-c2 cells
transfected with normal CFTR gene (19). HBE1 cells are immortalized
human bronchial epithelial cells from a normal individual (27). Cells
were grown in serum-free Ham's F-12 medium supplemented with seven
additives (5 µg/ml insulin, 3.7 µg/ml endothelial cell growth
supplement, 25 ng/ml epidermal growth factor, 3 × 10
8 M triiodothyronine, 1 × 10
6 M
hydrocortisone, 5 µg/ml transferrin, and 10 ng/ml cholera toxin) in
T-75cm2 collagen-coated flasks. Cells were passaged two
times without cholera toxin to eliminate its effects.
Subsequently, cells (0.5 × 106 cells/4.2 cm2) were seeded on collagen-coated permeable supports. One milliliter of F-12 medium was added to the inner well (apical side), and 2 ml medium were added to the outer well (basolateral side). After 24 h, cells were exposed to a medium mixture (50% F-12-50% NIH/3T3 cell-conditioned DMEM that contained 2% FBS). Transepithelial resistance (Rt) was measured to monitor the development of tight junctions. When cultures developed a maximum Rt (in ~7-10 days; see RESULTS), the medium was removed and replenished with 0.5 and 1.5 ml of fresh medium mixture on the apical and basolateral sides, respectively. In some studies, 0.2 mg/ml acivicin was added to medium to inhibit GGT. Media and cells were collected at different time points for GGT activity, ATP content, and GSH content measurement.
GSH content measurement. At different
time points, 0.5 ml of apical and basolateral media were collected and
centrifuged to spin down any detached cells that might be present. The
cells were washed one time with ice-cold PBS and scraped into the same buffer. Protein from either media or cells was precipitated in 10%
perchloric acid containing 2 mM EDTA and 10 nmol/ml
-glutamyl-glutamic acid as an internal standard. On centrifugation,
GSH, glutathione disulfide (GSSG), and cysteine glutathione disulfide
(Cys-SG) content in the supernatant was derivatized with
2,4-dinitrofluorobenzene and determined by HPLC, as previously
described by Fariss and Reed (8). Protein concentration was measured
with the bicinchoninic acid protein assay kit (Pierce, Rockford, IL).
GGT activity assay. Cells were scraped
into ice-cold PBS buffer. After centrifugation, the cell pellets were
suspended in PBS containing 0.1% Triton X-100 and sonicated briefly on
ice. GGT activity was measured with the fluorescent substrate
-glutamyl-7-amino-4-methyl-coumarin, as described previously (11).
ATP assay. To determine the cytotoxic effect of acivicin, intracellular ATP content was measured with a somatic cell ATP assay kit. After the cells were treated with 0.2 mg/ml of acivicin for 18 h, cells grown on the permeable support were washed one time with PBS. ATP was released with 0.5 ml of somatic cell ATP-releasing solution. ATP concentration was determined by firefly luciferin-luciferase reaction with a Perkin-Elmer LS-5 fluorescence spectrophotometer in the phosphorescence mode. The ATP content was calculated based on the ATP standard curve and was normalized by protein concentration.
Statistics. Data are expressed as means ± SE and were evaluated by one-way ANOVA; n is the number of observations. Statistical significance was then determined by Tukey's test. P < 0.05 was considered significant.
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RESULTS |
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Apical medium GSH content was decreased with
CFTR-deficient cells. Cells grew to confluence at
~7-8 days after seeding. Both CFT1-c2 and CFT1-c9 cells
developed a higher
Rt (>1,000
· cm2)
compared with other cells (~20
· cm2).
Both apical and basolateral media and cells were collected 18 h after
media were changed. Intracellular GSH content in both CFT1-c2 and
CFT1-c9 cells was not different from that in CFT1-c2 cells stably
transfected with either
-galactosidase (CFT1-LC3) or normal CFTR
(CFT1-LCFSN; P > 0.05;
Fig. 1). Intracellular GSH content in HBE1
cells was similar to that of CFT1-LCFSN cells (88.3 ± 7.1%
CFT1-LCFSN; P > 0.05). Thus
intracellular GSH content was not significantly affected by the defect
in CFTR function.
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In contrast, GSH found on the apical surface of both CFT1-c2 and
CFT1-c9 cells was markedly lower than that of the "CFTR-repaired" CFT1-LCFSN cells (P < 0.001; Fig.
2). CFT1-c2 cells transfected with
-galactosidase (CFT1-LC3) had no significant difference versus
CFT1-c2 cells (Fig. 2). GSH content in the apical fluid of HBE1 cells
was similar to that of CFT1-LCFSN cells (113.4 ± 8.5% CFT1-LCFSN;
P > 0.05). GSH concentration on the
basolateral side was variable but lower than that of apical GSH
concentration for each cell line (data not shown). Because of the high
cystine content in cell culture medium, most GSH in the medium reacted with cystine and formed Cys-SG. Therefore, extracellular GSH content was calculated from GSH equivalents (= GSH + 2 GSSG + Cys-SG).
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Increased GGT activity did not explain the abnormal
apical GSH content in CFTR-deficient cells. To study
whether the low apical GSH content in CFT1 and CFT1-LC3 cells was
caused by a rapid degradation of GSH outside the cells, GGT activity
was measured. GGT activity in CFT1-c9 cells was higher than in all
other cells tested (P < 0.001), as
shown in Table 1. Nonetheless, GGT activity
in CFT1-c2 cells was not different from either CFT1-c2 cells
transfected with -galactosidase (CFT1-LC3) or CFT1-c2 cells
transfected with normal CFTR gene (CFT1-LCFSN;
P > 0.05). GGT activity in HBE1 cells was similar to that of CFT1-LCFSN cells (91.8 ± 15.0%
CFT1-LCFSN; P > 0.05).
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When cells were incubated for 18 h with 0.2 mg/ml acivicin to inhibit
GGT activity, GGT activity decreased to <1.1
pmol · min1 · mg
protein
1 in all cells
tested. As expected, inhibition of GSH degradation by GGT with acivicin
caused a slight increase in apical GSH content in all cell lines
tested. However, apical GSH content of CFT1-c2, CFT1-c9, and CFT1-LC3
cells was still substantially lower than for CFT1-LCFSN cells, as shown
in Fig. 3. Apical GSH content of HBE1 cells
was higher than that for CFT1-LCFSN cells (146.5 ± 6.2%
CFT1-LCFSN; P < 0.05). With acivicin
treatment, the pattern of intracellular GSH content in all cell lines
did not change compared with that without acivicin treatment. These
results suggested that the differences in GGT activity among the
cell lines studied here did not significantly contribute to the
decrease in apical GSH content in CFTR-deficient cells. Intracellular
ATP content was not decreased after acivicin treatment for 18 h in all
cell lines tested, suggesting that acivicin was not toxic to the cells under the present condition (data not shown).
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Decreased GSH secretion from CFTR-deficient
cells. Another possible mechanism that was examined as
a possible explanation for the differences in the apical GSH content
was the release of cellular GSH. GSH efflux into the apical fluid was
examined in the presence of the GGT inhibitor acivicin to inhibit any
extracellular GSH degradation (Fig. 4). The
efflux rates (in
pmol · min1 · mg
cellular protein
1) were
20 ± 3 from CFT1-c9 cells, 29 ± 2 from CFT1-LC3 cells (CFT1-c2 cells transfected with
-galactosidase), 50 ± 2 from CFT1-LCFSN cells (CFT1-c2 cells transfected with normal CFTR), and 60 ± 2 from
HBE1 cells. The efflux rate of CFT1-c2 cells was similar to that of
CFT1-LC3 cells. Three separate experiments of GSH efflux were done, and
similar results were observed. Thus the rate of efflux of GSH to the
apical medium was significantly impaired by the CFTR defect and was
markedly elevated by transfection with normal CFTR.
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Basolateral GSH accumulation was slower than for apical GSH accumulation. In contrast to the accumulation in the apical medium, which was linear for >10 h, there was a lag in the accumulation of GSH in the basolateral fluid. Initially, the GSH concentration was very low, and there was no clear difference in accumulation between the cell lines. After 2 h, the basolateral GSH concentration began to approach the apical concentration. The lag in basolateral accumulation may result from paracellular transport.
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DISCUSSION |
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Antioxidants in ELF are important in protection of the integrity of the lung epithelium against oxidative damage. In CF, patients are exposed to chronic infection and inflammation. Therefore, maintenance of sufficient antioxidant levels in ELF in CF patients is crucial. The most abundant antioxidants in ELF are GSH, ascorbic acid, and uric acid (6). GSH content has been reported to be decreased in plasma and lavaged ELF of CF patients (22). Yet, others have found no such change in the GSH content in lung lavage from pediatric patients with CF, especially those without inflammation (12). One major problem with determining the extracellular GSH in CF lungs by lavage is what it represents. The bronchotracheal surface is very small compared with the alveolar surface such that what is obtained by filling a lung segment probably largely represents GSH content on the alveolar rather than tracheobronchial surface. Another problem is that a small amount of damage to tissue can result in release of significant GSH during cell lysis. Recently, Linsdell and Hanrahan (17) demonstrated that CFTR itself might transport GSH using isolated membrane from kidney cells. This further raised the question of whether the CFTR defect affected GSH transport and GSH content in ELF from CF patients.
In the present study, GSH content was determined to be lower in the apical medium of CFTR-deficient cells than CFTR-sufficient cells (Fig. 2). This was the first study to use confluent airway epithelium and show such differences in the extracellular GSH content. By growing cells on a permeable support, cells were allowed to form tight junctions, as determined by Rt. This mimicked the physiological condition in the lung. In addition, these cells were derived from the human airway where inhaled oxidants such as nitrogen dioxide and ozone have their initial effects (5) and where the consequence of inflammation in CF patients probably originates. Lung damage in CF patients mainly results from ion transport abnormalities in airway epithelia that lead to mucus accumulation and bacterial colonization (13). Although a recent paper suggested that CFTR is widely expressed, including bronchi, distal airway, and alveoli of human lungs (7), rat alveolar type II cells have relatively low CFTR (28). Thus the question of the involvement of the alveolus as a primary site of damage from the CFTR defect remains unresolved. Apical GSH is physiologically important because GSH in the epithelial lining layer serves as an initial defense against oxidative stress. Some oxidants are so insoluble (i.e., ozone and nitrogen dioxide) or so reactive (i.e., hypochlorous acid, peroxynitrite, ozone, and nitrogen dioxide) that they would most rapidly react with the extracellular fluid contents or outer leaflet of the cell membrane. This does not imply that these toxicants cannot cause damage by entering cells but suggests the value of extracellular GSH in lessening that possibility. GSH reacts directly and rapidly with hypochlorite to produce GSSG and chloride (10) and with peroxynitrite to form GSSG and nitrate (21). Although reaction with the oxidant gases may be more controversial, GSH will rapidly reduce carbon-centered radicals formed by their reactions (24).
We explored three possible explanations for the lower apical surface GSH content of the CFT1 cells compared with the CFTR-transfected CFT1 cells. These are increased extracellular degradation, decreased intracellular GSH and decreased export of GSH. The last is considered the most likely for the following reasons.
One possible explanation for reduced extracellular GSH content that was
considered was degradation of GSH outside the cells. GGT activity was
found to be higher in CFT1-c9 cells than in CFT1-LCFSN cells (Table 1).
However, when GGT activity was inhibited, GSH content in apical medium
was still lower in CFT1 and CFT1-LC3 (-galactosidase-transfected)
cells than in CFT1-LCFSN cells (Fig. 3). This suggested that such
increased GGT activity in CFT1-c9 cells did not result in the
abnormally low GSH content in the apical medium. Furthermore, the GGT
activity of the CFT1-c9 cells was actually small relative to the apical
GSH content. Indeed, if GGT was acting at its maximum rate and no GSH
continued to be released from the cells, it would have required ~13 h
to have degraded enough of the GSH found in the CFT1-LCFSN fluid to the level found in the CFT1-c9 apical fluid. An explanation for the high
GGT activity in CFT1-c9 cells may have been clonal variation.
A second possibility for the lower GSH on the apical surface of CFT1 cells that was examined was decreased intracellular GSH by either decreased GCS activity or decreased cysteine availability. Decreased GSH biosynthesis seems unlikely, as the intracellular GSH content in CFT1 cells was not decreased.
On the basis of the evidence provided here, the most likely mechanism for the difference in apical GSH content between CFTR-deficient and -sufficient cells was abnormally low GSH export by CFT1 cells. As shown in Fig. 4, GSH efflux from CFTR-deficient cells was lower than that from CFTR-restored cells. Therefore, the differences in apical GSH content among the cell lines were probably caused by differences in GSH efflux. A recent study has shown that CFTR was permeable to GSH and to a lesser extent to GSSG (17). From the present study, it appeared that the CFTR mutation directly affected GSH transport in CF cells. It is also possible that GSH transport was indirectly regulated by CFTR. In rat hepatocytes and isolated kidney membrane, GSH transport has been shown to be dependent on membrane potential (9, 14, 15). It is possible that the GSH transporter in lung is similar to that in hepatocytes in being membrane potential dependent. Therefore, such decreased CFTR function, which affects the membrane potential, could indirectly regulate GSH transport.
Studies have demonstrated a contribution of oxidative stress to the pathophysiology of CF (26). Because GSH is an antioxidant, the decrease in apical GSH content would make CF cells more susceptible to oxidative stress as found in the chronic inflammation associated with CF. Potentially, modulation of GSH content in ELF might provide a partial remedy to alleviate inflammation in CF patients.
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ACKNOWLEDGEMENTS |
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We thank Jinah Choi and Drs. Rui-Ming Liu, Huanfang Zhou, Beth Schomer, and Timothy Robison for technical and other expert advice. We also thank Ronald Kim (University of North Carolina) for assistance with establishing the cell lines in our laboratory.
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FOOTNOTES |
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This work was supported by the Berger-Webb Foundation and National Institutes of Health Grants ES-05511, HL-38658, and HL-46943.
Part of this work was presented at the North American Cystic Fibrosis Conference in Montreal, Canada, 1998.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. J. Forman, Dept. of Molecular Pharmacology and Toxicology, School of Pharmacy, PSC 516, Univ. of Southern California, 1985 Zonal Ave., Los Angeles, CA 90033 (E-mail: hforman{at}hsc.usc.edu).
Received 27 August 1998; accepted in final form 12 March 1999.
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