Journal of Applied Physiology Watch the video to see how APS reaches out to developing nations.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

J Appl Physiol 91: 748-754, 2001;
8750-7587/01 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 ISI Web of Science
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 (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Roum, J. H.
Right arrow Articles by Borok, Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Roum, J. H.
Right arrow Articles by Borok, Z.
Vol. 91, Issue 2, 748-754, August 2001

Extracellular glutathione inhibits oxygen-induced permeability changes in alveolar epithelial monolayers

J. H. Roum1, A. S. Aledia1, L. A. Carungcong1, K.-J. Kim2,3,4,5,6, and Z. Borok2,3

1 Department of Medicine, University of California Irvine Medical Center, Orange 92868; and 2 Will Rogers Institute Pulmonary Research Center and Departments of 3 Medicine, 4 Physiology and Biophysics, 5 Biomedical Engineering, and 6 Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, California 90033


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Exposure to high fractional inspired oxygen for 24 h increases permeability of the alveolar epithelium, contributing to the clinical manifestations of oxygen toxicity. Utilizing a model of the alveolar epithelium in which isolated rat type II cells form polarized monolayers on polycarbonate filters [transepithelial resistance (Rt) > 1 kOmega · cm2 by day 4], we evaluated the ability of reduced glutathione (GSH) to ameliorate these changes. On day 4, apical fluid was replaced with culture medium containing 1) no additives, 2) GSH (500 µM), or 3) GSH (500 µM) + glutathione reductase (0.5 U/ml) + nicotinamide adenine dinucleotide phosphate (250 µM). Monolayers were exposed (for 24 h) to room air (control) or 95% O2, each containing 5% CO2. After 24 h of hyperoxia, Rt for condition 1 decreased by 45% compared with control (P < 0.001). In conditions 2 and 3, Rt did not decrease significantly (P = not significant). Hyperoxia-induced decreases in active ion transport were observed for conditions 1 and 2 (P < 0.05), but not for condition 3 (P = not significant). These findings indicate that extracellular GSH may protect the alveolar epithelium against hyperoxia-induced injury. Addition of glutathione reductase and nicotinamide adenine dinucleotide phosphate may further augment these protective effects of GSH.

hyperoxia; rat; oxidant; antioxidant; glutathione reductase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE RESPIRATORY SURFACE of the lung provides the functional interface for gas exchange, making it susceptible to pulmonary toxicity caused by exposure to high concentrations of oxygen (20, 34). Pulmonary toxicity following hyperoxic exposure is mediated by the production of highly reactive oxygen species (ROS), which leads to structural alterations in proteins, lipids, and DNA (27). Although the normal alveolar epithelium of the mammalian lung constitutes a tight barrier characterized by low paracellular permeability (30, 38, 43), studies in animal models (45, 57) and in humans (23, 36) indicate that hyperoxic exposure alters alveolar epithelial barrier properties as a result of increases in permeability. In addition, hyperoxia leads to decreases in Na+ pump mRNA and protein levels in alveolar epithelial cell (AEC) monolayers accompanied by reductions in transepithelial active ion transport (8).

The alveolar epithelium is normally covered by a thin layer of epithelial lining fluid (ELF), which contains a number of antioxidants (13), including a tripeptide, reduced glutathione (GSH) (15, 46). Antioxidants provide protection against oxidative damage by preventing the accumulation of ROS. ELF normally contains high concentrations of GSH compared with plasma (15) and pulmonary interstitial fluid (11). The normal ELF glutathione concentration in healthy nonsmoking individuals is 424 ± 34 µM (15), ranging between 150 and 600 µM (56). Two additional components, glutathione reductase (GR) and nicotinamide adenine dinucleotide phosphate (NADPH), which are important for maintenance of glutathione in its active (reduced) form, are also present in ELF (15).

Barrier properties of the alveolar epithelium can be evaluated using an in vitro model, in which changes in transepithelial resistance (Rt) (17) across AEC monolayers reflect the integrity of the tight junctions between AEC. We hypothesized that exogenous GSH, by virtue of its antioxidant properties, would prevent permeability changes and alterations in ion transport caused by exposure to high concentrations of oxygen in rat AEC monolayers. We further hypothesized that the presence of GR and NADPH, which maintain glutathione in its reduced form, could potentiate the protective effect of glutathione. To evaluate this hypothesis, rat AEC grown to confluence on polycarbonate membranes were exposed for 24 h to 95% O2-5% CO2 or room air with 5% CO2 (21% O2-5% CO2). The effects of GSH, GR, and NADPH on Rt and equivalent short-circuit current (Ieq) were evaluated.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Isolation and Culture

Rat type II cells were isolated from specific pathogen-free, male Sprague-Dawley rats weighing 150-200 g (Simonsen Laboratories, Gilroy, CA) by elastase disaggregation (0.7-1.5 U/ml elastase; Worthington Biochemical, Freehold, NJ) and further enriched by "panning" on bacterial plates coated with rat IgG with use of methods previously described in detail (7, 26). Purified type II pneumocytes were resuspended in medium consisting of Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture supplemented with 1.25 mg/ml bovine serum albumin, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 0.1 mM nonessential amino acids, 2.0 mM glutamine, 100 U/ml sodium penicillin G, 100 µg/ml streptomycin, and 10% newborn calf serum (all obtained from Sigma Chemical, St. Louis, MO, with the exception of bovine serum albumin, which was obtained from Collaborative Biomedical Research, Bedford, MA). Type II cell purity (90 ± 10%) and viability (>99%) were assessed by tannic acid staining and trypan blue dye exclusion, respectively, as previously described (7, 44). Type II cells were plated on tissue culture-treated polycarbonate membranes (6.5 mm, 0.4 µm pore size; Transwell, Corning Costar, Cambridge, MA) at a density of 1.5 × 106 cells/cm2 and incubated in a humidified 5% CO2 incubator at 37°C. Apical and basolateral media were changed on day 2 and every other day thereafter. Rt (kOmega · cm2) and potential difference (PD, mV; apical side as reference) of AEC monolayers were measured on day 4 and also after 24 h of exposure to room air or 95% O2 (see below) with a rapid screening device (Millicell-ERS, Millipore, Bedford, MA), as previously described (6). Background Rt and PD contributed by an empty Transwell membrane were estimated separately and used for correction of the observed Rt and PD from AEC monolayers. An equivalent active ion transport rate (i.e., Ieq, µA/cm2) was calculated using Ohm's law as follows: Ieq = PD/Rt.

Oxygen Exposure

Previous studies indicate that, in vivo, concentrations of GSH are substantially higher in apical alveolar fluid than in basolateral (interstitial) fluids (11). Additionally, recent preliminary data indicate that AEC monolayers are relatively impermeable to GSH (54). Therefore, before exposure on day 4, apical fluid was replaced with 1) culture medium alone or culture medium supplemented with 2) GSH (500 µM; Sigma Chemical) or 3) GSH (500 µM) + GR (0.5 U/ml; Sigma Chemical) + NADPH (250 µM; Sigma Chemical). Concentration of GSH (15, 56) and GR activity (15) were chosen to approximate reported normal ELF concentrations. Concentration of NADPH was chosen to give an excess (~10×) over known ELF concentrations (15). Basolateral fluid was replaced with culture medium alone. AEC monolayers were then exposed in parallel incubator chambers (Lab-line dual CO2 Imperial II incubator 422, Melrose Park, IL) circulated with 21% O2-5% CO2 or 95% O2-5% CO2 for 24 h. Rt and PD were measured before and after the 24-h exposure as described above.

Statistical Analysis

Values are means ± SE; n is number of monolayers. Differences among group means (e.g., media conditions due to room air or oxygen exposure) were evaluated by one-way ANOVA. Post hoc analyses using Student-Newman-Keuls procedure were performed to contrast group means. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Baseline Resistances, PD, and Ieq

On day 4 of culture (before exposure to room air or hyperoxic conditions), the Rt of monolayers was 1.73 ± 0.05 kOmega · cm2 (n = 46). With the apical side as reference, the PD of monolayers was 5.6 ± 0.3 mV before exposure, with a calculated Ieq of 3.22 ± 0.11 µA/cm2. There were no differences in the Rt or Ieq among individual preexposure groups [P = not significant (NS)]. Because there were no differences in the Rt and Ieq among the preexposure groups, postexposure values on day 5 were compared.

Oxygen Exposure

After 24 h of room air exposure, slight elevations in Rt and Ieq relative to day 4 values were noted, although the changes were not statistically significant (P = NS; Figs. 1 and 2). Similar increases in Rt and Ieq from days 4 through 5 under baseline conditions have been previously reported for rat AEC monolayers over time in culture (16). In contrast, monolayers exposed to 95% O2 for 24 h showed significant decreases in Rt (45%, P < 0.001; Fig. 1) and Ieq (37%, P < 0.05; Fig. 2) compared with room air controls.


View larger version (16K):
[in this window]
[in a new window]
  		Fig. 1.   Transepithelial resistance (Rt) of rat alveolar epithelial cell monolayers exposed to room air-5% CO2 or 95% O2-5% CO2 for 24 h. Each bar represents mean ± SE (n >=  7). Dashed line, mean preexposure Rt. *Significantly different from 24-h room air exposure (P < 0.001).



View larger version (17K):
[in this window]
[in a new window]
  		Fig. 2.   Equivalent short-circuit current (Ieq) of rat alveolar epithelial cell monolayers exposed to room air-5% CO2 or 95% O2-5% CO2 for 24 h. Each bar represents mean ± SE (n >=  7). Dashed line, mean preexposure Ieq. *Significantly different from 24-h room air exposure (P < 0.05).

Effect of Apical GSH, GR, and NADPH

Room air exposure. After 24 h of exposure to room air, monolayers showed no differences in Rt when media conditions 1, 2, and 3 were compared (P = NS; Fig. 3). Likewise, no statistical differences in Ieq were found when conditions 1, 2, and 3 were compared after room air exposure for 24 h (P = NS; Fig. 4). These data demonstrate that individual media conditions did not differentially affect Rt or Ieq after 24 h of room air exposure.


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 3.   Effect of reduced glutathione (GSH), glutathione reductase (GR), and NADPH on Rt of cell monolayers exposed to room air (21% O2-5% CO2) or 95% O2-5% CO2 for 24 h. Each bar represents mean ± SE (n >=  7). Dashed line, mean preexposure Rt. *Significantly different from 24-h room air exposure of monolayers in medium alone (P < 0.01).



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 4.   Effect of GSH, GR, and NADPH on Ieq of cell monolayers exposed to room air (21% O2-5% CO2) or 95% O2-5% CO2 for 24 h. Each bar represents mean ± SE (n >=  7). Dashed line, mean preexposure Ieq. *Significantly different from 24-h room air exposure of monolayers in medium alone (P < 0.05).

Oxygen exposure. In contrast to the marked decrease in Rt after 24 h of exposure to 95% O2 seen in cells bathed in media alone compared with room air controls, Rt did not significantly decrease in the presence of GSH in the apical fluid (P = NS compared with media alone after 24 h of hyperoxia). Likewise, when GSH, GR, and NADPH were present in the apical fluid, there was no significant decrease in Rt compared with room air controls (P = NS; Fig. 3).

The Ieq observed in hyperoxia-treated monolayers in the absence or presence of GSH in the apical fluid was significantly less than that in monolayers exposed to room air for 24 h (P < 0.01; Fig. 4). In contrast, when GSH, GR, and NADPH were present in the apical fluid, no significant decrement in Ieq was observed in response to hyperoxia compared with 24-h room air control (P = NS).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Exposure of AEC monolayers in primary culture on polycarbonate filters to 95% O2 for 24 h resulted in decreases in Rt and Ieq. These findings are consistent with previous animal studies, in which hyperoxia causes an increase in the permeability of the air-blood barrier as reflected by an increase in the movement of small solutes across the lung epithelial barrier (45, 57). Previous studies of epithelial cells in culture have demonstrated similar increases in epithelial permeability after oxidative stress (42, 53). The increase in epithelial permeability and subsequent decrease in Rt caused by hyperoxia observed in this study are accompanied by a significant decrease in Ieq. These findings are consistent with a previous study, in which hyperoxia-induced decreases in Ieq were accompanied by reduced Na+ pump mRNA and protein levels (8).

Although there appears to be some variability in the effects of oxygen depending on species (29, 45), exposure duration (8, 22, 45, 48), and exposure methods or models used (47), in this study we demonstrate that 24 h of oxygen exposure leads to acute permeability changes reflected by decrements in Rt in this in vitro model of the alveolar epithelium. The present data are inconsistent with the occurrence of major cell death or "desquamation" from the polycarbonate filters, since relatively high resistance (>1 kOmega · cm2) was still maintained after oxygen exposure in the absence of exogenous glutathione. Sprague-Dawley rats are reportedly more resistant to oxygen toxicity than other strains (32, 60), and it is conceivable that the oxygen-induced effect on permeability presented here might have been greater if a different strain was used.

Consistent with the role of GSH as an antioxidant, we demonstrate that the presence of GSH in apical fluid protects against oxygen-induced changes in Rt and Ieq, suggesting that glutathione ameliorates oxidant-induced permeability changes. GSH is a tripeptide antioxidant, normally present in high concentrations in ELF (15, 46). In ELF, glutathione is found predominantly in the reduced form (15), which is its active antioxidant form. Among the antioxidant properties of GSH is its ability to protect cells against free radicals and ROS because of its strongly reducing thiol group (46). Furthermore, glutathione in ELF has been reported to be deficient in a number of disease states that are characterized by alterations of the alveolar epithelium [e.g., adult respiratory distress syndrome (49)] or feature an increased oxidant burden on the alveolar epithelial surface [e.g., cystic fibrosis (CF) and idiopathic pulmonary fibrosis (IPF) (14, 56)]. These diseases often place the patient at risk for oxygen toxicity because of the need for supplemental oxygen. The possibility of potentiating acute lung injury by oxygen toxicity is supported by results of studies in animal models (59).

In addition to changes in permeability and active ion transport with hyperoxia, other measurements of alveolar epithelial dysfunction due to hyperoxia have been reported previously for similar exposures (24 h, >90% O2). These include a decrease in ATP content (1), decreased cell proliferation (21), decreased DNA, thymidine, GSH, and superoxide dismutase levels, decreased synthesis of dipalmitoylphosphatidylcholine, and increased lactate dehydrogenase levels (33). Although they are not evaluated here, it may be of interest to further study effects of GSH supplementation on these and other hyperoxia-induced processes.

Analysis of the role of glutathione in oxygen toxicity is complex; this complexity is related to the unique characteristics of GSH and its metabolism: 1) it likely functions as an intracellular and an extracellular antioxidant (5, 11, 14, 15, 49, 56); 2) it interacts with several other molecules in the extracellular milieu (other thiols including thiolated proteins, nitric oxide, and other nitrogen oxides, e.g., S-nitrosothiols); 3) it has a complete system of enzymes and substrates [GR, glutathione peroxidase (GPx), and NADPH], extracellularly (ELF) (15) and intracellularly, to enhance its activity and restore it to its reduced (active antioxidant) form (46); 4) it has complex means of cellular uptake, involving other substrates such as cysteine (25) and cellular enzymes such as gamma -glutamyl transpeptidase (28); and 5) it has a multienzyme intracellular synthesis pathway (46). These multiple characteristics have led to the study of the role of GSH from multiple different aspects including oxidative-reductive state, activity of related enzymes, absolute concentrations intracellularly and extracellularly, synthesis, cellular uptake, and release. In this study, the effects of exogenous extracellular GSH were investigated on the basis of the knowledge that ELF normally contains large GSH concentrations (15), deficiencies in ELF GSH in disease have been demonstrated (14, 49, 56), and the feasibility of in vivo ELF supplementation with GSH has been demonstrated (5, 56). Likewise, study of extracellular GR and NADPH was pursued because of their known presence at high concentrations in normal ELF (15) and to evaluate the hypothesis that maintaining glutathione in its reduced form would improve its antioxidant protective role. It is, however, conceivable that extracellular glutathione or GR and NADPH may also in part exert their effect by affecting any of the characteristics listed above.

Several investigative approaches have supported an important role for intracellular and extracellular GSH in preventing lung oxygen toxicity. Studies based on whole animal and in vitro models suggest that depletion of intracellular GSH (24, 40, 58) or blocking of extracellular GSH uptake (28, 50, 58) potentiates oxygen toxicity, suggesting mechanisms by which sufficient extracellular GSH concentrations may be important in preventing oxygen toxicity. Furthermore, a greater susceptibility of Fischer 344 than of Sprague-Dawley rat lung to hyperoxic damage has been attributed to significantly lower levels of GSH in bronchoalveolar lavage fluids (60). With regard to direct effects of GSH, studies show that 95% O2 for 24 h leads to a decrease in GSH content of AEC (33), accompanied by an increased sensitivity to oxygen toxicity compared with fibroblasts. Additional studies showed a correlation between an increase in markers of AEC damage due to hyperoxia and decreased GSH cell content (1).

Other studies have demonstrated that perfusion of isolated rat lungs with GSH or N-acetylcysteine enhanced intracellular synthesis of glutathione (3). In addition, several investigators have shown that extracellular application of GSH or its synthetic substrates can enhance intracellular levels of GSH (25, 61, 62). Inhibition of GSH synthesis enhances oxygen toxicity (19), while supplementation with N-acetylcysteine protects against oxygen toxicity in distal fetal rat lung epithelial cells (19) and preterm guinea pig lung (41), as does GSH supplementation in preterm rabbits (10). Extracellular exogenous GSH also appears to preserve rat type II cell signal transduction pathways in the face of oxidant stress (9). Thus the literature supports the concept that depletion of cellular GSH increases oxygen toxicity, extracellular supplementation with exogenous GSH or its analogs can enhance extracellular GSH and thiol content as well as intracellular GSH, and these interventions may be protective against oxidant-induced damage.

Studies of nutritional supplementation with GSH also indicate protection against oxygen toxicity (10, 61). Supplementation with antioxidant analogs of GSH, such as N-acetylcysteine (19, 41) or other nutritional substrates that could enhance GSH synthesis (18), confers protection against hyperoxic lung injury. Also, direct administration of extracellular GSH appears to provide an antioxidant effect in response to other oxidants. For example, bathing type II cells in 1 mM GSH protected cells against t-butyl hydroperoxide (9). In addition, extracellular GSH protected alveolar type II cells against paraquat toxicity (31). GSH also has been shown to decrease the release of oxidants from bronchoalveolar lavage inflammatory cells from patients with CF (55).

Our findings that GR and NADPH added to the apical fluid appear to further enhance the protective effect of GSH against oxygen-induced epithelial dysfunction support the importance of GSH as an antioxidant defense against epithelial injury. Normal ELF contains substantial activity of GR and GPx, as well as NADPH (15). In the present study, we chose activities of GR to approximate physiological conditions (15) and concentrations of NADPH (a substrate for GR-dependent reduction of oxidized glutathione) to be in excess of physiological concentrations (~10× concentration) (15). Together, intracellular GR and NADPH help maintain glutathione in the reduced form, thus keeping the concentration of the oxidized form low (46). Extracellular glutathione in ELF collected from normal individuals is also found to be predominantly in its reduced state (15). Because GR activity and substantial NADPH are present in ELF from normal individuals (15), these are thought to be important in maintaining glutathione in the reduced form at steady state in ELF.

In hyperoxia, the presence of adequate extracellular GR and NADPH may become particularly important. Although it appears from the present study that the presence of GSH alone in the apical surface of the alveolar epithelium may be capable of partially abrogating the harmful effects of hyperoxia, GR and NADPH appear to further enhance the protective effect. In an in vitro model, inactivation of GR appears to exacerbate the effect of oxygen toxicity (51). Moreover, glutathione content and GR activity appear to be upregulated in the lung in animal models of prolonged hyperoxia (37, 52), suggesting a compensatory antioxidant response to hyperoxia.

Another important enzyme in the glutathione redox cycle, GPx (46), has been studied extensively with regard to its role in prevention of oxygen toxicity. Several lines of evidence with regard to GPx provide additional support for the role of GSH as an important antioxidant in ELF and in the intracellular compartment. First, ELF has been shown to contain substantial amounts of GPx (15). Also, alveolar macrophages and lung cell lines (A549) express the extracellular-type GPx (2). Furthermore, whole lung homogenates demonstrate an increase in GPx after 100% O2 for 72 h (39), while GPx activity more than doubled in isolated type II pneumocytes from adult and fetal rats exposed to 95% O2 (4). Moreover, older rats had elevated GPx activity in whole lung homogenates compared with younger rats, suggesting a mechanism for their increased resistance to oxygen toxicity (12). Finally, 1,3-bis(2-chloroethyl)-1-nitrosourea, an inhibitor of GPx activity, increases susceptibility of rat type II pneumocytes to oxidant stress (35). Thus, although the present study specifically addressed the role of GR and NADPH in maintenance of GSH to protect against oxygen toxicity, it is conceivable that addition of exogenous GPx activity to ELF may further potentiate the function of GSH in preventing alveolar epithelial oxygen-induced damage.

The site of action of exogenously administered GSH/GR/NADPH is suggested by the properties of the alveolar epithelial model employed in this study. The transepithelial permeability to GSH in this model appears to be negligible (regardless of whether GSH is added to the apical or basolateral surface). Therefore, because GSH was added to the apical bathing fluid in the present study, it is unlikely that its primary protective effect was on the extracellular basolateral cell surface. An intracellular effect is a possibility, however, since increased extracellular GSH may increase intracellular GSH concentrations (25). With regard to GR activity, it is unlikely that this relatively large protein enzyme substantially crosses the epithelial monolayer or gains access to the intracellular space. Thus it is likely that GR added to the apical bathing fluid maintained the glutathione contained in this fluid in its reduced form (GSH), and the GR-provided protection for Ieq was dependent on these changes in the apical bathing fluid compartment (ELF).

The finding that protection of Ieq required the presence of GR and NADPH in addition to GSH suggests that at least two separate oxidant or oxygen-related "injuries" occur. For example, Rt, which is considered to be primarily dependent on the integrity of the paracellular tight junctions (17), may have a high threshold for oxygen toxicity, and therefore the presence of GSH alone may be sufficient for protection. Ieq, on the other hand, which is dependent on Rt, as well as ion transport mechanisms (apical surface channels and basolateral Na+-K+-ATPase active transport) (17), may have a lower threshold for oxygen toxicity and, thus, may require additional GR and NADPH activity to provide adequate antioxidant protection.

A deficiency of GSH in ELF is found in a number of diseases characterized by an increased oxidant burden on the alveolar epithelial surface, including the adult respiratory distress syndrome (49), CF (56), and IPF (14); this deficiency may play an important role in the pathophysiology of these disorders. The increased oxidant burden that is characteristic of these pulmonary diseases, together with the hyperoxia encountered during oxygen therapy, may make the GSH deficiency even more consequential to the respiratory epithelial injury associated with these disease states. Aerosol delivery of GSH to the lower respiratory tract has been used to correct some of the oxidant-antioxidant imbalance in CF and IPF (5, 55). In this regard, supplementation of GSH levels in ELF may be a viable therapeutic approach in augmenting the antioxidant defense of the respiratory epithelial surface to protect against oxidant-induced damage to the alveolar epithelial barrier. Codelivery of GR and NADPH may further enhance therapeutic effects of GSH against oxygen toxicity in the respiratory epithelial tract.


    ACKNOWLEDGEMENTS

This work was supported in part by the Baxter Foundation, Hastings Foundation, American Lung Association, American Heart Association-National Center Grants-in-Aid 9950172N and 9950442N, and National Heart, Lung, and Blood Institute Research Grants HL-38658, HL-62569, and HL-64365.


    FOOTNOTES

Address for reprint requests and other correspondence: J. H. Roum, University of California Irvine Medical Center, Bldg. 53, Rm. 119, 101 City Dr. South, Orange, CA 92868 (E-mail: jhroum{at}uci.edu).

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.

Received 7 July 2000; accepted in final form 5 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aerts, C, Wallaert B, and Voisin C. In vitro effects of hyperoxia on alveolar type II pneumocytes: inhibition of glutathione synthesis increases hyperoxic cell injury. Exp Lung Res 18: 845-861, 1992[ISI][Medline].

2.   Avissar, N, Finkelstein JN, Horowitz S, Willey JC, Coy E, Frampton MW, Watkins RH, Khullar P, Xu YL, and Cohen HJ. Extracellular glutathione peroxidase in human lung epithelial lining fluid and in lung cells. Am J Physiol Lung Cell Mol Physiol 270: L173-L182, 1996[Abstract/Free Full Text].

3.   Berggren, M, Dawson J, and Moldeus P. Glutathione biosynthesis in the isolated perfused rat lung: utilization of extracellular glutathione. FEBS Lett 176: 189-192, 1984[ISI][Medline].

4.   Bhandari, V, Maulik N, and Kresch M. Hyperoxia causes an increase in antioxidant enzyme activity in adult and fetal rat type II pneumocytes. Lung 178: 53-60, 2000[ISI][Medline].

5.   Borok, Z, Buhl R, Grimes GJ, Bokser AD, Hubbard RC, Holroyd KJ, Roum JH, Czerski DB, Cantin AM, and Crystal RG. Effect of glutathione aerosol on oxidant-antioxidant imbalance in idiopathic pulmonary fibrosis. Lancet 338: 215-216, 1991[ISI][Medline].

6.   Borok, Z, Danto SI, Zabski SM, and Crandall ED. Defined medium for primary culture de novo of adult rat alveolar epithelial cells. In Vitro Cell Dev Biol Anim 30A: 99-104, 1994.

7.   Borok, Z, Hami A, Danto SI, Zabski SM, and Crandall ED. Rat serum inhibits progression of alveolar epithelial cells toward the type I cell phenotype in vitro. Am J Respir Cell Mol Biol 12: 50-55, 1995[Abstract].

8.   Borok, Z, Mihyu S, Fernandes VF, Zhang XL, Kim KJ, and Lubman RL. KGF prevents hyperoxia-induced reduction of active ion transport in alveolar epithelial cells. Am J Physiol Cell Physiol 276: C1352-C1360, 1999[Abstract/Free Full Text].

9.   Brown, LA. Glutathione protects signal transduction in type II cells under oxidant stress. Am J Physiol Lung Cell Mol Physiol 266: L172-L177, 1994[Abstract/Free Full Text].

10.   Brown, LA, Perez JA, Harris FL, and Clark RH. Glutathione supplements protect preterm rabbits from oxidative lung injury. Am J Physiol Lung Cell Mol Physiol 270: L446-L451, 1996[Abstract/Free Full Text].

11.   Buhl, R, Vogelmeier C, Critenden M, Hubbard RC, Hoyt RF, Jr, Wilson EM, Cantin AM, and Crystal RG. Augmentation of glutathione in the fluid lining the epithelium of the lower respiratory tract by directly administering glutathione aerosol. Proc Natl Acad Sci USA 87: 4063-4067, 1990[Abstract/Free Full Text].

12.   Canada, AT, Herman LA, and Young SL. An age-related difference in hyperoxia lethality: role of lung antioxidant defense mechanisms. Am J Physiol Lung Cell Mol Physiol 268: L539-L545, 1995[Abstract/Free Full Text].

13.   Cantin, AM, Fells GA, Hubbard RC, and Crystal RG. Antioxidant macromolecules in the epithelial lining fluid of the normal human lower respiratory tract. J Clin Invest 86: 962-971, 1990.

14.   Cantin, AM, Hubbard RC, and Crystal RG. Glutathione deficiency in the epithelial lining fluid of the lower respiratory tract in idiopathic pulmonary fibrosis. Am Rev Respir Dis 139: 370-372, 1989[ISI][Medline].

15.   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].

16.   Cheek, JM, Evans MJ, and Crandall ED. Type I cell-like morphology in tight alveolar epithelial monolayers. Exp Cell Res 184: 375-387, 1989[ISI][Medline].

17.   Cheek, JM, Kim KJ, and Crandall ED. Tight monolayers of rat alveolar epithelial cells: bioelectric properties and active sodium transport. Am J Physiol Cell Physiol 256: C688-C693, 1989[Abstract/Free Full Text].

18.   Chessex, P, Lavoie JC, Laborie S, and Vallee J. Survival of guinea pig pups in hyperoxia is improved by enhanced nutritional substrate availability for glutathione production. Pediatr Res 46: 305-310, 1999[ISI][Medline].

19.   Christie, NA, Slutsky AS, Freeman BA, and Tanswell AK. A critical role for thiol, but not ATP, depletion in 95% O2-mediated injury of preterm pneumocytes in vitro. Arch Biochem Biophys 313: 131-138, 1994[ISI][Medline].

20.   Clark, JM, and Lambertsen CJ. Pulmonary oxygen toxicity: a review. Pharmacol Rev 23: 37-133, 1971[Free Full Text].

21.   Clement, A, Edeas M, Chadelat K, and Brody JS. Inhibition of lung epithelial cell proliferation by hyperoxia. Posttranscriptional regulation of proliferation-related genes. J Clin Invest 90: 1812-1818, 1992.

22.   Crapo, JD, Hayatdavoudi G, Knapp MJ, Fracica PJ, Wolfe WG, and Piantadosi CA. Progressive alveolar septal injury in primates exposed to 60% oxygen for 14 days. Am J Physiol Lung Cell Mol Physiol 267: L797-L806, 1994[Abstract/Free Full Text].

23.   Davis, WB, Rennard SI, Bitterman PB, and Crystal RG. Pulmonary oxygen toxicity. Early reversible changes in human alveolar structures induced by hyperoxia. N Engl J Med 309: 878-883, 1983[Abstract].

24.   Deneke, SM, Lynch BA, and Fanburg BL. Transient depletion of lung glutathione by diethylmaleate enhances oxygen toxicity. J Appl Physiol 58: 571-574, 1985[Abstract/Free Full Text].

25.   Deneke, SM, Susanto I, Vogel KA, Williams CE, and Lawrence RA. Mechanisms of use of extracellular glutathione by lung epithelial cells and pulmonary artery endothelial cells. Am J Respir Cell Mol Biol 12: 662-668, 1995[Abstract].

26.   Dobbs, LG, Gonzalez R, and Williams MC. An improved method for isolating type II cells in high yield and purity. Am Rev Respir Dis 134: 141-145, 1986[ISI][Medline].

27.   Fisher, AB, Forman HJ, and Glass M. Mechanisms of pulmonary oxygen toxicity. Lung 162: 255-259, 1984[ISI][Medline].

28.   Forman, HJ, and Skelton DC. Protection of alveolar macrophages from hyperoxia by gamma -glutamyl transpeptidase. Am J Physiol Lung Cell Mol Physiol 259: L102-L107, 1990[Abstract/Free Full Text].

29.   Frank, L, Bucher JR, and Roberts RJ. Oxygen toxicity in neonatal and adult animals of various species. J Appl Physiol 45: 699-704, 1978[Abstract/Free Full Text].

30.   Goodman, BE, Kim KJ, and Crandall ED. Evidence for active sodium transport across alveolar epithelium of isolated rat lung. J Appl Physiol 62: 2460-2466, 1987[Abstract/Free Full Text].

31.   Hagen, TM, Brown LA, and Jones DP. Protection against paraquat-induced injury by exogenous GSH in pulmonary alveolar type II cells. Biochem Pharmacol 35: 4537-4542, 1986[ISI][Medline].

32.   He, LS, Chang SW, Ortiz de Montellano P, Burke TJ, and Voelkel NF. Lung injury in Fischer but not Sprague-Dawley rats after short-term hyperoxia. Am J Physiol Lung Cell Mol Physiol 259: L451-L458, 1990[Abstract/Free Full Text].

33.   Housset, B, Hurbain I, Masliah J, Laghsal A, Chaumette-Demaugre MT, Karam H, and Derenne J. Toxic effects of oxygen on cultured alveolar epithelial cells, lung fibroblasts and alveolar macrophages. Eur Respir J 4: 1066-1075, 1991[Abstract].

34.   Jackson, RM. Molecular, pharmacologic, and clinical aspects of oxygen-induced lung injury. Clin Chest Med 11: 73-86, 1990[ISI][Medline].

35.   Jenkinson, SG, Lawrence RA, Zamora CA, and Deneke SM. Induction of intracellular glutathione in alveolar type II pneumocytes following BCNU exposure. Am J Physiol Lung Cell Mol Physiol 266: L125-L130, 1994[Abstract/Free Full Text].

36.   Katzenstein, AL, Bloor CM, and Leibow AA. Diffuse alveolar damage---the role of oxygen, shock, and related factors. A review. Am J Pathol 85: 209-228, 1976[Medline].

37.   Kennedy, KA, and Lane NL. Effect of in vivo hyperoxia on the glutathione system in neonatal rat lung. Exp Lung Res 20: 73-83, 1994[ISI][Medline].

38.   Kim, KJ, Cheek JM, and Crandall ED. Contribution of active Na+ and Cl- fluxes to net ion transport by alveolar epithelium. Respir Physiol 85: 245-256, 1991[ISI][Medline].

39.   Kim, KK, Whitin JC, Sukhova NM, and Cohen HJ. Increase in extracellular glutathione peroxidase in plasma and lungs of mice exposed to hyperoxia. Pediatr Res 46: 715-721, 1999[ISI][Medline].

40.   Langley, SC, and Kelly FJ. Depletion of pulmonary glutathione using diethylmaleic acid accelerates the development of oxygen-induced lung injury in term and preterm guinea-pig neonates. J Pharm Pharmacol 46: 98-102, 1994[ISI][Medline].

41.   Langley, SC, and Kelly FJ. N-acetylcysteine ameliorates hyperoxic lung injury in the preterm guinea pig. Biochem Pharmacol 45: 841-846, 1993[ISI][Medline].

42.   Li, XY, Donaldson K, Rahman I, and MacNee W. An investigation of the role of glutathione in increased epithelial permeability induced by cigarette smoke in vivo and in vitro. Am J Respir Crit Care Med 149: 1518-1525, 1994[Abstract].

43.   Lubman, RL, Kim K-J, and Crandall ED. Alveolar epithelial barrier properties. In: The Lung: Scientific Foundations (2nd ed.), edited by Crystal RG, West JB, Weibel E, and Barnes P.. Philadelphia, PA: Lippincott-Raven, 1997, p. 585-602.

44.   Mason, RJ, Walker SR, Shields BA, Henson JE, and Williams MC. Identification of rat alveolar type II epithelial cells with a tannic acid and polychrome stain. Am Rev Respir Dis 131: 786-788, 1985[ISI][Medline].

45.   Matalon, S, and Egan EA. Effects of 100% O2 breathing on permeability of alveolar epithelium to solute. J Appl Physiol 50: 859-863, 1981[Abstract/Free Full Text].

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

47.   Nici, L, Dowin R, Gilmore-Hebert M, Jamieson JD, and Ingbar DH. Upregulation of rat lung Na-K-ATPase during hyperoxic injury. Am J Physiol Lung Cell Mol Physiol 261: L307-L314, 1991[Abstract/Free Full Text].

48.   Olivera, WG, Ridge KM, and Sznajder JI. Lung liquid clearance and Na,K-ATPase during acute hyperoxia and recovery in rats. Am J Respir Crit Care Med 152: 1229-1234, 1995[Abstract].

49.   Pacht, ER, Timerman AP, Lykens MG, and Merola AJ. Deficiency of alveolar fluid glutathione in patients with sepsis and the adult respiratory distress syndrome. Chest 100: 1397-1403, 1991[Abstract/Free Full Text].

50.   Peacock, MD, Schenk DA, Lawrence RA, Morgan JA, and Jenkinson SG. Elimination of glutathione-induced protection from hyperbaric hyperoxia by acivicin. J Appl Physiol 76: 1279-1284, 1994[Abstract/Free Full Text].

51.   Pietarinen, P, Raivio K, Devlin RB, Crapo JD, Chang LY, and Kinnula VL. Catalase and glutathione reductase protection of human alveolar macrophages during oxidant exposure in vitro. Am J Respir Cell Mol Biol 13: 434-441, 1995[Abstract].

52.   Rister, M, and Wustrow C. Effect of hyperoxia on reduced glutathione in alveolar macrophages and polymorphonuclear leukocytes. Res Exp Med (Berl) 185: 445-450, 1985[Medline].

53.   Rochat, T, Burkhard C, Finci-Cerkez V, and Meda P. Oxidative stress causes a protein kinase C-independent increase of paracellular permeability in an in vitro epithelial model. Am J Respir Cell Mol Biol 9: 496-504, 1993.

54.   Roum, JH, Borok Z, and Kim KJ. Transalveolar epithelial permeability to glutathione (Abstract). Am J Respir Crit Care Med 155: A189, 1997.

55.   Roum, JH, Borok Z, McElvaney NG, Grimes GJ, Bokser AD, Buhl R, and Crystal RG. Glutathione aerosol suppresses lung epithelial surface inflammatory cell-derived oxidants in cystic fibrosis. J Appl Physiol 87: 438-443, 1999[Abstract/Free Full Text].

56.   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].

57.   Royston, BD, Webster NR, and Nunn JF. Time course of changes in lung permeability and edema in the rat exposed to 100% oxygen. J Appl Physiol 69: 1532-1537, 1990[Abstract/Free Full Text].

58.   Smith, LJ, Horcher P, Anderson J, and Shamsuddin M. Effect of fasting on the lung glutathione redox cycle in air- and oxygen-exposed mice: beneficial effects of sugar. J Lab Clin Med 116: 717-723, 1990[ISI][Medline].

59.   Smith, RA, Venus B, Masood S, and Carter MC. Effects of hyperoxia in the presence of acute lung injury. Crit Care Med 18: 198-202, 1990[ISI][Medline].

60.   Stenzel, JD, Welty SE, Benzick AE, Smith EO, Smith CV, and Hansen TN. Hyperoxic lung injury in Fischer-344 and Sprague-Dawley rats in vivo. Free Radic Biol Med 14: 531-539, 1993[ISI][Medline].

61.   Taylor, CG, Bauman PF, Sikorski B, and Bray TM. Elevation of lung glutathione by oral supplementation of L-2-oxothiazolidine-4-carboxylate protects against oxygen toxicity in protein-energy malnourished rats. FASEB J 6: 3101-3107, 1992[Abstract].

62.   Tsan, MF, Danis EH, Del Vecchio PJ, and Rosano CL. Enhancement of intracellular glutathione protects endothelial cells against oxidant damage. Biochem Biophys Res Commun 127: 270-276, 1985[ISI][Medline].

J APPL PHYSIOL 91(2):748-754
8750-7587/01 $5.00 Copyright © 2001 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Respir. Cell Mol. Bio.Home page
P. J. O'Reilly, J. M. Hickman-Davis, I. C. Davis, and S. Matalon
Hyperoxia Impairs Antibacterial Function of Macrophages Through Effects on Actin
Am. J. Respir. Cell Mol. Biol., April 1, 2003; 28(4): 443 - 450.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
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 ISI Web of Science
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 (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Roum, J. H.
Right arrow Articles by Borok, Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Roum, J. H.
Right arrow Articles by Borok, Z.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online