Superoxide released from neutrophils causes a reduction in nitric oxide gas

Kimberly L. Jones, Ty W. Bryan, Patricia A. Jinkins, Keith L. Simpson, Matthew B. Grisham, Michael W. Owens, Shawn A. Milligan, Boaz A. Markewitz, and Richard A. Robbins

Departments of Medicine and Physiology and Biophysics, Overton Brooks Veterans Affairs and Louisiana State University Medical Centers, Shreveport, Louisiana 71101

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Exhaled nitric oxide (NO) is increased in some inflammatory airway disorders but not in others such as cystic fibrosis and acute respiratory distress syndrome. NO can combine with superoxide (O-2) to form peroxynitrite, which can decompose into nitrate. Activated polymorphonuclear neutrophils (PMNs) releasing O-2 could account for a reduction in exhaled NO in disorders such as cystic fibrosis. To test this hypothesis in vitro, we stimulated confluent cultures of LA-4 cells, a murine lung epithelial cell line, to produce NO. Subsequently, human PMNs stimulated to produce O-2 were added to the LA-4 cells. A gradual increase in NO in the headspace above the cultures was observed and was markedly reduced by the addition of PMNs. An increase in nitrate in the culture supernatant fluids was measured, but no increase in nitrite was detected. Superoxide dismutase attenuated the PMN effect, and xanthine/xanthine oxidase reproduced the effect. No changes in epithelial cell inducible NO synthase protein or mRNA were observed. These data demonstrate that O-2 released from PMNs can decrease NO by conversion to nitrate and suggest a potential mechanism for modulation of NO levels in vivo.

oxidants; oxygen radicals; nitrogen oxides; cystic fibrosis; lung disease

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

NITRIC OXIDE (NO) is endogenously produced in lung epithelial cells, alveolar macrophages, neutrophils, and mast cells (4, 16, 18, 23, 26, 31). It is synthesized from L-arginine by NO synthase (NOS) and several cofactors (flavones, NADPH, and tetrahydrobiopterin) (22). NOS has several isoforms, including inducible NOS (iNOS) (22). iNOS is expressed in response to inflammatory cytokines, such as tumor necrosis factor-alpha , interleukin-1beta , and interferon-gamma , and endotoxin. iNOS generates increased amounts of NO compared with the constitutive isoforms of NOS (22). NO is involved in the regulation of numerous pulmonary functions including upregulation of ciliary motility, host inflammatory responses, and antibacterial and antiviral activity (25).

Exhaled NO concentrations have been measured in various disease states and have been shown to be elevated in patients with asthma and bronchiectasis (14, 15). On the basis of prior data showing increased amounts of inflammatory cytokines in the cystic fibrosis (CF) lung (1, 11) and acute respiratory distress syndrome (ARDS) lung (7), exhaled NO would be expected to be increased in patients with CF and ARDS. However, several studies (3, 6, 9, 17) have measured exhaled NO in CF and ARDS patients and found these levels to be comparable with or less than normal control subjects. One possible mechanism for these findings is increased numbers of activated neutrophils in the CF or ARDS lungs releasing superoxide. Superoxide may then combine with NO to produce an unstable intermediate, peroxynitrite, which can form nitrotyrosine or decompose to nitrate (2, 13). On the basis of this premise, one would expect increased levels of nitrate and/or increased peroxynitrite in CF or ARDS lungs, which have been found in prior studies (9, 12).

We tested this hypothesis in vitro by incubating lung epithelial cells with polymorphonuclear neutrophils (PMNs) that had been stimulated to generate increased amounts of superoxide. Measurements of NO, nitrite, and nitrate as well as expression of iNOS mRNA and protein were subsequently made from the incubated cultures.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

LA-4 cultures. The clonal murine epithelial cell line LA-4 was purchased from the American Type Culture Collection (Manassas, VA) (29). LA-4 cells were grown in 25-cm2 tissue culture flasks (Costar, Cambridge, MA) in Ham's F-12 medium containing 10% fetal calf serum, L-glutamine (2 mM), and penicillin-streptomycin (100 U/ml and 100 mg/ml, respectively) until confluent. After the cells were washed with serum-free Ham's F-12 medium, they were stimulated for 24 h to express iNOS by culturing with a combination of recombinant human tumor necrosis factor-alpha , human interleukin-1beta , and murine interferon-gamma (Cytomix; all at a final concentration of 10 ng/ml; Sigma) (28).

PMNs. Samples of heparinized peripheral blood were obtained after informed consent from normal individuals. PMNs were isolated by dextran sedimentation (Dextran T-500, Pharmacia, Uppsala, Sweden) for 1 h at 1 g followed by Ficoll-Hypaque density centrifugation (Histopaque 1077, Sigma). The PMNs (>90% purity, >90% viability) were placed in serum-free Ham's F-12 medium and subsequently stimulated with phorbol 12-myristate 13-acetate (PMA; 10-7 M) to produce superoxide (19). Additional studies were done in which the PMNs were incubated with LA-4 cells without initial stimulation by PMA.

Addition of PMNs to LA-4 cultures. PMNs were added at a concentration of 0.05-10 × 106 cells/flask to the LA-4 cells and incubated for 0.25-4 h. All flasks were tightly sealed to minimize any loss of NO gas. In some cultures, cellular viability was assessed by measuring lactate dehydrogenase (LDH; Sigma) in the culture supernatant fluids. Further assessment was done by measuring the pH of the medium before and after incubation (2). To some cultures of LA-4 cells with added PMNs, superoxide dismutase (SOD; 10-1,000 U/ml; Sigma) was added to decrease superoxide (10), and to additional cultures, the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) was added. In addition, some cultures had peroxynitrite (final concentration of 50 µM; Calbiochem, La Jolla, CA) added to determine whether there was a rise in nitrate levels.

NO, nitrite, and nitrate measurements. From each flask, ~12 ml of gas were aspirated from the headspace, and NO was measured by a sensitive NO chemiluminescence analyzer (model 270B, Sievers, Boulder, CO). Nitrites in the supernatant were converted to NO under acidic conditions, and NO was measured (28). The amount of nitrite was calculated by comparison to a standard curve of sodium nitrite, with sensitivity of the assay <20 nM. Nitrite + nitrate was measured by reducing nitrate to nitrite with the use of Aspergillus nitrate reductase (final concentration of 0.1 U/ml; Boehringer Mannheim, Indianapolis, IN) in the presence of NADPH (final concentration of 0.086 mM), KH2PO4 (final concentration of 0.062 M), and FAD (final concentration of 0.011 mM) for 60 min at room temperature (27). The nitrate + nitrite was then measured as nitrite with the above techniques. Nitrate was calculated by subtracting nitrite from nitrite + nitrate.

Xanthine/xanthine oxidase. To some Cytomix-stimulated LA-4 cultures without added PMNs, xanthine (X; 1-1,000 µM, Sigma) and xanthine oxidase (XO; 0.00003-0.003 U/ml) were added to produce superoxide (10). The LA-4 cells were cultured for 30 min and NO, nitrite, and nitrate were determined as in NO, nitrite, and nitrate measurements.

Measurement of nitrotyrosine. Formation of nitrotyrosine has been hypothesized to be secondary to peroxynitrite formation (12). To quantitate nitrotyrosine, we used an inhibition enzyme-linked immunosorbent assay (ELISA). LA-4 cells were stimulated as in LA-4 cultures, and PMNs (10 × 106 cells) were added to some cultures. After 4 h, the cell culture supernatant fluids were removed, and the cells were harvested by scraping in PBS-0.075% Tween 20 (PBS-Tween; Sigma) and kept frozen until assayed. Human serum albumin was incubated with peroxynitrite for 2 h at 37°C. The nitrated albumin was then dissolved in Voller's buffer (1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, and 0.2 g of sodium azide in 1 liter of distilled water, pH 9.6, at a final concentration of 25 µg/ml), and 200 µl were added to flat-bottomed 96-well plates (Costar). The nitrated albumin was allowed to adsorb to the plastic overnight at 4°C. In addition, 110 µl of the culture supernatant fluids, scraped cells, or standards were dissolved in PBS-Tween and combined in round-bottomed 96-well plates with 110 µl of a 1:100 dilution of goat polyclonal anti-nitrotyrosine antibody (Calbiochem). The round-bottomed plates were also incubated overnight at 4°C. After the flat-bottomed plate was washed three times with PBS-Tween, 200 µl of the samples containing the antibody were transferred from the round-bottomed plate to the flat-bottomed plate and incubated for 33 min at room temperature. After three more washes with PBS-Tween, 200 µl of a 1:500 dilution of peroxidase-conjugated rabbit anti-goat IgG antibody were added to the wells and incubated for 90 min. After three more washes with PBS-Tween, 200 µl of o-phenylenediamine (100 µg/ml; Sigma) in 0.003% H2O2 were added and visually monitored. The reaction was terminated by the addition of 25 µl of 8 N H2SO4, and the absorbance was read at 490 nm. The amount of material in the standards was calculated by comparison to a standard curve of the nitrated albumin with a nonlinear regression analysis. The results were expressed relative to albumin in micromolar concentrations.

iNOS protein. iNOS protein was quantified by Western blotting as previously described (21). Briefly, the epithelial cells were lysed in 10 mM Tris · HCl (pH 7.4)-1% sodium dodecyl sulfate (SDS) buffer containing protease inhibitors. Samples were electrophoresed on 7.5% SDS-polyacrylamide gels and then were blotted to polyvinylidene difluoride membranes. The membranes were probed with a monoclonal goat anti-macrophage iNOS antibody (Transduction Laboratories, Lexington, KY). The blots were then washed and incubated with an anti-mouse alkaline phosphatase-conjugated antibody. After the blots were washed, they were developed with 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium. After the blots were dried, they were photographed, and the intensity of the bands was quantified by densitometry.

iNOS mRNA. iNOS mRNA was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Total cellular RNA was extracted from adherent cells with a modification of the method of Chomczynski and Sacchi (5). The RNA was reverse transcribed with a commercially available kit (Promega). One microgram of the reverse-transcribed DNA was then mixed with Ready-to-Go RT-PCR Beads (Pharmacia), and the front and back primers (Table 1) were added at a final concentration of 0.2 µM. The PCR for iNOS was then carried out in a Perkin-Elmer model 480 thermal cycler: 94°C for 2 min and 28 cycles consisting of 94°C for 45 s, 60°C for 45 s, and 72°C for 2 min, followed by 72°C for an additional 7 min. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a "housekeeping gene" with PCR under conditions as for iNOS except for an annealing temperature of 50°C. The DNA was then subjected to agarose gel and quantitated by densitometry. The results are expressed as the ratio of intensity of iNOS to that of GAPDH.

                              
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Table 1.   Primers

As a positive control, human chorionic DNA (Sigma) was used. Increasing PCR cycles gave increasing amounts of DNA through a fairly broad range, with linearity obtained from approximately cycles 24-32, beginning with 1 µg of DNA (r = 0.99 for iNOS and GAPDH).

Statistical analysis. All experiments were performed in triplicate, and data are means ± SE. Comparisons were performed with the use of Scheffé's analysis of variance. A P value of <0.05 was considered significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of PMNs on NO production by lung epithelial cells. Stimulated LA-4 cells were incubated with and without PMNs, and levels of headspace NO, nitrite, and nitrate were measured at intervals of 30 min to 4 h. There was a time-dependent increase in NO, nitrite, and nitrate (Fig. 1). At each of the time points tested, headspace NO was less in the presence of PMNs (P < 0.05 for all comparisons; Fig. 1A). However, there was no significant change in nitrite (P > 0.05 for all comparisons; Fig. 1B), but there was a significant increase in the amount of nitrate at each of the time points tested (P < 0.05 for all comparisons; Fig. 1C). The reduction in NO approximated the increase in nitrate (4-h time point, 10.9 ± 1.7 vs. 12.7 ± 1.8 µM; P > 0.05). There was also a dose-dependent decrease in headspace NO and an increase in nitrate with increasing numbers of PMNs (1-10 × 106 cells; Fig. 2). PMNs not stimulated with PMA still produced a decrease in NO and a rise in nitrate; however, these changes were decreased compared with stimulated cells [NO with PMA dropped by 1,256 vs. 864 parts/billion (ppb) without PMA]. Addition of the NOS inhibitor L-NMMA (10-4 M) decreased NO approximately the same as the PMNs (770 ppb with L-NMMA vs. 864 ppb with PMNs). Nitrite and nitrate formation by PMNs alone (10 × 106 cells, 4 h) was low at 1.4 ± 0.2 and 2.1 ± 1.9 µM, respectively. NO production by the PMNs alone was also low at 46 ± 8 ppb. LA-4 cultures with up to 50 µM nitrite, nitrate, or peroxynitrite added did not affect the NO concentrations in the headspace at 4 h (data not shown).


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Fig. 1.   Response of nitric oxide (NO; A), nitrite (B), and nitrate (C) in LA-4 cells with (bullet ) and without (open circle ) addition of polymorphonuclear neutrophils (PMNs). A shows elevation in NO in system without PMNs and significantly lower response after addition of PMNs. ppb, Parts/billion. B shows nitrite in culture supernatant fluids. C shows rise in nitrate after addition of PMNs. n = 3 experiments. * P < 0.05 compared with epithelial cells without PMNs.


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Fig. 2.   Response with increasing concentration (Conc) of PMNs. A shows drop in NO observed from baseline (without PMNs) with increasing concentration of PMNs. B shows no significant change in nitrite. C shows rise in nitrate with increasing concentration of PMNs. n = 3 experiments. * P < 0.05 compared with epithelial cells without PMNs.

Inhibition of superoxide formation. The studies with the LA-4 cells and PMNs were repeated with PMNs at a steady concentration (10 × 106 cells/flask) and increasing concentrations of SOD (10-1,000 U/ml). The effect seen on NO and nitrate previously by the addition of PMNs was reversed when SOD was added (Fig. 3).


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Fig. 3.   Addition of superoxide dismutase (SOD) to cultures of epithelial cells and PMNs (10 × 106 cells/flask). Concentrations of NO (A), nitrite (B), and nitrate (C) were measured against increasing SOD concentration. n = 3 experiments. * P < 0.05 compared with epithelial cells without SOD.

X/XO. Additional studies were then done with X/XO to reproduce the effect on LA-4 cells by PMNs. Stimulated LA-4 cells were incubated for 30 min with increasing concentrations of X/XO (10-1,000 µM and 0.00003-0.003 U/ml, respectively). Measurements of headspace NO fell from 306 ppb at baseline to 26 ppb with 1 mM X/XO, with an accompanying rise in nitrate at the highest concentration of X/XO used (Fig. 4).


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Fig. 4.   Addition of xanthine/xanthine oxidase (X/XO) to cytokine-stimulated epithelial cells releasing NO. Concentrations of NO (A), nitrite (B), and nitrate (C) were measured against increasing X/XO concentrations. n = 3 experiments. * P < 0.05 compared with epithelial cells without X/XO.

Peroxynitrite and nitrotyrosine. Addition of peroxynitrite (50 µM) to unstimulated LA-4 cells resulted in an increase in nitrate of 2.2 ± 0.6 and 6.1 ± 0.6 µM at 0.5 and 4 h, respectively, compared with the amount of nitrate contained in the added peroxynitrite (n = 3 experiments at each time point). There was no change in nitrite at 0.5 or 4 h (P > 0.05; n = 3 experiments).

The nitrotyrosine ELISA gave the expected S-shaped curve seen with inhibition ELISAs (r = -0.97 after log-log transformation), with an estimated sensitivity of >200 nM. Nitrotyrosine was not detectable by ELISA in unstimulated LA-4 cells. There was detectable nitrotyrosine in the cells obtained from cytokine-stimulated LA-4 cells cultured for 4 h without PMNs (12.3 ± 6.2 µM relative to nitrated albumin as a standard; n = 5 experiments) but no detectable nitrotyrosine in cell culture supernatant fluids. In contrast, there was a marked increase in the amount of nitrotyrosine in the cells obtained from cytokine-stimulated LA-4 cells cultured with added PMNs (10 × 106 cells) for 4 h (911 ± 211 µM; n = 5 experiments) and detectable nitrotyrosine in the cell culture supernatant fluids (33.4 ± 16.3 µM; n = 5 experiments). PMNs (10 × 106 cells) cultured for 4 h without epithelial cells also had nitrotyrosine in the cell layer (518 ± 159 µM; n = 5 experiments) and in their culture supernatant fluids (10.9 ± 2.1 µM; n = 5 experiments), consistent with their known capacity to produce nitrotyrosine from nitrite and myeloperoxidase (8).

iNOS protein and mRNA. Incubation with stimulated PMNs did not alter expression of iNOS protein as shown by Western blot analysis (P > 0.05; Fig. 5). Similarly, iNOS mRNA analysis by RT-PCR did not show appreciable differences in iNOS mRNA between cultures with and without PMNs (10,600 ± 1,843 vs. 10,084 ± 973; P > 0.05; n = 3 experiments; Fig. 6A) or when expressed as a ratio to GAPDH mRNA (1.64 ± 0.21 vs. 1.00 ± 0.33; P > 0.05; n = 3 experiments; Fig. 6B).


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Fig. 5.   Lack of effect of PMNs on inducible NO synthase (iNOS) protein expression. LA-4 cells were incubated with PMNs for 30 min. After epithelial cells were washed to remove PMNs, epithelial cells were lysed, and amount of iNOS was assessed by Western blotting. Amount of iNOS was quantified by scanning densitometry. A: representative Western blot of separate cultures from LA-4 cells stimulated with Cytomix with (lanes 1-3) and without (lanes 4-6) addition of PMNs. Left, molecular-weight markers. B: summary data for LA-4 cells stimulated with Cytomix but without PMNs or stimulated with Cytomix and PMNs for 30 min. n = 3 experiments. The 2 groups did not differ significantly (P > 0.05). LA-4 cells cultured in medium alone were undetectable.


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Fig. 6.   Lack of effect of PMNs on iNOS mRNA expression. LA-4 cells were incubated with PMNs for 30 min. After PMNs were removed by washing, RNA was extracted, reverse transcribed, and subjected to PCR. Amount of iNOS mRNA was quantified by scanning densitometry. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was similarly quantified and used as a "housekeeping" gene. A: results with RT-PCR for iNOS and GAPDH with (lanes 1-3) and without (lanes 4-6) addition of PMNs. B: densitometry data with iNOS expressed as ratio of iNOS mRNA to GADPH mRNA (iNOS mRNA/GAPDH mRNA). n = 3 experiments.

Cellular viability and pH. Measurements of LDH to determine cellular viability showed no significant change with the addition of PMNs (10 × 106 cells/flask after 4 h; P > 0.05; n = 3 experiments). Similarly, pH was unchanged from the initial value of 8-8.5 (P > 0.05; n = 3 experiments).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The goal of this study was to demonstrate in an in vitro cell system that superoxide generated by PMNs causes a decrease in detectable NO gas. These studies have demonstrated that when PMNs are stimulated to generate superoxide, there was a decrease in NO and an equivalent rise in nitrate that were both time and dose dependent. Nitrite levels remained unchanged. Furthermore, these effects could be blunted by the addition of SOD, and addition of X/XO could reproduce this effect. These changes were not secondary to altered iNOS protein expression because the total amount of NO + nitrite + nitrate did not change and there was no significant change in the amount of iNOS protein or mRNA expression.

The mechanism of action of these results is consistent with the reduction in NO by superoxide to peroxynitrite and subsequently to nitrate. The finding that iNOS protein and mRNA expression was not significantly changed in either culture system is consistent with this statement. Furthermore, the reduction in the effect with SOD and the reproduction of the effect with X/XO are consistent with this hypothesis.

A prior study (2) demonstrated conversion of nitrite to nitrate under acidic conditions. Acidic conditions might be found in the setting of cell death and lysis. For these reasons, LDH was tested in some cell systems as a marker of cell death but was not found to be elevated compared with baseline. The actual pH of the medium before incubation with PMNs and after incubation was also tested, and no appreciable change from baseline was found.

Exhaled NO levels are not elevated in CF or ARDS patients as seen in some other inflammatory diseases (3, 6, 9, 17). The findings of this study are in agreement with the hypothesis that the preponderance of neutrophils in the airway of CF patients are converting NO to nitrate, resulting in a reduction in NO and an increase in nitrate. These in vitro data suggest a plausible explanation for the alteration of exhaled NO or tissue nitrate levels in those disorders, such as CF, being associated with a large influx of PMNs releasing superoxide. Our findings are consistent with recent reports (9, 12) demonstrating an increase in nitrate in the sputum of subjects with CF and formation of nitrotyrosine by peroxynitrite in the lungs of patients with ARDS.

SOD attenuated the effect of PMNs on NO production. Although a likely explanation for this effect is a reduction in superoxide, SOD may also directly interact with peroxynitrite (24). This interaction could potentially affect the NO and nitrate levels. Another factor known to affect formation of peroxynitrite and nitrate is pH (2). However, pH was unaltered during these experiments, suggesting that this is not a likely explanation for the change in NO and nitrate levels.

A previous study (16) investigated the fate of NO derived from macrophages. It is apparent that, depending on the relative rate of production of NO and superoxide, the ratio of nitrite to nitrate will change due to secondary reactions of both with peroxynitrite. In the conditions of this study, nitrite and nitrate appeared to be formed as the principal end products. However, in stimulated macrophages, addition of SOD actually resulted in an increase in the ratio of nitrate to nitrite (16). The differences between the studies are unknown but likely are a result of the complex oxidant chemistry of NO with different cells and the amounts of NO and superoxide formed.

The cell and tissue toxicity from NO has been hypothesized to be due to formation of peroxynitrite, which has strong oxidizing properties (2). Our results are consistent with peroxynitrite formation from an interaction of NO and superoxide and a subsequent nitration of tyrosine residues. However, the reactivity of peroxynitrite may be dependent on the amounts of superoxide and NO present because excess superoxide and NO may interact with peroxynitrite, altering its reactivity (20, 30).

A recent study (8) demonstrated an alternative mechanism for tyrosine nitration via nitrite and myeloperoxidase without peroxynitrite formation. Our findings with PMNs alone are consistent with this mechanism where only a small amount of nitrite, nitrate, and NO was detected, but large amounts of nitrotyrosine residues were detected on PMNs and in PMN culture supernatant fluids.

The data from the present paper demonstrate that NO levels can be modulated by superoxide, which, in turn, can form peroxynitrite and subsequently nitrate. Clinically, these data imply that exhaled NO levels may not necessarily reflect NO production in the lower respiratory tract. This may be the explanation for exhaled NO levels being comparable to control subjects in CF patients or lower than control subjects in ARDS patients. Furthermore, these data imply that an absence of an increase in exhaled NO does not exclude the possibility of NO participating in the inflammation and tissue toxicity in lung disorders such as CF and ARDS.

    ACKNOWLEDGEMENTS

This work was supported by a Merit Review Grant from the Veterans Administration.

    FOOTNOTES

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: R. A. Robbins, Overton Brooks Veterans Affairs Medical Center, 510 E. Stoner Ave., Shreveport, LA 71101.

Received 16 March 1998; accepted in final form 19 August 1998.

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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Lung Cell Mol Physiol 275(6):L1120-L1126
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