Inhibition of amiloride-sensitive sodium-channel activity in distal lung epithelial cells by nitric oxide

Jin Wen Ding1, John Dickie1, Hugh O'Brodovich2, Yutaka Shintani2, Bijan Rafii2, David Hackam1, Yoshinori Marunaka2, and Ori D. Rotstein1

1 Department of Surgery and Medical Research Council of Canada Group in Mechanisms of Organ Injury, Toronto Hospital, University of Toronto, and Toronto Hospital Research Institute, Toronto M5G 2C4; and 2 Medical Research Council of Canada Group in Lung Development and Department of Pediatrics (Respiratory Research), University of Toronto and The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Distal lung epithelial cells (DLECs) play an active role in fluid clearance from the alveolus by virtue of their ability to actively transport Na+ from the alveolus to the interstitial space. The present study evaluated the ability of activated macrophages to modulate the bioelectric properties of DLECs. Low numbers of lipopolysaccharide (LPS)-treated macrophages were able to significantly reduce amiloride-sensitive short-circuit current (Isc) without affecting total Isc or monolayer resistance. This was associated with a rise in the flufenamic acid-sensitive component of the Isc. The effect was reversed by the addition of N-monomethyl-L-arginine to the medium, implying a role for nitric oxide. We hypothesized that macrophages exerted their effect by expressing inducible nitric oxide synthase (iNOS) in DLECs. The products of LPS-treated macrophages increased the levels of iNOS protein and mRNA transcripts in DLECs as well as causing a rise in iNOS activity. Immunofluorescence microscopy of LPS-stimulated macrophage-DLEC cocultures with anti-nitrotyrosine antibodies provided evidence for the generation of peroxynitrite in macrophages but not in DLECs. These data indicate that activated macrophages in the lung may contribute to impaired resolution of acute respiratory distress syndrome and suggest a novel mechanism whereby nitric oxide might alter cell function by altering its ion-transporting phenotype.

distal lung epithelium; macrophages; lung injury

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

ACUTE RESPIRATORY DISTRESS SYNDROME (ARDS) is characterized by the presence of hypoxemia, reduced lung compliance, and increased permeability pulmonary edema that results in diffuse alveolar infiltrates on chest radiographs. This pathological process develops as a result of the disruption of the alveolar-capillary membrane with leakage of protein-rich fluid exudate and migration of inflammatory cells (neutrophils and macrophages) into the air space (reviewed in Ref. 40). Under normal circumstances, the distal lung epithelial cells (DLECs) that line the terminal airways and alveoli provide a tight physical barrier to the movement of interstitial fluid into the alveolar space. Injury to this layer may result in alveolar edema that contributes to the clinical characteristics of ARDS.

Recent studies have demonstrated that the DLECs also play an active role in fluid clearance from the alveolus by virtue of their ability to actively transport Na+ from the alveolus to the interstitial space. Vectorial transport of Na+ (with Cl- and water following) across monolayers of these cells is mediated by the presence of Na+ channels on their apical surface that permit Na+ entry down their electrochemical gradient into the cell and Na+-K+-adenosinetriphosphatase (ATPase) on the basolateral surface of the cell that extrudes Na+ (3, 30). Both in vitro and in vivo experiments suggest a physiological role for this transport mechanism. Studies performed under conditions where the bioelectric properties may be measured show that monolayers grown on porous supports exhibit unidirectional apical-to-basolateral transport of Na+ and are able to establish a significant potential difference (apical negative) across the monolayer. These processes are inhibited by the Na+-channel blockers amiloride and benzamil as well as by the Na+-K+-ATPase inhibitor ouabain and are stimulated by beta -adrenergic agonists and membrane-permeant analogs of adenosine 3',5'-cyclic monophosphate (3, 27-30). The existence of this process in vivo is supported by several lines of evidence. First, tracer studies in isolated perfused rat lung preparations using 22Na+ have clearly demonstrated active Na+ reabsorption from the alveolar spaces (9). Second, physiological studies in animals have shown a role for an amiloride-sensitive process in fluid clearance from the alveolus (1, 2, 20). Finally, there is evidence supporting the existence of a comparable system in humans. Sakuma et al. (38) have reported amiloride- and ouabain-inhibitable alveolar fluid clearance in resected human lung at rates comparable to those observed in animal experiments. Considered together with the in vitro data, these observations suggest that this fluid-resorptive mechanism may play an active role in the resolution phase of ARDS. In this regard, a study by Matthay and Wiener-Kronish (26) correlated the clinical resolution of ARDS with the ability of patients to concentrate alveolar fluid protein, an indirect measure of Na+ transport-mediated fluid clearance from the alveolar space.

Our laboratory has previously investigated the ability of alveolar macrophages (Mphi s) to modulate DLEC Na+-transport activity as a model whereby activated alveolar Mphi s might contribute to the development and persistence of ARDS in the septic patient. These studies demonstrated that lipopolysaccharide (LPS)-treated alveolar Mphi s were able to reduce total and amiloride-sensitive short-circuit current (Isc) in primary cultures of DLECs (7). The observation that this inhibitory effect was dependent on L-arginine metabolism suggested a role for nitric oxide (NO) as a key mediator of this effect. Because DLECs incubated with the supernatants of LPS-treated Mphi s exhibited alterations in Isc similar to those seen in the coculture system, we hypothesized that NO derived from DLECs might, at least in part, be responsible for the effect. The present studies demonstrate that the soluble products of LPS-treated Mphi s decrease the amiloride-sensitive Na+ transport of the DLECs without affecting total Isc. These changes correlated with an increase in the expression of inducible NO synthase (iNOS) at both the mRNA and protein levels in DLECs. However, treatment of DLECs with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) alone did not reproduce the effect, suggesting that NO production is necessary but not sufficient to account for the observed alterations in amiloride-sensitive Isc in DLECs exposed to Mphi products. These data therefore suggest a mechanism whereby NO might contribute to altered cell function by altering its ion-transporting phenotype.

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

Materials and Solutions

Tissue culture media and additives, including RPMI 1640, Hanks' balanced salt solution (HBSS; with and without Ca2+ and Mg2+), Dulbecco's phosphate-buffered saline (PBS), fetal bovine serum, Eagle's minimum essential medium (MEM), MEM selectamine kits, trypsin, and penicillin-streptomycin, were all obtained from GIBCO BRL (Life Technologies, Grand Island, NY). Collagenase and deoxyribonuclease for epithelial cell harvest were purchased from Worthington Biochemical (Freehold, NJ). Heparin (Hepalean; 1,000 U/ml) was from Organon Teknika (Toronto, Canada), and pentobarbital sodium (Somnotol) was from MTC Pharmaceuticals (Cambridge, Canada). Endotoxin (Escherichia coli O111:B4) was from Difco Laboratories (Detroit, MI). NG-monomethyl-L-arginine (L-NMMA) was purchased from Calbiochem Behring (La Jolla, CA). Amiloride, bumetanide, and flufenamic acid were from Sigma (St. Louis, MO).

Alveolar Mphi Isolation

Adult male Wistar rats (300-350 g) were anesthetized with halothane-nitrous oxide and then exsanguinated after the administration of intravenous heparin (500 U). The tracheae were then cannulated (14-gauge catheter; Becton Dickinson Vascular Access, Sandy, UT), and heart-lung blocks were extracted for ex vivo bronchoalveolar lavage. Bronchoalveolar lavage was performed by instilling 10-ml aliquots of PBS with 1 mM EDTA and recovering the fluid by gentle suction as previously described (6). This was repeated five times to yield a recovered volume of ~45 ml. Lavaged cells were pelleted by centrifugation (200 g for 10 min), pooled, and resuspended in RPMI medium containing 10% fetal bovine serum. Wright's staining of cytospin-prepared cell populations demonstrated >92% alveolar Mphi s by morphology. Cell viability was routinely >95% as determined by trypan blue exclusion. Total cell numbers were counted with a hemocytometer (Improved Neubauer, American Optical, Buffalo, NY).

Epithelial Cell Isolation and Culture

DLECs were harvested from late-gestation fetal rats and grown in primary culture according to methods previously described (28). In brief, the lungs were excised from timed-gestation 20- or 21-day (term = 22 days) Wistar rat fetuses and minced into 1-mm3 pieces. The lung fragments were incubated at 37°C with 0.125% trypsin and 0.002% deoxyribonuclease, and the dissociated cells were then passed through a Nitex 100 (B. and S. H. Thompson, Scarborough, Canada) mesh filter. The cells were then incubated with 0.1% collagenase and purified with differential adhesion techniques. These cells are >99% pure epithelial cells and consist of mature and precursor type II epithelial cells or distal airway cells, and hence we refer to these cells as DLECs (30). Previous experiments have demonstrated that epithelial cells cultured in this manner transport Na+ via amiloride-sensitive and -insensitive mechanisms (28). These cells possess amiloride-sensitive whole cell Na+ currents but no detectable Cl- currents (43) and have amiloride-sensitive nonselective cation (NSC) and Na+-selective channels on their apical membranes (31, 42).

The harvested epithelial cells were immediately seeded (1 × 106 cells/cm2) onto Transwell tissue culture-treated porous polycarbonate filters (total surface area 4.7 cm2; Costar, Cambridge, MA). All cells were grown in MEM with 10% fetal bovine serum and penicillin-streptomycin at 37°C in a humidified 95% air-5% CO2 environment. Nonadherent epithelial cells were removed 24 h after they were seeded. Epithelial monolayers were subsequently studied 3 or 4 days after they were seeded. Cells used in patch-clamp experiments were seeded (5 × 105 cells/cm2) onto translucent porous Nunc filter inserts (Whatman Scientific) and were confluent when studied 4 days later.

Epithelial Cell Monolayer Bioelectric Properties

The bioelectric properties of epithelial cell monolayers were studied by placing the filters in Ussing chambers (MRA International, Clearwater, FL) that contained warmed HBSS and 22.4 mM NaHCO3 that was circulated with an air lift of a 95% air-5% CO2 gas mixture (7, 30). Measurements of the bioelectric properties of the monolayer were made with KCl agar-calomel half-cells and silver-silver chloride electrode-saline agar bridges that were connected to a high-impedance millivoltmeter that could function as a voltage-current clamp with automatic fluid-resistance compensation (VCC 600 Physiologic Instruments, San Diego, CA). The transepithelial monolayer potential difference (PD) was recorded continuously under open-circuit conditions with a linear-chart recorder (Linear Recorder 585, Baxter, Toronto, Canada). Every 10 s, a 0.5-s duration 1-µA pulse of current was delivered across the monolayer so that the measured change in PD enabled the calculation of resistance (R) using Ohm's law. Transepithelial R is a sensitive measurement of epithelial monolayer permeability to ions and in large part reflects the barrier function of epithelial tight junctions. Every 15 min, the transepithelial PD was temporarily clamped to 0 mV so that Isc could be recorded. Isc represents the net movement of positive charge from the apical to the basolateral side of the epithelial membrane, with the basolateral side of the monolayer having a positive PD relative to the apical side of the monolayer.

Four groups of epithelial cell monolayers were studied to determine the effect of alveolar Mphi s and/or LPS on monolayer permeability (R) and ion transport (Isc). The apical side of confluent epithelial monolayers was exposed to control medium, LPS alone (10 µg/ml), Mphi s alone (1-6 × 105 cells/cm2), or both LPS (10 µg/ml) and Mphi s (1-6 × 105 cells/cm2) for varying times. In some studies, the NO donor SNAP (0.1 mM) was added to the monolayers every 4 h for four doses before evaluation of the bioelectric properties. Monolayers were then rinsed with MEM and placed in Ussing chambers containing freshly prepared HBSS. After baseline bioelectric properties were determined, the Na+-transport blocker amiloride (0.1 mM apically), the Na+-K+-2Cl- cotransport inhibitor bumetanide (0.1 mM basally), and the NSC inhibitor flufenamic acid (0.45 mM apically) were added sequentially to the monolayer, thus enabling the calculation of amiloride-sensitive current and nonselective current as a percentage of the total current in all experimental groups. The amiloride-sensitive Isc likely reflects blockade of Na+ channels because our laboratory has previously shown that amiloride blocks 12- and 25-pS Na+-permeant channels (31, 42) and whole cell cation conductances (43) in the apical membrane of these epithelial cells. This amiloride dose was chosen based on previous dose-response studies performed on similar cells (28). Furthermore, dimethylamiloride, an amiloride analog with high potency for the Na+/H+ antiport, does not affect in vivo lung water clearance (28), DLEC Isc (30) or whole cell Na+ currents (43).

In some studies, alveolar Mphi s were incubated in coculture but were physically separated from the DLEC monolayer. To accomplish this, alveolar Mphi s (2 × 106) were added to the bottom of the Ussing chambers and allowed to adhere for 2 h before insertion of the filters coated with the epithelial cell monolayers into the Ussing apparatus. This coculture setup was incubated for a further 16-24 h in the presence or absence of LPS, at which time the bioelectric properties were investigated.

iNOS Expression in Alveolar Mphi s and DLECs

To evaluate separately iNOS expression in alveolar Mphi s and DLECs, a coculture system in which these cells were physically separated was established. A petri dish (60 mm; Falcon) was secured with silicone in the center of a larger petri dish (150 mm). Epithelial cells were seeded and grown to confluence over a 3-day period in the outer well. At this time, alveolar Mphi s (1.0 × 107) were added to the central well and allowed to adhere for 2 h at 37°C before being washed to remove nonadherent cells. The level of the culture medium was then raised so that the inner and outer wells were bathing within the same medium. The double-well system was slowly agitated on a plate shaker, gently enough to prevent Mphi s from detaching and reaching the outer well. DLECs were studied with or without Mphi s in the center well in the presence or absence of LPS. At various time points, the cells were separately recovered and extracted for use in either Northern or Western blot analysis.

Northern blot analysis. Total RNA was extracted with the method of Chomczynski and Sacchi (4). Briefly, cells were washed with cold Ca2+- and Mg2+-free HBSS and lysed with guanidium thiocyanate. The cell lysate was then recovered from the dish with a sterile cell scraper and transferred to Eppendorf tubes. After RNA extraction and spectrophotometric quantitation, 10 µg of total RNA were electrophoresed in a 1.2% agarose gel containing formaldehyde, blot transferred to a nylon membrane, and then ultraviolet cross-linked. Membranes were hybridized with the 32P random-labeled cDNA probe for the murine Mphi iNOS (kindly provided by Dr. Dennis Stuehr, Cleveland Clinic, OH; see Ref. 10), washed at room temperature in 1× sodium chloride-sodium phosphate-EDTA and 0.1% sodium dodecyl sulfate for 30 min, and exposed overnight to Kodak film at -70°C. Comparable RNA loading between lanes was assessed by probing with a cDNA probe for rat alpha -tubulin.

Western blot analysis. Alveolar Mphi s and DLECs were recovered separately for Western blot analysis by lysing cells in the inner and outer wells, respectively, with boiling Laemmli buffer (22). Cell lysates (50 µg for DLECs and 5 µg for Mphi s) were separated with 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to nitrocellulose with the Bio-Rad Mini Trans-Blot system. The blot was blocked with 5% nonfat milk (Bio-Rad) in tris(hydroxymethyl)aminomethane-buffered saline for 30 min at room temperature and then exposed for 1 h while being shaken at room temperature to a 1:2,500 dilution of a rabbit polyclonal antibody generated against a synthetic peptide derived from the COOH-terminal sequence of the mouse Mphi iNOS protein (kindly provided by Dr. Carl Nathan, Cornell University Medical College, New York, NY). The blot was then washed three times with antibody buffer solution and incubated with a 1:25,000 dilution of goat anti-rabbit immunoglobulin G conjugated to horseradish peroxidase. The blots were washed, dried, and quantitated with an enhanced chemiluminescence detection system (ECL, Amersham).

Nitrite Analysis

The nitrite content of the cell-free supernatants was measured by reacting samples (100 µl) with the Griess reagent (100 µl) for 10 min at 37°C according to established methods (12). Absorbance at 540 nm was then measured, and nitrite concentrations were calculated from a linear standard curve generated between 0 and 128 mM sodium nitrite.

Detection of Nitrotyrosine Residues

Cells were cultured on glass coverslips in six-well tissue culture dishes. Coverslips were then washed three times with ice-cold PBS and fixed in 100% methanol (-20°C for 10 min). Coverslips were blocked in 5% donkey serum-PBS for 2 h at room temperature, then incubated with primary antibody overnight at 4°C (titer of 1:100 diluted in 1% bovine serum albumin-PBS). To demonstrate the specificity of immunofluorescence, the antibodies were coincubated with the antigenic fusion protein (1 mg fusion protein/mg antibody) in a volume of 100 µl of antibody buffer (1% bovine serum albumin-PBS) overnight at 4°C on a rotating shaker. Coverslips were then washed three times in ice-cold PBS, and secondary antibodies were applied (6 µg/ml of fluorescence-labeled anti-rabbit immunoglobulin G) for 2 h at room temperature. The cells were washed and mounted for confocal-microscopic visualization with Slow Fade (Molecular Probes, Eugene, OR).

Statistics

The results are means ± SE of n experiments unless otherwise indicated. The statistical significance of the differences between the means of multiple groups or the means of an individual group at multiple time points was determined by one-way analysis of variance followed by Newman-Keuls multiple intergroup comparisons. Student's unpaired two-tailed t-test was used to assess significance between two groups. P < 0.05 was considered statistically significant.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of Mphi Number on the Bioelectric Properties of the Epithelium

In a previous study, Compeau et al. (7) demonstrated that treatment of a Mphi -DLEC coculture system with LPS significantly reduced both Isc and R of the DLEC monolayers. To study Isc independent of alterations in monolayer R, we performed initial studies in which we varied the number of Mphi s in the coculture. As reported, high numbers of Mphi s (3 × 106/filter) significantly reduced Isc compared with control monolayers or monolayers exposed to LPS or Mphi s in the absence of LPS (Fig. 1A). By contrast, lesser numbers of Mphi s (0.5-2.0 × 106/filter) stimulated with LPS had no effect on total Isc. Similarly, LPS-stimulated Mphi s at concentrations <=  2 × 106/filter did not alter baseline resistance of the DLEC monolayers (Fig. 1B). These studies thus established conditions in which we were able to study the effect of stimulated Mphi s on DLEC monolayer Isc independent of an alteration in monolayer R.


View larger version (14K):
[in this window]
[in a new window]
 


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1.   Effect of varying macrophage (Mphi ) numbers on distal lung epithelial cell (DLEC) short-circuit current (Isc; A) and baseline resistance (B). DLECs were cultured alone (Control), with lipopolysaccharide (LPS; 10 µg/ml), with Mphi s (3 × 106), or with LPS+Mphi s at varying numbers for 16 h and were then evaluated for bioelectric properties. Mphi s were cocultured either in direct contact (C) or physically separated (S) during incubation period. Data are means ± SE of 12-43 studies/group. * P < 0.001 vs. all other groups.

Because our previous studies had demonstrated that LPS-stimulated Mphi s induced a reduction in amiloride-sensitive Isc several hours before having an effect on total Isc, we postulated that the lower numbers of Mphi s might exert a selective effect on amiloride-sensitive Isc. To test this possibility, amiloride was added after the measurements had stabilized to determine the amiloride-sensitive component of the Isc. O'Brodovich and colleagues (28, 30) and others (3, 34) have previously demonstrated that amiloride decreases Na+ transport without affecting total transepithelial R. Although total Isc did not differ among groups, DLEC monolayers incubated with LPS-treated Mphi s (2 × 106/filter) demonstrated a marked reduction in the amiloride-sensitive component of the Isc compared with all other groups (Fig. 2A, Table 1). A time course of this effect demonstrated that some degree of inhibition occurred by 4 h and then was progressive over the next several hours (Fig. 2B). By 16 h, DLECs exposed to LPS-treated Mphi s exhibited essentially no measureable amiloride-sensitive Isc. This effect was present whether Mphi s were in direct contact with the DLECs (contact) or were physically separated from the DLEC monolayer. To investigate the source of the amiloride-insensitive Isc, monolayers were treated with flufenamic acid (0.45 mM) and bumetanide (0.1 mM). As shown in Table 1, DLECs exposed to LPS-treated Mphi s, either in direct contact or physically separated, exhibited a significant increase in the flufenamic acid-sensitive component. Bumetanide-sensitive Isc remained low (<1%) in all groups. Considered together, these data show that soluble products derived from LPS-treated Mphi s are able to induce significant qualitative and quantitative alterations in DLEC ion transport. Specifically, they are able to virtually obliterate the amiloride-sensitive Isc and increase the amount of flufenamic acid-sensitive Isc while having no effect on bumetanide-sensitive Isc, total Isc, or monolayer R.


View larger version (12K):
[in this window]
[in a new window]
 


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 2.   A: effect of Mphi s and LPS on amiloride-sensitive Isc. DLECs were cultured alone (Control), with LPS (10 µg/ml), with Mphi s (2 × 106), or with LPS+Mphi s (2 × 106) for 16 h and were then evaluated for bioelectric properties. Mphi s were cocultured either in direct contact or physically separated during incubation period. Open bars, before amiloride; solid bars, after amiloride. Data are means ± SE of 12-43 studies/group. * P < 0.001 vs. after-amiloride groups for Control, LPS alone, and Mphi s alone. B: time course of effect of LPS-stimulated Mphi s on amiloride-sensitive (Amil Sens) Isc. DLECs were cultured alone (control; hatched bars), with Mphi s (2 × 106; solid bars), or with LPS+Mphi s (2 × 106; open bars) for 2-6 h and were then evaluated for bioelectric properties. Data are expressed as percent amiloride-sensitive Isc and are means ± SE of 4 studies/group at each time point. * P < 0.01 vs. control at indicated time point.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effect of Mphi s on short-circuit current in DLECs

Induction of DLEC iNOS Expression by Stimulated Mphi s

Figure 3 demonstrates the ability of L-NMMA to prevent the loss of amiloride-sensitive Isc caused by LPS-treated Mphi s in coculture as previously reported (7). L-NMMA also reversed the flufenamic acid-inhibitable component back toward control levels [18.0 ± 2.1% (SE); n = 8]. The ability of Mphi products to alter amiloride-sensitive Isc in DLEC monolayers combined with our previous studies indicating a role for NO in this process suggested the possibility that soluble Mphi factors might upregulate iNOS expression in DLECs. To test this hypothesis, Mphi s and DLECs were cultured physically separated but bathing within the same medium in the presence or absence of LPS (see MATERIALS AND METHODS). At various time points, Mphi s and DLECs were separately recovered and analyzed for iNOS mRNA and protein expression. Figure 4A demonstrates the level of iNOS mRNA at 16 h in DLEC monolayers cultured in the presence or absence of Mphi s with or without LPS treatment. iNOS mRNA was detected in DLEC monolayers exposed to both Mphi s and LPS but not in any of the other groups. The time course of this induction is shown in Fig. 4B. iNOS expression in DLECs cultured in the presence of Mphi s and LPS was evident by 2 h, reached a maximum at 6 h, and began to fall by 12 h. LPS-treated Mphi s recovered from the same study showed clear evidence of iNOS mRNA expression at 2 h, and this increased steadily over the 12-h time course studied (Fig. 4C).


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of NG-monomethyl-L-arginine (L-NMMA) on amiloride-sensitive component of Isc in DLECs. DLECs were cultured alone (Control) and with LPS (10 µg/ml)+Mphi s (2 × 106) with or without L-NMMA for 16 h and were then evaluated for bioelectric properties. A: Isc expressed as percent change (%Delta ) in amiloride-sensitive Isc in each treatment group. B: resistance. Data are means ± SE of 10 studies/group. L-NMMA alone had no effect on total Isc or amiloride-sensitive component of Isc in control DLECs (not shown). * P < 0.001 vs. Control. + P < 0.001 vs. LPS+Mphi +L-NMMA.


View larger version (30K):
[in this window]
[in a new window]
 


View larger version (78K):
[in this window]
[in a new window]
 


View larger version (60K):
[in this window]
[in a new window]
 
Fig. 4.   Inducible nitric oxide synthase (iNOS) gene expression in DLECs. A: level of iNOS mRNA expression in DLECs cultured in presence (+) or absence (-) of Mphi s with or without LPS treatment. In DLE-Mphi cocultures, cells were physically separated to permit recovery of DLECs alone. At 16 h, DLECs were recovered and subjected to Northern analysis for iNOS as described in MATERIALS AND METHODS. Blots were stripped and reprobed with cDNA probe for alpha -tubulin. Data are representative of 3 separate studies. B: time course of induction of iNOS mRNA in control DLECs or DLECs incubated in presence of LPS and Mphi . In DLEC-Mphi cocultures, cells were physically separated to permit recovery of DLECs alone. Data are representative of 3 separate studies. C: time course of induction of iNOS mRNA in Mphi s exposed to LPS. Mphi s were treated with LPS for the indicated times and recovered for Northern blot analysis as described in MATERIALS AND METHODS. Data are representative of 3 separate studies.

The time course of iNOS protein expression is illustrated in Fig. 5. No detectable iNOS protein was seen in control DLECs over the 10-h study period. By contrast, epithelial cells cultured in the presence of Mphi s and LPS demonstrated a small amount of iNOS protein by 4 h, which increased to maximum values between 6 and 10 h. At 6 h, Mphi s recovered from the same study expressed a large amount of iNOS protein. The induction of iNOS protein activity was reflected in increased nitrite release by the Griess reaction. To detect nitrite produced by DLECs, two approaches were taken. First, we generated Mphi supernatants by treating cells with LPS or vehicle overnight and then added this cocktail to DLECs. The cocktail was washed away after 6 h, the DLECs were thoroughly washed, and then the DLECs were further incubated in fresh medium for 16 h. Nitrite levels in the supernatant were then measured. The nitrite level increased from undetectable in studies in which the cocktails were derived from unstimulated Mphi s to 14.6 ± 3.9 µM (n = 6 experiments) in studies in which the cocktails were from LPS-treated Mphi s. Second, Mphi s were cocultured physically separated from DLECs but bathing in the same medium in the presence or absence of LPS for 6 h. The cells were then washed, and the production of nitrite was detected in DLECs cultured separately in fresh medium for 16 h. Exposure of DLECs to LPS-stimulated Mphi s increased nitrite release to 71.6 ± 4.2 µM (n = 6 experiments). By contrast, LPS-treated Mphi s (3 × 106) released 204.6 ± 3.0 µM (n = 3 experiments). LPS-treated DLECs in the absence of Mphi s repeatedly released small concentrations of nitrites (<5 µM). These data thus support the iNOS gene and protein expression studies, demonstrating that iNOS activity is less in DLECs than in Mphi s.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5.   Time course of induction of iNOS protein in control (CTRL) DLECs or DLECs incubated in presence of LPS and Mphi s. In DLEC-Mphi cocultures, cells were physically separated to permit recovery of DLECs alone. At the indicated times, DLECs were recovered and subjected to Western blot analysis with rabbit anti-iNOS antibody as described in MATERIALS AND METHODS. Mphi s stimulated with LPS for 6 h are shown in the far right lane for comparison. Protein loading was 50 µg in DLEC lanes and 5 µg in Mphi lane. Data are representative of 3 studies.

Role of NO as a Mediator of Altered Bioelectric Properties in DLECs

After iNOS expression in LPS-stimulated DLECs was demonstrated, studies were performed to discern the role of NO as the effector molecule of the observed changes in amiloride-sensitive Isc. A previous study (7) showed that L-NMMA was able to prevent Mphi -mediated inhibition of DLEC Isc, suggesting that NO was involved in the inhibitory effect. To determine whether NO generation was sufficient to account for the reduction in amiloride-sensitive Isc, DLEC monolayers cultured on porous substrata were treated with the NO donor SNAP and evaluated for alterations in bioelectric properties. SNAP (0.1 mM) had no effect on amiloride-sensitive Isc, whereas higher concentrations of SNAP (1 mM) reduced baseline R to <50 Omega  · cm2. At the higher concentration of SNAP, nitrite release by DLECs approximated that generated by DLECs exposed to LPS-treated Mphi s. Combined with the findings from using L-NMMA (Fig. 3A), these data suggest that NO is necessary but not sufficient to account for the changes in amiloride-sensitive Isc seen in DLECs after exposure to LPS-treated Mphi s.

A recent study (17) has implicated peroxynitrite as playing an important role in NO-mediated effects on target cells including DLECs. To discern the generation of peroxynitrite in DLECs when cultured with LPS-stimulated Mphi s, cocultures were blotted with anti-nitrotyrosine antibodies and studied by confocal microscopy. As shown in Fig. 6, nitrotyrosine residues were detected in Mphi s in LPS-treated Mphi -DLEC cocultures but not in DLECs. Specificity was confirmed by the lack of fluorescence when the primary antibody was omitted (Fig. 6C) or competed with peptide (Fig. 6D).


View larger version (69K):
[in this window]
[in a new window]
 
Fig. 6.   Nitrotyrosine residues in Mphi -DLEC cocultures. DLEC+Mphi s were cultured without (A) or with (B) LPS on glass coverslips in 6-well tissue culture dishes for 16 h. Coverslips were then washed 3 times with ice-cold phosphate-buffered saline and fixed in 100% methanol (-20°C for 10 min). Coverslips were blocked in 5% donkey serum-phosphate-buffered saline for 2 h at room temperature, then incubated with primary antibody (Ab). Coverslips were then washed 3 times in ice-cold phosphate-buffered saline, and secondary antibodies were applied (6 µg/ml fluorescence-labeled anti-rabbit immunoglobulin G) for 2 h at room temperature. To demonstrate specificity of immunofluorescence, primary antibody was omitted (C) or antibodies were preincubated with antigenic fusion protein (D). Cells were washed and mounted for confocal-microscopic visualization.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Resolution of pulmonary edema after acute lung injury is dependent on the successful treatment of the primary process responsible for fluid leakage into the air space coupled with efficient fluid reabsorption from the alveolar space. Fluid transport out of the alveolar space is largely mediated by epithelial cell vectorial transport of Na+ from the apical to the basolateral surface (reviewed in Ref. 27). The clinical importance of this epithelial Na+ transport mechanism is illustrated by data showing that survival from either high pressure (congestive heart failure) or high permeability (ARDS) correlated with the ability of the lungs to concentrate its air space fluid (26), presumably via the active absorption of Na+ and Cl-, with water following. Such a speculation was supported by a recent report (38) that demonstrated that the distal lung regions of the human lung also absorb fluid by active Na+ transport. In the present study, we have utilized an in vitro model to investigate cellular interactions that might contribute to reduced Na+ transport by DLECs, leading to impaired recovery from pulmonary edema. We originally chose to study Mphi s because of data showing their potential role in the pathogenesis of lung injury during the early stages before neutrophil influx. In addition, their ability to release a diverse range of inflammatory mediator molecules coupled with their close apposition to DLECs in vivo suggested the possibility that they might be capable of modulating DLEC function. Using a Mphi -DLEC coculture system, we demonstrated that Mphi products released in response to LPS are able to induce iNOS gene and protein expression in DLECs and stimulate iNOS activity in these cells. The NO generated in this system is necessary but not sufficient to cause the observed reduction in the amiloride-sensitive Isc and the increase in the flufenamic acid-sensitive component of total Isc in DLECs. This occurred without affecting total Isc or the R of the monolayer. These observations suggest a mechanism whereby NO produced locally in the lung might interact with other proinflammatory molecules to exert effects that impair Na+ transport during acute lung inflammation.

Increased iNOS expression with enhanced local production of NO in the lung has been demonstrated after systemic LPS administration in rats (21, 25, 41, 46). Several possible cellular sources for NO generation in this setting have been suggested, including both interstitial and alveolar Mphi s, DLECs, bronchial epithelial cells, fibroblasts, and pulmonary vascular smooth muscle cells. The present study confirms the ability of Mphi s and DLECs to express iNOS in response to appropriate stimuli. Importantly, they provide a physiological model whereby cells might interact to augment NO production. Specifically, although LPS alone was unable to induce iNOS in DLECs, coculture with Mphi s in the presence of LPS resulted in iNOS expression. This occurred when Mphi s and DLECs were physically separated to allow evaluation of the cell source of iNOS. These data suggest that Mphi s products released in response to LPS treatment induced iNOS expression in the DLECs. This is supported by the temporal delay in iNOS expression by the DLECs, presumably the time required for LPS to stimulate the synthesis and release of Mphi products into the medium. The nature of these products was not evaluated in the present study. However, other investigators have defined various cytokines that either alone or in combination induce DLEC iNOS expression. These include interleukin-1beta , tumor necrosis factor-alpha , and interferon-gamma (13, 33, 35, 36). A recent study by Gutierrez et al. (13) reported that LPS alone is capable of stimulating a modest amount of NO release from DLECs. However, this effect was observed at a later time point (>24 h) than that evaluated in the present study. In relative terms, Mphi s appear to be much more potent generators of NO than DLECs in the in vitro setting. In the context of the in vivo setting, the close apposition of Mphi s to DLECs in vivo would readily permit Mphi -derived NO as well as endogenous production by DLECs to contribute to NO-mediated events occurring in the DLECs.

Although NO appears to be necessary for the reduction in amiloride-sensitive Isc in DLEC monolayers, the data suggest that it must act in concert with other cellular products to exert its effects. One possible candidate is peroxynitrite. Peroxynitrite is generated through a chemical reaction of NO with superoxide anion (18). In separate studies, this molecular species was shown to be secreted by activated Mphi s into the epithelial cell lining fluid (19) and also to inhibit amiloride-sensitive 22Na+ uptake in alveolar type II cells (17). The half-life of this molecule is <1 s, making it unlikely that Mphi -derived peroxynitrite is the active species when the cells are physically separated in the in vitro separate-culture model. However, this does not preclude an effect in vivo where the cells are closely apposed. In this regard, the presence of nitrotyrosine residues has been reported in histological sections of lung tissue with ARDS due to sepsis (14). Localization along the blood-gas barrier is consistent with an effect of peroxynitrite on DLECs (14). In the present study, we were unable to detect nitrotyrosine residues in DLECs as evidence of their exposure to peroxynitrite. Although this suggests that peroxynitrite was not involved, we cannot totally rule out the possibility of an inadequate sensitivity of the immunohistochemistry in the DLECs. Alternatively, S-nitrosothiols produced by the interaction of NO with thiols may mediate the effect of NO on DLECs. S-nitrosoglutathione, the predominant form found in alveolar lining fluid, has a prolonged half-life (~3 h) and bioactivity that includes bronchodilation, inhibition of receptor-ligand interaction, and inhibition of enzyme function (11). Importantly, S-nitrosoglutathione levels were found to be markedly increased in the lavage fluid of patients with pneumonia, suggesting a possible role for this product in vivo (11).

The mechanism whereby NO and its cofactor might exert their effects on DLECs requires further study. Many of the effects of NO and its by-products are known to be mediated via a guanosine 3',5'-cyclic monophosphate (cGMP)-dependent pathway. There are well-characterized NSC channels in epithelial cells of the rod retina (8) and inner medulla collecting duct of the kidney (24) that are respectively upregulated and downregulated by cGMP. Studies from our group demonstrated that neither atrial natriuretic peptide (the second messenger of which is cGMP) nor 8-bromo-cGMP (a membrane-permeant analog of cGMP) altered the bioelectric properties of DLECs within 30 min of exposure (29). However, our present and previous work has demonstrated that the LPS-treated Mphi effect requires several hours of incubation. This longer time-dependent effect may indicate additional mechanisms, possibly related to cGMP generation. Another possible explanation relates to the recent report by Rotin et al. (37) indicating that cytoskeleton-Na+-channel interactions determine the apical localization of the alpha -subunit of the epithelial cell Na+ channel in DLECs. Coupled with a previous study by Compeau et al. (7) demonstrating that LPS-treated Mphi s induced cytoskeletal changes in DLECs, these data suggest an effect related to altered traffic to or retention of Na+ channels at the apical membrane. In this regard, S-nitrosoglutathione has been shown to stimulate ADP ribosylation of F-actin in neutrophils (5). Finally, NO or its metabolites may modulate both transcriptional and posttranscriptional events in the cell (16, 44), suggesting a possible effect on expression of the Na+ channel itself or of its regulatory proteins.

The Ussing chamber studies demonstrated that the ion-transport characteristics of the DLECs were markedly changed as a result of exposure to LPS-stimulated Mphi s; there was a virtual disappearance of amiloride-sensitive Isc with a concomitant increase in its flufenamic acid sensitivity. Normally, ~70% of the Isc of the DLEC monolayer is amiloride and benzamil sensitive (28-30). It has been previously demonstrated that the amount of amiloride-sensitive Isc is increased when DLECs are cultured in serum-free medium (29), whereas it is decreased when DLECs are cultured from more immature fetal rat lungs (34) or when DLECs are grown on an immature fetal lung cell-derived matrix (32). Exposure of rats to sublethal hyperoxia has also been shown to upregulate non-amiloride-inhibitable Isc (15, 47). The ionic nature of the amiloride-insensitive Isc is not completely understood. The weight of evidence suggests that the amiloride-insensitive Isc is Na+ transport. Previous studies have demonstrated that the Isc of the DLECs is entirely dependent on the presence of Na+ in the bathing medium (28), that amiloride-insensitive 22Na+ transport is present in tracheal epithelium (23), and that flufenamic acid, which inhibits NSC channels in colonic epithelium (39), markedly decreased the Isc in LPS-Mphi -exposed DLEC monolayers. In addition, as previously described (28, 34), the classic inhibitor of Cl- secretion, bumetanide, had no influence on DLEC Isc. However, Pitkanen et al. (32) have noted that Cl- depletion has a modest effect on the DLEC Isc, and flufenamic acid has also been reported to block Ca2+-activated Cl- channels in Xenopus laevis oocytes (45). Therefore, one cannot rule out the possibility that some Cl- secretion is present in the DLECs but that the basolateral entry pathway for Cl- is via an Na+-dependent bumetanide-insensitive transporter.

In summary, the present study demonstrates that cell-cell interactions in the lung during inflammation may impair fluid resorption and thus resolution of pulmonary edema. The central role for NO generation in this process suggests alternative approaches to the prevention and treatment of lung injury.

    ACKNOWLEDGEMENTS

We acknowledge the contributions of Jeffrey R. Weidner and Richard A. Mumford (Merck Research Laboratory, Rathway, NJ), who were responsible for the preparation of the synthetic peptide used to develop the anti-inducible nitric oxide synthase rabbit serum. Anti-nitrotyrosine antibodies were kindly provided by Dr. H. Ischiropoulos of the University of Pennsylvania (Philadelphia).

    FOOTNOTES

This work was supported by the Medical Research Council (MRC) of Canada Group in the Mechanisms of Organ Injury, the MRC Group in Lung Development, and the Physicians' Services Incorporated Foundation.

H. O'Brodovich is a career scientist of the Heart and Stroke Foundation of Ontario (Canada).

Address for reprint requests: O. D. Rotstein, Toronto Hospital, 200 Elizabeth St. EN9-232, Toronto, Ontario, Canada M5G 2C4.

Received 21 February 1996; accepted in final form 3 December 1997.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Berthiaume, Y., V. C. Broaddus, M. A. Gropper, T. Tanita, and M. A. Matthay. Alveolar liquid and protein clearance from normal dog lungs. J. Appl. Physiol. 65: 585-593, 1988[Abstract/Free Full Text].

2.   Berthiaume, Y., N. C. Staub, and M. A. Matthay. Beta-adrenergic agonists increase lung liquid clearance in anesthetized sheep. J. Clin. Invest. 79: 335-343, 1987[Medline].

3.   Cheek, J. M., K. Kim, and E. D. Crandall. Tight monolayers of rat alveolar epithelial cells: bioelectric properties and active sodium transport. Am. J. Physiol. 256 (Cell Physiol. 25): C688-C693, 1989[Abstract/Free Full Text].

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

5.   Clancy, R. M., J. Leszcynska-Piziak, and S. B. Abramson. Nitric oxide stimulates the ADP-ribosylation of actin in human neutrophils. Biochem. Biophys. Res. Commun. 191: 847-852, 1993[Medline].

6.   Compeau, C. G., J. Ma, K. N. Decampos, T. K. Waddell, G. F. Brisseau, A. S. Slutsky, and O. D. Rotstein. In situ ischemia and hypoxia enhance alveolar macrophage tissue factor expression. Am. J. Respir. Cell Mol. Biol. 11: 446-455, 1994[Abstract].

7.   Compeau, C. G., O. D. Rotstein, H. Tohda, Y. Marunaka, B. Rafii, A. S. Slutsky, and H. M. O'Brodovich. Endotoxin-stimulated rat alveolar macrophages impair distal lung epithelial sodium transport by an L-Arg-dependent mechanism. Am. J. Physiol. 266 (Cell Physiol. 35): C1330-C1341, 1994[Abstract/Free Full Text].

8.   Cook, N. J., W. Hanke, and U. B. Kaupp. Identification, purification, and functional reconstitution of the cyclic GMP-dependent channel from rod photoreceptors. Proc. Natl. Acad. Sci. USA 84: 585-589, 1987[Abstract].

9.   Crandall, E. D., T. A. Heming, R. L. Palombo, and B. E. Goodman. Effects of terbutaline on sodium transport in isolated perfused rat lung. J. Appl. Physiol. 60: 289-294, 1986[Abstract/Free Full Text].

10.   Deng, W., B. Thiel, C. S. Tannenbaum, T. A. Hamilton, and D. J. Stuehr. Synergistic cooperation between T cell lymphokines for induction of the nitric oxide synthase gene in murine peritoneal macrophages. J. Immunol. 151: 322-329, 1993[Abstract/Free Full Text].

11.   Gaston, B., J. Reilly, J. M. Drazen, J. Fackler, P. Ramdev, D. Arnelle, M. E. Mullins, D. J. Sugerbaker, C. Chee, D. J. Singel, J. Loscalzo, and J. S. Stamler. Endogenous nitrogen oxides and bronchodilator S-nitrosothiols in human airways. Proc. Natl. Acad. Sci. USA 90: 10957-10961, 1993[Abstract].

12.   Green, L. C., D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok, and S. R. Tannenbaum. Analysis of nitrite, nitrate, and [15N]-nitrate in biological fluids. Anal. Biochem. 126: 131-138, 1982[Medline].

13.   Gutierrez, H. H., B. R. Pitt, M. Schwarz, S. C. Watkins, C. Lowenstein, I. Caniggia, P. Chumley, and B. A. Freeman. Pulmonary alveolar epithelial inducible NO synthase gene expression: regulation by inflammatory mediators. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L501-L508, 1995[Abstract/Free Full Text].

14.   Haddad, I. Y., G. Pataki, C. Gallini, J. S. Beckman, and S. Matalon. Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury. J. Clin. Invest. 94: 2407-2413, 1994[Medline].

15.   Haskell, J. F., G. Yue, D. J. Benos, and S. Matalon. Upregulation of sodium conductive pathways in alveolar type II cells in sublethal hyperoxia. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L30-L37, 1994[Abstract/Free Full Text].

16.   Henderson, S. A., P. H. Lee, E. E. Aeberhard, J. W. Adams, L. J. Ignarro, W. J. Murphy, and M. P. Sherman. Nitric oxide reduces early growth response-1 gene expression in rat lung macrophages treated with interferon gamma and lipopolysaccharide. J. Biol. Chem. 269: 25239-25242, 1994[Abstract/Free Full Text].

17.   Hu, P., H. Ischiropoulos, J. S. Beckman, and S. Matalon. Peroxynitrite inhibition of oxygen consumption and sodium transport in alveolar type II cells. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L628-L634, 1994[Abstract/Free Full Text].

18.   Huie, R. E., and S. Padmaja. Reaction of nitric oxide with superoxide. Free Radic. Res. Commun. 18: 195-199, 1993[Medline].

19.   Ischiropoulos, H., L. Zhu, and J. S. Beckman. Peroxynitrite formation from macrophage-derived nitric oxide. Arch. Biochem. Biophys. 298: 446-451, 1992[Medline].

20.   Jayr, C., C. Garat, M. Meignan, J. F. Pittet, M. Zelter, and M. A. Matthay. Alveolar liquid and protein clearance in anesthetized ventilated rats. J. Appl. Physiol. 76: 2636-2642, 1994[Abstract/Free Full Text].

21.   Knowles, R. G., M. Merrett, M. Salter, and S. Moncada. Differential induction of brain, lung, and liver nitric oxide synthase by endotoxin in the rat. Biochem. J. 270: 833-836, 1990[Medline].

22.   Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[Medline].

23.   Langridge-Smith, J. E. Interaction between sodium and chloride transport in bovine tracheal epithelium. J. Physiol. (Lond.) 376: 299-319, 1986[Abstract].

24.   Light, D. B., E. M. Schwiebert, K. H. Karlson, and B. A. Stanton. Atrial natriuretic peptide inhibits cation channel in renal inner medullary collecting duct cells. Science 243: 383-385, 1989[Medline].

25.   Liu, S., I. M. Adcock, R. W. Old, P. J. Barnes, and T. W. Evans. Lipopolysaccharide treatment in vivo induces widespread tissue expression of inducible nitric oxide synthase mRNA+. Biochem. Biophys. Res. Commun. 196: 1208-1213, 1993[Medline].

26.   Matthay, M. A., and J. P. Wiener-Kronish. Intact epithelial barrier function is critical for the resolution of alveolar edema in humans. Am. Rev. Respir. Dis. 142: 1250-1257, 1990[Medline].

27.   O'Brodovich, H. M. The role of active Na+ transport by lung epithelium in the clearance of airspace fluid. New Horiz. 3: 240-247, 1995[Medline].

28.   O'Brodovich, H. M., V. Hannam, and B. Rafii. Sodium channel but neither Na+-H+ nor Na-glucose symport inhibitors slow neonatal lung water clearance. Am. J. Respir. Cell Mol. Biol. 5: 377-384, 1991[Medline].

29.   O'Brodovich, H. M., B. Rafii, and P. Perlon. Arginine vasopressin and atrial natriuretic peptide do not alter ion transport by cultured fetal distal lung epithelium. Pediatr. Res. 31: 318-322, 1992[Abstract].

30.   O'Brodovich, H., B. Rafii, and M. Post. Bioelectric properties of fetal alveolar epithelial monolayers. Am. J. Physiol. 258 (Lung Cell. Mol. Physiol. 2): L201-L206, 1990[Abstract/Free Full Text].

31.   Orser, B. A., M. Bertlik, L. Fedorko, and H. M. O'Brodovich. Non-selective cation channel in fetal alveolar type II epithelium. Biochim. Biophys. Acta 1094: 19-26, 1991[Medline].

32.   Pitkanen, O., A. K. Tanswell, and H. O'Brodovich. Fetal lung cell-derived matrix alters distal lung epithelial ion transport. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L762-L771, 1995[Abstract/Free Full Text].

33.   Punjabi, C. J., J. D. Laskin, K. J. Pendino, N. L. Goller, S. K. Durham, and D. L. Laskin. Production of nitric oxide by rat type II pneumocytes: increased expression of inducible nitric oxide synthase following inhalation of a pulmonary irritant. Am. J. Respir. Cell Mol. Biol. 11: 165-172, 1994[Abstract].

34.   Rao, A. K., and G. R. Cott. Ontogeny of ion transport across fetal pulmonary epithelial cells in monolayer culture. Am. J. Physiol. 261 (Lung Cell. Mol. Physiol. 5): L178-L187, 1991[Abstract/Free Full Text].

35.   Robbins, R. A., P. J. Barnes, D. R. Springall, J. B. Warren, O. J. Kwon, L. D. Buttery, A. J. Wilson, D. A. Geller, and J. M. Polak. Expression of inducible nitric oxide in human lung epithelial cells. Biochem. Biophys. Res. Commun. 203: 209-218, 1994[Medline].

36.   Robbins, R. A., D. R. Springall, J. B. Warren, O. J. Kwon, L. D. K. Buttery, A. J. Wilson, I. M. Adcock, V. Riveros-Moreno, S. Moncada, J. Polak, and P. J. Barnes. Inducible nitric oxide synthase is increased in murine lung epithelial cells by cytokine stimulation. Biochem. Biophys. Res. Commun. 198: 835-843, 1994[Medline].

37.   Rotin, D., D. Bar-Sagi, H. O'Brodovich, J. Merilainen, V. Lehto, C. M. Canessa, B. C. Rossier, and G. Downey. A SH3 binding region in the epithelial Na+ channel (arENaC) mediates its localization at the apical membrane. EMBO J. 13: 4440-4450, 1994[Abstract].

38.   Sakuma, T., G. Okaniwa, T. Nakada, T. Nishimura, S. Fujimura, and M. A. Matthay. Alveolar fluid clearance in the resected human lung. Am. J. Respir. Crit. Care Med. 150: 305-310, 1994[Abstract].

39.   Siemer, C., and H. Gogelein. Activation of nonselective cation channels in the basolateral membrane of rat distal colon crypt cells by prostaglandin E2. Pflügers Arch. 420: 319-328, 1992[Medline].

40.   Spragg, R. G., and R. M. Smith. Biology of acute lung injury. In: The Lung: Scientific Foundations, edited by R. G. Crystal, and J. B. West. New York: Raven, 1991, p. 2003-2017.

41.   Stewart, T. E., F. Valenza, S. P. Ribeiro, A. D. Wener, G. Volgyesi, B. M. Mullen, and A. S. Slutsky. Increased nitric oxide in exhaled gas as an early marker of lung inflammation in a model of sepsis. Am. J. Respir. Crit. Care Med. 151: 713-718, 1995[Abstract].

42.   Tohda, H., and Y. Marunaka. Insulin-activated amiloride-blockable nonselective cation and Na+ channels in fetal distal lung epithelium. Gen. Pharmacol. 26: 755-763, 1995[Medline].

43.   Wang, X., T. Kleyman, H. Tohda, Y. Marunaka, and H. M. O'Brodovich. EIPA sensitive Na+ currents in intact fetal distal lung epithelial cells. Can. J. Physiol. Pharmacol. 71: 58-62, 1993[Medline].

44.   Weiss, G., B. Goosen, W. Doppler, D. Fuchs, K. Pantopoulos, G. Werner-Felmayer, H. Wachter, and M. W. Hentze. Translational regulation via iron-responsive elements by nitric oxide/NO-synthase pathway. EMBO J. 12: 3651-3657, 1993[Abstract].

45.   White, M. M., and M. Aylwin. Niflumic and flufenamic acids are potent reversible blockers of Ca2+-activated Cl- channels in Xenopus oocytes. Mol. Pharmacol. 37: 720-724, 1990[Abstract].

46.   Wizemann, T. M., C. R. Gardner, J. D. Laskin, S. Quinones, S. K. Durham, N. L. Goller, S. T. Ohnishi, and D. L. Laskin. Production of nitric oxide and peroxynitrite in the lung during acute endotoxemia. J. Leukoc. Biol. 56: 759-768, 1994[Abstract].

47.   Yue, G., W. J. Russell, D. J. Benos, R. M. Jackson, M. A. Olman, and S. Matalon. Increased expression and activity of sodium channels in alveolar type II cells of hyperoxic rats. Proc. Natl. Acad. Sci. USA 92: 8418-8422, 1995[Abstract].


AJP Lung Cell Mol Physiol 274(3):L378-L387
1040-0605/98 $5.00 Copyright © 1998 the American Physiological Society