O2-induced ENaC expression is associated with NF-kappa B activation and blocked by superoxide scavenger

Bijan Rafii1,2, A. Keith Tanswell1,3, Gail Otulakowski1,2, Olli Pitkänen4, Rose Belcastro-Taylor1,3, and Hugh O'Brodovich1,2

1 Medical Research Council Group in Lung Development and Divisions of 2 Respiratory and 3 Neonatal Research, Hospital for Sick Children Research Institute and Department of Paediatrics, University of Toronto, Toronto, Ontario, Canada M5G 1X8; and 4 Hospital for Children and Adolescents, University of Helsinki, 00290 Helsinki, Finland

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Cultured rat fetal distal lung epithelial cells (FDLEs), when switched from fetal (3%) to postnatal (21%) O2 concentrations, have increased epithelial Na+ channel (ENaC) mRNA levels and amiloride-sensitive Na+ transport [O. Pitkänen, A. K. Tanswell, G. Downey, and H. O'Brodovich. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L1060-L1066, 1996]. The mechanisms by which O2 mediates these effects are unknown. After isolation, FDLEs were kept at 3% O2 overnight, then switched to 21% O2 (3-21% O2 group) or maintained at 3% O2 (3-3% O2 group) for 48 h. The amiloride-sensitive short-circuit current (Isc) in the 3-21% O2 group was double that in the 3-3% O2 group. Amiloride-sensitive Isc could not be induced by medium conditioned by 21% O2-exposed FDLEs but was reversed by returning the cells to 3% O2. Neither the cyclooxygenase inhibitor ibuprofen, liposome-encapsulated catalase, nor hydroperoxide scavengers (U-74389G or Trolox) blocked the O2-induced amiloride-sensitive Isc. In contrast, the cell-permeable superoxide scavenger tetramethylpiperidine-N-oxyl (TEMPO) eliminated the O2-induced increases in amiloride-sensitive Isc and ENaC mRNA levels. The switch from 3 to 21% O2 induced the transcription factor nuclear factor-kappa B, which could also be blocked by TEMPO. We conclude that 1) the O2-induced increase in amiloride-sensitive Isc is reversible and 2) the O2-induced increase in amiloride-sensitive Isc and ENaC mRNA levels is associated with activation of nuclear factor-kappa B and may be mediated, at least in part, by superoxide.

epithelial sodium channel; nuclear factor-kappa B; amiloride; type II epithelium; tetramethylpiperidine-N-oxyl; reactive oxygen species

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

BEFORE BIRTH, THE FETAL LUNG is filled with liquid that has been secreted by its epithelium. This liquid secretion results from the active transport of Cl- from the interstitium to the alveolar lumen, a phenomenon that is necessary for the normal development of the lung (1). At birth, however, fluid secretion must cease, and the lung liquid must be cleared before effective gas exchange can take place. The major component of this liquid clearance results from active epithelial Na+ transport from the lumen to the interstitium (5, 25-27). Indeed, a transgenic mouse deficient in the amiloride-sensitive epithelial Na+ channel (ENaC) fails to clear its fetal lung liquid and dies shortly after birth (17).

How the lung converts from a fluid-secreting to an absorbing organ is incompletely understood and has been the subject of many studies. There is general agreement that increases in the circulating levels of vasoactive compounds such as catecholamines (5) and arginine vasopressin (30) can initiate this conversion in mature lungs in utero. However, as soon as the levels of these vasoactive agents return to basal values, the fetal lung reverts to fluid secretion. The change in O2 concentration from the fetal (~3%) to the postnatal (21%) environment represents another potential signal for the perinatal lung. Others (3) have previously demonstrated that the ability of late-gestation fetal distal lung explants to form fluid-filled cysts is dependent on O2 concentration. Our laboratory (31) has also shown that when primary cultures of fetal distal lung epithelial cells (FDLEs) were switched from 3 to 21% O2, there was an increase in the amount of amiloride-sensitive short-circuit current (Isc) and mRNA coding for the alpha -, beta -, and gamma -subunits of ENaC. This effect was observed within 18 h of incubation of FDLEs in 21% O2. Although a 48-h incubation led to a further augmentation in amiloride-sensitive Isc, an 8-h incubation had no effect.

In the present study, we determined whether the induction of FDLE Na+ transport by postnatal O2 was permanent or reversible. In addition, we investigated possible mechanisms for the increased gene expression and functional activity of ENaC in FDLEs. This included experiments to determine whether the change in O2 concentration resulted in the release of a stable compound into the cell medium that could induce amiloride-sensitive Isc and whether the increase in amiloride-sensitive Isc could be prevented by various inhibitors, including blockers of prostaglandin production and the inducible form of nitric oxide synthase, or by scavengers of reactive oxygen species (ROS). Because our experiments demonstrated that the cell-permeable superoxide scavenger tetramethylpiperidine-N-oxyl (TEMPO) blocked the postnatal O2-mediated induction of ENaC mRNA expression, we performed additional experiments to determine whether the ROS-sensitive transcription factor nuclear factor (NF)-kappa B (for a review, see Ref. 14) was activated during exposure of our primary cultures of FDLEs to postnatal O2 concentrations.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Primary cell culture. FDLEs were isolated and cultured as previously described (28). In brief, 20-day-gestation Wistar rats (breeding day = day 0, term = 22 days; Charles River, St. Constant, PQ) were killed with an anesthetic overdose. Fetal lung tissue was dispersed with 0.125% (wt/vol) trypsin, and the resulting cell pellet was further incubated with 0.1% collagenase (wt/vol) to separate associated fibroblasts from epithelial cells. Fibroblasts were separated from epithelial cells with a differential adherence and centrifugation procedure. FDLEs were then seeded at 1 × 106 cells/cm2 onto 0.4-µm pore size Snapwell cell culture inserts (Corning Costar, Cambridge, MA) for Ussing chamber studies and at 0.5 × 106 cells/cm2 onto 75-mm-diameter, 0.4-µm pore size Transwell cell culture inserts (Corning Costar) for subsequent RNA isolation, preparation of conditioned medium (CM), and nuclear extract preparations. All cells were submersion cultured in Dulbecco's modified Eagle's medium (DMEM; 4.5 g/l of glucose with 2 mM L-glutamine and 110 mg/l of sodium pyruvate) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS), 100 U/ml of penicillin G sodium, and 100 µg/ml of streptomycin sulfate. All cell culture reagents were purchased from GIBCO BRL (Burlington, ON).

O2 environment and interventions. After being seeded, FDLEs were returned to an incubator containing 3% O2-5% CO2-balance N2 for ~20 h. Then the medium was replaced with fresh medium (containing inhibitors or vehicle alone; see Inhibitors and ROS scavengers). The monolayers were then either transferred to 21% O2 or kept at 3% O2 for approximately the next 48 h.

Reversibility of O2 induction. To test the reversibility of O2-induced Na+ transport, we placed some of the monolayers at 3% O2 after seeding (3-21% group) and some directly into 21% O2 (21-3% O2 group). After remaining at the initial O2 concentration for ~48 h, the 3-21% O2 group was moved to 21% O2 and the 21-3% O2 group was moved to 3% O2. These monolayers stayed at the final O2 concentration for an additional 48 h. A third group remained in 3% O2 throughout the entire 4-day experimental period (3-3% O2 group).

Studies with CM. To determine whether FDLEs exposed to postnatal O2 concentrations released a soluble factor that was capable of stimulating amiloride-sensitive Isc, FDLEs were seeded onto 75-mm Transwell membranes and left in 3% O2 overnight. After the addition of fresh medium, one-half of the monolayers were returned to 3% O2, whereas the other half were transferred to 21% O2. After 48 h, CM was collected from above the monolayers and centrifuged at 200 g for 5 min, and the supernatant was stored at -80°C. To test the CM, fresh FDLEs were seeded as described above and incubated in 3% O2. Approximately 20 h after seeding, the medium was changed to either fresh DMEM + 10% FBS (vol/vol) or a 1:1 dilution in DMEM + 10% (vol/vol) FBS of the 3% O2 or the 21% O2 CM.

Inhibitors and ROS scavengers. To determine whether cyclooxygenase-derived prostaglandins were involved in the O2-mediated induction of FDLE amiloride-sensitive Isc, ibuprofen [50 µM, a concentration previously shown to inhibit prostaglandin synthesis by these cells (38)] was added 4 h before exposure of FDLEs to 21% O2.

To evaluate the potential role of ROS in the induction of Na+ transport, a variety of compounds were tested: Trolox (100 µM), U-74389G (30 µM), N-acetyl-L-cysteine (NAC; 20 mM), EUK-8 (30 µM), and TEMPO (1 mM). These concentrations were selected based on preliminary cytotoxicity assays to define the maximum tolerated concentration (data not shown). Unless indicated otherwise, these compounds were added when the FDLEs were switched from 3 to 21% O2. In addition, the effect of exogenous catalase or superoxide dismutase (SOD) enzymes on O2 induction of amiloride-sensitive Isc was assessed. For catalase treatment, cationic liposomes [1:1 dioleoyldimethylammonium chloride-dioleoylphosphatidylethanolamine; INEX, Vancouver, BC] containing succinylated catalase were prepared as previously described (38). After the initial incubation in 3% O2 for 20 h, FDLEs were exposed to 150 mU/ml of catalase encapsulated in cationic liposomes (10 nmol/cm2) in DMEM + 2% FBS (vol/vol) for 2 h. This concentration was designed to increase cell catalase activity by 50%, which was confirmed by assay (37). After this 2-h incubation, the monolayers were rinsed, the medium was replaced with DMEM + 10% FBS (vol/vol), and the FDLEs then remained at 3% O2 for an additional 24 h before induction at 21% O2. SOD without liposomes (500 U/ml) was added to the medium 2 h before induction and again after 24 h. Alternatively, SOD was encapsulated in pH-sensitive liposomes (1:1 1,2-dioleoyl-sn-glycero-3-succinate-dioleoylphosphatidylethanolamine) and added at a concentration of 875 mU/ml (5 nmol/cm2 liposome) to the cell medium at the time of O2 induction. This concentration was based on studies using direct measurements of enzyme activity (37) to define the concentration required to achieve a 50% increase in enzyme activity. Preparation of pH-sensitive liposomes has been described elsewhere (4). As a control in liposome-mediated studies, an equivalent mass of bovine serum albumin (Sigma-Aldrich, Mississauga, ON) was used instead of the antioxidant enzymes in the preparation of the liposomes.

To determine the possible role of nitric oxide (NO) as a mediator in O2-induced amiloride-sensitive Isc, we incubated the cells with the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 0.2 mM) for the duration of the induction of amiloride-sensitive Isc by 21% O2.

U-74389G was a gift from Upjohn Scientific (Kalamazoo, MI). TEMPO, ibuprofen, and Trolox were purchased from Sigma-Aldrich; NAC was from Roberts Pharmaceutical (Mississauga, ON); L-NAME was from Calbiochem (San Diego, CA); catalase was from Cooper Biomedical (Malvern, PA); SOD was from Boehringer Mannheim (Laval, PQ); and EUK-8 was a gift from Eukarion (Bedford, MA).

Ussing chamber studies. Methods for Ussing studies have been previously described (28). The bioelectric properties of the FDLE monolayers were studied in modified Ussing chambers (World Precision Instruments, Sarasota, FL) while being bathed in 37°C Hanks' balanced salt solution (GIBCO BRL) supplemented with 1.8 g/l of sodium bicarbonate and equilibrated with a 5% CO2-balance air gas mixture. FDLEs were maintained under open-circuit conditions, and Isc was determined every 10 min with a voltage-current clamp (Physiologic Instruments, San Diego, CA) until stabilized. The amiloride-sensitive Isc was determined by the addition of 0.1 mM amiloride (Sigma-Aldrich) to the apical side of monolayers. Resistance (R) was calculated by dividing the transepithelial potential difference by the Isc.

Northern analyses. RNA was extracted from FDLE monolayers that had been grown on permeable supports with 4 ml of Trizol (GIBCO BRL) according to the manufacturer's instructions. The final pellet was dissolved in water treated with dimethyl pyrocarbonate (Sigma-Aldrich), and then 20 µg of total RNA were size fractioned on a 1% agarose (wt/vol)-MOPS-2% (vol/vol) formaldehyde gel. The RNA was subsequently transferred to Hybond N+ nylon membranes (Amersham, Oakville, ON). The blots were then ultraviolet cross-linked and hybridized with 32P random-primed alpha -rENaC and gamma -rENaC cDNA fragments [base pairs 74-403 for alpha -rENaC and 2161-2520 for gamma -ENaC (6)] in Expresshyb solution (Clontech, Palo Alto, CA) following the manufacturer's instructions. After being washed in 0.1× saline-sodium citrate (SSC; 1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) + 0.1% SDS at 50°C for 1 h, the blots were exposed to autoradiography film at -80°C. Autoradiographic bands were quantified with an LKB Ultrascan XL enhanced laser densitometer (Pharmacia, Montreal, PQ). All mRNA expression was normalized to 18S rRNA content by hybridizing the blots with a full-length mouse 18S rRNA 32P random-primed cDNA probe (American Type Culture Collection, Manassas, VA).

Electrophoretic mobility shift assay. Nuclear extract preparations and binding reactions were performed according to the protocol of Kazmi et al. (18) without further modification. Either 2 or 5 µg of nuclear extract and 10,000 counts/min of 32P end-labeled oligonucleotide were used for each binding reaction. The binding reaction mixture was run on a 4 or 6% nondenaturing acrylamide gel in 0.5× Tris-borate-EDTA buffer, which, after being dried, was exposed to autoradiography film at -80°C. The oligo containing the consensus sequence for NF-kappa B was 5'-AGC TTC AGA G<UNL>GG GAC TTT CC</UNL> GAGAGG-3', and the one containing the activator protein (AP)-1 consensus sequence was 5'-AGC TTT CC<UNL>A AAG AGT CA</UNL>T CAG G-3'.

Statistics. We used the INSTAT statistical package (GraphPad, San Diego, CA) to perform repeated-measures ANOVA followed by Tukey's post hoc test to examine significant differences between experimental groups. P values < 0.05 were considered to be significant. Unless stated otherwise, all data are expressed as means ± SE, and comparisons are for treatment vs. 21% O2-untreated control groups (received vehicle only). From each primary culture, we studied two to four Snapwell filters mounted in our Ussing chambers, and the results were pooled so that each n = 2-4 monolayers from a single primary culture of FDLEs.

    RESULTS
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Introduction
Methods
Results
Discussion
References

Postnatal O2 stimulates amiloride-sensitive Na+ transport. Consistent with a previous report by Pitkänen et al. (31), when we switched the FDLE monolayers from 3 to 21% O2, the amiloride-sensitive Isc increased from 1.3 ± 0.1 to 2.5 ± 0.1 µA/cm2, baseline Isc increased from 2.7 ± 0.1 to 4.0 ± 0.2 µA/cm2, and baseline R increased from 1,245 ± 66 to 1,687 ± 99 Omega  · cm2 (P < 0.05 for all changes; n = 50 monolayers). There was no change in amiloride-insensitive Isc (1.4 ± 0.1 vs 1.5 ± 0.1 µA/cm2; P > 0.05).

O2-induced increase in amiloride-sensitive Isc is reversible. In this series of experiments, the amiloride-sensitive Isc increased by an average of 60% after the FDLEs were switched from 3 to 21% O2 (Fig. 1). When FDLEs were switched from 21 to 3% O2 (21-3% O2 group), the amiloride-sensitive Isc declined to the same level as the 3-3% O2 group (P > 0.05 between the 2 groups; n = 3).


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Fig. 1.   Increase in O2 concentration reversibly induces amiloride-sensitive short-circuit current (Isc) in fetal distal lung epithelial cells (FDLEs). FDLEs (n = 3) were incubated under 3% O2 for 96 h (3-3% O2), 3% O2 for 48 h followed by 21% O2 for a further 48 h (3-21% O2), or 21% O2 for 48 h followed by 3% O2 for a further 48 h (21-3% O2). Amiloride-sensitive Isc was measured at end of 4-day experimental period. * P < 0.05 relative to other groups.

O2-induced FDLEs do not release a stable soluble factor capable of inducing amiloride-sensitive Isc. Figure 2 illustrates that CM derived from FDLEs maintained in 21% O2 did not induce amiloride-sensitive Isc in FDLEs that were kept at 3% O2 compared with the group that received the noninduced CM. Furthermore, the groups that received CM had amiloride-sensitive Isc values similar to those of O2-matched control groups receiving fresh medium, indicating that the process of CM preparation did not deprive the cell culture medium of factors that may have been essential for the ability of the cell to sustain ion transport.


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Fig. 2.   Ability of postnatal O2 to increase FDLE amiloride-sensitive Isc cannot be transferred by conditioned medium (CM). All cells were initially incubated at 3% O2 for 20 h, and then medium was changed to fresh DMEM + 10% fetal bovine serum for control groups (3% O2 and 21% O2) and to a 1:1 dilution of CM with fresh DMEM + 10% fetal bovine serum for remaining groups. CM was prepared as described in METHODS. CM from 21% O2-exposed FDLEs [21% O2 (CM21% O2)] did not stimulate amiloride-sensitive Isc in FDLEs that had been kept at fetal PO2 [3% O2 (CM21% O2)] compared with group that was also maintained at 3% O2 but received 3% O2 CM [3% O2 (CM3% O2); P > 0.05; (n = 3)]. * P < 0.05 relative to other groups.

Inhibitors and ROS scavengers. We used a number of different inhibitors and ROS scavengers to further investigate the mechanism of FDLE amiloride-sensitive Isc induction by O2. Inhibitors of prostaglandin (ibuprofen) and NO synthesis (L-NAME) did not impair the ability of O2 to increase the amiloride-sensitive Isc of FDLEs (Table 1). In contrast, 1 mM TEMPO (a superoxide scavenger) prevented both the increase in O2-induced amiloride-sensitive Isc (Fig. 3) and the concomitant increase in alpha -ENaC mRNA levels (Fig. 4). TEMPO similarly prevented O2 induction of gamma -rENaC mRNA expression (1 mM; P > 0.05 for 3% O2 vs. treatment; data not shown; n = 5). EUK-8, a catalytic scavenger of superoxide and hydrogen peroxide (13) also prevented induction of amiloride-sensitive Isc (Table 1). However, SOD alone, SOD encapsulated in pH-sensitive liposomes, liposomes containing catalase, NAC, and the hydroperoxide scavengers U-74389G and Trolox had no effect on the O2-induced amiloride-sensitive Isc in FDLEs (Table 1). In none of the experiments above did the treatment alter R; therefore, at the concentrations used, these drugs were not likely to be cytotoxic to FDLEs.

                              
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Table 1.   Effects of enzyme inhibitors and ROS scavengers on Isc


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Fig. 3.   O2-mediated induction of amiloride-sensitive Isc in FDLEs is blocked by superoxide scavenger tetramethylpiperidine-N-oxyl (TEMPO). TEMPO (1 mM) was added to FDLEs (n = 6) at time of switching the cells from a 3% to a 21% O2 environment. Control FDLEs received vehicle (ethanol) only. * P < 0.05 relative to other groups.


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Fig. 4.   Superoxide scavenger TEMPO blocks O2-induced increase in alpha -subunit of rat epithelial Na+ channel (alpha -rENaC) mRNA expression in FDLEs. Cells (n = 5) were treated with 1 mM TEMPO at time of O2 induction. Twenty-one percent O2 control cells received vehicle (ethanol) only. A: representative Northern blot probed sequentially with cDNA to alpha -rENaC and 18S rRNA (2 replicates/lane are shown). B: densitometric quantitation of alpha -rENaC expression normalized to 18S rRNA (alpha -ENaC/18S) from Northern blots. * P < 0.05 relative to other groups. Our group has previously demonstrated that a shift from 3 to 21% O2 equally increases mRNA concentration of alpha -, beta -, and gamma -ENaC subunits (31).

Postnatal O2 concentration activates the nuclear transcription factor NF-kappa B. Because the transcription factors NF-kappa B and AP-1 are known to be redox sensitive (22) and NF-kappa B is known to be activated by ROS (35), we tested nuclear extracts from FDLEs grown under 3 or 21% O2 using electrophoretic mobility shift assays with radiolabeled oligonucleotides corresponding to consensus binding sites for these two transcription factors. Three hours after FDLEs were switched from 3 to 21% O2, there was increased binding activity to an NF-kappa B consensus oligonucleotide (Fig. 5A). This activation was prevented when FDLEs were incubated with 1 mM TEMPO during the O2 induction period. In contrast, AP-1-binding activity was not induced under these conditions (Fig. 5B).


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Fig. 5.   Postnatal O2 activates nuclear factor (NF)-kappa B but not activator protein (AP)-1. A: 1 mM TEMPO prevents NF-kappa B activation induced by switch from 3 to 21% O2 concentration. Nuclear extracts from FDLEs cultured on permeable supports under 21% O2 (lane 2) show increased binding to an end-labeled NF-kappa B consensus oligo compared with cells grown under 3% O2 (lane 1). Specificity of binding is demonstrated by competition with a 200-fold excess of cold NF-kappa B oligo (lane 5) but not with an unrelated oligonucleotide (AP-1; lane 6). Induction of NF-kappa B binding activity by 21% O2 can be blocked with 1 mM TEMPO (lane 3). Lane 4, positive control in which NF-kappa B was induced with 1 ng/ml of tumor necrosis factor (TNF)-alpha for 4 h. B: no activation of AP-1 occurs when FDLEs are switched from 3 to 21% O2 concentration (lanes 1 and 2). Lane 4, positive control in which AP-1 was activated with 0.1 µM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 3 h. AP-1 activation was also absent in longer induction times of up to 5 h (data not shown). TEMPO did not activate AP-1. Each gel shift assay was repeated at least once.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

This study demonstrated that a change from a fetal (3%) to a postnatal (21%) O2 concentration induces a reversible increase in amiloride-sensitive Isc of FDLEs. We demonstrated that this increase in Na+ transport was associated with an increase in the levels of mRNAs that code for the subunits of ENaC. This induction of Na+ transport could not be reproduced with CM from FDLEs that had been exposed to postnatal O2, and it was not prevented by pharmacological blockers of cyclooxygenase (ibuprofen) or NO synthase (L-NAME). The induction of FDLE Na+ transport is due, at least in part, to an alteration in intracellular ROS, specifically superoxide, because the superoxide scavenger TEMPO prevented both the O2 induction of FDLE Na+ transport and increases in the level of ENaC mRNA. Our experiments also show that this perinatal physiological change in O2 concentration is associated with activation of the redox-sensitive transcription factor NF-kappa B and that this activation of NF-kappa B could also be prevented with TEMPO. Together these results suggest a possible mechanism by which an increase in O2 concentration and a concomitant change in the intracellular redox state of FDLE at birth may result in increased Na+ transport in the distal regions of the lung.

We tested the reversibility of O2 induction of amiloride-sensitive Isc by returning FDLE monolayers to 3% O2 after an initial incubation in 21% O2. After the switch back to 3% O2, amiloride-sensitive Na+ transport in this group declined to the same level as that of the group that had remained in 3% O2 throughout the study. This observation is in agreement with recent studies that have shown that Na+ uptake (21, 32) and mRNA expression for all three ENaC subunits (32) decline in adult rat type II epithelial cells when these cells were switched from 21% to hypoxic O2 concentrations.

Any increase in O2 concentration leads to an increase in intracellular ROS levels due to an increased rate of respiration. Because an increased O2 concentration, ROS, and hydroperoxides are known to stimulate prostaglandin synthesis and secretion (9, 16, 38, 39) and prostaglandins alter Na+ transport by otic epithelium (16), we treated FDLEs with ibuprofen before O2 induction. Ibuprofen, at concentrations known to inhibit cyclooxygenase (38), did not prevent 21% O2 induction of amiloride-sensitive Isc, thus making cyclooxygenase-derived prostaglandins unlikely mediators in the process of 21% O2-induced Na+ absorption.

It is believed that lung epithelium may regulate its biological character by secreting autocrine factors (7). To determine whether changing FDLEs from a 3% to a 21% O2 environment produced factors that could stimulate amiloride-sensitive Isc, we incubated FDLE in 3 or 21% O2-derived CM. Medium derived from FDLEs grown in 21% O2 was ineffective in stimulating amiloride-sensitive Isc in FDLEs cultured in 3% O2. Because prostaglandins are secreted molecules, these experiments further enforced the notion that prostaglandins do not mediate 21% O2-induced Na+ transport. We cannot, however, rule out the possibility that an unstable factor was secreted by FDLEs under the 21% O2 conditions because such factors would not be detected with our experimental protocol.

NO is an essential mediator in many cellular processes. It can play a role as both an oxidant and an antioxidant molecule (12). The mechanism by which NO acts varies from cell to cell, but, in general, it acts through a cGMP pathway (24). Atrial naturetic peptide acts via cGMP to regulate the 25-pS nonselective cation channel in kidney epithelium (20). Although a study (27) from our laboratory has shown that a brief (<1-h) exposure to atrial naturetic peptide is ineffective in regulating amiloride-sensitive Isc in FDLEs, Compeau et al. (10) also observed that activated macrophages reduce amiloride-sensitive Isc in FDLEs by an NO-dependent mechanism. To test the possible role NO may play in O2-induced Na+ transport in FDLEs, we incubated FDLE monolayers with an inhibitor of the inducible form of NO synthase (L-NAME) for the duration of the O2 induction protocol. L-NAME was unable to block the O2-induced increase in amiloride-sensitive Isc. Therefore, we do not believe 21% O2 induction of amiloride-sensitive Isc is mediated by NO.

There has been an increasing recognition of the role of ROS in signal transduction and gene regulation (reviewed in Ref. 19). Our results demonstrated that the superoxide scavenger TEMPO inhibited the 21% O2-induced amiloride-sensitive Na+ transport and ENaC mRNA expression. We chose TEMPO for this study because of its high cellular permeability and its lack of hydroperoxide scavenging activity (8, 34; Rafii, Tanswell, and O'Brodovich, unpublished observations). The SOD-catalase mimic EUK-8, at a concentration that was much lower than we used for TEMPO, also diminished the O2-induced amiloride-sensitive Isc to the 3% O2 level. On the other hand, neither catalase nor the cell-permeable hydroperoxide scavengers U-74389G and Trolox were able to block O2 induction of amiloride-sensitive Isc. NAC, a substrate source for the synthesis of glutathione (cofactor for the glutathione peroxidase that detoxifies H2O2), was equally ineffective, indicating that the change in the overall intracellular redox balance was not a factor in inducing amiloride-sensitive Isc on the switch of FDLEs from 3 to 21% O2. SOD, either alone or when encapsulated with pH-sensitive liposomes, did not block O2-induced amiloride-sensitive Isc. The failure of SOD to block O2-induced amiloride-sensitive Isc may have been due to its instability or its lack of uptake by FDLEs under these culture conditions for the 48-h O2 induction period. Unlike the small and diffusible TEMPO, large proteins such as SOD may also have stearic limitations in reaching relevant sites of superoxide production. Indeed, exogenous SOD has been shown to be an ineffective antioxidant in other whole cell systems (8, 23).

Together, these data suggest that superoxide anion and not H2O2 (the product of dismutated superoxide) is the ROS mediating the 21% O2 induction of amiloride-sensitive Na+ transport in FDLEs. These results are in contrast with other studies suggesting H2O2 as the ROS capable of modulating ion transport. Catalase, and not SOD, inhibits Na+ current in gerbil ear epithelium (16) and Na+-K+-ATPase activity in adult lung type II epithelial cells (15) that have been stimulated with the xanthine/xanthine oxidase ROS-generating system. However, these studies were conducted at ROS concentrations that may have been higher than those predicted to be generated by exposing FDLEs to postnatal 21% O2.

Our study shows that the change in O2 concentration was associated with increased levels of mRNA coding for ENaC. Although we did not directly assess transcription, it is likely that there was increased synthesis of ENaC mRNA. Others (11) have described O2-responsive elements on the human glutathione peroxidase promoter. In addition to the potential for an O2-responsive element in the ENaC promoter, it is also possible that there are other genes in the FDLEs that are O2 responsive and alter ENaC expression through their expressed proteins or metabolic by-products.

The transcription factors NF-kappa B and AP-1 are known to be sensitive to the intracellular redox state and ROS (14, 22). Both NF-kappa B and AP-1 are key players in the regulation of gene expression by cellular redox state (14, 22, 36) and the promoter for alpha -rENaC contains an NF-kappa B consensus binding element (GGGGAGTTCC at -519 to -510) and a 12-O-tetradecanoylphorbol 13-acetate (CTAGTCA at -284 to -278) response element (29). In the present study, we have provided preliminary data indicating that NF-kappa B is activated by a postnatal 21% O2 concentration and that this NF-kappa B activation can be blocked by the superoxide scavenger TEMPO. This is in agreement with another study (33) that has shown that NF-kappa B is activated in tumor cells on posthypoxic reoxygenation. Because catalase and hydroperoxide scavengers were ineffective in blocking O2-induced amiloride-sensitive Isc, it is likely that superoxide acts directly and not through its dismutated by-product H2O2.

In conclusion, our experiments have demonstrated that the physiological increase in O2 concentration at the time of birth increases distal lung epithelial Na+ transport. This increase is a reversible phenomenon and is likely mediated by an increase in ROS that cause increased gene expression of ENaC. Because FDLEs (28) and human distal lung epithelial cells (2) have similar bioelectric properties, these findings may be relevant to the normal transition from fetal to postnatal life and in the recovery of patients with pulmonary edema.

    ACKNOWLEDGEMENTS

We thank Eukarion for providing EUK-8 and Dr. Susan Doctrow for helpful comments.

    FOOTNOTES

This research was supported by the Medical Research Council Group in Lung Development.

A. K. Tanswell is the Hospital for Sick Children Women's Auxiliary and University of Toronto Chair in Neonatology. O. Pitkänen was supported by a grant from the Finnish Cultural Foundation.

Preliminary results of this study were presented in abstract form (Am. J. Respir. Crit. Care Med. 155: A648, 1997).

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: H. O'Brodovich, Hospital for Sick Children, Univ. of Toronto, 555 Univ. Ave., Toronto, Ontario, Canada M5G 1X8.

Received 22 January 1998; accepted in final form 12 June 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Alcorn, D., T. M. Adamson, T. F. Lambert, J. E. Maloney, B. C. Ritchie, and P. M. Robinson. Morphological effects of chronic tracheal ligation and drainage in the fetal lamb lung. J. Anat. 123: 649-660, 1977[Medline].

2.   Barker, P. M., R. C. Boucher, and J. R. Yankaskas. Bioelectric properties of cultured monolayers from epithelium of distal human fetal lung. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L270-L277, 1995[Abstract/Free Full Text].

3.   Barker, P. M., and J. T. Gatzy. Effect of gas composition on liquid secretion by explants of distal lung of fetal rat in submersion culture. Am. J. Physiol. 265 (Lung Cell. Mol. Physiol. 9): L512-L517, 1993[Abstract/Free Full Text].

4.   Briscoe, P., I. Caniggia, A. Graves, B. Benson, L. Huang, A. K. Tanswell, and B. A. Freeman. Delivery of superoxide dismutase to pulmonary epithelium via pH-sensitive liposomes. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L374-L380, 1995[Abstract/Free Full Text].

5.   Brown, M. J., R. A. Olver, C. A. Ramsden, L. B. Strang, and D. V. Walters. Effects of adrenaline and of spontaneous labour on the secretion and absorption of lung liquid in the fetal lamb. J. Physiol. (Lond.) 344: 137-152, 1983[Abstract].

6.   Canessa, C. M., L. Schild, G. Buell, B. Thorens, I. Gautschi, J.-D. Horisberger, and B. C. Rossier. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367: 463-467, 1994[Medline].

7.   Caniggia, I., I. Tseu, R. N. N. Han, B. T. Smith, K. Tanswell, and M. Post. Spatial and temporal differences in fibroblast behavior in fetal rat lung. Am. J. Physiol. 261 (Lung Cell. Mol. Physiol. 5): L424-L433, 1991[Abstract/Free Full Text].

8.   Caraceni, P., H. S. Ryu, D. H. Van Thiel, and A. B. Borle. Source of oxygen free radicals produced by rat hepatocytes during postanoxic reoxygenation. Biochim. Biophys. Acta 1268: 249-254, 1995[Medline].

9.   Chakraborti, S., G. H. Gurtner, and J. R. Michael. Oxidant-mediated activation of phospholipase A2 in pulmonary endothelium. Am. J. Physiol. 257 (Lung Cell. Mol. Physiol. 1): L430-L437, 1989[Abstract/Free Full Text].

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

11.   Cowan, D. B., R. D. Weisel, W. G. Williams, and D. A. G. Mickle. Identification of oxygen responsive elements in the 5' flanking region of the human glutathione peroxidase gene. J. Biol. Chem. 268: 26904-26910, 1993[Abstract/Free Full Text].

12.   Darley-Usmar, V., H. Wiseman, and B. Halliwell. Nitric oxide and oxygen radicals: a question of balance. FEBS Lett. 369: 131-135, 1995[Medline].

13.   Doctrow, S. R., K. Huffman, C. B. Marcus, W. Musleh, A. Bruce, M. Baudrey, and B. Malfroy. Salen-manganese complexes: combined superoxide dismutase/catalase mimics with broad pharmacological efficacy. Adv. Pharmacol. 38: 247-269, 1997[Medline].

14.   Flohe, L., R. Brigelius-Flohe, C. Saliou, M. G. Traber, and L. Packer. Redox regulation of NF-Kappa B activation. Free Radic. Biol. Med. 22: 1115-1126, 1997[Medline].

15.   Gonzalez-Flecha, B., P. Evelson, K. Ridge, and J. I. Sznaider. Hydrogen peroxide increases Na+/K+-ATPase function in alveolar type II cells. Biochim. Biophys. Acta 1290: 46-52, 1996[Medline].

16.   Herman, P., T. Y. Tu, A. Loiseau, C. Clerici, R. Cassingena, A. Grodet, G. Friedlander, C. Amiel, and P. Tran Ba Huy. Oxygen metabolites modulate sodium transport in gerbil middle ear epithelium: involvement of PGE2. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L390-L398, 1995[Abstract/Free Full Text].

17.   Hummler, E., P. Barker, J. Gatzy, F. Beermann, C. Verdumo, A. Schmidt, R. Boucher, and B. C. Rossier. Early death due to defective neonatal lung liquid clearance in ENaC-deficient mice. Nat. Genet. 12: 325-328, 1996[Medline].

18.   Kazmi, S. M., R. K. Plante, V. Visconti, G. R. Taylor, L. Zhou, and C. Y. Lau. Suppression of NF kappa B activation and NF kappa-dependent gene expression by tepoxalin, a dual inhibitor of cyclooxygenase and 5-lipoxygenase. J. Cell. Biochem. 57: 299-310, 1995[Medline].

19.   Lander, H. M. An essential role for free radicals and derived species in signal transduction. FASEB J. 11: 118-124, 1997[Abstract/Free Full Text].

20.   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].

21.   Mairbaurl, H., R. Wodopia, S. Eckes, S. Schulz, and P. Bartsch. Impairment of cation transport in A549 cells and rat alveolar epithelial cells by hypoxia. Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17): L797-L806, 1997[Medline].

22.   Meyer, M., H. L. Pahl, and P. A. Baeuerle. Regulation of the transcription factors NF-kappaB and AP-1 by redox changes. Chem. Biol. Interact. 91: 91-100, 1994[Medline].

23.   Mitchell, J. B., A. Samuni, M. C. Krishna, W. G. DeGraff, M. S. Ahn, U. Samuni, and A. Russo. Biologically active metal-independent superoxide dismutase mimics. Biochemistry 29: 2802-2807, 1990[Medline].

24.   Moncada, S., R. M. J. Palmer, and E. A. Higgs. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43: 109-142, 1991[Medline].

25.   O'Brodovich, H., V. Hannam, M. Seear, and J. B. M. Mullen. Amiloride impairs lung water clearance in newborn guinea pigs. J. Appl. Physiol. 68: 1758-1762, 1990[Abstract/Free Full Text].

26.   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].

27.   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].

28.   O'Brodovich, H. M., 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].

29.   Otulakowski, G., and H. O'Brodovich. Genomic reorganization and promoter analysis of the subunit of the rat amiloride-sensitive epithelial sodium channel (Abstract). Am. J. Respir. Crit. Care Med. 157: A853, 1998.

30.   Perks, A. M., and S. Cassin. The effect of arginine vasopressin and epinephrine on lung liquid production in fetal goats. Can. J. Physiol. Pharmacol. 67: 491-498, 1989[Medline].

31.   Pitkänen, O., A. K. Tanswell, G. Downey, and H. O'Brodovich. Increased PO2 alters the bioelectric properties of fetal distal lung epithelium. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L1060-L1066, 1996[Abstract/Free Full Text].

32.   Planes, C., B. Escoubet, M. Blot-Chabaud, G. Friedlander, N. Farman, and C. Clerici. Hypoxia downregulates expression and activity of epithelial sodium channels in rat alveolar epithelial cells. Am. J. Respir. Cell Mol. Biol. 17: 508-518, 1997[Abstract/Free Full Text].

33.   Rupec, R. A., and P. A. Baeuerle. The genomic response of tumor cells to hypoxia and reoxygenation: differential activation of transcription factors AP-1 and NF-kB. Eur. J. Biochem. 234: 632-640, 1995[Abstract].

34.   Samuni, A., C. M. Krishna, J. B. Mitchell, C. R. Collins, and A. Russo. Superoxide reactions with nitroxides. Free Radic. Res. Commun. 9: 241-249, 1990[Medline].

35.   Schreck, R., P. Rieber, and P. A. Baeuerle. Reactive oxygen intermediates as apparently widely used messengers in the activation of NF-kB transcription factor and HIV-1. EMBO J. 10: 2247-2258, 1991[Abstract].

36.   Sen, C. K., and L. Packer. Antioxidant and redox regulation of gene transcription. FASEB J. 10: 709-720, 1996[Abstract/Free Full Text].

37.   Tanswell, A. K., D. M. Olson, and B. A. Freeman. Response of fetal rat lung fibroblasts to elevated oxygen concentrations after liposome-mediated augmentation of antioxidant enzymes. Biochim. Biophys. Acta 1044: 269-274, 1990[Medline].

38.   Tanswell, A. K., D. M. Olson, and B. A. Freeman. Liposome-mediated augmentation of antioxidant defenses in fetal rat pneumocytes. Am. J. Physiol. 258 (Lung Cell. Mol. Physiol. 2): L165-L172, 1990[Abstract/Free Full Text].

39.   Taylor, L., M. J. Menoconi, and P. Polgar. The participation of hydroperoxides and oxygen radicals in the control of prostaglandins synthesis. J. Biol. Chem. 258: 6855-6857, 1983[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 275(4):L764-L770
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