Centre de recherche, Centre hospitalier de l'Université de Montréal-Hôtel-Dieu, and Département de médecine, Université de Montréal, Montreal, Quebec, Canada H2W 1T7
Submitted 25 September 2002 ; accepted in final form 20 September 2003
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ABSTRACT |
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sodium ion channel; transepithelial current; cytokine; inflammation
Tumor necrosis factor- (TNF-
) is a proinflammatory cytokine that plays an important role in the activation of host defense by promoting the production of a wide spectrum of cytokines (IL-1, IL-6, granulocyte-monocyte colony-stimulating factor, IL-8) in the inflammatory process (27, 63). Although the main sources of TNF-
in the lungs are the monocytes and macrophages, alveolar epithelial cells have been found recently to release TNF-
upon stimulation with LPS (50). TNF-
exerts a vital function in protecting the lung against Pseudomonas infection in the mouse (31). High or chronic TNF-
expression can be associated, however, with pulmonary dysfunction. An elevated TNF-
level has been detected in bronchoalveolar lavage (BAL) of patients with acute respiratory distress syndrome (ARDS) (62), and TNF-
injection can lead to septic shock symptoms with respiratory distress and lung edema (66).
By stimulating the ubiquitous receptors TNFRI or TNFRII, TNF- can elicit a wide spectrum of cellular responses, ranging from apoptosis to activation of gene expression by nuclear mobilization of transcription factors, such as NF-
B or c-jun (42, 63). In the lung, TNFRI mediates signaling between the alveolar space and the endothelium upon TNF-
infusion in alveoli (39). Several studies suggest that TNF-
modulates liquid transport in the alveolus. It has been found to alter alveolar liquid clearance in bacterial pneumonia (58) and in septic peritonitis (6). Direct instillation of TNF-
in the lung also affects lung liquid clearance (28). Although TNF-
has been shown to downregulate the steady-state mRNA level of some ionic channels such as the CFTR (52), nothing is known about its impact on sodium transport expression and activity in alveolar epithelial cells.
Because TNF- is a major cytokine produced during lung infection, we tested the hypothesis that it could modulate Na+ transport in alveolar epithelial cells isolated from the rat lung. We found that TNF-
decreases amiloride-sensitive current as well as the steady-state mRNA and protein levels of ENaC in adult alveolar epithelial cells.
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MATERIALS AND METHODS |
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Northern blotting. To study the influence of TNF- on the steadystate mRNA level for ENaC and Na+-K+-ATPase subunits, we treated alveolar epithelial cells cultured for 3 days with 100 ng/ml of recombinant mouse TNF-
(Calbiochem, La Jolla, CA) for 4, 6, 8, or 24 h. Total mRNA was isolated and quantified by Northern blotting. Total RNA from alveolar epithelial cells was extracted by a modifi-cation of the guanidinium-phenol technique (19). For Northern blotting, 10 µg of total RNA were electrophoresed on 1% agaroseformaldehyde gel and transferred to GeneScreen nylon membranes (NEN, Boston, MA) after overnight blotting with 10x SSC. Hybridization was performed, as reported previously, in Church buffer [0.5 M sodium phosphate, pH 7.2, 7% SDS (wt/vol), 1 mM EDTA, pH 8] (19). The nylon membranes were hybridized successively with different cDNA probes (
-,
-, and
-ENaC;
1 and
1 Na+-K+-ATPase; 18S rRNA).
-ENaC mRNA was detected with a 764-bp mouse
-ENaC cDNA probe (His445 to the stop codon), which has high homology with rat
-ENaC cDNA (19).
- and
-ENaC mRNAs were detected with the complete cDNA clone, a gift from Dr. B. C. Rossier (Institut de pharmacologie et de toxicologie, Université de Lausanne, Switzerland). The
1 and
1 Na+-K+-ATPase probes were gifts from Dr. J. Orlowski (Physiology Department, McGill University, Montreal, Quebec, Canada). The
1 Na+-K+-ATPase probe consisted of a NarI-StuI 332-bp cDNA fragment coding from nt 89 to 421 (from the 5'-untranslated region to Arg61). The
1 Na+-K+-ATPase probe consisted of a NcoI-SspI 750-bp cDNA fragment, which encompasses the entire coding region (70). The RNA loaded on each lane was normalized to 18S rRNA and detected by fluorescence scanning of the gels after staining with SYBR green II (Molecular Probes, Eugene, OR) and before blotting RNA on nylon membrane. The SYBR green-stained gels as well as the Northern blots were scanned and analyzed with a Typhoon PhosphorImager (Molecular Dynamics, Sunnyvale, CA). For the actinomycin D series, the blots were hybridized with an 18S rRNA probe as reported previously (18). mRNA expression was always compared with matched, untreated cells for each time period of the study. For reproducibility and statistical reasons, Northern blotting was repeated several times with RNA extracted from cells isolated from different rats. At least three animals (n = 3) were used for each time point.
Actinomycin D treatment and -ENaC mRNA stability. To investigate the importance of transcription in the modulation of
-ENaC mRNA by TNF-
, we cultured alveolar epithelial cells for 3 days and treated them for 6 h with 5 µg/ml actinomycin D, an inhibitor of transcription, in the presence or absence of 100 ng/ml TNF-
.
-ENaC mRNA expression was studied by Northern blotting as stated above. The experiment was repeated three times with cells purified from different rats.
To assess -ENaC mRNA stability after TNF-
treatments, alveolar epithelial cells cultured for 3 days were treated or not for 16 h with 100 ng/ml TNF-
and incubated thereafter with 5 µg/ml actinomycin D for 0, 4, 6, 8, 10, or 12 h. Total RNA was extracted, and the amount of
-ENaC was quantified by Northern blotting as above.
Western blotting. Alveolar epithelial cells were cultured in 25-cm2 flasks for 3 days and treated for 24 h with 100 ng/ml TNF-. We harvested them by scraping the monolayer with a rubber policeman, followed by 15-min centrifugation at 2,800 g in PBS. The cell pellet was solubilized in 60 µl of lysate solution (250 mM sucrose, 10 mM Tris·HCl, pH 7.4, 1 mM EGTA, 0.5% vol/vol Triton X-100, 1 mM PMSF, and 25 µg/ml leupeptin) for 30 min on ice. Protein concentration was evaluated by the Bradford method (8) (Pierce, Rockford, IL). Total proteins (50 µg) were subjected to SDS-polyacrylamide gel electrophoresis and transferred electrophoretically onto Bio-Rad polyvinylidene difluoride membranes (Hercules, CA). The membranes were blocked for 16 h at 4°C with 5% wt/vol skim milk in PBST buffer (137 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8.1 mM Na2HPO4, 0.05% vol/vol Tween 20, and 0.02% Thimerosal) before incubation in the same buffer for 16 h at 4°C with a 1:1,000 dilution of the PA1-920 antibody (Affinity Bioreagent, Golden, CO), an affinity-purified antibody directed against the L20-C42 NH2-terminal portion of the rat
-ENaC subunit. After 2 h of incubation at room temperature with a 1:4,000 dilution of horseradish peroxidase-linked secondary antibody (Cell Signaling Technology, Beverly, MA) in 5% skim milk-PBST, the bands were detected by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ) on Kodak X-K1 films. For quantification, the X-ray films were scanned on a Hewlett-Packard ScanJet, and the bands were quantified by NIH ImageJ software. ENaC antibody specificity was tested on 10-µg cell lysates of Madin-Darby canine kidney cells transfected with rat
-ENaC tagged with the influenza hemagglutinin (HA) epitope, a gift from Dr. D. Rotin (Sick Children's Hospital, Toronto, Ontario, Canada). For the detection of the HA-rat
-ENaC construct, the cell lysate was tested with an anti-HA mouse monoclonal antibody (clone HA-7) at a 1:10,000 dilution (Sigma, Oakville, Ontario, Canada) followed by an incubation with a 1:4,000 dilution of an anti-mouse IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA) linked to peroxidase. The HA and
-ENaC antibodies detected the same broad band of 82-96 kDa, the molecular mass expected for glycosylated ENaC (Fig. 4) (59). The
-ENaC antibody detected a band at the same molecular mass in alveolar epithelial cells. Western blots were repeated seven times (n = 7) with lysates extracted from cells purified from different rats.
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Electrophysiology. The impact of TNF- on the electrophysiology of alveolar epithelial cells was studied, as reported previously (18), by successively recording potential differences (Pd, mV) and transepithelial resistance (Rte,
·cm2) across the cell monolayers with an epithelial voltohmeter (EVOM; World Precision Instruments, Sarasota, FL). Alveolar cells plated at 1 x 106 cells/cm2 density on polycarbonate membranes (catalog no. 3401, 1.0 cm2; Costar Transwell, Toronto, Ontario, Canada) were cultured for 3 days until they formed a tight epithelium. The monolayers were then treated for 24 h at the apical and basolateral sides with 100 ng/ml TNF-
. Transepithelial current across the monolayers was calculated according to the following formula: Ite = Pd/Rte, where Ite is transepithelial current generated by the cell monolayer, and Rte is its transepithelial resistance. To quantify the amount of amiloride-sensitive current generated by alveolar cells treated for 24 h with TNF-
, Ite was measured successively before and after 5-min incubation with 1 µM amiloride at 37°C. At this concentration, amiloride is a specific inhibitor of ENaC. At least 18 filters (n = 18), all coming from different rats, were used for these experiments.
Apical membrane permeabilization by amphotericin B. The apical surface of alveolar cells was permeabilized to determine if the decrease in Ite evoked by TNF- also involved a reduction in Na+-K+-ATPase activity at the basolateral surface. Cells grown on 24-mm Costar filters (catalog no. 3412, 4.5 cm2; Costar Transwell) were cultivated for 3 days and treated for 24 h with 100 ng/ml TNF as above. For short-circuit current (Isc) measurements, filters were mounted in the Ussing chamber and bathed on the apical and basolateral sides with warm physiological buffer (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM glucose, and 10 mM TES, pH 7.4). Isc was determined with a VCC MC2 voltage clamp amplifier (Physiological Instruments, San Diego, CA) linked to a 4sp PowerLab data acquisition and analysis system (ADInstruments, Grand Junction, CO). When the Isc was stabilized, the cell monolayers were treated with 10 µM amiloride on the apical side, to quantify the amiloride-sensitive current in control and TNF-
-treated cells. The cells were then treated with 10 µM amphotericin B added on the apical side of the monolayers. This treatment gradually raised the Isc to reach a plateau after
5 min. At that time, the basolateral membrane was bathed with 2 mM ouabain in warm physiological solution to inhibit Na+-K+-ATPase activity. Amphotericin B and ouabain solution were freshly prepared before the experiment. Amphotericin B was diluted as a concentrated 20 mM solution in DMSO, whereas ouabain was dissolved directly as a 2 mM solution in physiological solution. For each condition tested, five filters (n = 5) seeded with cells coming from different animals were analyzed in these experiments.
Dose-dependent modulation of amiloride-sensitive current and -ENaC mRNA expression by TNF-
in cells cultured on filters. Amiloride-sensitive current and
-ENaC mRNA expression were measured in alveolar epithelial cells treated with different TNF-
concentrations. The cells were cultured on 4-cm2 Transwell membranes for RNA extraction. After 3 days of culture, they were treated for 24 h with 0, 5, 10, 20, 50, or 100 ng/ml TNF-
added to the apical and basolateral medium bathing the cell monolayers. The transepithelial amiloride-sensitive current generated by the monolayers was assessed as described above with the EVOM. After these measurements, we isolated total RNA by directly lysing the cells on filters with Trysol reagent according to the manufacturer's protocol (Invitrogen).
-ENaC mRNA expression was examined by semiquantitative reverse transcription-polymerase chain reaction. Total RNA (5 µg) was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase (Invitrogen) as reported previously (19). Of the cDNA reaction, 1.5 µl out of 20 µl was subjected to PCR amplification with Taq polymerase as described elsewhere (19). For
-ENaC amplification, 50 pmol (1 µM) of the sense primer 5'-GAG CCT GCC TTT ATG GAT GA-3' located in exon 5 and 50 pmol (1 µM) of the antisense primer 5'-gag ctt tgc aac tcc gtt tc-3' present in exon 11 were used for PCR. For
-actin cDNA amplification, 12.5 pmol (250 nM) of the sense primer 5'-CTA AGG CCA ACC GTG AAA AG located in exon 3 and 12.5 pmol (250 nM) of the antisense primer 5'-GCC ATC TCT TGC TCG AAG TC-3' present in exon 4 were used. The amplification conditions were: 1 min at 94°C for denaturation of DNA, 2 min at 58°C for annealing of the primers, and 2-min incubation at 72°C for elongation. PCR amplifications for
-ENaC and
-actin were done side by side. The reactions were stopped after 21 and 20 cycles, respectively, when amplification was still in the exponential phase. Fifteen microliters of the reactions were run on 1% agarose gel. After electrophoresis, the gels were stained by ethidium bromide, and the amplified products were quantified by scanning with a Typhoon fluorescence scanner (Molecular Dynamics). The results were reported as percentages of control expression after normalization with the
-actin signal. The experiments were repeated four times (n = 4) on alveolar epithelial cells purified from different rats.
Rubidium uptake assays. Alveolar epithelial cells were cultured for 3 days in 24-well plates seeded with 3 x 105 cells/cm2. Rubidium (Rb+) uptake was studied as shown previously with some modifications (60). Control cells or cells treated for 24 h with 100 ng/ml TNF- were incubated for 30 min at 37°C in medium A (140 mM NaCl, 1 mM CaCl2, 5 mM KCl, 1 mM MgCl2, 5 mM glucose, 20 mM HEPES). The medium was replaced by medium A in the presence or absence of 2 mM ouabain, and the cells were incubated for 5 min at 37°C. An equivalent volume of medium A containing Rb86 at 0.5 µCi/ml final concentration was then added to the wells. After 15-min incubation at 37°C, the Rb+ uptake was stopped by four volumes of medium B (10 mM HEPES, pH 7.4, 100 mM MgCl2) chilled at 0°C and three successive washings with the same medium. The cells were lysed in 1 ml of lysate solution [1% SDS (wt/vol), 4 mM EDTA], and radioactivity was measured with 3 ml of scintillation fluid in a
-counter (TriCarb 1600; Packard Instrument, Downers Grove, IL). Protein concentration was estimated by the Lowry method. Rb+ uptake was calculated by the following formula: Rb uptake = Ax/a/p, where Ax is the cpm of the sample, a is the Rb cpm added (total), and p is the amount of protein (mg). The experiments were performed seven times (n = 7) on alveolar epithelial cells purified from different animals.
Statistics. The data are presented as means ± SE. Comparisons between groups were made by the unpaired t-test, one group t-test, analysis of variance, and post hoc analysis (Fisher paired least significant difference) with Statsview software (SAS Institute, Cary, NC). A probability of P < 0.05 was considered to be significant. The -ENaC mRNA decay curves, amiloride-sensitive current, and
-ENaC mRNA expression after TNF-
treatment were compared by multiple regression analysis.
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RESULTS |
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Actinomycin D inhibits TNF- downregulation of
-ENaC mRNA. TNF-
has been shown to exert its action by activating a wide range of genes. Therefore, we investigated the role of transcription in the downregulation of
-ENaC mRNA expression. Alveolar epithelial cells were treated for 6 h with TNF-
in the presence of 5 µg/ml actinomycin D, a transcription inhibitor. Treatment abolished the downregulation of ENaC mRNA by TNF-
, suggesting that transcription is involved in this process (Fig. 2).
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-ENaC mRNA stability in TNF-
-treated cells. The cellular pool of a given mRNA is modulated by the regulated balance between gene transcription and mRNA degradation. Because actinomycin D as cotreatment for 6 h abolished the decrease elicited by TNF-
on
-ENaC mRNA, we investigated the impact of TNF-
on
-ENaC mRNA stability after 16 h of treatment, at a time where ENaC mRNA expression has reached its lower steady-state equilibrium. There was a significant difference (P < 0.05) in
-ENaC mRNA stability in TNF-
-treated cells (half-life 10.4 h) compared with the controls (half-life 15.1 h) (Fig. 3).
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Modulation of -ENaC protein expression by TNF-
. We investigated whether TNF-
has an impact on the total cellular pool of
-ENaC protein in alveolar epithelial cells treated with 100 ng/ml TNF-
for 24 h. Immunodetection by Western blotting revealed a broad band of 82-96 kDa at the molecular mass expected for native and glycosylated
-ENaC (59). TNF-
significantly decreased to 50.0 ± 4.8% the amount of
-ENaC protein in treated cells compared with untreated controls (Fig. 4).
Influence of TNF- on Ite. The bioelectric properties of alveolar epithelial cells were tested under control conditions and TNF-
treatment. In unstimulated cells, an Ite of 6.02 µA/cm2 was detected across the monolayer (Table 1). This current was inhibited by 65% with 1 µM amiloride (Table 1), indicating that the cells were actively involved in Na+ transport. Treatment for 24 h with 100 ng/ml TNF-
decreased the Ite to 2.23 µA/cm2, a 63% drop compared with untreated cells (Table 1). TNF-
had a major impact on the amiloride-sensitive portion of the current, which dropped from 3.93 µA/cm2 in control cells to 1.09 µA/cm2 after treatment. This represents a 72% difference between control and TNF-
-treated cells for the amiloride-sensitive portion of the current. The same results were obtained with a higher amiloride concentration (10 µM) or with benzamil, a specific ENaC inhibitor (data not shown). TNF-
also affected the proportions of amiloride-sensitive and -insensitive current detected. After treatment, there was a decline in the amiloride-sensitive portion of the current, which fell to 49% of the total current. TNF-
also evoked a 45% decrease in the amiloride-insensitive portion of the current that went from 2.09 µA/cm2 in control cells to 1.14 µA/cm2 after treatment. There were no significant differences, however, in Rte between control and TNF-
-treated cells (Table 1).
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Dose-dependent modulation of amiloride-sensitive current and -ENaC mRNA expression by TNF-
. To establish whether there was a possible correlation between
-ENaC mRNA expression and amiloride-sensitive current, we seeded alveolar epithelial cells on Costar filters and tested them successively for amiloride-sensitive current and
-ENaC mRNA expression after treatment for 24 h with different concentrations of TNF-
(5-100 ng/ml). TNF-
at 5 ng/ml reduced by 54% the amiloride-sensitive current generated by alveolar epithelial cells (Fig. 5). TNF-
concentration >10 ng/ml did not further decrease the current, which stabilized
30% of control values. TNF-
also significantly downregulated
-ENaC mRNA expression in alveolar epithelial cells grown on filter. As for amiloride-sensitive current, TNF-
concentration >10 ng/ml did not further reduce mRNA expression of the subunit, which stabilized
30% (Fig. 5). There was a good correlation (r = 99) between
-ENaC mRNA expression and amiloride-sensitive current for the different TNF-
concentrations (Fig. 5).
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Ouabain-sensitive Rb+ uptake after TNF- treatment. TNF-
could act through different pathways to reduce the Ite of alveolar epithelial cells. Although there was no modulation of
1 and
1 Na+-K+-ATPase mRNA expression by TNF-
, we tested whether the cytokine could affect the activity of the pump after treatment for 24 h with 100 ng/ml TNF-
. Sodium pump activity decreased by 61% in cells treated for 24 h with TNF-
compared with the controls (Fig. 6) (P < 0.05).
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Ite after apical surface permeabilization. The diminution of ouabain-sensitive Rb+ uptake after 24-h TNF- treatment could be secondary to a reduced ability of the apical membrane to transport Na+ or could be the result of a direct inhibiting effect of TNF-
on sodium pump activity. To test the latter hypothesis, we measured the ouabain-sensitive Isc of control and TNF-
-treated cells in the Ussing chamber following amphotericin B permeabilization of the apical membrane. In such conditions, there is no restriction in Na+ entry in the cytoplasm by the apical membrane, which allows an estimation of the activity of Na+-K+-ATPase. Typical traces, presented in Fig. 7, show that 10 µM apical amiloride treatment decreased the basal Isc of control and TNF-treated cells. Apical addition of amphotericin B led to a progressive increase in the current, in control and TNF-treated cells, that was inhibited by 2 mM basolateral application of ouabain. The different Isc for control and TNF-treated cells are compared in Fig. 8. As shown previously by EVOM measurement, TNF-
significantly decreased (P < 0.05) basal transepithelial current (basal Isc) from 7.01 ± 0.71 to 2.27 ± 0.24 µA/cm2 (Fig. 8A) and amiloride-sensitive Isc from 5.63 ± 0.71 to 1.08 ± 0.16 µA/cm2 (Fig. 8B). After amiloride treatment, apical membrane permeabilization with amphotericin B (see MATERIALS AND METHODS and Fig. 7) increases current of control cells (6.06 ± 0.44 µA/cm2). There was also a significant increase in current of TNF-treated cells (4.21 ± 0.46 µA/cm2), but it did not reach the level of control cells (P < 0.05) (Fig. 8C). Subsequent addition of ouabain led to the determination of ouabain-sensitive current (ouabain-sensitive Isc) corresponding to the current driven by Na+-K+-ATPase activity (Fig. 8D) after apical membrane permeabilization. Although ouabain-sensitive current was weaker in TNF-treated cells (2.45 ± 0.29 µA/cm2) than in the controls (3.15 ± 0.28 µA/cm2), there were no significant differences between control and TNF-treated cells.
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DISCUSSION |
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The treatment of alveolar epithelial cells with 100 ng/ml TNF- over a 24-h period produced a marked reduction in the expression level of
-,
-, and
-ENaC mRNA. However, expression of the
1 and
1 Na+-K+-ATPase subunits was not affected, suggesting that sodium channel but not sodium pump mRNAs were affected by TNF-
action. Several studies have revealed that hormones, such as steroids (12, 18) or vasopressin (24, 53); oxygen tension (54, 56, 69, 71); and growth factors, such as keratinocyte growth factor (7, 73), transforming growth factor-
(36), and epithelial growth factor (37) can modulate ENaC mRNA expression. Few data are available concerning the effect of proinflammatory cytokines on ENaC mRNA expression. IL-4, a cytokine involved in asthma pathogenesis, has been shown to decrease
-ENaC mRNA expression in human bronchial epithelial cells (30). To the best of our knowledge, our results are the first to establish that TNF-
can downregulate ENaC mRNA expression in alveolar epithelial cells.
To study further how TNF- affects the steady-state mRNA level of ENaC expression, we decided to focus on the
-ENaC subunit. First, we tested the importance of transcription in the downregulation of
-ENaC mRNA expression by TNF-
. We found that actinomycin D, a drug that blocks the transcription of new RNA, abolished the reduction of
-ENaC mRNA in cells treated for 6 h with TNF-
. These results suggest that
-ENaC mRNA downregulation by TNF-
is a complex process that could require the transcription of one or several genes to decrease
-ENaC mRNA expression. This is similar to what has been reported in vascular smooth muscle cells where actinomycin D blocks the TNF-
downregulation of insulin-like growth factor-1 mRNA (2). In addition to actinomycin D, several inhibitors of putative TNF-activated pathways were tested (data not shown). Inhibitors to P38, inducible nitric oxide synthase, and NF-
B, as well as
-glutathione reduced ethyl ester, a membrane-soluble form of glutathione, all failed to inhibit
-ENaC mRNA downregulation by TNF. The pathway by which TNF modulates ENaC expression is therefore not elucidated yet and could involve a complex process.
TNF- has been demonstrated to downregulate the transcript level of several genes by affecting mRNA stability (52, 57, 68). For this reason, we tested its influence on the stability of
-ENaC transcripts. As shown above, actinomycin D used in cotreatment abolishes the downregulation of
-ENaC mRNA by TNF-
. Therefore, we chose to study the effect of TNF-
on
-ENaC mRNA stability by adding actinomycin D after 16-h TNF-
incubation, since, at this time, the
-ENaC mRNA level has already reached its low steady-state mRNA level. We found that TNF-
significantly decreases
-ENaC mRNA stability. This result suggests that TNF-
could act in part through a posttranscriptional mechanism to downregulate
-ENaC mRNA in alveolar epithelial cells.
Thereafter, we tested the impact of TNF- on the amount of
-ENaC protein in the cells and found that it significantly decreased the protein pool in treated cells. The reduction of
-ENaC protein by TNF-
is in the same range as that observed for
-ENaC mRNA. Although no data are available concerning the stability of
-ENaC protein in alveolar epithelial cells, several studies have reported its short half-life (1
2 h) in cultured cells (34, 49, 61, 67). The results presented here suggest that the half-life of
-ENaC protein could be in the same range in alveolar epithelial cells, since a reduction in
-ENaC mRNA brings down the amount of
-ENaC protein.
The bioelectric properties of alveolar epithelial cells were also affected by TNF- treatment. Amiloride-sensitive current is the main current detected when unstimulated alveolar epithelial cells are cultured on a permeable membrane. This is similar to what has been reported elsewhere for these cells (14, 16). Upon TNF-
treatment, there was a pronounced reduction of amiloride-sensitive and -insensitive current. The effect was more marked for amiloride-sensitive current, which declined by 72% compared with untreated cells. In the experimental conditions used for this study, TNF-
had no toxicity in alveolar epithelial cells since it had no influence on Rte. Furthermore, we could not detect significant necrosis or apoptosis induced in the cells by the cytokine (unpublished results). Very few studies have been reported on the effect of TNF-
on ionic transport in alveolar epithelial cells. Our results confirm the work of Zhang et al. (72), who showed that 48 or 72 h treatment with TNF-
decreases the Ite across alveolar epithelial cells. The data reported here further reveal that TNF-
can decrease the Ite after 24-h treatment and that amiloride-sensitive current is the main current affected by this agent. We did not, however, find a decline in Rte as reported by them (72). The difference could be explained by the diversity of experimental protocols in the two studies, especially the TNF-
concentration administered and the length of treatment, since the authors followed a 48-h treatment protocol. In support of this, Coyne et al. (17) reported that in human primary airway cells, TNF-
has no impact on Rte at 24 h but decreases it by 40% after 48 h of treatment.
In A549 cells, an alveolar epithelial cell line, TNF- has been shown to increase the amiloride-sensitive current within minutes (28). In this model, however, no study was performed after 24-h treatment. It is possible that acute and chronic treatments with TNF-
have different effects on alveolar epithelial cell bioelectric properties.
There was a good correlation (r = 0.99) between -ENaC mRNA expression and the amiloride-sensitive current of alveolar epithelial cells exposed to different TNF-
concentrations. The amiloride-sensitive current depends on several factors, including gene transcription, translation, membrane insertion, and channel activation. The short half-life of ENaC protein in mammalian cells (34, 49, 61, 67) and the relatively long half-life of ENaC mRNA suggest that the pool of
-ENaC mRNA could be very important in modulating the amount of active ENaC at the membrane. These results indicate that downregulation of
-ENaC mRNA expression is probably one of the mechanisms involved in the decrease of sodium transport in alveolar epithelial cells. The 100 ng/ml TNF-
concentration chosen for our study, although used by other investigators, is fairly high. Our data show, however, that 5 ng/ml TNF-
is sufficient to induce significant declines in ENaC activity and expression. This concentration is in the order of magnitude detected in the BAL of patients with cystic fibrosis (5), pneumonia (21), and ARDS (51, 62), as well as in the BAL of mice infected with Streptococcus pneumoniae (4). Our findings indicate that, at the TNF-
concentration found in several lung pathologies, there could be a significant decrease of ENaC expression and activity in the distal lung.
The ability of alveolar epithelial cells to transport sodium depends on ENaC expression and activity at the apical membrane as well as Na+-K+-ATPase expression and activity at the basolateral membrane. Although TNF- did not decrease
1 and
1 Na+-K+-ATPase expression, we tested whether 24-h TNF-
treatment affected the activity of the pump. There was a significant reduction of ouabain-sensitive Rb+ uptake, indicating that the activity of the pump was reduced after 24-h treatment with TNF-
. Two distinct mechanisms could explain such reduction. TNF-
could somehow affect the pump's ability to extrude Na+ at the basolateral membrane, or the reduced pump activity could be secondary to a restriction of Na+ entry at the apical membrane. In several Na+-reabsorbing epithelia, ENaC is a limiting factor for transepithelial sodium transport (3). Therefore, to evaluate the possibility that the decrease in the ouabain-sensitive Rb+ uptake observed (Fig. 6) could be related to a decrease in Na+ entry at the apical membrane, we measured sodium pump activity in control and TNF-treated cells following amphotericin B permeabilization of the apical membrane. Ussing chamber measurements of control and TNF-treated cells gave Isc in the range of what has been reported in the literature (32, 44, 55). Apical membrane permeabilization with amphotericin B following amiloride treatment increased the Isc in control and TNF-treated cells similarly to what has been described elsewhere (44). In TNF-treated cells, the Isc doubles after apical membrane permeabilization, compared with basal Isc, showing that TNF seems to decrease apical membrane permeability to ion. Although ouabain-sensitive Isc, the portion of the current driven by Na+-K+-ATPase, was slightly smaller in TNF-treated cells compared with the controls, a statistically significant difference could not be found. Although it is not possible at this point to exclude any effect of TNF on Na+-K+-ATPase activity at the basolateral membrane, the permeabilization experiments revealed that, in regard to the effect of TNF on Ite, the apical membrane of the alveolar epithelial cells seems to be the major target of TNF-
action.
There are discrepancies in the literature on the role of TNF- in the modulation of Na+ and water transport in the lung. TNF-
has been demonstrated to increase lung liquid clearance, a process involving amiloride-sensitive transport, during acute bacterial pneumonia (58), intestinal ischemia-reperfusion (6), and direct instillation into the air space (28). However, several reports, including the work presented here, indicate that TNF-
decreases the current and permeability of alveolar epithelial cells (72) and human bronchial epithelial cells (29) but elevates lung permeability in a rat model of acute lung inflammation (43). TNF-
also reduces the expression of aquaporin-5, a water channel involved in transepithelial water transport, in a lung epithelial cell line (65), and in adenovirus-infected lungs (64). As stated above, TNF-
concentration and length of treatment could be important factors in the ability of TNF-
to decrease Na+ transport in alveolar epithelial cells. Rezaiguia et al. (58) have reported that in a model of Pseudomonas lung infection, TNF-
is produced very rapidly with a sharp peak at 4 h. At 12 h, the TNF-
level practically returns to baseline, and at 24 h, they found an increase in lung liquid clearance (58). Towne et al. (64) report, however, that lung infection with adenovirus leads to lung edema with sustained TNF-
expression still detectable after 14 days. In such a model, aquaporin-1 expression and aquaporin-5 and
-ENaC mRNA levels are reduced on days 7 and 14 postinfection (64). In vitro, the infection of alveolar epithelial cells with Mycobacterium tuberculosis also leads to sustained TNF-
expression, still detectable after 4 days, with a decline in Ite generated by the cells (72). Altogether, these results suggest that the nature of sodium transport modulation by TNF-
on the distal lung could vary greatly with the length of treatment.
The data presented in this paper show that sustained TNF- treatment can downregulate ENaC expression and decrease amiloride-sensitive current in alveolar epithelial cells. These observations suggest that a proinflammatory cytokine, by influencing ENaC expression in the lung, could play an important role during sustained lung inflammation.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported in part by the Canadian Cystic Fibrosis Foundation and the Canadian Institutes of Health Research. Y. Berthiaume is a "Chercheur national" of the Fonds de la recherche en santé du Québec. E. Brochiero is a scholar from the Fonds de la relève of the Université de Montréal.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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