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Respir ResRespiratory Research1465-99211465-993XBioMed Central London 1465-9921-6-21563663510.1186/1465-9921-6-2ResearchModulation of epithelial sodium channel (ENaC) expression in mouse lung infected with Pseudomonas aeruginosa Dagenais André 1andre.dagenais.chum@ssss.gouv.qc.caGosselin Diane 2dgosselin@fondsftq.comGuilbault Claudine 3claudine.guilbault@mail.mcgill.caRadzioch Danuta 3danuta.radzioch@muhc.mcgill.caBerthiaume Yves 1yves.berthiaume@umontreal.ca1 Centre de recherche, Centre hospitalier de l'Université de Montréal/ Hôtel-Dieu, Département de médecine, Université de Montréal, Montreal, Quebec, Canada2 Present address: Fonds de solidarité FTQ, Montreal, Quebec, Canada3 Departments of Experimental Medicine and Human Genetics, McGill University, Montreal, Quebec, Canada2005 6 1 2005 6 1 2 2 21 11 2003 6 1 2005 Copyright © 2005 Dagenais et al; licensee BioMed Central Ltd.2005Dagenais et al; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background
The intratracheal instillation of Pseudomonas aeruginosa entrapped in agar beads in the mouse lung leads to chronic lung infection in susceptible mouse strains. As the infection generates a strong inflammatory response with some lung edema, we tested if it could modulate the expression of genes involved in lung liquid clearance, such as the α, β and γ subunits of the epithelial sodium channel (ENaC) and the catalytic subunit of Na+-K+-ATPase.

Methods
Pseudomonas aeruginosa entrapped in agar beads were instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains. The mRNA expression of ENaC and Na+-K+-ATPase subunits was tested in the lung by Northern blot following a 3 hours to 14 days infection.

Results
The infection of the different mouse strains evoked regulation of α and β ENaC mRNA. Following Pseudomonas instillation, the expression of αENaC mRNA decreased to a median of 43% on days 3 and 7 after infection and was still decreased to a median of 45% 14 days after infection (p < 0.05). The relative expression of βENaC mRNA was transiently increased to a median of 241%, 24 h post-infection before decreasing to a median of 43% and 54% of control on days 3 and 7 post-infection (p < 0.05). No significant modulation of γENaC mRNA was detected although the general pattern of expression of the subunit was similar to α and β subunits. No modulation of α1Na+-K+-ATPase mRNA, the catalytic subunit of the sodium pump, was recorded. The distinctive expression profiles of the three subunits were not different, between the susceptible and resistant mouse strains.

Conclusions
These results show that Pseudomonas infection, by modulating ENaC subunit expression, could influence edema formation and clearance in infected lungs.
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Background
The epithelial sodium channel (ENaC) is expressed in epithelial cells of several tissues involved in salt and water reabsorption. The channel is composed of three related subunits (α, β, γ) that are able to reconstitute a functional channel when expressed in Xenopus laevis oocytes [1,2]. ENaC is expressed in a wide range of tissues, including the kidney [1,3-5], distal colon [1,3,5], lung [6-8], ear epithelium [9,10], papilla of the tongue [11-13], eyes [14], chondrocytes [15] and differentiating epithelia [16]. ENaC synthesis and activity are highly regulated by hormones, such as aldosterone, vasopressin and catecholamines, by intracellular pH, feedback inhibition and extracellular proteases [17,18]. In the lung, vectorial Na+ transport from the alveoli to the interstitium is the main force that drives water out of the alveoli [19,20]. This transport mechanism plays a crucial role late in gestation and at birth when sodium transport is involved in lung liquid clearance [21]. Its importance at birth has been shown unambiguously in αENaC gene knockout mice, where the inability to clear lung water rapidly leads to hypoxemia and death [22]. Na+ transport is also important in adults for lung liquid clearance [19,23].

Increased ENaC expression has been detected in the lung and in alveolar epithelial cells in vitro, following stimulation with steroids, β-agonists, catecholamines, and agents that increase cAMP concentration [24-27]. αENaC expression in the lung is modulated at birth when considerable liquid clearance is required [3,6,27]. It is also upregulated during hyperoxia [28,29] and downregulated during hypoxia, which could explain high altitude lung edema (HALE) [30,31]. Several lines of evidence suggest that up-regulation or downregulation of ENaC activity in the lung could be associated with lung infection. In type I pseudohypoaldosteronism, a recessive genetic disease leading to a non-functional ENaC, susceptibility to lung infection has been reported [32-34]. Although ENaC is not the primary defect associated with cystic fibrosis (CF), airway cells from CF patients show a 2–3-fold increase in Na+ transport compared to normal cells [35,36]. This sodium hyperabsorption results from the inability of cystic fibrosis transmembrane regulator (CFTR) in CF cells to downregulate ENaC activity [37,38].

Pseudomonas aeruginosa is a bacterium occuring naturally in a wide range of environments such as in soil, fresh and seawater, plants and decomposing organic matter [39]. Although not usually pathogenic, this common bacterium can evoke opportunistic infections in immunodeficient persons, such as patients with severe burns [39]. Pseudomonas can promote nosocomial lung infection after artificial ventilation [40] and is also present in patients with bronchiectasis [39]. Chronic lung infections are the major cause of morbidity and mortality in CF patients [41] where Pseudomonas aeruginosa is the main source of chronic lung infection in CF patients [42].

Instillation of Pseudomonas aeruginosa in the lung of anaesthetised rabbits has been reported to promote acute pneumonia, resulting in alveolar epithelial injury, loss of epithelial barrier integrity, lung edema, pleural empyema and pleural effusions within 8 h of infection [43]. A more chronic pneumonia model has been developed in the mouse by the intratracheal instillation of P. aeruginosa entrapped in agar beads. In this model, the lungs of susceptible mouse strains develop severe lung infection with a strong inflammatory response and some lung edema [44,45]. Pseudomonas by itself has been shown to inhibit active sodium absorption in cultured airway epithelial cells [46]. Here, we studied its impact on the expression of genes involved in the modulation of liquid absorption in alveolar and airway epithelium, namely the three ENaC subunits and the catalytic subunit of Na+-K+-ATPase.

Pseudomonas entrapped in agar beads was instilled in the lung of resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) mouse strains, and the expression of α, β, γENaC and α1 Na+-K+-ATPase mRNA was studied by Northern blotting in lungs infected between 3 hours and 14 days.

Methods
Infection of mice with P. aeruginosa
Clinical strain 508 of P. aeruginosa (provided by Dr. Jacqueline Lagacé, Université de Montréal, Montreal, Canada) was entrapped in agar beads, and 50-μl suspensions containing 2 × 105 to 1 × 106 CFU/ml were instilled intratracheally in male mice of resistant (BALB/c) or susceptible (DBA/2, C57BL/6 and A/J) strains as described previously [44,45].

Macrophage and polymorphonuclear (PMN) counts in bronchoalveolar lavage (BAL)
BAL were performed as described elsewhere with a few modifications [44]. The infected mice were sacrificed by CO2 inhalation at different time points after P. aeruginosa instillation in the lungs. The trachea was cannulated, and the lungs were washed seven times with 1 ml PBS. Total cell counts were conducted in a hemacytometer. Differential cell counts were made by Diff-Quick staining (American Scientific Products) of Cytospin preparations. Number of animals: day 1, n = 6; day 4, n = 16; day 6, n = 6; day 14, n = 3.

Northern blotting
The lungs from infected mice were harvested between 3 h to 14 days after infection, homogenized in 5 ml of 4 M guanidine isothiocyanate, and centrifuged on a cesium chloride gradient [44]. Fifteen to 20 μg of total RNA purified from the lungs were electrophoresed on 1% agarose-formaldehyde gel and transferred to Nytran membranes (Schleicher & Schuell, Keene, NH, USA) by overnight blotting with 10 X SSC. Hybridization was performed, as reported previously [3], in Church buffer (0.5 M Na phosphate, pH 7.2, 7% SDS (w/v), 1 mM EDTA, pH 8) [47]. The nylon membranes were hybridized successively with different cDNA probes. (αENaC, βENaC and γENaC, α1Na+-K+-ATPase, glyceraldehyde-3-phosphate dehydrogenase (GADPH) or 18S rRNA). To detect αENaC mRNA, the blots were hybridized with 764-bp mouse αENaC cDNA (His-445 to stop codon) [3]. The probes for rat β and γENaC cDNA were gifts from Dr. B.C. Rossier (Institut de pharmacologie et de toxicologie de l'Université de Lausanne, Lausanne, Switzerland) and coded for the entire cDNA [2]. The α1Na+-K+-ATPase probe was a gift from Dr. J. Orlowski (Physiology Department, McGill University, Montreal, Quebec, Canada) and consisted of a NarI-StuI 332-bp fragment coding from nucleotide 89 to 421 (from the 5'UTR to Arg-61) of the rat kidney and brain α isoform [48]. For quantitative study, αENaC mRNA expression was normalized to murine GADPH with a 455 bp cDNA probe cloned between nucleotide 146 and 601 [44] or with 18S rRNA, using a 640-bp cDNA probe between nucleotidet 852 and 1492 of the rat 18S rRNA sequence [26]. The blots were exposed to Kodak Xar-film with an intensifying screen, or to a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA) for densitometric analysis. Because different strains of mice were investigated in this study (BalB/c, DBA/2, C57BL/6 and A/J), the expression of the different mRNA was calculated at each time point as the % of expression relative to an untreated control from the same strain. The data from the different strains were pooled and subjected to statistical analysis.

Between each round of hybridization, the membranes were stripped by treatment with 0.1 X SSC, 1% SDS and 2.5 mM EDTA at 95°C. The blots were allowed to cool gradually with agitation for 30 min at room temperature. The membranes were then rinsed with 5 X SSC and rehybridized. Number of animals: n = between 6 and 8 animals for each time point and each mRNA studied.

Statistics
For the BAL cell count, the data are presented as means ± SE (standard error). For ENaC and Na+, K+-ATPase mRNA expresion, the comparisons between groups were analyzed by Wilcoxon signed rank non-parametric test using Statsview software (SAS Institute, Inc., Cary, NC, USA). Probability p values < 0.05 were considered to be significant.

Results
Inflammation in mice infected with P. aeruginosa
The inflammation process evoked by Pseudomonas instillation in the lung of C57BL/6 mice was monitored by studying the number of total cells in BAL at different times after infection. As shown in Figure 1, the inflammation process was more pronounced on days 1 and 4 post-infection. Significant PMN recruitment was noted on day 1 after infection since these cells constituted 90% of the cell population in BAL at that time (Fig. 1). The proportion of PMN decreased gradually over time. On days 6 and 14, there was a significant reduction of PMN in BAL (p < 0.05) compared to day 1. PMN still constituted 18% of the cells in BAL on day 14. The infection also led to modulation in the number of macrophages with a significant increase (p < 0.05) on day 4 post-infection (Fig. 1).

Figure 1 Differential cell counting in C57BL/6 mice infected with 1–2 × 105 Pseudomonas aeruginosa embedded in agar beads. Pseudomonas infection leads to strong inflammation with recruitment of PMN and macrophages in bronchoalveolar lavage on days 1 and 4 post-infection. Day 1, n = 6; day 4, n = 16; day 6, n = 6; day 14, n = 3. Differential cell counting: PMN in light grey, macrophages in dark grey.

Modulation of α, β and γENaC expression following lung infection with P. aeruginosa
Pseudomonas embedded in agar beads was administered intratracheally in resistant (BALB/c) and susceptible strains of mice (DBA/2, C57BL/6, A/J) as described previously [44,49]. α, β and γENaC expression in infected lungs was measured by Northern blot hybridization (Fig. 2). Expression of the three subunits was highly modulated in time after lung infection, but showed a similar pattern between the four mouse strains tested. The BALB/c strain that is resistant to Pseudomonas infection [45], as well as the DBA/2, C57BL/6 and A/J susceptible strains, showed increased α, β and γENaC expression at 24 h, followed by a decrease on day 3 post-infection. The GADPH standard gene did not manifest any modulation of its expression. Densitometric quantitative analyses of the Northern blots were performed for the four mouse strains. The relative expression at each time point was determined relative to uninfected animals of the same strain and the data from the 4 strains were pooled for analysis (Fig. 3). αENaC mRNA expression presented a significant decline to a median of 43% on days 3 and 7 post-infection, and was still decreased to a median of 45% on day 14 post-infection compared to uninfected controls (p < 0.05, Fig. 3). βENaC mRNA expression was increased to a median of 241% of uninfected control values, 24 h post-infection (p < 0.05), and was followed by a decrease to medians of 42% and 54% on day 3 and 7 post-infection (p < 0.05) (Fig. 3). Although the expression of γ ENaC mRNA showed an expression pattern very similar to the α and βENaC subunits, with an increased expression at 24 h (median of 171%) followed by a decreased expression on day 3 (median of 53%) and 7 (median of 66%) of infection, these changes failed however to reach significance (Fig. 3). No modulation of α, β or γENaC mRNA was detected when the lungs were instilled with agarose beads only (data not shown). We also investigated the expression of α1 Na+-K+-ATPase mRNA coding for the catalytic domain of the sodium pump, but could not find any significant change during infection (Fig. 3).

Figure 2 Expression of α, β and γENaC mRNA in the lung following infection with Pseudomonas aeruginosa. Representative Northern blot of α, β and γENaC mRNA expression following infection with Pseudomonas in resistant (BalB/c) and susceptible (DBA/2, C57BL/6 and A/J) strains of mice. There is a characteristic modulation of the three ENaC subunits that is not different between strains.

Figure 3 Densitometric analysis of the modulation of αENaC, βENaC, γENaC and α1Na+-K+-ATPase mRNA following Pseudomonas infection. The modulation of αENaC, βENaC, γENaC and α1Na+-K+-ATPase mRNA by Northern blots hybridization was subjected to a densitometric analysis. Because different strains of mice were investigated in this study (BalB/c, DBA/2, C57BL/6 and A/J), the expression of the different mRNA was calculated at each time point as the % of expression relative to an untreated control coming from the same strain. The α and βENaC mRNA were modulated at some time point by Pseudomonas infection compared to uninfected animals. There was no modulation for γENaC or α1Na+-K+-ATPase mRNA. αENaC mRNA was downregulated compared to uninfected controls on days 3, 7 and 14 post-infection (*, p < 0.05). βENaC mRNA was elevated at 24 h post-infection (*, p < 0.05) compared to uninfected controls and was downregulated thereafter on day 3 and 7 post-infection (*, p < 0.05). Number of animals: n = between 6 and 8 animals for each time point and each mRNA studied.

Discussion
The instillation of Pseudomonas enmeshed in agarose beads in the lung is a good model to study lung inflammation [44,45] and lung injury [43] secondary to an infection. For this study, P. aeruginosa enmeshed in agarose beads was instilled into the mouse lung because the model allows the development of chronic lung infection in susceptible mouse strains [44,45]. The infection leads to cellular infiltration and alveolar edema that stand on day 3 post-infection and that can be still demonstrated on day 14 post-infection in Pseudomonas-susceptible mouse strains [45]. Because the lung inflammation associated with Pseudomonas infection is accompanied by lung injury [43], and because we have shown recently that ENaC expression can be modulated under conditions that promote lung injury [50], we tested here if Pseudomonas was affecting the mRNA expression level of the three ENaC subunits as well as the catalytic subunit of the Na+ pump since these elements are involved in lung liquid balance across the alveolar epithelium [19,23]. The results reported here indicate that Pseudomonas infection modulated the expression of the three ENaC mRNA with a characteristic pattern. There was no significant difference, however, in the expression profile of ENaC mRNA between the Pseudomonas-resistant (Balb/C) and -susceptible (DBA/2, C57BL/6, A/J) mouse strains. Modulation of ENaC expression is therefore most likely not a genetic marker linked to the susceptibility of mouse strains to establishment of a chronic infection with Pseudomonas.

Pseudomonas infection affected ENaC mRNA with a pattern consisting of increased expression at 24 h, followed by a marked decrease on day 3 post-infection. The change in ENaC mRNA was related to bacterial infection, since agarose beads alone failed to evoke any modulation of these RNA. The three ENaC subunits were modulated with a similar profile, with some noticeable differences, however. Although αENaC mRNA expression tends to increase by day 1, the most noticeable feature brought by Pseudomonas infection to αENaC mRNA was the significant decreases after 3 days, 7 days and 14 days post-infection. To the best of our knowledge, this is the first report demonstrating that bacterial infection in vivo can lead to modulation of ENaC mRNA expression. Recently, αENaC mRNA expression was found to be downregulated in the mouse lung after 7 and 14 days of adenoviral infection [51]. Furthermore, there is some evidence that αENaC expression is also decreased in other models of lung injury. Folkesson et al. [52] reported a decline in ENaC expression following subacute lung injury, 10 days after intratracheal administration of bleomycin. More recently, we recorded a decrease in ENaC expression after ischemia-reperfusion lung injury [50]. All these results, and the results reported in the present report, suggest that the modulation of ENaC expression associated with lung infection could be a widespread mechanism, not specific to a given pathogen or injury process, but a general response of the lung to inflammation and injury.

The β ENaC subunit was also modulated by Pseudomonas infection. There was a significant increase in the mRNA expression on day 1 post-infection, followed, as for αENaC, by a decreased expression on day 3 and day 7 post-infection. Different stoichiometries have been proposed for ENaC. One model suggests a 2α, β, γ ratio [53,54] whereas others postulate an octomeric [55] or nonameric structure [55,56]. Although the expression of the α subunit alone is sufficient to allow ENaC activity [1], the three subunits are needed to get a fully functional channel [2]. The expression of the three subunits in Xenopus laevis oocytes increases amiloride-sensitive Na+ current by 100% compared to αENaC alone [2]. The α, β and α, γ channels are 20 times less effective in driving amiloride-sensitive current than the native channels and show differences in their biophysical properties [57,58]. Gene inactivation or over-expression of the different ENaC subunits has revealed important differences in the role each subunit plays in lung liquid management. αENaC knockout mice develop respiratory distress and die within 40 h from birth because of their inability to clear lung liquid [22]. Lung liquid clearance at birth is also slower in γENaC knockout mice [59], but is not affected in βENaC knockouts [60]. Increased transgenic expression of βENaC targeted in the airway epithelia, but not α or γ subunits, showed an increase Na+ transport across the airway epithelium and a reduced height of the airway surface liquid [61]. For all these reasons, it is difficult to predict how the modulation of ENaC mRNA expression and its effect on the ratio of the three subunits, would have an impact on ENaC activity. In addition, ENaC mRNA content also does not necessarily reflect the amount of active channel at the membrane. One thing seems clear however, because of its prominence in the lung, the diminution of αENaC expression that we detected in the lung following Pseudomonas infection, could certainly influence amiloride-sensitive current and lung liquid clearance as in αENaC KO mice rescued by transgenic expression of αENaC that has a lower expression of ENaC in the lung [62,63]. In such model, there is a reduced ENaC current in tracheal cells [63], and a much slower lung liquid clearance following thiourea or hyperoxia-induced lung edema [64].

The general biphasic modulation of ENaC mRNA expression with an increase at 24 h followed by a decrease thereafter is an interesting finding that could explain some contradictory reports concerning ENaC expression in lung following Pseudomonas infection. Acute bacterial pneumonia in rats has been shown to increase alveolar epithelial fluid clearance [65,66] when in late pneumonia, there is a decrease in the lung liquid clearance ability of the lung [66]. These contradictory results could be well explained by the modulation of ENaC expression reported here. The long term ENaC downregulation by Pseudomonas infection could be of potential clinical significance to understand the slow improvement in some ARDS patients.

In contrast to α and β ENaC, α1 Na+-K+-ATPase mRNA was unaffected in the course of lung infection. This is similar to what has been reported during adenovirus lung infection [51] where αENaC, aquaporin 1 (AQP1) and AQP5 mRNA show decreased expression, but not α1 Na+-K+-ATPase. In ischemia reperfusion injury, there was also no modulation of α1 Na+-K+-ATPase expression despite significant ENaC downregulation [50]. These results, as well as the data reported here, suggest that the inflammatory process seems to selectively affect, and not in a non-specific way, some elements of lung liquid clearance. It would be difficult at this time to speculate on the reasons for this modulation. Na+-K+-ATPase is an important element in lung liquid clearance, however, by being one of the key generator of membrane potential, the enzyme also affects other channels and ion transport process. It is possible that by modulating ENaC expression and not α1 Na+-K+-ATPase, Pseudomonas infection alters the Na+ transport system but does not change other important cell functions meditated by Na+-K+-ATPase. Furthermore, despite a similar mRNA expression level, there could be a fall in protein content or activity of the sodium pump. Additional experiments are necessary to answer this question.

Several studies report that in lung epithelial cells, viral infection [67,68], mycoplasma [69], bacterial infection [70,71], and inflammatory cytokines such as tumor necrosis factor-α (TNF-α) [70,72], interleudin-1β (IL-1β) [73], or TGF-β [74] decrease the expression of water channels, such as AQP1 and AQP5 and reduce the short circuit current generated by cells. Adenoviral lung infection in mice results in pulmonary inflammation and lung edema with lowered expression of AQP1, AQP5 and αENaC [51]. All these data, including the results presented here, suggest that lung inflammation, by decreasing the expression of αENaC and water channels, could hamper the liquid clearance ability of the lungs and favour edema formation.

Conclusions
We have shown in this report that Pseudomonas infection modulates ENaC mRNA expression. This modulation is independent of mouse strain susceptibility to establishment of chronic infection with Pseudomonas. Although there is an elevation of ENaC expression after 24 h, the most important feature is probably the long-lasting decrease of αENaC transcripts on days 3 and 7 post-infection. The lung inflammation induced by Pseudomonas infection therefore seems to favour a reduction in the expression of an essential element involved in lung liquid clearance as well as the regulation of airway surface liquid volume.

Authors' contributions
AD performed the hybridization, the statistical analysis of the blots and wrote the manuscript. DG performed the Pseudomonas instillation, RNA extraction and Northern blotting of RNA sample. The BAL recovery as well as PMN and macrophage counting was performed by CG. YB and DR designed and co-ordinated the study. All authors read and approved the final manuscript.

Acknowledgements
Dr Yves Berthiaume and Dr. Danuta Radzioch are Chercheur-Nationaux from Fonds de la recherche en santé du Québec. This work was supported in part by the Canadian Cystic Fibrosis Foundation and the Canadian Institutes of Health Research. The authors acknowledge the editorial work on this manuscript by Ovid Da Silva, éditeur/rédacteur of the Research Support Office of the CHUM Research Center.
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Canessa CM Horisberger J-D Rossier BC  Epithelial sodium channel related to proteins involved in neurodegeneration Nature 1993 361 467 470 8381523 10.1038/361467a0 
Canessa CM Schild L Buell G Thorens B Gautschi I Horisberger J-D  Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits Nature 1994 367 463 467 8107805 10.1038/367463a0 
Dagenais A Kothary R Berthiaume Y  The α subunit of the epithelial sodium channel in the mouse: developmental regulation of its expression Pediatr Res 1997 42 327 334 9284273 
Dijkink L Hartog A Deen PM Van Os CH Bindels RJ  Time-dependent regulation by aldosterone of the amiloride-sensitive Na+ channel in rabbit kidney Pflugers Arch 1999 438 354 360 10398866 10.1007/s004240050920 
Renard S Voilley N Bassilana F Lazdunski M Barbry P  Localization and regulation by steroids of the alpha, beta and gamma subunits of the amiloride-sensitive Na+ channel in colon, lung and kidney Pflügers Arch 1995 430 299 307 10.1007/BF00373903 
Voilley N Lingueglia E Champigny G Mattéi M-G Waldmann R Lazdunski M  The lung amiloride-sensitive Na+ channel: Biophysical properties, pharmacology, ontogenesis, and molecular cloning Proc Natl Acad Sci USA 1994 91 247 251 8278374 
O'Brodovich H Canessa C Ueda J RafII B Rossier BC Edelson J  Expression of the epithelial Na+ channel in the developing rat lung Am J Physiol 1993 265 C491 C496 7690185 
Champigny G Voilley N Lingueglia E Friend V Barbry P Lazdunski M  Regulation of expression of the lung amiloride-sensitive Na+ channel by steroid hormones EMBO J 1994 13 2177 2181 8187771 
Herman P Tan C-T Van den Abbeele T Escoubet B Friedlander G Tran Ba Huy P  Glucocorticosteroids increase sodium transport in middle ear epithelium Am J Physiol 1997 272 C184 C190 9038824 
Portier F Van den Abbeele T Lecain E Sauvaget E Escoubet B Tran Ba Huy P  Oxygen modulates Na+ absorption in middle ear epithelium Am J Physiol 1999 276 C312 C317 9950758 
Li XJ Xu RH Guggino WB Snyder SH  Alternatively spliced forms of the alpha subunit of the epithelial sodium channel: distinct sites for amiloride binding and channel pore Mol Pharmacol 1995 47 1133 1140 7603452 
Kretz O Barbry P Bock R Lindemann B  Differential expression of RNA and protein of the three pore-forming subunits of the amiloride-sensitive epithelial sodium channel in taste buds of the rat J Histochem Cytochem  1999 47 51 64 9857212 
Lin W Finger TE Rossier BC Kinnamon SC  Epithelial Na+ channel subunits in rat taste cells: Localization and regulation by aldosterone J Comp Neurol 1999 405 406 420 10076935 10.1002/(SICI)1096-9861(19990315)405:3<406::AID-CNE10>3.0.CO;2-F 
Mirshahi M Nicolas C Mirshahi S Golestaneh N d'Hermies F Agarwal MK  Immunochemical analysis of the sodium channel in rodent and human eye Exp Eye Res 1999 69 21 32 10375446 10.1006/exer.1999.0675 
Trujillo E Alvarez dlR Mobasheri A Gonzalez T Canessa CM Martin-Vasallo P  Sodium transport systems in human chondrocytes. II. Expression of ENaC, Na+/K+/2Cl- cotransporter and Na+/H+ exchangers in healthy and arthritic chondrocytes Histol Histopathol 1999 14 1023 1031 10506918 
Brouard M Casado M Djelidi S Barrandon Y Farman N  Epithelial sodium channel in human epidermal keratinocytes: expression of its subunits and relation to sodium transport and differentiation J Cell Sci 1999 112 3343 3352 10504339 
Garty H Palmer LG  Epithelial sodium channels: function, structure, and regulation Physiol Rev 1997 77 359 396 9114818 
Fyfe GK Quinn A Canessa CM  Structure and function of the Mec-ENaC family of ion channels Semin Nephrol 1998 18 138 151 9541269 
Matthay MA Folkesson HG Verkman AS  Salt and water transport across alveolar and distal airway epithelia in the adult lung Am J Physiol 1996 270 L487 L503 8928808 
Berthiaume Y  Weir EK, Reeves JT  Mechanisms of edema clearance In Pulmonary edema 1998 Armonk: Futura Publishing Company, inc 77 94 
Folkesson HG Norlin A Baines DL  Salt and water transport across the alveolar epithelium in the developing lung: correlations between function and recent molecular biology advances (review) Int J Mol Med 1998 2 515 531 9858647 
Hummler E Barker P Gatzy J Beermann F Verdumo C Schmidt A  Early death due to defective neonatal lung liquid clearance in alphaENaC-deficient mice Nature Genet 1996 12 325 328 8589728 10.1038/ng0396-325 
Berthiaume Y Folkesson HG Matthay MA  Lung edema clearance: 20 years of progress: invited review: alveolar edema fluid clearance in the injured lung J Appl Physiol 2002 93 2207 2213 12433940 
Dumasius V Sznajder JI Azzam ZS Boja J Mutlu GM Maron MB  Beta(2)-adrenergic receptor overexpression increases alveolar fluid clearance and responsiveness to endogenous catecholamines in rats Circ Res 2001 89 907 914 11701618 
Minakata Y Suzuki S Grygorczyk C Dagenais A Berthiaume Y  Impact of the beta-adrenergic agonist terbutaline on Na+ channel and Na+-K+-ATPase expression in alveolar type II cells Am J Physiol 1998 275 L414 L422 9700104 
Dagenais A Denis C Vives MF Girouard S Masse C Nguyen T  Modulation of alpha-ENaC and alpha(1)-Na+-K+-ATPase by cAMP and dexamethasone in alveolar epithelial cells Am J Physiol Lung Cell Mol Physiol 2001 281 L217 L230 11404265 
Finley N Norlin A Baines DL Folkesson HG  Alveolar epithelial fluid clearance is mediated by endogenous catecholamines at birth in guinea pigs J Clin Invest 1998 101 972 981 9486967 
Pitkänen O Tanswell AK Downey G O'Brodovich H  Increased PO2 alters the bioelectric properties of fetal distal lung epithelium Am J Physiol 1996 270 L1060 L1066 8764233 
Yue G Russel WJ Benos DJ Jackson RM Olman MA Matalon S  Increased expression and activity of sodium channels in alveolar type II cells of hyperoxic rats Proc Natl Acad Sci U S A 1995 92 8418 8422 7667305 
Planès C Escoubet B Blot-Chabaud M Friedlander G Farman N Clerici C  Hypoxia downregulates expression and activity of epithelial sodium channels in rat alveolar epithelial cells Am J Respir Cell Mol Biol 1997 17 508 518 9376126 
Wodopia R Ko HS Billian J Wiesner R Bartsch P Mairbaurl H  Hypoxia decreases proteins involved in epithelial electrolyte transport in A549 cells and rat lung Am J Physiol Lung Cell Mol Physiol 2000 279 L1110 L1119 11076801 
Hanukoglu A Bistritzer T Rakover Y Mandelberg A  Pseudohypoaldosteronism with increased sweat and saliva electrolyte values and frequent lower respiratory tract infections mimicking cystic fibrosis J Pediatr 1994 125 752 755 7965429 
Marthinsen L Kornfalt R Aili M Andersson D Westgren U Schaedel C  Recurrent Pseudomonas bronchopneumonia and other symptoms as in cystic fibrosis in a child with type I pseudohypoaldosteronism Acta Paediatr 1998 87 472 474 9628311 10.1080/08035259850157147 
Schaedel C Marthinsen L Kristoffersson AC Kornfalt R Nilsson KO Orlenius B  Lung symptoms in pseudohypoaldosteronism type 1 are associated with deficiency of the α-subunit of the epithelial sodium channel J Pediatr 1999 135 739 745 10586178 
Boucher RC Cotton CU Gatzy JT Knowles MR Yankaskas JR  Evidence for reduced Cl- and increased Na+ permeability in cystic fibrosis human primary cell cultures J Physiol (Lond) 1988 405 77 103 3255805 
Boucher RC Stutts MJ Knowles MR Cantley L Gatzy JT  Na+ transport in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation J Clin Invest 1986 78 1245 1252 3771796 
Stutts MJ Canessa CM Olsen JC Hamrick M Cohn JA Rossier BC  CFTR as a cAMP-dependent regulator of sodium channels Science 1995 269 847 850 7543698 
Stutts MJ Rossier BC Boucher RC  Cystic fibrosis transmembrane regulator inverts protein kinase A-mediated regulation of epithelial sodium channel single channel kinetics J Biol Chem 1997 272 14037 14040 9162024 10.1074/jbc.272.22.14037 
Wilson R Dowling RB  Lung infections. 3. Pseudomonas aeruginosa and other related species Thorax 1998 53 213 219 9659359 
Craven DE Steger KA  Ventilator-associated bacterial pneumonia: challenges in diagnosis, treatment, and prevention New Horiz 1998 6 S30 S45 9654310 
Bye MR Ewig JM Quittel LM  Cystic Fibrosis Lung 1994 172 251 270 7934150 10.1007/BF00164308 
Collins FS  Cystic fibrosis: molecular biology and therapeutic implications Science 1992 256 774 779 1375392 
Wiener-Kronish JP Sakuma T Kudoh I Pittet JF Frank D Dobbs L  Alveolar epithelial injury and pleural empyema in acute P. aeruginosa pneumonia in anesthetized rabbits J Appl Physiol 1993 75 1661 1669 8282618 
Gosselin D DeSanctis J Boulé M Skamene E Matouk C Radzioch D  Role of tumor necrosis factor alpha in innate resistance to mouse pulmonary infection with Pseudomonas aeruginosa Infect Immun 1995 63 3272 3278 7642255 
Morissette C Skamene E Gervais F  Endobronchial inflammation following Pseudomonas aeruginosa infection in resistant and susceptible strains of mice Infect Immun 1995 63 1718 1724 7729877 
Evans DJ Matsumoto PS Widdicombe JH Li-Yun C Maminishkis aA Miller SS  Pseudomonas aeruginosa induces changes in fluid transport across airway surface epithelia Am J Physiol 1998 275 C1284 C1290 9814977 
Church GM Gilbert W  Genomic sequencing Proc Natl Acad Sci USA 1984 81 1991 1995 6326095 
Shull GE Greeb J Lingrel JB  Molecular cloning of three distinct forms of the Na+, K+-ATPase alpha-subunit from rat brain Biochemistry 1986 25 8125 8132 3028470 
Gosselin D Stevenson MM Cowley EA Griesenbach U Eidelman DH Boulé M  Impaired ability of Cftr knockout mice to control lung infection with Pseudomonas aeruginosa Am J Respir Crit Care Med 1998 157 1253 1262 9563748 
Sugita M Ferraro P Dagenais A Clermont ME Barbry P Michel RP  Alveolar liquid clearance and sodium channel expression are decreased in transplanted canine lungs Am J Respir Crit Care Med 2003 167 1440 1450 12738601 10.1164/rccm.200204-312OC 
Towne JE Harrod KS Krane CM Menon AG  Decreased expression of aquaporin (AQP)1 and AQP5 in mouse lung after acute viral infection Am J Respir Cell Mol Biol 2000 22 34 44 10615063 
Folkesson HG Nitenberg G Oliver BL Jayr C Albertine KH Matthay MA  Upregulation of alveolar epithelial fluid transport after subacute lung injury in rats from bleomycin Am J physiol 1998 275 L478 L490 9728042 
Firsov D Gautschi I Merillat A-M Rossier BC Schild L  The heterotetrameric architecture of the epithelial sodium channel (ENaC) EMBO J 1998 17 344 352 9430626 10.1093/emboj/17.2.344 
Kosari F sheng S Li J Mak D-OD Foskett JK Kleyman TR  Subunit stoichiometry of the epithelial sodium channel J Biol Chem 1998 273 13469 13474 9593680 10.1074/jbc.273.22.13469 
Eskandari S Snyder PM Kreman M Zampighi GA Welsh MJ Wright EM  Number of subunits comprising the epithelial sodium channel J Biol Chem 1999 274 27281 27286 10480948 10.1074/jbc.274.38.27281 
Snyder PM Cheng C Prince LS Rogers JC Welsh MJ  Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits J Biol Chem 1998 273 681 684 9422716 10.1074/jbc.273.2.681 
McNicholas CM Canessa CM  Diversity of channels generated by different combinations of epithelial sodium channel subunits J Gen Physiol 1997 109 681 692 9222895 10.1085/jgp.109.6.681 
Fyfe GK Canessa CM  Subunit composition determines the single channel kinetics of the epithelial sodium channel J Gen Physiol 1998 112 423 432 9758861 10.1085/jgp.112.4.423 
Barker PM Nguyen MS Gatzy JT Grubb B Norman H Hummler E  Role of gamma-ENaC subunit in lung liquid clearance and electrolyte balance in newborn mice. Insights into perinatal adaptation and pseudohypoaldosteronism J Clin Invest 1998 102 1634 1640 9788978 
McDonald FJ Yang B Hrstka RF Drummond HA Tarr DE McCray PB Jr Disruption of the beta subunit of the epithelial Na+ channel in mice: Hyperkalemina and neonatal death associated with pseudohypoaldosteronism phenotype Proc Natl Acad Sci USA 1999 96 1727 1731 9990092 10.1073/pnas.96.4.1727 
Mall M Grubb BR Harkema JR O'Neal WK Boucher RC  Increased airway epithelial Na(+) absorption produces cystic fibrosis-like lung disease in mice Nat Med 2004 10 487 493 15077107 10.1038/nm1028 
Hummler E Barker P Talbot C Wang Q Verdumo C Grubb B  A mouse model for the renal salt-wasting syndrome pseudohypoaldosteronism Proc Natl Acad Sci USA 1997 94 11710 11715 9326675 10.1073/pnas.94.21.11710 
Olivier R Scherrer U Horisberger JD Rossier BC Hummler E  Selected contribution: limiting Na(+) transport rate in airway epithelia from alpha-ENaC transgenic mice: a model for pulmonary edema J Appl Physiol 2002 93 1881 1887 12381779 
Egli M Duplain H Lepori M Cook S Nicod P Hummler E  Defective Respiratory Amiloride Sensitive Sodium Transport Predisposes To Pulmonary Oedema and Delays Its Resolution In Mice J Physiol 2004 560 857 865 15308680 10.1113/jphysiol.2004.066704 
Rezaiguia S Garat C Delclaux C Meignan M Fleury J Legrand P  Acute bacterial pneumonia in rats increases alveolar epithelial fluid clearance by a tumor necrosis factor-alpha-dependent mechanism J Clin Invest 1997 99 325 335 9006001 
Viget NB Guery BP Ader F Neviere R Alfandari S Creuzy C  Keratinocyte growth factor protects against Pseudomonas aeruginosa- induced lung injury Am J Physiol Lung Cell Mol Physiol 2000 279 L1199 L1209 11076810 
Kunzelmann K Beesley AH King NJ Karupiah G Young JA Cook DI  Influenza virus inhibits amiloride-sensitive Na+ channels in respiratory epithelia Proc Natl Acad Sci U S A 2000 97 10282 10287 10920189 10.1073/pnas.160041997 
Cloutier MM Wong D Ogra PL  Respiratory syncytial virus alters electrophysiologic properties in cotton rat airway epithelium Pediatr Pulmonol 1989 6 164 168 2654848 
Lambert LC Trummell HQ Singh A Cassell GH Bridges RJ  Mycoplasma pulmonis inhibits electrogenic ion transport across murine tracheal epithelial cell monolayers Infect Immun 1998 66 272 279 9423868 
Zhang M Jion Kim K IYer D Lin Y Belisle J McEnery K  Effects of Mycobacterium tuberculosis on the bioelectric properties of the alveolar epithelium Infect Immun 1997 65 692 698 9009332 
Stutts MJ Schwab JH Chen MG Knowles MR Boucher RC  Effects of Pseudomonas aeruginosa on bronchial epithelial ion transport Am Rev Respir Dis 1986 134 17 21 2425672 
Towne JE Krane CM Bachurski CJ Menon AG  Tumor necrosis factor-alpha inhibits aquaporin 5 expression in mouse lung epithelial cells J Biol Chem 2001 276 18657 18664 11279049 10.1074/jbc.M100322200 
Galietta LJ Folli C Marchetti C Romano L Carpani D Conese M  Modification of transepithelial ion transport in human cultured bronchial epithelial cells by interferon-gamma Am J Physiol Lung Cell Mol Physiol 2000 278 L1186 L1194 10835324 
Frank J Roux J Kawakatsu H Su G Dagenais A Berthiaume Y  TGF-β1 Decreases αENaC Expression and alveolar Epithelial Vectorial sodium and Fluid Transport via an ERK 1/2-dependent Mechanism J Biol Chem 2003 278 43939 43950 12930837 10.1074/jbc.M304882200

