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Am J Physiol Lung Cell Mol Physiol 294: L409-L416, 2008. First published November 16, 2007; doi:10.1152/ajplung.00307.2007
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EDITORIAL FOCUS

Low expression of the β-ENaC subunit impairs lung fluid clearance in the mouse

Nadia Randrianarison,1,2 Christine Clerici,1,2 Chrystophe Ferreira,3 Alexandre Fontayne,1,2 Sylvain Pradervand,4 Nicole Fowler-Jaeger,4 Edith Hummler,4 Bernard C. Rossier,4 and Carole Planès1,5

1Institut National de la Santé et de la Recherche Médicale U773, CRB3, 2Université Denis Diderot-Paris 7 and 3IFR02, Université Denis Diderot-Paris 7, Paris, France; 4Département de Pharmacologie et de Toxicologie, Université de Lausanne, Lausanne, Switzerland; and 5Université de Versailles Saint-Quentin, Versailles, France

Submitted 2 August 2007 ; accepted in final form 15 November 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transepithelial alveolar sodium (Na+) transport mediated by the amiloride-sensitive epithelial sodium channel (ENaC) constitutes the driving force for removal of fluid from the alveolar space. To define the role of the β-ENaC subunit in vivo in the mature lung, we studied a previously established mouse strain harboring a disruption of the β-ENaC gene locus resulting in low levels of β-ENaC mRNA expression. Real-time RT-PCR experiments confirmed that β-ENaC mRNA levels were decreased by >90% in alveolar epithelial cells from homozygous mutant (m/m) mice. β-ENaC protein was undetected in lung homogenates from m/m mice by Western blotting, but {alpha}- and {gamma}-ENaC proteins were increased by 83% and 45%, respectively, compared with wild-type (WT) mice. At baseline, Na+-driven alveolar fluid clearance (AFC) was significantly reduced by 32% in m/m mice. Amiloride at the concentration 1 mM inhibited AFC by 75% and 34% in WT and m/m mice, respectively, whereas a higher concentration (5 mM) induced a 75% inhibition of AFC in both groups. The β2-agonist terbutaline significantly increased AFC in WT but not in m/m mice. These results show that despite the compensatory increase in {alpha}- and {gamma}-ENaC protein expression observed in mutant mouse lung, low expression of β-ENaC results in a moderate impairment of baseline AFC and in decreased AFC sensitivity to amiloride, suggesting a possible change in the stoichiometry of ENaC channels. Finally, adequate β-ENaC expression appears to be required for AFC stimulation by β2-agonists.

pneumocytes; alveolar sodium transport; amiloride; cation channels

ACTIVE TRANSCELLULAR SODIUM (Na+) transport by alveolar epithelial cells (AEC) is a driving force for the reabsorption of fluid from the alveolar space and represents the primary mechanism for the resolution of alveolar edema (2, 3, 30, 31, 37). Sodium entry at the apical side of AEC is usually considered as the rate-limiting step for this process. There is abundant evidence that the apical amiloride-sensitive epithelial Na+ channels (ENaC; Refs. 5, 6) represent the major pathway for apical Na+ entry (18, 29–31), although other transporters such as the Na+-glucose cotransporter or cyclic nucleotide-gated (CNG) channels have also been involved (2, 23, 24). The three ENaC subunits, {alpha}-, β-, and {gamma}-, are expressed in both alveolar type I and type II epithelial cells (22, 23, 32), but the exact stoichiometry of the subunits forming a channel is controversial. Patch-clamp analysis established the presence of highly selective cation (HSC) channels in the apical membrane of both alveolar type I and type II cells (21, 23). These HSC channels have a unit conductance of 4–6 pS, a very high selectivity for Na+ over K+, and are inhibited by a low concentration of amiloride (<0.1 µM), consistent with the properties of ENaC channels composed of {alpha}-, β-, and {gamma}-subunits (14). However, alveolar type I and type II cells also contain nonselective cation (NSC) channels with a unit conductance of 19–21 pS, equal selectivity to Na+ and K+, and a lower sensitivity to amiloride (21, 23, 29). Studies using antisense oligonucleotides to any of the three ENaC subunits suggested that these NSC channels could be made of {alpha}-ENaC subunits alone (20, 21).

The pivotal role of {alpha}-ENaC in the distal lung epithelium has been confirmed by animal studies. Newborn mice with complete inactivation of Scnn1a ({alpha}-ENaC) gene exhibit abolition of lung Na+ transport and subsequent fatal respiratory distress at birth from failure to clear their lungs from fluid (18), whereas adult mice with reduced {alpha}-ENaC expression exhibit decreased alveolar fluid clearance (AFC) and delayed resolution of alveolar edema (11, 25). By contrast, the relative importance of β- or {gamma}-ENaC subunits in the lung is less well-established. Complete inactivation of either Scnn1b (β-ENaC; Ref. 27) or Scnn1c ({gamma}-ENaC; Ref. 1) genes in the mouse only mildly delayed fetal lung fluid clearance at birth but induced severe renal dysfunction leading to neonatal death. This indicates that both β- and {gamma}-ENaC subunits are essential for renal function but not for the transition from fluid-filled to air-filled lung at birth. Of course, the mature lung phenotype of these knockout mice could not be analyzed due to their premature death. A new insight in the role of β- and {gamma}-ENaC in the lung was recently provided by the study by Mall et al. (28) in which any of ENaC subunit genes was specifically overexpressed in mouse airways. Interestingly, it was found that overexpression of the Scnn1b gene, but not of Scnn1a or Scnn1c genes, led to increased ENaC activity and transepithelial Na+ transport in the airways. This suggests that β-ENaC subunit expression could be rate-limiting in the mouse lung, at least in the airways.

Therefore, the aim of the present in vivo study was to clarify the functional role of β-ENaC subunit in the mature lung alveolar Na+ and water transport. To do so, we used a previously established knock-in mouse strain expressing low levels of β-ENaC subunit (34). This mouse strain was obtained by B. Rossier and E. Hummler's group in the course of generating a mouse model for Liddle's syndrome (35) by inserting a stop codon (corresponding to residue R566 in human β-ENaC) and the selection marker neomycin in the exon 13 of Scnn1b gene. Unexpectedly, the disruption of the Scnn1b gene locus resulted in a >90% decrease in β-ENaC mRNA levels in all organs tested including the lung, kidney, and colon (34). Homozygous β-ENaC mutant (m/m) mice only show a small delay in lung fluid clearance at birth but grow normally when fed with normal salt diet. However, they exhibit a severe pseudohypoaldosteronism type 1 phenotype with renal salt loss on low-salt diet. Their mature lung phenotype has not been studied to date. Therefore, we investigated whether low expression of β-ENaC subunit in adult mutant m/m mice would affect lung fluid balance and Na+-driven AFC under basal and β2-agonist-stimulated conditions. Our data show that low expression of β-ENaC 1) induces a compensatory increase in {alpha}- and {gamma}-ENaC subunits in the distal lung, 2) moderately impairs AFC at baseline but decreases AFC sensitivity to amiloride, and 3) abolishes AFC stimulation by β2-agonists.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transgenic mice. The generation of mice with a disruption of the Scnn1b gene locus due to the insertion of a stop codon and the selection marker neomycin has been described previously (34). Animals were housed in standard cages and light conditions and fed standard diet and water ad libitum. Heterozygous mutant +/m mice were intercrossed, and the resulting offspring were genotyped by PCR on genomic tail DNA at 3 wk of age as previously described (34). Experiments were performed on litter-matched wild-type (WT; β-ENaC +/+) and β-ENaC m/m mice aged 2–6 mo. The investigators were blinded to genotype information for all comparative measurements. The total number of mice used in this study was 145. The experiments were approved by Université Paris 7 review board on animal experimentation and were in accordance with animal welfare guidelines (Ministère Français de la Pêche et de l'Agriculture, agreement no. 75-1045).

Lung histology. Lung histology was performed as previously described (9). Animals were euthanized with intraperitoneal pentobarbital (250 mg/kg). The trachea was cannulated and connected to a syringe before a thoracotomy was performed. Lungs were inflated with 4% paraformaldehyde at a pressure of 20 cmH2O before the trachea was tied. The heart and lungs were removed en bloc and placed in 20 ml of 4% paraformaldehyde overnight. The lungs were embedded with paraffin, and sections were cut at 4-µm thickness stained with hematoxylin and eosin.

Determination of the bloodless wet-to-dry lung weight ratio. The amount of extravascular lung water (i.e., interstitial and alveolar water) was assessed at baseline by the bloodless wet-to-dry lung weight ratio as previously described (36). Briefly, mice were anesthetized as described above and killed by exsanguination. A blood sample was obtained for measurement of hemoglobin concentration. Lungs were removed and homogenized for measurement of lung homogenate supernatant hemoglobin concentration, and lung homogenate was placed in an incubator at 80°C for 24 h for desiccation. The bloodless wet-to-dry lung weight ratio was calculated using standard methods (4, 13).

Isolation of mouse bronchiolar-alveolar epithelial cells. Mouse bronchiolar-alveolar epithelial cells (BAEC) were isolated from mice aged 2 mo by dispase digestion of lung tissue followed by sequential filtration and differential adherence on culture dishes coated with rat anti-mouse CD45 and rat anti-mouse CD16/32 (BD Pharmingen) as previously described (8, 36). The yield was 4–5 x 106 BAEC per mouse with a percentage of alveolar type II cells (as assessed by phosphine 3R staining) ≥78% and a cell viability >95%. BAEC were used immediately after isolation for RNA extraction.

Reverse transcription and real-time RT-PCR analysis. Total RNA was prepared from freshly isolated mouse BAEC using the RNeasy extraction kit (Qiagen, Hilden, Germany) and processed as previously described (36). The RNAs (2.5 µg per sample) were reverse-transcribed at 37°C for 1 h using the SuperScript II RNase H reverse transcriptase (Invitrogen, Basel, Switzerland) and random primers. Two microliters of the first-strand cDNA reaction was amplified using a Roche LightCycler and the 2x QuantiTect SYBR Green PCR Master Mix (Qiagen) according to the manufacturer's guidelines. β-Actin was used as internal control. The sequences of the primers were as follows: mouse {alpha}-ENaC, sense (S): 5'-CGGAGTTGCTAAACTCAACATC-3' (position +1638 to +1659; 3' of the translation start); antisense (AS): 5'-TGGAGACCAGTACCGGCT-3' (position +1849 to +1866; 3' of the translation start); mouse β-ENaC, S: 5'-ATGTGGTTCCTGCTTACGCTG-3' (position +151 to +171; 3' of the translation start); AS: 5'-GTCCTGGTGGTGTTGCTGTG-3' (position +397 to +416; 3' of the translation start); mouse {gamma}-ENaC, S: 5'-CCAAAGCCAGCAAATAAACAAA-3' (position +1479 to +1500; 3' of the translation start); AS: 5'-GCGGCGGGCAATAATAGAGA-3' (position +1691 to +1710; 3' of the translation start); mouse β-actin, S: 5'-CGGAGTTGCTAAACTCAACATC-3' (position +412 to +432; 3' of the translation start); AS: 5'-TGTCACGCACGATTTCCC-3' (position +697 to +714; 3' of the translation start). As negative control, reverse transcription was performed in the absence of enzyme and amplified by PCR.

Western blot analysis. Animals were euthanized with intraperitoneal pentobarbital (250 mg/kg) before a thoracotomy was performed. The lungs were removed and immediately homogenized for 3 min in ice-cold lysis RIPA buffer (pH 8) containing 20 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, and protease inhibitors. The lysate was centrifuged (15,000 rpm, 10 min) at 4°C, and supernatants were aliquoted and immediately frozen before use. For Western blotting, samples of protein extracts (100–200 µg) were diluted in 1 volume of sample Laemmli buffer [10% glycerol, 6.25 mM Tris·HCl (pH 6.8), 0.1% SDS, 5% 2-mercaptoethanol, and 0.01% bromophenol blue in H2O], resolved through 8% acrylamide gels, electrically transferred to nitrocellulose paper, and subsequently probed for {alpha}-, β-, or {gamma}-ENaC subunits. The following two antibodies against β-ENaC were used: a previously characterized rabbit polyclonal antibody against the COOH terminus of rat β-ENaC (dilution 1:2,000; Ref. 10) and an affinity-purified rabbit polyclonal antibody against a fragment of the NH2 terminus of rat β-ENaC showing 100% sequence homology with corresponding mouse fragment (dilution 1:120; Alpha Diagnostic International, San Antonio, TX). Rabbit polyclonal anti-rat {alpha}-ENaC and anti-rat {gamma}-ENaC antibodies (10, 36) were used at the dilution 1:2,000, and mouse monoclonal anti-β-actin were used at the dilution 1:1,000 (Sigma, St. Louis, MO). The anti-rabbit IgG secondary antibody (Amersham Pharmacia Biotech) was used at dilution 1:5,000, and the anti-mouse IgG (Sigma) was used at the dilution 1:10,000. The signal was developed with the ECL Plus system (Amersham Pharmacia Biotech). Quantification of ENaC subunits and β-actin levels was obtained using NIH Image software.

Measurement of AFC. Sodium-driven AFC was measured in vivo using an in situ model of mouse lung as previously described (15, 33, 36). This model has been shown to give AFC values similar to those obtained with the ventilated mouse model over a 15-min period (15). Briefly, male or female WT or m/m mice aged 2–5 mo were euthanized with intraperitoneal pentobarbital (250 mg/kg) and maintained at 37–38°C using a heating pad, an infrared lamp, and an intraabdominal monitoring thermistor. A 20-gauge venous catheter was inserted in the trachea through a tracheotomy and tightly fixed. The lungs were inflated with 100% O2 at 7 cmH2O continuous positive airway pressure throughout the experiment. Then, 10 ml/kg of instillate was delivered to the lungs over 30 s through the tracheal catheter. The instillate consisted of Ringer lactate solution (pH 7.4) adjusted to 325 mosmol/kgH2O with NaCl and containing 5% BSA and 0.1 µCi/ml 125I-albumin (Cis Bio International, Gif-sur-Yvette, France) as a labeled alveolar fluid volume tracer. An alveolar fluid sample (50–100 µl) was aspirated 1 min after instillation and at the end of experiment (15 min later). The aspirates were centrifuged at 3,000 g for 10 min, and the radioactivity in supernatants was counted in duplicate. AFC (percentage fluid absorption at 15 min) was calculated from the increase in alveolar fluid albumin as AFC (%) = (Cf – Ci)/Cf x 100, where Ci and Cf represent the initial and final concentrations of 125I-albumin in the aspirate at 1 and 15 min, respectively, as assessed by radioactivity measurements.

In some experiments, amiloride (final concentration 1 or 5 mM), pimozide (final concentration 1.5 10–4 M), or terbutaline (final concentration 10–4 M) were added to the instillate, and AFC was measured at 15 min as described above.

Statistical analysis. Results are presented as means ± SD. For functional data, one-way or two-way variance analyses (ANOVA) were performed, and, when allowed by the F value, results were compared by the modified least significant difference (StatView software; Abacus Concepts, Berkeley, CA). For Western blot experiments, differences between groups were evaluated with unpaired t-test performed on raw densitometric data. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Lung histology. Lung macroscopic appearance was similar in WT and β-ENaC m/m mice (n = 3 per group). Histological examination of the lungs revealed no morphological difference between the two groups (Fig. 1). In particular, the histological aspect of the bronchioles, alveolar ducts, alveolar epithelium, and blood vessels appeared normal in β-ENaC m/m mice. No aspect of interstitial or alveolar edema could be detected in m/m mouse lung.


Figure 1
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  		Fig. 1. A and B: photomicrographs of the distal lung in wild-type and β-amiloride-sensitive epithelial sodium channel (β-ENaC) homozygous mutant (m/m) mice. Photomicrographs of representative lung sections of wild-type (+/+; A) and m/m (B) littermates. The morphological aspect of blood vessels (V), bronchioles (BR), alveolar ducts (AD), and alveoli was normal in m/m mice.

 
Lung fluid balance at baseline. Lung fluid balance at baseline was estimated in WT and β-ENaC m/m mice by calculation of the bloodless wet-to-dry lung weight ratio, reflecting the amount of extravascular lung water. The bloodless wet-to-dry lung weight ratio was not significantly different (NS) in β-ENaC m/m mice compared with WT mice (3.60 ± 1.43 vs. 3.54 ± 1.06 in WT and m/m, respectively; n = 4–5 per group; NS), indicating the absence of pulmonary edema in mutant mice.

Expression of ENaC subunit mRNA transcripts in BAEC. The levels of expression of ENaC subunit mRNA transcripts were evaluated by real-time RT-PCR in BAEC freshly isolated from β-ENaC m/m or WT mice. As expected, the expression level of β-ENaC mRNA transcripts (relative to β-actin mRNA) was dramatically decreased in β-ENaC m/m BAEC, representing 7% of control (Fig. 2). The levels of {alpha}-ENaC mRNA transcripts expressed in BAEC were not significantly different in β-ENaC m/m and WT mice (Fig. 2). By contrast, the expression level of {gamma}-ENaC mRNA transcripts was significantly decreased by 50% in BAEC from β-ENaC m/m mice compared with WT (Fig. 2).


Figure 2
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  		Fig. 2. Expression of ENaC mRNA transcripts in bronchiolar-alveolar epithelial cells (BAEC) from wild-type and β-ENaC m/m mice. BAEC were isolated from wild-type (+/+) and m/m littermates. Total RNA was extracted from freshly isolated cells and reverse-transcribed. Each sample was amplified by real-time RT-PCR with sets of primers specific for mouse {alpha}-ENaC, β-ENaC, {gamma}-ENaC, and β-actin as described in MATERIALS AND METHODS. Data were normalized for the corresponding β-actin signal in each sample. Results are expressed as the ratio of {alpha}-, β-, or {gamma}-ENaC mRNA-to-β-actin mRNA and represent means ± SD (4–5 mice per group). Black bars, wild-type (+/+) group; gray bars, m/m group. ***Significantly different from +/+ group (P < 0.001).

 
Expression of ENaC subunit proteins in the distal lung. A rabbit polyclonal affinity-purified antibody raised against a fragment of NH2 terminus of rat β-ENaC was used to study the expression of both WT β-ENaC protein and mutant β-ENaC that lacks part of the COOH terminus. As shown in Fig. 3A, both this NH2 terminus antibody and a well-characterized anti-rat β-ENaC rabbit polyclonal antibody raised against the COOH terminus of the protein (10) detected a band of molecular mass 95 kDa in WT lung homogenate extracts, corresponding to full-length glycosylated β-ENaC protein (lanes 1 and 2). This 95-kDa band was not detected with the NH2 terminus antibody in m/m lung homogenates, and no additional band could be seen (lanes 3 and 4), indicating that expression of mutant β-ENaC protein was very low, below the threshold of detection by immunoblotting.


Figure 3
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  		Fig. 3. A–C: Western blot analysis of ENaC subunit proteins in distal lung homogenates from wild-type and β-ENaC m/m mice. Lung homogenate extracts from wild-type (+/+) and m/m littermates were submitted to SDS-PAGE followed by immunoblotting with antibodies raised against ENaC subunits as described in MATERIALS AND METHODS. A: representative immunoblots showing the expression of β-ENaC subunit and of the intracellular protein β-actin. The gel was probed with either a rabbit polyclonal antibody raised against a fragment of rat β-ENaC COOH terminus (anti-β-ENaC C-term Ab, lane 1) or a rabbit polyclonal affinity-purified antibody raised against a fragment of rat β-ENaC NH2 terminus (anti-β-ENaC N-term Ab, lanes 2–4). A band of molecular mass 95-kDa (indicated by *) was detected by both antibodies in +/+ extracts (lanes 1 and 2). No specific band was detected in m/m extracts using the anti-β-ENaC N-term Ab (lanes 3 and 4). B: representative immunoblots showing the expression of {alpha}-ENaC subunit (left), {gamma}-ENaC subunit (right), and the intracellular protein β-actin. C: quantification of {alpha}-ENaC (left) and {gamma}-ENaC (right) signals in distal lung homogenates from +/+ (black bars) and m/m (gray bars) mice was obtained using NIH Image software, and data were normalized for the corresponding β-actin signal. Results are expressed as the ratio of {alpha}- or {gamma}-ENaC-to-β-actin and represent means ± SD (4–6 animals per group). * And **, significantly different from +/+ group (P < 0.05 and P < 0.01, respectively).

 
Protein expression of {alpha}- and {gamma}-ENaC subunits was also evaluated in distal lung homogenates from β-ENaC m/m or WT mice by Western blotting using previously characterized rabbit polyclonal antibodies anti-rat {alpha}- and {gamma}-ENaC (10, 36). As shown in Fig. 3B, immunoblotting with anti-{alpha}-ENaC revealed a major band migrating at 85–90 kDa and a minor band at 65 kDa, the specificity of which had been previously verified (32). Immunoblotting with anti-{gamma}-ENaC revealed a major band migrating at 75 kDa and a minor band around 90 kDa as previously reported (17, 36). Quantitative analysis showed a significant 1.83-fold increase in the amount of {alpha}-ENaC protein (relative to β-actin protein) and a significant 1.45-fold increase in the amount of {gamma}-ENaC protein in the distal lung from β-ENaC m/m mice compared with WT mice (Fig. 3C).

AFC under basal condition. Sodium-driven AFC was assessed under basal condition in WT and β-ENaC m/m mice using an in situ nonventilated mouse lung model (Fig. 4). Basal AFC was significantly decreased by 32% in β-ENaC m/m mice compared with WT mice (P < 0.01). Addition of amiloride in the alveolar instillate (final concentration 1 mM) decreased AFC by 75% in the WT group but only by 34% in the β-ENaC m/m group. The amiloride-insensitive component of AFC was significantly increased in β-ENaC m/m mice compared with WT mice (5.3% ± 1.76% vs. 2.9% ± 1.64% fluid cleared at 15 min; P < 0.05; Fig. 4A). The amiloride-sensitive component of AFC (calculated as the difference between mean basal AFC value and mean AFC value in the presence of 1 mM amiloride) was 8.9% ± 2.87% and 2.7% ± 2.10% of fluid cleared at 15 min in WT and m/m mice, respectively (P < 0.001).


Figure 4
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  		Fig. 4. Alveolar fluid clearance under basal condition in wild-type and β-ENaC m/m mice. Sodium-driven alveolar fluid clearance was measured at baseline (Basal) over a 15-min period in wild-type (+/+; black bars) and m/m (gray bars) littermates aged 2–5 mo at 37°C using an in situ nonventilated model in which the air space was instilled with an isoosmolar Ringer lactate solution containing 125I-albumin as a volume marker as described in MATERIALS AND METHODS. Where indicated, amiloride (final concentration 1 or 5 mM; A) or pimozide (final concentration 1.5 10–4 M; B) were added to the instillate. Results are expressed as percentage fluid absorption at 15 min and represent means ± SD of 6–13 mice per condition for basal and pimozide experiments and of 4–6 mice per condition for amiloride experiments. *, **, And ***, significantly different from wild-type (+/+) group (P < 0.05, P < 0.01, and P < 0.001, respectively); # and ###, significantly different from corresponding basal value (P < 0.05 and P < 0.001, respectively).

 
This increase in the amiloride (1 mM)-insensitive part of AFC could be due either to a decrease in ENaC sensitivity to amiloride in the lungs from m/m mice or to the compensatory increase of a Na+-transport system distinct from ENaC like, for instance, the CNG cationic channels (23, 24). To address this question, we first tested the effect of 5 mM amiloride (final concentration) in the instillate. Amiloride (5 mM) reduced AFC by 75% in WT mice, an inhibition of the same magnitude as 1 mM amiloride, and by 74% in m/m mice (Fig. 4A). The amiloride (5 mM)-insensitive component of AFC was comparable in the two groups (2.9% ± 1.62% vs. 2.2% ± 2.28% fluid cleared at 15 min in WT and m/m mice, respectively; NS). Two-way ANOVA showed a significant interaction between the genotype and the effect of amiloride concentration (P < 0.05). The amiloride (5 mM)-sensitive part of AFC was 8.9% ± 2.97% and 5.8% ± 2.24% of fluid cleared at 15 min in WT and m/m mice, respectively (P < 0.01).

Second, we tested the effect of the CNG blocker pimozide. Pimozide (final concentration in the instillate 1.5 10–4 M) significantly decreased AFC in WT and m/m mice (Fig. 4B). The pimozide-sensitive component of AFC (calculated as the difference between mean basal AFC value and mean AFC value in the presence of pimozide) was comparable in both groups (4.0% ± 2.78% vs. 4.23% ± 2.96% of fluid cleared at 15 min in WT and m/m mice, respectively; NS).

Effect of β2-agonist stimulation on AFC. AFC was also measured under β2-agonist-stimulated condition (Fig. 5). Terbutaline (10–4 M in the alveolar instillate) increased AFC by almost 60% in WT mice (P < 0.001). By contrast, in β-ENaC m/m mice, the value of AFC following terbutaline treatment was not significantly different from the basal value (9.7% ± 2.08% vs. 8.0% ± 2.23% fluid cleared at 15 min under terbutaline-treated and basal conditions, respectively; NS).


Figure 5
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  		Fig. 5. Alveolar fluid clearance under β2-agonist-stimulated condition in wild-type and β-ENaC m/m mice. Sodium-driven alveolar fluid clearance was measured at baseline (Basal) over a 15-min period in wild-type (+/+; black bars) and m/m (gray bars) littermates aged 2–5 mo at 37°C using an in situ nonventilated model in which the air space was instilled with an isoosmolar Ringer lactate solution containing 125I-albumin as a volume marker as described in MATERIALS AND METHODS. When indicated, terbutaline (final concentration 10–4 M) was added to the instillate. Results are expressed as percentage fluid absorption at 15 min and represent means ± SD of 6–13 mice per condition. ** And ***, significantly different from respective control group as indicated (P < 0.01 and P < 0.001, respectively); NS, not significantly different.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study was designed to evaluate in vivo the functional role of β-ENaC subunit in the mature distal lung by analyzing mice expressing low levels of the β-ENaC gene. Our data show that the dramatic decrease in β-ENaC mRNA and protein levels observed in the distal lung from adult β-ENaC mutant m/m mice was associated with a compensatory increase in {alpha}- and {gamma}-ENaC proteins. Distal lung histology and fluid homeostasis appeared normal at baseline in mutant mice. However, Na+-driven AFC was reduced by one-third in m/m mice compared with WT due to a decrease in the amiloride-sensitive component of the clearance reflecting impaired ENaC activity. Pharmacological experiments revealed that the sensitivity of AFC to amiloride was decreased in m/m mice, suggesting a change in the stoichiometry of ENaC channels expressed in AEC. Finally, the stimulation of AFC by the β2-agonist terbutaline was abolished in mice expressing low levels of the β-ENaC subunit.

β-ENaC mutant m/m mice have been previously shown to express very low levels of β-ENaC mRNA (34). The most plausible explanation for this is that the disruption of the Scnn1b gene resulting from the insertion of the neomycin-resistance gene is responsible for destabilization and/or degradation of mutated β-ENaC mRNA transcripts (34). Our molecular data confirmed that β-ENaC expression was dramatically decreased at both the mRNA and the protein level in mutant mouse distal lung, representing <10% of WT. Thus β-ENaC mutant mice represent an interesting animal model to study specifically the functional role of the β-ENaC subunit in the mature distal lung. To characterize the lung phenotype of these mice, we first studied distal lung histology and lung fluid homeostasis. Distal lung histology appeared normal in β-ENaC mutant mice. Likewise, the amount of extravascular lung water as depicted by the bloodless wet-to-dry lung weight ratio was similar in WT and mutant mice, indicating that low expression of β-ENaC did not compromise lung fluid balance at baseline. We also evaluated whether β-ENaC gene mutation would modify the expression of other ENaC subunits in distal lung epithelial cells. At the mRNA level, our data show that expression of {alpha}-ENaC mRNA transcripts was comparable in WT and mutant AEC, but, surprisingly, the level of {gamma}-ENaC mRNA transcripts was reduced by one-half in mutant AEC. This finding is in sharp contrast with the strong upregulation of {gamma}-ENaC mRNAs previously reported in the colon from these mice (34), confirming that the regulation of ENaC mRNA expression is tissue specific. At the protein level, both {alpha}- and {gamma}-ENaC subunits appeared to be markedly increased in mutant mouse distal lung, representing 183% and 145% of WT, respectively. The discrepancy between {alpha}- or {gamma}-ENaC mRNA levels, which are unchanged or even decreased, and the protein levels, which are clearly increased, indicates that ENaC subunit expression is mostly regulated at the posttranscriptional level in mutant mouse distal lung. Indeed, our protein data raise the possibility that increased expression of {alpha}- and {gamma}-ENaC subunits could, in some way, compensate for the decrease in β-ENaC subunit expression.

Next, we investigated whether low expression of β-ENaC subunit would affect Na+ and water transport across the alveolar epithelium at baseline. Functional in vivo experiments revealed that baseline AFC was reduced by one-third in β-ENaC m/m mice, the decrease being only due to a decrease in the amiloride-sensitive component of AFC reflecting ENaC-mediated fluid transport. Amiloride at the concentration 1 mM inhibited AFC by 75% in WT mice but by 34% only in mutant mice, so that the amiloride (1 mM)-insensitive AFC was almost doubled in m/m mice. Several mechanisms could be involved in this increase in amiloride-insensitive AFC in mutant mice. First, the impairment of ENaC activity due to β-ENaC mutation could result in a compensatory increase in Na+-transport pathways distinct from ENaC and not inhibited by amiloride. Among these alternative pathways are the Na+-glucose cotransporter, which could be responsible for as much as one-half of apical Na+ entry in rat AEC (2), and the pimozide-sensitive CNG channels, which are functionally expressed in rat type I AEC (23, 24). A role of the Na+-glucose cotransporter could be excluded here since there was no glucose in the solution instilled in distal air spaces. Moreover, a compensatory increase in CNG channel activity appeared very unlikely inasmuch as the pimozide-sensitive component of AFC was similar in WT and m/m mice. The second explanation for the increase in amiloride (1 mM)-insensitive AFC observed in mutant mice could be a change in Na+-channel sensitivity to amiloride block. In accordance with this hypothesis, we found that 5 mM amiloride inhibited AFC significantly more potently (–75%) than did 1 mM amiloride (–34%) in m/m mice. By contrast, 1 and 5 mM amiloride induced the same degree of AFC inhibition (–75%) in WT mice. These observations indicate that Na+-driven AFC in mutant mice was less sensitive to amiloride than in WT mice. Of course, amiloride concentrations at the millimolar range are far above the ENaC K0.5 for amiloride determined in vitro (<50 nM; Refs. 21, 23) and could block other Na+ transporters such as the Na+-proton antiporter. High concentrations are needed in vivo because an important fraction of the amiloride added to the alveolar instillate binds to the BSA present in the solution so that the effective concentration of free amiloride is much lower than 1 mM (~10–4 M; Ref. 16). Also, the Na+-proton antiport activity has been previously shown to be located exclusively on the basolateral surface of polarized rat AEC (26). Therefore, we assume that the Na+-proton antiporter was not involved in Na+-driven AFC and that the change in AFC inhibition by amiloride in mutant mice most likely reflected a change in Na+-channel sensitivity to the inhibitor.

Interestingly, former patch-clamp studies in rat lung using freshly isolated AEC or fresh lung slices demonstrated that alveolar type I and type II cells both express two types of functional Na+ channels differing by their amiloride sensitivity: the HSC channels and the NSC channels. HSC channels exhibit a high sensitivity to amiloride (K0.5 < 50 nM) and biophysical characteristics consistent with those of heterotetrameric channels composed of {alpha}-, β-, and {gamma}-ENaC subunits (14, 21, 23), whereas NSC channels have a much lower sensitivity to amiloride (K0.5 > 2 µM and up to ~1 mM in a recent study, Ref. 23). Jain et al. (20, 21) previously reported that treatment of cultured rat alveolar type II cells with {alpha}-ENaC antisense oligonucleotides markedly reduced the frequency of observing both HSC and NSC channels by patch-clamp. By contrast, treatment with β- or {gamma}-ENaC antisense reduced the frequency of observing HSC channels but also increased frequency of observing NSC channels (21). The authors therefore proposed that the combination of the three subunits was required to form HSC channels, whereas NSC channels could be composed of {alpha}-ENaC subunits alone. This theory is consistent with initial observations showing that {alpha}-ENaC subunits alone (or in association with β- or {gamma}-ENaC) were able to form amiloride-sensitive cation channels in the Xenopus laevis oocyte expression system, although the Na+ currents produced by these channels were much smaller than the currents obtained when the three subunits were coexpressed (6). Accordingly, we propose that the apparent change in AFC sensitivity to amiloride observed in mutant m/m mice could be due to a change in ENaC stoichiometry in the distal lung. Low expression of the β-ENaC subunit most likely decreased the number of HSC heterotetrameric ENaC channels at the surface of AEC and could lead to a compensatory increase in the number of NSC channels (made of {alpha}-ENaC alone or in combination with {gamma}-ENaC or another unidentified protein) less sensitive to amiloride. The increase in {alpha}-ENaC and {gamma}-ENaC protein expression observed in m/m mouse distal lung homogenates supports this hypothesis, which remains, however, clearly speculative inasmuch as we did neither perform patch-clamp experiments nor direct quantification of {alpha}- and {gamma}-ENaC at the cell surface of AEC.

The last point of the present study was to investigate whether the β-ENaC mutation would affect the response of AFC to β2-agonists. Our data show that the β2-agonist terbutaline increased AFC by 60% in WT mice but failed to significantly stimulate AFC in m/m mice. Stimulation of β2-adrenergic receptors, either by endogenous catecholamines or by β2-agonist drugs, induces a marked increase in alveolar Na+ transport and fluid clearance in vivo in most mammalian species (4, 30). In vitro it was demonstrated that β-adrenergic agonists stimulate Na+ channels in native rat AEC through a dual effect by increasing the number of HSC channels at the cell surface on one hand and by increasing the open probability of NSC channels on the other hand (7). Indeed, we previously reported that terbutaline increased cell surface expression of ENaC subunits in cultured rat AEC, especially that of β- and {gamma}-ENaC subunits, consistent with an increase in the number of HSC channels (32). Therefore, one plausible explanation for the lack of response to terbutaline in β-ENaC mutant mice is that the recruitment of new β-ENaC subunits at the cell surface was compromised due to the very low expression of the protein and that, as a result, the number of HSC channels could not increase. It was previously shown that decreased expression of the {alpha}-ENaC subunit abolished the stimulation of AFC by β2-agonists (25), which is not really surprising, owing to the pivotal role of {alpha}-ENaC in the distal lung. Our data demonstrate that appropriate expression of β-ENaC is also required for optimal AFC response to β-adrenergic receptor stimulation.

In conclusion, this study shows that a severe decrease in β-ENaC subunit expression in the mature lung as encountered in β-ENaC mutant mice impaired total and ENaC-mediated AFC without affecting distal lung fluid homeostasis at baseline. Low expression of β-ENaC in the alveolar epithelium was associated with a compensatory increase in {alpha}- and {gamma}-ENaC proteins and with a decrease in alveolar Na+-channel sensitivity to amiloride, suggesting that Na+ channels made of {alpha}-ENaC eventually associated with {gamma}-ENaC or another protein could compensate for the decrease in classical ENaC channels composed of {alpha}-, β-, and {gamma}-ENaC. Finally, low expression of β-ENaC subunit in the alveolar epithelium blunted the stimulation of AFC by β2-agonists, providing additional evidence for the functional importance of ENaC in the distal lung.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Institut National de la Santé et de la Recherche Médicale (INSERM), by Fondation du Legs Poix, and by the Swiss National Foundation (Grant FNRS no. 3100AO-102125/1 to E. Hummler and B. C. Rossier).


   ACKNOWLEDGMENTS
 
We thank Drs. Paul Soler and Evelyne Ferrary for helpful suggestions, Sylviane Couette and Céline Leyvraz for technical assistance, and Olivier Thibaudeau (CEFI IFR02, Faculté de Médecine Bichat, Université Denis Diderot-Paris 7) for help with histology.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Planès, INSERM 773, CRB3, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France (e-mail: carole.planes{at}apr.aphp.fr)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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H. G. Folkesson
Variations in ENaC subunit composition may determine amiloride sensitivity and {beta}-adrenergic stimulation of lung fluid absorption
Am J Physiol Lung Cell Mol Physiol, March 1, 2008; 294(3): L399 - L400.
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