Hypoxia and β2-Agonists Regulate Cell Surface Expression of the Epithelial Sodium Channel in Native Alveolar Epithelial Cells

Alveolar hypoxia may impair sodium-dependent alveolar fluid transport and induce pulmonary edema in rat and human lung, an effect that can be prevented by the inhalation of β2-agonists. To investigate the mechanism of β2-agonist-mediated stimulation of sodium transport under conditions of moderate hypoxia, we examined the effect of terbutaline on epithelial sodium channel (ENaC) expression and activity in cultured rat alveolar epithelial type II cells exposed to 3% O2 for 24 h. Hypoxia reduced transepithelial sodium current and amiloride-sensitive sodium channel activity without decreasing ENaC subunit mRNA or protein levels. The functional decrease was associated with reduced abundance of ENaC subunits (especially β and γ) in the apical membrane of hypoxic cells, as quantified by biotinylation. cAMP stimulation with terbutaline reversed the hypoxia-induced decrease in transepithelial sodium transport by stimulating sodium channel activity and markedly increased the abundance of β-and γ-ENaC in the plasma membrane of hypoxic cells. The effect of terbutaline was prevented by brefeldin A, a blocker of anterograde transport. These novel results establish that hypoxia-induced inhibition of amiloride-sensitive sodium channel activity is mediated by decreased apical expression of ENaC subunits and that β2-agonists reverse this effect by enhancing the insertion of ENaC subunits into the membrane of hypoxic alveolar epithelial cells.

The amiloride-sensitive epithelial sodium channel (ENaC) 1 located in the apical membrane of alveolar epithelial type II (ATII) cells constitutes a rate-limiting step for sodium absorption across alveolar epithelium (1)(2)(3)(4). The critical role of ENaC in alveolar fluid homeostasis has been highlighted by the fact that newborn ␣-ENaC knockout mice died shortly after birth, primarily from failure to clear their lungs of fluid (5). Despite its physiological importance, little is known about the regulation of ENaC processing, trafficking to, and stability at the cell surface of alveolar epithelial cells under physiological or pathological conditions and in response to hormonal and pharmacological stimuli. ␤-Adrenergic agonists have been reported to stimulate active sodium transport across alveolar epithelium in various species in vivo and in vitro (4), but the mechanism(s) whereby these agents increase apical sodium channel activity in native ATII cells remains unclear. Interestingly, a recent study performed in a model of Fischer rat thyroid epithelial cells cotransfected with ␣-, ␤-, and ␥-ENaC suggested that cAMP agonists stimulate sodium current by enhancing the translocation of ENaC subunit proteins from intracellular pools to the plasma membrane, thus increasing the number of sodium channels at the cell surface (6).
The alveolar epithelium is normally exposed after birth to a mean alveolar oxygen (O 2 ) pressure of 100 mm Hg, but alveolar hypoxia may occur in many physiological or pathological conditions, such as ascent to high altitude, alveolar hypoventilation, or pulmonary edema from heart failure or acute lung injury. Rapid ascent to high altitude may induce in some subjects the development of pulmonary edema. Although the initial cause of alveolar flooding is likely related to altered hemodynamics or increased lung microvascular permeability (7,8), new data from human and animal studies support the concept that defective alveolar fluid clearance could have a pathogenic role in the development of high altitude pulmonary edema (HAPE) and could be a potential target for therapy. Sartori et al. (9) recently reported that HAPE-sensitive subjects at low altitude have a decrease in nasal transepithelial potential difference compared with HAPE-insensitive subjects, suggesting that these subjects may have a genetically determined impairment of transepithelial sodium and liquid clearance in the lungs. Interestingly, the risk of HAPE was clearly reduced in these susceptible subjects by prophylactic inhalation of ␤ 2agonists, which are known to up-regulate sodium-dependent alveolar fluid transport in the human lung (10). In accordance with these findings, a recent animal study revealed that in vivo hypoxia (8% O 2 ) reduced sodium and fluid transport across rat alveolar epithelium (11). This hypoxia-induced decrease in transepithelial sodium transport occurred with no concomitant decrease in the quantity of sodium transport proteins expressed in alveolar epithelial cells from hypoxic rats, suggesting that inhibition of sodium transport was related to posttranslational mechanism(s) altering the trafficking, stability at the cell surface, or the biophysical properties of transport proteins. Moreover, in this rat model, intra-alveolar administration of the ␤ 2 -agonist terbutaline rapidly and completely reversed the hypoxia-induced decrease in transepithelial sodium transport and alveolar liquid clearance.
Therefore, we hypothesized that one mechanism whereby hypoxia may reduce transepithelial sodium transport across alveolar epithelial cells would be by decreasing the number of sodium channels at the cell surface of ATII cells and that ␤ 2 -agonists could reverse this effect by promoting insertion of new sodium channel subunits in the apical membrane. To test this hypothesis, we used an in vitro model of rat ATII cells in primary culture exposed to 3% O 2 , equivalent to an O 2 pressure of 45 mm Hg in culture medium and corresponding to the degree of alveolar hypoxia used in the in vivo model of previously studied rats. The effects of hypoxia and ␤ 2 -agonist treatment on transepithelial sodium transport and specifically on rat ENaC (rENaC) expression and activity were evaluated. Using an approach of apical cell surface biotinylation, we also quantified, for the first time in native mammalian epithelial cells, the abundance of the three rENaC subunits in the plasma membrane of ATII cells and evaluated whether it was altered by moderate hypoxia or ␤ 2 -agonists.

MATERIALS AND METHODS
Cell Isolation and Culture-The procedure of ATII cell isolation from pathogen-free male Sprague-Dawley rats accorded with legislation currently in force in France and animal welfare guidelines (Ministère de la Pêche et de l'Agriculture, agreement 5669). ATII cells were isolated from adult rats (200 -250 g) by elastase digestion of lung tissue followed by sequential filtration and differential adherence on bacteriological dishes as previously described (12). Cells (purity, Ͼ80%; viability, Ͼ95%) were seeded either onto Transwell/Snapwell (polycarbonate membrane with a pore size of 0.4 m; Costar, Cambridge, MA) filters or onto 6-or 12-well plastic culture dishes and cultured in a 5% CO 2 , 95% air atmosphere in DMEM containing 25 mM D-glucose, 10 mM Hepes, 23.8 mM NaHCO 3 , 2 mM L-glutamine, 10% fetal bovine serum, 50 units/ml penicillin, 50 g/ml streptomycin, and 10 g/ml gentamycin. On day 3 after isolation, 1 M dexamethasone was added to the culture medium, and fetal bovine serum concentration was reduced to 5%. The experiments were performed 4 -6 days after isolation.
Exposure to Hypoxia-4 -5 days after plating, the cell medium was removed and replaced by 0.2 ml/cm 2 of fresh medium to decrease the diffusion distance of the ambient gas. The cells were placed in a humidified airtight incubator with inflow and outflow valves, and the hypoxic gas mixture containing 3% O 2 , 5% CO 2 , 92% N 2 was flushed at 5 liters/min for 20 min before the airtight incubator was sealed and kept at 37°C for 24 h. Control normoxic cells were maintained in a 21% O 2 , 5% CO 2 , 74% N 2 humidified incubator for the same period of time. Oxygen tensions assayed in cell culture medium were ϳ45 and 140 mm Hg for 3 and 21% O 2 , respectively.
Electrophysiological Studies-Measurements of short circuit current (I sc ), transepithelial potential difference, and transepithelial resistance (R te ) were performed on day 5 or 6 using a voltage-clamp system (Costar Corp.) with the apical and basolateral surfaces bathed in DMEM thermostated at 37°C. I sc was measured every 5 min by clamping potential difference to 0 mV for 1 s, and R te was calculated from I sc and potential difference using Ohm's law. Snapwell filters with unstable I sc or with R te Ͻ 300 ⍀.cm 2 were discarded. Amiloride-sensitive I sc was determined as the difference in current with and without amiloride (AML, 10 M).
In some experiments, the ␤ 2 -agonist terbutaline (TB, 100 M) or vehicle was added into the apical compartment, and I sc was monitored for 30 -40 min before apical addition of 10 M amiloride.
Experiments were also undertaken to measure sodium influx through apical amiloride-sensitive channels in basolaterally permeabilized cells, as previously described by Guo et al. (13). ATII cell monolayers were bathed with an apical compartment solution containing 145 mM Na ϩ , 5 mM K ϩ , 125 mM Cl Ϫ , 1. , 10 mM glucose (pH 7.4) and 120 N-methyl-D-glucamine (an impermeant cation) for 20 min, before the basolateral membrane was permeabilized by basolateral addition of amphotericin B (10 M), a monovalent ionophore. This induced a slowly developing (3-6 min) rise in I sc . Once the I sc reached a new steady state (I ampho,max ), amiloride was added to the apical bathing solution, and the difference current, representing the amiloride-sensitive component of the Na ϩ current across the apical membrane (I ampho,AML ), was calculated.
RNase Protection Assay-Cells in 35-mm plastic dishes were lysed in a buffer containing 4 M guanidium thiocyanate and 25 mM sodium citrate (pH 7.0, 0.5% sarcosyl, and directly used for RNase protection assay as previously described (12). Briefly, total RNA equivalent of 10 6 cells or 20 g of yeast tRNA (Roche Molecular Biochemicals) were cohybridized with 5 ϫ 10 5 cpm for rENaC probes and 5 ϫ 10 4 cpm for ␤-actin probes 50°C overnight. RNase digestion (RNase A, 40 g/ml and T1, 2 g/ml; Roche Molecular Biochemicals) was performed at 30°C for 60 min, followed by digestion with proteinase K (12.5 g/ml; Roche Molecular Biochemicals) at 37°C for 30 min. After phenol extraction and ethanol precipitation, the protected fragments were separated by urea-polyacrylamide gel electrophoresis. The signal was quantitated from the gel using direct radioactivity measurement with an Instant Imager (Packard Instrument Company, Meriden, CT). Actin expression was used as an internal standard because hypoxia did not significantly modify the level of actin mRNA (12). The results were expressed as the ratios of expression of the mRNA of interest to actin mRNA (arbitrary units).
Biotinylation of Apical rENaC Subunits-Biotinylation and recovery of apical membrane proteins were performed with a method adapted from Gottardi et al. (15) and Hanwell et al. (16). Briefly, ATII cells grown on Transwell filters were placed on ice and washed three times with ice-cold PBS-Ca 2ϩ -Mg 2ϩ (PBS with 0.1 mM CaCl 2 and 1 mM MgCl 2 ). Apical membrane proteins were then biotinylated by a 15-min incubation at 4°C with NHS-ss-biotin 1.25 mg/ml (Pierce) freshly diluted into biotinylation buffer (10 mM triethanolamine, 2 mM CaCl 2 , 150 mM NaCl, pH 7.5) with gentle agitation. ATII cells were rinsed with PBS-Ca 2ϩ -Mg 2ϩ ϩ glycine (100 mM) and washed in this buffer for 20 min at 4°C to quench unreacted biotin. The cells were then rinsed twice with PBS-Ca 2ϩ -Mg 2ϩ , scraped in cold PBS, and pelleted at 2,000 rpm at 4°C. The pellets were solubilized for 45 min in 20 l of lysis buffer (1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.5) containing protease inhibitors. The lysates were clarified by centrifugation at 14,000 ϫ g for 10 min at 4°C, and the supernatants were incubated overnight with packed streptavidin-agarose beads to recover biotinylated proteins. The beads were then pelleted by centrifugation, and aliquots of supernatants were taken to represent the unbound, intracellular pool of proteins. Biotinylated proteins were eluted from the beads by heating to 100°C for 5 min in SDS-PAGE sample buffer before loading onto a 10% SDS-PAGE gel as described above. To ensure the TABLE I Effect of moderate hypoxia on bioelectric measurements in rat ATII cell monolayers Confluent rat ATII cells grown for 4 -5 days on snapwell filters were exposed to either 21 or 3% O 2 for 24 h before bioelectric measurements were performed using a voltage clamp system. The data are the means Ϯ S.E. from six or seven filters for each condition, obtained from at least three separate primary cultures. AML-sensitive I sc was the difference between the I sc value in the absence of amiloride (baseline I sc ) and the I sc value measured after the addition of 10 M amiloride into the apical bath.

Regulation of ENaC Cell Surface Expression in Alveolar Cells
absence of leakage of biotin into the cells, we systematically verified the absence of the intracellular protein actin in biotinylated extracts. Statistical Analysis-The results are presented as the means Ϯ S.E. For functional data, one-way or two-way variance analyses were performed, and when allowed by the F value, the results were compared by the modified least significant difference. For Western blot experiments, differences between groups were evaluated with paired t tests. p Ͻ 0.05 was considered significant.

Effect of Hypoxia on ATII Cell Sodium Transport
Properties-The effects of hypoxia (3% O 2 ) on sodium transport across ATII cell monolayers were evaluated by electrophysiological studies. Snapwell filters were mounted immediately at the end of normoxic/hypoxic exposure into a voltage clamp system, and electrophysiological measurements were performed. The mean electrophysiological values obtained from filters exposed to normoxia or hypoxia are given in Table I. Hypoxia significantly decreased base-line I sc and amiloride-sensitive I sc by 37 and 45%, respectively, but did not change the amiloride-insensitive part of I sc . To determine whether hypoxia affected apical sodium channel activity, I sc was measured in the presence of a sodium concentration gradient across the monolayers (mucosal to serosal: 145:25 mM) after permeabilization of the basolateral membrane with amphotericin B. The addition of amphotericin B to the basolateral bath rapidly increased I sc to a peak value (I ampho,max ), as shown in Fig. 1. The peak value I ampho,max and the amiloride-sensitive part of this current (I ampho,AML ), reflecting sodium influx through amiloride-sensitive channels located in the apical membranes, were both significantly decreased in cells exposed to hypoxia as compared with normoxic controls (Fig. 1).
Effect of Hypoxia on rENaC mRNA Expression- Fig. 2A shows that transcripts encoding for the three rENaC subunits were detected by RNase protection assays in ATII cells exposed to either normoxia or moderate hypoxia (3% O 2 for 24h). Exposure to hypoxia did not significantly modify the levels of ␣-, ␤-, or ␥-rENaC mRNAs in ATII cells, as compared with normoxic controls (Fig. 2B).
Effect of Hypoxia on rENaC Subunit Protein Levels-The protein levels of the three rENaC subunits were determined in whole ATII cell extracts grown on filters by Western blot ex-periments using rabbit polyclonal antibodies (Fig. 3A). The ␣-rENaC antibody revealed two bands, a main band at 85 kDa (the expected size of ␣-rENaC) and a smaller band that migrated at 65 kDa. The ␤and ␥-rENaC subunits were detected FIG. 1. Effect of moderate hypoxia on amiloride-sensitive apical membrane Na ؉ channels in rat ATII cell monolayers. Rat ATII cells grown on Snapwell filters were exposed to either 21 or 3% O 2 for 24 h and immediately mounted into a voltage-clamp system in the presence of a Na ϩ concentration gradient (mucosal to serosal, 145:25 mM). The addition of 10 M amphotericin B (Ampho B) into the basolateral bath increased the I sc to a peak value (I ampho,max ), before AML (10 M) was added into the apical bath. I ampho,AML represents the amiloride-sensitive component of I ampho,max . The values are the means Ϯ S.E. of six filters for each condition. * and **, significantly different from normoxic control (p Ͻ 0.05 and p Ͻ 0.01, respectively). as two single bands that migrated at 100 and 90 kDa, respectively, which represent the expected sizes of ␤and ␥-rENaC proteins. The protein levels of ␣and ␥-rENaC subunits (normalized to the corresponding actin signal) were not significantly modified by exposure to 3% O 2 hypoxia, but the quantity of ␤-rENaC subunit was decreased by almost 30% (p Ͻ 0.05) as compared with normoxic controls (Fig. 3B).
Effect of Hypoxia on the Abundance of rENaC Subunits Expressed at the Cell Surface-Biotinylation of ATII cell apical membranes was used to estimate the effect of moderate hypoxia on the amount of rENaC subunit proteins expressed at the plasma membrane. The absence of any labeling of actin, an abundant intracellular protein, in biotinylated extracts indicated that biotin did not enter the cells during the experimental procedures. As shown in Fig. 4A, biotinylated ␣-rENaC subunit protein was detected on Western blot as a single band of 65 kDa, a molecular mass clearly lower than that of the ␣-rENaC protein detected in the corresponding intracellular pool of proteins (85 kDa), as previously reported in A6 cells derived from Xenopus laevis kidney (17). The specificity of the signal was assessed by the absence of any signal with the preimmune serum and by competition experiments showing a decrease of the 65-kDa band in biotinylated extracts when the antiserum was preincubated with an excess of ␣-rENaC fusion protein. Crude estimation revealed that ␣-rENaC expressed at the apical membrane of normoxic ATII cells represented ϳ20 -25% of total ␣-rENaC pool. Biotinylated ␤-rENaC and ␥-rENaC proteins were also detected as single bands with molecular masses similar to the signals obtained in corresponding intracellular extracts (Fig. 4A). Biotinylated ␤-rENaC and ␥-rENaC expressed at the cell membrane represented ϳ5% of total ␤-rENaC and ␥-rENaC cellular pools, respectively.
Exposure to moderate hypoxia significantly decreased the amount of biotinylated ␣-, ␤-, and ␥-rENaC subunit proteins (normalized to the content of ␤-actin in corresponding intracellular pool of proteins), as compared with normoxic condition (Fig. 4B). The decrease was moderate for ␣-rENaC but much more severe for ␤-rENaC and ␥-rENaC, which was sometimes undetectable in hypoxic biotinylated extracts. Hypoxia did not modify the amount of ␣-rENaC and ␥-rENaC in intracellular extracts but decreased by ϳ25% the amount of intracellular ␤-rENaC.
Effect of the ␤ 2 -Agonist Terbutaline on Hypoxia-decreased Sodium Transport across ATII Cells-The effect of TB was tested on the bioelectric properties of normoxic and hypoxic

FIG. 3. Effect of moderate hypoxia on the protein level of ␣-, ␤-, and ␥-rENaC subunits in rat ATII cells. Rat ATII cells grown on
Transwell filters were exposed to either 21 or 3% O 2 for 24 h, and Western blot experiments were performed on whole cell extracts using anti-rENaC subunit rabbit polyclonal antibodies. As shown in A, the ␣-rENaC antibody revealed two bands, a main band at 85 kDa and a smaller one at 65 kDa, whereas the ␤and ␥-rENaC antibodies revealed two single bands at 100 and 90 kDa, respectively. Quantification of ␣-, ␤-, and ␥-rENaC levels was obtained using NIH image software, and the data were normalized for the corresponding actin signal in each lane. The results are expressed as the ratios of ␣-, ␤-, and ␥-rENaC/ actin and represent the means Ϯ S.E. of four or five separate experiments (B). *, significantly different from normoxic control (p Ͻ 0.05).

FIG. 4. Effect of moderate hypoxia on the abundance of ␣-, ␤-, and ␥-rENaC subunits accessible to biotinylation in rat ATII cells.
Rat ATII cells grown on Transwell filters were exposed to either 21 or 3% O 2 for 24 h and immediately processed for apical biotinylation experiments as described under "Materials and Methods." A, validity of the surface biotinylation approach in normoxic ATII cells, showing cell surface expression of ␣-, ␤-, and ␥-rENaC but not of the intracellular protein actin. For ␣-rENaC, control experiments were performed using either the preimmune serum or the specific antiserum ϩ ␣-rENaC fusion protein (f.p., 100 g/ml). No band was visible in the presence of the preimmune serum. The band visible in surface extracts in the presence of the antiserum was displaced when the antiserum was preincubated with ␣-rENaC fusion protein. B, cell surface expression of ␣-, ␤-, and ␥-rENaC subunits in ATII cells exposed to 21 or 3% O 2 . Quantification of biotinylated ␣-, ␤-, and ␥-rENaC signals was obtained using NIH image software, and the data were normalized for the actin signal in corresponding intracellular extracts. The results are expressed as the ratios of ␣-, ␤-, and ␥-rENaC/actin and represent the means Ϯ S.E. of three to five separate experiments. Statistical significance was calculated from the raw data by paired t tests. * and **, significantly different from normoxic control (p Ͻ 0.05 and p Ͻ 0.01, respectively). Fig. 5A, the addition of 100 M terbutaline into the apical bath induced a transient decrease in I sc that was of greater magnitude in normoxic and than in hypoxic filters (0.9 Ϯ 0.10 versus 0.4 Ϯ 0.07 A/cm 2 , respectively; p Ͻ 0.001). This decrease was followed by a gradual increase in I sc to steady state levels (I TB,max ), surpassing the base line in both normoxic and hypoxic monolayers. The TB-induced increase in I sc was of greater magnitude in hypoxic cells than in normoxic cells because I TB,max represented 142 Ϯ 4.8% versus 117 Ϯ 3.8% of base-line I sc value in hypoxic and normoxic filters, respectively (p Ͻ 0.01). As a consequence, although base-line I sc was significantly lower in hypoxic filters, I TB,max was not significantly different in normoxic and hypoxic filters (Fig. 5B). Indeed, the amiloride-sensitive component of I TB,max was comparable in cells exposed to 3% O 2 and in control cells (5.1 Ϯ 0.26 versus 5.9 Ϯ 0.51 A/cm 2 , respectively, nonsignificant), indicating that the ␤ 2 -agonist completely reversed the hypoxia-induced decrease in sodium transport across ATII cell monolayers.

ATII cells grown on filters. As shown in
To determine whether the TB-induced increase in I sc was associated in hypoxic ATII cells with an increase in apical sodium channel activity, hypoxic ATII cell monolayers treated for 40 min with 100 M TB (or vehicle) in the presence of a mucosal to serosal sodium concentration gradient were basolaterally permeabilized with amphotericin B. Terbutaline induced as expected a progressive increase in I sc , and basolateral application of amphotericin B further increased I sc . Terbutaline increased by 48% I ampho,AML , the amiloride-sensitive part of I sc peak value following amphotericin addition, which reflects sodium influx through apical amiloride-sensitive channels, as compared with vehicle (I ampho,AML: 8.7 Ϯ 0.56 versus 5.8 Ϯ 0.87 A/cm 2 , for TB and vehicle, respectively; n ϭ 5-6 filters for each conditions. p Ͻ 0.05). This result indicates that the reversal by TB of the hypoxia-induced decrease in transepithelial sodium transport is, at least in part, accounted for by an increase in amiloride-sensitive sodium channel activity.
To determine whether the reversal of the hypoxia-induced decrease in amiloride-sensitive sodium transport by ␤ 2 -agonists could partly result from the translocation of sodium transport proteins from cytoplasmic storage to the plasma membrane (6), we studied the effect of brefeldin A (BFA), which inhibits intracellular trafficking of membrane proteins from the cytosolic pool to the cell surface, on TB-induced I sc response in hypoxic ATII cells. Alveolar type II cells exposed to 3% O 2 for 24 h were incubated for 20 min with or without BFA (1 g/ml) before TB (or vehicle) was added to the apical bath (Fig. 6A). Brefeldin A had no significant effect per se on the basal I sc or on amiloride-sensitive I sc measured in the absence of TB (Fig. 6B). Pretreatment with BFA completely abolished the stimulatory effect of TB on I sc . Indeed, amiloride-sensitive I sc in hypoxic cells treated with BFA and TB was not significantly different from I sc in control hypoxic cells or in hypoxic cells treated with BFA alone. Additional experiments showed that preincubation with BFA also completely inhibited the TB-induced I sc increase in normoxic cells (data not shown).
Effect of Terbutaline on the Abundance of rENaC Subunits Expressed at the Cell Surface-Incubation of hypoxic ATII cells with TB immediately at the end of the 3% O 2 exposure significantly increased the amount of biotinylated ␤and ␥-rENaC subunit proteins by 1.9-and 2.9-fold, respectively, as compared with unstimulated hypoxic controls (Fig. 7). Terbutaline did not significantly increase the amount of biotinylated ␣-rENaC subunit protein in hypoxic cells. In normoxic ATII cells, cell surface expression of rENaC subunits was not significantly modified by TB, because biotinylated ␣-, ␤-, and ␥-rENaC in treated cells represented 114 Ϯ 16, 126 Ϯ 20, and 126 Ϯ 36% of untreated normoxic controls, respectively (n ϭ 3-4, non-significant).

DISCUSSION
The present study shows that reduction of amiloride-sensitive sodium channel activity in native rat ATII cells by moderate hypoxia is related to decreased expression of rENaC subunits, especially that of ␤and ␥-rENaC, at the apical plasma membrane. The ␤ 2 -agonist terbutaline rapidly reversed the hypoxia-induced inhibition of amiloride-sensitive sodium channel activity, at least in part by increasing the abundance of ␤and ␥-rENaC subunits in the apical membrane of hypoxic ATII cells. These results provide direct evidence that moderate hypoxia reduces the apical expression of sodium channels in ATII cells, either by decreasing the translocation of rENaC subunits from cytoplasmic pools to the plasma membrane or by increasing their rate of internalization. The results also suggest that ␤ 2 -agonists stimulate sodium absorption by increasing the delivery of rENaC subunits into the plasma membrane of alveolar epithelial cells.
A previous study reported that in vivo exposure of rats to moderate hypoxia (8% O 2 for 24 h, equivalent to an alveolar O 2 FIG. 5. Effect of terbutaline on I sc of rat ATII cells exposed to moderate hypoxia or normoxia. Rat ATII cells grown on Snapwell filters were exposed to either 21 or 3% O 2 for 24 h before the bioelectric measurements were performed. A, representative traces of normoxic (black squares) and hypoxic (white squares) filters treated with TB (100 M into the apical bath). The addition of terbutaline increased the short circuit current (I sc ), which reached a plateau (I TB,max ) after 35-40 min, and AML (10 M) was then added into the apical bath. B, effect of 21 and 3% O 2 exposure on mean values of base-line I sc and I TB,max . The stimulation of I sc was of greater magnitude in hypoxic filters than in normoxic controls so that I TB,max was not significantly different in normoxic and hypoxic cells. The values are the means Ϯ S.E. of six or seven filters for each condition. *, p Ͻ 0.05; **, p Ͻ 0.01; NS, not significantly different from the normoxic value. pressure of 45-50 mm Hg) reduced alveolar fluid clearance through a decrease of active sodium transport across alveolar epithelium (11). This hypoxia-induced decrease in alveolar transepithelial sodium transport occurred with no change in gene expression or protein abundance of ␣-, ␤-, and ␥-rENaC subunits in ATII cells, suggesting that hypoxia modifies rENaC expression at the post-translational level. Interestingly, intraalveolar administration of the ␤ 2 -agonist terbutaline rapidly and completely reversed the decrease in transepithelial sodium transport in that in vivo study.
In the present in vitro study, rat ATII cell monolayers were exposed to a moderate hypoxia (3% O 2 , equivalent to an O 2 pressure of 45 mm Hg in culture medium) for 24 h. Hypoxia resulted in an inhibition of active transepithelial sodium transport as assessed by a 45% decrease in amiloride-sensitive I sc , caused, at least in part, by a decrease in sodium channel activity because the amiloride-sensitive part of I sc in basolaterally permeabilized monolayers decreased by ϳ40% in hypoxic ATII cells as compared with normoxic controls. In this condition of moderate hypoxia, ␣-, ␤-, and ␥-rENaC mRNA levels as well as ␣and ␥-rENaC subunit proteins in whole ATII cell extracts were unchanged, and only a mild decrease in the amount of ␤-rENaC subunit was observed. These in vitro results mimicked those obtained in vivo in rats exposed to 8% O 2 (11). They strongly suggest that inhibition of amiloride-sensitive sodium channels by moderate hypoxia occurs at the posttranslational level and may involve either a decrease in the number of sodium channels in the apical membrane of hypoxic ATII cells or direct alteration of intrinsic sodium channel properties.
To assess rENaC subunit protein expression at the plasma membrane of normoxic and hypoxic ATII cells, we performed experiments of biotinylation of proteins expressed at the apical cell surface. This approach was original inasmuch as, to our knowledge, it constitutes the first example of direct quantification of rENaC subunit apical expression in mammalian epithelial cells endogenously expressing ENaC. In normoxic ATII cells under steady state conditions, the apical expression of ␣-rENaC represented ϳ20 -25% of total ␣-rENaC cellular pool, a proportion similar to that obtained in renal amphibian A6 cells (18), and apical expression of ␤and ␥-rENaC subunits was only 5% of total cellular pools. Although biotinylated ␤and ␥-rENaC subunit bands migrated at the same level as intracellular ␤and ␥-rENaC, biotinylated ␣-rENaC had an apparent molecular mass (65 kDa) lower than intracellular ␣-rENaC (85 kDa). A similar observation has been previously made in X. laevis kidney-derived A6 cells in which the prominent fraction of ␣-xENaC expressed at the cell surface also had an apparent molecular mass of 65 kDa (17). This finding has been attributed to the fact that during its maturation, ␣-xE-NaC is prone to form disulfide bridges resistant to reducing agents, a phenomenon that induces a faster migration of the protein on SDS-PAGE and changing the apparent molecular mass to 65 kDa.
Hypoxia significantly decreased the abundance of ␣-, ␤-, and ␥-rENaC subunits expressed at the apical membrane of ATII cells. Interestingly, the decrease was greater for ␤and ␥-rENaC subunits than for the ␣-rENaC subunit. Although moderate, the decrease in apical ␣-rENaC might participate per se in the functional impairment of sodium transport, inasmuch as the expression of ␣-rENaC subunit is required to obtain significant channel activity in Xenopus oocytes (2). In-  7. Effect of terbutaline on the abundance of ␣-, ␤-, and ␥-rENaC subunits accessible to biotinylation in rat ATII cells exposed to hypoxia. Rat ATII cells grown on Transwell filters were exposed to 3% O 2 for 24 h and incubated with vehicle (Control) or 100 M TB for 30 min immediately at the end of hypoxic exposure. Apical cell surface biotinylation experiments were then performed as described under "Materials and Methods" to assess the cell surface expression of ␣-, ␤-, and ␥-rENaC. Quantification of biotinylated ␣-, ␤-, and ␥-rENaC signals was obtained using NIH image software, and the data were normalized for the actin signal in corresponding intracellular extracts. The results are expressed as the ratios of ␣-, ␤-, and ␥-rENaC/actin and represent the means Ϯ S.E. of three or four separate experiments. Statistical significance was calculated from the raw data by paired t test. *, significantly different from control (p Ͻ 0.05). deed, the dramatic decrease in ␤and ␥-rENaC subunit expression at the cell surface suggests that the trafficking of these proteins is highly sensitive to hypoxia and that the density of ␤and ␥-rENaC subunits at the apical membrane of ATII cells could become critical when O 2 availability is reduced. Insufficient abundance of ␤and ␥-rENaC subunits may lead to a decreased number of highly selective sodium channels typical of ENaC, inasmuch as the presence of the three subunits is required to form complete ENaC channels with optimal activity (2). In support of this hypothesis, a previous patch-clamp study (19) reported that in cultured rat ATII cells, the level of oxygenation positively influenced the expression of highly selective sodium channels over nonselective sodium channels presumably made of ␣-rENaC subunits alone or in association with proteins other than ␤and ␥-rENaC subunits (20).
In the present study, the ␤ 2 -agonist terbutaline added at the end of hypoxic exposure rapidly and completely reversed the hypoxia-induced decrease in transepithelial sodium transport by increasing apical sodium channel activity, in line with results obtained in hypoxic rats in vivo (11). Up to now, most studies trying to address the mechanism(s) whereby ␤ 2 -agonists increase sodium absorption in native epithelial cells have been limited to a functional approach, because the very small number of ENaC channels expressed at the plasma membrane has hampered the use of traditional biochemical techniques. In native ATII cells, functional studies have provided conflicting results, attributing the effect of ␤ 2 -agonists to either an increase in the number of highly selective sodium channels in the apical membrane (21), an increase in sodium channel open probability (22,23), or an indirect stimulation of transcellular sodium movement through the stimulation of apical chloride conductance (24). Recently, using a novel approach based on the covalent modification of cotransfected ENaC subunits at the cell surface of Fischer rat thyroid epithelial cells, Snyder (6) provided convincing evidence that the cAMP-mediated stimulation of I sc resulted from an increase in the ratio of translocation versus internalization of ENaC subunits to the cell surface. That such a phenomenon could also take place in alveolar epithelial cells endogenously expressing rENaC was supported herein by biotinylation experiments and functional data. In hypoxic ATII cells, terbutaline largely increased the abundance of ␤and ␥-rENaC subunit proteins at the apical cell surface, and preincubation of ATII cells with brefeldin A, a blocker of anterograde transport, completely abolished the terbutaline-induced stimulation of I sc . Moreover, the fact that the transient I sc decrease following the addition of terbutaline, which reflects chloride influx across the apical membrane (24), was clearly smaller in hypoxic than in normoxic monolayers rules out the possibility that the stimulation of sodium transport induced by ␤ 2 -agonists in hypoxic cells was due solely to an increased driving force secondary to activation of apical chloride channels.
It is interesting to note that the levels of expression of ␤and ␥-rENaC at the plasma membrane, which were the most affected by hypoxia, were also the most responsive to terbutaline in hypoxic cells. This finding suggests that the regulation of rENaC subunit apical expression in response to hypoxic challenge and ␤ 2 -agonists was noncoordinate and that the apical expression of ␤and ␥-rENaC, but not of ␣-rENaC, may play a critical role in the regulation of sodium channel activity under hypoxic conditions. In normoxic ATII cells, only a mild and insignificant increase in rENaC subunit apical expression was evident following treatment with terbutaline, which was not surprising in view of the small magnitude of the I sc increase induced by terbutaline under normoxic conditions. This mild increase in rENaC subunit apical expression may be partly responsible for the terbutaline-induced stimulation of I sc , but we cannot exclude that additional effect(s) of ␤ 2 -agonists, such as augmented sodium channel open probability, might also be involved, at least under normoxic conditions (22,23).
The present study has important pathophysiological and clinical implications. First, moderate to severe alveolar hypoxia occurs in virtually all critically ill patients who develop pulmonary edema. Second, alveolar hypoxia caused by exposure to high altitude may induce in susceptible subjects the development of pulmonary edema resulting from imbalance between forces that drive liquid into the airspaces and mechanisms of alveolar fluid clearance that are primarily driven by active transepithelial alveolar sodium transport. In fact, it has been recently reported that prophylactic inhalation of a ␤ 2 -agonist significantly reduces the risk of high altitude pulmonary edema in susceptible subjects, most likely by up-regulating alveolar sodium and fluid transport (9). The experimental data, demonstrating that terbutaline increases apical expression of sodium channel subunits in hypoxic ATII epithelial cells, provide one mechanism whereby ␤ 2 -agonists may prevent the hypoxia-induced decrease in vectorial sodium transport across alveolar epithelium. They do not exclude, of course, an additional effect of ␤ 2 -agonists on basolateral Na,K-ATPase activity, as previously reported (25). Finally, because terbutaline, although administered at the end of a 24-h exposure to hypoxia, was able to reverse rapidly and completely the hypoxiainduced inhibition of transepithelial sodium transport in ATII cells, it is quite possible that ␤ 2 -agonists could be useful not only in the prevention of high altitude pulmonary edema but also in the treatment of other causes of pulmonary edema associated with moderate to severe hypoxia.