Abstract
In cultured alveolar epithelial cells, hypoxia induces a downregulation of the two main Na proteins, the epithelial Na channel (ENaC) and the Na,K-ATPase. However, the in vivo effects of hypoxia on alveolar epithelial transport have not been well studied. Therefore, the objectives of this study were to investigate in an in vivo rat model if hypoxia induces a reduction in vectorial Na and fluid transport across the alveolar epithelium in vivo, and if a change in net fluid transport is associated with modification in the expression and/or activity of Na transport proteins. Rats were exposed to 8% O2 from 3 to 24 h. Hypoxia induced a progressive decrease in alveolar liquid clearance (ALC) reaching 50% at 24 h, an effect that was related primarily to a decrease in amiloride-sensitive transepithelial Na transport. On RNase protection assay of alveolar type II (ATII) cells isolated immediately after hypoxic exposure, steady state levels of mRNA were increased for α -rENaC and β1-Na, K-ATPase, whereas the levels of γ -rENaC and α1-Na,K-ATPase were unchanged. On Western blots of ATII cell membranes, α -ENaC subunit protein slightly increased, whereas the amount of α1- and β1-Na,K-ATPase protein were unchanged with hypoxia. Thus, the decrease in transepithelial Na transport was not explained by a parallel change in gene expression or the quantity of transport proteins. Interestingly, hypoxia-induced decrease in ALC was completely reversed by intra-alveolar administration of the β2 agonist, terbutaline (10− 4 M). These results suggest that hypoxia-induced decrease in Na transport is not simply related to a downregulation of Na transport proteins but rather to a decrease in Na protein activity by either internalization of the proteins and/or direct alteration of the protein in the membrane. The dramatic increase of ALC with β2-agonist therapy indicates that the decrease of transepithelial Na and fluid transport during hypoxia is rapidly reversible, a finding of major clinical significance.
Several animal and human studies have established active sodium transport as the primary mechanism driving alveolar liquid clearance (ALC) in the normal lung. Na enters the apical membranes of alveolar type II (ATII) cells mainly through amiloride-sensitive ion channels and is actively transported across the basolateral membranes of these cells by the ouabain-sensitive Na/K-ATPase (1). Alveolar fluid is reabsorbed across the epithelium by the resulting osmotic gradient. The alveolar epithelial barrier may play a critical role in regulating alveolar liquid clearance under certain pathologic situations. For example, an upregulation of alveolar epithelial fluid transport secondary to increased vectorial transport of Na was observed after subacute lung injury in rats from bleomycin (2), several acute injury models of bacterial pneumonia (3), or chronic exposure to hyperoxia. In hyperoxic lung injury, some investigators have found that increased transepithelial Na transport matches with an enhanced expression and activity of Na channel and Na,K-ATPase proteins in ATII cells, suggesting that molecular homeostatic mechanisms are upregulated to prevent alveolar flooding (4-6). However, others have found that changes in protein levels and Na transport did not change in parallel (7).
Alveolar hypoxia occurs in many physiologic and pathologic conditions. For example, during ascent to high altitude, a decrease in alveolar oxygen tension (PaO2) occurs in direct relation to the decline in barometric pressure. Also, alveolar hypoxia may be the consequence of hypoventilation related to either a central nervous disorder, obstructive airway disease, or pulmonary edema from heart failure or acute lung injury. In some humans, a decrease in ambient O2 tension at high altitude can be associated with development of pulmonary edema, although the initial cause of the edema may be related to altered hemodynamics or an increase in lung microvascular permeability. Several in vitro studies demonstrated that hypoxia impaired both amiloride-sensitive Na channels and Na,K-ATPase activity in isolated or cultured alveolar epithelial cells (8, 9). More recently, Planes and coworkers (10) reported that hypoxia-induced decrease of Na transport activity in ATII cells occurred concurrently with the decrease of mRNA level expression of Na channels and Na,K-ATPase proteins; both were fully reversed by reoxygenation. Taken as a whole, these results indicate that hypoxia may impair the ability of the alveolar epithelium to prevent alveolar flooding.
Therefore, the objective of this study was to use an in vivo rat model to investigate (1) if hypoxia induced a change in vectorial Na and fluid transport across the alveolar epithelium and (2) if a change in net alveolar epithelial sodium and fluid transport is associated with modification in the expression of Na transport proteins. Since hypoxia markedly decreased in vivo alveolar fluid clearance, we also tested the hypothesis that β-agonist therapy could restore fluid clearance to normal level.
Pathogen-free male Sprague Dawley rats (mean weight 220 g) were exposed to 21% O2 (control normoxic rats) or 8% O2 (hypoxic rats) from 3 to 24 h in specially designed environmental chambers. The chamber was flushed with O2 at 15 l/min for 5 min and oxygen flow was then maintained at 8 l/min. The O2 concentrations were continuously monitored with an O2 analyzer. Carbon dioxide was trapped by soda lime granules in the box. The rats had access to food and water ad libitum throughout the exposure period. The procedure was the same for control normoxic rats, except that the chamber was ventilated with 21% O2 at the same flow rate.
At the end of normoxic or hypoxic exposure, rats were exsanguinated and their lungs were removed and processed for gravimetric determination of extravascular lung water (EVLW) and dry weight. EVLW was determined by calculating the wet-to-dry weight ratio (QW/QD) as previously described (3).
Preparation of the instillate. A 5% bovine serum albumin (Sigma, St. Louis, MO) was prepared using Ringer's lactate, adjusted with NaCl to be isosmolar with the animal circulating plasma and 1.5 μCi of 125I-labeled human albumin (CIS Bio International, Saclay, France) was added. A sample of the instillate was saved for total protein measurement and radioactivity counts.
General protocol.Rats were anesthetized with pentobarbital sodium (50 mg/kg intraperitoneally). A tracheostomy was performed and a tube (3 mm diameter) was inserted via the tracheostomy and advanced to the carina. Body temperature was monitored and maintained at 38°C with a thermostatically controlled pad placed under the animal. The instillate (10 ml/kg of 5% bovine serum albumin and of 125I-labeled human albumin) was heated to 38°C and then delivered via the tracheostomy over 1 min into both lungs using a 1-ml syringe. To keep the rats under hypoxic conditions and because 8% O2 ventilation induced hemodynamic disturbances, the instillation was performed in nonventilated animals as soon as possible after hypoxic exposure. A cardiac arrest occurred within a few minutes of instillation so that perfusion ceased at the beginning of the experiment; therefore, alveolar liquid clearance was measured with a nonrecirculating system. The animals were studied for 30 to 120 min, after which the abdomens were opened and the animals exsanguinated. The lungs were removed via a midline sternotomy and a sample of alveolar fluid (0.3 to 0.5 ml) was aspirated with a silastic tubing that was passed into a wedged position in both lungs. Alveolar fluid samples were centrifuged at 3,000 × g for 10 min and the supernatant was obtained for measurement of total protein concentration and radioactivity. Radioactivity counts were also measured in the lungs.
Protocol to validate measurement of ALC. The details of the methods for calculation of ALC are provided below. To validate the method in this experimental protocol in which ALC was measured without blood flow, ALC was determined 30, 60, and 90 min after the instillation. The rate of ALC was linear up to 60 min, then slowed between 60 and 90 min. Therefore, 30 min was chosen for the time course of these studies. Slowing of ALC in an in situ model may be related to accumulation of interstitial fluid (11).
Specific protocol. Specific protocols for measurement of ALC were normoxic group (n = 6) and hypoxic (8% O2 for 24 h) group (n = 6). Additionally, ALC was measured immediately after instillation of either 10−3 M amiloride (normox + amiloride, n = 5 and hypox + amiloride, n = 6), 10−4 M terbutaline (normox + terbutaline, n = 6 and hypox + terbutaline, n = 5), or 10−3 M amiloride plus 10−4 M terbutaline (normox + amiloride + terbutaline, n = 6 and hypox + amiloride + terbutaline, n = 5).
Measurement of alveolar liquid clearance. ALC (percent loss over 30, 60, or 90 min from alveolar space of the volume of liquid instilled) was measured by the increase in the final unlabeled alveolar protein concentration, compared with the initial instilled alveolar protein concentration. ALC was calculated as ALC = (Vi × Fwi − Vf × Fwf)/(Vi × Fwi) × 100.
Fw is the water fraction of the initial (i) and final (f) alveolar fluid. The water fraction is the volume of water per volume of solution measured by gravimetric method. V is the volume of the initial (i) and the final (f) alveolar fluid. Vf is estimated as Vf = (Vi × 125cpmi × Fr)/125cpmf.
125cpm is the counts per minute per milliliter of initial (i) and final (f) alveolar fluid. Vi is the volume of the initial alveolar fluid. Fr is the fraction of alveolar tracer (125I-albumin) that remains in the lung at the end of the experiments.
The term “alveolar,” however, does not imply that all reabsorption of fluid occurs at the alveolar level; some fluid reabsorption may occur across distal bronchial epithelium (12).
After exposure to normoxic or hypoxic (8% O2) conditons for 24 h, rats were anesthetized with intramuscular ketamine (100 mg/kg) and xylazine (15 mg/kg). 125I-albumin (1.5 μCi) was injected intravenously and a blood sample was obtained 5 min later. The animals were replaced in either normoxic or hypoxic conditions. Two hours later, rats were anesthetized with pentobarbital (50 mg/kg intraperitoneally) and a blood sample was obtained for measurement of 125I-albumin radioactivity. Rats were exsanguinated and the lungs were removed via a sternotomy and homogenized. Radioactivity counts were measured on lung homogenates and plasma samples. The clearance of 125I-albumin (as the vascular protein tracer) in the extravascular fraction of the lung was used as an index of lung endothelial permeability. To calculate the amount of 125I-albumin present in the extravascular spaces of the lung, the counts of the blood in the lung were deducted from the 125I-albumin counts in the entire lung. The clearance of plasma into the extravascular spaces of the lung was estimated by the following equation: 125cpm lung − (125cpm blood × QB)/125cpm plasma, where 125cpm lung is the total counts per minute in lung, 125cpm blood is the counts per minute per milliliter of plasma in the final blood sample, 125cpm plasma is the counts per minute per milliliter of plasma averaged over the time of the experiment, and QB is the blood volume in the lung.
QB = 1.039 × (QH × FWH × HbS)/(FWS × HbB), where 1.039 is the density of blood, QH the weight of the lung homogenate, FWH the water content of lung homogenate, HbS the hemoglobin concentration of supernatant of lung homogenate, FWS the water content in supernatant of lung homogenate, and HbB the hemoglobin concentration of the blood.
As an index of alveolar barrier integrity, we used the residual 125I-albumin in the lung at the end of the experiment. In each experiment, > 92% of the total radioactivity that was instilled at the beginning of the experiment was recovered from the final aspirate, lungs, and tracheal cannula. This indicates that the alveolar epithelial barrier remained intact (13).
Protein concentration was measured by the Bradford method. Hemoglobin was measured spectrophotometrically on the blood sample and the supernatant obtained after centrifugation of the lung homogenate (10,000 × g for 20 min).
Transmission electron microscopy was performed in normoxic and hypoxic (8% O2 for 24 h) rats. The lungs were fixed in situ by inflating 10 ml of 2.5% glutaraldehyde in Cacodylate buffer followed by immersion in fixative. Blocks of tissue were then postfixed in osmium tetraoxide and embedded in epon as usual. Ultrastructural sections were observed under an EM 301 microscope (Philips, Eindhoven, The Netherlands).
At the end of normoxic or hypoxic (8% O2 for 24 h) exposure, ATII cells were isolated by elastase digestion of lung tissue followed by sequential filtration and differential adherence on bacteriologic dishes as previously described (10). Each rat lung yielded ∼ 5 × 106 cells. Phosphine 3R indicated that > 80% of the cells were ATII cells. Viability, assessed by the ability of the cells to exclude trypan blue, was > 95%.
Total mRNA from lung tissue and ATII cells was isolated according to the method of Chomczynski and Sacchi and used for RNase protection assay as previously described (10). Ten micrograms of RNA or 20 μg of yeast tRNA (Boehringer Mannheim, Indianapolis, IN) were cohybridized with 5 × 105 counts per minute (cpm) for rENaC or rat Na,K-ATPase probes and 5.104 cpm for β-actin probes in 80% formamide, 1,4-piperazine-diethanesulphonic acid 40 mM (PIPES, pH 7.4), 400 mM NaCl, 1 mM EDTA at 50°C overnight. RNase digestion (RNAse A, 40 mg/ml and T1, 2 mg/ml, from Boehringer) was performed at 30°C for 60 min. Then, digestion with proteinase K (12.5 mg/ml, Boehringer) was done at 37°C for 30 min. After phenol extraction and ethanol precipitation, protected fragments were separated by urea-polyacrylamide gel electrophoresis. Gels were fixed with 10% acetic acid and vacuum dried prior to exposure to Kodak X-OMAT AR 5 film (Eastman Kodak, Rochester, NY), and 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 neither hypoxia nor reoxygenation significantly modified the level of actin mRNA. Results were expressed as the ratio of expression of the mRNA of interest to actin mRNA (arbitrary units).
The 3′-untranslated regions of α- and γ-rENaC subunit cDNA were used. The length of rENaC probe subunits were 361 nt for α (protected fragment 317 nt, corresponding to nt 2458–2775) and 385 nt for γ (protected fragment 316 nt, corresponding to nt 2594– 2911). Na,K-ATPase probes for α1 or β1 subunits were also localized in the 3′-untranslated region. The length of the Na,K-ATPase probes was 240 nt for α1 (protected fragment 202 nt, corresponding to nt 3434–3636) and 395 nt for β1 (protected fragment 337 nt, corresponding to nt 1263–1600). Mouse β-actin was synthesized using a cDNA insert in PGEM-3. The probe was 190 nt long with a protected fragment of 135 nt (nt 696–831). Antisense RNA probes were synthesized using a T7 in vitro synthesis kit (Promega, Madison, WI) in the presence of [32P]UTP (15 TBq/mmol).
At the end of ATII cell isolation, cells were washed in PBS, harvested in a solution containing 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), and 50 mM Tris HCl (pH 7.4), and homogenized manually on ice with 20 strokes in a glass potter homogenizer. The homogenates were centrifuged for 5 min at 500 × g to remove nuclei and the supernatant was then centrifuged for 100,000 × g for 1 h at 4°C. The pellets were resuspended in a buffer containing (in mM) 150 NaCl, 2 EDTA, 50 Tris HCl (pH 7.4), and protease inhibitors. Protein content was determined by the method of Bradford.
The supernatants were frozen at −80°C until used. Samples (40 μg), used without deglycosylation, in one volume of sample buffer (10% glycerol, 12.5% 0.5 M Tris HCl [pH 6.8], 10% of 20% sodium dodecyl sulfate, 5% β-mercaptoethanol, and 2.5 of 0.05% [wt/vol] bromophenol blue) were loaded per lane. Samples were to be subsequently probed for the α-ENaC subunit (molecular mass ≈ 90 kD) (10, 14), α1-Na,K-ATPase subunit (molecular mass ≈ 100 kD) and β1-Na,K-ATPase subunit (molecular mass ≈ 50 kD) (15) were resolved through 7%, 7%, and 10% acrylamide gels, respectively, electroblotted, electrically transferred to nitrocellulose paper, and blocked 1 h at 37°C in TBS-milk (137 mM NaCl, Tris, pH 7.4, containing 50 mg/ml nonfat dry milk). Blots were then washed in TBS-Tween (in mM): 137 NaCl Tris, as well as 0.25% Tween-20, pH 7.4, and were subsequently incubated with anti–α-ENaC, anti–α1- and anti–β1 Na,K-ATPase antibodies in TBST for 16 h at 4°C. The following antibodies and dilutions were used: rabbit polyclonal anti-αENaC 1:3,000, rabbit polyclonal anti-α1 Na,K-ATPase 1:3,000, and rabbit polyclonal anti–β1 Na,K-ATPase 1:5,000 (Upstate Biotechnology, Lake Placid, NY), and mouse monoclonal anti-actin 1:5,000. The specificity of the antibody against αENaC was demonstrated by the displacement of the immunoprecipitated product (90 kD) with α-fusion protein (14, 15). Characterization of the antibody specificity was reported by Carranza and coworkers for the rabbit polyclonal anti-Na,K-ATPase α1 subunit antibody (16) and by Shyjan and colleagues for the rabbit polyclonal anti-Na,K-ATPase β1 subunit antibody (17). Blots were washed and incubated with an antirabbit antibody for 2 h at 23°C. Blots were washed three times in PBS-Tween for 15 min each. The antigenic sites were visualized with enhanced chemiluminescence detection (Amersham, UK) on Kodak X-Omat AR film (Eastman Kodak). Quantification of ENaC, Na,K-ATPase and actin levels was obtained using NIH image software.
Results are presented as means ± SE. One-way or two-way variance analyses were performed and, when allowed by the F value, results were compared by the modified least significant difference. P < 0.05 was considered significant.
Ultrastructural examination of the lungs of hypoxic rats (n = 2) did not provide any evidence of endothelial or alveolar epithelial barrier injury (Figure 1).
Fig. 1. Ultrastructural examination of hypoxic rat lung. Rats were exposed for 24 h to hypoxia (8% O2) (original magnification: ×7,100). Alveolar type II cells (PII), alveolar type I cells (PI), and endothelial cells (EC) are intact. lb: lamellar bodies.
[More] [Minimize]In rats exposed to 24 h hypoxia (8% O2) a mild but significant increase in the wet-to-dry weight ratio was observed compared with controls (Figure 2). This result is consistent with the presence of mild interstitial edema. The wet-to-dry weight ratio did not increase further when the rats were exposed for 48 h to 8% O2 compared with 24 h exposure.
Fig. 2. Extravascular water content of normoxic rats and hypoxic rats exposed for 24 and 48 h to 8% O2 (mean ± SE, n = 4). The extravascular lung water content of hypoxic rats was significantly increased (*P < 0.05) compared with that measured in normoxic rats.
[More] [Minimize]The lung endothelial protein flux was measured in specific experiments by the use of the vascular protein tracer 125I-albumin, a method which can detect a very small change in lung vascular permeability (13). The endothelial protein flux is expressed as extravascular plasma equivalents in the lung, representing milliliters of plasma that had moved from blood vessels into the extravascular compartment of the lung. In rats exposed for 24 h to 8% O2, extravascular plasma equivalents in the lung over 2 h was not significantly different compared with control rats (Table 1).
The effect of hypoxia on alveolar protein flux was evaluated by the measurement of residual 125I-albumin in the lung at the end of the experiments performed for ALC measurement. The quantities of 125I-albumin are expressed as the percentage of the instilled amounts (in cpm). The alveolar protein flux over 30 min across the epithelial barrier was not different in hypoxic and in control rats (Table 1).
In this experimental model, the animal died a few minutes after instillation and ALC was then measured in the absence of ventilation and blood flow. Although several studies in rats (3), sheep (18), and humans (19) have shown that absence of blood flow does not modify fluid reabsorption, we performed preliminary experiments in control rats to determine the time course of alveolar fluid clearance over 90 min. The volume removed was linear over 60 min; ALC was measured at 30 min in this study.
ALC measured by the increase of labeled alveolar protein tracer concentration was significantly decreased in hypoxic rats compared with the normoxic controls. Hypoxia-induced decrease of ALC was time-dependent, being significant at 6 h of exposure, and reached 50% of the controls at 24 h exposure (Figure 3). This effect was completely reversed when hypoxic rats were replaced in normoxic atmosphere for 24 h.
Fig. 3. ALC (percent of liquid instilled) in rats for different times of hypoxic exposure (8% O2) (hatched bars) and after 24 h of reoxygenation (stippled bars) of rats exposed to 24 h hypoxia. Results are expressed as means ± SE of three to five experiments. The ALC was significantly decreased (*P < 0.05, **P < 0.01, ***P < 0.001) compared with that measured in normoxic rats.
[More] [Minimize]To determine whether the decrease in ALC in hypoxic rats was mediated by a decrease in the uptake and transport of sodium across the lung epithelial barrier, 10−3 M amiloride, an inhibitor of the epithelial sodium channel, was added at the instilled solution for the 30-min experiments. We select this concentration because previous studies indicated that instilled amiloride is cleared rapidly across the airspaces (20, 21). In control rats, amiloride reduced ALC by ∼ 60% compared with basal conditions, as has been previously reported in other rat in vivo studies (22). In hypoxic rats (8% O2, 24 h), the addition of amiloride did not further reduce the hypoxia-induced decrease of ALC (Figure 4). This result indicates that hypoxia-induced inhibition of ALC was related primarily to a decrease in transepithelial Na transport.
Fig. 4. ALC (percent of instilled liquid) is shown for rats exposed to normoxia (21% O2) and hypoxia (8% O2). Rats were pretreated with either amiloride (10−3 M) or terbutaline (10−4 M) or amiloride plus terbutaline added to the alveolar instillate. Results are expressed as means ± SE of four to six different experiments for each condition. The ALC was significantly different (*P < 0.001) compared with that measured in control conditions.
[More] [Minimize]Terbutaline (10−4 M), a β2 agonist, which is known to stimulate fluid reabsorption, was added to the instilled solution. Terbutaline increased ALC by 40% in control rats. In hypoxic rats, terbutaline completely reversed the hypoxia-induced ALC decrease; under these conditions, ALC reached the same value as in controls. When terbutaline and amiloride were added simultaneously in the instilled solution, the stimulation of ALC induced by terbutaline was completely abolished either in control or hypoxic rats (Figure 4). This result suggests that terbutaline-induced increase in ALC was due to stimulation of transepithelial Na transport in normoxic as well as in hypoxic rats. The calculated concentration of terbutaline in alveolar fluid was similar to recent measured levels of an areosolized β2 agonist in the pulmonary alveolar fluid of critically ill patients (23).
RNAse protection assays were performed to determine the level of α- and γ-rENaC and α1- and β1-Na,K-ATPase mRNA transcripts in ATII cells obtained from normoxic and hypoxic rats exposed to 8% O2 for 24 h. In ATII cells from hypoxic rats compared with normoxic rats, α-rENaC and β1-Na,K-ATPase mRNA were increased by 3- and 4-fold, respectively, while no significant change was observed for γ-rENaC and α1-Na,K-ATPase mRNA levels (Figures 5 and 6).
Fig. 5. mRNA expression of α- and γ-rENaC subunits in freshly isolated ATII cells from normoxic (21% O2) or hypoxic (8% O2, 24 h) rats. RNA protection assays were performed on total mRNA as described in Materials and Methods. Quantification was performed using an Instant Imager. Data were normalized for the corresponding actin signal in each lane. Results are expressed as the ratio of α-, γ-rENaC mRNA/actin mRNA and represent means ± SE of four to five experiments. *Significantly different from the normoxic value (P < 0.01).
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Fig. 6. mRNA expression of α1- and β1-Na,K-ATPase subunits in freshly isolated ATII cells from normoxic (21% O2) or hypoxic (8% O2, 24 h) rats. RNA protection assays were performed on total mRNA as described in Materials and Methods. Quantification was performed using an Instant Imager. Data were normalized for the corresponding actin signal in each lane. Results are expressed as the ratio of α1, γ-Na,K-ATPase mRNA/actin mRNA and represent means ± SE of four to five different experiments. *Significantly different from the normoxic value (P < 0.01).
[More] [Minimize]Western blot analyses were performed on freshly isolated ATII cell membranes from different hypoxic and normoxic rats (Figure 7). α-ENaC protein level, normalized by the corresponding β-actin signal, was slightly but significantly increased in hypoxic rats compared with control rats, while α1- and β1-Na,K-ATPase protein levels were unaffected by hypoxia (Figure 8).
Fig. 7. Protein expression of α-ENaC and α1- and β1-Na,K-ATPase in freshly isolated ATII cells from normoxic (21% O2) or hypoxic (8% O2, 24 h) rats determined by Western blot analysis on crude cell membranes using α-ENaC (∼ 90 kD) and α1-Na,K-ATPase (∼ 100 kD) and β1-Na,K-ATPase (∼ 50 kD) polyclonal rabbit antibodies as described in Materials and Methods. Lanes 1 and 2: samples from two different normoxic (N) rats; lanes 3 and 4: samples from two different hypoxic (H) rats.
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Fig. 8. Protein level of α-ENaC and α1- and β1-Na,K-ATPase in freshly isolated ATII cells from normoxic (21% O2) or hypoxic (8% O2, 24 h) rats. Quantification of ENaC, Na,K-ATPase and actin levels was obtained using NIH image software. Data were normalized for the corresponding β-actin signal in each lane. Results are expressed as the ratio of α-ENaC and α1- and β1-Na,K-ATPase/actin and represent means ± SE of three to four different samples provided from different animals. *Significantly different from the normoxic value (P < 0.05).
[More] [Minimize]Hypoxia is a common feature of many physiologic and pathologic situations. This study demonstrated that in vivo exposure of rats to 8% O2, equivalent to an alveolar O2 pressure of 50 mm Hg, substantially reduces ALC, an effect that was fully reversed when the animals recovered in normoxic conditions for 1 d. Under these experimental conditions, hypoxia did not alter the integrity of the endothelial and epithelial barriers: lung histologic examination did not show interstitial edema, capillary endothelial and alveolar epithelial cells were intact, and there was no change in endothelial and epithelial permeability as measured by our vascular protein tracer studies, a method which can detect a very small change in lung vascular permeability (13). Despite the lack of morphologic and functional changes of the lung endothelial barrier, hypoxia induced a slight increase in extravascular lung water content, indicating that probably a small amount of interstitial edema was present, perhaps from a small increase in postcapillary vascular pressure as shown in micropunctured isolated hypoxic lungs (24). These results agree with previous reports in rats and sheep, which indicated that the structural and functional integrity of lung capillary endothelial cells and alveolar epithelial cells are preserved during prolonged and severe hypoxia (25, 26).
In this study, exposure to hypoxia induced a progressive decrease of ALC as a function of time with a maximal inhibition of 50% after 24 h of hypoxic exposure, an effect that was fully reversed when the rats were allowed to recover in normoxic conditions for 1 d. The hypoxia-induced decrease in ALC resulted in a major inhibition of transepithelial Na transport across the alveolar epithelium, because hypoxia abrogated the amiloride-sensitive component of ALC. Similar to our result, a recent study indicated that hypobaric hypoxia induced a decrease in nasal transepithelial potential difference in rats that was not additive to the changes in nasal potential difference produced by amiloride (27). Together, these results indicate that hypoxia inhibits Na transport throughout the pulmonary epithelium in rats. In this study, the hypoxia-induced decrease in transepithelial Na transport may be the result of either an inhibitory effect of hypoxia on Na entry through apical epithelial Na channels and/or Na extrusion at the basolateral side through Na,K-ATPase, since amiloride at 1 mM concentration can cause some degree of Na pump inhibition, or both. In fact, previous in vitro findings have demonstrated that exposure to hypoxia alters the activity of the two main Na proteins involved in alveolar transepithelial Na transport. In alveolar epithelial cells, hypoxia induced a decrease of both amiloride-sensitive 22Na influx and Na,K-ATPase activity (10). Interestingly, the temporal inhibition was quite similar to the effect that we observed in vivo, reaching 30% inhibition after 18 h to 50 mm Hg O2 tension and reversed after the cells were allowed to reoxygenate in normoxic conditions. Also, Suzuki and colleagues (26) reported that hypoxia decreased Na,K-ATPase activity measured on lung homogenates from rats exposed to 10% O2 for 72 h.
What are the mechanisms by which hypoxia decreases transepithelial Na transport in the intact alveolar epithelium in vivo? One possibility is that the hypoxia-induced decrease in transepithelial Na transport was due to reduced lung cellular energy production secondary to O2 deprivation. However, several studies indicate that O2 deprivation does not induce a significant change in lung ATP content. In vitro, alveolar epithelial cells exposed to severe hypoxia (0% O2 for 18 h) maintain their ATP content close to that of normoxic conditions (28-30), and, in vivo, lung ATP content of rats exposed to hypoxia increased by 20% (26). Moreover, ATP depletion achieved by metabolic inhibitors does not affect Na transport or alveolar fluid absorption in vivo (31) and Na,K-ATPase activity in alveolar epithelial cells in vitro (9). A second possible mechanism by which hypoxia could decrease transepithelial Na transport is by downregulating Na transport proteins at the transcriptional or posttranscriptional levels. Exposure of cultured adult ATII cells to severe hypoxia resulted in a decrease of mRNA levels of α-, β-, and γ-rENaC subunits and α1-, β1-Na,K-ATPase associated with a significant decrease in α-rENaC protein synthesis (10). However, in the current in vivo study, hypoxia (8% O2 24 h) induced a significant increase of α-rENaC and β1-Na,K-ATPase mRNA transcripts with either a little increase or no change in protein levels. These results clearly indicate that the hypoxia-induced decrease in transepithelial Na transport could not be attributed to a downregulation of Na transport protein expression. The difference between in vitro and in vivo results could be explained by the fact that in vivo, hypoxia is associated with systemic hormonal changes which could counteract the direct effect of hypoxia on gene regulation. Exposure to hypoxia induces hormonal changes such as increase in plasma cortisol (32), and glucocorticoids upregulate both rENaC subunits and Na,K-ATPase subunits in alveolar epithelial cells (33, 34).
Finally, a third potential mechanism is that the hypoxia-induced decrease in transepithelial Na transport resulted from a decrease of functioning Na proteins in the plasma membranes. This effect could be brought about by alteration in the rate of protein synthesis, consequently leading to changes in enzymatic activity. However, altered protein synthesis cannot account for the effect in this study, since protein levels for ENaC and Na,K-ATPase subunits were not decreased. Moreover, instillation of a β2-adrenergic agonist induced a dramatic and rapid (30 min) increase in transepithelial Na transport in hypoxic rats, so that the alveolar clearance reached the value obtained in normoxic rats treated by β2 agonist. β2 agonists can increase Na transport in alveolar epithelial cells by increasing sodium pump activity and/or apical epithelial Na channel uptake. In alveolar epithelial cells, administration of β2 agonists favors the traffic of Na pump subunits from late endosomes to the plasma membrane, a process which is inhibited by stabilizing actin skeleton with phallacidin (35). Also, Snyder and coworkers (36) recently reported that cAMP agonists induced an increase in the number of Na channels at the cell surface, resulting from an increase in the ratio of translocation versus internalization of channels to the cell surface; alternatively, β2 agonists may increase the sodium channel activity directly by increasing the open probability (37) or indirectly through the stimulation of Cl channel activity (38). Because, in renal epithelial cells, hypoxia induces a disruption of actin filaments which are necessary for Na pump activity (39), it could also be hypothesized that hypoxia-induced decrease in Na transport reflects internalization of Na proteins, which are then recruited to the membrane level by administration of β2 agonists.
In summary, this study demonstrated that exposure to 24 h of hypoxia in rats is associated with a 50% decrease of alveolar epithelial Na and fluid transport, an effect that is explained by a decrease in Na transport protein activity and not by transcriptional or translational mechanisms. This observation has clinical relevance because subclinical pulmonary edema is a common finding in newcomers to high altitude without prior history of high altitude pulmonary edema (HAPE) (40). Interestingly, a recent preliminary report indicates that HAPE-sensitive subjects have a decrease in nasal potential difference compared with HAPE-insensitive subjects, a finding that seems to parallel our finding that hypoxia reduced basal alveolar epithelial fluid transport. Furthermore, β-agonist inhalation therapy may prevent HAPE in mountain climbers, suggesting that β-agonist stimulation might upregulate alveolar fluid transport under hypoxic conditions at high altitude in humans (41). This clinical data matches well with this experimental study because β-agonist therapy, at a concentration that gives similar alveolar concentration to that reported in alveolar fluid of critically ill patients who received aerosolized β2 agonist (23), completely and rapidly overcame the hypoxia-induced decrease in alveolar epithelial fluid clearance in rats.
The authors wish to thank Dr. Carole Planes and Dr. Hans G. Folkesson for their help in the preparation of the manuscript. This work was supported by grants from the University Paris 13 and Fondation pour la Recherche Médicale.
| 1. | Matalon S.Mechanisms and regulation of ion transport in adult mammalian alveolar type II pneumocytes. Am. J. Physiol.2611991C727C738 |
| 2. | Folkesson H., Nitenberg G., Oliver B., Jayr C., Albertine K., Matthay M.Upregulation of alveolar epithelial fluid transport after subacute lung injury in rats from bleomycin. Am. J. Physiol.2751998L478L490 |
| 3. | Rezaiguia S., Garat C., Delclaux C., Meignan M., Fleury J., Legrand P., Matthay M. A., Jayr C.Acute bacterial pneumonia in rats increases alveolar epithelial fluid clearance by a tumor necrosis factor-alpha-dependent mechanism. J. Clin. Invest.991997325335 |
| 4. | Yue G., Russell W., Benos D., Jackson R., Olman M., Matalon S.Increased expression and activity of sodium channels in alveolar type II cells of hyperoxic rats. Proc. Natl. Acad. Sci. USA92199584188422 |
| 5. | Nici L., Dowin R., Gimore-Hebert M., Jamieson J., Ingbar D.Upregulation of rat lung Na,K-ATPase during hyperoxic injury. Am. J. Physiol.2611991L307L314 |
| 6. | Olivera W., Ridge K., Wood L., Sznajder J.Active sodium transport and alveolar epithelial Na,K-ATPase increase during subacute hyperoxia in rats. Am. J. Physiol.2661994L577L584 |
| 7. | Carter E. P., Wangensteen O. D., O'Grady S. M., Ingbar D. H.Effects of hyperoxia on type II cell Na-K-ATPase function and expression. Am. J. Physiol.2721997L542L551 |
| 8. | Mairbaurl H., Wodopia R., Eckes S., Schulz S., Bartsch P.Impairment of cation transport in A549 cells and rat alveolar epithelial cells by hypoxia. Am. J. Physiol.2731997L797806 |
| 9. | Planes C., Friedlander G., Loiseau A., Amiel C., Clerici C.Inhibition of Na-K-ATPase activity after prolonged hypoxia in an alveolar epithelial cell line. Am. J. Physiol.2711996L70L78 |
| 10. | Planes 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.171997508518 |
| 11. | Fukuda N., Folkesson H., Matthay M.Relationship of interstitial fluid volume to alveolar fluid clearance in mice. J. Appl. Physiol.892000672679 |
| 12. | Matthay M. A., Folkesson H. G., Verkman A. S.Salt and water transport across alveolar and distal airway epithelia in the adult lung. Am. J. Physiol.2701996L487L503 |
| 13. | Pittet J., Lu L., Morris D., Modelska K., Welch W., Carey H., Roux J., Matthay M.Reactive nitrogen species inhibit alveolar epithelial fluid transport after hemorrhagic shock in rats. J. Immunol.166200163016310 |
| 14. | Duc C., Farman N., Canessa C., Bonvalet J. P., Rossier B.Cell-specific expression of epithelial sodium channel a, b and g subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry. J. Cell Biol.127199419071921 |
| 15. | Djelidi C., Fay M., Cluzeaud F., Escoubet B., Eugene E., Capurro C., Bonvalet J., Farman N., Blot-Chabaud M.Transcriptional regulation of sodium transport by vasopressin in renal cells. J. Biol. Chem.27219973291932924 |
| 16. | Carranza M., Féraille E., Favre H.Protein kinase C-dependent phosphorylation of Na, K-ATPase a-subunit in rat kidney cortical tubules. Am. J. Physiol.2711996C136C143 |
| 17. | Shyjan A. W., Levenson R.Antisera specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the Na,K-ATPase: differential expression of alpha and beta subunits in rat tissue membranes. Biochemistry28198945314535 |
| 18. | Sakuma T., Pittet J. F., Jayr C., Matthay M. A.Alveolar liquid and protein clearance in the absence of blood flow or ventilation in sheep. J. Appl. Physiol.741993176185 |
| 19. | Sakuma, T., G. Okaniwa, T. Nakada, T. Nishimura, S. Fujimura, and M. A. Matthay. 1994. Alveolar fluid clearance in the resected human lung. Am. J. Respir. Crit. Care Med. 150:305–310. [see comments] |
| 20. | Garty H., Benos D. J.Characteristics and regulatory mechanisms of the amiloride-blockade sodium channels. Physiol. Rev.681988309373 |
| 21. | O'Brodovich H., Hannam V., Rafii B.Sodium channel but neither Na-H nor Na-glucose symport inhibitors slow neonatal lung water clearance. Am. J. Respir. Cell Mol. Biol.51995377384 |
| 22. | Garat C., Rezaiguia S., Meignan M., D'Ortho M. P., Harf A., Matthay M. A., Jayr C.Alveolar endotoxin increases alveolar liquid clearance in rats. J. Appl. Physiol.79199520212028 |
| 23. | Atabai K., Ware L., Snider M., Koch P., Daniel B., Nuckton T., Matthay M.Aerosolized β2 adrenergic agonists achieve therapeutic levels in the pulmonary edema fluid of ventilated patients with acute respiratory failure. Am. J. Respir. Crit. Care Med.1632001A618 |
| 24. | Nagasaka Y., Bhattacharya G., Nanjo S., Gropper M., Staub N.Micropuncture measurement of lung microvascular pressure profile during hypoxia in cats. Circ. Res.5419849095 |
| 25. | Bland R. D., Demling R. H., Selinger S. L., Staub N. C.Effects of alveolar hypoxia on lung fluid and protein transport in unanesthetized sheep. Circ. Res.401977269274 |
| 26. | Suzuki, S., M. Noda, M. Sugita, S. Ono, K. Koike, and S. Fujimura. 1999. Impairment of transalveolar fluid transport and lung Na(+)-K(+)- ATPase function by hypoxia in rats. J. Appl. Physiol. 87:962–968 [In Process Citation]. |
| 27. | Tomlinson L. A., Carpenter T. C., Baker E. H., Bridges J. B., Weil J. V.Hypoxia reduces airway epithelial sodium transport in rats. Am. J. Physiol.2771999L881L886 |
| 28. | Fisher A. B., Itakura N., Dodia C., Thurman R. G.Pulmonary mixed-function oxidation: stimulation by glucose and the effects of metabolic inhibitors. Biochem. Pharmacol.301981379383 |
| 29. | Ouiddir A., Planes C., Fernandes I., VanHesse A., Clerici C.Hypoxia upregulates activity and expression of the glucose transporter GLUT1 in alveolar epithelial cells. Am. J. Respir. Cell Mol. Biol.211999710718 |
| 30. | Clerici C., Matthay M. A.Hypoxia regulates gene expression of alveolar epithelial transport proteins. J. Appl. Physiol.88200018901896 |
| 31. | Saumon G., Martet G.Effect of metabolic inhibitors on Na+ transport in isolated perfused rat lungs. Am. J. Respir. Cell Mol. Biol.91993157165 |
| 32. | Ou L. C., Tenney S. M.Adrenocortical function in rats chronically exposed to high altitude. J. Appl. Physiol.47197911851187 |
| 33. | Tchepichev S., Ueda J., Canessa C., Rossier B. C., O'Brodovich H.Lung epithelial Na channel subunits are differentially regulated during development and by steroids. Am. J. Physiol.2691995C805C812 |
| 34. | Barquin N., Ciccolella D. E., Ridge K. M., Sznajder J. I.Dexamethasone upregulates the Na-K-ATPase in rat alveolar epithelial cells. Am. J. Physiol.2731997L825L830 |
| 35. | Bertorello A., Ridge K., Chibalin A., Katz A., Sznajder J.Isoproterenol increases Na-K-ATPase activity by membrane insertion of a-subunits in lung alveolar cells. Am. J. Physiol.2761999L20L27 |
| 36. | Snyder P.Liddle's syndrome mutations disrupt cAMP-mediated translocation of the epithelial Na channel to the cell surface. J. Clin. Invest.10520004553 |
| 37. | Matalon S., O'Brodovich H.Sodium channels in alveolar epithelial cells: molecular characterization, biophysical properties, and physiological significance. Annu. Rev. Physiol.611999627661 |
| 38. | Jiang X., Ingbar D. H., O'Grady S. M.Adrenergic stimulation of Na+ transport across alveolar epithelial cells involves activation of apical Cl- channels. Am. J. Physiol.2761998C1610C1620 |
| 39. | Racusen L.Alterations in human proximal tubule cell attachment in response to hypoxia: role of microfilaments. J. Lab. Clin. Med.1231994357364 |
| 40. | Jaeger J. J., Sylvester J. T., Cymerman A., Berberich J. J., Denniston J. C., Maher J. T.Evidence for increased intrathoracic fluid volume in man at high altitude. J. Appl. Physiol.471976670676 |
| 41. | Sartori C., Lipp E., Duplain H., Egli M., Hutter D., Alleman Y., Nicod P., Scherrer U.Prevention of high altitude pulmonary edema by beta-adrenergic stimulation of the alveolar transepithelial sodium transport. Am. J. Respir. Crit. Care Med.1612000415A |
Abbreviations: alveolar liquid clearance, ALC; alveolar type II, ATII; ethylenediaminetetraacetic acid, EDTA; messenger RNA, mRNA.
(Received in original form November 2, 2000 and in revised form June 28, 2001)
