Hypoxia decreases proteins involved in epithelial electrolyte transport in A549 cells and rat lung

Ralf Wodopia1, Hyun Soo Ko1, Javiera Billian1, Rudolf Wiesner2, Peter Bärtsch1, and Heimo Mairbäurl1

1 Medical Clinic, Section Sports Medicine, University of Heidelberg, 69115 Heidelberg; and 2 Department of Vegetative Physiology, University of Cologne, 50931 Cologne, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Fluid reabsorption from alveolar space is driven by active Na reabsorption via epithelial Na channels (ENaCs) and Na-K-ATPase. Both are inhibited by hypoxia. Here we tested whether hypoxia decreases Na transport by decreasing the number of copies of transporters in alveolar epithelial cells and in lungs of hypoxic rats. Membrane fractions were prepared from A549 cells exposed to hypoxia (3% O2) as well as from whole lung tissue and alveolar type II cells from rats exposed to hypoxia. Transport proteins were measured by Western blot analysis. In A549 cells, alpha 1- and beta 1-Na-K-ATPase, Na/K/2Cl cotransport, and ENaC proteins decreased during hypoxia. In whole lung tissue, alpha 1-Na-K-ATPase and Na/K/2Cl cotransport decreased. alpha - and beta -ENaC mRNAs also decreased in hypoxic lungs. Similar results were seen in alveolar type II cells from hypoxic rats. These results indicate a slow decrease in the amount of Na-transporting proteins in alveolar epithelial cells during exposure to hypoxia that also occurs in vivo in lungs from hypoxic animals. The reduced number of transporters might account for the decreased transport activity and impaired edema clearance in hypoxic lungs.

hypoxic pulmonary edema; sodium-potassium-adenosine 5'-triphosphatase; sodium/potassium/2 chloride cotransport; epithelial sodium channel; alveolar epithelium; expression


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ALVEOLAR GAS EXCHANGE might be limited by the thickness of the diffusion barrier, which consists of alveolar lining fluid and alveolar epithelial and capillary endothelial cells as well as their basal membrane. The thickness of the film of lining fluid is determined by the balance between fluid filtration into the alveolar space, e.g., by hydrostatic pressure gradients, and fluid reabsorption across the alveolar epithelium (24, 34). Reabsorption of fluid is coupled to the reabsorption of NaCl; Na enters alveolar epithelial cells via cation channels such as the epithelial Na channel (ENaC) as well as via Na-coupled transporters (e.g., Na/glucose cotransport) and is extruded by the Na-K pump located in the basolateral plasma membrane. Transcellular Na movement then causes chloride and, subsequently, water to follow (34).

Hypoxia, e.g., by rapid ascent to high altitude or acute respiratory distress syndrome, can cause alveolar flooding (2, 25). Alveolar fluid accumulation has been attributed to hypoxic pulmonary vasoconstriction, increased vascular permeability, and an inhibition of fluid reabsorption from the alveolar space (31). Only an intact alveolar barrier allows clearance of alveolar edema (27). Evidence for impairment of transepithelial electrolyte and water movement has accumulated as a result of studies on cultured alveolar epithelial cells (21, 28-30), which show that both Na entry pathways and Na-K-ATPase are inhibited by hypoxia. Flux measurements to assess transport activity indicate a very fast response to hypoxia, probably by inactivation (21) that is maintained by decreasing transport capacity, possibly by a decreased rate of expression (28) when exposure to hypoxia continues.

Severe hypoxia also causes a decrease in mitochondrial ATP production. It is therefore possible that ATP itself serves as the signal to trigger hypoxia-related cellular responses such as inhibition of ion transport by means of an insufficient energy supply, effects on protein synthesis, or modification of membrane-located transporters. The latter is indicated by results showing that chemical ATP depletion causes internalization and subsequent degradation of membrane transport proteins such as Na-K-ATPase in renal epithelial cells (22).

The present experiments were performed to study whether the reduction in transport activity of alveolar epithelial cells during prolonged hypoxia is associated with a decrease in the number of copies of transport proteins and whether effects observed in cultured cells also occur in vivo in the lung tissue of rats exposed to hypoxia.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Exposure of Cultured Cells and Rats to Hypoxia

Most experiments were performed on 2- to 8-day-old confluent A549 cells passaged 3-12 times after purchase (American Type Culture Collection, Manassas, VA), a human adenocarcinoma cell line with characteristics of primary cultured alveolar type II (ATII) cells that also shows hypoxic transport inhibition (21). The A549 cells were exposed to normoxic or hypoxic atmospheres (3% O2) for 1-24 h, respectively, in an O2 and CO2 (5%) controlled incubator (NUNC, Wiesbaden, Germany) as indicated in Figs. 1-10. Before hypoxic exposure, the culture medium (Ham's F-12 medium containing 10 mM HEPES, 15 mM NaHCO3, and 7% FCS; Life Technologies, Karlsruhe, Germany) was replaced with one equilibrated to the respective atmosphere.


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Fig. 1.   Western blots of alpha 1- and beta 1-Na-K-ATPase (A and B, respectively), Na/K/2Cl cotransport (C), and alpha -, beta -, and gamma -epithelial Na channel (ENaC; D-F, respectively) proteins in A549 cells. From confluent monolayers of A549 cells, membrane fractions were prepared after cell lysis in a high-pressure disruption bomb and differential centrifugation in the presence of protease inhibitors. Western blots were obtained by ligation with specific antibodies and peroxidase-labeled secondary antibodies for detection of chemiluminescence (see MATERIALS AND METHODS for details). A-C, lanes 1 and 2, scanned images from directly stained blots (3,3',5,5'-tetramethylbenzidine membrane peroxidase substrate) that allow visualization of prestained standards. Insets, typical blots from membranes of normoxia (N)- and hypoxia (H; 3% O2)-exposed A549 cells. Nos. on left, molecular mass in kDa.



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Fig. 2.   Hypoxia-induced decrease in alpha 1- and beta 1-Na-K-ATPase proteins (A and B, respectively) and Na-K-ATPase activity (C) in A549 cells. After exposure of confluent monolayers of A549 cells to normoxia and hypoxia for the indicated time periods, membrane fractions were prepared, and Western blots were obtained (see MATERIALS AND METHODS for details). The relative amounts of alpha 1- and beta 1-subunit proteins of Na-K-ATPase were determined from band density measurements after normalization to normoxic control values. Activity of Na-K-ATPase was measured in the same membrane preparations used for Western blot analysis. Values are means ± SD from 11 normoxia- and 4-7 hypoxia-exposed batches of cells for each exposure period. * Significant difference from normoxic control cells, P <=  0.05.



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Fig. 3.   Hypoxia-induced decrease in ENaC proteins of A549 cells. Preparations and quantification were performed as in Figs. 1 and 2. co, Control. Values are means ± SD from 4 Western blots for alpha - and beta -subunits and 2 Western blots for the gamma -subunit for each time period of exposure to hypoxia. * Significant difference from normoxic control cells, P <=  0.05.



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Fig. 4.   Hypoxia-induced decrease in Na/K/2Cl (NKCC) cotransport protein in A549 cells. Preparations and band evaluations were performed as in Figs. 1 and 2. Values are means ± SD from 13 Western blots for normoxic cells and 4, 4, and 5 Western blots for hypoxic cells (1, 4, and 24 h, respectively). * Significant difference from normoxic control cells, P <=  0.05.



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Fig. 5.   Lung water content in normoxia- and hypoxia-exposed rats. Lungs were extracted after pentobarbital sodium anesthesia and removal of major airways. Water content was determined from wet weight and dry weight after 15 h of drying at 85°C. Values are means ± SD expressed as water-to-dry weight ratio (gwater/gsolids) from 6 rats/group.



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Fig. 6.   Effects of hypoxia on alpha 1- and beta 1-Na-K pump proteins in whole lung tissue of hypoxia-exposed rats. After exposure of rats to normoxia or hypoxia for the indicated time periods, membranes were prepared from excised lungs of anesthetized rats (see MATERIALS AND METHODS for details). Western blot analysis was performed as in Figs. 1 and 2. A, lanes 1 and 2: typical Western blots of alpha 1- and beta 1-Na-K pump proteins showing the bands stained with the antibodies used (see Fig. 1). Nos. on left, molecular mass in kDa. Insets: typical results from 1 normoxic control rat and 1 rat exposed to 10% O2 for 24 h. B and C: band density measurements of Western blots of alpha 1- and beta 1-subunits, respectively, of Na-K-ATPase. Values are means ± SD from 10-12 normoxic rats and 5-6 hypoxic rats for each O2 concentration and time of exposure. * Significant difference from normoxic control rats, P <=  0.05.



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Fig. 7.   Northern blot analysis of the alpha - and beta -subunits of ENaCs in whole lung tissue of hypoxia-exposed rats. Treatment of rats was the same as in Fig. 4. Total RNA was prepared, and Northern blot analysis was performed with full-length cDNAs for hybridization. A: typical Northern blots of alpha - and beta -ENaC RNAs and 28S rRNA from lungs of normoxic and hypoxic (24 h at 10% O2) rats. B and C: band density measurements of alpha -ENaC and beta -ENaC mRNAs, respectively, after normalization for band densities of 28S rRNA. Values are means ± SD from 6-9 normoxic rats and 3 hypoxic rats for each O2 concentration and time of exposure. * Significant difference from normoxic control rats, P <=  0.05.



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Fig. 8.   Effects of hypoxia on NKCC cotransport protein of whole lung tissue of hypoxia-exposed rats. Exposure to hypoxia, sample preparation, and Western blot analysis were the same as in Figs. 4 and 1. A, lanes 1 and 2: typical Western blots showing the bands stained with the antibody used (see Fig. 1). Nos. on left, molecular mass in kDa. Inset: typical results from a normoxic control rat and a rat exposed to 10% O2 for 24 h. B: band density measurements of Western blots. Values are means ± SD from 10-12 normoxic rats and 3-7 hypoxic rats for each O2 concentration and time of exposure. * Significant difference from normoxic control rats, P <=  0.05.



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Fig. 9.   Energy status of whole lung tissue of hypoxia (10% O2)-exposed rats. Lung tissue was frozen in liquid nitrogen immediately on removal, pulverized at -196°C in a mortar, and precipitated in 0.6 N perchloric acid for metabolite measurements. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was evaluated by Northern blot analysis. A: ATP, ADP, and phosphorylation potential (ATP/ADP*Pi). B: lactate concentration (n = 6 normoxic and 2 hypoxic rats at each time of exposure). C: GAPDH mRNA band densities (n = 9 normoxic and 3 hypoxic rat lungs at each time of exposure). * Significant difference from normoxic control rats, P <=  0.05.



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Fig. 10.   Effects of hypoxia on alpha 1- and beta 1-Na-K-ATPase proteins (A and B, respectively) of alveolar type II cells from hypoxia-exposed rats. After exposure of rats to normoxia or hypoxia for the indicated time periods, the rats were anesthetized, and alveolar type II cells were prepared according to Dobbs (9) with medium equilibrated with normoxic and hypoxic gases. Total cell proteins were extracted immediately after the cell preparation was finished. Western blot analysis was performed as in Fig. 1. Values are means ± SD from 10 normoxic rats and 5 hypoxia-exposed rats of each time of exposure.

Male Sprague-Dawley rats (150-200 g) were exposed to normoxia or hypoxia (14, 12, and 10% O2) for 1, 4, or 24 h in an acrylic glass box (160-liter volume), with free access to water and standard rat chow. The gas flow was adjusted to maintain 20°C and to prevent an increase in CO2 inside the box. The gas composition in the box was checked with a blood gas analyzer (model 278, Ciba-Corning, Fernwald, Germany). Whole lungs were taken after pentobarbital sodium anesthesia, frozen in liquid nitrogen immediately after resection, and stored frozen at -80°C until further processing.

ATII cells were prepared according to Dobbs (9) from the lungs of normoxic male control rats (Sprague-Dawley, 150-200 g) and of rats that were exposed to hypoxia (10% O2) for 1, 4, and 24 h. Briefly, the lungs from rats anesthetized with pentobarbital sodium were perfused with ice-cold PBS while being ventilated with air or the hypoxic gas. ATII cells were isolated with elastase (Elastin Products, Owensville, MS) digestion, filtration, and differential adhesion. Cell purity and vitality typically were >90% as judged from Nile red staining and trypan blue exclusion, respectively. After being washed, the cells were suspended in lysis buffer [in mM: 250 sucrose and 50 Tris, pH 7.4, at room temperature containing the protease inhibitors N-tosyl-L-phenylalanine chloromethyl ketone, pepstatin, chymostatin, aprotinin, leupeptin, and 4-(2-aminoethylbenzenesulfonyl fluoride); Sigma, Deisenhofen, Germany] and were stored frozen (-80°C).

Preparation of Cell Membranes and Whole Cell Protein

Frozen rat lungs were homogenized with a mortar cooled with liquid nitrogen. Rat lung homogenates and A549 cells were lysed in a cell disruption bomb (Parr Instrument, Moline, IL) after release of the N2 pressure. The membranes were isolated by differential centrifugation (5 min at 1,000 g, 5 min at 5,000 g, and 20 min at 45,000 g) at 4°C and stored at -80°C. Total cell protein and total RNA of ATII cells were isolated with Tristar (Hybaid, Heidelberg, Germany). The protein concentration in lung and cell membrane preparations and whole cell lysates was measured with a protein assay with human serum albumin and globulin standards (Bio-Rad Laboratories, Hercules, CA).

Western Blots

Plasma membrane or total cell proteins were separated electrophoretically by SDS-PAGE with the Laemmli buffer system. Proteins in the gel were transferred onto a polyvinylidene difluoride or nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) in a semidry blotter (Bio-Rad Laboratories). Monoclonal Na-K-ATPase antibodies were provided by M. Caplan [6H antibody for alpha 1 (26); Department of Cellular and Molecular Physiology, Yale University Medical School, New Haven, CT] and Upstate Biotechnology (beta 1; Lake Placid, NY), and monoclonal Na/K/2Cl cotransporter antibodies were provided by C. Lytle [T4 (20); Department of Biomedical Sciences, University of California, Riverside, CA]. Polyclonal antibodies used for the detection of ENaC subunits were kindly provided by C. Canessa (Department of Cellular and Molecular Physiology, Yale University Medical School, New Haven, CT) (10). Horseradish peroxidase-linked secondary antibodies and enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Freiburg, Germany) were used for autoradiographic detection. Exposed X-ray films were scanned for evaluation of band densities (see Quantification of Western and Northern Blots).

Northern Blots

Total RNA from frozen rat lung homogenate was isolated according to Chomczynski and Sacchi (7). Northern blots were performed with nylon membranes (Schleicher & Schuell) according to Ausubel et al. (1). Full-length cDNAs for all three subunits of the ENaC were kindly provided by C. Canessa and were radiolabeled with 32P (random-primed DNA-labeling kit, Hybaid) for autoradiographic evaluation. Blots of ENaC and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were washed twice at 40°C with 2× saline-sodium citrate containing 0.1% SDS after hybridization, and blots of 28S rRNA were washed four times with 0.1× saline-sodium citrate-0.1% SDS. Bands of ENaC mRNA were normalized to the density of the 28S rRNA signal.

Quantification of Western and Northern Blots

Because each sample represented one individual rat, each gel was loaded with samples of six normoxic control rats and up to eight samples from lungs of hypoxic rats. Band densities were measured with the Scion Image software (Scion, Frederick, MD). For each gel, background-corrected band densities of all normoxic control rats were averaged, and each individual band density (from both normoxic and hypoxic rats) is expressed as a percentage of this normoxic mean.

Energy Status

Perchloric acid (0.6 M) extracts were obtained from the aliquots of lung tissue from hypoxic rats that were also used for Western blot analysis. In these extracts, ATP and ADP were measured as described earlier (21). Phosphate and lactate concentrations were measured with routine assays (Sigma). All concentrations were related to the amount of protein in the extracts, which, after the precipitate was dissolved with NaOH, was measured with a test kit (see Preparation of Cell Membranes and Whole Cell Protein).

Na-K-ATPase Activity

To compare the amount of Na-K-ATPase protein with transport capacity, Na-K-ATPase activity was measured according to Forbush (11) after the cell membrane preparations were pretreated with 0.65 mg/ml of SDS and 1% BSA. Na-K-ATPase activity was taken as the ouabain-sensitive portion of phosphate production; the ouabain concentration was 0.1 mM for A549 cells and 3 mM for rat tissue.

Lung Water Content

To detect edema formation, lung water was determined from the wet-to-dry weight ratio. Lungs from rats exposed to hypoxia (10, 12, and 14% O2 for 1, 4, or 24 h) were extracted, and the trachea and major bronchi were removed. The wet weight of the upper right lung lobe was determined. Dry weight was measured after the lungs were dried to a constant weight (at least 15 h) at 85°C.

Statistics

Results are means ± SD from the number of experiments given in Figs. 1-10. Statistical analysis was done by one-way analysis of variance and Student's t-test with the SigmaStat and SigmaPlot software packages from SPSS Science (Erkrath, Germany). Level of significance was P <=  0.05.


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A549 Cells

Experiments on A549 cells were performed to test whether a decrease in the amount of protein might contribute to the hypoxia-induced decrease in transport activity. In Western blots, the alpha 1-Na-K-ATPase protein was seen as a single band with a molecular mass of ~97 kDa (Fig. 1A). Figure 2A summarizes the results of band density measurements and shows that the amount of alpha 1-Na-K-ATPase protein in the membranes of A549 cells had decreased by ~13 (P < 0.08), 27 (P < 0.006), and 37% (P < 0.001) after 1, 4, and 24 h, respectively, of exposure to hypoxia (3% O2). Of the typically found 50- and 35-kDa bands of the Na-K-ATPase beta 1-subunit protein, only the 35-kDa band was found in A549 cells with this antibody (Fig. 1B). As shown in Fig. 2B, the 35-kDa band density had decreased during hypoxia, although the response was less pronounced than changes in the beta 1-subunit, resulting in reductions of 9, 14 (P < 0.017), and 14% after 1, 4, and 24 h, respectively, at 3% O2.

Approximately 20% of the total ATPase activity was inhibited by ouabain, suggesting that this portion represented Na-K-ATPase (data not shown). Its activity decreased during exposure to hypoxia (P < 0.001; Fig. 2C).

Typical Western blots of ENaC proteins in A549 cells are shown in Fig. 1, D-F. The antibodies stain single bands of ~115 (alpha -subunit), 90 (beta -subunit), and 65 (gamma -subunit) kDa. Those values differ slightly from others reported in the literature (23). The differences might be due to different levels of glycosylation. Figure 3 summarizes the results of band density measurements. It shows that both the alpha - and beta -subunits decreased progressively during exposure of A549 cells to hypoxia by ~20% (P < 0.017) after 4 h and 40-50% (alpha , P < 0.018; beta , P < 0.001) after 24 h. The amount of gamma -subunit protein was decreased by ~20% after 24 h of hypoxia. It has to be pointed out that the gamma -subunit of ENaC could not always be detected.

Bands in Western blots of the Na/K/2Cl cotransport protein in A549 cell membranes were usually quite wide (Fig. 1C), probably due to a variable degree of glycosylation (no deglycosylation was performed). The molecular mass was in the range of 150-190 kDa. The amount of Na/K/2Cl cotransport protein was unchanged after 1 h of hypoxia but decreased during prolonged exposure to 3% O2, which amounted to almost 30% after 24 h (P < 0.043; Fig. 4).

Lungs of Hypoxia-Exposed Rats

Water content. Lung water content was measured to obtain an indicator of the formation of pulmonary edema during exposure to hypoxia. Figure 5 shows that during exposure to 10% O2 for 1, 4 and 24 h, no change in lung water was detectable. There was also no change in cell water during exposure to 12 or 14% O2 (data not shown).

Western blots of transport proteins. Figure 6A shows typical Western blots of the alpha 1- and beta 1-Na-K-ATPase proteins. Whereas alpha 1-Na-K-ATPase typically gave a single band of ~100 kDa, beta 1-Na-K-ATPase sometimes showed several bands of low intensity besides the major band visible at ~50 and 35 kDa. However, in rat lung tissue, the 35-kDa band often was barely visible. The amount of transport proteins determined by Western blot analysis varied considerably with the degree of hypoxia and the time of hypoxic exposure. Band density measurements showed that 24 h of exposure of rats to hypoxia caused a significant decrease (by ~25%) in the amount of the alpha 1-subunit of Na-K-ATPase regardless of the degree of hypoxia (P < 0.03; Fig. 6B), whereas short-term exposure (1 or 4 h) had no significant effect. However, after 1 h of exposure to 14% O2, there was a slight but not significant increase in the alpha 1-Na-K-ATPase protein. There was a tendency toward similar changes in the amount of the 50-kDa beta 1-Na-K-ATPase band (Fig. 6C), but the changes were not significant.

Due to problems obtaining good signals on Western blots of ENaC, mRNA levels of this channel protein were determined by Northern blot analysis. Figure 7A shows typical Northern blots of alpha - and beta -ENaC mRNAs. Levels of alpha -ENaC mRNA were slightly elevated after 1 h of hypoxia (not significant) but decreased with prolonged exposure to hypoxia. After 24 h of hypoxia, the normalized alpha -ENaC mRNA-to-28S rRNA ratio had decreased significantly by ~25% (P < 0.034). In contrast, beta -ENaC mRNA levels varied considerably in the lungs from rats exposed to 12 and 14% O2, whereas during exposure to 10% O2, a steady decrease in beta -ENaC mRNA was found (worst case of significance, P < 0.043). gamma -ENaC mRNA could not be detected.

The amount of Na/K/2Cl cotransport protein in whole lung tissue, seen as two bands at ~130 and 180 kDa (no deglycosylation has been performed; Fig. 8A), was increased by >30% with a 1-h exposure to 10% O2 (P < 0.08) However, it was decreased significantly by almost 40% after 24 h of exposure to 12 (P < 0.008) and 10% O2 (P < 0.072; Fig. 8B)

Lung energy status. To test whether energy depletion precedes the changes in membrane transport proteins, measurements of the cell energy status were obtained on whole lung tissue of normoxic and hypoxic (10% O2) rats. Figure 9A shows a pronounced decrease in ATP after 1 and 4 h of hypoxia and a concomitant decrease in the phosphorylation potential as indicated by the decreased ATP/ADP*Pi quotient. However, values were normal after 24 h of hypoxia. No clear change in ADP was seen, whereas lactate (Fig. 9B) had decreased. Neither of these parameters changed significantly in the lungs of rats that were exposed to hypoxia of 12 and 14% O2 (data not shown). The mRNA level of the glycolytic enzyme GAPDH, used as a marker for one enzyme involved in energy metabolism that is known to be upregulated during hypoxia in various tissues (e.g., Ref. 12), increased by up to 50% (P < 0.034) during 4 and 24 h of exposure to hypoxia of 10% O2 (Fig. 9C) but not at the higher levels of O2 (data not shown).

ATII Cells of Hypoxia-Exposed Rats

Because whole lungs contain a variety of different cell types, we also studied the membranes of ATII cells from hypoxic rats. It needs to be pointed out that their preparation includes the complicated procedure of cell isolation that was carried out immediately after exposure of the rats to hypoxia. The procedure consists of a series of steps in which it is almost impossible to control the PO2. Due to the small sample size, whole cell protein rather than membrane protein was studied. Despite these limitations, the results from Western blot analysis appeared similar to those obtained from whole lung membrane protein preparations. The amount of the alpha 1- and beta 1-subunits of Na-K-ATPase in ATII cells of hypoxic rats decreased by ~35 (P < 0.036) and 24%, respectively (P < 0.068), after 24 h (Fig. 10, A and B, respectively). In contrast, Na/K/2Cl cotransport showed a greater variability but no significant hypoxia-related changes (data not shown).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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The goal of this work was to evaluate whether hypoxia changes the number of copies of transport proteins such as the Na-K pump, Na/K/2Cl cotransport, and ENaC in cultured lung alveolar epithelial cells and the rat lung during in vivo hypoxia. The results of the present study confirm some of the results by Planes and colleagues (28, 29) and show that not only in rat alveolar type II cells but also in A549 cells, the amount of immunoreactive protein in the above-mentioned transporters decreases slowly during exposure to hypoxia. Our results extend beyond those because we found similar changes in expression also in lung tissue and in freshly isolated ATII cells of rats that were exposed to hypoxia. These results demonstrate for the first time that the effects of hypoxia on alveolar ion transporters (21, 29, 30) are not a tissue culture phenomenon but also occur in vivo. They point out the potential clinical significance because the vital role of these transporters in the maintenance of alveolar fluid balance has clearly been demonstrated (5). A reduction in the number of copies of these transporters could reduce the capacity of alveolar ion transport. Subsequently, the reabsorption of fluid filtered into alveolar space might be reduced, thus favoring formation of alveolar edema.

Results from tracer flux measurements indicate that inhibition of Na transporters of A549 cells by hypoxia occurs within ~30 min, stays at this level of inhibition for at least 24 h while hypoxia continues, and can be reversed by reoxygenation (21). Flux studies on actual transport activity allow no direct conclusion on the mechanisms that lead to transport inhibition. Planes et al. (28) showed reduced levels of Na-K-ATPase and ENaC mRNAs and ENaC protein in hypoxia-exposed rat ATII cells, which also reverse on reoxygenation. Their results indicate for the first time that inactivation of transport by the inhibition of gene transcription could lead to a sustained transport inhibition during hypoxia (28). However, changes in expression cannot explain the fast inhibition of transport (21). Here we show that the number of copies of the alpha 1- and beta 1-subunits of Na-K-ATPase decreases slowly during hypoxia both in cultured alveolar epithelial cells and in lung tissue. However, there is some inconsistency in those results in that the magnitude of change in pump flux exceeded the change in amount of protein. It is possible that regulatory changes in pump activity, which could explain the rapid inactivation reported earlier (21), overlap with changes in pump expression. Our results on ENaC and Na/K/2Cl cotransport indicate that other ion transporters are affected similarly. It can therefore be concluded that the inhibition of Na-K-ATPase during prolonged hypoxia might be caused by a decreased rate of expression of transporters or a stimulation of proteolysis that causes the number of active transporters to decrease. Rafii et al. (30) showed recently that fetal distal lung epithelial cells that were cultured at 21% O2 decrease their amiloride-sensitive short circuit current as well as the expression of ENaC with 48 h of exposure to hypoxia (3% O2), which is in support of the results indicated above.

The mechanisms that control the expression of transport proteins in hypoxic lung epithelial cells are not understood. An increased expression of many hypoxia-induced proteins involves the transcription factor hypoxia-inducible factor-1alpha (36). It is, however, not known yet which factor mediates inactivation of expression by hypoxia. Rafii et al. (30) suggested a role for nuclear factor-kappa B and reactive oxygen species (ROS), which fits other reports on an involvement of ROS in the O2-sensing process (36). Lowering cellular ROS levels with scavengers mimics the hypoxia-induced decrease in ENaC expression and amiloride-sensitive short circuit current of fetal alveolar epithelial cells (30). Results from tracer flux measurements on A549 cells also show ROS scavenger-induced reduction in transport similar to the changes observed in hypoxia (14). The hyperoxia-induced upregulation of the expression of Na-K-ATPases (37) is not necessarily in support of this hypothesis because it might represent a mechanism compensating for cell damage (8). There are indications that the activity of transepithelial Na transport per se, possibly via changes in the intracellular ion concentration, might regulate the expression of Na-transporting systems (4, 32). These studies show that increased Na entry activates, whereas inhibition of Na uptake by epithelial cells reduces the expression of Na transporters such as Na-K-ATPase. However, we have no indication that similar mechanisms are involved in the expression of Na-K-ATPase, ENaC, and Na/K/2Cl cotransport in alveolar epithelial cells.

The decrease in synthesis of new transporters in hypoxia is in accordance with the decreased overall rate of synthesis of total membrane protein in hypoxic A549 cells as reported earlier (21). Also the expression of GAPDH (Fig. 9) is stimulated in alveolar epithelial cells by hypoxia (36). Taken together, these results support the notion that exposure to hypoxia induces a controlled up- and downregulation of the expression of certain proteins, resulting in adjustments of ATP-producing pathways and inactivation of energy-consuming processes such as ion transport, which may not necessarily be vital for maintaining basal cell functions (15). It has not been evaluated experimentally to what degree cellular O2 consumption can actually be lowered by inactivation of transepithelial transporter by hypoxic alveolar epithelial cells, but a tight coupling between metabolic activity and ion transport has been observed in various cell types (13, 35).

A possibility to initiate both rapid transport inhibition and inhibition of synthesis of transporters by hypoxia might be the decreased ATP availability. In A549 cells and homogenized lung tissue of hypoxia-exposed rats, ATP decreased within the first 2 h of hypoxia, but normal ATP levels were regained during continued exposure to hypoxia for several hours. The decrease in ATP clearly preceded the slow decrease in the number of copies of transporters. The degree of ATP depletion should not cause transport inhibition because Na-K-ATPase can operate with unchanged capacity at ~25% of normal ATP levels, whereas secondary active transporters such as Na/K/2Cl cotransport require even less ATP for maintaining basal activity (16). The increase in ATP on prolonged exposure to hypoxia might already be the result of adjustments in the metabolic pathways and the reduction in ion transport activity. It needs to be evaluated whether a transient ATP decrease or another signal related to diminished oxidative ATP production might induce adjustments of ion transport activity and transport protein expression in hypoxia (6). In support of this idea are the results on renal epithelial cells by Mandel et al. (22), who showed that on ATP depletion by substrate depletion, ion transporters such as Na-K-ATPase are internalized, thus reducing transport capacity (22). In contradiction to the "ATP hypothesis" are the results by Planes et al. (29) showing that chemical ATP depletion of SV40 virus-transformed ATII cells does not mimic inhibition of Na-K-ATPase by hypoxia.

Hypoxic transport inhibition observed in cultured alveolar epithelial cells has been discussed as a possible factor that might contribute to the formation of hypoxic pulmonary edema such as high-altitude pulmonary edema (31). Here we show for the first time that changes similar to those observed in hypoxic cultured cells also occur in vivo in the lungs of hypoxia-exposed rats as well as in their alveolar epithelial cells. However, we could not demonstrate that the decrease in the number of copies of transport proteins causes formation of pulmonary edema. Recent results (18) indicate that in hypoxic rats edema formation can only be observed after at least 5 days of exposure to an atmosphere containing 8% O2. The changes observed in the present study might therefore indicate a state of initial adjustments of the alveolar epithelium to hypoxia that is yet too weak to cause inhibition of transport sufficient to induce edema formation. Based on the above-mentioned findings, it is possible that this effect might be exaggerated by a prolonged time of exposure and/or an increased degree of hypoxia. Transgenic mice with a significantly reduced number of copies of alpha -ENaC protein develop pulmonary edema when exposed to hypoxia, whereas mice with a normal density of ENaC do not (19). Taken together, both results imply that an increased risk for developing hypoxic alveolar edema might exist in subjects with a reduced number of Na transporters already in normoxia. This "defect" might not affect the clearance of alveolar fluid (17) in normoxia (3), whereas in hypoxia, an impairment of the transport capacity by transport inhibition and/or reduction in the number of copies of ion transporters now becomes sufficient to cause alveolar flooding. Sartori et al. (33) presented evidence for reduced activity of lung Na transport in subjects susceptible to high-altitude pulmonary edema from measurements of nasal potentials. However, experiments to demonstrate reduced reabsorption of alveolar fluid in hypoxic human subjects caused by ion transport inhibition are still lacking.


    ACKNOWLEDGEMENTS

We thank Christiane Herth and Sonja Engelhardt for excellent technical assistance.


    FOOTNOTES

The project was supported by Grant MA 1503/11-1 from the German Research Foundation (DFG).

Address for reprint requests and other correspondence: H. Mairbäurl, Medical Clinic Section VII, Sports Medicine, Univ. of Heidelberg, Hospitalstr. 3 Geb. 4100, 69115 Heidelberg, Germany (E-mail: heimo_mairbaeurl{at}med.uni-heidelberg.de).

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.

Received 28 October 1999; accepted in final form 3 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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