Department of Sports Medicine, University of Heidelberg, D-69115 Heidelberg, Germany
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ABSTRACT |
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A reduced cation reabsorption across the alveolar epithelium decreases water reabsorption from the alveoli and could diminish clearing accumulated fluid. To test whether hypoxia restricts cation transport in alveolar epithelial cells, cation uptake was measured in rat lung alveolar type II pneumocytes (AII cells) in primary culture and in A549 cells exposed to normoxia and hypoxia. In AII and A549 cells, hypoxia caused a PO2-dependent inhibition of the Na-K pump, of Na-K-2Cl cotransport, and of total and amiloride-sensitive 22Na uptake. Nifedipine failed to prevent hypoxia-induced transport inhibition in both cell types. In A549 cells, the inhibition of the Na-K pump and Na-K-2Cl cotransport occurred within ~30 min of hypoxia, was stable >20 h, and was reversed by 2 h of reoxygenation. There was also a reduction in cell membrane-associated Na-K-ATPase and a decrease in Na-K-2Cl cotransport flux after full activation with calyculin A, indicating a decreased transport capacity. [14C]serine incorporation into cell proteins was reduced in hypoxic A549 cells, but inhibition of protein synthesis with cycloheximide did not reduce ion transport. In AII and A549 cells, ATP levels decreased slightly, and ADP and the ATP-to-ADP ratio were unchanged after 4 h of hypoxia. In A549 cells, lactate, intracellular Na, and intracellular K were unchanged. These results indicate that hypoxia inhibits apical Na entry pathways and the basolateral Na-K pump in A549 cells and rat AII pneumocytes in culture, indicating a hypoxia-induced reduction of transepithelial Na transport and water reabsorption by alveolar epithelium. If similar changes occur in vivo, the impaired cation transport across alveolar epithelial cells might contribute to the formation of hypoxic pulmonary edema.
lung; sodium-potassium pump; sodium-potassium-chloride cotransport; sodium channels; energy metabolism; protein synthesis; pulmonary edema
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INTRODUCTION |
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IN THE NORMAL LUNG, an equilibrium exists between the
entry of fluid into the alveolar space and fluid reabsorption in order to keep the film of alveolar lining fluid thin and to allow an adequate
diffusion of respiratory gases. In hypoxia, e.g., at high altitude,
this equilibrium can be disturbed by pulmonary edema and alveolar
flooding (10, 32) that, by thickening of the diffusion barrier, worsens
the degree of hypoxemia. The mechanisms that lead to the accumulation
of fluid in the alveolar space in hypoxia can be manifold
(32). Hypoxic vasoconstriction of pulmonary blood vessels
causes pulmonary hypertension and increases the amount of fluid that
leaves the vascular bed by filtration. Nifedipine prevents this effect
presumably by reducing the blood pressure in the pulmonary artery (1).
Mediators released from lung cells, like alveolar macrophages and
endothelial cells, which can be activated by hypoxia, by exercise, or
by infections, might alter the permeability of the endothelium and the
alveolar barrier, allowing plasma or plasma water to leave the
vasculature and to enter the interstitium and alveolar space.
Furthermore, cation transport processes across alveolar epithelial
cells, which in normoxia mediate Na-coupled water reabsorption in the
adult lung (22), might be disturbed, resulting in an impaired clearance of alveolar fluid, and might contribute to edema formation if this
transport is reduced in hypoxia. However, the role of ion transport in
the removal of accumulated edema fluid in hypoxia is unclear. Evidence
demonstrating the significance of alveolar Na reabsorption for fluid
reabsorption from the alveolar space comes from studies on the
clearance of lung liquid after birth (25). These studies indicate that
hormone-activated and amiloride-inhibitable pathways mediate the
removal of lung liquid (3, 28). In support of that are observations on
a knockout mouse that lacks the -subunit of the epithelial Na
channel (ENaC; see Ref. 11). These animals die within a few hours after
birth since they are unable to remove the fluid that is contained in
the lung airspace during prenatal development (11). Results by Planes
et al. (31) on virus-transformed rat alveolar epithelial cells indicate
a reduced activity of the Na-K pump when these cells are exposed to
hypoxia for at least 12 h. The basolaterally located Na-K pump mediates
the removal of Na that entered the cells across the alveolar interface.
Therefore, inhibiting the Na-K pump without reducing apical Na entry
would cause the alveolar epithelial cells to swell and lyse. Because there are no reports on the destruction of alveolar epithelium in
subjects exposed to hypoxia at high altitude and because cells exposed
to hypoxia in tissue culture tolerate hypoxia well, there have to be
other mechanisms that preserve the cellular integrity.
This study was performed to investigate effects of hypoxia on ion transport systems in alveolar epithelial cells that are involved in Na entry and Na exit and to evaluate possible mechanisms accounting for the hypoxia-induced changes found in cultured alveolar epithelial cells. Our results of flux measurements on A549 cells, a human lung-derived carcinoma cell line that shows most functions of alveolar type II cells (17), and on rat alveolar type II cells in primary culture indicate that not only the Na-K pump activity but also Na entry via the Na-K-2Cl cotransporter and Na channels is reduced already after short-term exposure to hypoxia in a reversible manner. This reduction in ion transport seems to be caused by a decreased transport capacity and appears to be independent of protein synthesis and of the energy status of the cells.
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MATERIALS AND METHODS |
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Reagents
All media were prepared from deionized water and analytical grade reagents. Ouabain, amiloride, trypsin, and soybean trypsin inhibitor as well as various protease inhibitors (see below) were from Sigma Chemical. Phosphate-buffered saline (PBS), Dulbecco's modified Eagle's medium (DMEM), Ham's F-12, penicillin/streptomycin (PenStrep), fetal calf serum (FCS), and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) were from GIBCO. Calyculin A and okadaic acid were from Calbiochem. HOE-694 [(3-methylsulphonyl-4-piperidino-benzoyl)guanidine methanesulfonate] was a gift from Hoechst, and bumetanide was a gift from Hofmann LaRoche.Preparation of Rat Alveolar Type II Cells
Alveolar type II (AII) cells for primary culture were prepared similar to the method described by Richards et al. (33). Briefly, lungs from male Sprague-Dawley rats (100-200 g) were extracted after Trapanal (Byk Gulden) anesthesia and perfusion of the ventilated lung with cold PBS to remove blood cells. The lungs were lavaged five times with PBS, filled with trypsin (0.25% in Hanks' balanced salt solution) to its maximal capacity, and incubated at 37°C for 20 min. Trypsin digestion was stopped by instillation of trypsin inhibitor in DMEM. Major blood vessels and bronchi were removed, and the tissue was microdissected under a stereomicroscope. The minced tissue was vortexed with deoxyribonuclease for 10 min and was passed through filters of 150 and 17 µm pore size. The cells were washed and preplated (60 min) in AII medium (33). Nonadhering cells were seeded on untreated 24-well plates (Costar) at a density of ~300,000 cells/well. The viability assessed by trypan blue exclusion was usually >90%, and >80% of the cells resembled AII pneumocytes, as judged from tannic acid stain. Experiments were either performed on freshly prepared cells in suspension or on primary cultured cells after reaching confluence (3-4 days after seeding) but before differentiation as indicated by the loss of lamellar bodies (1-2 days after reaching confluence).A549 Cells
Most of the experiments presented were performed on A549 cells (American Type Culture Collection) that were derived from human lung carcinoma cells and that resemble many features of AII cells (17). The cells were grown on untreated 24- or 96-well plates (Costar) in Ham's F-12 medium substituted with 7% FCS and PenStrep and buffered with HEPES and sodium bicarbonate. Confluence was reached ~3-4 days after seeding. All experiments were performed on 2- to 8-day-old confluent monolayers.Tissue Culture, Hypoxia
Both rat AII cells in primary culture and A549 cells were kept in an incubator (Heraeus) under the usual tissue culture conditions at 37°C and 5% CO2. For exposure to hypoxia, cells in suspension (AII cells) or cells grown to confluence in normoxia in the incubator (AII and A549 cells) were transferred into a thermostatized (37°C) acrylic glass box (volume ~8 liters) that was flushed with sterile-filtered (0.22 µm) humidified gas composed of varying concentrations of O2, 5% CO2, and N2 for varying periods of time. Hypoxia was initiated by replacing the tissue culture medium with one previously equilibrated to the appropriate gas. After an initial flushing with the appropriate gas at a high rate for 5 min, the gas flow was kept constant at a rate of ~0.2 l/min during the incubation. Based on the results by Wolff et al. (41) on O2-dependent erythropoietin production by hepatocytes, which indicate that the oxygen supply to the cultured cells might be limited even in normoxia when no adequate equilibration is guaranteed, shaking of the box appeared necessary. The acrylic glass box was therefore placed on a shaker (12 per min). Experiments to control for the shaking procedure indicated that ion fluxes were the same regardless of whether cells were kept without shaking in an O2-CO2 controlled incubator or in the rocked acrylic glass box under normoxic and hypoxic conditions. Therefore, especially for long-term incubations, an O2-CO2 controlled incubator (Nunc) was used to expose cells to hypoxia. PO2 and PCO2 in the atmosphere of the incubator and the acrylic glass box and in the tissue culture medium supernatant to the cells was checked with a blood gas analyzer (model 278; Corning).Flux Measurements
The activity of ion transport pathways was determined by unidirectional tracer uptake measurements. The cells were washed two times with washing medium [150 mM NaCl and 2 mM HEPES, pH 7.4 at room temperature (RT)] to remove tissue culture medium and were equilibrated to the flux medium for 15-40 min at RT under either normoxic or hypoxic conditions (CO2 and bicarbonate free). The flux medium was usually composed of (in mM) 140 NaCl, 5 KCl, 1 Na2HPO4, 1 MgCl2, 0.2 CaCl2, 10 glucose, and 20 HEPES, pH 7.4, at RT. Both media were equilibrated to the respective gas.After equilibration, the medium was replaced with fresh flux medium
containing either 86Rb (used as
tracer for K) or 22Na, both at
final activities of 2 µCi/ml. The tracer uptake into the cells,
measured at RT, was terminated with six washes with washing medium to
remove contaminating radioactivity. The cells were lysed with 0.1 M
NaOH. The radioactivity in the lysate was measured in a -counter
(model TR 2100; Canberra Packard), and the protein content (see below)
was used for standardization.
In control experiments, the tracer uptake was found to be linear over a period of at least 10 min. Therefore, in most experiments, only one 6-min time point was taken unless otherwise stated. All fluxes were measured in duplicate or triplicate on batches of cells from different passages.
The Na-K pump was taken as the ouabain-inhibitable portion of 86Rb uptake. Because of the low affinity for ouabain, the concentration used was 3 mM in rat AII pneumocytes but 0.1 mM in the human-derived A549 cells. The Na-K-2Cl cotransport was taken as the portion of 86Rb or 22Na uptake inhibited by 50 µM bumetanide. Amiloride inhibition of 22Na uptake was complete at an inhibitor concentration of 100 µM, but HOE-694 had no effect. The maximal effective concentration of all inhibitors used was verified with dose-response curves only in A549 cells and not in AII cells due to the limited amount of material.
Preparation of Cell Membranes
For the measurement of Na-K-ATPase activity and the electrophoretic separation of membrane proteins, A549 cells grown to confluence in 80-cm2 tissue culture flasks in normoxia were exposed to normoxia or hypoxia and then were washed free of culture medium with a lysis medium composed of 250 mM sucrose and 50 mM tris(hydroxymethyl)aminomethane base, pH 7.4 at RT, containing the protease inhibitors N-tosyl-L-phenylalanine chloromethyl ketone, pepstatin, chymostatin, aprotinin, leupeptin, and 4-(2-aminoethyl)benzenesulfonyl fluoride. The cells were scrubbed off of the tissue culture flask with a rubber spatula and were lysed in a cell disruption bomb (Parr Instruments) after releasing the N2 pressure (1,200 psi). Membranes were isolated from the lysate by differential centrifugation and were stored frozen atNa-K-ATPase Assay
Membranes prepared from A549 cells as described above were assayed for Na-K-ATPase activity to obtain a measure of Na-K pump capacity. ATPase activity was measured according to Forbush (6). The Na-K-ATPase activity was taken as the activity inhibited by 0.1 mM ouabain.Measurements of Cell Metabolites and Cations
Freshly isolated AII cells kept in suspension and A549 cells grown to confluence in normoxia in 80-cm2 culture flasks were exposed to either normoxia or hypoxia as described above. Supernatant culture medium was removed by two washes with ice-cold washing medium. The cells were lysed with ice-cold 0.6 N perchloric acid (PCA). Precipitated protein was removed by centrifugation (3 min, 13,000 g, RT) in a microfuge. The pH of the supernatant was adjusted to seven with K2CO3. ATP and ADP were measured by high-performance liquid chromatography according to Weicker et al. (39). Lactate in supernatant tissue culture medium (A549 cells only) was measured using a test kit from Sigma Chemical. Intracellular Na (Nai) and K (Ki) concentrations were measured by flame photometry (model 410; Corning) in PCA extracts of confluent A549 cells.Protein Synthesis
The amount of [14C]serine incorporated into cell protein was determined as a measure of protein synthesis. [14C]serine was added to A549 cells grown to confluence in 3-cm dishes in normoxia and then was exposed to either normoxia or hypoxia. After incubation, the culture medium and contaminating [14C]serine were removed by four washes with washing medium. The cells were lysed with 0.6 N PCA. The precipitated protein was collected on cellulose filters (GF/C, average pore size 0.22 µm; Whatman). The radioactivity on the filters was determined by liquid scintillation counting.Other Measurements
Nai and Ki were measured by flame photometry after washing the cells two times with a medium composed of 150 mM choline chloride and 2 mM HEPES, pH 7.4 at RT, and subsequent lysis with 0.1% lithium dodecyl sulfate in water. The protein concentration was measured according to Bradford using a test kit from Bio-Rad and standards composed of human serum albumin and globulin in saline diluted with 0.1 M NaOH.Statistical evaluation. All
measurements were performed in duplicates to quadruplicates on cells
from at least three different passages. Results are presented either as
mean values ± SD of repeated measurements from one of several
experiments with similar results or as mean values ± SD of several
experiments on different batches of cells as indicated. Comparisons
were made using Student's t-tests.
Dose-response curves and time courses were tested by one-way analysis
of variance and linear correlation analysis, respectively. The level of
significance was P 0.05. Graphical and statistical evaluation was performed with the SigmaPlot (Jandel Scientific) and Systat (Systat) software packages.
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RESULTS |
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A549 Cells
Exposure of A549 cells to hypoxia (3% O2, ~21 mmHg) for 4 h caused a 14% decrease in the ATP content (Fig. 1). No statistically significant change was found in the ADP content in the cell lysate and the ATP-to-ADP ratio. The lactate concentration in supernatant culture medium was 4.8 ± 0.8 mM in cells cultured in normoxia and 4.7 ± 0.6 mM in cells exposed to hypoxia. The total amount of protein per well was not different between normoxic cells and cells exposed to 3% O2 for 4 h. Also, the Na and K content of the cells remained unchanged during hypoxia (results not shown).
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Figure 2 shows the time course of changes in the total 86Rb uptake by A549 cells upon exposure to 3% O2. Figure 2 shows that, within 15 min of hypoxia, the total 86Rb uptake was decreased by ~40% and that this decrease was maintained for 20 h. Reoxygenation for 2 h of cells exposed to 3% O2 for 2, 4, and 20 h was sufficient to restore the normal 86Rb uptake almost completely.
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In normoxia, ~40% of the total 86Rb uptake by A549 cells can be attributed to the Na-K pump. Exposure to hypoxia reduced the Na-K pump. This inhibition was seen already after 15 min of exposure to hypoxia (3% O2) and was stable for 20 h. A higher degree of hypoxia (1.5% O2) caused a further reduction of the pump flux (Fig. 3). Also, the activity of the Na-K-ATPase measured on plasma membranes prepared from cells exposed to 3% O2 for 4 h decreased by ~55% (Fig. 4).
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About 45% of the total 86Rb uptake was inhibited by 50 µM bumetanide (Fig. 5A). The half-maximal inhibitory concentration (IC50) for the bumetanide inhibition of 86Rb uptake was 1.1 ± 0.05 µM. These values are comparable to results obtained on different cell types (9). Bumetanide-sensitive 86Rb uptake and bumetanide-sensitive 22Na uptake (not shown) were about of the same magnitude. Therefore, it appears likely that this flux represents Na-K-2Cl cotransport. Na-K-2Cl cotransport is reduced by ~40% already within 15 min of exposure of A549 cells to hypoxia (3% O2; n = 2; not shown). Inhibition (~50%) was stable over the period of 4-20 h of hypoxia (Fig. 6). At 1.5% O2 the inhibition was more pronounced. As with the Na-K pump, upon reoxygenation for 2 h, the inhibition of cotransport was reversed (not shown). As a measure for cotransport capacity, 86Rb uptake was measured in A549 cells treated with 0.1 µM calyculin A before the flux measurement but after exposure to hypoxia. Thereby, the exposure to calyculin A was limited in time, which was important, since it is not clear whether calyculin A affects the cells response to hypoxia. This experiment is based on the observation that Na-K-2Cl cotransport is active in a phosphorylated state (18) and that, with maximal phosphorylation by inhibition of its dephosphorylation with inhibitors of the protein phosphatases PP1 and/or PP2a, also the cotransport flux reaches its maximal activity (30). Control experiments on normoxic cells indicated that it was necessary to apply calyculin A at least 10 min before the flux measurement to achieve complete activation of the cotransport flux (Fig. 5B). The IC50 for calyculin A activation of 86Rb uptake was 10 ± 3 nM, and this value was not changed by hypoxia. Values reported on the Na-K-2Cl cotransport activation by calyculin A obtained on endothelial cells are in the same range (15) and are similar to values for inhibition of type I and IIa protein phosphatases measured in cell-free systems (13). Figure 6 shows that the cotransport activity measured after treatment with calyculin A was inhibited when A549 cells were exposed to hypoxia for 4 and 20 h. As in untreated cells, this inhibition was more pronounced when the PO2 was decreased from 21 to 12 mmHg.
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Nifedipine at a concentration of 10 µM reduced total 86Rb uptake in normoxic and hypoxic A549 cells by ~10% and did not prevent the hypoxia-induced inhibition of transport (Fig. 7A). It did not affect the activity of the Na-K pump.
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The uptake of 22Na was measured to assess the activity of transport systems in which Na transport is not coupled to the transport of K. In normoxia, ~25% of the 22Na uptake into A549 cells was inhibited by bumetanide. The hypoxia effect on Na-K-2Cl cotransport measured as 22Na uptake was the same as for 86Rb uptake (result not shown). There was also a significant portion of 22Na uptake in normoxic cells that was inhibited by amiloride. However, this fraction varied quite widely from 25 to 65% of the total 22Na uptake. Because amiloride inhibits Na-H exchange and Na channels (16), it was important to determine which of these transport systems mediated the amiloride-sensitive 22Na uptake in A549 cells and how they were affected by hypoxia. In normoxia, amiloride inhibited 22Na uptake with an IC50 of 1.2 ± 0.2 µM in the high-Na flux medium used in all experiments, and 100 µM amiloride gave complete inhibition. Neither dimethylamiloride nor HOE-694 (Fig. 8), both specific inhibitors of Na-H exchange (16, 37), significantly inhibited 22Na uptake by A549 cells exposed to normoxia and hypoxia. This indicates that the amiloride-sensitive portion of 22Na uptake might be mediated by Na channels. Figure 8 shows that exposure to 3% O2 for 4 h decreased the total 22Na uptake by ~40%. Addition of HOE-694 had no effect. The amiloride-sensitive portion of 22Na uptake was reduced by ~25% in hypoxia.
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Protein synthesis. It was of interest whether prolonged exposure to hypoxia inhibited the protein synthesis of A549 cells and whether any reduction in protein synthesis might explain the hypoxia-induced reduction in the activity of the ion transport systems shown above. The uptake of [14C]serine into the cells was linear over several hours. In normoxia, ~90% of the rate of protein synthesis is inhibited by 50 µM of cycloheximide (IC50 = 0.42 ± 0.03 µM). Figure 9A shows that the amount of [14C]serine incorporated into protein was reduced after 4 h of hypoxia at 3% O2 but that the cycloheximide-insensitive portion remained unchanged. Figure 9B shows the results of flux measurements of cells exposed to normoxia and 3% O2 for 4 h in the absence or presence of 50 µM cycloheximide. Cycloheximide inhibition of protein synthesis did not affect 86Rb uptake in cells exposed to normoxia or hypoxia.
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Rat AII Pneumocytes
A549 cells appear to have many functions of AII cells but are derived from carcinogenous tissue and were passaged many times. It was therefore unclear whether ion transport processes studied on these cells reflect those of the lung alveolar epithelium. To test if A549 cells can be used as a model cell for AII cells, thus substituting for animal experiments, subsets of the experiments shown before were also performed on rat AII cells. These cells were used for experiments either freshly after isolation or after growing to confluence in normoxia under the usual tissue culture conditions.As with A549 cells, AII cells also decreased their ATP content upon 4 h of exposure to hypoxia (3% O2). The ATP concentration of freshly isolated AII cells that were kept suspended in normoxia- or hypoxia-equilibrated media was ~20% lower in the hypoxic AII cells than in normoxic controls (Fig. 1). No change in AII cell ADP was found, but the ATP/ADP fell significantly by ~25%.
The results of measurements of the Na-K pump activity and Na-K-2Cl cotransport as ouabain-sensitive and bumetanide-sensitive 86Rb uptake, respectively, and as amiloride-sensitive 22Na uptake are shown in Table 1. In normoxia, the Na-K pump mediated ~65% of the total 86Rb uptake, and ~12% was mediated by Na-K-2Cl cotransport. Amiloride (0.2 mM) inhibited ~50% of the total 22Na uptake measured in the high-Na flux medium. The total 86Rb uptake was significantly higher in AII cells grown to confluence than in freshly isolated cells; the Na-K pump was 6-fold and the Na-K-2Cl cotransport was 20-fold increased in confluent AII cells. In both freshly isolated and confluent AII cells, all flux components were about one order of magnitude lower than in confluent A549 cells. As in A549 cells and in AII cells, the Na-K pump, Na-K-2Cl cotransport, and the amiloride-inhibitable portion of 22Na uptake were reduced after 4 h of hypoxia at 3% O2. The percent change in 86Rb uptake in normoxia was about the same in both cell types, whereas the hypoxia-induced decrease in the amiloride-inhibitable portion of 22Na uptake was greater in AII cells.
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Figure 7 shows that, similar to the results obtained from A549 cells, the hypoxia (3% O2, 4 h)-induced inhibition of 86Rb uptake by freshly isolated AII cells could not be prevented by pretreatment with nifedipine. On the contrary, when nifedipine was present over the 4-h incubation period, it caused a slight inhibition of transport in both the normoxic and hypoxic cells. In contrast to A549 cells, nifedipine inhibited the Na-K pump in normoxic AII cells.
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DISCUSSION |
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The results indicate that the activity of transport pathways involved in the transepithelial transport of Na+ is reduced in both a human lung carcinoma cell line derived from alveolar epithelium and rat AII cells when these cells were exposed to hypoxia under tissue culture conditions. The Na+ transported by these mechanisms is assumed to contribute to the generation of an electrochemical and osmotic gradient that can be utilized for the reabsorption of water across the alveolar epithelium. Any reduction in Na transport can therefore be taken as an indication of an impaired water reabsorption from the alveolar space that might cause an accumulation of water in the alveoli and therefore contribute to the formation of lung edema in hypoxia.
In the adult lung, AII pneumocytes are thought to be the cells responsible for keeping the water film in the subphase between the surfactant layer and the epithelium thin by controlling cation and water reabsorption (22). The basis for this assumption is 1) the observation of active transport in AII cells (7, 21) and 2) the observation that alveolar type I cells, despite the fact that they cover >90% of the alveolar surface but represent only ~10% of all lung cells, do not contribute to Na and water reabsorption across the alveolar epithelium (22), probably because of a very low density of Na-K pumps (12). However, direct proof for the exclusive role of either of the two cell types in fluid reabsorption is lacking.
Water reabsorption by AII cells is, as in other reabsorptive epithelia,
coupled to the transepithelial transport of Na, which is driven by a
basolateral Na-K pump and apical pathways for Na entry into the cell.
The latter ones shown to be present in AII cells are ENaC (23), Na-H
exchange, Na-K-2Cl cotransport, and probably also Na-coupled glucose
and amino acid transporters (24, 35). The rate-limiting steps are
assumed to be the Na entry pathways, whereas the Na-K pump seems to
operate at a high rate. Our own results obtained on rat AII cells in
primary culture and on A549 cells indicate that up to ~80 or 90% of
the Na entry into the cell is mediated by amiloride- and
bumetanide-sensitive pathways. Based on the observation that the
bumetanide-sensitive portion of Na and Rb uptake are of the same size
and that the IC50 for bumetanide
is in the range of 1 µM, it is reasonable to assume that this
transport resembles Na-K-2Cl cotransport. Evidence for the presence of
Na-K-2Cl cotransport in A549 cells comes also from preliminary
experiments in which the presence of an Na-K-2Cl cotransport protein
with a molecular weight of ~156,000 was identified (not shown) with
the T4 antibody (gift of C. Lytle, University of California, Riverside,
CA) directed against the cotransport protein of T84 cells (19). The
portion of Na uptake inhibited by a high concentration of amiloride
(0.2 mM) appears to be mediated by Na channels rather than by Na-H
exchange. In A549 cells, evidence for Na channel-mediated Na uptake
comes from the IC50 for amiloride inhibition of ~1.2 µM, which is lower than the
IC50 for Na-H exchange, and the
lack of effects of dimethylamiloride and HOE-694, which are both
specific inhibitors of Na-H exchange (16, 37). Further evidence comes
from results showing that about the same portion of Na uptake that is
inhibited by amiloride can also be inhibited with benzamil (40) and the
presence of ENaC proteins in plasma membranes of A549 cells (40)
detected with polyclonal antibodies against ENaC subunits ,
, and
(collaboration with C. Canessa, Dept. Cellular and Molecular
Physiology, Yale University; see Ref. 2). However, additional work
needs to be done to specify the type of Na channel. The presence of
apical Na channels in AII cells has been shown by various authors (26,
27, 29) but has not been demonstrated in A549 cells before. The
significance of amiloride-sensitive pathways and of the Na-K pump for
lung liquid reabsorption has been demonstrated in studies on the
removal of fluid instilled into the intact lung by Ussing chamber
measurements on AII cells in primary culture (24, 35) and in a knockout mouse in which the
-subunit of ENaC has been deleted (11). The
contribution of other Na-coupled transport systems to the removal of
alveolar fluid is less clear.
Whereas it is evident from the above-mentioned reports that
transepithelial ion transport is involved in lung liquid clearance and
that, in alveolar epithelial cells, hypoxia seems to impair ion
transport, there are no reports that, in hypoxia, an impairment of lung
liquid clearance by reducing transalveolar ion transport contributes to
the formation of hypoxic pulmonary edema. This issue appears rather
controversial. In vitro, Planes et al. (31) demonstrated a reduction in
Na-K pump activity in an established cell line of SV40
virus-transformed rat AII cells after exposing these cells to hypoxia
for at least 12 h. Inhibition appears to be independent of the degree
of hypoxia (31). In A549 cells, a human lung carcinoma cell line
resembling several functions of AII cells, we show a rapid inhibition
of several cation uptake systems that varies with the degree of hypoxia
(see Figs. 2, 3, and 6) and that is reversible upon reoxygenation. We
also report that, in rat AII cells in primary culture, the Na-K pump,
Na-K-2Cl cotransport, and (presumably) Na channel-mediated flux are
reduced significantly after 4 h of hypoxia (Table 1). Although we
cannot rule out some damage and therefore alterations in function of rat primary AII cells in culture, these results are in agreement with
reports on a reduced dome formation by confluent rat AII cells upon
treatment with metabolic inhibitors (8). Information obtained on intact
lungs is sparse and less clear (32). Any maneuver known to stimulate
transepithelial ion transport in vitro also enhances the reabsorption
of liquid instilled into lungs (35). However, the absence of blood flow
and ventilation did not prevent the clearance of instilled serum in
sheep lungs (34), but ouabain still inhibited all fluid reabsorption
(34). Although no pulmonary edema was observed in isolated, perfused
rat lungs, inhibitors of aerobic metabolism decreased the rate of
reabsorption of Na+ and instilled
fluid (36). Furthermore, it has never been shown that inhibition of ion
transport by applying blockers of cation transport systems to the
intact lung causes pulmonary alveolar edema. The -ENaC knockout
mouse model (11) provides some evidence but has not been studied enough
to characterize effects that might occur secondary to the deletion.
The mechanisms causing the hypoxia-induced inhibition of ion transport in alveolar epithelial cells in tissue culture are not understood. The reduction in ATP in the SV40 transformed cells points to a metabolic limitation of active transport in hypoxia in this cell type (31), which agrees with the previously mentioned reduction of dome formation by metabolic inhibitors (8). In primary cultured AII cells and A549 cells, hypoxia decreased cellular ATP levels by 20% or less, which appears too small to cause inhibition of active transport and points to a mechanism that might be directly dependent on the level of oxygen. Metabolic inhibition might therefore have synergistic effects. A decrease in ATP would primarily affect active ion transport systems. An inhibition of the Na-K pump would cause Nai and cell volume to increase and, subsequently, would lead to cell lysis. The lack of increase in Nai in A549 cells implies therefore a coordinate inhibition of both apical Na entry and basolateral Na-K pumps. This might be a protective mechanism to conserve energy and to prevent cell destruction in hypoxia.
The hypoxia-induced reduction in the activity of the transporters studied might be due to inactivation or reduction of the number of active transporters in the plasma membrane. However, signaling pathways are unclear. Planes et al. (31) found that inhibiting Ca entry with nifedipine also prevents the hypoxia-induced inhibition of Na-K pumps in SV40 virus-transformed alveolar epithelial cells. In contrast, in primary cultured rat AII cells and in the human lung A549 cell, nifedipine did not prevent the inhibition of 86Rb uptake caused by hypoxia (Fig. 7). Rather, nifedipine reduced transport in both normoxic and hypoxic cells, indicating some Ca-dependent regulation of ion transport but a lack of involvement of nifedipine-sensitive Ca entry in the inhibition of transport by hypoxia.
Our results indicate that the transport capacity, a measure of the number of active transporters in the plasma membrane, is reduced in A549 cells made hypoxic. The reduction in the number of Na-K pumps and Na-K-2Cl cotransporters in the plasma membrane can be caused by internalization of active transporters into vesicles, an increased rate of degradation, and a reduced synthesis and/or translocation to the membrane. We measured protein synthesis and effects of protein synthesis inhibitors on the ion transport of A549 cells in hypoxia. The result of a reduced amount of protein containing [14C]serine indicates that hypoxia causes an inhibition of the overall protein synthesis in A549 cells. Other reports also indicate a reduction of protein synthesis by hypoxia (14), but there is also upregulation of the transcription of certain proteins like the hypoxia-inducible factor or hypoxia-associated proteins (4). However, the application of cycloheximide, which inhibits ~90% of the protein synthesis in A549 cells, did not alter the total Na-K pump and Na-K-2Cl cotransport-mediated 86Rb uptake by A549 cells, neither in normoxia nor in hypoxia. The turnover of the studied transporters appears to be too slow to be affected by a 4-h inhibition of protein synthesis. Therefore, it appear unlikely that a reduced rate of protein synthesis causes the effect of hypoxia on ion transport activity. The failure to prevent the hypoxia-induced reduction of ion transport with cycloheximide indicates further that a factor regulated by transcription, as in the case in the production of erythropoietin (38), is not involved in transducing a signal that mediates the inhibition of ion transport in hypoxia. It also indicates that hypoxia does not accelerate the degradation of transport proteins.
Taken together, these results show that hypoxia affects ion transport in lung alveolar epithelial cells of different origin. The mechanisms causing this effect are unclear and appear to differ depending on the system studied. There are distinct differences in the response to hypoxia between SV40 virus-transformed rat alveolar epithelial cells (31) and primary culture rat AII cells or human A549 lung carcinoma cells, raising the question of which cell type is the better model. Differences include the time course of inhibition of ion transport by hypoxia, which appears much slower in SV40-transformed cells, indicating a greater resistance to oxygen deprivation, as well as the dependency of this inhibition on extracellular Ca and the involvement of nifedipine-sensitive Ca channels in Ca entry in hypoxia. The latter aspect points to differences in the regulation of transport by Ca between these cells. Nevertheless, comparative studies on cultured cells and animal models are required to test whether cell lines can be used as a model system to simulate and study appropriately hypoxia effects on lung alveolar epithelium.
In conclusion, our results obtained in cultured alveolar epithelial cells indicate a hypoxia-induced reduction in the activity of apical Na entry and basolateral Na exit pathways that usually generate the gradients required for the reabsorption of excess water accumulated in the alveolar space. If under hypoxia similar changes occur in vivo, an impaired reabsorption of fluid accumulated in the alveolar space may be another pathophysiological factor that contributes to the formation of pulmonary edema when the capillary pressure and/or the capillary permeability are increased as in high altitude pulmonary edema or adult respiratory distress syndrome.
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FOOTNOTES |
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Preliminary results were presented in abstract form (Pflügers Arch. 431: R106/P-229, 1996 and Eur. J. Clin. Invest. 26: A60/347, 1996).
Address for reprint requests: H. Mairbäurl, Institut für Sportmedizin, Medizinische Klinik und Poliklinik, Universität Heidelberg, Hospitalstrasse 3/4100, D-69115 Heidelberg, Germany.
Received 3 October 1996; accepted in final form 23 June 1997.
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