1 Medical Clinic, Section Sports Medicine, University of Heidelberg, 69115 Heidelberg; and 2 Department of Vegetative Physiology, University of Cologne, 50931 Cologne, Germany
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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,
1- and
1-Na-K-ATPase, Na/K/2Cl
cotransport, and ENaC proteins decreased during hypoxia. In whole lung
tissue,
1-Na-K-ATPase and Na/K/2Cl cotransport decreased.
- and
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
|
|
|
|
|
|
|
|
|
|
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 atWestern 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 forNorthern 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 ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, theApproximately 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 (-subunit), 90 (
-subunit), and 65 (
-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
- and
-subunits decreased progressively during exposure of A549 cells to
hypoxia by ~20% (P < 0.017) after 4 h and
40-50% (
, P < 0.018;
, P < 0.001) after 24 h. The amount of
-subunit protein was
decreased by ~20% after 24 h of hypoxia. It has to be pointed out that the
-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 1- and
1-Na-K-ATPase
proteins. Whereas
1-Na-K-ATPase typically gave a single band of ~100 kDa,
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
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
1-Na-K-ATPase protein. There was a tendency toward similar changes in the amount of the 50-kDa
1-Na-K-ATPase band (Fig. 6C), but the changes
were not significant.
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 ![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 1- and
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-1 (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-
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 -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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ausubel, FM,
Brent R,
Kingston RE,
Moore DD,
Smith JA,
Seidman JG,
and
Struhl K.
Current Protocols in Molecular Biology. New York: Wiley, 1987.
2.
Bärtsch, P.
High altitude pulmonary edema.
Respiration
64:
435-443,
1997[ISI][Medline].
3.
Bärtsch, P,
Maggiorini M,
Ritter M,
Noti C,
Vock P,
and
Oelz O.
Prevention of high-altitude pulmonary edema by nifedipine.
N Engl J Med
325:
1284-1289,
1991[Abstract].
4.
Bowen, JW,
and
McDonough AA.
Pretranslational regulation of Na-K-ATPase in cultured canine kidney cells by low K+.
Am J Physiol Cell Physiol
252:
C179-C189,
1987
5.
Carter, EP,
Garat C,
Sakuma T,
Wang Y,
Campbell AR,
and
Matthay MA.
Resolution of alveolar edema: in vivo studies.
In: Pulmonary Edema, edited by Matthay MA,
and Ingbar DH.. New York: Dekker, 1998, vol. 116, p. 431-456. (Lung Biol Health Dis Ser)
6.
Chandel, NS,
Maltepe E,
Goldwasser E,
Mathieu CE,
Simon MC,
and
Schumacker PT.
Mitochondrial reactive oxygen species trigger hypoxia-induced transcription.
Proc Natl Acad Sci USA
95:
11715-11720,
1998
7.
Chomczynski, P,
and
Sacchi N.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:
159-165,
1987.
8.
Clerici, C.
Sodium transport in alveolar epithelial cells: modulation by O2 tension.
Kidney Int
65:
S79-S83,
1998.
9.
Dobbs, LG.
Isolation and culture of alveolar type II cells.
Am J Physiol Lung Cell Mol Physiol
258:
L134-L147,
1990
10.
Duc, C,
Farman N,
Canessa C,
Bonvalet JP,
and
Rossier BC.
Cell-specific expression of epithelial sodium channel ,
, and
subunits in aldosterone-responsive epithelia from rat: localization by in situ hybridization and immunocytochemistry.
J Cell Biol
127:
1907-1921,
1994[Abstract].
11.
Forbush, B.
Assay of Na,K-ATPase in plasma membrane preparations: increasing the permeability of membrane vesicles using sodium dodecyl sulfate buffered with bovine serum albumin.
Anal Biochem
128:
159-163,
1983[ISI][Medline].
12.
Graven, KK,
McDonald RJ,
and
Farber HW.
Hypoxic regulation of endothelial glyceraldehyde-3-phosphate dehydrogenase.
Am J Physiol Cell Physiol
274:
C347-C355,
1998
13.
Harris, SI,
Balaban RS,
and
Mandel LJ.
Oxygen consumption and cellular ion transport: evidence for adenosine triphosphate to O2 ratio near 6 in intact cell.
Science
208:
1148-1150,
1980[ISI][Medline].
14.
Heberlein, W,
Wodopia R,
Bärtsch P,
and
Mairbäurl H.
Possible role of ROS as mediators of hypoxia-induced ion transport inhibition of alveolar epithelial cells.
Am J Physiol Lung Cell Mol Physiol
278:
L640-L648,
2000
15.
Hochachka, PW.
Defense strategies against hypoxia and hypothermia.
Science
231:
234-241,
1986[ISI][Medline].
16.
Hoffman, JF,
and
Kregenow FM.
The characterization of new energy dependent cation transport processes in red blood cells.
Ann NY Acad Sci
137:
566-576,
1966[ISI][Medline].
17.
Hultgren, HN,
Lopez CE,
Lundberg E,
and
Miller J.
Physiologic studies of pulmonary edema at high altitude.
Circulation
29:
393-408,
1964[ISI].
18.
Jones, HA,
Krausz T,
and
Morrell NW.
Accumulation and activation of macrophages (M), but not neutrophils (PMN), in the lungs of hypoxic rats (Abstract).
Am J Respir Crit Care Med
159:
A163,
1999[ISI].
19.
Lepori, M,
Hummler E,
Feihl F,
Sartori C,
Nicod P,
Rossier B,
and
Scherrer U.
Amiloride sensitive sodium transport dysfunction augments susceptibility to hypoxia-induced lung edema (Abstract).
FASEB J
12:
231,
1998
20.
Lytle, C,
Xu JC,
Biemesderfer D,
and
Forbush B.
Distribution and diversity of the Na-K-Cl cotransport protein: a study with monoclonal antibodies.
Am J Physiol Cell Physiol
269:
C1496-C1505,
1995
21.
Mairbäurl, H,
Wodopia R,
Eckes S,
Schulz S,
and
Bärtsch P.
Impairment of cation transport in A549 cells and rat alveolar epithelial cells by hypoxia.
Am J Physiol Lung Cell Mol Physiol
273:
L797-L806,
1997[ISI][Medline].
22.
Mandel, LJ,
Doctor RB,
and
Bacallao R.
ATP depletion: a novel method to study junctional properties in epithelial tissues. 2. Internalization of Na+,K+-ATPase and E-cadherin.
J Cell Sci
107:
3315-3324,
1994
23.
Matalon, S,
Benos DJ,
and
Jackson RM.
Biophysical and molecular properties of amiloride-inhibitable Na+ channels in alveolar epithelial cells.
Am J Physiol Lung Cell Mol Physiol
271:
L1-L22,
1996
24.
Matthay, MA.
Function of the alveolar epithelial barrier under pathologic conditions.
Chest
105:
S67-S74,
1994[ISI].
25.
Matthay, MA,
and
Ingbar DH.
Pulmonary Edema. New York: Dekker, 1998, vol. 116. (Lung Biol Health Dis Ser)
26.
Pietrini, G,
Matteoli M,
Banker G,
and
Caplan MJ.
Isoforms of the Na,K-ATPase are present in both axons and dendrites of hippocampal neurons in culture.
Proc Natl Acad Sci USA
89:
8414-8418,
1992[Abstract].
27.
Pittet, JF,
Wiener-Kronish JP,
McElroy MC,
Folkesson HG,
and
Matthay MA.
Stimulation of lung epithelial liquid clearance by endogenous release of catecholamines in septic shock in anesthetized rats.
J Clin Invest
94:
663-671,
1994[ISI][Medline].
28.
Planes, C,
Escoubet B,
Blot-Chabaud M,
Friedlander G,
Farman N,
and
Clerici C.
Hypoxia downregulates expression and activity of epithelial sodium channels in rat alveolar epithelial cells.
Am J Respir Cell Mol Biol
17:
508-518,
1997
29.
Planes, C,
Friedlander G,
Loiseau A,
Amiel C,
and
Clerici C.
Inhibition of Na-K-ATPase activity after prolonged hypoxia in an alveolar epithelial cell line.
Am J Physiol Lung Cell Mol Physiol
271:
L70-L78,
1996
30.
Rafii, B,
Tanswell AK,
Otulakowski G,
Pitkanen O,
Belcastro Taylor R,
and
O'Brodovich H.
O2-induced ENaC expression is associated with NF-B activation and blocked by superoxide scavenger.
Am J Physiol Lung Cell Mol Physiol
275:
L764-L770,
1998
31.
Richalet, J-P.
High altitude pulmonary oedema: still a place for controversy?
Thorax
50:
923-929,
1995[ISI][Medline].
32.
Rokaw, MD,
Sarac E,
Lechman E,
West M,
Angeski J,
Johnson JP,
and
Zeidel ML.
Chronic regulation of transepithelial Na+ transport by the rate of apical Na+ entry.
Am J Physiol Cell Physiol
270:
C600-C607,
1996
33.
Sartori, C,
Lepori M,
Maggiorini M,
Allemann Y,
Nicod P,
and
Scherrer U.
Impairment of amiloride-sensitive sodium transport in individuals susceptible to high altitude pulmonary edema (Abstract).
FASEB J
12:
328,
1998.
34.
Saumon, G,
and
Basset G.
Electrolyte and fluid transport across the mature alveolar epithelium.
J Appl Physiol
74:
1-15,
1993[Abstract].
35.
Saumon, G,
and
Martet G.
Effect of metabolic inhibitors on Na+ transport in isolated perfused rat lungs.
Am J Respir Cell Mol Biol
9:
157-165,
1993[ISI][Medline].
36.
Semenza, GL.
Hypoxia-inducible factor 1: master regulator of O2 homeostasis.
Curr Opin Genet Dev
8:
588-594,
1998[ISI][Medline].
37.
Wendt, CH,
Towle H,
Sharma R,
Duvick S,
Kawakami K,
Gick G,
and
Ingbar DH.
Regulation of Na-K-ATPase gene expression by hyperoxia in MDCK cells.
Am J Physiol Cell Physiol
274:
C356-C364,
1998