HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 293/5/L1332    most recent 00338.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (5) Citing Articles via Google Scholar Google Scholar Articles by Güney, S. Articles by Mairbäurl, H. Search for Related Content PubMed PubMed Citation Articles by Güney, S. Articles by Mairbäurl, H.

Dexamethasone prevents transport inhibition by hypoxia in rat lung and alveolar epithelial cells by stimulating activity and expression of Na⁺-K⁺-ATPase and epithelial Na⁺ channels

evin Güney,^2,* Akelei Schuler,^1,* Alexandra Ott,^1,* Sabine Höschele,¹ Stefanie Zügel,¹ Emel Balolu,¹ Peter Bärtsch,¹ and Heimo Mairbäurl¹

¹Medical Clinic VII, Sports Medicine, University Hospital Heidelberg, University of Heidelberg, Heidelberg, Germany; and ²Department of Physiology, Gazi University School of Medicine, Ankara, Turkey

Submitted 31 August 2006 ; accepted in final form 11 September 2007

	ABSTRACT

TOP ABSTRACT MATERIAL AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Hypoxia inhibits Na and lung fluid reabsorption, which contributes to the formation of pulmonary edema. We tested whether dexamethasone prevents hypoxia-induced inhibition of reabsorption by stimulation of alveolar Na transport. Fluid reabsorption, transport activity, and expression of Na transporters were measured in hypoxia-exposed rats and in primary alveolar type II (ATII) cells. Rats were treated with dexamethasone (DEX; 2 mg/kg) on 3 consecutive days and exposed to 10% O₂ on the 2nd and 3rd day of treatment to measure hypoxia effects on reabsorption of fluid instilled into lungs. ATII cells were treated with DEX (1 µM) for 3 days before exposure to hypoxia (1.5% O₂). In normoxic rats, DEX induced a twofold increase in alveolar fluid clearance. Hypoxia decreased reabsorption (–30%) by decreasing its amiloride-sensitive component; pretreatment with DEX prevented the hypoxia-induced inhibition. DEX increased short-circuit currents (ISC) of ATII monolayers in normoxia and blunted hypoxic transport inhibition by increasing the capacity of Na⁺-K⁺-ATPase and epithelial Na⁺ channels (ENaC) and amiloride-sensitive ISC. DEX slightly increased the mRNA of - and -ENaC in whole rat lung. In ATII cells from DEX-treated rats, mRNA of ₁-Na⁺-K⁺-ATPase and -ENaC increased in normoxia and hypoxia, and -ENaC was increased in normoxia only. DEX stimulated the mRNA expression of ₁-Na⁺-K⁺-ATPase and -, -, and -ENaC of A549 cells in normoxia and hypoxia (1.5% O₂) when DEX treatment was begun before or during hypoxic exposure. These results indicate that DEX prevents inhibition of alveolar reabsorption by hypoxia and stimulates the expression of Na transporters even when it is applied in hypoxia.

hypoxic pulmonary edema; alveolar fluid reabsorption; mRNA expression; ion transport

Adjustments in the rate of Na and water reabsorption are required in situations of increased fluid filtration, e.g., when the pulmonary capillary pressure or the permeability of the alveolar barrier is increased (2). Among many other pathological conditions, this might be caused by alveolar hypoxia (8, 17). A variety of hormones and growth factors stimulate Na transport and fluid reabsorption. Among the best characterized are -adrenergic agents such as terbutaline and glucocorticoids (for review, see Ref. 24).

Glucocorticoids stimulate alveolar fluid clearance in a time-dependent manner. Noda et al. (27) have shown that reabsorption was stimulated 24 h after a single application of the glucocorticoid dexamethasone (DEX) to rats and observed a concomitant increase in the expression of Na transporters such as Na⁺-K⁺-ATPase and ENaC. Similar results were obtained in cultured alveolar epithelial cells (3, 7, 24). Whereas in human A549 cells, particularly, the expression of the - and -subunits of ENaC has been found to be upregulated by DEX (15), Dagenais et al. (7) reported also an increase in the mRNA expression of the -ENaC subunit on DEX treatment of primary alveolar type II (ATII) cells from adult rats.

Based on these results, it is conceivable that treatment with DEX might blunt the hypoxic inhibition of alveolar Na transport and fluid reabsorption by increasing expression and activity of Na transporters in alveolar epithelial cells and might thus prevent the formation and stimulate the clearance of pulmonary edema. This hypothesis appears to be supported by the prevention of pulmonary edema in high altitude hypoxia by prophylactic DEX in individuals with a history of high altitude pulmonary edema (16). It is not known whether hypoxia affects glucocorticoid hormone-dependent intracellular signaling. The main action of glucocorticoids on alveolar epithelial cell ion transport seems to be the synthesis of new transporters (15). Interference with hypoxia seems likely, since inhibition of protein synthesis is a common strategy to adjust to hypoxia (13). Also, in A549 cells, hypoxia has been shown to inhibit protein synthesis (21).

It was, therefore, studied whether DEX prevents the inhibition of alveolar reabsorption by hypoxia. To answer this question, fluid reabsorption was measured in rats treated with DEX before being exposed to hypoxia. Ion transport activity and the capacity of Na⁺-K⁺-ATPase and ENaC were measured in rat primary ATII cells. Human A549 alveolar cells were studied to test whether treatment with DEX before exposure to hypoxia was required to stimulate the expression of transporters.

	MATERIAL AND METHODS

TOP ABSTRACT MATERIAL AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Fluid Reabsorption in Hypoxia-Exposed Rats

Male Sprague-Dawley rats with an average weight of 220 g with free access to standard rat chow and tap water were randomly assigned to treatment groups. DEX (2 mg·kg^–1·day^–1) or an equivalent volume of saline was administered intraperitoneally on 3 consecutive days. Beginning on the 2nd day of treatment, rats were continued in normoxia or exposed normobaric hypoxia (10% O₂, rest N₂) for 48 h while DEX/sham injections continued. Rats were anesthetized by intraperitoneal injection of sodium thiopental (100 mg/kg; Trapanal, Altana) and were anticoagulated with heparin-Na (1,500 IU/kg). For the measurement of alveolar reabsorption, rats were placed on a heating pad (39°C), and 2.5 ml of prewarmed medium (in mM: 135 NaCl, 5 KCl, 1 MgCl₂, 1 CaCl₂, 1 NaH₂PO₄, 5 glucose, 10 HEPES) containing 5% fatty acid-free BSA (Sigma) without or with 1 mM amiloride was instilled through a tracheal cannula. Thirty minutes after instillation, 250 µl of the instillate was removed to account for dead space, and a sample of instillate was collected and centrifuged (10,000 g for 10 s at room temperature) for protein measurements. Fluid reabsorption was calculated from the increase in the protein concentration in the instillate measured with a test kit from Bio-Rad. An aliquot of lung tissue was removed and lysed in TriFast reagent (PeqLab) for RNA isolation. Animal experiments were approved by the Animal Protection Committee of the University of Heidelberg and by the Regierungspräsidium Karlsruhe.

Experiments on Rat ATII Cells

Experiments on primary rat ATII cells were performed to test whether hypoxia and DEX directly affected alveolar epithelial Na transport. ATII cells were isolated after treatment of the animals as described above. Directly after isolation, cells were transferred into lysis buffer and processed for measuring the mRNA expression of Na transporters. ATII cells were also prepared from untreated rats and cultured in normoxia (room air supplemented with 5% CO₂). On day 3 after preparation, cells were treated with DEX (24 h; 1 µM) and exposed to hypoxia (48 h; 1.5% O₂, 5% CO₂, rest N₂).

Cell isolation and culture. Experiments were performed on primary cultures of ATII cells isolated from lungs of normoxic male rats (Sprague-Dawley; 150–200 g) as described elsewhere (19). Briefly, lungs from rats anesthetized (intraperitoneal injection with 100 mg/kg pentobarbital; Trapanal, Byk Gulden) were perfused with PBS while being ventilated with air. ATII cells were isolated by elastase digestion, mincing of lung tissue, filtration, and differential adhesion on IgG-coated plates (21). Nonadherent cells were dissolved in RLT (Qiagen) for isolation of total RNA or suspended in DMEM supplemented with 10% neonatal calf serum, glutamine (4 mM), and gentamycin (50 µg/ml) and plated on tissue culture-treated Nuclepore filters (pore size 0.4 µm, diameter 12 mm, Transwell, Costar, Cambridge, MA) at a seeding density of 1 x 10⁶ cells/cm² for functional analysis. Both purity and viability of ATII cells were >85%. For tissue culture, cells were maintained in normoxia (room air/5% CO₂) until they had reached confluence (typically on day 3 after plating). Formation of tight monolayers was tested by measuring transepithelial resistance using the epithelial voltohmeter (EVOM) device and chopstick electrodes (World Precision Instruments, Sarasota, FL).

Ussing chamber measurements. For measuring transport activity, cell monolayers were typically used on day 3–5 after plating. After mounting in modified Ussing chambers, the monolayers were bathed with media composed of (in mM) 141 NaCl, 5.4 KCl, 0.78 NaH₂PO₄, 1.8 CaCl₂, 0.8 MgCl₂, 5 glucose, and 15 HEPES, pH 7.4, at 37°C. After equilibration to the medium (10 min; 37°C; open-circuit conditions), short-circuit current (ISC) was recorded with an automated voltage clamp unit (W. Nagel, Munich, Germany) by clamping the transepithelial potential to 0 mV and stored in a computer for offline analysis. Amiloride (final concentration 10 µM) was used to inhibit the activity of apical Na channels (ISC_amil).

To assess changes in the number of transport proteins in the plasma membrane of primary ATII cells, we chose to measure the capacity of the Na⁺-K⁺-ATPase (ISC_C-Na/K) and of amiloride-sensitive current (ISC_C-amil) after permeabilization of the apical and basolateral membrane, respectively (19). This method provides a measure of the number of active transporters, whereas Western blots show total protein without reference to its activity. To measure ISC_C-Na/K in the basolateral membrane, ISC was recorded after permeabilization of the apical membrane with amphotericin B (final concentration 7.5 µM) using the above described bathing medium. To measure ISC_C-amil, monolayers were bathed with the above mentioned medium at the apical membrane, whereas the basolateral side was bathed with this medium, but the concentration of Na⁺ was reduced to 25 mM by replacement with N-methyl-D-glucamine to establish a Na gradient before permeabilization of the basolateral membrane (19).

Experiments on A549 Cells

Since the action of DEX on Na reabsorption seems to depend mainly on stimulation of the expression of Na transport proteins (7, 27), it was tested on the human A549 alveolar epithelial cell line whether pretreatment with DEX is required to blunt hypoxia effects or whether DEX also exerts its action when it is applied during hypoxia. Although the basal mRNA expression of ENaC is lower in A549 cells than in primary ATII cells, these cells appear a valid model since they show many characteristics of alveolar epithelial cells, and they also show inhibition of ion transport by hypoxia similar to primary rat ATII cells (21, 36). It has not been studied, however, whether human A549 and primary rat ATII cells respond quantitatively similar to stimuli that affect the expression of Na transporters.

A549 cells were grown in tissue culture flasks (25 cm²; Costar) in Ham's F-12 medium substituted with 10 mM HEPES, 15 mM NaHCO₃, penicillin/streptomycin, and 7% FCS (Life Technologies) and cultured in room air with 5% CO₂ (normoxia) until reaching confluence (typically on day 4 after plating).

In a first series of experiments, cells were treated with DEX before exposure to hypoxia. DEX (1 µM) was added to the culture medium on days 4 and 6 after plating. On day 7, cells were exposed to hypoxia (1.5% O₂, 5% CO₂, rest N₂) in an O₂- and CO₂-controlled tissue culture incubator (Nunc); control cells continued to stay in normoxia for 24 h. In a second series of experiments, cells were exposed to hypoxia for 48 h. After 24 h of exposure, cells were treated with DEX (1 µM). Cells were then lysed for isolation of total RNA using the TriFast reagent (PeqLab) for RT-PCR.

RNA Isolation and RT-PCR

Total RNA was isolated from the TriFast lysates according to the manufacturer's instructions, and 1 µg of RNA was transcribed with SuperScript II RT (Life Technologies) using random hexamer primers (Roche, Mannheim, Germany). Real-time quantitative PCR was performed in the LightCycler using the LC Fast Start PCR Mix (Roche) and the primers (MWG Biotech) listed in Table 1. To test for the specificity of PCR amplification, PCR products were separated by agarose gel electrophoresis and stained with Gelstar (BMA). PCR products showed single bands of the predicted size (data not shown). Sequencing (MWG Biotech) of bands eluted from agarose gels of PCR products confirmed the predicted base sequence.

Statistical Evaluation

Results are shown as means ± SD or SE as indicated. Data were analyzed by ANOVA followed by Tukey's test for pairwise comparisons. Level of significance was P 0.05.

	RESULTS

TOP ABSTRACT MATERIAL AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
DEX Effects on Alveolar Fluid Reabsorption and Gene Expression in Rat Lung

Reabsorption of fluid instilled into the lungs of anesthetized rats was measured after treatment with DEX before the animals were exposed to hypoxia (10% O₂). Results are summarized in Fig. 1. Treatment of rats with DEX for 3 days stimulated alveolar reabsorption by 60% (P = 0.002) in normoxia. Exposure of rats without DEX to hypoxia for 48 h decreased reabsorption significantly by 30% (P = 0.001). When rats were treated with DEX and then exposed to hypoxia for 48 h with continued DEX treatment, only a slight stimulation of reabsorption with DEX was detectible relative to hypoxic control rats (P = 0.07), and the reabsorption rate was significantly lower than after DEX treatment in normoxia (P = 0.03). Thus, after treatment with DEX and exposure to hypoxia, the reabsorption rate was not different from untreated rats in normoxia (P = 0.73). In normoxic control rats, amiloride inhibited 60% of reabsorption. Significant inhibition of fluid reabsorption by amiloride was seen in all conditions except for hypoxia-exposed rats without DEX. The amiloride-insensitive portion of reabsorption was not affected by DEX and hypoxia.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Effects of hypoxia and dexamethasone (DEX) on lung fluid reabsorption are shown. Rats received DEX (2 mg/kg) on 3 consecutive days; 1 group of animals was exposed to hypoxia (10% O2) on days 2 and 3, and the other remained in normoxia. Reabsorption of fluid instilled into the lungs was measured by instillation of a medium composed of (in mM) 135 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 1 NaH2PO4, 5 glucose, 10 HEPES, containing 5% BSA without or with 1 mM amiloride. Values are means ± SE of 5–12 experiments. *P < 0.05 relative to normoxic controls; #P < 0.05 between normoxia and hypoxia of DEX-treated rats; +P < 0.05 of the amiloride effect.

Primary rat ATII cell monolayers were treated with DEX and hypoxia to test whether DEX blunts the hypoxic transport inhibition we described earlier (19). Figure 2A shows that total ISC (ISC_tot) was inhibited by hypoxia by 30% (P = 0.008). Also, ISC_amil was decreased by 44% (P = 0.046). In normoxia, a 3-day treatment with DEX increased ISC_tot by almost 50% (P = 0.001) and ISC_amil by +63% (P = 0.022). A 24-h DEX treatment before exposure to hypoxia increased transport activity above hypoxic values as indicated by an increase in ISC_tot by 43% (P = 0.029), whereas ISC_amil was not affected (P = 0.486).

View larger version (30K): [in this window] [in a new window]   Fig. 2. A and B: effects of hypoxia and DEX on short-circuit currents (ISC) in primary rat alveolar epithelial cells. Primary rat alveolar type II cells were prepared from untreated rats and grown on Nuclepore filters and exposed to DEX (1 µM) and/or hypoxia (1.5% O2) as described in MATERIALS AND METHODS. A: ISCamil is the portion of total ISC (ISCtot) inhibited by amiloride (10 µM) in intact cells measured in a medium composed of (in mM) 141 NaCl, 5.4 KCl, 0.78 NaH2PO4, 1.8 CaCl2, 0.8 MgCl2, 5 glucose, and 15 HEPES, pH 7.4, at 37°C. B: the capacity of the Na+-K+-ATPase (ISCC-Na/K) is the ouabain-sensitive portion of ISC after permeabilization of the apical membrane with 7.5 µM amphotericin B in cells bathed in the above mentioned medium. C: for the measurement of the capacity of the amiloride-sensitive component of ISC (ISCC-amil), cells were bathed with the above mentioned medium on the apical surface, whereas at the basolateral side, Na+ was reduced to 25 mM by replacement with N-methyl-D-glucamine. The basolateral membrane was permeabilized with amphotericin B, and amiloride (10 µM) was added apically. Values are means ± SD of 7–12 experiments from at least 3 different cell preparations. *P < 0.05 difference to normoxic controls; +P < 0.05 of DEX effect; #P < 0.05 difference between DEX in normoxia and hypoxia. N- and H-control, normoxic and hypoxic control cells; N- and H-DEX, DEX-treated cells in normoxia and hypoxia.

C-amil

C-amil

C-amil

mRNA Expression in A549 Cells

A twofold (P = 0.005; data not shown) increase in the expression of glyceraldehyde phosphate dehydrogenase (GAPD) mRNA confirmed that the exposure of the cultured cells to hypoxia resulted in an upregulation of typically hypoxia-induced genes. DEX did not affect the hypoxia-induced increase in GAPD mRNA.

DEX is known to stimulate alveolar Na transport by stimulating the expression of transport proteins (24). Since it is not known whether hypoxia affects DEX-stimulated gene expression, the mRNA expression of ENaC and Na⁺-K⁺-ATPase was measured in two sets of experiments: 1) A549 cells were treated with DEX before hypoxia; and 2) cells were exposed to hypoxia before DEX treatment. The results are summarized in Table 3. In both experimental conditions, DEX stimulated the mRNA expression of all three ENaC subunits and of the ₁-Na⁺-K⁺-ATPase. There was no effect of DEX on the ₁-Na⁺-K⁺-ATPase mRNA expression. The DEX-induced stimulation of - and -ENaC expression was significantly decreased when cells were exposed to hypoxia before DEX treatment. Hypoxia alone did not affect the mRNA expression of - and -ENaC and of ₁-Na⁺-K⁺-ATPase but significantly increased the mRNAs of -ENaC and ₁-Na⁺-K⁺-ATPase. The stimulatory effect of hypoxia on ₁-Na⁺-K⁺-ATPase mRNA expression was not seen after DEX treatment.

	DISCUSSION

TOP ABSTRACT MATERIAL AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

DEX Stimulates Alveolar Ion Transport in Normoxia

Our results in DEX-induced stimulation of rat alveolar fluid clearance confirm earlier findings obtained on rats pretreated by intramuscular injections on 2 consecutive days (11). Also, other results indicate that pretreatment might be required for stimulation of alveolar transport by DEX since it occurred with a time lag of 24–48 h on a single DEX application (27). Here, we show that repeated injections of DEX also increased reabsorption (Fig. 1), although the degree of stimulation of fluid reabsorption was not as high as that reported by Noda et al. (27). Most of the DEX effect appears to be due to stimulation of Na reabsorption, since the amiloride-insensitive component of alveolar fluid clearance was not affected. Our results using primary cultured rat alveolar epithelial cells support this notion since DEX treatment of normoxic cells stimulates ISC_tot and ISC_amil. Also, an increased amiloride-sensitive component of the transepithelial potentials (7) across ATII cell monolayers by DEX points to a stimulation of Na transport. Taken together, these results clearly show an increase in alveolar fluid and epithelial Na reabsorption on stimulation with DEX.

Several mechanisms may account for the stimulated transport activity. Patch-clamp analysis indicates that DEX increases the open probability of Na channels (15). We show here that DEX increased the capacity of amiloride-sensitive Na transport (ISC_C-amil) across the apical membrane of ATII cell monolayers (Fig. 2C), which is consistent with not only an increased open probability of Na channels, but also with an increased expression and an increased insertion of endogenous Na channels. The parallel increase in the expression of Na⁺-K⁺-ATPase and ENaC in the lung and the stimulation of alveolar clearance found by Noda et al. (27) implies that a stimulation of expression is required for DEX-stimulated transport activity (15, 22, 23, 27). Our results show that DEX treatment increased the mRNA expression of - and -ENaC both in whole lung tissue and in ATII cells isolated from DEX-treated rats (Table 2). However, changes in mRNA levels of ENaC were not as pronounced as those reported by Noda et al. (27).

DEX has also been reported to increase the activity of Na⁺-K⁺-ATPase (27) in whole lung, which indicates an increased capacity to remove Na taken up by the cells via Na channels. We confirm this result by showing an increased capacity of Na⁺-K⁺-pumping (ISC_C-Na/K) in ATII cell monolayers after permeabilizing the apical plasma membrane with amphotericin B (Fig. 2B). Also, in this case, it is not possible to discriminate between stimulated membrane insertion and increased expression. The increased mRNA levels of the ₁-subunit of the Na⁺-K⁺-ATPase we found in alveolar epithelium on DEX treatment support the latter (Table 2). Thus our results indicate an increase in alveolar clearance by stimulation of Na reabsorption, which is due to an increase in the capacity of apical, amiloride-sensitive Na uptake and Na removal across the basolateral membrane by Na⁺-K⁺-ATPase.

DEX Stimulates Transport in Hypoxia

Hypoxia has been shown to decrease alveolar fluid clearance of hypoxia-exposed rats (34) and to inhibit ion transport activity of cultured alveolar epithelial cells (21, 29, 30). Vivona et al. (34) reported an almost 60% inhibition of alveolar fluid clearance in rats exposed to 8% O₂, whereas inhibition was only 25% in our experiments (Fig. 1) where rats were exposed to 10% O₂. The difference probably relates to the different degree of hypoxia. In both experimental settings, mainly the amiloride-sensitive component of alveolar fluid clearance was affected by hypoxia, possibly due to a decreased transport activity (19), decreased expression (29), and decreased membrane insertion (28). In primary alveolar epithelial monolayers (Fig. 2), not only amiloride-sensitive transport, but also the capacity of the Na⁺-K⁺-ATPase is decreased by hypoxia as shown previously (19). It has to be pointed out that a higher degree of hypoxia was used in tissue culture experiments than in the in vivo rat experiments and that changes induced by hypoxia become smaller with increased oxygenation (19).

Hypoxic transport inhibition in cultured alveolar epithelial cells has been associated also with the inhibition of expression of Na transporters (29, 36). Since hypoxia decreases overall protein synthesis in lung alveolar epithelial cells (12, 21), it was not clear whether DEX, for which stimulation of fluid clearance and Na transport seems to depend mainly on the synthesis of new Na transporters (15, 27), would increase the expression of transport proteins also in hypoxia. Our results indicate that treatment with DEX of rats before exposure to hypoxia stimulated alveolar fluid clearance in hypoxia. This result was verified in primary rat ATII cells, where we found an increase in ISC_tot and ISC_amil by DEX. However, DEX-stimulated reabsorption and transport in hypoxia just gained the values measured without treatment in normoxia both in the intact lung and in cultured ATII cells, and in neither system was the normoxic maximal level of activity achieved. This is in contrast to results on terbutaline stimulation of alveolar fluid clearance, where the same maximal values were reached in either oxygenation state (34). Similar to normoxia, also in hypoxia, DEX stimulation of reabsorption and transport activity can be explained by an increased capacity of the Na⁺-K⁺-ATPase and of amiloride-sensitive transport (Fig. 2). The change in transport activity and capacity is paralleled by an increase in the mRNA expression of the ₁-subunit of Na⁺-K⁺-ATPase and of the -ENaC subunits, whereas - and -ENaC expression was not affected. These results indicate that DEX stimulates alveolar Na transport and fluid clearance also in hypoxia and thus blunts the hypoxic inhibition of reabsorption, which can, in part, be explained by increased expression of Na transporters.

Pathological conditions associated with alveolar hypoxia might favor the formation of pulmonary edema by decreasing the rate of alveolar Na transport and fluid reabsorption (18, 20, 26, 32, 35). It might, therefore, be of clinical significance to prevent the hypoxia-induced transport inhibition to reduce the risk of alveolar edema by prophylaxis or treatment with DEX. This appeared reasonable since prophylactic DEX intake has recently been shown to completely prevent high altitude pulmonary edema (16). Similarly, inhaled -adrenergics reduced the risk of high altitude pulmonary edema presumably by stimulation of alveolar reabsorption (32).

Since stimulation of reabsorption and Na transport in hypoxia was observed when treatment with DEX was begun before exposure to hypoxia, it was important to know whether treatment is also effective when cells are already hypoxic. We used the human alveolar epithelial A549 cell line to test this effect. These cells show hypoxic inhibition of Na transport (21) and stimulation of Na transport with DEX (15) similar to that found in primary cells despite the fact that their basal level of expression of Na transporters appears very low. The results of these measurements show that DEX increased the amounts of - and -ENaC mRNA much more than -ENaC mRNA. Most importantly, we found that the stimulation of mRNA expression was seen regardless of whether cells were DEX-treated before exposure to hypoxia or whether DEX was applied to already hypoxic cells, although the degree of stimulation of mRNA formation was smaller in the latter protocol.

In summary, our results indicate that DEX prevents the hypoxia-induced inhibition of alveolar fluid clearance and Na transport and stimulates the mRNA expression of Na⁺-K⁺-ATPase and ENaC, which leads to an increased capacity of alveolar epithelial amiloride-sensitive Na uptake and Na removal by Na⁺-K⁺-ATPases not only in normoxia, but also in hypoxia. These results also indicate that glucocorticoid-dependent signaling and gene expression are not impaired by hypoxia. If this is also the case in vivo in humans, stimulation of alveolar fluid clearance might, in part, explain the prevention of hypoxic edema at high altitude by prophylactic DEX (16) and might point to a possible therapeutic approach to prevent or treat pulmonary edema in pathological states of alveolar hypoxia. The results presented here as well as results on gene therapy aimed to increase alveolar fluid reabsorption are in support of this hypothesis (1, 10, 33).

	GRANTS

TOP ABSTRACT MATERIAL AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by German Research Foundation (DFG) Grant Ma 1503/18-1 to H. Mairbäurl and by a Scientific and Technological Research Council of Turkey (TÜBITAK-BAYG) Grant to . Güney.

	ACKNOWLEDGMENTS

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

	FOOTNOTES

 

Address for reprint requests and other correspondence: H. Mairbäurl, Medical Clinic VII, Sports Medicine, Univ. of Heidelberg, Im Neuenheimer Feld 410, 69120 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.

^* . Güney, A. Schuler, and A. Ott contributed equally to this study.

	REFERENCES

TOP ABSTRACT MATERIAL AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Azzam ZS, Dumasius V, Saldias FJ, Adir Y, Sznajder JI, Factor P. Na,K-ATPase overexpression improves alveolar fluid clearance in a rat model of elevated left atrial pressure. Circulation 105: 497–501, 2002.[Abstract/Free Full Text]
2. Bhattacharya J. The alveolar water gate. Am J Physiol Lung Cell Mol Physiol 286: L257–L258, 2004.[Free Full Text]
3. Champigny G, Voilley N, Lingueglia E, Friend V, Barbry P, Lazdunski M. Regulation of expression of the lung amiloride-sensitive Na⁺ channel by steroid hormones. EMBO J 13: 2177–2181, 1994.[Web of Science][Medline]
4. Clerici C. Sodium transport in alveolar epithelial cells: Modulation by O₂ tension. Kidney Int 65: S79–S83, 1998.
5. Clerici C, Matthay MA. Hypoxia regulates gene expression of alveolar epithelial transport proteins. J Appl Physiol 88: 1890–1896, 2000.[Abstract/Free Full Text]
6. Dada LA, Chandel NS, Ridge KM, Pedemonte C, Bertorello AM, Sznajder JI. Hypoxia-induced endocytosis of Na,K-ATPase in alveolar epithelial cells is mediated by mitochondrial reactive oxygen species and PKC-zeta. J Clin Invest 111: 1057–1064, 2003.[CrossRef][Web of Science][Medline]
7. Dagenais A, Denis C, Vives MF, Girouard S, Masse C, Nguyen T, Yamagata T, Grygorczyk C, Kothary R, Berthiaume Y. Modulation of -ENaC and ₁-Na⁺-K⁺-ATPase by cAMP and dexamethasone in alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 281: L217–L230, 2001.[Abstract/Free Full Text]
8. Dehler M, Zessin E, Bärtsch P, Mairbäurl H. Hypoxia causes permeability edema in the constant-pressure perfused rat lung. Eur Respir J 27: 600–606, 2006.[Abstract/Free Full Text]
9. Egli M, Duplain H, Lepori M, Cook S, Nicod P, Hummler E, Sartori C, Scherrer U. Defective respiratory amiloride sensitive sodium transport predisposes to pulmonary oedema and delays its resolution in mice. J Physiol 560: 857–865, 2004.[Abstract/Free Full Text]
10. Factor P, Saldias F, Ridge K, Dumasius V, Zabner J, Jaffe HA, Blanco G, Barnard M, Mercer R, Perrin R, Sznajder JI. Augmentation of lung liquid clearance via adenovirus- mediated transfer of a Na,K-ATPase beta(1) subunit gene. J Clin Invest 102: 1421–1430, 1998.[Web of Science][Medline]
11. Folkesson HG, Norlin A, Wang YB, Abedinpour P, Matthay MA. Dexamethasone and thyroid hormone pretreatment upregulate alveolar epithelial fluid clearance in adult rats. J Appl Physiol 88: 416–424, 2000.[Abstract/Free Full Text]
12. Heerlein K, Schulze A, Hotz L, Bärtsch P, Mairbäurl H. Hypoxia decreases cellular ATP-demand and inhibits mitochondrial respiration of A549 cells. Am J Respir Cell Mol Biol 32: 44–51, 2005.[Abstract/Free Full Text]
13. Hochachka PW. Defense strategies against hypoxia and hypothermia. Science 231: 234–241, 1986.[Abstract/Free Full Text]
14. Hummler E, Barker P, Gatzy J, Beermann F, Verdumo C, Boucher R, Rossier BC. Early death due to defective neonatal lung liquid clearance in -ENaC-deficient mice. Nat Genet 13: 325–328, 1996.[CrossRef][Web of Science][Medline]
15. Lazrak A, Samanta A, Venetsanou K, Barbry P, Matalon S. Modification of biophysical properties of lung epithelial Na⁺ channels by dexamethasone. Am J Physiol Cell Physiol 279: C762–C770, 2000.[Abstract/Free Full Text]
16. Maggiorini M, Brunner-La Rocca H, Peth S, Fischler M, Böhm T, Bernheim A, Kiencke S, Bloch KE, Dehnert C, Naeije R, Lehmann T, Bärtsch P, Mairbäurl H. Both tadalafil and dexamethasone may reduce the incidence of high-asltitude pulmonary edema. Ann Intern Med 145: 497–506, 2006.[Abstract/Free Full Text]
17. Maggiorini M, Melot C, Pierre S, Pfeiffer F, Greve I, Sartori C, Lepori M, Hauser M, Scherrer U, Naeije R. High-altitude pulmonary edema is initially caused by an increase in capillary pressure. Circulation 103: 2078–2083, 2001.[Abstract/Free Full Text]
18. Mairbäurl H. Role of alveolar epithelial sodium transport in high altitude pulmonary edema (HAPE). Respir Physiol Neurobiol 151: 178–191, 2006.[CrossRef][Web of Science][Medline]
19. Mairbäurl H, Mayer K, Kim KJ, Borok Z, Bärtsch P, Crandall ED. Hypoxia decreases active Na transport across primary rat alveolar epithelial cell monolayers. Am J Physiol Lung Cell Mol Physiol 282: L659–L665, 2002.[Abstract/Free Full Text]
20. Mairbäurl H, Weymann J, Möhrlein A, Swenson ER, Maggiorini M, Gibbs JS, Bärtsch P. Nasal epithelium potential difference at high altitude (4559 m): evidence for secretion. Am J Respir Crit Care Med 167: 862–867, 2003.[Abstract/Free Full Text]
21. Mairbäurl H, Wodopia R, Eckes S, Schulz S, 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.[Abstract/Free Full Text]
22. Matalon S. Mechanisms and regulation of ion transport in adult mammalian alveolar type II pneumocytes. Am J Physiol Cell Physiol 261: C727–C738, 1991.[Abstract/Free Full Text]
23. Matthay MA, Clerici C, Saumon G. Active fluid clearance from distal airspaces. J Appl Physiol 93: 1533–1541, 2002.[Abstract/Free Full Text]
24. Matthay MA, Folkesson HG, Clerici C. Lung epithelial fluid transport and the resolution of pulmonary edema. Physiol Rev 82: 569–600, 2002.[Abstract/Free Full Text]
25. Matthay MA, Folkesson HG, Verkman AS. Salt and water transport across alveolar and distal airway epithelia in the adult lung. Am J Physiol Lung Cell Mol Physiol 270: L487–L503, 1996.[Abstract/Free Full Text]
26. Matthay MA, Wiener-Kronish JP. Intact epithelial barrier function is critical for the resolution of alveolar edema in humans. Am Rev Respir Dis 142: 1250–1257, 1990.[Web of Science][Medline]
27. Noda M, Suzuki S, Tsubochi H, Sugita M, Maeda S, Kobayashi S, Kubo H, Kondo T. Single dexamethasone injection increases alveolar fluid clearance in adult rats. Crit Care Med 31: 1183–1189, 2003.[CrossRef][Web of Science][Medline]
28. Planes C, Blot-Chabaud M, Matthay MA, Couette S, Uchida T, Clerici C. Hypoxia and beta(2)-agonists regulate cell surface expression of the epithelial sodium channel in native alveolar epithelial cells. J Biol Chem 277: 47318–47324, 2002.[Abstract/Free Full Text]
29. Planes C, Escoubet B, Blot-Chabaud M, Friedlander G, Farman N, Clerici C. Hypoxia downregulates expression and activity of epithelial sodium channels in rat alveolar epithelial cells. Am J Respir Cell Mol Biol 17: 508–518, 1997.[Abstract/Free Full Text]
30. Planes C, Friedlander G, Loiseau A, Amiel C, Clerici C. Inhibition of Na-K-ATPase activity after prolonged hypoxia in an alveolar epithelial cell line. Am J Physiol Lung Cell Mol Physiol 271: L70–L78, 1996.[Abstract/Free Full Text]
31. Richalet JP. High altitude pulmonary oedema: still a place for controversy? Thorax 50: 923–929, 1995.[Free Full Text]
32. Sartori C, Allemann Y, Duplain H, Lepori M, Egli M, Lipp E, Hutter D, Turini P, Hugli O, Cook S, Nicod P, Scherrer U. Salmeterol for the prevention of high-altitude pulmonary edema. N Engl J Med 346: 1631–1636, 2002.[Abstract/Free Full Text]
33. Stern M, Ulrich K, Robinson C, Copeland J, Griesenbach U, Masse C, Cheng S, Munkonge F, Geddes D, Berthiaume Y, Alton E. Pretreatment with cationic lipid-mediated transfer of the Na⁺K⁺-ATPase pump in a mouse model in vivo augments resolution of high permeability pulmonary oedema. Gene Ther 7: 960–966, 2000.[CrossRef][Web of Science][Medline]
34. Vivona ML, Matthay MA, Chabaud MB, Friedlander G, Clerici C. Hypoxia reduces alveolar epithelial sodium and fluid transport in rats: reversal by beta-adrenergic agonist treatment. Am J Respir Cell Mol Biol 25: 554–561, 2001.[Abstract/Free Full Text]
35. Ware LB, Matthay MA. Alveolar fluid clearance is impaired in the majority of patients with acute lung injury and the acute respiratory distress syndrome. Am J Respir Crit Care Med 163: 1376–1383, 2001.[Abstract/Free Full Text]
36. Wodopia R, Ko HS, Billian J, Wiesner R, Bärtsch P, Mairbäurl H. Hypoxia decreases proteins involved in transepithelial electrolyte transport of A549 cells and rat lung. Am J Physiol Lung Cell Mol Physiol 279: L1110–L1119, 2000.[Abstract/Free Full Text]

This article has been cited by other articles:

				J. W. Lee, X. Fang, N. Gupta, V. Serikov, and M. A. Matthay Allogeneic human mesenchymal stem cells for treatment of E. coli endotoxin-induced acute lung injury in the ex vivo perfused human lung PNAS, September 22, 2009; 106(38): 16357 - 16362. [Abstract] [Full Text] [PDF]

				E. Baloglu, A. Ke, I. H. Abu-Taha, P. Bartsch, and H. Mairbaurl In vitro hypoxia impairs {beta}2-adrenergic receptor signaling in primary rat alveolar epithelial cells Am J Physiol Lung Cell Mol Physiol, March 1, 2009; 296(3): L500 - L509. [Abstract] [Full Text] [PDF]

				V. Bhalla and K. R. Hallows Mechanisms of ENaC Regulation and Clinical Implications J. Am. Soc. Nephrol., October 1, 2008; 19(10): 1845 - 1854. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 293/5/L1332    most recent 00338.2006v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (5) Citing Articles via Google Scholar Google Scholar Articles by Güney, S. Articles by Mairbäurl, H. Search for Related Content PubMed PubMed Citation Articles by Güney, S. Articles by Mairbäurl, H.