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Published ahead of print on October 24, 2003, doi:10.1164/rccm.200304-472OC
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American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 407-412, (2004)
© 2004 American Thoracic Society

Leukotriene D4 Activates Alveolar Epithelial Na,K-ATPase and Increases Alveolar Fluid Clearance

Daniel E. Sloniewsky, Karen M. Ridge, Yochai Adir, Francine P. Fries, Arturo Briva, Jacob I. Sznajder and Peter H. S. Sporn

Divisions of Pulmonary and Critical Care Medicine, Departments of Pediatrics and Medicine, Feinberg School of Medicine, Northwestern University; and Medical Service, Veteran Affairs Chicago Health Care System-Lakeside Division, Chicago, Illinois

Correspondence and requests for reprints should be addressed to Peter H. S. Sporn, M.D., Division of Pulmonary and Critical Care Medicine, The Feinberg School of Medicine, Northwestern University, 303 East Chicago Avenue, Tarry 14-707, Chicago, IL 60611-3008. E-mail: p-sporn{at}northwestern.edu


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cysteinyl leukotrienes are increased during acute lung injury in animals and humans. In this study, we determined the effect of leukotriene D4 (LTD4) on the function of Na,K-ATPase in alveolar epithelial cells and on alveolar fluid clearance in rat lungs. LTD4 (1 x 10-7 M) increased Na,K-ATPase activity at 1 and 5 minutes by 14% (p < 0.05) and 31% (p < 0.001), respectively, in A549 alveolar epithelial cells. This was accompanied by recruitment of Na,K-ATPase {alpha}1 subunits from intracellular compartment(s) to the basolateral plasma membrane. LTD4-induced {alpha}1 Na,K-ATPase membrane translocation was blocked by the dual cysteinyl LT1 (cysLT1)/ cysteinyl LT2 (cysLT2) receptor antagonist BAY-u9773, but not by the cysLT1 antagonist MK571, implicating the cysLT2 receptor. Expression of mRNA for cysLT2, but not cysLT1, was confirmed in A549 cells and rat alveolar type 2 cells by reverse transcriptase-polymerase chain reaction. Finally, compared with control, LTD4 (1 x 10-11 M) increased alveolar fluid clearance by 41% (p < 0.001) in isolated, perfused rat lungs; this was also blocked by BAY-u9773 but not MK571. By activating alveolar epithelial Na,K-ATPase and increasing alveolar fluid reabsorption, cysteinyl leukotrienes may, in part, have a beneficial role in the acute respiratory distress syndrome.

Key Words: leukotriene D4 • cysteinyl leukotriene receptors • Na(+)-K(+)–exchanging ATPase • pulmonary edema • adult respiratory distress syndrome

The cysteinyl leukotrienes (LTs) LTC4, LTD4, and LTE4 are potent proinflammatory lipid mediators derived from arachidonic acid (1, 2). Studies in animals and humans have suggested a role for cysteinyl LTs in acute lung injury. Increased lung levels of LTC4 and LTD4 have been detected in various animal models of acute lung injury (3–6), and a cysteinyl LT receptor antagonist blocked endotoxin-induced pulmonary edema in pigs (6). Elevated amounts of cysteinyl LTs have been also been detected in pulmonary edema and bronchoalveolar lavage fluid (7, 8) and in the urine (9) of patients with acute respiratory distress syndrome (ARDS). These findings, along with observations that LTC4 and LTD4 can increase microvascular permeability (10–12), suggest that cysteinyl LTs play a role in the pathogenesis of noncardiogenic pulmonary edema.

A major determinant of the accumulation of pulmonary edema is the rate of alveolar fluid reabsorption across the alveolar epithelium (13–16). Alveolar fluid reabsorption occurs as a result of active Na+ transport across the epithelium, which creates an osmotic gradient, causing water to also move out of the alveolar space. Na+ enters alveolar epithelial cells via apical Na+ channels (17, 18) and is actively transported out of the cell by Na,K-ATPase, which is active in the basolateral membrane (19, 20). The Na,K-ATPase is a ubiquitous heterodimeric transmembrane ion transporter that maintains Na+ and K+ gradients across cell membranes. It is composed of an {alpha} subunit that hydrolyzes ATP and exchanges intracellular Na+ for extracellular K+, and a ß subunit that controls enzyme assembly and insertion into the plasma membrane (21).

The effect of cysteinyl LTs on the Na,K-ATPase in alveolar epithelial cells has not been studied. Indeed, whether epithelial cells in the lung or other tissues express either of the two cysteinyl LT receptors, cysLT1 (22) or cysLT2 (23), is unknown. Epithelial expression of cysteinyl LT receptors seems likely, however, as it has been shown that LTD4 triggers rapid signal transduction events in human epithelial cells (24–27).

In this study, we determined the effects of LTD4 on the activity of Na,K-ATPase in cultured alveolar epithelial cells and on alveolar fluid clearance in the isolated, perfused rat lung. We report here, for the first time, that LTD4 increases Na,K-ATPase activity and protein abundance in the basolateral membrane of A549 human alveolar epithelial cells. Using the selective cysLT1 antagonist MK571 (22) and the dual cysLT1/cysLT2 antagonist BAY-u9773 (23, 28), we provide evidence that LTD4 exerts its effects through the cysLT2 receptor in A549 cells. We also document the presence of mRNA for cysLT2, but not cysLT1, receptors in A549 cells and in rat alveolar type 2 (AT2) cells by reverse transcriptase-polymerase chain reaction. Finally, we show that LTD4 increases lung liquid clearance, without changing permeability for small and large solutes, in the isolated, perfused rat lung. Together, these observations indicate that LTD4 acts via the cysLT2 receptor to upregulate Na,K-ATPase function in alveolar epithelial cells, and thereby increase lung liquid clearance. Some of the results of these studies have been previously reported in the form of an abstract (29).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Specific pathogen-free male Sprague-Dawley rats (Harlan Inc., Indianapolis, IN) were used for isolation of AT2 cells and for isolated, perfused lung experiments. Additional details are provided in the online supplement.

Culture of A549 Cells
A549 cells, a human lung adenocarcinoma-derived line with alveolar epithelial cell properties, were cultured in Dulbecco's modified Eagle medium containing 10% fetal calf serum. Additional details are provided in the online supplement.

Isolation and Culture of Rat AT2 Cells
Rat AT2 cells were isolated as previously described (30). Additional details are provided in the online supplement.

Human Eosinophils
Eosinophils were purified from venous blood of human volunteers, as described previously (31). Additional details are provided in the online supplement.

LTD4 Stimulation and Cysteinyl LT Receptor Antagonists
In experiments involving cysteinyl LT stimulation, A549 cells were serum-starved for 24 hours before the addition of LTD4. Cells were stimulated with LTD4 (Cascade Biochem Ltd., Berkshire, UK) at various concentrations or exposed to vehicle (dimethyl sulfoxide) alone for 1 to 5 minutes before the assessment of Na,K-ATPase activity, subcellular fractionation and Western blotting, or fixation for confocal microscopy. In selected experiments, A549 cells were incubated with the cysteinyl LT receptor antagonists MK571 (100 nM; Cayman Chemical, Ann Arbor, MI) or BAY-u9773 (3 µM; Biomol, Plymouth Meeting, PA) for 15 minutes before and during exposure to LTD4.

Na,K-ATPase Activity Assay
The activity of Na,K-ATPase in A549 cells was determined as the ouabain-sensitive uptake of 86Rb, as described (20). Additional details are provided in the online supplement.

Basolateral Plasma Membrane Isolation
After stimulation with LTD4 in the absence or presence of receptor antagonists, A549 cells were homogenized, and basolateral plasma membranes were isolated, as described (20). Additional details are provided in the online supplement.

Immunoblotting of Na,K-ATPase
Immunoblotting was performed using a specific monoclonal antibody against the {alpha}1 subunit of Na,K-ATPase. Additional details are provided in the online supplement.

Confocal Immunofluorescence Microscopy
A549 cells stably transfected with a rat green fluorescent protein (GFP)-{alpha}1 Na,K-ATPase plasmid (32) were cultured on glass coverslips and then stimulated with 1 x 10-7 M LTD4 for 1 or 5 minutes, or exposed to vehicle alone. Cells were fixed, permeabilized, and immunostained with a rabbit monoclonal anti-GFP antibody (Molecular Probes, Eugene, OR), followed by Alexa-488–conjugated goat anti-rabbit IgG (Molecular Probes). Additional details are provided in the online supplement.

Reverse Transcriptase-Polymerase Chain Reaction
RNA was isolated from cultured cells and reverse transcribed. Polymerase chain reaction for the cysLT1 and the cysLT2 receptors was performed as detailed in the online supplement.

Isolated, Perfused Rat Lung Model
Active Na+ transport and alveolar fluid reabsorption were measured in isolated, perfused, fluid-filled rat lungs, as described previously (15, 33). Additional details are provided in the online supplement.

Statistical Analysis
Data are presented as means ± SEM. Differences between group means were analyzed by the unpaired Student's t test or by one-way analysis of variance with Tukey's multiple comparison test, as appropriate (34). Significance was determined at the p < 0.05 level. Statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of LTD4 on Alveolar Epithelial Na,K-ATPase Activity and {alpha}1 Protein Abundance in the Basolateral Membrane
First, we evaluated the effect of LTD4 on Na,K-ATPase function in alveolar epithelial cells. A549 cells were incubated with LTD4 at concentrations from 1 x 10-11 to 1 x 10-7 M, or vehicle alone, for 1 or 5 minutes at 37°C. As shown in Figure 1A, 1 x 10-7 M LTD4 significantly increased Na,K-ATPase activity by 14% at 1 minute and by 31% at 5 minutes. Lower concentrations of LTD4 did not significantly increase Na,K-ATPase activity (data not shown). At the same time that it increased Na,K-ATPase activity, LTD4 (1 x 10-7 M) increased the amount of Na,K-ATPase {alpha}1 protein in the basolateral membrane of A549 cells at both 1 and 5 minutes (Figure 1B). Total {alpha}1 protein expression in whole-cell lysates from LTD4-stimulated and control cells was not different (data not shown), indicating that LTD4 caused recruitment/translocation of Na,K-ATPase subunits from intracellular compartment(s) to the basolateral plasma membrane.



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  		Figure 1. Activation of Na,K-ATPase in A549 cells by leukotriene D4 (LTD4). A549 cells were exposed to LTD4 (1 x 10-7 M), or vehicle alone (control), for 1 or 5 minutes before determination of Na,K-ATPase activity or fractionation and determination of {alpha}1 subunit protein abundance in the basolateral membrane. (A) Na,K-ATPase ion transport activity, determined as ouabain-sensitive 86Rb uptake. *p < 0.05 versus control (n = 4), {dagger}p < 0.001 versus control (n = 12), by analysis of variance (ANOVA) with the Tukey's multiple comparison test. (B) {alpha}1 Subunit protein abundance in the basolateral membrane, determined by immunoblotting. Bands from a representative immunoblot are shown above the bar graph, which depicts densitometry data from multiple experiments. *p < 0.05 versus control (n = 4), {dagger}p < 0.001 versus control (n = 7), by ANOVA with Tukey's multiple comparison test.

 
The effect of LTD4 on the subcellular distribution of the Na, K-ATPase was further examined by immunofluorescence laser-scanning confocal microscopy. A549 cells expressing GFP-tagged rat Na,K-ATPase {alpha}1 subunit (32) were incubated with LTD4 (1 x 10-7 M), or vehicle control, for 1 or 5 minutes before fixation and immunostaining. Figure 2 shows that LTD4 caused a time-dependent increase in Na,K-ATPase {alpha}1 subunit abundance in the plasma membrane, which is best seen at the junctions between adjacent cells. These microscopic images thus corroborate the results obtained by immunoblotting of basolateral membrane fractions, as shown in Figure 1B.



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  		Figure 2. Confocal immunofluorescence microscopy of A549 cells transfected with green fluorescent protein (GFP)-{alpha}1 Na, K-ATPase. Cells were exposed to vehicle alone (A) or to LTD4 (1 x 10-7 M) for 1 minute (B) or 5 minutes (C) and then fixed and immunostained for GFP. Arrowheads indicate the location of junctions between adjacent epithelial cells, where the GFP-{alpha}1 subunit can be seen to concentrate at the plasma membrane in time-dependent fashion after exposure to LTD4.

 
Role of the CysLT2 Receptor
To examine whether the effects of LTD4 on Na,K-ATPase were mediated by cysLT1 or cysLT2 receptors, we exposed A549 cells to LTD4 in the absence and presence of MK571, a potent and selective cysLT1 antagonist, and BAY-u9773, a dual cysLT1/cysLT2 antagonist. As shown in Figure 3 , the increase in basolateral membrane Na,K-ATPase {alpha}1 protein stimulated by LTD4 (1 x 10-7 M) was blocked by 3 µM BAY-u9773, but not by 100 nM MK571. In a previous report (22), this concentration of MK571 was sufficient to completely block LTD4-induced signaling in cells expressing human cysLT1 and was 10-fold greater than the IC50 for LTD4 binding to cysLT1 in radioligand-binding studies. LTD4-induced translocation of the Na,K-ATPase {alpha}1 subunit was also inhibited by pobilukast, another potent and selective cysLT1 inhibitor (data not shown). Our data therefore strongly suggest that stimulation of Na,K-ATPase function by LTD4 is mediated by cysLT2, and not cysLT1, receptors in A549 cells.



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  		Figure 3. Effect of cysteinyl LT receptor antagonists on LTD4-induced translocation of the Na,K-ATPase {alpha}1 subunit to the basolateral membrane. A549 cells were stimulated with LTD4 for 5 minutes in the absence of presence of either BAY-u9773 (3 µM) or MK571 (100 nM), after which cells were fractionated and the abundance of {alpha}1 subunit protein in the basolateral membrane was determined by immunoblotting. Bands from a representative immunoblot are shown above the bar graph, which depicts densitometry data from three or more independent experiments per condition. *p < 0.05 versus control, {dagger}p < 0.05 versus LTD4 alone, by ANOVA with Tukey's multiple comparison test.

 
To assess further the role of the two cysteinyl LT receptors in alveolar epithelial cells, we used reverse transcriptase-polymerase chain reaction to determine the expression of mRNA for cysLT1 and cysLT2 in A549 and rat AT2 cells, as compared with human eosinophils, which are known to express both receptor isoforms (23, 35). Figure 4 shows that both A549 and rat AT2 cells expressed mRNA for cysLT2, but neither expressed mRNA for cysLT1. Eosinophils expressed mRNA for both receptor isoforms, as expected. The finding that alveolar epithelial cells express cysLT2 and not cysLT1 receptors is consistent with the results of our experiments with inhibitors (Figure 3).



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  		Figure 4. Reverse transcriptase-polymerase chain reaction analysis of cysteinyl LT1 (cysLT1) and cysteinyl LT2 (cysLT2) receptor mRNA expression in A549 cells, rat alveolar type 2 (AT2) cells, and human eosinophils. Bands for cysLT2, but not cysLT1, are present in samples from A549 and rat AT2 cells, whereas eosinophils express mRNA for both receptors. Gels shown are representative of three independent experiments with identical results.

 
Effect of LTD4 on Alveolar Fluid Clearance in the Isolated, Perfused Rat Lung
Finally, we examined the effect of LTD4 on alveolar fluid clearance in the isolated, perfused rat lung model. LTD4 at various concentrations, or vehicle alone, was added to the airspace instillate and to the perfusate of isolated, perfused rat lungs, and alveolar liquid clearance was determined after 1 hour. As shown in Figure 5 , LTD4 (1 x 10-11 M) increased lung liquid clearance by 41%, as compared with the control. Higher concentrations of LTD4 (up to 1 x 10-7 M) did not significantly increase alveolar fluid clearance (data not shown). LTD4 at 1 x 10-11 M did not alter permeability for fluorescein isothiocyanate-labeled albumin, [3H]mannitol, or 22Na (data not shown). Figure 5 also illustrates that BAY-u9773 (3 µM) completely blocked the LTD4-induced increase in alveolar fluid clearance, whereas MK571 had no effect. These findings suggest that, like the effect of LTD4 on Na,K-ATPase in A549 cells, the LTD4-induced increase in alveolar fluid clearance in rat lungs is mediated by cysLT2 and not cysLT1.



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  		Figure 5. Effect of LTD4 and cysteinyl leukotriene receptor antagonists on alveolar liquid clearance in the isolated, perfused rat lung. LTD4 (1 x 10-11 M), without or with BAY-u9773 (3 µM) or MK571 (100 nM), or vehicle alone (control), was added to the airspace instillate and perfusate of isolated, perfused rat lungs, and alveolar liquid clearance was determined after 1 hour. In the absence of LTD4, neither BAY-u9773 nor MK571 had any effect on alveolar fluid clearance (data not shown). Results shown are from three or more rats per experimental condition. *p < 0.001 versus control, {dagger}p < 0.01 versus LTD4 alone, {ddagger}p < 0.05 versus control and p > 0.05 versus LTD4 alone, by ANOVA with Tukey's multiple comparison test.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study is the first to examine the effects of a cysteinyl LT on Na,K-ATPase function in alveolar epithelial cells and on alveolar fluid clearance in rat lungs. We have found that LTD4 rapidly (within 1 to 5 minutes) increases the activity of Na,K-ATPase in cultured A549 alveolar epithelial cells (Figure 1A). This observation provides a mechanistic basis for our additional finding that LTD4 increases the rate of alveolar liquid clearance in isolated, perfused rat lungs (Figure 5). These results were not expected because they reveal a potential protective role for cysteinyl LTs in relationship to the accumulation of pulmonary edema during acute lung injury.

At the same time that it increased Na,K-ATPase activity, LTD4 increased the abundance of Na,K-ATPase {alpha}1 subunits at the basolateral membrane of alveolar epithelial cells. This was documented by immunoblotting of basolateral membrane fractions (Figure 1B) and by confocal immunofluorescence microscopy (Figure 2), using an antibody directed at the Na,K-ATPase {alpha}1 subunit. There was no change in total {alpha}1 subunit protein detected in immunoblots of whole-cell lysates after LTD4 stimulation, indicating that the increase in {alpha}1 subunits in the basolateral membrane resulted from redistribution of the pump from intracellular compartment(s). In previous studies, we have shown that isoproterenol (19) and dopamine (20, 36) rapidly activate Na,K-ATPase in alveolar epithelial cells by recruiting enzyme subunits from intracellular pools (late endosomes) to the basolateral membrane. Thus, LTD4, which like isoproterenol and dopamine, acts through G-protein–coupled receptors (22, 23), upregulates alveolar epithelial Na,K-ATPase by a mechanism similar to that triggered by catecholamines.

The dual cysLT1/cysLT2 receptor antagonist BAY-u9773, but not the highly selective cysLT1 receptor antagonist MK571, blocked LTD4-induced translocation of the Na,K-ATPase {alpha}1 subunit to the basolateral membrane in A549 cells (Figure 3) and the increase in alveolar fluid clearance stimulated by LTD4 in isolated, perfused rat lungs (Figure 5). These results indicate that LTD4 acts via the cysLT2 rather than the cysLT1 receptor in human and rat alveolar epithelial cells. The fact that A549 cells and rat AT2 cells both expressed mRNA for cysLT2 and not cysLT1 (Figure 4) further supports this conclusion. To our knowledge, this is the first documentation that cysteinyl LT receptors are present and functional in epithelial cells of any type. This finding is not without precedent, however, because cysteinyl LTs have previously been shown to affect epithelial cell function. For example, Leikauf and colleagues observed that cysteinyl LTs enhance the growth of human airway epithelial cells (37), and more recently, LTD4 has been shown to rapidly trigger G-protein activation (24), cytoskeletal reorganization (25), calcium mobilization (26), and activation of RhoA (27) in human intestinal epithelial cells.

As noted, LTD4 (1 x 10-11 M) did not alter the flux of fluorescein isothiocyanate-labeled albumin, [3H]mannitol, and 22Na in the isolated, perfused rat lung (data not shown), indicating that it did not affect alveolocapillary permeability at the concentration that increased alveolar fluid clearance in our model. Although the ability of cysteinyl LTs to promote microvascular leak has been established (10–12), LTC4 and LTD4 did not increase lung microvascular permeability in a number of reports (38–40). Our findings and those of these latter studies may reflect the possibility that LTD4 affects permeability at only relatively high concentrations, while augmenting alveolar fluid reabsorption at lower levels. Also, it is important that we maintained pulmonary perfusion pressure at a constant low level in our model, thereby avoiding the pitfall of misinterpreting a change in transvascular fluid flux that would accompany a pulmonary vasopressor response to LTD4 as a change in microvascular permeability.

LTD4 increased alveolar fluid clearance in the isolated, perfused rat lung at a concentration (1 x 10-11 M) substantially lower than that that maximally increased Na,K-ATPase activity in A549 cells (1 x 10-7 M). This suggests the possibility that LTD4 may affect alveolar fluid clearance in the rat by other mechanism(s), in addition to activation of Na,K-ATPase. Among these, another potentially important target of LTD4's action may be the alveolar epithelial apical Na+ channel. It is also possible that the difference in effective LTD4 concentrations between our in vitro and isolated, perfused lung experiments might reflect alterations in cysLT2 receptor expression, ligand affinity, or downstream signaling in A549 cells as a result of malignant transformation or due to in vitro culture conditions. Of note, the concentrations of LTD4 detected in the pulmonary edema fluid of ARDS patients by Matthay and colleagues (7) were from 10 to 30 pM, in the same range as the concentration that we found to increase alveolar fluid clearance maximally in the isolated rat lung.

Our results suggest that the role of cysteinyl LTs in the pathophysiology of acute lung injury and ARDS is complex. Cysteinyl LTs may promote lung inflammation, microvascular permeability, and even fibroproliferation (41), but at the same time, they may decrease pulmonary edema by increasing fluid clearance across the alveolar epithelium. In this regard, LTD4 is similar to tumor necrosis factor-{alpha}, another proinflammatory mediator in ARDS with the capacity to increase both vascular permeability (42) and alveolar fluid clearance (43, 44). Furthermore, given the recent observation of Ware and Matthay (45) that higher pulmonary edema clearance rates correlated with a greater likelihood of recovery from ARDS, our findings suggest the possibility that cysteinyl LTs in the lung may actually have a salutary effect on patient outcome. Our results may also explain, in part, why ketoconazole, a LT synthesis inhibitor, failed to show benefit in a recent randomized, multicenter trial (46).

In summary, we have shown that the cysteinyl LTD4 stimulates Na,K-ATPase function in alveolar epithelial cells and increases alveolar fluid reabsorption in the rat lung. LTD4 activates the Na,K-ATPase by stimulating translocation/redistribution of the enzyme from intracellular compartment(s) to the basolateral membrane in a manner similar to catecholamines. LTD4 exerts its effects on alveolar epithelial cells through the cysLT2 receptor, whose expression we document here for the first time in epithelial cells of any origin. Our results suggest that cysteinyl LTs play a complex and, in part, beneficial role in the pathophysiology of acute lung injury and ARDS.


   Acknowledgments
 
The authors thank James Anderson, Zaher Azzam, and Aisha Nair for technical assistance. They also thank Dr. M. Caplan for the Na,K-ATPase {alpha}1 subunit antibody and Dr. A. Bertorello for A549 cells stably transfected with GFP-{alpha}1 Na,K-ATPase.


   FOOTNOTES
 
Supported by the National Institutes of Health (HL48129, HL65161), the Department of Veterans Affairs, the Crane Asthma Center of Northwestern University, and the Parker B. Francis Foundation (Fellowship Award to K.M.R.).

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: D.E.S. has no declared conflict of interest; K.M.R. has no declared conflict of interest; Y.A. has no declared conflict of interest; F.P.F. has no declared conflict of interest; A.B. has no declared conflict of interest; J.I.S. has no declared conflict of interest; P.H.S.S. has no declared conflict of interest.

Received in original form April 2, 2003; accepted in final form October 23, 2003


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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