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Circulation Research. 2003;92:453-460
Published online before print January 30, 2003, doi: 10.1161/01.RES.0000059414.10360.F2
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(Circulation Research. 2003;92:453.)
© 2003 American Heart Association, Inc.

Cellular Biology

Alveolar Type 1 Cells Express the {alpha}2 Na,K-ATPase, Which Contributes to Lung Liquid Clearance

K.M. Ridge, W.G. Olivera, F. Saldias, Z. Azzam, S. Horowitz, D.H. Rutschman, V. Dumasius, P. Factor, J.I. Sznajder

From the Division of Pulmonary and Critical Care Medicine (K.M.R., W.G.O., F.S., Z.A., V.D., P.F., J.I.S.), Northwestern University Medical School, Chicago, Ill; Jewish Hospital Heart & Lung Institute and the Departments of Medicine and Pharmacology & Toxicology (S.H.), University of Louisville, Louisville, Ky; and Department of Mathematics (D.H.R.), Northeastern Illinois University, Chicago, Ill.

Correspondence to J.I. Sznajder, MD, Northwestern University Medical School, Pulmonary and Critical Care Medicine, 303 East Chicago, Tarry Building 14-707, Chicago, IL 60611. E-mail j-sznajder{at}northwestern.edu


*    Abstract
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 *Abstract
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The alveolar epithelium is composed of alveolar type 1 (AT1) and alveolar type 2 (AT2) cells, which represent {approx}95% and {approx}5% of the alveolar surface area, respectively. Lung liquid clearance is driven by the osmotic gradient generated by the Na,K-ATPase. AT2 cells have been shown to express the {alpha}1 Na,K-ATPase. We postulated that AT1 cells, because of their larger surface area, should be important in the regulation of active Na+ transport. By immunofluorescence and electron microscopy, we determined that AT1 cells express both the {alpha}1 and {alpha}2 Na,K-ATPase isoforms. In isolated, ouabain-perfused rat lungs, the {alpha}2 Na,K-ATPase in AT1 cells mediated 60% of the basal lung liquid clearance. The ß-adrenergic agonist isoproterenol increased lung liquid clearance by preferentially upregulating the {alpha}2 Na,K-ATPase protein abundance in the plasma membrane and activity in alveolar epithelial cells (AECs). Rat AECs and human A549 cells were infected with an adenovirus containing the rat Na,K-ATPase {alpha}2 gene (Ad{alpha}2), which resulted in the overexpression of the {alpha}2 Na,K-ATPase protein and caused a 2-fold increase in Na,K-ATPase activity. Spontaneously breathing rats were also infected with Ad{alpha}2, which increased {alpha}2 protein abundance and resulted in a {approx}250% increase in lung liquid clearance. These studies provide the first evidence that {alpha}2 Na,K-ATPase in AT1 cells contributes to most of the active Na+ transport and lung liquid clearance, which can be further increased by stimulation of the ß-adrenergic receptor or by adenovirus-mediated overexpression of the {alpha}2 Na,K-ATPase.


Key Words: alveolar type 2 cells • alveolar type 1 cells • {alpha}1 and {alpha}2 Na,K-ATPase • alveolar fluid reabsorption


*    Introduction
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 *Introduction
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Active Na+ transport is critical for maintaining the alveolar space "dry" to ensure normal gas exchange.1,2 Sodium is actively transported across the alveolar epithelium; it enters via the apical Na+ channels and is actively extruded by basolaterally located Na,K-ATPase3–5 (see review6). The alveolar fluid is reabsorbed by the resulting osmotic gradient. The Na,K-ATPase is a heteromeric enzyme composed of an {alpha} isoform and a glycosylated ß isoform. The {alpha} isoform is the catalytic component of the holoenzyme, containing the cation and nucleotide binding sites as well as the receptor site for the cardiac glycoside ouabain.7–9 Four {alpha} isoforms have been identified ({alpha}1, {alpha}2, {alpha}3, and {alpha}4), each with a unique tissue distribution.10–12 The {alpha}1 and {alpha}2 Na,K-ATPase have been reported to be expressed in the lung.13,14 However, there are no previous reports on the precise cellular function or location of the {alpha}2 Na,K-ATPase in the lung.

In the human lung, the alveolar surface area is {approx}100 m2 and is composed of two morphologically distinct epithelial cells, type 1 and type 2.15 Alveolar type 2 (AT2) cells are small cuboidal cells that account for {approx}5% of the alveolar surface area. AT2 cells terminally differentiate into alveolar type 1 (AT1) cells, which are elongated, flattened cells that cover {approx}95% of the alveolar surface.15 Previously, it was thought that lung liquid clearance is effected by {alpha}1 Na,K-ATPase in AT2 cells, based on reports using ultrastructural immunocytochemistry6,16 and that AT1 cells lacked the proteins required for fluid and ion transport. However, two recent reports17,18 and the present study provide evidence to support a new paradigm that AT1 cells do indeed express the Na,K-ATPase and are capable of active Na+ transport and fluid reabsorption.

We provide evidence that both the {alpha}1 and {alpha}2 Na,K-ATPase contribute to active Na+ transport and alveolar fluid reabsorption under basal conditions, after catecholamine stimulation and adenoviral-mediated overexpression of the {alpha}2 Na,K-ATPase. The results of these studies demonstrate that (1) AT1 cells express the {alpha}1 and {alpha}2 Na,K-ATPase isoforms; (2) the {alpha}2 Na,K-ATPase is responsible for {approx}60% of the basal alveolar fluid reabsorption in the lung; (3) catecholamine-mediated increases in alveolar fluid reabsorption are due mostly to the {alpha}2 Na,K-ATPase in the AT1 cells; and (4) adenoviral-mediated overexpression of the {alpha}2 Na,K-ATPase increases alveolar fluid reabsorption by {approx}250%.


*    Materials and Methods
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 *Materials and Methods
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In Situ Hybridization
Methods were essentially as described previously.19 Complementary RNA probes were prepared as described previously.19

Immunoelectron Microscopy
Expression of Na,K-ATPase {alpha}2 protein in alveolar type 1 was assessed by immunoelectron microscopy using the {alpha}2 Na,K-ATPase, McB2, antibody and goat anti-mouse antibody, conjugated to colloidal gold particles. Gold particles were counted on mitochondria, nuclei, and the cytoplasm/plasma membrane.

Isolation and Culture of Alveolar Epithelial Cells (AECs)
AT2 cells were isolated from pathogen-free male Sprague-Dawley rats (200 to 225 g), as previously described.14 Cells were cultured in DMEM containing 10% fetal bovine serum with 2 mmol/L L-glutamine, 40 µg/mL gentamicin, 100 U/mL penicillin, and 100 µg/mL streptomycin and placed in culture for 7 days before the start of all experimental conditions.

The use of animals for the present study was approved by the Northwestern University Institutional Animal Care and Use Committee. Specific pathogen-free male Sprague-Dawley rats were purchased from Harlan Inc, Indianapolis, Ind.

Na,K-ATPase Activity
Ouabain-sensitive 86Rb+ uptake was used to estimate the rate of K+ transport by Na,K-ATPase in AECs.14,21 86Rb+ influx was quantified in aliquots of the SDS extract by liquid scintillation counting.

Preparation of Basolateral Plasma Membranes
Basolateral membranes (BLMs) were purified as previously described.21 For Western blot analysis, equal amounts of protein from BLMs were resolved by 10% SDS-PAGE and analyzed by immunoblotting with Na,K-ATPase anti-{alpha}2 monoclonal antibody (McB2, generous gift from K. Sweadner, Harvard University, Boston, Mass) or anti-{alpha}1 (generous gift from M. Caplan, Yale University School of Medicine, New Haven, Conn) monoclonal antibody.

Adenoviruses
The pCMV vector, which contains the immediate-early promoter and enhancer from CMV, a full-length cDNA for Escherichia coli lacZ and the SV40 t intron polyadenylation signal, was used. The ß-galactosidase cDNA was excised from pMRCMVß-gal and replaced with full-length cDNA for the rat {alpha}2 Na,K-ATPase (pMRCMV{alpha}2) or no cDNA (pCMVNull). AECs or rats were infected as previously reported.24,41–44

Isolated Perfused Lungs
The isolated perfused lung preparation used in our laboratory has been described in detail.5,22–24 Ten rat lungs were treated as control. Seventy rat lungs were perfused with 14 different concentrations of ouabain (10-11 to 7.5x10-4 mol/L) to determine the contribution of the {alpha}1 and {alpha}2 Na,K-ATPase to alveolar fluid reabsorption (AFR). Sixty-six rat lungs were perfused with 1 µmol/L isoproterenol in the presence or absence of ouabain (the same 14 concentrations of ouabain were used).

Data Analysis
Probability values were obtained by ANOVA followed by contrast analysis and considered significant below 0.05.

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
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Expression and Function of the {alpha}2 Na,K-ATPase
A strong hybridization signal corresponding to the {alpha}2 Na,K-ATPase mRNA was evident in the alveolar epithelium of normal rat lung tissue (Figure 1A). No signal was detected in sections hybridized with the sense probe (Figure 1B). Human lung, rat lung, and rat alveolar epithelial type 1-like (AT1-like) cells expressed both {alpha}1 and {alpha}2 Na,K-ATPase (Figure 2, lanes 3, 4, and 5, respectively) as determined by Western blot analysis. {alpha}1 Na,K-ATPase protein was detected in both rat brain and kidney tissue (Figure 2, lanes 1 and 2, respectively), but the {alpha}2 Na,K-ATPase was only detected in rat brain, as expected (Figure 2, lane 1), demonstrating the specificity of the Na,K-ATPase antibodies. Additionally, surfactant protein C (SP-C) and aquaporin 5 (AQ5) protein expression were determined in total cells lysates prepared from alveolar type 2 and AT1-like cells. As shown in Figure 2B, AT1-like cells express the AQ5, but not SP-C.



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  		Figure 1. Representative photomicrograph of {alpha}2 Na,K-ATPase mRNA, detected by in situ hybridization, in normal rat lung tissue. Digoxigenin-labeled, antisense cRNA probe specific for the {alpha}2 isoform was hybridized under high-stringency conditions to sections of the alveolar epithelium (A). Sense cRNA probe was used to detect nonspecific hybridization (B). Hematoxylin and eosin staining of a serial section (C).



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  		Figure 2. A, Expression of {alpha}1 and {alpha}2 Na,K-ATPase protein in human and rat lung tissue. Representative Western blot of the {alpha}1 and {alpha}2 Na,K-ATPase is shown. rB indicates rat brain; rK, rat kidney; hL, human lung; rL, rat lung; and rAT1, alveolar type 1-like cell. Immunoblotting was performed using a monoclonal antibody for {alpha}1 (McK1) or {alpha}2 (McB2) isoform. B, Surfactant protein C (SP-C) and aquaporin 5 (AQ5) protein expression in total cells lysates prepared from alveolar type 2 and AT1-like cells. Equal amounts of protein (12 µg) were loaded in each lane. Representative Western blot for SP-C and AQ5 is shown.

AT1 cells, but not AT2 cells, expressed the {alpha}2 Na,K-ATPase in normal rat and human lung tissue as determined by coimmunofluorescence (Figures 3A through 3I). AT2 cells were identified by staining with anti-SP-C antibody (Figures 3B and 3E). SP-C did not colocalize with {alpha}2 Na,K-ATPase (Figure 3C [rat] and Figure 3F [human]), indicating the AT1 cells, but not AT2 cells, express the {alpha}2 Na,K-ATPase. The {alpha}2 Na,K-ATPase protein was expressed in AT1 cells in normal rat lung tissue (Figure 3I) as determined by indirect immunofluorescence. T1{alpha} was used as a specific marker for AT1 cells (Figure 3H).15 Finally, the {alpha}1 Na,K-ATPase was ubiquitously expressed throughout the alveolar epithelium. As shown in Figures 3K and 3N, both human and rat lung tissue expressed the {alpha}1 Na,K-ATPase in both AT1 and AT2 cells. The {alpha}1 Na,K-ATPase colocalized with SP-C in AT2 cells (Figures 3M and 3P) as indicated by the yellow fluorescence.



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  		Figure 3. Representative photomicrograph coimmunofluorescence localization of {alpha}1 or {alpha}2 Na,K-ATPase normal human and rat lung tissue is shown. SP-C or T1{alpha} antibodies were used to identify AT2 cells and AT1 cells, respectively. A and G, Rat lung tissue expressing {alpha}2 Na,K-ATPase (green). D, Human lung tissue expressing {alpha}2 Na,K-ATPase (green). B and E, Identification of AT2 cells within lung tissue by SP-C staining (red). H, Identification of AT1 cells within lung tissue by T1{alpha} staining (red). C, F, and I, Composite panels demonstrating that the {alpha}2 Na,K-ATPase is expressed in AT1 cells and not AT2 cells. K and N, Rat and human lung tissue expressing {alpha}1 Na,K-ATPase (red). L and O, Identification of AT2 cells within lung tissue by SP-C staining (green). M and P, Composite panels demonstrating that the {alpha}1 Na,K-ATPase is expressed in AT2 cells (yellow) and AT1 cells (red).

The expression of the {alpha}2 Na,K-ATPase protein in AT1 cells was confirmed by immunoelectron microscopy. Figure 4A depicts the alveolus from which the AT1 cells in Figures 4B and 4C were magnified. The analysis of gold particle distribution demonstrated that immunogold labeling was specific to the cytoplasm/plasma membrane (Figure 4D). Labeling over mitochondria and nuclei was marginal, ie, equivalent to <6% of cytoplasm/plasma membrane labeling density, and was similar to nonspecific labeling.



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  		Figure 4. Immunoelectron microscopy. Electron micrographs of lung sections stained by immunogold technique for Na,K-ATPase using {alpha}2 monoclonal antibody (McB2). A, Normal rat alveolus. AT2 indicates alveolar type 2 cells; AT1, alveolar type 1 cells; and RBC, red blood cell (magnification x9000). B through C, Enlargement of AT1 cells from panel A (magnification, x92 400). Note the gold labeling on the cell indicating the presence of {alpha}2 isoform protein. *P<0.01. D, Labeling density for Na,K-ATPase {alpha}2 subunit over components of AT1 cells. Mean±SD, n=19 cells from lung tissue obtained from 6 animals.

In rodent tissue, it is possible to distinguish physiologically between the {alpha}1 and {alpha}2 Na,K-ATPase because of the large difference in their affinity/sensitivity for ouabain. The {alpha}2 Na,K-ATPase, but not the {alpha}1 Na,K-ATPase, is inhibited by 100 nmol/L ouabain,14,25 whereas >1 mmol/L ouabain is needed to inhibit the rat {alpha}1 Na,K-ATPase.14,25 We determined the contribution of the {alpha}1 and {alpha}2 Na,K-ATPases to AFR by perfusing, in different experiments, increasing concentrations of ouabain through the pulmonary circulation of the isolated rat lung model. As depicted in Figure 5A, the ouabain inhibition curve, obtained by least squares fit, of AFR was biphasic, indicating the presence of both {alpha}1 and {alpha}2 Na,K-ATPase. A class of low-affinity ouabain-insensitive {alpha}1 Na,K-ATPase constituted {approx}40% of AFR. A second class of high-affinity ouabain-sensitive {alpha}2 Na,K-ATPase constituted {approx}60% of AFR. The IC50 values calculated from the data in Figure 5A are consistent with the {alpha}1 Na,K-ATPase (2.3x10-4 mol/L) and the {alpha}2 Na,K-ATPase (2.5x10-7 mol/L).



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  		Figure 5. A, Ouabain inhibition curve of AFR in the isolated rat lung model. 10-11 to 7.5x10-4 mol/L ouabain was perfused through the pulmonary circulation to assess the effect on AFR (mL/h). B, Ouabain inhibition curve of ISO-mediated increase in AFR in the isolated rat lung model. 10-11 to 7.5x10-4 mol/L ouabain and 1 µmol/L ISO were perfused through the pulmonary circulation to assess the effect on AFR (mL/h). Data are presented as mean±SEM. Curve is nonlinear least square fit of data shown. C, ISO-mediated stimulation of Na,K-ATPase activity in AECs. Cells were treated with 1 µmol/L ISO for 15 minutes. 86Rb+ uptake was measured in the presence or absence of 5x10-5 mol/L ouabain. Each bar represents the mean±SEM of 3 independent cell isolations. Determinations were performed in quadruplicate. *P<0.01. The open bars indicate the contribution to the activity by {alpha}1 Na,K-ATPase; the filled bars indicate the contribution to the activity by {alpha}2 Na,K-ATPase. D, Na,K-ATPase {alpha}1 subunit abundance in the BLM prepared from ISO-treated primary AECs. Cells were incubated with 1 µmol/L ISO for 15 minutes at room temperature. Equal amounts of protein (10 µg) were loaded in each lane. Representative Western blot of the {alpha}1 and {alpha}2 Na,K-ATPase protein abundance is shown, (n=3).

The accuracy of the two-site binding model (eg, expression of {alpha}1 and {alpha}2 Na,K-ATPase) result was validated using an F test, which quantifies the relationship between the relative increase in the sum of squares and the relative increase in the degrees of freedom. If the one-site model (eg, expression of either {alpha}1 or {alpha}2 Na,K-ATPase) were correct, we would expect to get an F ratio near 1.0. Our F ratio was 9.9, which suggests two possibilities: (1) The two-site model is correct. (2) The one-site model is correct, but random scatter allowed the two-site model to fit better. However, the probability value was 0.0002. Thus, if the one-site model is correct, there is only a 0.02% chance that we randomly obtained data that fits the two-site model better. Based on these analyses, we conclude that there are two ouabain-binding sites in the alveolar epithelium, {alpha}1 Na,K-ATPase and {alpha}2 Na,K-ATPase.

The passive movement of small solutes ([3H]mannitol and 22Na+) and FITC-albumin across the epithelial barrier of the rat lung did not change when ouabain was perfused through the pulmonary circulation of the isolated rat lungs compared with control (data not shown), which validates the use of the model in assessing the role of {alpha}1 and {alpha}2 Na,K-ATPase in lung liquid clearance. The pulmonary circulation flow was measured periodically during the experiments and was similar ({approx}14 mL/min) in all groups (data not shown).

Catecholamine-Mediated Increase in {alpha}2 Na,K-ATPase
We have previously reported that isoproterenol (ISO) increased AFR in rat lungs.5,22 In this report, we determined the contribution of the {alpha}1 and {alpha}2 Na,K-ATPase to the ISO-mediated increase in AFR by perfusing, in different experiments, increasing concentrations of ouabain through the pulmonary circulation of the isolated rat lung model. As depicted in Figure 5B, the ouabain inhibition curve of ISO-stimulated AFR was biphasic but was shifted to the left. Scatchard plot analysis of the data in Figures 5A and 5B demonstrated that ISO did not affect the binding affinity of ouabain to either the {alpha}1 nor {alpha}2 Na,K-ATPase (data not shown). The F test was 8.9, and the probability value was 0.0005 for the comparison of one- versus two-site model. These results indicate that {approx}80% of the increase in AFR was due to the high-affinity ouabain-sensitive {alpha}2 Na,K-ATPase (Figure 5B) located in AT1 cells (Figure 4).

AT1-like cells,14 which express both the {alpha}1 and {alpha}2 Na,K-ATPase (Figure 2), were treated with 1 µmol/L ISO for 15 minutes. ISO increased the Na,K-ATPase activity by {approx}4.0-fold (Figure 5C) as measured by 86Rb+ uptake in the absence or presence of either 100 nmol/L ouabain to inhibit the {alpha}2 Na,K-ATPase or 5 mmol/L ouabain to inhibit the {alpha}1 Na,K-ATPase. The increase in Na,K-ATPase activity was mediated by both {alpha}1 and {alpha}2 Na,K-ATPase; however {approx}80% of this increase was due to the {alpha}2 Na,K-ATPase whereas <20% was due to the {alpha}1 Na,K-ATPase. The increase in activity was associated with an increase in {alpha}1 and {alpha}2 Na,K-ATPase protein abundance in the BLM of ISO-treated cells compared with control AT1-like cells (Figure 5D).

Adenovirus-Mediated Overexpression and Function of the Rat {alpha}2 Na,K-ATPase
Spontaneously breathing rats were infected with 109 to 1010 pfu of Ad{alpha}2; maximal clearance was noted with 2x109 pfu (data not shown). Lung liquid clearance was increased {approx}250% in rats infected with Ad{alpha}2 compared with Adnull-infected or sham-infected rats (Figure 6A). As shown in Figure 6A, 10-5 mol/L ouabain (which inhibits only the {alpha}2 Na,K-ATPase) decreased AFR by {approx}70% in Ad{alpha}2-infected rat lungs, suggesting that the observed increase in clearance was due to the increased {alpha}2 Na,K-ATPase protein abundance.



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  		Figure 6. A, AFR was increased by {approx}250% in rats infected with Ad{alpha}2 compared with sham- and Adnull-infected rats during the first hour of the isolated perfused rat lung model. In the second hour, 10-5 mol/L ouabain perfused through the pulmonary circulation decreased alveolar fluid clearance by {approx}70%, to a level not different from sham- and Adnull-infected rat lungs. *P<0.01 compared with control or sham-infected rat lungs. Bars indicate mean±SE, n=6. B, top, Representative Western blot of the {alpha}2 Na,K-ATPase protein abundance in lung tissue from control, Adnull-, and Ad{alpha}2-treated rats. B, bottom, Photomicrograph immunofluorescence localization of {alpha}2 Na,K-ATPase protein. Right, Adnull-infected rat lung tissue expressing {alpha}2 Na,K-ATPase. Left, Ad{alpha}2-infected rat lung tissue expressing {alpha}2 Na,K-ATPase.

Increased abundance of the {alpha}2 Na,K-ATPase protein was observed in the alveolar epithelium in Ad{alpha}2-infected rat lungs but not in Adnull-infected control rat lungs (Figure 6B), as determined by indirect immunofluorescence and Western blot analysis. No change in the {alpha}1 Na,K-ATPase protein expression was observed in any of the rat lungs (data not shown).

Rat AT1-like cells and human A549 cells were infected with multiplicity of infection (MOI) of 1 to 25 of Ad{alpha}2 for 24 hours. Both rat AT1-like cells and A549 cells infected with Ad{alpha}2 (MOI 10) had a {approx}2-fold increase in Na,K-ATPase activity compared with Adnull-infected cells (Figure 7). The increase in Na,K-ATPase activity was associated with an increase in {alpha}2 Na,K-ATPase protein abundance in the BLMs of Ad{alpha}2-infected cells (Figure 7, top panel). There was no change in the {alpha}1 Na,K-ATPase protein abundance in either A549 cells or AT1-like cells (data not shown). The use of adenoviruses to overexpress the {alpha}2 Na,K-ATPase was not cytotoxic to the cells, given that there was no increase in K+ or LDH concentrations in the supernatant (data not shown). Gene transfer efficiency and distribution were homogeneous in rat lungs and AECs by infection with Adß-Gal, which produced lacZ expression in all regions of the lungs and cell monolayer (data not shown).



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  		Figure 7. Top, {alpha}2 Na,K-ATPase protein abundance in the BLM prepared from AT1-like cells (AEC) and A549 cells infected with Adnull or Ad{alpha}2. Cells were infected with 1 to 25 MOI of the respective virus for 24 hours. Equal amounts of protein (7.5 µg) were loaded in each lane. Representative Western blot of the {alpha}2 Na,K-ATPase protein abundance is shown (n=3). Bottom, Na,K-ATPase activity, as measured by ouabain-inhibitable 86Rb+ uptake, in AT1-like cells (AEC) infected with 1 to 25 MOI Ad{alpha}2. Each bar represents the mean±SEM of 3 independent cell isolations. Determinations were performed in quadruplicate. *P<0.01; +P<0.001 compared with untreated control cells.


*    Discussion
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 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
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It is well established that AFR is regulated by active Na+ transport mechanisms.3,5,17,22–24,26–28 Specifically, the Na,K-ATPase generates the electrochemical gradient for transcellular transport of sodium, with water following the osmotic gradient across the alveolar epithelium. These processes were thought to occur in alveolar type 2 cells, which account for <5% of the alveolar surface area, whereas the alveolar type 1 cells, which account for >95% of the alveolar surface area, were thought to lack the molecular machinery required for active Na+ transport.16,29 Our data and two very recent reports17,18 provide evidence that alveolar type 1 cells participate in active Na+ transport. In this report, we provide the first evidence that {alpha}2 Na,K-ATPase is expressed in alveolar type 1 cells and is an important contributor to lung liquid clearance. The importance of the {alpha}2 Na,K-ATPase is further emphasized by our studies where AFR was significantly increased in rat lungs that overexpressed the {alpha}2 Na,K-ATPase.

{alpha}2 Na,K-ATPase mRNA was detected in the alveolar epithelium by in situ hybridization (Figure 1), and {alpha}2 Na,K-ATPase protein was localized to AT1 cells in situ (Figures 3 and 4). AT1 cells were identified by electron microscopy or by immunofluorescent staining with antibodies against either T1{alpha} or aquaporin 5, AT1 cell-specific phenotype markers. The alveolar surface area in situ varies considerably; AT2 cells are {approx}500 µm2, whereas the surface are of AT1 cells is {approx}4500 µm2.15 Therefore, the density of Na,K-ATPase molecules per unit membrane surface area of an AT1 cell in situ is relatively low, as demonstrated by immunoelectron microscopy (Figure 4). Quantitative analysis of the number of {alpha}2 Na,K-ATPase molecules detected by immunoelectron microscopy was somewhat limited because some antibody sites were lost on exposure of the tissue to the fixative, despite that a comparatively gentle fixative was used.20 Additionally, this fixative did not preserve well the membrane structures. Therefore, we used semiquantitative analysis of gold particle distribution30 to determine in which regions of the cell that the {alpha}2 Na,K-ATPase protein was expressed (Figure 4E). As anticipated, the {alpha}2 Na,K-ATPase proteins were expressed mostly at the membrane/cytoplasm of AT1 cells (Figure 4). The pattern of Na,K-ATPase protein expression in lung appears similar to the expression patterns in the heart.31,32 The {alpha}1 isoform is uniformly distributed within the plasma membrane of most cells,31–33 whereas the {alpha}2 isoform localized to microdomains within the membrane.31,32

A recent report17 established that isolated rat AT1 and AT2 cells are capable of active Na+ transport and that AT1 cells transport more Na+ per microgram of protein than AT2 cells. The interpretation of these experiments is limited by their in vitro design. Therefore, we assessed the contribution of the {alpha}2 Na,K-ATPase in AT1 cells in situ and the contribution of ubiquitously expressed {alpha}1 Na,K-ATPase to AFR using an isolated, perfused rat lung model. In rodent tissues, the {alpha}1 and {alpha}2 Na,K-ATPase can be biochemically distinguished by their low and high affinity for ouabain, respectively.14,34,35 As shown in Figure 5A, we demonstrated that there are two distinct binding sites for ouabain in the alveolar epithelium: {alpha}1 Na,K-ATPase (low affinity, IC50: 2.3x10-4 mol/L,) and {alpha}2 Na,K-ATPase (high affinity, IC50: 2.5x10-7 mol/L). Similar IC50 values have been reported for the {alpha}1 and {alpha}2 Na,K-ATPases in rat AECs, cardiac muscle, adipocytes, and brain.14,34,35

The {alpha}2 Na,K-ATPase accounts for {approx}60% of the basal lung liquid clearance (Figure 5A). In accordance with recent reports, these data suggest that AT1 cells play an important role in maintaining the fluid balance in the lung.17,18 It has been clearly established that catecholamines markedly increase alveolar fluid clearance in normal and injured lungs5,22–24,36 and that catecholamine-mediated increases in AFR are dependent on normal expression levels of Na,K-ATPase proteins.37 A recent report demonstrated that in transgenic mice that overexpressed the ß2-adrenergic receptor there was a 40% increase in AFR, which was associated with increased {alpha}2 Na,K-ATPase protein abundance, but not {alpha}1 Na,K-ATPase, in lung homogenates.38 Our data provide evidence that ß-adrenergic agonists increase AFR by regulating the {alpha}2 Na,K-ATPase in AT1 cells. As shown in Figure 5B, the ouabain inhibition curve of ISO-stimulated AFR was biphasic, indicating involvement of both {alpha}1 and {alpha}2 Na,K-ATPase, but was clearly shifted to the left. These results suggest that {approx}80% of the ISO-mediated increase in lung liquid clearance is regulated by the activity of the {alpha}2 Na,K-ATPase (Figure 5B). These results suggest that ISO preferentially regulates the {alpha}2 Na,K-ATPase, thus increasing the rate of lung liquid clearance across the AT1 cell.

To assess the mechanisms by which ISO increased AFR, we treated rat AT1-like cells, which express similar amounts of {alpha}1 and {alpha}2 Na,K-ATPase,39 with ISO. As shown in Figure 5D, these results suggest that ISO increases both {alpha}1 and {alpha}2 Na,K-ATPase protein abundance in the BLM of rat AT1-like cells. This increase in Na,K-ATPase protein abundance is not likely to represent increased de novo synthesis of Na,K-ATPase molecules, because the ISO effect occurred within 15 minutes, which concord with previous reports.4,21 The turnover rate of the Na,K-ATPase was then measured in the presence of ouabain at concentrations to selectively inhibit the {alpha}2 Na,K-ATPase. As shown in Figure 5C, both the {alpha}1 and {alpha}2 Na,K-ATPase contributed to the overall increase in activity; however, the majority of the increase was due to the {alpha}2 Na,K-ATPase (Figure 5). Under basal conditions, the {alpha}1 Na,K-ATPase is pumping at approximately one-half its maximum capacity,40 whereas the {alpha}2 Na,K-ATPase is pumping at one-twentieth of its Vmax.25 Therefore, even similar amounts {alpha}1 and {alpha}2 Na,K-ATPase protein abundance, as previously reported in rat AT1-like cells, would not translate into equal pumping capacity.14 ISO increased the {alpha}1 Na,K-ATPase activity from 5.2±1.0 to 12.0±2.7 nmol K+/mg protein per minute, suggesting that the {alpha}1 Na,K-ATPase is functioning at Vmax. In contrast, the {alpha}2 Na,K-ATPase activity increased 6-fold from 6.4±1.5 to 36.8±4.9 nmol K+/mg protein per minute, suggesting that its pumping capacity has not yet been maximized. These results suggest that both the {alpha}1 and {alpha}2 Na,K-ATPase can be regulated by ß-adrenergic agonists, but that the {alpha}2 Na,K-ATPase regulation might be physiologically more significant.

We have previously reported that adenovirus-mediated transfer of Na,K-ATPase isoform genes increases Na,K-ATPase expression and function in human and rat lung epithelial cells and the alveolar epithelium of rats.24,36 Specifically, we demonstrated that the overexpression of the ß1 Na,K-ATPase subunit gene, but not the {alpha}1 Na,K-ATPase, increases AFR by >100% in normal rats,41,42 mitigates oxidant-mediated decreases in active Na+ transport in rat fetal distal lung epithelialcells,43 and increases AFR and survival of rats exposed to 100% oxygen.44 In the present study, we explored whether overexpression of the {alpha}2 Na,K-ATPase in the alveolar epithelium could increase AFR. We instilled a surfactant-based vehicle to achieve a homogeneous delivery of our adenoviral vectors to the lung as previously reported and used the isolated rat lung model to assess AFR 7 days after adenoviral infection.24,36 As shown in Figure 6A, overexpression of the {alpha}2 Na,K-ATPase markedly increased the ability of the lung to reabsorb fluid. This increase in clearance was inhibited by 10-5 mol/L ouabain, a concentration that inhibits the {alpha}2 Na,K-ATPase, but not the rat {alpha}1 Na,K-ATPase. These findings are consistent with our in vitro data showing that the {alpha}2 Na,K-ATPase overexpression increases ouabain-inhibitable Na,K-ATPase activity in human A549 cells and rat AT1-like cells (Figure 7). The increases in {alpha}2 Na,K-ATPase protein after Ad{alpha}2 infection and the absence of functional change after Adnull infection suggest that the observed increases are specific responses to adenoviral-mediated gene transfer. Thus, these results suggest that in rat AT1-like cells and human A549 cells, the rate-limiting Na,K-ATPase subunit is the {alpha} subunit and not the ß1 subunit. Therefore, by overexpressing either {alpha}2 we can increase Na,K-ATPase activity.

Taken together, these studies demonstrate that (1) both {alpha}2 and {alpha}1 Na,K-ATPase are necessary for clearing fluid from the alveolar epithelium and (2) the {alpha}2 Na,K-ATPase, which is located in the alveolar epithelial type 1 cell, serves a specialized function, being responsible for {approx}60% of basal AFR and {approx}80% of the ß-adrenergic-mediated increase in AFR. (3) Adenovirus-mediated overexpression of the {alpha}2 Na,K-ATPase markedly increases Na,K-ATPase activity and lung liquid clearance. These data provide evidence to support the newly developing hypothesis that the {alpha}2 Na,K-ATPase expressed within alveolar type 1 cells is the primary contributor to lung liquid clearance.


*    Acknowledgments
 
This study was supported in part by NIH NHLBI HL48129 and the Parker B. Francis Fellowship (K.M.R.).

Received June 7, 2002; revision received January 21, 2003; accepted January 21, 2003.


*    References
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