INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The Na,K-ATPase is a transmembrane protein composed of an - and -polypeptide chain, which couples the transport of Na⁺ and K⁺ ions across the plasma membrane to the enzymatic hydrolysis of ATP, thus maintaining cellular ionic gradients and osmotic balance (1). The catalytic -subunit contains the binding sites for Na⁺, K⁺, and ATP. The -subunit is a glycosylated polypeptide whose function includes the incorporation of the -subunit into the cell membrane (2). The genes that encode for Na,K-ATPase are purported to be under hormonal control, as analysis of the 5' flanking regions of the subunits reveals the presence of hormonal response elements (3, 4). Previous studies have reported that dexamethasone increased Na,K-ATPase mRNA and protein abundance in AT2 cells, resulting in increased Na⁺ pump function (5, 6).

Specific receptors for aldosterone have been reported in the rat lung (7), suggesting the lung as a potential target organ. In cultured cardiocytes and renal cells, aldosterone increased Na,K-ATPase gene expression and Na,K-ATPase function (8, 9). One of the important functions of alveolar epithelial Na,K-ATPase is to effect active Na⁺ transport and lung edema clearance (10, 11). We reasoned that delivering aldosterone to the lungs could increase active Na⁺ transport and the ability to clear edema fluid. Thus, we set out to determine whether aldosterone regulates Na,K-ATPase activity in cultured AT2 cells and whether rats treated with aldosterone aerosol delivered to the lungs had increased active Na⁺ transport and edema clearance.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals and Reagents

Specific pathogen-free, adult, male Sprague-Dawley rats were purchased from Charles River Laboratories (Charles River, MA). Aldosterone (Sigma Chemical Co., St. Louis, MO) was dissolved in ethyl alcohol and then in 0.9% saline for a final stock concentration of 100 µg/ml in 1.0% ethyl alcohol/0.9% saline vehicle solution. Fetal bovine serum was obtained from Hyclone (Login, UT) and porcine pancreatic elastase was obtained from Worthington Biochemical Company (Freehold, NJ). Dulbecco's modified Eagle medium (DMEM), Ham's F-12 media, antibiotic solution, vitamins, linoleic acid-albumin, L-glutamine, transferrin, and sodium selenite were all obtained from Sigma Chemical.

Alveolar Type 2 Cell Isolation and Experimental Protocol

Alveolar type 2 cells were isolated from pathogen-free male Sprague- Dawley rats weighing 200 to 225 g, as previously described (5). Briefly, the lungs were perfused via the pulmonary artery, lavaged, and digested with elastase (30 U/ml; Worthington Biochemical). The AT2 cells were purified by differential adherence to IgG pretreated dishes. Cells were suspended in DMEM (Irving Scientific, Santa Ana, CA) containing 10% charcoal-stripped fetal bovine serum with 2 mM L-glutamine, 40 µg/ml gentamicin, 100 U/ml penicillin, and 100 µg/ml streptomycin. For Na,K-ATPase transport measurements (⁸⁶Rb⁺ uptake) in AT2 cells, 1 × 10⁶ cells were plated into each well of 24-well tissue culture plates (Becton-Dickinson, Rutherford, NJ). For mRNA preparation and Na,K-ATPase activity (³²P-ATP hydrolysis), 7 × 10⁶ cells/well were plated in 10 cm² tissue culture plates (Becton-Dickinson). Cells were incubated in a humidified atmosphere of 5% CO₂/ 95% air at 37° C. After 24 h, nonadherent cells were removed by rinsing the monolayers. Plating efficiency was ~ 60%. The cells were then cultured for 3, 6, 12, and 24 h in serum-free medium consisting of DMEM/Ham's F-12 medium containing 0.5 mg/ml BSA-linoleic acid, 4 ng/ml selenic acid, 5 µg/ml transferrin, and 2 mM glutamine in the absence or presence of 300 nM aldosterone.

RNA Isolation and Northern Analysis

Total RNA was isolated by guanidine thiocyanate-phenol-chloroform extraction methods as previously described (12). Quantity and purity were determined spectrophotometrically. Ten micrograms per sample was size-fractionated in 2.2 M formaldehyde 1% agarose gel by electrophoresis at 20 V for 14 to 16 h. Ethidium bromide staining of ribosomal 28S and 18S bands on the gel was visualized with ultraviolet light and recorded photographically to assure equal lane loading. RNA was electrotransferred (20 V for 6 h at 4° C) to nylon membranes. The blotting was verified as uniform across the paper by UV transillumination of the nylon filter and recording of the 18S and 28S ribosomal bands photographically. Membranes were baked for 2 h at 80° C and UV cross-linked. ³²P-CTP-labeled riboprobes (~ 1.0 kb) were prepared by SP6-mediated in vitro transcription from cDNAs spanning the phosphorylation and ATP-binding site sequences of each subunit that had been subcloned in pGEM3Z (Promega Corp., Madison, WI) generously supplied by J. Emanuel (13). Membranes were prehybridized in 50% formamide, 10% dextran sulfate, 0.5% nonfat dry milk, 1% SDS, 250 µg sheared salmon sperm DNA, and 250 µg yeast tRNA in 6× SSC overnight followed by hybridization at 57° C. They were then washed at 65° C in 0.1× SSC, 0.1% SDS for 1 h, and exposed to X-ray film at 70° C. Multiple exposures of the autoradiograms were made to ensure that signals were within the linear range of the film. Bands on autoradiograms were quantitated with a GS300 scanning densitometer (Hoeffer Scientific) and the area of the peak was determined with Gelscan Software (Lakeshore Technologies Inc.). In all cases, triplicate RNA samples from aldosterone-treated cells and time-matched controls were analyzed simultaneously on the same nylon membrane.

Na,K-ATPase Transport Measurements

Ouabain-sensitive ⁸⁶Rb⁺ uptake was used to assess the rate of K⁺ transport by the Na,K-ATPase in alveolar epithelial cells. AT2 cells were incubated at 37° C with and without 1 mM ouabain for 5 min. This medium was then removed, and otherwise identical fresh medium containing 1 µCi/ml ⁸⁶Rb⁺ was added. After a 5-min incubation, uptake was terminated by aspirating the assay medium and washing the plates in ice-cold MgCl₂. Plates were allowed to air-dry and cells were solubilized in 1 N NaOH. ⁸⁶Rb⁺ influx was quantitated in aliquots of the NaOH extract by liquid scintillation counter. Protein was quantitated in aliquots by the Bradford method. ⁸⁶Rb⁺ uptake was calculated as the slope of the uptake versus time plot as described previously. The K+ influx data were expressed as nanomoles of K⁺/min/ mg protein and are reported as ouabain-sensitive ⁸⁶Rb⁺ uptake determined from the total ⁸⁶Rb⁺ uptake minus ⁸⁶Rb⁺ uptake in the presence of 1 mM ouabain.

Membrane Preparation for Na,K-ATPase Hydrolytic Activity

Briefly, alveolar epithelial cells were washed three times with PBS, scrapped in 1 ml of homogenization buffer (5 mM histamine-imidazole, 2 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 60 µg/ml soybean trypsin inhibitor) and rinsed with an additional 1 ml of the same buffer. Alveolar epithelial cells were resuspended in 1 ml of homogenization buffer and disrupted in a Teflon homogenizer for 20 strokes. The homogenate was centrifuged (1,000 g for 5 min at 4° C) to remove unbroken cells and debris. The supernatant was then centrifuged at 60,000 g at 4° C for 1 h. The high speed supernatant was designated the cytosolic fraction. The high speed pellet was resuspended in 500 µl homogenization buffer and designated the membrane fraction. Total protein concentrations of cytosol and membrane fractions were determined by BioRad protein assay with BSA as standard (Bio-Rad Laboratories, Richmond, CA).

Preparation of AT2 Cell Basolateral Plasma Membranes

After incubation with the desired agonists, AT2 cells were homogenized and basolateral membranes were prepared as described by Chibalin and colleagues (14). Briefly, after several centrifugations to discard the nuclear and mitochondrial pellets, the remaining supernatant was centrifuged at 48,000 g for 30 min. Basolateral membrane (BLM) fractions were recovered after centrifugation of the membrane pellet in a Percoll gradient (16%) at 48,000 g for 30 min.

Preparation of AT2 Cell Whole Membranes

After several centrifugations to discard the nuclear and mitochondrial pellets, the remaining supernatant was centrifuged at 100,000 g for 60 min.

Western Blot Analysis

Na,K-ATPase 1- and -subunit abundance was determined by Western blot analysis. Proteins were resolved by SDS-PAGE on a 6 to 10% polyacrylamide gradient gel and transferred to nitrocellulose membranes (Hybond C; Amersham, Arlington Heights, IL). After transfer was completed (3 h at 1 A), the membranes were quenched at room temperature for 1 h with 7.5% casein in PBS containing 0.1% Tween 20. Incubation with specific Na,K-ATPase antibodies was performed overnight at 4° C (1-antibody was a gift from M. Caplan, 1 antibody was a gift from P. Martin-Vasallo). The membranes were rinsed five times with PBS, 1% Tween 20 followed by incubation with a horseradish peroxide-conjugated secondary antibody (Bio-Rad) for 1 h. Blots were developed as previously described with an ECL detection kit (Amersham) used as recommended by the manufacturer.

Na,K-ATPase Hydrolytic Activity

Na,K-ATPase activity was determined in membrane fractions of alveolar epithelial cells as previously described by Barquin and colleagues (5). Briefly, the total activity reaction mixture contained (in a final volume of 500 µl) 130 mM NaCl, 20 mM KCl, 3 mM ATP, 3 mM MgCl₂, and 30 mM imidazol. The ouabain-sensitive reaction mixture contained the same with 3 mM ouabain. Na,K-ATPase activity was determined by preincubating the microsomal fraction at 37° C in buffer for 30 min. The results were corrected for spontaneous hydrolysis of ATP. The reaction was stopped with 1% trichloroacetic acid, and the precipitated proteins were pelleted. Inorganic phosphate was determined by utilizing molybdate-H₂SO₄ solution with Fiske reducing agent (15). The specific activity of the enzyme is expressed as nmol P_i/mg protein/h.

Physiologic Studies

Nineteen pathogen-free male Sprague-Dawley rats weighing 330 to 360 g were purchased from Charles River Laboratories (Raleigh, NC). Nine rats were exposed to aldosterone aerosols, five rats were exposed to saline aerosols, and both groups were studied 24 h after the delivery of the aerosols and compared with five untreated control rats. Four additional rats were utilized to estimate alveolar aldosterone deposition after aerosolization.

Aerosolization Protocol

Aerosols were delivered to the respiratory tract of two groups of the rats using a nose-only aerosol delivery system as previously described (16). Each rat was lightly intramuscularly sedated with 0.08 g/kg ketamine and placed in a body suit (Alice Chatham King, Los Angeles, CA) suspended from two rails in a plethysmograph. The head was immobilized around the neck. The sling support system was advanced such that the rat's nose fit snugly through a small hole in a latex diaphragm stretched over a nose cone. The rat was able to spontaneously breath air flowing through the zero-dead-space aerosol delivery port. The cover was placed on the plethysmograph. Pressure in the plethysmograph was monitored with a Validyne MP-45 pressure transducer connected to its carrier amplifier (Model CD12; Validyne, Northridge, CA). The pressure tracing was monitored on chart recorder. Aerosols of aldosterone were generated from 100 µg aldosterone in 1 ml saline by Aerotech II nebulizer (Cadema, New York, NY) operated at 9 L/ min. An aerosol of either aldosterone (Sigma Chemical) or saline was delivered to the respiratory tract for 20 min.

Isolated Lungs

Briefly, rats were anesthetized with 30 mg/kg/body weight of pentobarbital and anticoagulated with 1,000 U heparin administered intraperitoneally. A tracheotomy was performed and the rats were mechanically ventilated with a tidal volume of 2.5 ml, peak airway pressure of 8 to 10 cm H₂O and 100% O₂ for 5 min. After exsanguination, the heart and lungs were removed en bloc. The pulmonary artery and the left atrium were cannulated with triple lumen catheters. The pulmonary circulation was flushed of remaining blood by perfusing with 30 ml buffer salt albumin (BSA) solution: 135 mM Na⁺, 120 mM Cl, 25 mM HCO₃, 4.1 mM K⁺, 2.8 nM Mg⁺, 2.5 mM CaCl₂, 0.8 mM SO₄, 8.3 mM glucose, 3% bovine albumin at pH 7.45, 300 mosm/kg H₂O. Two sequential bronchoalveolar lavages (BAL) were performed with 5 ml of BSA solution containing 0.1 mg/ml Evans blue dye tagged albumin (EBD; Sigma); 0.02 Ci/ml ²²Na⁺ (Amersham, Chicago, IL) and 0.12 Ci/ml ³H-mannitol (Dupont, N.E.N., Boston, MA). The volume of the epithelial lining fluid (ELF) was estimated by the dilution of EBD in the first BAL. The lungs were then instilled with the volume necessary to leave 5 ml in the alveolar space. Finally, the lungs were immersed in a covered "pleural bath" reservoir containing 100 ml of the BSA solution maintained at 37° C. This allowed us to follow markers that had moved across the pleural membrane or were drained by the lung lymphatics (17, 18).

Lungs were perfused with 90 ml of the same BSA solution containing 0.16 mg/ml fluorescein-tagged albumin (FITC-albumin; Sigma). The perfusate was pumped from a lower reservoir to an upper reservoir by a peristaltic pump and from there flowed through the pulmonary artery and exited via the left atrium. Left atrial and pulmonary artery pressures were maintained at 0 and 8 mm Hg with a pressure transducer with a zero reference point at the level of the left atrium. The perfusate was maintained at pH 7.45 by bubbling a mixture of 5% CO₂ and 95% O₂. Samples were drawn from the three reservoirs: air-space instillate, "pleural bath," and the perfusate at 10 and 70 min after starting the experiment protocol. To ensure homogeneous sampling from the air spaces, 2 ml of instillate were aspirated and reintroduced into the air spaces three times before removing each sample. All samples were centrifuged at 1,000 × g for 15 min. Colorimeric analysis of the supernatant for EBD (absorbance at 620 nm of 0.1 ml sample diluted 1:10 in BSA) was done in a Hitachi Model U2000 (Hitachi, Tokyo, Japan) or a Beckman spectrophotometer Model 35 (Beckman Instruments, Irvine, CA) and for FITC-albumin (excitation 487 nm and emission 520 nm of 0.10 ml sample diluted 1:10 in BSA) in a Perkin Elmer fluorescence spectometer Model LS-3B (Perkin Elmer Medical Instruments, Oakbrook, IL). ²²Na⁺ and ³H-mannitol were measured in a betacounter (Packard Tricarb, Downers Grove, IL) in an 0.2-ml sample diluted 1:25 in Budget Solve (Research Products International, Mount Prospect, IL).

Calculations for Isolated Perfused Rat Model

The derivation of all equations involved in the mathematical model of edema clearance has been previously described in detail (18). Concentration of EBD-albumin was used to estimate air-space volume. As virtually all EBD-albumin remains in the air space, we may calculate instillate volume (V) at a given time t as:

(1)

where V₀ is the initial volume instilled and [EBD]₀ and [EBD]_t are the concentrations of EBD-albumin at times 0 and t, respectively. The removal of sodium from the alveolar space during a defined period of time is assumed to be accompanied by isotonic water flux, which is given by:

(2)

where J_Na,net is the net or active Na⁺ transport, J_Na,out is the total or unidirectional Na⁺ outflux caused by active and passive Na⁺ transport, and J_Na,in is the back flux of Na⁺ into the alveolar fluid by passive movement. Because Na⁺ concentration remains constant in all compartments, the net Na⁺ flux (which we refer to as active Na⁺ transport) from the air space is:

(3)

The unidirectional fluxes of Na⁺ from the alveolar space, a result of active transport and passive movement, was calculated as:

(4)

Similar, the (undirectional) volume flux of ³H-mannitol (typically expressed as PA, permeability of surface area) was calculated as:

(5)

Albumin flux from the pulmonary circulation into the alveolar space was determined from the fraction of FITC-albumin that appears in the alveolar space during the experimental protocol. For reasons of comparison, fluxes are reported as volume fluxes (volume/time) by using the appropriate solute concentrations.

Data Analysis

The in vitro AT2 cell studies were performed after two or three separate cell isolations. Experimental conditions and assays were then performed in triplicate or as indicated in the figure legend. All of the data were analyzed by ANOVA and group comparison t tests. Duncan's multiple range test was used to compare experimental groups. Probability values < 0.05 were considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effect of Aldosterone on Na,K-ATPase mRNA Steady-state Levels

The effects of aldosterone exposure on AT2 cell Na,K-ATPase ₁- and ₁-subunits steady-state mRNA levels are shown in Figure 1. The steady-state Na,K-ATPase ₁-mRNA transcript levels in AT2 cells exposed to aldosterone were not different from those in time-matched control AT2 cells (Figure 1A). However, the steady-state Na,K-ATPase ₁-mRNA transcript levels were significantly increased after incubation with aldosterone at 3 and 6 h as compared with control levels (Figure 1B).

View larger version (21K): [in this window] [in a new window]   Figure 1.   Effects of 300 nM aldosterone on the Na,K-ATPase 1- (A) and 1- (B) mRNA steady-state transcript levels in cultured AT2 cells. Each point (n = 3, from three separate cell isolations done in triplicate) represents the mean ± SEM. * indicates significant difference (p < 0.05) as compared with time-matched controls. A value of 1 was arbitrarily assigned to the control mRNA for each time point, and the message levels of cells treated with aldosterone were compared with this value. There was a significant increase in the Na,K-ATPase 1-subunit mRNA signal compared with the baseline and the time-paired control.

Effect of Aldosterone on Na,K-ATPase Protein Abundance

The effects of aldosterone exposure on AT2 cell Na,K-ATPase ₁- and ₁-subunit protein abundance are shown in Figure 2. The Na,K-ATPase ₁-subunit protein abundance in basolateral membranes isolated from AT2 cells exposed to aldosterone was significantly increased at 12 and 24 h as compared with time-matched control AT2 cells (Figure 2A). Likewise, the Na,K-ATPase ₁-subunit protein abundance in BLM was also significantly increased after incubation with aldosterone at 12 and 24 h as compared with control time-matched AT2 cells (Figure 2B). The increase in the ₁-subunit protein abundance in the BLM of aldosterone treated AT2 cells was probably due to a recruitment/translocation of ₁-subunits from intracellular pools, as there was no increase in transcription (Figure 1A) nor translation as depicted in Figure 2C. Treatment with aldosterone did increase the ₁-subunit abundance in both whole membrane and BLM isolated from AT2 cells as depicted in a representative autoradiogram in Figure 2C.

View larger version (20K): [in this window] [in a new window]   View larger version (33K): [in this window] [in a new window]   Figure 2.   Effects of aldosterone on the Na,K-ATPase 1- (A) and 1- (B) subunit protein in basolateral membranes of AT2 cells. Densitometric data are from Western blot analysis. Each point (n = 6, from two separate cell isolations done in triplicate) represents the mean ± SEM. * indicates difference (p < 0.05) as compared with time-matched controls. A value of 1 was arbitrarily assigned to the control protein for each time point, and the protein levels of cells treated with aldosterone were normalized to this value. A representative Western blot (C) of whole cell homogenates (WM) and basolateral membranes (BLM) prepared from AT2 cells treated for 12 h in the absence or presence of 300 nM aldosterone is shown. Aldosterone increased the 1-subunit protein expression in BLM prepared from aldosterone-treated AT2 cells, but not in membranes prepared from whole cell homogenates. Aldosterone increased the 1-subunit protein expression in membranes prepared from whole cell homogenates and BLM prepared from aldosterone-treated AT2 cells (45 µg protein loaded per lane for WM; 12.5 µg protein loaded per lane for BLM).

Effect of Aldosterone on Na,K-ATPase Hydrolytic Activity and ⁸⁶Rb⁺ Uptake

As shown in Figure 3A, the Na,K-ATPase hydrolytic activity, measured under Vmax conditions, increased as much as 4-fold in AT2 cells by 12 and 24 h after incubation with aldosterone. Figure 3B depicts that the ouabain-sensitive ⁸⁶Rb⁺ uptake in AT2 cells increased 3.8-fold when incubated for 12 h with aldosterone.

View larger version (18K): [in this window] [in a new window]   Figure 3.   (A) Effects of 300 nM aldosterone on Na,K-ATPase hydrolytic activity of AT2 cell homogenates. Each point represents the mean ± SEM (n = 6 from three separate cell isolations). * indicates significant difference (p < 0.05) as compared with the time-matched control. (B) Effects of 300 nM aldosterone on 86Rb+ uptake of cultured AT2 cells. At the indicated time points 86Rb+ uptake was determined in the absence and presence of ouabain. Each point (n = 6) represent the mean ± SEM. * indicates significant difference (p < 0.05) compared with time-paired control.

Physiologic Studies

In rats exposed to aerosols containing ⁹⁹Tc-sulphur colloidal particles, we observed that 1.6 ± 0.35 µl of the 1-ml aerosolized solution was deposited in the lungs; ~ 24% of the amount was deposited in the alveolar space (16). Thus, in rats exposed to aerosols generated from 100 µg aldosterone dissolved in 1 ml saline, we estimated that 400 ng were deposited in the alveolar space. As the alveolar lining fluid volume, estimated by bronchoalveolar lavage, of isolated rat lungs is ~ 50 µl, aldosterone concentration in the alveolar lining fluid after aerosol exposure was ~ 8 ng/µl, assuming a uniform distribution of the aerosols in the alveolar space.

Twenty-four hours after aldosterone aerosols were delivered to live rats we found that in the isolated lungs the unidirectional Na⁺ flux (UNa⁺) increased to 64.3 ± 3.5 nM/s as compared with 45.4 ± 3 nM/s in the saline-treated rats and 45.4 ± 1.8 in the control rats (p < 0.01). In the rats exposed to aldosterone aerosols, the increased UNa⁺ flux was due in part to an increase in active Na+ transport (27 ± 0.9 nM/s) compared with saline (18 ± 0.6 nM/s) and control (18 ± 1.0 nM/s) rats. This change in active Na⁺ transport resulted in an increase in lung liquid clearance (estimated by the concentration of EBD) in rat lungs to which aerosolized aldosterone was delivered as compared with all other groups (Figure 4).

View larger version (10K): [in this window] [in a new window]   Figure 4.   Active Na+ transport increased 24 h after delivery of aldosterone aerosols. The bars represent means ± SE. * represents difference from all other groups (p < 0.01).

As shown in Figure 5, the passive Na⁺ movement calculated as the difference between the UNa⁺ flux and the active Na⁺ transport also increased in rats exposed to aldosterone aerosols from 16.0 ± 1.8% to 22.5 ± 1.8%, as compared with control and saline-treated rats (p < 0.05). The passive movement of small solutes across the alveolar epithelium assessed by ³H-mannitol flux increased somewhat to 18.3 ± 0.3% in rats exposed to aldosterone aerosols from 11.8 ± 0.3% and 12.3 ± 0.3% in control and saline-treated rats (p < 0.01).

View larger version (13K): [in this window] [in a new window]   Figure 5.   The y-axis represents the percentage of control passive Na+ (open bars) and mannitol movement (closed bars). Twenty-four hours after aldosterone aerosolization the passive movement of 22Na+ and 3H-mannitol flux were increased. The bars represent means ± SEM. * represents difference from all the other groups.

Alveolar capillary membrane integrity in the lungs was determined by albumin permeability. The appearance of FITC-albumin in the alveolar space serves as an index of capillary leak in this model. As shown in Figure 6, FITC-albumin flux was unchanged in all three experimental groups.

View larger version (9K): [in this window] [in a new window]   Figure 6.   Albumin movement from the pulmonary circulation into the alveolar space (µg/h) remained unchanged 24 h after aldosterone aerosolization. The bars represent means ± SE.

In this isolated-perfused, fluid-filled lung model, samples measured from the air space, pulmonary vascular compartment, and "pleural bath" were used to determine the mass balance for each tracer at each sampling time. We calculated that more than 95% of ²²Na, ³H-mannitol, EBD, and FITC- albumin was recovered from the three measured compartments (perfusate, air space, and pleural bath) in all the experimental groups, which indicates that only a small fraction of these tracers remained in the interstitium. The concentration of sodium was constant  136 mEq/L in all compartments and all experimental groups. The pulmonary circulation flow, measured periodically during the experiments, was 16 to 20 ml/min and was not different between the three experimental groups.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study has demonstrated that aldosterone increases the steady-state levels of Na,K-ATPase ₁-mRNA, in the absence of other hormones or growth factors, and leads to an increase in Na,K-ATPase protein abundance and activity in AT2 cells. In physiologic studies, 24 h after aldosterone aerosols were delivered to spontaneously breathing rats, Na⁺ transport and lung edema clearance increased.

Aldosterone increased Na,K-ATPase activity in AT2 cells at 12 and 24 h (Figure 3). Increases in the Na,K-ATPase activity can be mediated by changes in the activity of preexisitng Na,K-pumps, recruitment/translocation of Na,K-pumps from intracellular pools to the basolateral membrane, or by transcription/translation, resulting in an increase in the number of active Na,K-pumps on the cell plasma membrane (19, 20). The increase in steady-state levels of ₁-subunit mRNA at 3 and 6 h (Figure 1C) was associated with an increase in ₁-subunit protein abundance at 12 and 24 h (Figures 2B and 2C), in both whole cell membranes and basolateral membranes isolated from aldosterone-treated AT2 cells. In contrast, aldosterone had no effect on the steady-state levels of the Na,K-ATPase ₁-subunit mRNA. Additionally, there was no increase in ₁-subunit protein abundance in whole membranes isolated from aldosterone-treated AT2 cells. However, aldosterone did increased the ₁-subunit protein abundance in a basolateral membrane of AT2 cells Thus, we speculate that aldosterone differentially regulates the - and -subunits in order to increase Na,K-ATPase activity. Aldosterone modifies the transcription/translation of the ₁-subunit, whereas the ₁-subunit is probably recruited from intracellular pools to the basolateral membrane. Combined, these effects yield an increase in overall protein abundance in the plasma membrane and function of the Na,K-ATPase in aldosterone-treated AT2 cells.

In the basal state, the disproportional abundance of the -and -mRNAs may suggest that the mRNA stability and/or transcriptional rates may be different. This disproportional abundance has been reported in several types of cultured cells. Cultured chicken skeletal muscle cells (21) and fetal alveolar type 2 cells (22) show a differential expression of the Na,K-ATPase ₁-subunit mRNA as compared with the Na,K-ATPase ₁-subunit mRNA, whereas rat cardiocytes show a predominance of the -subunit mRNA (23). Because - dimer formation is required for functional activity, the increased ₁-subunit abundance may be rate-limiting for dimer formation in the upregulated state.

The effect of aldosterone on the Na,K-ATPase mRNA subunit abundance has been previously examined. Changes in mRNA have varied in terms of amount, subunit type, and time course. Exposure of rat cardiocytes to aldosterone induced a 3-fold increase in the Na,K-ATPase ₁-subunit mRNA within 6 h (8). In cultured kidney (A6) cells, aldosterone induced a 2.5-fold increase in the ₁-subunit mRNA, but without an increase in the ₁-subunit mRNA after a 3-h exposure (24).

In response to other stimuli, markedly disproportionate increases in the Na,K-ATPase ₁-subunit mRNA have been found to occur. In cultured liver cells, dexamethasone increased the Na,K-ATPase ₁-subunit mRNA with only a small increase in the ₁-subunit mRNA (25). In AT2 cells dexamethasone increased ₁-mRNA and protein, resulting in increased Na,K-ATPase function (5). In our study, after 3 h of exposure to aldosterone the Na,K-ATPase ₁-subunit mRNA levels were elevated 2.4-fold, whereas the ₁-subunit mRNA levels were unchanged. Thus, the disproportionately low basal level of the Na,K-ATPase ₁-subunit mRNA and its rise after aldosterone treatment suggests the ₁-subunit can be rate-limiting in AT2 cells (2, 5, 26).

Aldosterone was delivered to the alveolar epithelium by aerosolization, which provides a uniform distribution of the hormone to spontaneously breathing rats. As the nebulizer is one of the important determinants of aerosol delivery to the lungs, we used an Aerotech II nebulizer that has been proven to increase significantly pentamidine deposition in the lungs of patients with HIV infection (27).

In previous studies, the estimated volume of rat epithelial lining fluid was  50 µl as calculated by dilution of EBD-albumin in the BAL of isolated lungs (10). We delivered aerosols containing ⁹⁹Tc-sulphur colloidal particles to spontaneously breathing rats. Lung radioactivity was measured immediately after the exposure and 24 h later. After a methodology previously described (16), the amount of aldosterone that reached the lung was estimated to be 1.6 µg, whereas the amount deposited in the alveolar space was estimated to be ~ 400 ng after the aerosol exposure. Assuming a uniform distribution of the aerosols in the alveolar space, we estimated that ~ 8 ng/µl aldosterone was present in the alveolar lining fluid. In a previous study it was shown that 1 µM concentration of aldosterone was enough to elicit a stimulating response in rat cardiocytes (9). Similarly, an 0.1-µM aldosterone concentration increased active Na⁺ transport in the frog lung epithelium (28) and 300 nM aldosterone upregulated Na,K-ATPase activity in AT2 cells.

Saline aerosols induced no changes in active and passive solute movement across the alveolar epithelium as compared with controls, indicating that the aerosolization procedure was not injurious. Aldosterone increased active Na⁺ transport in the isolated lung, probably by increasing Na,K-ATPase activity as was observed in the AT2 cell experiments (Figure 3). Additionally, the time frame in the AT2 cell experiments was reproduced in the isolated lungs, suggesting that aldosterone increased active Na⁺ transport by regulating alveolar epithelial cell Na,K-ATPases. Whereas previous studies showed that aldosterone in epithelial cells increased Na⁺ channel density (28, 29), the effects of aldosterone on apical Na⁺ channels (30- 33) and other Na⁺ transporting mechanisms such as the Na⁺/H exchanger (34) and the Na/K/2Cl cotransporter (35) were not explored in this study.

We observed that aldosterone aerosols delivered to the rats increased the passive movement of small solutes across the alveolar epithelial membrane. This is consistent with previous studies using isoproterenol in the isolated-perfused rat lung model (36) and aldosterone-treated cultured monolayers of epithelial A6 cells (3). In that study aldosterone simultaneously effected both active Na⁺ transport and transepithelial membrane permeability Our results suggest that the increase in passive solute movement after aldosterone aerosols exposure may not be solely a solvent drag phenomenon induced by increased active Na⁺ transport. As shown in Figure 5, the passive movement of Na⁺ and mannitol, two different size molecules, is proportionally increased as it has been reported in lungs treated with isoproterenol (36). After aldosterone aerosolization there was no change in the albumin permeability in all experiments (Figures 5 and 6). According to previous studies the alveolar epithelial barrier contains two different size pores (38). A small pore is permeable to Na⁺ and partially permeable to mannitol, whereas a larger pore is semipermeable to albumin (39). We speculate that aldosterone induced a very small increase in the radius of the small pore as shown by a proportional increase in permeability of Na⁺ and mannitol. The lack of change in permeability for albumin allows for an accurate calculation of the rate of active Na⁺ transport and lung edema clearance.

In summary, the data demonstrate that aldosterone increased the Na,K-ATPase function in cultured AT2 cells within 12 h. This was associated with an increase in the ₁-subunit mRNA levels and ₁-subunit protein abundance in AT2 cell plasma membranes, suggesting hormonally induced regulation of the Na,K-ATPase function. Within 24 h of rats breathing aldosterone aerosols, active Na⁺ transport and lung edema clearance were increased, which was probably mediated by the upregulation of alveolar epithelial Na,K-ATPases.