Impact of beta -adrenergic agonist on Na+ channel and Na+-K+-ATPase expression in alveolar type II cells

Yoshiaki Minakata, Satoshi Suzuki, Czeslawa Grygorczyk, André Dagenais, and Yves Berthiaume

Centre de Recherche, Centre Hospitalier de l'Université de Montréal, Montreal, Quebec H2W 1T8; and Department of Medicine, Université de Montréal, Montreal, Quebec, Canada H3C 3J7

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

It has been shown that short-term (hours) treatment with beta -adrenergic agonists can stimulate lung liquid clearance via augmented Na+ transport across alveolar epithelial cells. This increase in Na+ transport with short-term beta -agonist treatment has been explained by activation of the Na+ channel or Na+-K+-ATPase by cAMP. However, because the effect of sustained stimulation (days) with beta -adrenergic agonists on the Na+ transport mechanism is unknown, we examined this question in cultured rat alveolar type II cells. Na+-K+-ATPase activity was increased in these cells by 10-4 M terbutaline in an exposure time-dependent manner over 7 days in culture. This increased activity was also associated with an elevation in transepithelial current that was inhibited by amiloride. The enzyme's activity was also augmented by continuous treatment with dibutyryl-cAMP (DBcAMP) for 5 days. This increase in Na+-K+-ATPase activity by 10-4 M terbutaline was associated with an increased expression of alpha 1-Na+-K+-ATPase mRNA and protein. beta -Adrenergic agonist treatment also enhanced the expression of the alpha -subunit of the epithelial Na+ channel (ENaC). These increases in gene expression were inhibited by propranolol. Amiloride also suppressed this long-term effect of terbutaline and DBcAMP on Na+-K+-ATPase activity. In conclusion, beta -adrenergic agonists enhance the gene expression of Na+-K+-ATPase, which results in an increased quantity and activity of the enzyme. This heightened expression is also associated with augmented ENaC expression. Although the cAMP system is involved, the inhibition of enhanced enzyme activity with amiloride suggests that increased Na+ entry at the apical surface plays a role in this process.

adenosinetriphosphatase; epithelial sodium channel; pulmonary edema; gene expression; sodium pump; terbutaline; alveolar epithelium

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE ALVEOLAR EPITHELIUM is considered to be not only an important barrier to alveolar flooding but also the most likely site of fluid reabsorption after pulmonary edema (29). This vectorial fluid transport is dependent in part on active Na+ transport across the alveolar epithelium (29). Results obtained from alveolar epithelial cells in culture suggest that Na+ enters these cells by an apical Na+ channel (27) and is actively pumped out of the cells by basolateral Na+-K+-ATPase (31). This active Na+ transport can be modulated by beta -adrenergic agonists (6, 23, 40), probably via the cAMP second messenger system, since cAMP analogs increase lung liquid clearance in vivo (4, 22, 40) as well as Na+ transport in alveolar type II cells (24).

The exact mechanism of activation of Na+ transport by beta -adrenergic agonists is not fully understood. A paradigm can be developed based on data available in the literature. We know that acute exposure of alveolar type II cells to beta -adrenergic agonists augments short-circuit current over a period of 20-30 min (16). This rise in short-circuit current is secondary to heightened transepithelial Na+ transport (24), which is probably caused by activation of the Na+ channel on the apical surface of cells by protein kinase A (PKA; see Ref. 50). The increased activity of the Na+ channel could lead to an elevation in intracellular Na+ concentration, which could in turn stimulate Na+-K+-ATPase (38). Activation of Na+-K+-ATPase could also be secondary to direct or indirect stimulation of the enzyme by cAMP (41). This model of enhanced transcellular Na+ transport could explain the increased Na+ transport and lung liquid clearance seen in animals (5, 6, 40) and humans (39) after acute (1-4 h) exposure to beta -adrenergic agonists.

However, it has been shown recently that septic animals presenting enhanced lung liquid clearance also manifest heightened secretion of endogenous catecholamines (36). In this model, we can expect the alveolar epithelium to be exposed to an elevated circulating level of catecholamines for a prolonged period of time. The impact of chronic stimulation with beta -adrenergic agonists on the Na+ transport mechanism in alveolar epithelial cells is not known.

The purpose of this work was to study the effect of sustained exposure (days) of alveolar type II cells to a beta -adrenergic agonist on the Na+ transport mechanism and its modulation. After determining the impact of long-term beta -adrenergic agonist treatment on Na+-K+-ATPase activity and transepithelial current, we tried to dissect the response by evaluating the role of cAMP and establishing if it is associated with enhanced expression of Na+-K+-ATPase and the epithelial Na+ channel (ENaC). The possible involvement of Na+ entry in this response was then ascertained by studying the influence of amiloride.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Experimental Protocols

Effect of long-term exposure to terbutaline on Na+-K+-ATPase activity and transepithelial current. To determine whether long-term treatment with beta -adrenergic agonists modulates Na+-K+-ATPase activity and transepithelial current, we used three different experimental protocols. In the first experimental protocol, alveolar type II cells were exposed continuously to terbutaline (10-4 M; Sigma, St. Louis, MO) for 7 days starting from the day of cell isolation. The medium containing terbutaline was changed every 2 days. Control cells were treated in an identical fashion except that no terbutaline was present in the media. Na+-K+-ATPase activity was measured by radiometric phosphate release assay after day 0 (day of cell isolation; control: n = 9; 60 min exposure to terbutaline: n = 5), 2 days (control and terbutaline: n = 6), 5 days (control: n = 7; terbutaline: n = 6), and 7 days (control and terbutaline: n = 6) of treatment.

In the second protocol, we determined the minimal treatment period needed to stimulate Na+-K+-ATPase on day 5 in culture. The cells were treated continuously for 1 day (n = 4), 2 days (n = 4), or 5 days (n = 6) from the time of isolation. At the end of treatment, terbutaline was removed from the incubation media. Na+-K+-ATPase activity was measured for all of these treatment periods on day 5 in culture.

Finally, in the third experimental protocol, cell monolayers were maintained in culture in the presence of culture media (control, n = 24), culture media with 10 -4 M terbutaline (n = 18), 10-4 M amiloride (n = 13), or 10-4 M amiloride and 10-4 M terbutaline (n = 8). The cells were grown continuously in the appropriate experimental media from the time of cell isolation for either 4 or 6 days. At the end of the treatment period, we determined transepithelial current for monolayers with transepithelial resistance (Rte) >800 Omega  (control Rte = 1,420 ± 418 Omega , terbutaline Rte = 1,231 ± 417 Omega , amiloride Rte = 1,650 ± 528 Omega , amiloride + terbutaline Rte = 1,714 ± 420 Omega ). Because there was no significant difference between the data (transepithelial current) obtained on day 4 and day 6, the data were analyzed together.

Modulation of Na+-K+-ATPase activity by cAMP. To evaluate the role of the intracellular cAMP system in long-term enhancement of Na+-K+-ATPase activity by terbutaline, cultured cell monolayers were exposed continuously to 10-3 M dibutyryl-cAMP (DBcAMP; Sigma) for 5 days starting from the time of cell isolation. Na+-K+-ATPase activity was measured (n = 5) after 5 days of continuous treatment. To ascertain whether the long-term effect of terbutaline on Na+-K+-ATPase involves activation of cAMP-dependent PKA, we measured PKA activity in alveolar type II cells exposed to 10-4 M terbutaline. Treatment with terbutaline started at the end of the cell isolation period and lasted for 10 min (n = 4), 60 min (n = 6), or 24 h (n = 7).

Effect of long-term exposure to terbutaline on Na+ transporter mRNA and/or protein expression. To evaluate if long-term beta -adrenergic agonist treatment is associated with changes in expression of the Na+ channel and Na+-K+-ATPase, alveolar type II cells were exposed continuously to 10-4 M terbutaline for 6 days from the cell isolation period. The medium containing terbutaline was changed every 2 days. On day 2 (n = 8), day 4 (n = 8), and day 6 (n = 8) in culture, we determined mRNA expression of the alpha 1-subunit of Na+-K+-ATPase and of the alpha 1-subunit of ENaC. We also established if the changes in expression observed were related to beta -adrenergic agonist stimulation by continuously exposing cell monolayers to either 10-5 M propranolol alone (n = 8) or 10-5 M propranolol plus 10-4 M terbutaline (n = 8) from the cell isolation period for up to 2 days in culture. The mRNA expression of Na+-K+-ATPase and ENaC was determined by Northern blotting at the end of this continuous 2 days of treatment.

We also measured protein expression of the alpha 1-subunit of Na+-K+-ATPase by Western blotting on cell monolayers treated continuously with terbutaline from the cell isolation period for up to 7 days (day 2, n = 5; day 5, n = 6; day 7, n = 2).

Modulation by amiloride of increased Na+-K+-ATPase activity induced by beta -adrenergic stimulation. To determine whether the long-term effect of terbutaline or DBcAMP on Na+-K+-ATPase activity was secondary to increased Na+ uptake, cell monolayers were treated continuously with either 10-4 M amiloride alone, 10-4 M amiloride plus 10-4 M terbutaline, or 10-4 M amiloride and 10-3 M DBcAMP continuously for 5 days from the cell isolation period. At the end of the 5-day treatment period, Na+-K+-ATPase activity was measured by phosphate release assay.

Methods

Cell isolation. Alveolar epithelial type II cells were isolated from male Sprague-Dawley rats weighing 175-200 g by enzymatic tissue digestion with elastase (Worthington Biochemical, Freehold, NJ) and purified by a differential adherence technique in rat IgG-coated bacteriological plastic plates (21). The cells were suspended in minimum essential medium (GIBCO, Burlington, Ontario, Canada) containing 10% fetal bovine serum (GIBCO), 0.08 mg/l gentamicin, 0.2% NaHCO3, 0.01 M HEPES, and 2 mM L-glutamine and then plated at a density of 4 × 105 cells/cm2 in plastic cell culture flasks (25 cm2) kept in a humidified 5% CO2-air incubator at 37°C.

Na+-K+-ATPase activity. Na+-K+-ATPase activity in alveolar type II cells was assessed as ouabain-inhibitable ATPase hydrolysis under maximal velocity conditions by radiometric monitoring as described previously (41). Exposure to the agents tested was terminated on day 2, day 5, and day 7 in culture by washing the cell monolayers with ice-cold phosphate-buffered saline. The cells were scraped to obtain a cell pellet and centrifuged at 3,500 rpm at 4°C for 15 min. The cell pellet was resuspended in 250 µl of ice-cold homogenate solution (250 mM sucrose, 10 mM Tris, and 1 mM EGTA, pH 7.4) and then treated with a 15-s burst of sonication at 60 W on ice using an ultrasound cell processor (Broun-Sonic, Allentown, PA). Whole cell homogenates containing 100-200 µg of protein were preincubated in a final volume of 1 ml of assay buffer (100 mM NaCl, 10 mM KCl, 5 mM MgCl2, 4 mM ATP, 1 mM EGTA, 5 mM sodium azide, and 50 mM Tris, pH 7.4) in the presence or absence of 1 mM ouabain for 30 min at 37°C. ATPase hydrolysis was then started by adding ATP solution containing 5-10 µCi of [gamma -32P]ATP (ICN Biochemicals, Montréal, Québec, Canada) as a tracer and terminated every 3 min by placing aliquots of the assay mixture into an ice-cold stop solution (1 M perchloric acid and 0.35 M NaH2PO4). The release of Pi was detected as Cerencov radiation after extraction of Pi by activated charcoal absorption. Na+-K+-ATPase activity was calculated as the difference between the slopes of regression lines adapted to Pi release in the presence or absence of ouabain. Data were standardized to cellular protein content using the method described by Bradford (10).

Northern blotting. mRNA expression of the alpha 1-subunit of Na+-K+-ATPase and of the alpha -subunit of ENaC was analyzed by Northern blotting. On day 2, day 4, and day 6 in culture, total cellular RNA was extracted by the acidic guanidium thiocyanate-phenol-chloroform extraction technique (17). The purity of the extracted RNA was assessed by the 260- to 280-nm absorbance ratio. Ten micrograms of total cellular RNA samples, quantified by 260-nm absorbance, were fractionated in formaldehyde-agarose gel (1%) and blotted on nylon membranes (Amersham, Oakville, Ontario, Canada) by vacuum or capillary transfer. The blotted membrane was hybridized with rat alpha 1-Na+-K+-ATPase cDNA fragment (Nar I to Stu I, 332 bp; a gift from Dr. J. Orlowski, McGill University, Montréal, Québec, Canada; see Ref. 34) or the mouse alpha -subunit of a Na+ channel probe, which was generated by polymerase chain reaction and consisted of a 764-bp fragment coding from His-445 to the stop codon (19). The probes were labeled with [32P]dCTP (GIBCO). After hybrid-ization, the membranes were washed successively for 30 min with 100 mM sodium phosphate (pH 7.2)-0.1% (wt/vol) sodium dodecyl sulfate (SDS); 40 mM sodium phosphate (pH 7.2)-0.1% (wt/vol) SDS; and 40 mM sodium phosphate (pH 7.2)-1% (wt/vol) SDS. The blots were then analyzed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) by comparing the reaction product with untreated cells isolated from the same animal. The data were normalized to 18S ribosomal RNA. All values of mRNA expression presented in Figs. 1-8 represent the ratio of normalized (to 18S) gene expression of treated cells to normalized gene expression of control cells kept in culture for the same period of time.

Western blotting. The protein expression of Na+-K+-ATPase in alveolar type II cells was analyzed by detecting the alpha 1-subunit on Western blots, since this isoform has been shown to be predominant in these cells (41). Cellular protein was solubilized with a detergent solution (0.5% Triton X-100, 250 mM sucrose, 10 mM Tris, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 25 µg/ml leupeptin, pH 7.4). Twenty micrograms of cellular protein samples were diluted in sample buffer (5% SDS, 10% glycerol, 1% 2-mercaptoethanol, 65 mM Tris, and 0.01% bromphenol blue, pH 6.8) at room temperature for 30 min, separated in polyacrylamide gel containing 10% SDS, and then transferred electrophoretically to a nitrocellulose membrane. After overnight blocking with 5% milk in 137 mM NaCl, 20 mM Tris, and 0.05% Tween 20, pH 7.6, the blotted membrane was reacted with 1:50 diluted McK1 (a mouse IgG monoclonal antibody directed against the alpha 1-subunit; a gift from Dr. K. Sweadner, Boston General Hospital, Boston, MA; see Ref. 42) and then incubated with 1:2,000 diluted anti-mouse-IgG-horseradish peroxidase conjugate (Amersham) for exposure to X-ray film using an enhanced chemiluminescence detection system (Amersham). Densitometric analyses were performed with a PhosphorImager (Molecular Dynamics) by comparing the reaction product with untreated cells isolated from the same animal.

PKA activity. PKA activity in alveolar type II cells was measured by detecting the phosphorylation of a peptide substrate for PKA (kemptide) using the PKA Assay System (GIBCO). The cells were scraped and homogenized with extraction buffer (5 mM EDTA and 50 mM Tris, pH 7.5). Ten microliters of cell homogenates were preincubated in the presence or absence of either PKA activator (40 µM cAMP and 50 mM Tris, pH 7.5) or PKA inhibitor (4 µM protein kinase A inhibitor and 50 mM Tris, pH 7.5) at room temperature for 15 min. Kemptide phosphorylation was started by adding 10 µl of PKA substrate solution (200 µM kemptide, 400 µM ATP, 40 mM MgCl2, 1 mg/ml BSA, and 50 mM Tris, pH 7.5) containing 0.2-0.5 µCi of [gamma -32P]ATP (Amersham) as tracer and terminated after 5 min by spotting aliquots of the assay mixture on disc paper. Kemptide-incorporated 32P was detected with a Liquid Scintillation Analyzer (Packard, Downers Grove, IL). The percent PKA activity was calculated according to Clegg and Ottey's (18) method. The data are presented as a ratio (%) of activated PKA (percentages of activated PKA = activated PKA/total PKA activity). The total PKA activity present in the sample was determined by incubating the cell lysate with 40 µM of cAMP. The data were standardized by cellular protein content measured according to Bradford's (10) method.

Electrophysiology. In the present study, alveolar epithelial type II cells were grown to confluence on permeable filters (Costar Transwell, Toronto, Ontario, Canada; 1.0 cm2). The cells were plated at a density of 1.5-2.0 × 106/cm2. The medium was changed on the second day after isolation. Monolayers of cells on filters were maintained in a humidified 5% CO2 incubator at 37°C for 4-6 days. Rte (Omega /cm2) and potential difference (PD) across monolayers (mV) were measured with an EVOM epithelial voltmeter (World Precision Instrument, Sarasota, FL). Transepithelial current (I; µA/cm2) was calculated from the relationship I = PD/Rte.

Statistics. Data are presented as means ± SE. Comparisons between groups were analyzed by paired t-test, unpaired t-test, or ANOVA and post hoc comparison (Fisher protected least significant difference) depending on the experimental design. We accepted P < 0.05 as indicating statistically significant differences.

    RESULTS
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Abstract
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Materials & Methods
Results
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References

Chronic stimulation with the beta -adrenergic agonist did not influence monolayer formation. Alveolar type II cells in both the presence and absence of 10-4 M terbutaline spread to form confluent cell monolayers by day 3 in culture. There were no significant differences in cellular protein content on day 2 and day 5 of culture for cells treated with terbutaline compared with nontreated cell monolayers.

Na+-K+-ATPase activity in cultured rat alveolar type II cells was augmented by continuous exposure to the beta -adrenergic agonist. It increased in an exposure time-dependent manner over 7 days in culture, when the cells were kept in the presence of 10-4 M terbutaline (Fig. 1). This increase in Na+-K+-ATPase activity was also paralleled by a rise in transepithelial current measured between day 4 and day 6 of continuous treatment with terbutaline (Fig. 2). At least 48 h of treatment with the beta -adrenergic agonist were necessary to stimulate Na+-K+-ATPase activity at day 5 in culture (Fig. 3).


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Fig. 1.   Effect of sustained treatment with terbutaline on alpha 1-Na+-K+-ATPase activity. alpha 1-Na+-K+-ATPase activity was increased by terbutaline (10-4 M) in an exposure time-dependent manner. Cont, control; terb, terbutaline (10-4 M). * P < 0.05 vs. day 0 by ANOVA and post hoc comparison.


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Fig. 2.   Effect of sustained treatment with terbutaline, amiloride (Amil), or terbutaline and amiloride on transepithelial current. Cells were exposed continuously from the time of cell isolation for either 4 or 6 days to the appropriate experimental media. Because there was no difference between the response seen at 4 or 6 days, the results were analyzed together. There was a significant increase in transepithelial current for cells exposed continuously to terbutaline for 4-6 days. This effect of terbutaline was inhibited by continuous coincubation of amiloride with terbutaline. * P < 0.05 vs. control; dagger  P < 0.05 vs. terbutaline.


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Fig. 3.   Effect of the duration of terbutaline treatment on Na+-K+-ATPase activity. Two days of terbutaline treatment (10-4 M) were sufficient to increase Na+-K+-ATPase activity on day 5 in culture. One day of treatment was not enough to augment the enzyme's activity. * P < 0.01 vs. control by the unpaired t-test.

To examine the possible role of the cAMP system in this response, we first determined if a permeable cAMP analog could also modulate Na+-K+-ATPase activity. After 5 days of continuous treatment with DBcAMP (10-3 M), Na+-K+-ATPase activity rose to 2.5 times the control value (Table 1). Furthermore, the percentage of activated PKA increased to two times the control value when the cells were treated continuously with 10-4 M terbutaline for 24 h after cell isolation (Fig. 4). This rise in PKA activity was not seen after 10 or 60 min of terbutaline treatment of freshly isolated alveolar type II cells.

                              
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Table 1.   Effect of amiloride on alpha 1-Na+-K+-ATPase activity augmented by terbutaline or DBcAMP


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Fig. 4.   Effect of terbutaline on protein kinase A (PKA) activity. %PKA activity was enhanced to 2-fold the control value by terbutaline treatment (10-4 M) for 24 h in culture. The enzyme's activity was not increased at 10 or 60 min. * P < 0.02 vs. control by the unpaired t-test.

We then determined if this sustained treatment with terbutaline is associated with increased expression of the two systems involved in Na+ transport in epithelial cells, the alpha -subunit of ENaC and the alpha 1-subunit of Na+-K+-ATPase. alpha -ENaC mRNA expression in cultured rat alveolar type II cells was increased by continuous exposure to terbutaline (10-4 M; Figs. 5 and 6A). Terbutaline treatment also elevated the expression of the alpha 1-subunit of Na+-K+-ATPase mRNA (Figs. 5 and 6B). The increased expression of alpha -ENaC and alpha 1-Na+-K+-ATPase was inhibited by continuous cotreatment with terbutaline and propranolol for 2 days, starting on the day of cell isolation (Figs. 5 and 7).


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Fig. 5.   Representative blot of the effect of terbutaline and/or propranolol (prop) on the expression of alpha -rat (r) epithelial Na+ channel (ENaC) or alpha 1-Na+-K+-ATPase. After 2 days of treatment, an increase in the expression of alpha -ENaC and alpha 1-Na+-K+-ATPase was inhibited by propranolol.


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Fig. 6.   Effect of terbutaline on alpha -rENaC (A) and Na+-K+-ATPase (B) mRNA expression. alpha -rENaC mRNA expression was enhanced by chronic terbutaline treatment (10-4 M) on day 2 in culture compared with day 6. alpha 1-Na+-K+-ATPase mRNA expression was also increased by terbutaline (10-4 M) on days 2 and 4 in culture. * P < 0.05, different from values on day 6 by ANOVA and post hoc comparison.


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Fig. 7.   Effect of propranolol on alpha -rENaC (A) and alpha 1-Na+-K+-ATPase (B) mRNA expression. Enhancement of both mRNA expressions by terbutaline (10-4 M) was inhibited by cotreatment with propranolol (10-5 M). * P < 0.05 vs. terbutaline by ANOVA and post hoc comparison.

This augmented activity and gene expression of alpha 1-Na+-K+-ATPase were also associated with heightened expression of alpha 1-Na+-K+-ATPase protein. The alpha 1-subunit of Na+-K+-ATPase detected at 96 kDa increased in a time-dependent manner over 7 days in culture when the cells were exposed continuously to 10-4 M terbutaline (Fig. 8).


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Fig. 8.   Effect of terbutaline on Na+-K+-ATPase protein. A: representative blot of Na+-K+-ATPase protein increased with terbutaline treatment. B: summary data also demonstrate that alpha 1-Na+-K+-ATPase protein was increased by chronic treatment with terbutaline (10-4 M) in an exposure time-dependent manner. * P < 0.05 vs. day 2.

To determine the possible role of Na+ entry in modulation of this response, we studied the effect of amiloride. When amiloride was added to the medium with terbutaline or DBcAMP, the rise in Na+-K+-ATPase activity seen after 5 days of continuous treatment was inhibited (Table 1). There was also significant inhibition of terbutaline-increased transepithelial current with amiloride (Fig. 2).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

This study provides novel evidence that sustained beta -adrenergic agonist stimulation can lead to enhanced Na+-K+-ATPase activity associated with increased expression of Na+-K+-ATPase and of the Na+ channel in alveolar epithelial cells.

We first tried to determine the effect of sustained stimulation with terbutaline on Na+-K+-ATPase activity. The continuous presence of terbutaline for up to 7 days produces a gradual increase in Na+-K+-ATPase activity (Fig. 1) and transepithelial current (Fig. 2). This response is mediated by cAMP, since it could be reproduced by long-term exposure to the membrane-permeable cAMP analog DBcAMP (Table 1). The role of the cAMP system is also suggested by the augmented activity of PKA, which we recorded 24 h after initiating treatment with the beta -adrenergic agonist (Fig. 4).

We thought it was important to evaluate the effect of long-term exposure to the beta -adrenergic agonist on the Na+ transport mechanism, even if short-term modulation of the Na+ channel (50) and Na+-K+-ATPase (41) is relatively well established, because long-term treatment could elicit not only changes in activity but also in the quantity of these molecules. This is especially possible since the beta -adrenergic agonist has been shown to modify the expression of other genes (26, 45). We then determined if the increased activity of Na+-K+-ATPase is associated with heightened gene expression of systems involved in transepithelial Na+ transport. We demonstrated that sustained stimulation with the beta -adrenergic agonist augmented the gene expression of ENaC, the Na+ channel that is responsible for apical entry of Na+ into the cell (Figs. 5 and 6). Expression of the Na+-K+-ATPase gene, the mechanism for Na+ extrusion on the basolateral side of the cell, was also enhanced by long-term beta -adrenergic stimulation (Figs. 5 and 6). This response was mediated by beta -receptors since it was inhibited by propranolol (Fig. 7).

Because the increase in gene expression was only transient, we also wanted to determine if it was associated with a greater level of expression of the protein. We only measured changes in alpha 1-Na+-K+-ATPase protein since we could not identify a functional ENaC antibody to work in Western blotting of proteins obtained from cell lysates. We used a monoclonal antibody against the alpha 1-subunit of Na+-K+-ATPase that is actively involved in the transport mechanism (38, 42). The increased expression of alpha 1-Na+-K+-ATPase protein (Fig. 8) suggests that the augmented activity measured on days 5 and 7 is partially related to an elevation in alpha 1-Na+-K+-ATPase protein. However, the sustained increment of protein expression (Fig. 8) in the presence of transient changes in mRNA (Fig. 6) probably indicates that other cellular mechanisms besides the rise in gene transcription are involved in this response. This increased expression of Na+-K+-ATPase protein probably also explains the persistent elevation in Na+-K+-ATPase activity on day 5, even when terbutaline treatment was stopped on day 2 (Fig. 3). It would have been interesting to evaluate if there was also an increase in alpha -ENaC protein. However, technical difficulties prevented us from performing this experiment. Other laboratories have been able to measure changes in expression of a Na+ channel protein in alveolar type II cells of hyperoxic rats (49). The antibodies used by these investigators were not directed against purified ENaC but were raised against an Na+ channel protein purified from the bovine kidney papilla. This polyclonal antibody mainly recognizes a protein of 135 kDa in membrane vesicles of alveolar type II cells (28). However, because it recognizes a protein that is of a different molecular weight than alpha -ENaC (11), it could not be used to determine if changes in alpha -ENaC mRNA are associated with an increase in alpha -ENaC protein. Furthermore, we did not determine if stimulation by the beta -adrenergic agonist was associated with an increase of other ENaC subunits (beta  and gamma ) or with an elevation in the beta -subunit of Na+-K+-ATPase. We decided to focus on the alpha -subunits of ENaC and Na+-K+-ATPase because they represent the most important subunits for the activity of these proteins (12, 42).

Because the increase in mRNA of ENaC and Na+-K+-ATPase as well as the changes in Na+-K+-ATPase protein were relatively modest, their physiological significance needed to be determined. To address this issue, we measured transepithelial current across monolayers treated continuously for 4-6 days with terbutaline. These treated cells showed a significant increase in transepithelial current that could be inhibited by the continuous presence of amiloride. Our results suggest that this modest increase in genes and protein expression had a significant physiological impact on the monolayer. However, it is also interesting to note that changes in transepithelial current, Na+-K+-ATPase activity, and protein level were persistent 5-6 days after the initiation of treatment (Figs. 1, 2, and 8) at a time when the mRNA levels of ENaC and Na+-K+-ATPase had returned to the value of nontreated cells (Fig. 6). These results indicate that modulation by cAMP is not limited to a transcriptional effect but could also involve some posttranslational modifications such as changes in protein synthesis or the protein degradation rate. Hence, although the data suggest that chronic stimulation with the beta -adrenergic agonist leads to enhanced transport capacity of the epithelium, further studies are needed to establish the mechanism involved in this process.

Our results are quite novel, since no previous publications have reported a role of beta -adrenergic agonists in modulating the gene expression of proteins involved in Na+ transport. However, chronic stimulation with beta -adrenergic agonists has been shown to enhance the expression of other genes. Chronic exposure to beta 1- or beta 2-adrenergic agonists stimulates the expression of the beta 3-receptor gene as well as protein expression (45). beta -Adrenergic stimulation also upregulates the activity and protein expression of phosphodiesterases (26). Furthermore, beta -adrenergic stimulation has been demonstrated to enhance myocardial actin gene expression (7). Thus, although beta -adrenergic agonists have not been reported to modulate the expression of Na+ transport protein in the lung, they alter the gene expression of other proteins by different mechanisms (7, 26, 45).

Because beta -adrenergic agonists have been shown to enhance the expression of different genes (7, 26, 45), it is possible that what we observed was a nonspecific effect of terbutaline. Although this question was not specifically addressed in these experiments, more recent preliminary work done by our group has revealed that, unlike the heart muscle, continuous treatment with cAMP does not increase the expression of beta -actin in alveolar type II cells (20). However, it is possible that continuous exposure to the beta -adrenergic agonist leads to the enhanced expression of other genes in alveolar type II cells. This would not be surprising, since continuous treatment of type II cells with the beta -adrenergic agonist is probably similar to the stimulation that could be observed in a stress situation. In fact, chronic elevation of catecholamines probably triggers the expression of multiple genes. Hence, the increased expression of Na+ transport protein probably represents only one system that responds to this stimulus.

To investigate the mechanism that could be involved in this response, we decided to evaluate first the potential role of Na+ entry. It has been demonstrated that beta -adrenergic treatment leads to activation of the Na+ channel (50). This would result in increased Na+ uptake (24) and would elevate intracellular Na+ concentration, which would stimulate Na+-K+-ATPase (38). Thus inhibition of Na+ entry by amiloride could then suppress the rise in Na+-K+-ATPase activity measured on day 5. Amiloride did indeed inhibit the increased activity of the enzyme (Table 1). Because a significant portion of the elevation in activity seen on day 5 is secondary to increased Na+-K+-ATPase protein expression, this suppression by amiloride indicates that Na+ itself could be an important modulator of Na+-K+-ATPase gene expression. Although it has been known for many years that chronic elevation of intracellular Na+ leads to an increased number of pumps (8), recent data obtained by Yamamoto et al. (47) suggest that this increase in the number of Na+-K+-ATPase could be related to stimulation of gene expression by a Na+-responsive element present in the Na+-K+-ATPase promoter. Hence it is possible that long-term treatment with beta -adrenergic agonists increases Na+ entry into cells, which could result in higher intracellular Na+ concentration and stimulate Na+-K+-ATPase gene expression and activity. This hypothesis is, in fact, supported by the study of Rokaw et al. (37), who showed that chronic stimulation of Na+ entry leads to elevated Na+-K+-ATPase activity that persists after stimulation is stopped. The increased activity they observed was also associated with a greater quantity of Na+-K+-ATPase on the cell surface. Thus these results confirm that modulation of Na+ entry in cells can cause changes in Na+-K+-ATPase expression. However, we have to be careful when interpreting experiments with amiloride. It has been suggested in the past that amiloride could directly inhibit DNA and protein synthesis (25, 46). This conclusion was reached in experiments performed on peripheral mononuclear cells (46) and 3T3 cells (25). However, the authors did not consider the possibility that changes in intracellular ion concentration could have led to their observations. This is particularly important in mononuclear cells, since the presence of ENaC has been demonstrated (1). Hence, it is possible that, in this cellular model, amiloride inhibits Na+ entry into cells and that its effect is not nonspecific but related to the suppression of Na+ entry. Recent work suggests that the effect of amiloride is probably not one of nonspecific toxicity. Yamamoto et al. (47), in experiments where they induced increased Na+-K+-ATPase expression with ouabain, were unable to demonstrate inhibition of this response with amiloride. If amiloride had a nonspecific toxic effect, its presence should have inhibited the increase in gene expression seen with ouabain. The study by Rokaw et al. (37) also supports the concept that the effect of amiloride is not toxic. They reported that chronic treatment of A6 cells with amiloride leads to a decrease in short-circuit current and a reduced number of Na+-K+-ATPase pumps on the cell surface. A similar response was observed when apical Na+ was replaced by tetramethylammonium, a nonpermeable cation. Interestingly, when amiloride was washed out, the authors noted a gradual recovery of short-circuit current over 100 min. Again, such a recovery would not be possible if amiloride had a toxic action. Thus, although we cannot exclude the possibility that the effect of amiloride may be related to nonspecific toxicity on protein synthesis, it is quite unlikely. In this series of experiments, we have, however, not determined if amiloride had an impact on alpha -ENaC expression. Further studies are needed to establish if there is also a modulation of ENaC expression by Na+.

However, the effect of the beta -adrenergic agonist could also be explained by other mechanisms. One of these possible mechanisms is that the increased activity of PKA, measured after 24 h of stimulation, leads to phosphorylation of cAMP-responsive element binding protein (9). This protein could then bind to the cAMP-responsive element (CRE) site on the gene promoter, which then causes activation of the gene transcript (9). Such a mechanism is possible for the Na+-K+-ATPase pump since the presence of a CRE has been demonstrated in the promoter region (2). Although we do not know if there is a CRE in the promoter of alpha -ENaC, a putative CRE has been found in the 5'-flanking region of gamma -ENaC (44). Other cAMP-independent pathways could also be involved in this response. For example, in the heart, chronic stimulation with beta -adrenergic agonists leads to an increase in actin mRNA that is mediated by G protein-coupled calcium channel activation (7). Although we would like to propose that the observed effect of the beta -adrenergic agonist is mediated by changes in Na+ entry into cells, we cannot exclude the possibility that other regulatory mechanisms, such as CRE and calcium, are involved in this process.

Although these results suggest that continuous stimulation with beta -adrenergic agonists could increase Na+-K+-ATPase activity, it is well known that this causes desensitization and downregulation of beta -receptor function (3). We therefore wanted to determine the minimal period of exposure to a beta - adrenergic agonist that would be necessary to stimulate Na+-K+-ATPase activity. Alveolar type II cells were exposed to terbutaline for 1, 2, or 5 days, and Na+-K+-ATPase activity was measured on day 5 in culture. Two days of treatment with the beta -adrenergic agonist were sufficient to increase Na+-K+-ATPase activity at day 5 in culture (Fig. 3). This observation coupled with results demonstrating that PKA activity is augmented at 24 h suggests that the response to the beta -adrenergic agonist occurs between days 1 and 2. The data indicate that a period of time is necessary for beta -adrenergic receptors to regain their function, since we could not demonstrate PKA activation at 10 and 60 min after cell isolation. The response observed is probably not due to continuous receptor stimulation for 5 days but to transient stimulation between days 1 and 2. Although beta -receptors were probably desensitized in our system, their transient stimulation may have occurred before desensitization.

Overall, the data presented in this paper as well as the data that we have published previously (41) suggest that there are at least two different mechanisms by which beta -adrenergic agonists can modulate lung liquid clearance. When cells or lung tissues are exposed for a short period of time to beta -adrenergic agonists (1-4 h), there is an acute stimulation of the Na+ channel (50) and of Na+-K+-ATPase (41) that is related to an increase in intracellular cAMP concentration. These modifications are not, however, associated with any changes in the expression of the two proteins (41, 50). This mechanism probably explains the results from experiments in which lung liquid clearance was stimulated in animals exposed to terbutaline for a few hours (6, 40). When cells or lung tissues are exposed for a more prolonged period of time (days) to beta -adrenergic agonists, not only is the activity of the Na+ channel or Na+-K+-ATPase augmented but the expression of these genes is also increased. Furthermore, it would seem that intracellular Na+ concentration could play a modulatory role in this response. These results could possibly explain the enhanced Na+ transport and liquid clearance (30, 32, 36, 48) as well as the augmented Na+ transport protein expression (13, 33, 49) seen in hyperoxic or thiourea- or sepsis-induced lung injury. This paradigm is interesting, but it is important to remember that, in these models of lung injury, the stress response could lead to changes in factors other than catecholamines. These other factors could also be involved in the regulation of expression of these genes. For example, hyperoxia itself is considered to be a potential stimulant of Na+-K+-ATPase expression (14). Corticosteroids, which could be released in response to stress, have been shown to be important stimulants of Na+ channel expression (15, 43) as well as Na+-K+-ATPase in certain tissues (35) but not in the fetal lung (43). All of these data suggest that modulation of Na+ transport in vivo probably depends on multiple mechanisms and that a better understanding of these mechanisms could lead to new treatment strategies for pulmonary edema.

In summary, the present study demonstrates that sustained treatment of cultured rat alveolar type II cells with a beta -adrenergic agonist enhances the gene expression of Na+-K+-ATPase, which results in an increase in the quantity and activity of the enzyme. This enhanced expression is associated with an increased expression of alpha -ENaC. Although the cAMP system is involved in this response, the inhibition of increased enzyme activity with amiloride suggests that augmented Na+ entry at the apical surface plays a role in the process.

    ACKNOWLEDGEMENTS

Y. Minakata and S. Suzuki contributed equally to this paper.

    FOOTNOTES

This work was supported in part by the Medical Research Council of Canada (Grant MT-1203) and by the Canadian Cystic Fibrosis Foundation.

S. Suzuki is the recipient of a fellowship from the Canadian Lung Association, and Y. Berthiaume is a Chercheur-Boursier Clinicien from Fonds de la Recherche en Santé du Québec.

Address for reprint requests: Y. Berthiaume, Centre de Recherche, Centre Hospitalier de l'Université de Montréal, Campus Hôtel-Dieu, 3850 St. Urbain, Montréal, Québec, Canada H2W 1T8.

Received 18 July 1997; accepted in final form 3 April 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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