Transepithelial Na+ transport through epithelial Na+ channels (ENaC) on the apical membrane and Na+-K+-ATPase activity on the basolateral membrane of distal lung epithelial cells are critical for alveolar fluid clearance. Acute exposure to β-adrenergic agonists stimulates lung fluid clearance by increasing Na+ transport. We investigated the effects of chronic exposure to the β2-adrenergic agonist terbutaline on the transepithelial Na+ transport in rat fetal distal lung epithelia (FDLE). FDLE monolayers exposed to 10−4 M terbutaline for 48 h had significantly increased propanolol-blockable transepithelial total and amiloride-sensitive short-circuit current (Isc); however, when these chronically exposed monolayers were acutely exposed to additional β-agonists and intracellular cAMP upregulators, there was no further increase in Isc. Monolayers exposed to terbutaline for >48 h had Isc similar to control cells. Ouabain-sensitive Na+-K+-ATPase activity was increased in 48-h terbutaline-exposed FDLE whose apical membranes were permeabilized with nystatin. In contrast, terbutaline did not increase amiloride-sensitive apical membrane Isc in FDLE whose basolateral membranes were permeabilized with nystatin. Terbutaline treatment did not affect α-, β-, or γ-ENaC mRNA or α-ENaC protein steady-state levels, but increased total cellular levels and rate of synthesis of α1-Na+-K+-ATPase protein in FDLE in the absence of any change in α1-Na+-K+-ATPase mRNA. Total cellular β1-Na+-K+-ATPase mRNA and protein levels were not affected by terbutaline. These data suggest that FDLE have different responses from adult type II epithelial cells when chronically exposed to terbutaline, and their increased transepithelial Na+ transport occurs via a posttranscriptional increase in α1-Na+-K+-ATPase expression.
- epithelial sodium channel
- sodium pump
- ion transport
- short-circuit current
active sodium transport across alveolar epithelium is the primary mechanism of fluid removal from the lung lumen into the interstitium in the adult (31) and fetal (11) lung. During fetal lung development, secretion of ions and fluid into the growing air spaces by the distal lung epithelia is crucial for normal lung development. However, for the newborn infant to breathe at birth and to survive, this excess liquid has to be removed from the alveolar air space of the developing lungs. The phenotypic switch of distal lung epithelia from secretion to absorption involves the regulation of key membrane transport proteins responsible for transepithelial Na+ transport. Labor itself (1, 12) and changes in oxygen partial pressure (44), epinephrine, steroids, and thyroid hormones (4, 5) all play important roles in this phenotypic switch of fetal lung. Thus, an understanding of active Na+ transport across alveolar epithelia is important for the therapeutic treatment of pulmonary edema.
Coordinated function of two key membrane proteins, the apical amiloride-sensitive epithelial Na+ channel (ENaC) and the basolateral ouabain-sensitive Na+-K+-ATPase, is required for transepithelial Na+ transport across lung epithelia (30, 40). ENaC is made up of at least three homologous subunits, α, β, and γ (15), and the Na+-K+-ATPase consists of α- and β-subunits, each with several isomers (24). Lung epithelia express the α1- and β1-subunit isoforms of Na+-K+-ATPase (22). The sharp increase of ENaC and Na+-K+-ATPase around the time of birth is associated with a surge in catecholamine, glucocorticoid, and thyroid hormone in fetal circulation (20, 39, 42). The elevated fetal catecholamine secretion at birth is associated with increased lung fluid clearance (13, 20, 42), and intravenous catecholamine (epinephrine and isoproterenol) administration results in rapid lung fluid reabsorption in fetal lambs (4). Thus, both endogenous and exogenous catecholamines can play a critical role in accelerating the lung liquid clearance. The role of alveolar epithelial β-adrenergic receptors in modulating active Na+ transport in the setting of excess alveolar edema has been recently reviewed (36), and acute exposure of adult alveolar or fetal distal lung epithelial (FDLE) cells in culture to β-adrenergic agonists augments transepithelial Na+ transport over a period of minutes (16, 41) via increases in ENaC and Na+-K+ ATPase activities (25, 61). β-agonists have been reported to enhance active Na+ transport in isolated rat lung (56, 61). In experimental animal models, acute introduction to β-agonists stimulated in vivo (6, 7, 38, 61) and in vitro (21, 54, 61) lung liquid clearance within minutes to hours, and a growing base of information confirmed that this lung liquid clearance was due to increased transepithelial Na+ transport (6, 7, 9, 21, 60). Acute exposure to epinephrine increases alveolar liquid clearance in the isolated human lung (55). β-agonists in clinical use have shown promising results in the prevention of high-altitude pulmonary edema (58) and in patients with acute lung injury/respiratory distress syndrome (46). β-receptor knockout mice exhibit decreased alveolar fluid clearance (35), and transgenic- and adenoviral-mediated β-receptor overexpressing mice (19, 32) show increased alveolar active Na+ transport and alveolar fluid clearance, indicating that β-agonists are essential for maximal Na+ transport.
The mechanisms by which acute exposure to β-agonists increase Na+ transport have been studied in considerable detail and found to be largely dependent on increased insertion of Na+-K+-ATPase (9) and ENaC (14, 26, 62) into the membrane. However, relatively less is known about the long-term effects of β2-agonists on active Na+ transport in fetal lung epithelial cells. This is an important consideration since desensitization to β2-agonists via receptor downregulation may limit therapeutic efficacy (37). Long-term administration in rats and mice has shown encouraging results, up to 6 days of continuous infusion (29, 59). Long-term effects on α-ENaC function and expression have been demonstrated in rat alveolar epithelial cells (18, 33), and a study in a rat submandibular gland cell line demonstrated transcriptional regulation via the cAMP response element binding protein (CREB) (34). Long-term β-agonist treatment of rat alveolar cells in culture results in a rapamycin-sensitive increase in Na+-K+-ATPase protein and function without increasing mRNA levels, an indication of translation regulation in this system (48).
In this study, we investigated the long-term effects of the specific β2-agonist terbutaline in rat FDLE, including effects on transepithelial short-circuit current (Isc) expression of ENaC and Na+-K+-ATPase mRNAs and proteins at 48 h. We found that terbutaline modulated Na+ transport via increases in α1-Na+-K+-ATPase protein synthesis rate.
MATERIALS AND METHODS
Cell culture media was purchased from Invitrogen (Burlington, ON, Canada). Snapwells and Transwells were from Costar (Corning, NY). Primary rabbit polyclonal α-ENaC antibody was purchased from Affinity BioReagents (Golden, CO), mouse monoclonal anti-β-actin antibody was from Sigma (Oakville, ON, Canada), and mouse monoclonal anti-α1- and β1-Na+-K+-ATPase primary antibodies were from Upstate Technology (Temecula, CA). Secondary antibodies and goat anti-rabbit horseradish peroxidase (HRP)-conjugated IgG was purchased from Roche Diagnostics (Laval, Quebec, Canada); goat anti-mouse-HRP IgG antibody was from Bio-Rad (Hercules, CA). All other reagents were from Sigma unless otherwise indicated.
Cell isolation and culture.
FDLE from 20-day gestation rat fetuses (breeding day = day 0, term = 22 days) were isolated and grown in primary culture as described earlier (51). All animal procedures were reviewed and approved by the Hospital for Sick Children Animal Care Committee. FDLE were seeded at a density of 1 × 106 cells/cm2 onto 0.4-μm pore size Snapwell cell culture inserts for Ussing chamber studies or onto 24-mm, 0.4-μm pore size Transwells for mRNA and protein isolation. All cells were grown as submersion cultures in DMEM-high glucose (4.5 g/l glucose with 2 mM l-glutamine and 110 mg/l sodium pyruvate) supplemented with 10% FBS (Cansera, Rexdale, ON, Canada), 100 U/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate. The culture media was replaced 24 h after seeding to remove unattached cells, at which time media (with or without 100 μM terbutaline) containing hormone-depleted FBS (stripped with charcoal and ion exchange resin) (57) was used, and cells were incubated in a humidified atmosphere of 5% CO2/95% air at 37°C. For long-term β2-agonist study, monolayers were incubated for up to 120 h in media alone or in the presence of terbutaline in both upper and lower compartments of the insert. Media and terbutaline were refreshed every 48 h. Where both terbutaline and propranolol were used, propranolol was added 30 min before addition of terbutaline.
Measurement of bioelectric properties in FDLE monolayers.
The bioelectric properties of the FDLE monolayers were determined as described previously (41) using modified Ussing chambers (World Precision Instruments, Sarasota, FL). The cells were bathed in 37°C Hanks' balanced salt solution (Gibco Burlington, ON, Canada) supplemented with 1.98 g/l sodium bicarbonate and equilibrated with a 5% CO2-95% O2 gas mixture. FDLE monolayers were first maintained under open-circuit conditions, while transepithelial potential difference (PD) was observed to reach a stable level and then Isc across monolayers was monitored continuously using a voltage-current clamp (VCC600; Physiologic Instruments, San Diego, CA). Once the bioelectric properties stabilized ∼10 min after insertion of monolayers into the Ussing chambers, amiloride-sensitive Isc was determined by the addition of 0.1 mM amiloride to the apical side of the monolayers. Transepithelial resistance (R) was calculated by dividing the PD by the transepithelial Isc. Data were excluded if transepithelial PD of monolayers into the Ussing chambers was <0.8 mV and R <500 Ω·cm2.
Measurement of apical membrane amiloride-sensitive Isc.
Apical membrane Isc was measured across a fixed Na+ gradient as reported earlier (3) with some modifications. At first, the cells were bathed in a low Na+ solution [mM: Na+ (11.5), K+ (136.4), Cl− (10.3), gluconate (122), HCO3− (25), Mg2+ (1.2), NaH2PO4− (1.2), Ca2+ (5.4)] in a Ussing chamber under Isc conditions. Once the monolayers are stabilized in ∼10 min, nystatin (75 μM) was added to the basolateral side of the monolayers. One earlier study has shown this concentration sufficient to fully permeabilize the cell membrane (28). After 3–4 min, the apical solution was changed so that the final concentration of ions would be [(mM): Na+ (55), K+ (92.9), Cl− (10.3), Mg2+ (1.2), NaH2PO4− (1.2), Ca2+ (5.4), HCO3− (25), and gluconate (123.3)]. Concurrently, the basolateral solution was replaced to the final concentration of [(mM): Na+ (11.5), K+ (92.9), Mg2+ (1.2), Ca2+ (5.4), NMDG (43.5), gluconate (123.3), Cl− (10.3), HCO3− (25), NaH2PO4− (1.2)]. After ∼10 min, 0.1 mM amiloride was added to the apical side of the membrane, and amiloride-insensitive Isc was measured 2 min later. The amiloride-sensitive apical Isc values were calculated by subtracting the difference between baseline Isc and postamiloride Isc.
Measurement of Na+-K+-ATPase activity.
FDLE monolayers mounted in Ussing chambers were bathed in 37°C Hanks' balanced salt solution (Gibco) supplemented with 1.98 g/l sodium bicarbonate and equilibrated with a 5% CO2-95% O2 gas mixture. To establish the maximum basolateral ouabain-sensitive Na+-K+-ATPase activity, bioelectric properties were allowed to stabilize ∼10 min, after which apical membranes were permeabilized by adding 50 μM nystatin to the apical side of the FDLE monolayers. After the transepithelial PD reached a plateau value, the basolateral side of the monolayers was exposed to 1 mM ouabain to inhibit Na+-K+-ATPase activity. The ouabain-sensitive basolateral Isc was calculated by subtracting the Isc present after application of ouabain from the Isc before application of ouabain.
Acute response to β-agonists and intracellular cAMP upregulators in chronically β2-agonist-exposed FDLE.
Control and 48-h terbutaline-treated monolayers inserted in Ussing chambers were exposed to 100 μM isoproterenol (β1,2-agonist), 100 μM terbutaline (β2-agonist), or combined 10 μM IBMX plus 10 μM forskolin (increase cAMP). Transepithelial Isc was monitored for up to 45 min followed by amiloride-sensitive Isc determination by the addition of 0.1 mM amiloride to the apical side of the monolayers. Measurement of cAMP levels was performed on lysates from parallel filters, rinsed in Hanks' balanced salt as for Ussing chamber studies, and treated with either 100 μM terbutaline or vehicle for 5 min before lysis in 0.1 M HCl. Lysates were cleared by centrifugation at 600 g for 10 min, and cAMP concentration in the supernatant was measured using the Cyclic AMP PLUS Enzyme Immunoassay Kit (Biomol International, Plymouth Meeting, PA). Data were analyzed using SOFTMax PRO Software (Molecular Devices, Sunnyvale, CA), and the standard curve was fitted to a 4-parameter logistical model and used to calculate cAMP levels in samples. Protein levels for each sample were measured using the Bio-Rad Protein Assay Reagent (Bio-Rad Laboratories, Hercules, CA) and used to normalize measured cAMP levels.
RNA was extracted from FDLE monolayers using RNeasy (Qiagen, Mississauga, ON, Canada) according to the manufacturer's instructions and quantitated through standard UV absorbance measurements. α-, β-, and γ-ENaC, and α1- and β1-Na+-K+-ATPase mRNA expressions were quantitated by slot blot analysis. Briefly, 3-μg aliquots of FDLE mRNA were applied to Hybond-N+ membranes (GE Healthcare, Baie d'Urfé, PQ, Canada) and fixed by UV crosslinking. α1- and α2-Na+-K+-ATPase mRNA expression was also examined in further n = 3 samples by Northern analysis. Blots were sequentially hybridized to 32P-labeled cDNA probes to rat α-, β-, and γ-ENaC and rat α1-, α2-, and β1-Na+-K+-ATPase with washing and stripping as described earlier (50). Quantitative analysis of probe hybridization was performed using a Molecular Dynamics PhosphorImager equipped with ImageQuant software (Amersham Biosciences, Baie d'Urfé, PQ, Canada). Hybridization to a 32P-labeled cDNA probe encoding 18S rRNA was used to normalize data.
Western blot analysis.
To harvest the cellular protein, the tissue culture dishes were placed on ice, media discarded, and monolayers were washed twice with ice-cold PBS, lysed by direct addition of whole cell extract buffer [150 mM NaCl; 50 mM Tris, pH 7.4; 5 mM EDTA; 0.1% SDS; 20 mM β-glycerophosphate; 10 mM NaF; 0.25 mM Na orthovanadate; 1× Roche complete protease inhibitor cocktail (Roche Applied Science, Laval, PQ, Canada)]. Protein concentrations were determined using the BioRad RC DC protein assay kit (BioRad Laboratories, Mississauga, ON, Canada). Thirty micrograms of total proteins were size fractionated on standard 7.5 or 10% polyacrylamide gels (SDS-PAGE) and transferred onto nitrocellulose membrane (Bio-Rad). After blocking with 5% nonfat dry milk in TBST buffer [20 mM Tris, pH 7.6; 140 mM NaCl; 0.1% (wt/vol) Tween20], membranes were incubated overnight at 4°C with diluted specific primary antibodies (αENaC, 1:1,000; α1-Na+-K+-ATPase, 1:10,000; β1-Na+-K+-ATPase, 1:1,000) in 5% BSA in TBST buffer. Secondary antibodies (HRP-conjugated goat anti-rabbit IgG, 1:20,000 or goat anti-mouse IgG, 1:40,000) were incubated for 1 h at room temperature in blocking buffer. All washes were in TBST buffer. For loading controls, membranes were immunoblotted with anti-β-actin antibody according to standard protocols. For detection of the protein bands, enhanced chemiluminescence detection reagents were used according to the manufacturer's recommendations (GE Healthcare). Quantitation of ECL signal was performed by scanning autoradiograms (Agfa DuoScanner and Agfa Fotolook 3.6v software) followed by quantification of the integrated optical band density using ImageJ software (NIH). Protein expression values were normalized to β-actin.
Polysome profiles were prepared as previously described (43). Briefly, six 10-cm plastic dishes containing confluent FDLE were used for each gradient. Cells were harvested on ice by washing twice with ice-cold PBS containing 100 μg/ml cycloheximide, followed by lysis in 100 μl per dish of polysome lysis buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES, pH 7.4, 100 μg/ml cycloheximide, 0.5% Nonidet P-40, and 1,000 U/ml placental RNase inhibitor). After centrifugation, postnuclear supernatants (10–12 A260nm units/gradient) were layered on linear 15–45% (wt/vol) sucrose gradients in polysome gradient buffer (100 mM KCl, 5 mM MgCl2, and 10 mM HEPES, pH 7.4). Gradients were centrifuged at 35,000 rpm for 2 h in a Beckman SW41 rotor and recovered in 13 equal fractions using a Brandel gradient fractionator equipped with an ISCO UA-6 flow cell set to 254 nm (Brandel, Gaithersburg, MD). RNA was isolated from individual sucrose density fractions by proteinase K digestion followed by phenol/chloroform extraction and ethanol precipitation as described (43). The entire RNA pellet from each fraction was analyzed by Northern blot using 32P-labeled random primed cDNA probes for rat α1-Na+-K+-ATPase as described (43).
Metabolic labeling and immunoprecipitation of α1-Na+-K+-ATPase.
FDLE cultured on permeable supports were rinsed and incubated for 30 min in labeling media (methionine-free, cysteine-free DMEM supplemented with 10% hormone-depleted, dialyzed FBS). Proteins were metabolically labeled for 30 min by carefully placing the basolateral side of each Transwell onto a 100-μl aliquot of labeling media supplemented to 1 mCi/ml with EasyTag 35S protein labeling mix (Perkin Elmer, Waltham, MA) resting on a square of Parafilm. For pulse-chase experiments, Transwells were rinsed three times, returned to six-well plates, and incubated in chase medium (DMEM supplemented with 10% hormone-depleted FBS, 2 mM l-cysteine, 2 mM l-methionine) for indicated times. Labeled cells were rinsed three times in ice-cold PBS and harvested by gentle scraping in nondenaturing lysis buffer (50 mM Tris, pH 7.4, 300 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1× Roche complete protease inhibitor cocktail). For assessment of synthesis rate, [35S]-amino acid incorporation was determined by TCA precipitation of 5-μl aliquots of the supernatants, and equal amounts of cpm were submitted to immunoprecipitation. Lysates were precleared with 25-μl packed volume of protein G-Sepharose before overnight incubation with 2 μg of monoclonal antibody recognizing α1-Na+-K+-ATPase. Immunoprecipitates were pulled down with protein G-Sepharose and washed extensively in 50 mM Tris, pH 7.4, 300 mM NaCl, 5 mM EDTA, and 0.1% Triton X-100. Immunoprecipitated proteins were recovered by heating for 30 min at 37°C in Laemmli sample buffer and separated on acrylamide SDS-polyacrylamide gels. Gels were fixed, incubated in Amplify (GE Healthcare), and dried for analysis on a Molecular Dynamics PhosphorImager equipped with ImageQuant software.
Data are expressed as means ± SE. Statistical significances for multiple comparisons were calculated using one-way ANOVA with Newman-Keuls posttest. Statistical significances between two groups were assessed by Mann-Whitney U-test. Statistical analysis was performed using GraphPad Instat version 3.01 and GraphPad Prism version 4.0 (GraphPad Software, San Diego, CA). P values <0.05 were considered statistically significant.
Long-term terbutaline exposure induces a time-limited increase in transepithelial Isc in FDLE.
To investigate the effects of prolonged terbutaline exposure on transepithelial ion transport in FDLE, FDLE monolayers were cultured in the absence or presence of 100 μM terbutaline for up to 120 h. Total transepithelial Isc in untreated control cells did not change significantly with time in culture up to 120 h; therefore, all control values were expressed together. Continuous terbutaline exposure initiated 24 h after seeding resulted in a transient increase in total transepithelial Isc that was maximal (2.3-fold increase over control) at 48 h (Fig. 1A). The amiloride-sensitive component of transepithelial Isc was similarly increased by culture in terbutaline up to 2.7-fold at 48 h (Fig. 1B). Since the maximum transepithelial Isc was observed with 48-h terbutaline treatment, this time point was used for all subsequent experiments. When we delayed the initiation of terbutaline exposure to 48 h after seeding and assayed Isc after a subsequent 48 h of exposure, the terbutaline-treated cells had no difference in total or amiloride-sensitive Isc relative to control monolayers (data not shown)
To demonstrate the specificity of terbutaline's effects on total and amiloride-sensitive Isc, we used propranolol, a competitive nonselective β-adrenergic receptor antagonist. FDLE monolayers exposed to 1 μM or 10 μM propranolol alone showed no effects on total or amiloride-sensitive Isc, whereas FDLE treated with 100 μM terbutaline significantly increased both total and amiloride-sensitive Isc. Terbutaline's effects on Isc measurements could be blocked by 10 μM (P < 0.001) but not by 1 μM propranolol (Fig. 2, A and B).
Long-term terbutaline exposure increases basolateral Na+-K+-ATPase activity.
To investigate effects on apical vs. basolateral components of Na+ transport, nystatin-permeabilized monolayers were used. Amiloride-sensitive Isc was not different between control and terbutaline-treated monolayers following basolateral permeabilization (Fig. 3A). In contrast, terbutaline-treated FDLE monolayers showed a significant increase (1.8-fold) in ouabain-sensitive basolateral Isc following apical permeabilization (Fig. 3B).
Effect of acute addition of β-agonists and intracellular cAMP upregulators on FDLE cultured long term in terbutaline.
To determine whether FDLE continuously cultured in the presence of terbutaline could increase Isc further in response to acute β-agonists or increased intracellular cAMP levels, we tested the effects of 100 μM isoproterenol (β1,2-agonist), 100 μM terbutaline (β2-agonist), or combined 10 μM IBMX plus 10 μM forskolin (increase cAMP) added in the Ussing chambers. FDLE monolayers cultured in the absence of terbutaline increased total Isc (Fig. 4A) and amiloride-sensitive Isc (Fig. 4B) when exposed to any of the three acute treatments. Although addition of IBMX plus forskolin resulted in a trend to higher Isc, none of these acute treatments significantly increased total or amiloride-sensitive Isc (Fig. 4, C and D) in FDLE that had been previously cultured in the presence of terbutaline for 48 h. Intracellular cAMP levels measured in control and chronic terbutaline-exposed cells following acute exposure to terbutaline showed that chronically treated cells had cAMP levels comparable to control cells. FDLE chronically exposed to terbutaline did not increase cAMP concentrations in response to acute treatment (Fig. 4E).
Effect of long-term terbutaline on Na+ transporter expression.
We examined the effects of culture in terbutaline on steady-state expression levels of ENaC and Na+-K+-ATPase mRNA and protein. Consistent with the apical membrane bioelectric measurements, long-term exposure to 100 μM terbutaline had no effect on FDLE total cellular α-ENaC mRNA (Fig. 5A) or protein levels (Fig. 5B). Nor were β- or γ-ENaC mRNA levels affected (Fig. 5, C and D). We found similar results when mRNA levels for α1- (Fig. 6A) and β1- (Fig. 6B) subunits of Na+-K+-ATPase were analyzed; that is, terbutaline treatment did not increase mRNA level for either subunit in FDLE. However, the total steady-state cellular content of α1-Na+-K+-ATPase protein was significantly increased (1.4-fold) in terbutaline-treated monolayers compared with control, and this effect was blocked by propranolol (Fig. 6C). We therefore confirmed the α1-Na+-K+-ATPase mRNA results using Northern blots and additionally confirmed that α2-Na+-K+-ATPase mRNA, which has been previously suggested to play a role in acute stimulation of alveolar fluid clearance by β-agonists (53), is not significantly expressed in control or chronic terbutaline-treated FDLE (Supplemental Fig. S1. Supplemental data for this article is available online at the AJP-Lung web site.). β1-Na+-K+-ATPase protein level was unchanged by terbutaline treatment (Fig. 6D).
The increase in α1-Na+-K+-ATPase protein detected in the absence of any change in the expression level of its mRNA suggested posttranscriptional regulation of the protein expression. Consequently, we analyzed the rate of synthesis in metabolically labeled FDLE. Immunoprecipitation of protein from cells pulse-labeled with [35S]-amino acids with antibody specific to the α1-subunit detected a statistically significant increase in the amount of this protein synthesized in 30 min (Fig. 7A). This increase represents specific changes in α1-Na+-K+-ATPase synthesis rate, not just an overall increase in rate of synthesis of all cellular proteins, since equal cpm of newly synthesized proteins were submitted to immunoprecipitation. Sucrose density gradient fractionation of polyribosomes followed by Northern blot detection of α1-Na+-K+-ATPase mRNA failed to detect significant changes in this mRNA's distribution among polyribosome fractions (Fig. 7B).
Chronic exposure of FDLE to terbutaline evoked a significant increase in amiloride-sensitive transepithelial ion transport, which peaked at 48 h and returned to baseline by 96 h. Complete blockade of this effect by propranolol confirmed that these effects were mediated via terbutaline's interaction with β-adrenergic receptors. Cells cultured in the presence of terbutaline for 48 h had intracellular cAMP levels that were similar to control cells and were unable to acutely increase cAMP in response to terbutaline or to increase ion transport in response to addition in the Ussing chamber of terbutaline or other agents (isoproterenol, IBMX + forskolin), which usually upregulate intracellular cAMP levels. These data, plus the trend to increased ion transport in response to IBMX + forskolin, suggest that β-agonist desensitization likely occurred when FDLE were chronically exposed to terbutaline.
We detected a significant increase in ouabain-sensitive Isc in apically permeabilized monolayers, consistent with an increase in basolateral Na+-K+-ATPase activity in terbutaline-cultured monolayers, but were unable to demonstrate any change in amiloride-sensitive Isc in basolaterally permeabilized monolayers exposed to a fixed Na+ gradient. Studies by Jiang et al. (23) also reported no increased apical Na+ conductance in basolaterally permeabilized adult cells in response to isoprenaline. However, this is in contrast to the results of Collett et al. (17) in FDLE exposed to isoproterenol in an acute fashion, which showed an increase in apical Na+ conductance in basolaterally permeabilized monolayers. It is also unexpected if chronic terbutaline were to promote the insertion of ENaC into the apical membrane as has been well established for acute β-adrenergic stimulation (14, 26, 62) or decreases retrieval of ENaC from the surface, as in Xenopus oocytes treated for 24 h with IBMX plus forskolin (63). We cannot rule out the possibility that the basolateral permeabilization resulted in the rapid loss of regulation of ENaC activity via changes in cytoplasmic components (as speculated by others, Ref. 17), resulting in an inability to detect any differences in apical amiloride-sensitive Na+ transport between cells cultured with and without terbutaline. Others have reported that growth of FDLE in defined media free of hormones and growth factors rendered the cells insensitive to isoprenaline-evoked stimulation of ENaC, which could be restored by addition of dexamethasone and triiodothyronine to the culture media (52). Our experiments were performed using hormone-depleted FBS that may similarly lack hormones that in some way limit potential ENaC stimulation by terbutaline.
Furthermore, we detected no change in mRNA expression of any ENaC subunit or in total cellular protein level of αENaC, suggesting long-term terbutaline treatment has no effect on ENaC transcription, translation, or stability in FDLE. Although this result is consistent with the lack of measured change in apical amiloride-sensitive Isc in basolaterally permeabilized monolayers, it contrasts again with results in ATII cells; an increase in αENaC mRNA was detected in ATII cultured for 48 h in terbutaline (33) or for 8 h in dibutyryl cAMP (DBcAMP) (18) (in both cases coinciding with an increase in ion transport). Increases in αENaC mRNA via activation of CREB-driven transcription have also been demonstrated in a submucosal gland cell line exposed to DBcAMP for 24 h (34). Together, the data indicate important differences between adult and fetal distal lung epithelia and among different cell types in their response to long-term β-agonist stimulation.
Expression of α1-, but not β1-, Na+-K+-ATPase was modulated by 48 h of terbutaline exposure. Although we did not examine translocation to the membrane, total cellular protein level of the α1-subunit was increased 1.4-fold, somewhat lower than the increase in ouabain-sensitive Isc (1.8-fold) and total transepithelial Isc (2.7-fold), thus suggesting that posttranslational regulation such as translocation also contributes to the increase in ion transport. Indeed, increased insertion of Na+-K+-ATPase into the basolateral membrane is an important mechanism in acute hormonal control of Na+ transport in lung epithelia in response to a variety of hormones, including β-agonists (2, 9), dopamine (8), and triiodothyronine (27). Again, our observation in FDLE contrasts with previous reports in ATII in which an increase in mRNA was seen following 48 h of treatment, with an increase in protein levels only after 7 days (33). The increase we observed in steady-state protein level in the absence of a change in mRNA suggests posttranscriptional regulation of α1-Na+-K+-ATPase protein expression. Indeed, others have suggested translational regulation of Na+-K+-ATPase, including evidence that long-term stimulation of ATII with isoproterenol increased Na+-K+-ATPase protein in the absence of changes in mRNA, and that these increases were sensitive to inhibition of the kinase mammalian target of rapamycin (mTOR), and dependent on phosphorylation of p70S6kinase, important regulators of translation initiation (47, 48). More recently, Bhargava et al. (10) demonstrated discordance between Na+-K+-ATPase mRNA and protein in acutely isolated ATII cells from rats treated with intraperitoneal triiodo-l-thyronine for three consecutive days, with increased α1-subunit protein in the presence of decreased mRNA. However, regulation of rate of synthesis of α1-Na+-K+-ATPase protein has not been directly assessed by these previous studies. We used two approaches to examine α1-Na+-K+-ATPase protein synthesis, directly measuring the rate of synthesis via immunoprecipitation of pulse-labeled protein from metabolically labeled cells, and indirectly by measuring distribution of α1-Na+-K+-ATPase mRNA among polysome fractions as a measure of translation initiation rate (under most circumstances, the rate-limiting step in protein translation is the initiation step, and most changes in the translational efficiency of a given mRNA can be assessed by determining the percentage of that mRNA associated with actively translating polysomes of various sizes) (49). We were able to detect an increase in rate of α1-Na+-K+-ATPase protein synthesized during a 30-min pulse label in FDLE (∼20% increase), but unable to detect a change in the polysome distribution of α1-Na+-K+-ATPase mRNA. There are two possible explanations for this: 1) the increase in synthesis rate detected by immunoprecipitation of pulse-labeled cells is small, and polysome gradient analysis shows that this mRNA is associated mainly with the largest polysomes even in control FDLE, thus the resolution available with this technique is probably insufficient to detect increased ribosomal loading, or 2) the increase in synthesis may be due to increases in translation elongation or translation termination rates instead of translation initiation. Nevertheless, this increase in α1-Na+-K+-ATPase protein synthesis would be expected to significantly contribute to the overall increase in steady-state protein level and consequent increase in ion transport activity. We were unable to determine whether a change in α1-Na+-K+-ATPase stability also contributed to higher steady-state protein levels using pulse-chase approaches due to the low signal-to-noise ratio in control cells (data not shown).
Concerns regarding the potential efficacy of β-agonists to treat pulmonary edema have arisen due to the potential for downregulation of β-adrenergic receptors. Encouraging results from both animal models (29) and a clinical trial (46) underline the need to understand the long-term effects of β-agonist stimulation, both on fluid clearance and on potential for alveolar epithelial repair (45). The study reported here highlights a number of differences in the ion transport responses of fetal (FDLE) cells vs. previously published studies using adult alveolar epithelial cells. Such differences might influence the clinical applications of β-agonist, for example, in treating neonatal vs. adult respiratory distress syndrome (ARDS).
This work was supported by the Canadian Institutes of Health Research Operating Grant MGP-25046 and Group Grant in Lung Development.
No conflicts of interest are declared by the author(s).
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