Na+-K+-ATPase pumps (Na+ pumps) in the alveolar epithelium create a transepithelial Na+ gradient crucial to keeping fluid from the pulmonary air space. We hypothesized that alveolar epithelial stretch stimulates Na+ pump trafficking to the basolateral membrane (BLM) and, thereby, increases overall Na+ pump activity. Alveolar type II cells were isolated from Sprague-Dawley rats and seeded onto elastic membranes coated with fibronectin or 5-day-conditioned extracellular matrix. After 2 days in culture, cells were uniformly stretched for 1 h in a custom-made device. Na+ pump activity was subsequently assessed by ouabain-inhibitable uptake of 86Rb+, a K+ tracer, and BLM Na+ pump abundance was measured. In support of our hypothesis, cells increased Na+pump activity in a “dose-dependent” manner when stretched to 12, 25, or 37% change in surface area (ΔSA), and cells stretched to 25% ΔSA more than doubled Na+ pump abundance in the BLM. Cells on 5-day matrix tolerated higher strain than cells on fibronectin before the onset of Na+ pump upregulation. Treatment with Gd3+, a stretch-activated channel blocker, amiloride, a Na+ channel blocker, or both reduced but did not abolish stretch-induced effects. Sustained tonic stretch, unlike cyclic stretch, elicited no significant Na+ pump response.
- ventilator-induced lung injury
- alveolar edema
- Na+-K+- ATPase
- mechanical deformation
although mechanical ventilation can afford life-saving ventilatory support for ill or injured patients, it can also potentiate further pulmonary dysfunction (15, 22, 24, 25, 39, 50, 54-56, 70). This phenomenon, aptly termed ventilator-induced lung injury (VILI), typically presents in patients ventilated with moderate-to-high tidal volume (Vt) and has been linked more closely to elevated lung volume than high ventilation pressure (23-25,49). In support of the case that high lung volume causes VILI, a recent National Institutes of Health-sponsored Ventilator Management Study in acute respiratory distress syndrome demonstrated a significant decrease in mortality in patients ventilated with low Vt(1). Alveolar edema, one of the primary hallmarks of VILI, is thought to result from high-volume overinflation of the alveoli, which in turn leads to overdistension of the alveolar epithelium (AE), disruption of the epithelial barrier, and increased epithelial permeability (22, 25). As the epithelium becomes more permeable, small solutes and, eventually, large proteins cross the epithelium to the alveolar air space, and fluids follow the resultant osmotic gradient.
Once edema occurs, the primary recovery mechanism for clearing fluids from the air space is active Na+ transport from the alveolar mucosa across the epithelium; Cl− follow the Na+-based charge gradient, and fluids exit the air space along the NaCl-based osmotic gradient (38, 43, 60). The primary components of this Na+ transport across the epithelium are passive epithelial Na+ channels (ENaCs), located in the apical membrane of the epithelial layer, and energy-dependent Na+-K+-ATPase pumps (Na+ pumps), found in the cells' basolateral membrane (BLM) (44, 51, 65, 66). These Na+ transport components are recognized and studied primarily in AE type II (AT2) cells but have also been found in alveolar type I (AT1) cells (58). In addition to these primary Na+transport elements, epithelial cells also contain a variety of Na+-transporting symports, antiports, and co-ports, especially in the cells' apical membrane. These proteins, however, use the Na+ gradient as a potential source for transporting other ions or molecules but typically play a negligible role in overall Na+ transport (45, 46, 51, 59). With Na+ pumps as the driving force behind vectorial Na+ transport across the epithelium, their proper function, activity levels, and overall population in the epithelial cell membrane are crucial factors in edema clearance. Indeed, studies have shown that disabling Na+ pumps with toxins such as ouabain can lead to edema (35, 37). On the other hand, stimulating Na+ pump activity, primarily through β-adrenergic stimulation, with aldosterone (52), catecholamines (4-6, 63), growth factors (32), or directly with β-adrenergic agonist (40, 62) or β-adrenergic receptor overexpression (48) can significantly improve edema clearance. Similarly, Na+ pump overexpression itself improves edema clearance (3, 28,29).
Given the interconnectedness of Na+ pump activity and pulmonary fluid balance, study of Na+ pump response to alveolar stretch during mechanical ventilation is central to understanding VILI-associated edema. Although it is clear that high Vt results in alveolar edema (22, 26, 36, 39,54, 71, 75), it is unclear whether edema worsens as a result of decreased Na+ transport or, conversely, occurs despite increased Na+ transport. In other words, does stretch prompt an increase in Na+ pump activity to counter the edema caused by increased AE permeability, or does stretch impair Na+ pump function, exacerbating edema by allowing unchecked fluid accumulation in the alveoli?
In recent years, investigators have produced evidence supporting both sides of this debate. Whole lung studies have reported significant decreases in edema clearance with high-volume mechanical ventilation and have attributed this dysfunction to a decrease in active Na+ transport (41, 42). However, investigators have reported increases (67) and decreases (41,42) in Na+ pump activity (as evidenced by ouabain-sensitive ATP hydrolysis) in cells isolated from whole lung preparations that had previously undergone high-volume ventilation. Meanwhile, cells from an immortal MLE-12 cell line exhibited increased Na+ pump activity (evidenced by86Rb+ uptake) after 30 or 60 min of cyclic loading in a nonuniform strain field (averaging ∼10% strain), and this increased activity was abolished by blocking amiloride- or Gd3+-sensitive cation channels (74).
Disagreement on the effect of stretch on Na+ pump activity could result from a number of factors, including differences in average strain magnitude among studies, nonuniform strain fields within studies, differences between primary cells and cell lines, and differences in timing and cell treatment between injury (in vivo ventilation or in vitro mechanical stretch) and Na+ pump activity assessment. Thus, to address the topic carefully, we stretched primary AT2 cells in uniform, well-defined strain fields at precisely chosen strain magnitudes, which enabled measurement of change in Na+ transport over a variety of strain levels and determination of the relative role of stretch magnitude. Furthermore, we measured Na+ pump activity immediately after stretch without further disturbance of the cells between stretch and Na+ pump activity assessment.
In addition to determining that Na+ pump activity is stimulated by epithelial stretch, we investigated some of the potential mechanisms responsible for translating the mechanical stimulus of stretch into a biochemical signal for Na+ pump regulation and the means by which AT2 cells modulate their Na+ pump activity in response to that signal. This included probing the potential stimulatory role of ion influx through apical stretch-activated channels (SACs), which open in response to mechanical loading, examining the influence of extracellular attachment on Na+ pump stimulation, assessing different cell sensitivities to cyclic and tonic stretch stimuli, measuring Na+ pump concentrations in the BLM immediately after stretch, and ascertaining levels of Na+ pump mRNA immediately after stretch.
AT2 cells were isolated from male Sprague-Dawley rats (Charles River, Wilmington, MA) according to a previously described protocol (68) approved by the University of Pennsylvania Institutional Animal Care and Use Committee. Briefly, animals were anesthetized with pentobarbital sodium (50 mg/kg ip). The trachea was cannulated, the lungs were mechanically ventilated, and the animal was exsanguinated by abdominal aortotomy. The heart was pierced, and the lungs were perfused via the pulmonary artery. The lungs were subsequently excised, and AT2 cells were isolated using an elastase digestion technique adapted from Dobbs et al. (19), in which the lungs are instilled and incubated with an elastase solution (3 U/ml; Worthington Biochemical) and then minced with a tissue sectioner (Sorvall, Newtown, CT). Cells were filtered through a series of sterile progressively finer Nitex mesh (Crosswire Cloth, Bellmawr, NJ) and plated on a suspension culture dish coated with rat IgG (Sigma, St. Louis, MO; 3 mg IgG per 5 ml Tris · HCl), incubated overnight, and rinsed. After a 1-h incubation at 37°C, gentle panning lifted AT2 cells from the macrophages, and other contaminating cells preferentially adhered to the culture dish. Finally, cells were spun down and resuspended in MEM supplemented with Earle's salts, 10% FCS, and 25 μg/ml gentamicin (Life Technologies, Rockville, MD).
All cells were seeded at a density of 1 × 106cells/cm2 onto flexible Silastic membranes (Specialty Manufacturing, Saginaw, MI) mounted in custom-made wells. Cells were seeded onto fibronectin-coated membranes (42 μg/ml; Boehringer Mannheim Biochemicals, Indianapolis, IN) or membranes coated with a 5-day AT2 extracellular matrix (ECM) prepared from cells from a previous isolation. Briefly, to create 5-day matrix, AT2 cells were seeded onto a fibronectin-coated membrane, maintained in culture for 5 days, lysed for 5 min in a bath of 0.5% Triton X-100 (Boehringer Mannheim)-5 mM EDTA (Sigma) in Hanks' balanced salt solution without CaCl2, MgCl2, MgSO4, and phenol red (Life Technologies), and rinsed with Hanks' balanced salt solution immediately before freshly isolated cells were seeded onto the remaining 5-day ECM. All cells, whether on fibronectin or 5-day matrix, were cultured for 48 h at 37°C under 5% CO2 in MEM supplemented with Earle's salts, 10% fetal bovine serum, and 25 μg/ml gentamicin (Life Technologies).
Before each study, MEM was aspirated from cells and replaced with a stretching medium of DMEM without NaHCO3, supplemented with 1% penicillin (1,000 U/ml)-streptomycin (10 mg/ml) solution (Life Technologies) with 20 mM HEPES (Sigma). For each cyclic strain experiment, six experimental wells were mounted in a custom-made stretching device (68), and the Silastic membrane and AE monolayer were stretched uniformly and equibiaxially for 1 h at 15 cycles/min to a peak strain of 12, 25, or 37% change in surface area (ΔSA; ∼60, ∼80, or ∼100% total lung capacity, respectively) (69). In tonic stretch experiments, six wells of AT2 cells were stretched to 12 or 25% ΔSA and held at that strain for 1 h. In experiments imposing cyclic stretch over a tonic stretch baseline, six wells of AT2 cells were stretched to a baseline deformation of 12% ΔSA and cycled between 12 and 25% ΔSA for 1 h at 15 cycles/min. In all experiments, six wells of unstretched cells from the same isolation remained as controls. All stretched and unstretched wells were maintained at 37°C in room air during the 1-h stretch period. For 86Rb+ uptake experiments, three wells from each of the stretched and the unstretched groups were pretreated with 1 mM ouabain (Sigma), a specific Na+ pump blocker, during the last 20 min of stretch.
A protocol for using 86Rb+, a radioactive K+ channel tracer, to monitor Na+ pump activity was adapted from Clerici et al. (16). Immediately after 1 h of stretch, cells from stretched and unstretched (control) groups were aspirated and bathed for 5 min in an uptake solution derived from DMEM and composed of (in mM) 120 NaCl, 5 RbCl, 1 MgSO4, 0.15 Na2HPO4, 0.2 NaH2PO4, 4 NaHCO3, 1 CaCl2, 5 glucose, 4 essential and nonessential amino acids, and 20 HEPES and supplemented with 1 μCi/ml 86RbCl (Amersham Pharmacia Biotech, Piscataway, NJ). The uptake solution was also supplemented with 1 mM ouabain in the same six wells pretreated with ouabain during the stretch protocol. Uptake was arrested by aspiration of the 86Rb+ solution and five rapid rinses with an ice-cold solution containing (in mM) 140N-methylglucamine, 1.2 MgCl2, 3 BaCl2, and 10 HEPES; this solution halts metabolic activity and blocks ion channels. Cells were then air-dried, solubilized in 0.1% Triton X-100, and spun down to separate cell membrane fragments.86Rb+ uptake was ascertained by liquid scintillation counting of a portion of the supernatant from each well, while the remaining supernatant was used for assessing each well's protein content using a Bradford-based (10) protein assay kit (Bio-Rad Laboratories, Hercules, CA). Na+ pump activity was defined as the difference between86Rb+ uptake (counts · min−1 · μg protein−1) in the absence and presence of 1 mM ouabain (see Data analysis).
Ion channel blocking with Gd3+ and amiloride.
To determine whether stretch-induced changes in Na+ pump activity stem from a biochemical cascade originating in ion flux through SACs, the above protocol was repeated, stretching cells at 25% ΔSA in stretching medium supplemented with 10 μM Gd3+(Sigma), a potent SAC blocker, 1 μM amiloride (Sigma), an apical Na+ channel blocker, or both. Unstretched, similarly treated wells served as controls. Because phosphates and bicarbonates found in most physiological media aggressively chelate Gd3+, disabling its SAC-blocking ability and often leading to false-negative results (11, 73), Gd3+studies were performed with bicarbonate- and phosphate-free HEPES-buffered media and solutions in all experimental and control groups.
Unlike our earlier studies with AT2 cells maintained for 24 h (1-day cells) or 5 days in culture (68), this study used cells cultured for 48 h (2-day cells), which exhibited greater substrate attachment and viability than 1-day cells while retaining desired type II characteristics such as surfactant synthesis. With the use of previously described epifluorescent microscopy methods (53, 68), cell viability was assessed for 2-day cells on fibronectin and on 5-day ECM stretched at 12, 25, and 37% ΔSA. Briefly, 0.23 μM ethidium homodimer-1, which enters through the damaged membrane of dead cells and binds to nucleic acids, and 0.12 μM calcein-AM, which is retained in live cells (Live/Dead kit, Molecular Probes, Eugene, OR), were added to cultures before stretch. After stretch, images were captured using a Nikon TE-300 inverted microscope with a ×20 objective, a Hamamatsu digital camera, and Metamorph 3.0 software (Universal Imaging, West Chester, PA). Live and dead cells were counted in three random locations in each well for six stretched and six unstretched wells in every experimental condition. Viability was calculated as a percentage of live cells per the sum of live and dead cells. Double-labeled cells, which indicated a damaged but resealed cell membrane, accounted for <0.5% of any culture and were counted as live cells.
Phenotype determination by immunocytochemistry.
AT2 cells, known to serve as AT1 cell progenitors in vivo, have been shown to differentiate in vitro, expressing AT1 phenotypic markers and ceasing AT2-specific functions, such as surfactant production, within 4–5 days in culture (9, 13, 17, 18, 20). To determine whether the shift from AT2 to AT1 phenotype occurred in the 2-day cells used in this experiment and to assess whether this shift was accelerated in cells seeded on 5-day matrix, we labeled cells against RTI-40, an AT1-specific antibody (21) (monoclonal anti-RTI-40 was a gift from Dr. Leland Dobbs, University of California San Francisco, San Francisco, CA), and pro-surfactant protein C (SP-C), a surfactant peptide precursor and, hence, an AT2 phenotype indicator (7) (polyclonal anti-pro-SP-C was a gift from Dr. Michael Beers, University of Pennsylvania, Philadelphia, PA). In addition, separate cells were labeled with antioccludin (Zymed, South San Francisco, CA). Occludin is a tight junction protein most commonly associated with AT1 cells and a mature AE barrier. Cells were fixed with 4% paraformaldehyde for 20 min and treated with sodium borohydride (1 mg/ml; Sigma) to reduce endogenous fluorescence. Normal goat serum (Life Technologies) was used to block nonspecific sites. Primary antibodies were incubated at room temperature for time periods recommended by the donor or manufacturer. Primary antibodies were fluorescently tagged with fluorescein- or red X-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA), and immunofluorescence was preserved using fluorescent mounting medium (DAKO, Carpinteria, CA). Slides were stored at 4°C and inspected by standard immunofluorescent microscopy.
BLM isolation and Western blotting.
To determine whether AT2 cells increase their Na+ pump activity by trafficking lipids from the intracellular stores, AT2 cells were harvested from rats in seven separate cell isolations and maintained for 48 h in culture as described above. Cells from four isolations were used to determine Na+ pump content in whole cell homogenates of stretched and unstretched cells. The remaining three isolations were BLMs isolated from stretched and unstretched cells using a protocol generously provided by Dr. Karen Ridge (Northwestern University). Specifically, after 1 h of cyclic stretch at 25% ΔSA, stretched cells and unstretched controls were rinsed and scraped in ice-cold PBS, collected by centrifugation at 4°C, and resuspended in a homogenization buffer consisting of 12 mM HEPES titrated with Tris · HCl to pH 7.6, 300 mM mannitol, 4.2 μM leupeptin, 600 μM phenylmethylsulfonyl fluoride, and 284 μMN-tosyl-l-phenylalanine chloromethyl ketone. BLMs were isolated using previously described techniques (14,34). Briefly, endosomes, ghost cells, and mitochondria were successively isolated and removed by differential centrifugation. The remaining fraction was rehomogenized and centrifuged through a 16% Percoll (Sigma) gradient at 48,000 g. The BLM ring was collected and solubilized in 1% Triton X-100, which was subsequently extracted with Detergent-OUT spin columns (Geno Technology, St. Louis, MO).
For whole cell homogenates and BLM fractions, a portion of each sample was used to assess total protein concentration with the Bio-Rad protein assay mentioned above. Another fraction of each sample was then loaded into duplicate 8% SDS-polyacrylamide gels. Equal amounts of total protein were loaded into the stretched and unstretched lanes within each pair. Proteins were separated by PAGE (200 V, 60 min) and transferred to polyvinylidene difluoride membranes (250 V, 60 min). Membranes were incubated for 1 h in 5% powdered milk and 0.1% Tween 20 in PBS (PBS-MT) to block nonspecific binding and then incubated overnight at 4°C with mouse Na+-K+-ATPase α1-specific antibody (Upstate Biotechnology, Lake Placid, NY) diluted 1:15,000 in PBS-MT. After three 15-min rinses in PBS containing 0.1% Tween 20 (PBS-T), membranes were incubated for 1 h with a horseradish peroxidase-conjugated donkey anti-mouse secondary antibody diluted 1:5,000 in PBS-MT. After the membranes were rinsed again three times for 15 min in PBS-T, blots were developed using an enhanced chemiluminescence preparation (Amersham) and recorded on Kodak film. Films were subsequently scanned with a Kodak Imaging System 1000 and analyzed with Kodak 1D Image Analysis Software (Eastman Kodak, Rochester, NY). Films from duplicate gels were compared to confirm consistent gel loading. Polyvinylidene difluoride membranes were also stained with Ponceau S to verify equal total protein loads within each pair of lanes.
RT-PCR and gel analysis.
Cells from four separate isolations were cultured on fibronectin, maintained for 2 days in culture, and cyclically stretched to 25% ΔSA for 1 h. Immediately after stretch, total RNA was isolated from eight pooled wells of stretched cells and eight pooled wells of unstretched controls using an RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. After assessment of the total RNA yield by light transmission at 260 nm, RNA samples were reverse transcribed into cDNA, which was amplified using a Superscript one-step RT-PCR with Platinum Taq kit (Life Technologies/GIBCO) and primers specific to the Na+ pump α1-subunit (Life Technologies/GIBCO) as used in similar studies (42). Briefly, 1 μg of RNA from each stretched and unstretched sample was combined with polymerase and sense and antisense primers. RT was carried out at 42°C for 50 min. Subsequent PCR consisted of a 2-min denaturation at 94°C, 25 amplification cycles of 60 s at 94°C, 90 s at 50°C, and 120 s at 72°C, and a final extension at 72°C for 7 min, all performed with a GeneAmp 9600 PCR system (Perkin-Elmer, Norwalk, CT). Glyceraldehyde-3-phosphate dehydrogenase (GAPD), a metabolic enzyme assumed to remain constant with stretch, was used as a control. GAPD was also reverse transcribed and amplified with specific primers through 25 PCR cycles of 45 s at 94°C, 45 s at 55°C, and 120 s at 72°C, with all other steps using the same protocol used above for the Na+ pump α1-subunit. Amplified cDNA samples of Na+ pump α1-subunit and GAPD were then isolated by electrophoresis on 12% agarose gels loaded with ethidium bromide. Polaroid photographs of the gels, taken on an ultraviolet light box, were digitized and densitometrically analyzed using MCID software (Imaging, St. Catharine, ON, Canada). Equal intensity of GAPD blots was used to confirm equal loading of stretched and unstretched mRNA samples before PCR and of cDNA into stretched and unstretched lanes after PCR. Separately, we verified that PCR amplification remained linear up to 30 cycles by running replicate RT-PCR reactions for 10, 15, 20, 25, and 30 cycles, performing agarose gel electrophoresis, and analyzing blot densitometry for the Na+ pump α1-subunit and GAPD.
Measurements from each well in the 86Rb+ uptake studies were recorded as counts per minute per microgram of protein. Within each experimental trial (1 isolation, 12 wells), equal variances were assumed among all four three-well groups: stretched and ouabain treated, unstretched and ouabain treated, stretched and untreated, and unstretched and untreated. Ouabain-treated groups were then averaged, and these averages were subtracted from each of three nontreated well values in the corresponding stretched or unstretched group. Thus each experiment yielded three values of ouabain-inhibitable (Na+pump-dependent) 86Rb+ uptake for stretched cells and three values for ouabain-inhibitable86Rb+ uptake in unstretched cells. Within each protocol, differences were evaluated by two-way ANOVA: between stretched and unstretched treatments and among isolations. After ANOVA, stretched values for each isolation were normalized by unstretched controls for that isolation and combined across isolations solely for graphical purposes. In Western blots, densities were measured using Kodak 1D Image Analysis Software and analyzed using paired Student'st-tests for stretched and unstretched pairs over several isolations (n = 4 for whole cell homogenates;n = 3 for BLM fractions).
Na+ pump activity increases with cyclic stretch in AE cells.
AE cells generally increase Na+ pump activity in response to mechanical strain, and they do so in a “dose-dependent” manner: the greater the applied strain, the greater the increase in Na+ pump activity. AT2 cells cultured on fibronectin and stretched cyclically at 12% ΔSA increased Na+ pump activity to 134 ± 11% (mean ± SE) over unstretched controls at 100 ± 18%, while cells stretched at 25% ΔSA more than doubled their Na+ pump activity to 236 ± 27% over control values of 100 ± 19% (Fig.1). In contrast, AT2 cells seeded onto 5-day AE ECM show no significant response to stretch at 25% ΔSA (91 ± 19% over controls of 100 ± 13%) but increased Na+ pump activity to 287 ± 65% over an unstretched control value of 100 ± 7% at 37% ΔSA (Fig.2). This finding corresponds with our own viability data (53), which reveal greater stretch tolerance, measured by percent cell viability, after stretch in cells grown on 5-day ECM than in cells grown directly on fibronectin.
Ionic fluxes through SACs and amiloride-sensitive ENaCs stimulate Na+ pump activity.
Blocking SACs with Gd3+ and/or amiloride attenuated stretch-induced increases in Na+ pump activity to 148 ± 24, 154 ± 10, and 153 ± 23%, respectively (normalized values), with stretch of 25% ΔSA—compared with 236 ± 27% for the same stretch without treatment—but did not abolish the increase completely (Fig. 3). Notably, treatment with either blocker individually or both in concert yielded similar degrees of attenuation, suggesting that both toxins target the same population of channels, at least in terms of channels associated with Na+ pump increases. In sum, this result implies that SACs equally sensitive to amiloride or Gd3+ open in response to cellular stretch and permit an influx of ions, which, in turn, stimulate increased Na+ pump activity. However, the observation that stretch could still induce a smaller but significant increase in Na+ pump activity despite carefully administered Gd3+ or amiloride treatment indicates that this SAC-originating signaling pathway may not account for the entire response.
Tonic stretch has little effect on Na+ pump activity.
In contrast to cyclically stretched cells, AT2 cells stretched to 25% ΔSA and held at that strain for 1 h showed no significant stretch response (103 ± 17% relative to control values of 100 ± 12%; Fig. 4). Furthermore, cells stretched cyclically with a baseline stretch of 12% ΔSA to a maximum deformation of 25% ΔSA for 1 h increased Na+ pump activity to 144 ± 12% over control of 100 ± 11% (Fig. 4). This Na+ pump response appears to correspond more closely to the cyclic fraction of this deformation (∼11.6% ΔSA from 12 to 25% ΔSA) than to the maximum stretch of 25% ΔSA (compare with cyclic stretch at 12 and 25% ΔSA in Fig.1).
2-Day cells are viable on fibronectin and 5-day ECM.
Unlike 1-day AT2 cells used in previous studies (68), 2-day AT2 cell monolayers showed very little denudation and suffered significantly less cell death after 1 h of cyclic stretch. 2-Day cells seeded on fibronectin and cyclically stretched at 12, 25, and 37% maintained viability of 93.9 ± 1.2, 86.5 ± 2.4, and 80.6 ± 2.1%, respectively. Even more robust, 2-day cells on 5-day ECM stretched at 25 and 37% ΔSA were 97.6 ± 0.9 and 94.6 ± 1.6% viable, respectively, i.e., significantly more viable than cells on fibronectin (P < 0.05). This agrees with previous studies, which reported a marked increase in viability for 1-day cells seeded on 5-day ECM over cells seeded on fibronectin (53). [For a full discussion of stretch and cell death see Tschumperlin and Margulies (68) and Oswari et al. (53).]
2-Day cells present a transitional phenotype independent of substrate.
After 48 h in culture, AT2 cells expressed the AT1 phenotypic indicator RTI-40 and the pro-SP-C surfactant peptide precursor (Fig.5). This transitional state was observed in all 2-day cells with no difference in the presence or amount of these phenotypic markers between cells on fibronectin and those on 5-day matrix. Occludin was also observed in equal abundance at the junctional boundaries of all 2-day cells regardless of substratum (Fig.5). The presence of occludin served as a phenotypic marker and also suggested that the cells had formed a barrier required for epithelial polarization and vectorial Na+ transport.
Cyclic stretch stimulates Na+ pump trafficking to the BLM.
To test the hypothesis that stretch increased Na+ pump activity by increasing the abundance of Na+ pumps in the plasma membrane, we compared Na+ pump content in BLMs of cells stretched cyclically for 1 h at 25% ΔSA with paired, unstretched controls. As shown in Fig. 6, Western blots of BLM Na+ pump α1-subunit from stretched and unstretched cells from three separate cell isolations show a marked increase (P < 0.01) in blot intensity with stretch with an average stretch-to-unstretched intensity ratio of 2.14. In contrast, whole cell homogenates of cells stretched cyclically for 1 h at 25% ΔSA showed no significant change in Na+ pump content. This indicates that stretch induces Na+ pump trafficking to the BLM from existing intracellular stores.
mRNA levels of the Na+-K+-ATPase α1-subunit diminish with stretch.
By isolating total cellular mRNA immediately after stretch to 25% ΔSA and amplifying the mRNA of the Na+ pump catalytic α1-subunit through relative RT-PCR, it was possible to capture a predictor of α1-subunit expression after AE stretch. Although blots for coamplified control samples of GAPD were unchanged, densities of Na+ pump α1-subunit cDNA blots diminished with stretch (P < 0.05) over four cell isolations, perhaps a premonition of a future decrease in Na+ pump population, and likewise Na+ pump activity, despite short-term increases.
Na+ pumps serve as the engine powering vectorial Na+ transport across the AE and thus play an integral role in alveolar edema prevention and, if alveolar edema occurs, fluid clearance (43, 47, 64). Because acute alveolar edema is known to be present with the onset of VILI (22, 26, 36, 39, 54,71, 75), it is important to understand how mechanical strain of the AE, as experienced in mechanical ventilation, impacts the Na+ pumps that are responsible for preventing and resolving alveolar fluid accumulation.
The present study advances understanding of mechanically induced effects with evidence that Na+ pumps are trafficked to the cell surface and Na+ pump activity increases in a “dose-dependent” manner with cyclic strain of the AE membrane within physiological ranges. This indicates that alveolar edema occurs during VILI, despite increased pump activity, not because of it, at least in the short term. Such a result helps define the focus of edema resolution strategies by reducing concerns of dysfunctional Na+ pumps and emphasizing the overall imbalance between Na+ pumps working to clear fluids from the air space on one side and an overwhelming increase in epithelial permeability, which allows fluids to leak into the air space, on the other. Of course, this change of focus by no means discounts the importance of Na+pumps. Studies have clearly shown that stimulation or overexpression of Na+ pumps can greatly improve edema clearance in injured lungs (5, 6, 30, 31, 35, 40, 52, 61-63) above and beyond natural stretch-induced increases in Na+ pump activity, tipping the balance in favor of clearance, despite greatly increased alveolar permeability. Furthermore, recent studies indicate that Na+ pump function plays a critical role in tight junction formation and maintenance, underscoring Na+ pump importance not only on the clearance side of the balance but also on the permeability side (57).
With evidence of a stretch-activated Na+ pump upregulation, we focused on determining how the mechanical stretch stimulus is translated into an intracellular biochemical signal. SACs, a subclass of mechanogated channels, have been implicated as the origin of many biochemical cascades (33), including stretch-induced Na+ pump upregulation in a transformed murine lung epithelial cell line (MLE-12) (74). Using Gd3+, a lanthanide metal with potent SAC blocking potential, Waters et al. (74) abolished the stretch-induced Na+ pump response, implying an integral role of SACs in mediating Na+ pump upregulation in the MLE-12 cell line. In contrast, the present study in primary AE cells found that Gd3+ blocking significantly attenuated Na+ pump upregulation but did not abolish the effect completely. Hence, we conclude that SACs play an important role in stretch-induced Na+ pump upregulation but do not account for the total response in primary cells, which often respond to the stretch stimulus differently from immortal, transformed cell lines (68). Given that the SAC-modulated signal may not completely account for observed Na+ pump upregulation, it is possible that mechanotransduction through focal adhesions and the cytoskeleton (CSK) could contribute to the remainder of the observed increases in Na+ pump activity.
We also found that blocking ENaCs with amiloride or blocking with both Gd3+ and amiloride yielded results similar to treating with Gd3+ alone. This similarity between Gd3+ and amiloride and the apparent redundancy of both suggest that both toxins may be targeting the same channels, at least within the population of channels involved in stretch-induced Na+ pump stimulation. Caution is warranted, however, in concluding precisely which channels these are. Although amiloride is generally enlisted for blocking ENaCs and Gd3+ for SACs, both toxins block a broad, overlapping spectrum of channels, including voltage-gated and mechanogated ENaCs, L- and T-type Ca2+ channels, and numerous nonselective cation channels (33). Because the Na+ pump changes are associated with a stretch stimulus, we can conclude that the channels are stretch activated, but it is impossible to pinpoint an ion such as Na+ or to rule out ions such as Ca2+ as potential signalers.
Once stretch stimulates the signaling cascade, Na+ pump activity may be upregulated by increased activity of individual pumps or by an increase in the number of functional pumps in the BLM. On one hand, it is plausible that Na+ entering the cell through apical SACs provides increased substrate to Na+ pumps, thus increasing their velocity. On the other hand, previous studies of the AE using pharmaceutical stimulation (8, 63) or stretch (74) linked increased Na+ pump activity to increased Na+ pump content in the BLM. In this study, we observed increases in BLM Na+ pump content of slightly over twofold (∼214%) with cyclic stretch to 25% ΔSA; this corresponds nicely to observed increases in Na+ pump activity of the same order (∼236%) under the same conditions. However, we cannot completely rule out the possibility that individual Na+pump velocity could have increased slightly in response to increased substrate as well. Cell permeabilization, a technique that would create high intracellular Na+ substrate and thus ensure maximum Na+ pump velocity, was not used in this study, because group I cation ionophores such as nystatin and gramicidin are also permeable to 86Rb+. Thus permeabilization would decrease the ratio of 86Rb+ passing through Na+ pumps (signal) to 86Rb+ passing through ionophoric channels (noise), making it difficult to interpret data we obtain by this technique. Furthermore, observed increases in BLM Na+ pump content suggest that ionic influxes trigger a complex trafficking mechanism. This contraindicates permeabilization, because, in addition to ensuring maximum Na+ pump velocity, it could also artificially overstimulate Na+ pump trafficking. Thus we conclude from our data that overall Na+ pump activity clearly increases in proportion to an increase in Na+ pumps in the BLM, but we cannot conclude definitively whether increased Na+ pump velocity might also augment this response.
We also found that although stretch increased Na+ pump content in the BLM, overall levels of the protein remained constant. This implies that stretch stimulates Na+ pump recruitment from existing intracellular stores rather than de novo transcription and reflects what other investigators have observed by stimulating Na+ pump activity pharmacologically or mechanically (8, 63, 74). Similar to these studies in the literature, we observed that Na+ pump activity increases within 60 min, which corroborates the theory of fast trafficking from existing stores rather than slower production of new proteins (8, 63, 74).
Differences in the Na+ pump response between cyclic and static stretch protocols may also provide insight into how Na+ pump activity increases with stretch and ultimately yield safer ventilation strategies. Our study shows that maintaining a static stretch of 25% ΔSA (80% total lung capacity) for 1 h has no significant impact on Na+ pump upregulation. Similarly, cyclic stretch from 12 to 25% ΔSA elicited a response similar to that caused by 12% ΔSA cyclic stretch and significantly less than that caused by 25% ΔSA cyclic stretch. This finding is consistent with viability results of Tschumperlin and Margulies (68), who observed a great reduction of AT2 cell mortality with tonic stretch relative to cyclic stretch to the same peak magnitude. Static deformations may provide an opportunity for the cell to reduce stress by remodeling the CSK via actin turnover (2) or the cell membrane via lipid trafficking (72). Cyclically stretched cells, in contrast, may not be held in the stretched position long enough to remodel their CSK or to traffic lipids and, thus, experience the full effect of stretch with each cycle. This implies that the application of a moderate tonic epithelial stretch, such as the baseline used in ventilation with positive end-expiratory pressure, has a small or, in the case of Na+ pump regulation, negligible effect on certain cell functions.
The present study also indicates that Na+ pump stretch response depends on the substratum on which the cells are tested. Although a positive correlation between Na+ pump activity and strain magnitude is evidenced on all substrata, cells grown on a 5-day ECM containing laminin, collagen, and fibronectin (27) tolerate a higher strain before the response appears than cells grown on fibronectin alone. Previous cell viability studies displayed a similar trend: cell mortality increased with strain, but cells grown on 5-day matrix tolerated higher strain than cells grown on fibronectin alone before similar mortality was observed (53). This apparent tolerance threshold may correspond to ECM-dependent modifications in the mechanical signal transduction path, such as different integrins involved, altered attachment protein density, or secondary CSK enhancements. An alternative explanation is that cells on 5-day matrix shifted phenotype more rapidly than cells on fibronectin and that the observed differences in Na+ pump response might reflect phenotype differences. However, because we detected no phenotype differences between cells on different substrata, it appears unlikely that phenotype accounts for substratum-dependent Na+ pump response.
The noted variation among cells on different substrata is indicative of the manifold challenges encountered in selecting a meaningful and tractable model. A variety of models have been used for testing the impact of stretch on Na+ pump regulation, but different models and methods have led to a variety of results. Sznajder et al. (67), looking at differences in ouabain-inhibitable ATP hydrolysis, reported an increase in Na+ pump activity in AT2 cells immediately after isolation from excised rat lungs that had been ventilated with high Vt for 25 min. However, in a similar study, Lecuona et al. (41, 42), using longer ventilation times (40 min), found a decrease in Na+ pump activity along with a corresponding decrease in edema clearance. On the other hand, Waters et al. (74) reported stretch-induced increases in Na+ pump activity in MLE-12 cells studied in vitro. The present study was designed to control for cell type, heterogeneity of applied deformation, and poststretch artifact and to focus on strain-magnitude effects. To that end, we used authentic primary cells, stretched the cells uniformly and equibiaxially at precise magnitudes, and assessed Na+ pump activity immediately after stretch with the cells still attached to the substratum.
Ultimately, the primary result of this study agrees with the studies of Waters et al. (74) with MLE-12 cells and with the studies of Sznajder et al. (67) with cells isolated from ventilated lungs but contrasts with the findings of Lecuona et al. (41, 42). Disparities possibly result from differences in the timing and treatment of cells after injury. Lecuona et al. stretched AT2 cells in situ by mechanical ventilation but then had to isolate AT2 cells from the lung before measuring uptake, whereas we were able to measure Na+ pump activity in an adherent, polarized epithelium immediately after stretch without otherwise disturbing the cells. Although the in vivo environment is clearly ideal for emulating VILI, it does not permit measurement of Na+pump activity immediately after stretch. Instead cells must undergo a lengthy and stressful isolation process, which removes them from the substratum completely, before Na+ pump activity can be evaluated. It has been shown that Na+ pumps are trafficked from intracellular stores and inserted into the cell membrane, and it is suggested that a reverse process of reabsorption from the membrane accounts for decreases in cellular Na+ pump activity (8); the stress of the isolation process and the disorientation of typically polar epithelial cells in suspension could possibly trigger such a reabsorption. Another possibility is that the two studies sample the same process at different time points. Lecuona et al. (42) note a decrease in Na+ pump activity but report no change in Na+ pump α1-subunit mRNA levels. The present study, evaluating the stretch effect immediately after stretch is ceased, notes increased pump activity but a reduction in mRNA, a possible prelude to the downstream scenario observed by Lecuona et al.
Although these differences in results primarily reflect disparity between the experimental models and methods used, they may also provide valuable insight into the underlying physiology. If Na+pumps are upregulated for the short term, but the intracellular mRNA is downregulated, the defense mechanism may be short lived. In addition, our data show that stretch reduces the intracellular store of ATP (12), the fuel required for the pumping process, so that stretch-induced Na+ pump upregulation may be a defense mechanism of only short-term efficacy.
In summary, we found that cyclic stretch of the AE stimulates ionic fluxes through SACs, which can be blocked by Gd3+ or amiloride. In response, the cell traffics Na+ pumps from intracellular stores to the cell membrane, where their augmented number serves to increase overall Na+ pump activity. Additionally, we report that stretch-induced upregulation of Na+ pump activity is subject to a number of factors, including substratum composition and stretch magnitude and mode. At the same time, we raise several questions that require further study, including why AT2 cells seem relatively insensitive to static stretch and what role integrins and CSK might play in signaling and Na+ pump trafficking. Finally, our study indicates that stretch-induced upregulation occurs but may not adequately counter the flux of fluids into the alveoli allowed by increased epithelial permeability or that the upregulation is possibly too short lived. Although it is not clear how this in vitro stretch-induced response translates into overall alveolar transport and why edema persists during VILI, our findings emphasize the importance of both sides of the balance, minimizing the initial disruption of epithelial integrity and sustaining Na+ pump upregulation, to reduce the incidence of alveolar edema.
We thank Dr. K. Ridge (Northwestern University) for providing a protocol for BLM isolation and for patient advice and suggestions on everything from antibodies to apparatus while we mastered the technique.
Support for this study was provided by National Heart, Lung, and Blood Institute Grant R01-HL-527204. J. L. Fisher is supported by a Graduate Fellowship from the Whitaker Foundation.
Address for reprint requests and other correspondence: S. S. Margulies, Dept. of Bioengineering, University of Pennsylvania, 3320 Smith Walk, Philadelphia, PA 19104 (E-mail:).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
May 17, 2001;10.1152/ajplung.00030.2001
- Copyright © 2002 the American Physiological Society