Differential effects of cyclic and constant stress on ATP release and mucociliary transport by human airway epithelia

Abstract

In the lungs, the first line of defence against bacterial infection is the thin layer of airway surface liquid (ASL) lining the airway surface. The superficial airway epithelium exhibits complex regulatory pathways that blend ion transport to adjust ASL volume to maintain proper mucociliary clearance (MCC). We hypothesized that stresses generated by airflow and transmural pressures during breathing govern ASL volume by regulating the rate of epithelial ATP release. Luminal ATP, via interactions with apical membrane P2-purinoceptors, regulates the balance of active ion secretion versus absorption to maintain ASL volume at optimal levels for MCC. In this study we tested the hypothesis that cyclic compressive stress (CCS), mimicking normal tidal breathing, regulates ASL volume in airway epithelia. Polarized tracheobronchial epithelial cultures from normal and cystic fibrosis (CF) subjects responded to a range of CCS by increasing the rate of ATP release. In normal airway epithelia, the CCS-induced increase in ASL ATP concentration was sufficient to induce purinoceptor-mediated increases in ASL height and MCC, via inhibition of epithelial Na(+)-channel-mediated Na(+) absorption and stimulation of Cl(-) secretion through CFTR and the Ca(2+)-activated chloride channels. In contrast, static, non-oscillatory stress did not stimulate ATP release, ion transport or MCC, emphasizing the importance of rhythmic mechanical stress for airway defence. In CF airway cultures, which exhibit basal ASL depletion, CCS was partially effective, producing less ASL volume secretion than in normal cultures, but a level sufficient to restore MCC. The present data suggest that CCS may (1) regulate ASL volume in the normal lung and (2) improve clearance in the lungs of CF patients, potentially explaining the beneficial role of exercise in lung defence.

Figure 1. System generating positive static and cyclic compressive stress on human airway cultures

A, a microprocessor controlled the pressure amplitude and timing (in the case of CCS) of transepithelial pressure to the apical surface of the cultured human airway epithelial cells. B, sample pressure tracing from a culture exposed to SCS (top) at 20 cmH₂O and CCS (bottom) at 20 cmH₂O for 1 s, every 3 s. See text for details.

Figure 2. Cyclic, but not static compressive stress increases steady-state ATP level in CF airway cultures

A, relationship between compressive stress pressure amplitude and apical ATP concentration during CCS (□, n=9) and SCS (▴, n=7). Measurements made from individual cultures after 30 min of compressive stress at the indicated pressure. B, mean basolateral ATP concentration obtained after 1 h of control conditions (n=7), CCS (20 cmH₂O, 20 CPM, n=7) or SCS (20 cmH₂O, n=5). C, ATP release rates in normal (open bars) and CF (filled bars) airway cultures under control (Ctrl) conditions, or after 30 min of CCS (20 cmH₂O, 20 CPM) or SCS (20 cmH₂O), in the presence of a cocktail of ATPase inhibitors (30 μm ebselen and 300 μm βγMe-ATP) (n=4 in each group). D, apical ectonucleotidase activities measured with 10 μm ATP after 1 h exposures to control, CCS (20 cmH₂O, 20 CPM) or SCS (20 cmH₂O) conditions (n=5 in each group). (*Significantly different from control cultures at ambient pressure.)

Figure 3. Compressive stress does not affect cell viability

A, representative fluorescence micrographs of CF airway cultures after 1 h under control (ambient pressure), CCS (20 cmH₂O, 20 CPM), or SCS (20 cmH₂O) after staining with calcein-AM (viable cells stain green) and ethidium homodimer-1 (non-viable cells stain red). As a positive control, cell permeabilization with digitonin (50 μm) produced a large change in fluorescence, consistent with cell death. B, summary of results for ethidium homodimer-1 labelled cells. (n=6 in each group.)

Figure 4. Compressive stress does not affect tight junctional integrity

A, mean transepithelial resistance, as a measure of tight junctional integrity, of CF cultures under CCS for 1 h at various pressure amplitudes (n=8). B, time course of transepithelial resistance over 24 h CCS (□, 20 cmH₂O, 20 CPM, n=8) or SCS (•, 20 cmH₂O, n=6), relative to controls at ambient pressure, relative to control cultures at ambient pressure. (*Significantly different from control cultures.)

Figure 5. CCS, but not SCS induces net liquid secretion in normal and CF airway epithelial cultures

A, XZ confocal images of ASL at 0, 6 and 48 h after mucosal addition of 30 μl TBR containing Texas red–dextran to normal and CF airway cultures under control conditions and CF cultures undergoing CCS (20 cmH₂O, 20 CPM) or SCS (20 cmH₂O). Scal bar=10 μm. B and C, time course of changes in ASL height on normal (B) and CF (C) cultures under control (•), CCS (□), or SCS (▴) conditions. (n=6 in each condition.) Dotted line denotes ASL height of normal airway cultures in steady-state conditions. D, mean ASL height of CF cultures (at 24 h) under control, SCS, or CCS conditions in the absence and presence of apyrase (Apy; 5 U ml⁻¹, n=9) or bumetanide (10⁻⁵m, n=6). (*Significantly different than control cultures. †Significantly different from CCS alone.)

Figure 6. CCS alters the pattern of Na⁺ and Cl⁻ bioelectrics in normal and CF airway cultures

Bar graphs depicting total potential difference (V_t) (open bars) and changes in V_t in response to sequential application of bumetanide (10⁻⁴m, basolateral; grey bars) and benzamil (10⁻⁵m, apical; black bars) in normal (A, n=5) and CF (B, n=6) cultures under control, CCS (20 cmH₂O, 20 CPM) or SCS (20 cmH₂O) at 0 and 24 h after mucosal addition of 30 μl TBR. (*Significantly different from control cultures (Ctrl) at 24 h.)

Figure 7. CCS, but not SCS, increases cilia beat frequency (CBF) and mucociliary transport (MCT) rates

A, summary of CBF measurements from CF cultures after 1 h control (Ctrl, n=10), SCS (20 cmH₂O, n=7) or CCS (20 cmH₂O, 20 CPM, n=10) alone or in the presence of apyrase (CCS + APY, n=5). B, rates of MCT after the addition of 30 μl TBR to CF cultures under control (•), CCS (□, 20 cmH₂O, 20 CPM), or SCS (▴, 20 cmH₂O) (n=7 in each group). (*Significantly different from control cultures. †Significantly different from CCS alone.)

Figure 8. Long-term preservation of MCT on cultured CF airway epithelium undergoing CCS

A, representative 2 s exposures of fluorescent microspheres on CF airway cultures 48 h after the addition of 30 μl TBR under control and CCS (20 cmH₂O). A similar exposure from a normal culture, at 48 h, is shown for comparison. Scale bar=100 μm. B, summary of MCT rates before (control; open bars) and after 48 h (filled bars) of control, SCS (20 cmH₂O), or CCS (20 cmH₂O, 20 CPM). n=5–7 in each group. (*Significantly different from control cultures (Ctrl) at 48 h.)