Physiological regulation of ATP release at the apical surface of human airway epithelia

Abstract

Extracellular ATP and its metabolite adenosine regulate mucociliary clearance in airway epithelia. Little has been known, however, regarding the actual ATP and adenosine concentrations in the thin ( approximately 7 microm) liquid layer lining native airway surfaces and the link between ATP release/metabolism and autocrine/paracrine regulation of epithelial function. In this study, chimeric Staphylococcus aureus protein A-luciferase (SPA-luc) was bound to endogenous antigens on primary human bronchial epithelial (HBE) cell surface and ATP concentrations assessed in real-time in the thin airway surface liquid (ASL). ATP concentrations on resting cells were 1-10 nm. Inhibition of ecto-nucleotidases resulted in ATP accumulation at a rate of approximately 250 fmol/min/cm2, reflecting the basal ATP release rate. Following hypotonic challenge to promote cell swelling, cell-surface ATP concentration measured by SPA-luc transiently reached approximately 1 microm independent of ASL volume, reflecting a transient 3-log increase in ATP release rates. In contrast, peak ATP concentrations measured in bulk ASL by soluble luciferase inversely correlated with volume. ATP release rates were intracellular calcium-independent, suggesting that non-exocytotic ATP release from ciliated cells, which dominate our cultures, mediated hypotonicity-induced nucleotide release. However, the cystic fibrosis transmembrane conductance regulator (CFTR) did not participate in this function. Following the acute swelling phase, HBE cells exhibited regulatory volume decrease which was impaired by apyrase and facilitated by ATP or UTP. Our data provide the first evidence that ATP concentrations at the airway epithelial surface reach the range for P2Y2 receptor activation by physiological stimuli and identify a role for mucosal ATP release in airway epithelial cell volume regulation.

FIGURE 1. Process of SPA-luc purification

Coomassie Blue-stained gels of fractions from the indicated purification steps (2 μl of protein/lane) are shown. Lanes MM and 1–4, molecular marker (MM), soluble fraction from E. coli crude lysate (lane 1), resuspended pellet of 55% ammonium sulfate solution (2), pooled fractions 13–31 of the first Ni²⁺ column (3), and flow-through of the third Ni²⁺ column (lane 4). Lanes 3 and 4 represent desalted and concentrated product.

FIGURE 2. Optimization of SPA binding to HBE cultures

The localization of KS (A), MUC1 (B), and WGA (C) were studied in primary HBE cells as indicated under “Experimental Procedures.” Anti-KS antibody and WGA bound to the apical plasma membrane (arrow) and cilia (arrowhead), while MUC1 antibody recognized mostly the apical plasma membrane (arrow). Bar = 10 μm. D, binding of SPA-HRP to apical HBE cell surface as quantitated with the colorimetric reaction of HRP and o-phenylenediamine under varied antibody/lectin concentrations. Live primary HBE cultures were incubated with anti-KS antibody (blue diamond), anti-MUC1 antibody (orange triangle), or biotinylated WGA (green circle) at indicated concentration. WGA binding quantitation required incubation with 2 μg/ml anti-biotin antibody. All samples were subsequently incubated with 1 μg/ml SPA-HRP (solid symbols) or buffer (open symbols). Values are mean ± S.E. of three Transwells/subject established from two different subjects.

FIGURE 3. ATP sensitivity of antibody/lectin-protein A-luciferase complexes

A, primary HBE cells were first incubated with buffer (open squares), anti-KS antibody (10 μg/ml) (solid diamonds), anti-MUC1 antibody (20 μg/ml) (gray triangles), or biotinylated WGA (4 μg/ml) and subsequent anti-biotin antibody (2 μg/ml) (open circles). The cultures were subsequently incubated with SPA-luc (0.5 mg/ml) and rinsed as described under “Experimental Procedures.” Changes in luminescence in response to the addition of ATP of varied concentrations were measured in real-time with 10-s integration. Values are mean ± S.E. of 3 Transwells/subject established from three different subjects. †, *, and ‡ indicates significant difference (p < 0.05) between specific (via KS, WGA, and MUC1, respectively) and nonspecific SPA-luc attachment. B, ATP calibration curves were generated with 500 μg/ml (gray circles), 50 μg/ml (open triangles), 25 μg/ml (gray squares), 10 μg/ml (open circles), 5 μg/ml (gray triangles), and 0.5 μg/ml (white squares) SPA-luc in solution (100 μl on 12-mm Transwell). Values are mean ± S.E. of two separate experiment with n = 3 for each. The ATP sensitivity curve from HBE cell-attached SPA-luc labeled with KS (solid diamonds, same as the “KS” curve in Fig. 3A) was aligned best with bulk 10 μg/ml. ALU values in Fig. 3 and those in Table 1 were obtained under different assay conditions (e.g. a different luminometer, buffer, luciferin/luciferase amount, and dilutions) and thus resulted in different scales of values.

FIGURE 4. ATP concentrations on resting HBE cell surface

ATP concentrations on resting primary HBE surfaces in varied ASL volumes, as measured by off-line measurement of pipetted or micro-sampled samples (gray triangles), real-time measurement by luciferase dissolved in bulk ASL (open circles), and real-time measurement by cell-attached SPA-luc (via KS, solid diamonds), respectively. Values are mean ± S.E. of 4 Transwells/subject established from three different subjects.

FIGURE 5. ATP release rates from resting HBE cells

A, following mucosal preincubation (2 min) with vehicle or ecto-nucleotidase inhibitors, 500 nM ATP was added to 300 μl of ASL on resting HBE cells and its hydrolysis rate measured by off-line luminometry of samples taken at indicated time points. Addition of inhibitors (β, γ-methylene-ATP (300 μM; open diamonds), levamisole (10 mM; solid triangles), ebselen (30 μM; open circles), or the three inhibitors together (solid circles)) resulted in significant delay in ATP hydrolysis compared with control (solid diamonds). B, ATP accumulation following addition of the inhibitor mixture (at t = 0) was measured by soluble luciferase (open symbols) or cell-attached SPA-luc (via KS, solid symbols) in 25 μl (circles) or 50 μl (diamonds) ASL. As controls, vehicle was added to cells (open triangles), or the inhibitor mixture was added to wells without cells (gray squares), and luminescence measured in 50 μl with soluble luciferase. C, summary data illustrating basal ATP accumulation (nM/min) following addition of the inhibitor mixture as measured by soluble luciferase (open circles) and cell-attached SPA-luc (diamonds) in varied ASL volumes. In A–C, values are mean ± S.E. of 3–4 Transwells/subject established from three different subjects. Data without cells are mean ± S.E. of two separate experiments with n = 3 for each.

FIGURE 6. Hypotonicity-induced ATP release

Primary HBE cultures were exposed to luminal 33% hypotonic challenge (A–D) or an isosmotic challenge (E–G). ATP concentrations were measured by different techniques and varied ASL volumes (i.e. 500 μl (open triangles), 300 μl (gray diamonds), 100 μl (solid triangles), 50 μl (open circles), and 25 μl (solid circles)); A and E, off-line luminometry of samples; B and F, real-time luminometry with soluble luciferase; C, and G, real-time luminometry with cell-attached SPA-luc via KS; D, real-time luminometry with cell-attached SPA-luc via MUC1 (gray triangles) or WGA (open circles) in 50 μl of ASL. H, summary data illustrating peak ATP concentration as measured by soluble luciferase (open circles) and cell-attached SPA-luc (via KS, solid diamonds) in varied ASL volumes. In A–H, values are mean ± S.E. of 4 Transwells/subject established from three to four different subjects.

FIGURE 7. Cell volume regulation and ATP release rates following hypotonic challenge

A, representative images of calcein-labeled primary HBE cells in swelling and RVD phases following hypotonic challenge. Top panels, 33% hypotonic challenge; second and third panels, cultures were pretreated with apyrase (10 units/ml) for 5 min or 8-SPT (100 μM) for 30 min, respectively, and H₂O (i.e. 33% hypotonic challenge) including those reagents added at t = 0; bottom panels, UTP (100 μM) was added at t = 12 s following hypotonic challenge at t = 0. Bar, 10 μm. B, quantitative data from protocols shown in A of HBE cells treated with vehicle (green diamonds), apyrase (red circles), 8-SPT (blue triangles) or UTP (black circles). * and † indicate significant difference (p < 0.05) between the indicated point and a time-matched vehicle point. C, to measure the accumulation rates of released ATP, the nucleotidase inhibitor mixture (i.e. 300 μM β, γ-methylene-ATP, 30 μM ebselen, and 10 mM levamisole) were added to ASL (50 μl) containing soluble luciferase at t = 0 and 33% hypotonic challenge applied at t = 5 min. The inset is the magnified scale for the ATP concentration at t = −1–5 min. D, initial rates of ATP release following hypotonic challenge (black circles) were calculated from ATP accumulation rates in the presence of the nucleotidase inhibitors (in C) and aligned in comparison with the rate of cell height change (green diamonds;+, increase;−, decrease) measured in calcein-labeled cells. In the inset, data points for ATP accumulation rates represent values of every 0.2 s for 2 s, every 0.4 s for next 4 s, then every second for next 9 s, calculated from the raw data acquired every 0.2 s; data points for cell height change represent values of every second from the raw data acquired every second. In B–D, values are mean ± S.E. of 4 Transwells/subject established from three different subjects.

FIGURE 8. Ca²⁺_i dependence of hypotonicity-induced ATP release

A, representative Ca ²⁺_i tracings depicting the effect of apical hypotonic challenge (33%) and UTP (100 μM) in Fura-2-loaded primary HBE cultures loaded with BAPTA (100 μM) (dotted line) or vehicle (solid line). B, hypotonicity (at t = 0) induced ATP release from HBE cultures loaded with BAPTA (100 μM) (open circles) or vehicle (solid circles) as measured by soluble luciferase in 50 μl of ASL. The inset is the magnified scale for ATP concentrations measured during t = −1 to 0 min. * indicates significant difference (p < 0.05) between values of BAPTA- and vehicle-loaded cultures at the indicated time point. C, the cell height change following hypotonic challenge (i.e. swelling and RVD properties) quantitated in HBE cultures loaded with BAPTA (100 μM) (open circles) or vehicle (solid circles) with the same protocol as Fig. 7. In B and C, values are mean ± S.E. of 4 Transwells/subject established from three different subjects.

FIGURE 9. Hypotonicity-induced ATP release from CF and normal (non-CF) HBE cells

A, hypotonicity (at t = 0) induced ATP release from CF (open circles), age-matched normal (solid circles), and inh-172-treated (10 μM, 30 min) normal (open triangles) HBE cultures as measured by soluble luciferase in 50 μl of ASL. The inset is the magnified scale for ATP concentrations measured during t = −1 to 0 min. B, the cell height change following hypotonic challenge was quantitated in CF (open circles) and normal (inh-172-untreated, solid circles) HBE cultures with the same protocol as Fig. 7. Values are mean ± S.E. of four Transwells/subject established from three different subjects.