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

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 (similar to 7 mu m) 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 ectonucleotidases resulted in ATP accumulation at a rate of similar to 250 fmol/min/cm(2), reflecting the basal ATP release rate. Following hypotonic challenge to promote cell swelling, cell-surface ATP concentration measured by SPA-luc transiently reached similar to 1 mu M 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 P2Y(2) receptor activation by physiological stimuli and identify a role for mucosal ATP release in airway epithelial cell volume regulation.