ATP release triggered by activation of the Ca2+-activated K+ channel in human airway Calu-3 cells

Airway mucociliary clearance is subject to the autocrine/paracrine regulation of extracellular nucleotides released from the airway epithelial cells. The present study was performed in pursuit of effective modulators of ATP release under physiologic conditions in polarized human airway epithelial cells (Calu-3). Neither isoproterenol, forskolin, nor ionomycin augmented extracellular ATP release detected by luciferase assay. However, direct activation of the human intermediate conductance, Ca2+-activated K+ channel (hlK-1) by 1-ethyl-2-benzimdazolinone (1-EBIO, 1 mM) and chlorzoxazone (CZ, 1 mM) increased ATP release predominantly in the apical compartment. Measurement of fluo-3 signals revealed that I-EBIO-and CZ-stimulated cytosolic Ca2+ mobilization was suppressed by the presence of MRS-2179, a specific P2Y(1) receptor antagonist. The hlK-1-mediated ATP release was inhibited by a hlk-1 blocker (charybdotoxin), and an Na+-K+-2CI(-) cotransport blocker (bumetanide) without interruption by GdCl3, an inhibitor of stretch-activated nonselective cation (SA) channels, or glybenclamide, a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR). These results suggest that a cell volume decrease via the hlk-1-mediated KCl loss and the resultant induction of a regulatory volume increase via the Nal(+)-K+-2Cl(-) transporter may trigger release of ATP, which causes P2Y(1)-mediated Ca2+ mobilization, through mechanisms unrelated to the CFTR and SA channels.