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ATP Release Triggered by Activation of the Ca²⁺-Activated K⁺ Channel in Human Airway Calu-3 Cells

Division of Respiratory Diseases, Department of Internal Medicine, and Department of Cellular Physiology, Nagoya University Graduate School of Medicine, Nagoya, Japan

Address correspondence to: Yasushi Ito, M.D., Division of Respiratory Diseases, Department of Internal Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: itoyasu{at}med.nagoya-u.ac.jp

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 (hIK-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 1-EBIO– and CZ-stimulated cytosolic Ca²⁺ mobilization was suppressed by the presence of MRS-2179, a specific P2Y₁ receptor antagonist. The hIK-1–mediated ATP release was inhibited by a hIK-1 blocker (charybdotoxin), and an Na⁺-K⁺-2Cl^- 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 hIK-1–mediated KCl loss and the resultant induction of a regulatory volume increase via the Na⁺-K⁺-2Cl^- transporter may trigger release of ATP, which causes P2Y₁-mediated Ca²⁺ mobilization, through mechanisms unrelated to the CFTR and SA channels.

Abbreviations: 1-ethyl-2-benzimidazolinone, 1-EBIO • bumetanide, BMT • cystic fibrosis, CF • cystic fibrosis transmembrane conductance regulator, CFTR • charybdotoxin, ChTx • chlorzoxazone, CZ • chronic obstructive lung disease, COPD • 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, DIDS • forskolin, FK • human intermediate conductance, Ca²⁺-activated K⁺ channel, hIK-1 channel • short-circuit current, I_sc • isoproterenol, ISO • intracellular Ca²⁺ concentration, [Ca²⁺]_i • G protein–coupled purinergic receptors, P2Y • potential difference, PD • physiologic saline solution, PSS • regulatory volume increase, RVI • regulatory volume decrease, RVD • stretch-activated nonselective cation channels, SA channels • volume-sensitive large conductance ATP-permeable anion channel, VDACL

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