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TOXICOLOGY

Dual Pathway Activated by tert-Butyl Hydroperoxide in Human Airway Anion Secretion

Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan

We analyzed the mechanisms underlying the ion transport induced by tert-butyl hydroperoxide (t-BOOH), a membrane-permeant oxidant that has been widely used as a model of oxidative stress, in human airway epithelial cells (Calu-3). We found that t-BOOH induced a short-circuit current that was composed of two distinct components, a peaked component (PC) and a sustained component (SC). Both components were reduced by the presence of H-89 (N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline) [10 µM, a protein kinase A (PKA) inhibitor] and clofilium (100 µM, a cAMP-dependent K⁺ channel inhibitor) but not by charybdotoxin (50 nM, a human intermediate conductance Ca²⁺-activated K⁺ channel inhibitor), suggesting that both PC and SC were generated through a common PKA-dependent/Ca²⁺-independent pathway. Notwithstanding, analyses of the physiological properties revealed that PC and SC were attributable to different pathways. PC, but not SC, was correlated with apical membrane Cl^- conductance and was inhibited by the cyclooxygenase (COX)-2 inhibitor NS-398 (N-[2-(cyclohexyloxyl)-4-nitrophenyl]-methane sulfonamide; 10 µM). In contrast, SC, but not PC, was composed of a component sensitive to bumetanide (50 µM), an inhibitor of the basolateral Na⁺-K⁺-2Cl^- cotransporter (NKCC1), and was abolished by the cytoskeleton dysfunction induced by cytochalasin D (10 µM) and (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexane carboxamide (Y-27632; 20 µM). Collectively, t-BOOH induces PKA-related anion secretion through two independent pathways: rapid activation of apical anion efflux through a COX-2-dependent/cytoskeleton-independent pathway and relatively delayed activation of NKCC1 for basolateral anion uptake through a COX-2-independent/cytoskeleton-dependent pathway.

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