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

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 Ca 2+ -activated K + channel inhibitor), suggesting that both PC and SC were generated through a common PKA-dependent/Ca 2+ -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.