Received for publication May 30, 2008.
Revised July 27, 2008.
Accepted for publication July 28, 2008.

Dual pathway activated by tert-butyl hydroperoxide in human airway anion secretion

Abstract

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 (I_SC) 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 (10 µM, a protein kinase A [PKA] inhibitor) and clofilium (100 µM, a cyclic AMP-dependent K⁺ channel [KCNQ1] inhibitor) but not by charybdotoxin (100 nM, a Ca²⁺-dependent K⁺ channel [KCNN4] 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 cycloxygenase-2 (COX-2) inhibitor NS-398 (10 µM). In contrast, SC, but not PC, was composed of a component sensitive to bumetanide, an inhibitor of the basolateral Na⁺-K⁺-2Cl^- cotransporter (NKCC1), and was abolished by the cytoskeleton dysfunction induced by cytochalasin D (20 µM) and 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.

Key words: NKCC1, airway anion secretion, cyclooxygenase, cytoskeleton, short-circuit current, tert-butyl hydroperoxide