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.
|Number of pages||11|
|Journal||Journal of Pharmacology and Experimental Therapeutics|
|Publication status||Published - 11-2008|
All Science Journal Classification (ASJC) codes
- Molecular Medicine