EGF mediates calcium-activated chloride channel activation in the human bronchial epithelial cell line 16HBE14o-: involvement of tyrosine kinase p60c-src.

Particulate atmospheric pollutants interact with the human airway epithelium, which releases cytokines, chemokines, and EGF receptor (EGFR) ligands leading to proinflammatory responses. There is little information concerning the short-term effects of EGFR activation by extracellular ligands on ionic regulation of airway surface lining fluids. We identified in the membrane of human epithelial bronchial cells (16HBE14o(-) line) an endogenous calcium- and voltage-dependent, outwardly rectifying small-conductance chloride channel (CACC), and we examined the effects of EGF on CACC activity. Ion channel currents were recorded with the patch-clamp technique. In cell-attached membrane patches, CACC were activated by exposure of the external surface of the cells to physiological concentrations of EGF without any change in cytosolic Ca(2+) concentration ([Ca(2+)](i)) and inhibited by tyrphostin AG-1478 (an inhibitor of EGFR that also blocks EGF-dependent Src family kinase activation). EGF activation of c-Src protein in 16HBE14o(-) cells was observed, and the signaling pathway elicited by EGFR was blocked by tyrphostin AG-1478. In excised inside-out membrane patches CACC were activated by exposure of the cytoplasmic face of the channels to the human recombinant Src(p60(c-src)) kinase with endogenous or exogenous ATP and inhibited by lambda-protein phosphatase. Secretion of EGFR ligands by epithelial airway cells exposed to pollutants would then elicit a rapid and direct ionic response of CACC mediated by EGFR activation via a Src kinase family-dependent signaling pathway.

Oxidant stress stimulates Ca2+-activated chloride channels in the apical activated membrane of cultured nonciliated human nasal epithelial cells.

Respiratory tissues can be damaged by the exposure of airway epithelial cells to reactive oxygen species that generate oxidative stress. We studied the effects of the hydroxyl radical *OH, for which there is no natural intra- or extracellular scavenger, on a Ca(2+)-activated chloride channel (CACC) that participates in Cl(-) secretion in the apical membrane of airway epithelial cells. We identified and characterized CACC in cell-attached and in inside-out excised membrane patches from the apical membrane of cultured nonciliated human nasal epithelial cells. In these cells, the CACC was outwardly rectified, Ca(2+)/calmodulin-kinase II, and voltage dependent. The channel was activated in cell-attached and inside-out patches in a bath solution containing millimolar [Ca(2+)] and ran down quickly. The channel was reversibly or irreversibly activated by exposure of the internal surface of the membrane to *OH, which depended on the concentration and the duration of exposure to H(2)O(2). CACC activity evoked by oxidative stress was inhibited by 1,3-dimethyl-2-thiurea, an antioxidant that scavenges hydroxyl radicals, and by the reduced form of glutathione. The oxidized SH residues could be close to the Ca(2+)/calmodulin kinase site. The reversible or irreversible activation of CACC after a period of oxidative stress without change in [Ca(2+)] is a new observation. CACC play a direct role in mucus production by goblet cells and may thus contribute to the pathogenesis of asthma.

Effects of hydrogen peroxide and hydroxyl radicals on the cytosolic side of a non-selective cation channel in the cultured human bronchial epithelial cell line 16HBE14o-.

Respiratory tissues can be damaged by the exposure of airway epithelial cells to reactive oxygen species (ROS) that generate an oxidative stress. We studied the effects of the hydroxyl radical *OH, for which there is no natural intra- or extracellular scavenger, on a Ca2+-activated, non-selective cation channel (NSC(Ca)) which might participate in the transepithelial Na+ fluxes involved in the maintenance of the cytosolic [Na+] ([Na+]i). We identified and characterized NSC(Ca) in inside-out excised membrane patches from cells of the human bronchial cell line 16HBE14o- and exposed the cytoplasmic side of NSC(Ca) to H2O2 or *OH created in front of the patch pipette. In these cells, the NSC channel was Ca2+ (above 0.1 microM) and voltage dependent. The channel's low open probability ( P(o)) at hyperpolarized voltages increased rapidly to a very high value upon short exposure (seconds) to *OH or to H2O2 in a bath solution containing millimolar [Ca2+]. This sensibility to ROS was reversible on wash-out of the oxidants. Long exposure (several minutes) to *OH in a bath solution containing 10(-7) M Ca2+ activated the channel irreversibly, a finding that has pathophysiological implications. The functioning of the NSC(Ca) protein channel may be modified by the intracellular redox status and the reversibility of the sensitivity to ROS depends on [Ca2+]i. The role of NSC(Ca) channels in airway epithelia is not yet understood, but might conform to what might be expected of an NSC channel: increase in [Na]i following intracellular exposure to *OH or H2O2. Reversible or irreversible excessive opening of cation channels may induce gradual cellular responses, including changes in Na+/K+-ATPase activity, cell volume regulation, membrane polarization, activation of transcription factors required for the expression of genes of cytokines or chemokines, apoptosis and necrosis, thus triggering inflammation.