Intracellular Hydrogen Peroxide-mediated Responses under Acidic Condition in Primary Cultured Feline Esophageal Epithelial Cells

Sun Young Park, Minji Sohn, Uy Dong Sohn. Chung-Ang University, Pharmacology 156-756, Republic of Korea

One component of the esophageal luminal surface layer is the epithelial lining. The epithelial lining serves not only as a barrier or protective layer, but also as a first damaged layer in response to various stresses such as food bolus, or gastric refluxates in gastroesophageal reflux diseases. In this study, we examined the responses of esophageal epithelial cells (EEC) when exposure to acidified medium at pH 4 (AM4) in the feline. Primary cultured EEC was used and MTT assay was carried out for measurement of cytotoxicity by AM4 compared to acidified medium at pH 5.5 (AM5.5) or at pH 6. Intracellular hydrogen peroxide production was measured, and activation of mitogen-activated protein kinase (MAPK) by AM4 was analyzed by Western blotting. Under acidic condition, cellular viability was shown pH-dependently, and was further fallen in the presence of N-acetyl-L-cysteine or 5-(N-ethyl-N-isopropyl)amiloride, an inhibitor of the Na+/H+ exchanger. The acid-induced generation of intracellular hydrogen peroxide was prevented by a nonspecific reactive oxygen species scavenger N-acetyl-L-cysteine, an inhibitor of NADPH oxidase diphenyleneiodonium or apocynine, but not by the mitochondrial complex I inhibitor rotenone. AM4 maximally induced phosphorylation of MAP/ERK Kinase 1/2, ERK1/2, p38 MAPK and Akt at 5 min exposure. Phosphorylation of MEK1/2, ERK1/2 and Akt induced by AM4 was declined by the N-acetyl-L-cysteine, diphenyleneiodonium or apocynine. Taken together, under conditions to low pH, H+ may enter across the cell membrane and intracellular pH of EEC may be regulated by acid-extruding mechanism via the Na+/H+ exchanger with contributing to cell survival. AM4-induced generation of intracellular hydrogen peroxide may originate from activation of NADPH oxidase and mediate the phosphorylation of MEK1/2, ERK1/2 or Akt, then subsequently participate in signaling of cell survival under condition of low pH.