Inhibitory effect of rosiglitazone on the acid-induced intracellular generation of hydrogen peroxide in cultured feline esophageal epithelial cells

Abstract

Peroxisome proliferator-activated receptor-gamma (PPAR gamma) agonists have been reported to enhance antioxidant defenses by increasing levels of catalase and copper-zinc superoxide dismutase (Cu/Zn SOD) in oligodendrocyte progenitor cells. In this study, we investigated the effects of the PPAR gamma agonist, rosiglitazone, on hydrogen peroxide (H2O2) generation by acidified medium at pH 5.5 (AM5.5), which is in the pH range of duodenogastric refluxates, in primary cultured feline esophageal epithelial cells (EEC). Successful isolation of EEC was identified by immunocytochemistry. AM5.5- and rosiglitazone-induced cell viabilities were determined using 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide assays. The NAD(P)H oxidase activity was measured, and expression of catalase or SOD protein by AM5.5 in the absence and presence of rosiglitazone was assessed using western blotting analysis. PPAR gamma protein and mRNA were constitutively expressed in EEC. In the incubation with rosiglitazone alone, cell viability was shown more than 90% at 0-10 mu M for 72 h. After exposure to AM5.5 for 8 h, intracellular H2O2 was significantly generated. Treatment with rosiglitazone prior to and during exposure to AM5.5 inhibited the H2O2 generation whereas the specific PPAR gamma antagonist GW9662 offsets the inhibitory action of rosiglitazone. H2O2 generation was also prevented by a nonspecific ROS scavenger N-acetylcysteine or an inhibitor of NADPH oxidase diphenyleneiodonium. The enhanced AM5.5-induced NAD(P)H oxidase activity was not suppressed by rosiglitazone. Instead, the pretreatment of rosiglitazone enhanced the protein expression of catalase, Cu/Zn SOD, and Mn SOD, which are endogenous antioxidative enzymes. These findings indicate that rosiglitazone inhibits AM5.5-induced intracellular H2O2 production, which occurs via NAD(P)H oxidase activation, by using a PPAR gamma-dependent pathway, and that the underlying mechanism involves an increase in the expression of catalase and SOD proteins.