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C. Hutnik, Q. Shao, H. Liu, C. Pocrnich, A. Mao, D. Laird; Gap Junctional Intercellular Communication Protects Human Retinal Pigment Epithelial Cells From Oxidative Stress-Induced Injury. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5054.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the roles of connexin 43 (Cx43) and gap junctional intercellular communication (GJIC) in oxidant-stressed cultured human retinal pigment epithelial (hRPE) cells.
Cultured hRPE cells were treated with the chemical oxidant tert-butyl hydroperoxide (t-BOOH). Cell viability was assessed by the MTT assay. GJIC was evaluated by scrape loading/dye transfer and microinjection assays. Cx43 expression was detected by Western blot and immunofluorescent staining combined with confocal microscopy analysis. Retroviral infection of hRPE cells with disease-linked dominant negative Cx43(G21R), and dominant active mutants of both Cx43 and Cx26 was performed and the effect on cell viability was assessed.
t-BOOH induced hRPE cell death by necrosis. GJIC was hindered by t-BOOH. Phosphorylation of Cx43 and a decrease in overall expression of Cx43 was observed following t-BOOH treatment. Retroviral infection with disease-linked dominant negative Cx43 increased t-BOOH-induced cell death, as did treatment with gap junction blockers 18b-glycyrrhetinic acid (GZA) and flufenamic acid (FFA). Conversely, over-expression of Cx26 and Cx43 increased the viability of oxidant-treated hRPE cells from 45.2% to 74.8% (p<0.01) and to 83.2% (p<0.01), respectively.
Connexin-mediated protection of hRPE cells from oxidative injury is GJIC-dependent.
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