Obligatory Role of Early Ca2+ Responses in H2O2-Induced β-Cell Apoptosis

Our previous study using apoptosis analysis suggested that Ca2+ release through inositol 1,4,5-trisphosphate (IP3) receptors and the subsequent Ca2+ influx through store-operated channels (SOCs) constitute a triggering signal for H2O2-induced beta-cell apoptosis. In the present study, we further examined the obligatory role of early Ca2+ responses in beta-cell apoptosis induction. H2O2 induced elevation of the cytosolic Ca2+ concentration ([Ca2+](c)) consisting of two phases: an initial transient [Ca2+](c) elevation within 30 mm and a slowly developing one thereafter. The first phase was almost abolished by 2-aminoethoxydiphenylborate (2-APB), which blocks IP3 receptors and cation channels including SOCs, while the second phase was only partially inhibited by 2-APB. The inhibition by 2-APB of the second phase was not observed when 2-APB was added 30 min after the treatment with H2O2. 2-APB also largely inhibited elevation of the mitochondrial Ca2+ concentration ([Ca2+](m)) induced by H2O2 when 2-APB was applied simultaneously with H2O2, but not when applied 30 min after H2O2 application. In addition, 2-APB inhibited the release of mitochondrial cytochrome c to the cytosol induced by H2O2 when 2-APB was applied simultaneously with H2O2 but not 30 mm post-treatment. H2O2-induced [Ca2+](m) elevation and cell death were not inhibited by Ru360, an inhibitor of the mitochondrial calcium uniporter (MCU). These results suggest that the H2O2-induced initial [Ca2+](c) elevation, occurring within 30 mm and mediated by Ca2+ release through IP3 receptors and subsequent Ca2+ influx through SOCs, leads to [Ca2+](m) elevation, possibly through a mechanism independent of MCU, thereby inducing cytochrome c release and consequent apoptosis.