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Reactive oxygen species, including hydrogen peroxide (H(2)O(2)), are known to induce β-cell apoptosis. The present study investigated the role of Ca(2+) in H(2)O(2)-induced apoptosis of the β-cell line INS-1. Annexin V assay with flow cytometry and DNA ladder assay demonstrated that treatment of INS-1 cells with 100 µM H(2)O(2) for 18 h significantly increased apoptotic cells. A comparable level of apoptosis was also observed after 18 h when the cells were treated with 100 µM H(2)O(2) only for initial 30 min. The H(2)O(2)-induced apoptosis was abolished by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA/AM), a chelator of intracellular Ca(2+), by 2-aminoethoxydiphenylborate (2-APB), a blocker of inositol 1,4,5-trisphosphate (IP(3)) receptors and cation channels, and by xestospongin D, a blocker of IP(3) receptors, and was partially blocked by SKF-96365, a non-selective cation channel blocker. However, nicardipine, an L-type voltage-dependent Ca(2+) channel blocker, or N-(p-amylcinnamoyl)anthranilic acid (ACA), a TRPM2 blocker, had little effect on the apoptosis. The inhibitory effect of BAPTA/AM or 2-APB on the H(2)O(2)-induced apoptosis was largely attenuated when the drug was added 30 min or 1 h after start of the treatment with H(2)O(2). These results suggest that the initial intracellular Ca(2+) elevation induced by H(2)O(2), which is mediated via IP(3) receptors and store-operated cation channels, plays an obligatory role in the induction of β-cell apoptosis.

biological and pharmaceutical bulletin

Inositol 1,4,5-Trisphosphate Receptor-Mediated Initial Ca2+ Mobilization Constitutes a Triggering Signal for Hydrogen Peroxide-Induced Apoptosis in INS-1 .BETA.-Cells