Intracellular-produced hydroxyl radical mediates H2O2-induced Ca2+ influx and cell death in rat β-cell line RIN-5F

The melastatin-related transient receptor potential channel TRPM2 is a Ca2+-permeable channel that is activated by H2O2, and the Ca2+ influx through TRPM2 mediates cell death. However, the responsible oxidants for TRPM2 activation remain to be identified. In the present study, we investigated the involvement of hydroxyl radical on TRPM2 activation in TRPM2-expressing HEK293 cells and the rat β-cell line RIN-5F. In both cell types, H2O2 induced Ca2+ influx in a concentration-dependent manner. However, the addition of hydroxyl radical, which was produced by mixing FeSO4 and H2O2, to the cells, did not increase intracellular Ca2+ concentration. Interestingly, when H2O2 was added to the cells under intracellular Fe2+-accumulated conditions, Ca2+ influx was markedly enhanced compared to H2O2 alone. In addition, the H2O2-induced Ca2+ influx was reduced by hydroxyl radical scavengers and an iron chelator. Under intracellular Fe2+-accumulated conditions, H2O2-induced RIN-5F cell death through TRPM2 activation was also markedly enhanced. Hydroxyl radical scavengers and an iron chelator suppressed the RIN-5F cell death by H2O2. These results strongly suggest that the intracellular hydroxyl radical plays a key role in the activation of TRPM2 during H2O2 treatment, and TRPM2 activation mediated by hydroxyl radical is implicated in H2O2-induced cell death in the β-cell line RIN-5F.