Hydrogen peroxide induces intracellular calcium overload by activation of a non-selective cation channel in an insulin-secreting cell line

Fura-2 fluorescence was used to investigate the effects of H2O2 on [Ca2+](i ) in the insulin-secreting cell line CRI-G1. H2O2 (1-10 mM) caused a biphas ic increase in free [Ca2+](i), an initial rise observed within 3 min and a second, much larger rise following a 30-min exposure. Extracellular calcium removal blocked the late, but not the initial, rise in [Ca2+](i), Thapsiga rgin did not affect either response to H2O2, but activated capacitive calci um entry, an action abolished by 10 mu M La3+, Simultaneous recordings of m embrane potential and [Ca2+](i) demonstrated the same biphasic [Ca2+](i) re sponse to H2O2 and showed that the late increase in [Ca2+](i) coincided tem porally with cell membrane potential collapse. Buffering Ca-i(2+) to low na nomolar levels prevented both phases of increased [Ca2+](i) and the H2O2-in duced depolarization. The H2O2-induced late rise in [Ca2+](i) was prevented by extracellular application of 100 mu M La3+. La3+ (100 mu M) inhibited t he H2O2-induced cation current and NAD-activated cation (NSNAD) channel act ivity in these cells. H2O2 increased the NAD/NADH ratio in intact CRI-G1 ce lls, consistent with increased cellular [NAD], These data suggest that H2O2 increases [NAD], which, coupled with increased [Ca2+](i), activates NSNAD, channels, causing unregulated Ca2+ entry and consequent cell death.