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 H(2)O(2) on [Ca(2+)](i) in the insulin-secreting cell line CRI-G1. H(2)O(2) (1-10 mM) caused a biphasic increase in free [Ca(2+)](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 [Ca(2+)](i), Thapsigargin did not affect either response to H(2)O(2), but activated capacitive calcium entry, an action abolished by 10 mu M La(3+), Simultaneous recordings of membrane potential and [Ca(2+)](i) demonstrated the same biphasic [Ca(2+)](i) response to H(2)O(2) and showed that the late increase in [Ca(2+)](i) coincided temporally with cell membrane potential collapse. Buffering Ca(i)(2+) to low nanomolar levels prevented both phases of increased [Ca(2+)](i) and the H(2)O(2)-induced depolarization. The H(2)O(2)-induced late rise in [Ca(2+)](i) was prevented by extracellular application of 100 mu M La(3+). La(3+) (100 mu M) inhibited the H(2)O(2)-induced cation current and NAD-activated cation (NS(NAD)) channel activity in these cells. H(2)O(2) increased the NAD/NADH ratio in intact CRI-G1 cells, consistent with increased cellular [NAD], These data suggest that H(2)O(2) increases [NAD], which, coupled with increased [Ca(2+)](i), activates NS(NAD), channels, causing unregulated Ca(2+) entry and consequent cell death.