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 biphasic 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. Thapsigargin did not affect either response to H₂O₂, but activated capacitive calcium entry, an action abolished by 10 µM La³⁺. Simultaneous recordings of membrane potential and [Ca²⁺]_i demonstrated the same biphasic [Ca²⁺]_i response to H₂O₂ and showed that the late increase in [Ca²⁺]_i coincided temporally with cell membrane potential collapse. Buffering Ca²⁺_i to low nanomolar levels prevented both phases of increased [Ca²⁺]_i and the H₂O₂-induced depolarization. The H₂O₂-induced late rise in [Ca²⁺]i was prevented by extracellular application of 100 µM La³⁺. La³⁺ (100 µM) inhibited the H₂O₂-induced cation current and NAD-activated cation (NS_NAD) channel activity in these cells. H₂O₂ increased the NAD/NADH ratio in intact CRI-G1 cells, consistent with increased cellular [NAD]. These data suggest that H₂O₂ increases [NAD], which, coupled with increased [Ca²⁺]i, activates NS_NAD channels, causing unregulated Ca²⁺ entry and consequent cell death.