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 microM 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 microM La3+. La3+ (100 microM) inhibited the H2O2-induced cation current and NAD-activated cation (NSNAD) 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 NSNAD channels, causing unregulated Ca2+ entry and consequent cell death.
Herson
P S
PS
Department of Biomedical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, United Kingdom.
Lee
K
K
Pinnock
R D
RD
Hughes
J
J
Ashford
M L
ML
D002118:Calcium / Q000378:metabolism*; D002412:Cations; D002460:Cell Line; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D015640:Ion Channel Gating; D007473:Ion Channels / Q000378:metabolism*; D017136:Ion Transport; D008564:Membrane Potentials; D018408:Patch-Clamp Techniques; D019284:Thapsigargin / Q000494:pharmacology
D002412:Cations; D007328:Insulin; D007473:Ion Channels; D019284:Thapsigargin; D006861:Hydrogen Peroxide; D002118:Calcium