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J Biol Chem, Vol. 274, Issue 2, 833-841, January 8, 1999

Hydrogen Peroxide Induces Intracellular Calcium Overload by Activation of a Non-selective Cation Channel in an Insulin-secreting Cell Line

Paco S. HersonDagger , Kevin Lee§, Rob D. Pinnock§, John Hughes§, and Michael L. J. AshfordDagger

From the Dagger  Department of Biomedical Sciences, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, United Kingdom and the § Parke-Davis Neuroscience Research Centre, Cambridge University, Forvie Site, Robinson Way, Cambridge CB2 2QB, United Kingdom

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 (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.

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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