The mechanisms of H2O2-induced elevated calcium baselines in PC12 cells wer
e investigated in the present study by using fura-2-fluorescent image analy
sis. The results showed that the calcium comes from both intracellular and
extracellular sources. Although the major intracellular source was mitochon
dria, only the extracellular calcium influx was responsible for the sustain
ed post-H2O2-exposure increases. This calcium influx was partially blocked
by calcium channel antagonists [verapamil (L-type) or mibefradil (nonselect
ive)] and was more effectively blocked by the sodium channel antagonist, te
trodotoxin (TTX). Membrane depolarization following H2O2 exposure contribut
ed to the opening of the ion channels. The H2O2-induced calcium influx was
blocked by TTX even in a sodium-free buffer, indicating that calcium direct
ly fluxed through sodium channels. Sodium-calcium exchangers (NCX) on the p
lasma membrane did not play a role, because use of a specific reverse mode
NCX inhibitor, No. 7943, was ineffective in blocking the influx. The H2O2-i
nduced calcium influx was mimicked by using a thiol-selective oxidizing rea
gent, 2',2/-dithiodipyridine, and in both situations, the calcium levels we
re completely reversed by a thiol-selective reducing reagent, dithiothreito
l. Our results indicated that mechanisms of oxidant-induced elevated calciu
m baselines in PC12 cells involved calcium influx through sodium and calciu
m channels that may be directly or indirectly attributed to thiol oxidation
. (C) 2000 Elsevier Science Inc.