Mechanisms of hydrogen peroxide-induced calcium dysregulation in PC12 cells

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.