Flow Cytometric Analysis of the H₂O₂-Induced Increase in Intracellular Ca²⁺ Concentration of Rat Thymocytes

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The effect of hydrogen peroxide (H₂O₂) on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) of rat thymocytes was examined by a flow cytometer and two fluorescent dyes, fluo-3-AM and ethidium bromide, a dye impermeant to intact membranes, to characterize the H₂O₂-induced increase in [Ca²⁺]_i. H₂O₂ at concentrations greater than 30 μM dose-dependently increased the [Ca²⁺]_i, of thymocytes which were not stained with ethidium. Removal of external Ca²⁺ greatly reduced the degree of H₂O₂-induced increase in [Ca²⁺]_i. However, H₂O₂ still increased the [Ca²⁺]_i under the external Ca²⁺-free condition. Diethylmaleate, which is known to produce a chemical depletion of cellular nonprotein thiol, significantly increased the [Ca²⁺]_i. Dithiothreitol, which is used to protect cellular nonprotein thiol, slightly decreased the [Ca²⁺]_i, but greatly reduced the H₂O₂-induced increase in [Ca²⁺]_i. Therefore, it is considered that H₂O₂ may increase the [Ca²⁺]_i through a mechanism related to the effects of H₂O₂ on the cellular nonprotein thiol.