Jpn. J. Pharmacol. 71 (4), 273-280 (1996)

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

Eisuke Okazaki (1), Lumi Chikahisa (1,2,3), Kaori Kanemaru (1) and Yasuo Oyama (1,*)

(1) Laboratory of Cell Signaling (Pharmacology), Faculty of Integrated Arts and Sciences, The University of Tokushima, Tokushima 770, Japan (2) Anticancer and Antimicrobials Research Laboratory, Taiho Pharmaceutical Co., Ltd., Tokushima 771-01, Japan (3) Cancer Research Laboratory, Taiho Pharmaceutical Co., Ltd., Hanno 357, Japan (*) To whom correspondence should be addressed.

Abstract: 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 microM 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.

Keywords: Flow cytometer, Nonprotein thiol, Hydrogen peroxide, Intracellular Ca2+, Oxidative stress

Copyright© The Japanese Pharmacological Society 1996

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