Zn2+, derived from cell preparation,partly attenuates Ca2+-dependent cell death induced by A23187, calcium ionophore,in rat thymocytes

A23187, a calcium ionophore, is used to induce Ca²⁺-dependent cell death by increasing intracellular Ca²⁺ concentration (Ca²⁺]_i) under in vitro condition. Since this ionophore also increases membrane permeability of metal divalent cations such as Zn²⁺ and Fe²⁺ rather than Ca²⁺, trace metal cations in cell suspension may affect Ca²⁺-dependent cell death induced by A23187. Therefore, the effects of chelators for divalent metal cations, EDTA and TPEN, on the A23187-induced cytotoxicity were cytometrically examined in rat thymocytes. The cytotoxicity of A23187 was attenuated by 1 mM EDTA while it was augmented by 50 μM EDTA and 10 μM TPEN. These changes were statistically significant. The A23187-induced increase in Fluo-3 fluorescence intensity, a parameter for Ca²⁺]_i, was significantly reduced by 1 mM EDTA while it was not the case for 50 μM EDTA and 10 μM TPEN. The intensity of FluoZin-3 fluorescence, a parameter for Zn²⁺]_i, increased by A23187 was respectively reduced by 50 μM EDTA and 10 μM TPEN. It is suggested that the attenuation of A23187-induced cytotoxicity by 1 mM EDTA is due to the chelation of extracellular Ca²⁺ and Zn²⁺ while the augmentation by 50 μM ETDA or 10 μM TPEN is due to the chelation of extracellular Zn²⁺. The Tyrode’s solution without thymocytes contained 32.4 nM of zinc while it was 216.9 nM in the cell suspension. In conclusion, trace Zn²⁺, derived from cell preparation, partly attenuates the Ca²⁺-dependent cell death induced by A23187.