Increased Intracellular Zn²⁺ Levels Suppress Ca²⁺-Dependent Cytotoxicity in Rat Thymocytes

Abstract Zinc is an essential trace element that plays catalytic, structural, and regulatory roles in cellular physiology. Intracellular free Zn²⁺ participates in various signaling pathways, including the regulation of cell survival and death. However, its role in Ca²⁺-dependent cytotoxicity remains unclear. We investigated the involvement of intracellular free Zn²⁺ in Ca²⁺-dependent cell death using rat thymocytes. In cells treated with the ionophore A23187, cell death was evaluated using propidium iodide staining, intracellular Ca²⁺ levels were assessed using Fluo-3 fluorescence, and membrane potential changes were monitored using oxonol fluorescence. Simultaneous treatment with low concentrations of Zn²⁺ (1–10 µM) significantly suppressed A23187-induced cell death, whereas extracellular Zn²⁺ chelation with DTPA did not attenuate cytotoxicity, indicating that extracellular Zn²⁺ is not required for A23187-induced cell death. The protective effect of Zn²⁺ was strongly dependent on the timing of administration and was most prominent when Zn²⁺ was added concurrently with A23187, gradually diminishing when Zn²⁺ addition was delayed. Zn²⁺ significantly suppressed the A23187-induced increase in intracellular Ca²⁺ concentration, and this effect was reversed by the Zn²⁺ chelator TPEN. Thus, Zn²⁺ is essential for the modulation of Ca²⁺ dynamics. Zn²⁺ attenuated A23187-induced membrane hyperpolarization, suggesting suppression of Ca²⁺-dependent membrane excitability. These findings indicate that intracellular Zn²⁺ acts as a negative regulator of early Ca²⁺-dependent cytotoxicity by suppressing intracellular Ca²⁺ elevation and membrane hyperpolarization in rat thymocytes. In conclusion, this study suggests that intracellular Zn²⁺ may act as a negative regulator of early-phase Ca²⁺-dependent cytotoxicity in rat thymocytes, although the detailed underlying mechanisms remain to be elucidated.