Estimation of increased concentration of intracellular Cd2+ by fluo-3 in rat thymocytes exposed to CdCl2

Abstract Cadmium, an environmental pollutant, has been reported to induce apoptosis in murine lymphocytes. To reveal the mechanism of cadmium-induced apoptosis, one of important questions is whether cadmium increases intracellular concentration of Ca 2+ ([Ca 2+ ] i ), Cd 2+ ([Cd 2+ ] i ) or both. It is difficult to detect the increase in [Ca 2+ ] i using Ca 2+ -chelator-based fluorescent Ca 2+ indicators in the presence of Cd 2+ because of their sensitivity to Cd 2+ . Therefore, the study on membrane response such as Ca 2+ -dependent hyperpolarization gives a clue to reveal whether the [Ca 2+ ] i or [Cd 2+ ] i is increased. Cadmium at concentrations of 3 μM or more dose-dependently augmented fluo-3 fluorescence in rat thymocytes, presumably suggesting an increased [Ca 2+ ] i . However, the membranes were not hyperpolarized although the cells possess Ca 2+ -dependent K + channels. One may argue that cadmium inhibits Ca 2+ -dependent K + channels so that cadmium fails to hyperpolarize the membranes. It is unlikely because the [Ca 2+ ] i increased by A23187, a calcium ionophore, elicited the hyperpolarization in the presence of Cd 2+ . Furthermore, the profile of cytotoxicity induced by cadmium, examined by ethidium bromide and annexin V-FITC, was different from that induced by A23187. Taken together, it is concluded that the application of cadmium increases the [Cd 2+ ] i rather than the [Ca 2+ ] i in rat thymocytes, resulting in the induction of cytotoxicity.