Increase in intracellular Cd2+ concentration of rat cerebellar granule neurons incubated with cadmium chloride: Cadmium cytotoxicity under external Ca2+-free condition

In order to examine the cadmium cytotoxicity unrelated to external Ca2+, the effects of micromolar CdCl2 on intracellular Cd2+ concentration, cellular content of glutathione, and cell viability of rat cerebellar granule neurons were examined under normal Ca2+ and external Ca2+-free conditions, using a laser confocal microscope with fluorescent probes, fluo-3-AM, 5-chloromethylfluorescein (CMF) diacetate, and propidium iodide. CdCl2 (10–300 μM) dose-dependently increased the intensity of fluo-3 fluorescence. Exposure to CdCl2 equally enhanced the fluo-3 fluorescence under both Ca2+ conditions and MnCl2 did not quench the CdCl2-enhanced fluorescence. The results indicate that the enhancement of fluo-3 fluorescence is due to the increase in intracellular Cd2+ concentration. CdCl2 at 100–300 μM decreased the intensity of CMF fluorescence, indicating the decrease in cellular content of glutathione. The population of cells stained with propidium (dead cells) was increased by 100–300 μM CdCl2. Similar results described above were also observed under external Ca2+-free condition. It is suggested that some of cytotoxic actions of CdCl2 on neurons are unrelated to external Ca2+, one of main sources for increasing intracellular Ca2+ concentration.