Heavy metal effects on cytosolic free Ca²⁺ level in the marine protozoan Euplotes crassus evaluated by confocal laser scanning microscopy

The Ca²⁺ concentration in the cytosol of the marine ciliate protozoan Euplotes crassus has been evaluated utilizing the dye Fluo3-AM and measuring the fluorescent response by confocal laser scanning microscopy (CLSM). Serial sections were piled-up in a three dimensional image of the cell to estimate the cation intracellular spatial distribution and subsequently processed in order to quantify the free Ca²⁺ content. The results clearly demonstrated that nano-to micromolar concentrations of Hg²⁺ added to seawater culture medium caused up to a 12-fold increase in the free cytosolic Ca²⁺ of Euplotes crassus. Exposure of the protozoa to Cu²⁺, an essential element, also caused a sustained increase in the free cytosolic Ca²⁺. Pretreatment of Euplotes crassus with W5, a voltage-dependent Ca²⁺-channel blocker, greatly reduced the deleterious effects of the heavy metals on Ca²⁺ concentration. These results indicate that the increase of free Ca²⁺ concentration in the cytosol of the protozoa, induced by micromolar heavy metal concentrations, could depend on an increased influx of extracellular Ca²⁺ not physiologically compensated by the activity of the differed mechanisms involved in Ca²⁺-homeostasis.