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.