Physiological and morphological evidences for the presence acidocalcisomes in Trypanosoma evansi: single cell fluorescence and 31P NMR studies.

A new Ca(2+) intracellular store, the acidocalcisome, has been reported in trypanosomatids. It has been characterized physiologically as a Ca(2+) store sensitive to nigericin. The Ca(2+)/H(+)-ATPase is the system responsible for Ca(2+) accumulation, which depends on a pH gradient formed by ATP- and PPi-dependent proton pumps. In this work we present physiological and morphological evidences for the presence of acidocalcisomes in Trypanosoma evansi. The parasites were purified and loaded with the fluorescent dye Fura 2-AM in order to detect the intracellular changes of Ca(2+) levels in individual cells. The simultaneous incubation of T. evansi cells with ionomycin and nigericin led to large release of Ca(2+) (ca. 200 nM) from intracellular stores, which was not observed with either agent alone. On the other hand, no enhancement of the nigericin-induced Ca(2+) release was observed in the presence of oligomycin. Additionally, the pretreatment with bafilomycin decreases the nigericin-induced Ca(2+) release. These results confirm the presence of an intracellular non-mitochondrial acidic Ca(2+) storage compartment. These results suggest that H(+)-ATPase is involved in the process of Ca(2+) accumulation into the acidocalcisomes. Furthermore, the cells loaded with acridine orange exhibited abundant fluorescent vacuoles, which were sensitive to nigericin or bafilomycin A(1). Electronic transmission microscopy observations demonstrated the presence of electron dense particles in the parasites. High levels of inorganic pyrophosphate and triphosphate were detected in perchloric acid extracts of T. evansi by high resolution 31P NMR. Taken together, these results present the first evidence for the presence of acidocalcisomes in T. evansi.