Trypanosoma evansi: A convenient model for studying intracellular Ca2+ homeostasis using fluorometric ratio imaging from single parasites

Abstract Mendoza, M., Mijares, A., Rojas, H., Ramos, M., and DiPolo, R. 2001. Trypanosoma evansi: A convenient model for studying intracellular Ca 2+ homeostasis using fluorometric ratio imaging from single parasites. Experimental Parasitology 99 , 213–219. The aim of this work was to measure, for the first time, the basal cytosolic Ca 2+ levels of Trypanosoma evansi and to explore the possibility of observing changes in the intracellular Ca 2+ concentration ([Ca 2+ ] i ) using fluorescence ratio imaging techniques in single isolated parasites of this species. Under appropriate loading conditions, the high intracellular levels of the Ca 2+ fluorescence probe Fura-2 permits resolution, in real time, of single parasite [Ca 2+ ] i signals. Measurements of the basal [Ca 2+ ] i indicate that homeostatic mechanisms maintain [Ca 2+ ] i at 106 ± 38 ( n = 32) nM in the presence of 2 mM extracellular calcium. The resting [Ca 2+ ] i was unaffected by changes in extracellular Ca 2+ in the range from 0 to 10 mM. The Ca 2+ ionophore A23187 induced a large increase in [Ca 2+ ] i which (i) reached a steady state value even in the simultaneous presence of both external calcium and ionophore and (ii) returned to base line upon removal of extracellular Ca 2+ . A dose-response curve of the protonophore nigericin shows that T. evansi contains an important pH-sensitive intracellular pool which may be released by this drug with a K 1/2 of 8 μM. These data demonstrate that this parasite contains highly efficient systems to control [Ca 2+ ] i . Finally, our results, with the use of sera as source of an antibody-complement to induce Ca 2+ entry, demonstrate that it is possible to resolve fast [Ca 2+ ] i signals in single parasites from T. evansi .