Intracellular Ca2+ homeostasis in trypomastigotes of Trypanosoma cruzi

Trypomastigotes of Trypanosoma cruzi maintain an intracellular Ca2+ concentration([Ca2+](i)) of 63 +/- 30 nM. Equilibration of trypomastigotes in an extracellular buffer containing 0.5 mM [Ca2+](o) (preloaded cells) increased [Ca2+](i) < 20 nM whereas total cell Ca2+ increased by 1.5 to 2.0 pmole/cell. This amount of Ca2+ would be expected to increase [Ca2+](i) to > 10 mu M suggesting active sequestration of Ca2+. We tested the hypothesis that maintenance of [Ca2+](i) involved both the sequestration into intracellular storage sites and extrusion into the extracellular space. Pharmacological probes known to influence [Ca2+](i) through well characterized pathways in higher eukaryotic cells were employed. [Ca2+](i) responses in the presence or absence of [Ca2+](o) were measured to asses the relative contribution of sequestration or extrusion processes in [Ca2+](i) homeostasis. In the presence of 0.5 mM [Ca2+](o), the ability of several agents to increase [Ca2+](i) was magnified in the order ionomycin >>> nigericin > thapsigargin > monensin > valinomycin. In contrast, preloading markedly enhanced the increase in [Ca2+](i) observed only in response to monensin. Manoalide, an inhibitor of phospholipase A(2), enhanced the accumulation of [Ca2+](i) due to all agents tested, particularly ionomycin and thapsigargin. Our results suggest that sequestration of [Ca2+](i) involved storage sites sensitive to monensin and ionomycin whereas extrusion of Ca2+ may involve phospholipase A(2) activity. A Na+/Ca2+ exchange mechanism did not appear to contribute to Ca2+ homeostasis.