Intracellular Ca²⁺ homeostasis in trypomastigotes of Trypanosoma cruzi

Trypomastigotes of Trypanosoma cruzi maintain an intracellular Ca²⁺ concentration([Ca²⁺](i)) of 64 ± 30 nM. Equilibration of trypomastigotes in an extracellular buffer containing 0.5 mM [Ca²⁺]₀ (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 μM 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⁺]₀ were measured to asses the relative contribution of sequestration or extrusion processes in [Ca²⁺](i) homeostasis. In the presence of 0.5 mM [Ca²⁺]₀, 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 A₂, 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 A₂ activity. A Na⁺/Ca²⁺ exchange mechanism did not appear to contribute to Ca²⁺ homeostasis.