Intracellular Ca²⁺ Homeostasis in Trypomastigotes of Trypanosoma cruzi

Abstract

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+]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 microM 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 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.

Characterization of Ca2+uptake in a subcellular membrane fraction ofHerpetomonassp. promastigotes

“…Thapsigargin and IP 3 were not able to affect the vanadate-sensitive Ca 2+ transport. Such an effect of digitonin has been attributed, in some cases, to the cell growth medium composition (Zhang et al 1998 ;Miranda et al 2005). No evidence of a third Ca 2+ compartment with the characteristics of the acidocalcisomes described by Vercesi et al (1994) was observed in those experiments.…”

“…Discrepancies between experiments have been attributed to an undesirable effect of digitonin solubilizing the membranes rather than the cytoplasm. Such an effect of digitonin has been attributed, in some cases, to the cell growth medium composition (Zhang et al 1998; Miranda et al 2005).…”