Trypanosoma cruzi: Mechanisms of intracellular calcium homeostasis

Regulation of intracellular Ca²⁺ homeostasis was characterized in epimastigote forms of Trypanosoma cruzi using the fluorescence probe Fura-2. Despite an increase in extracellular Ca^{2 +}, [Ca^{2 +}]_o, from 0 to 2 mM, cytosolic Ca²⁺, [Ca²⁺]_i, increased only from 85 ± 9 to 185 ± 21 nM, indicating the presence of highly efficient mechanisms for maintaining [Ca²⁺]_i. Exposure to monovalent Na⁺ (monensin)-, K⁺ (valinomycin, nigericin)-, and divalent Ca²⁺ (ionomycin)-specific ionophores, uncouplers of mitochondrial respiration (oligomycin), inhibitors of Na⁺/K⁺-ATPase (ouabain), and Ca²⁺-sensitive ATPase (orthovanadate) in 0 or 1 mM [Ca²⁺ ]_o resulted in perturbations of [Ca²⁺]_i, the patterns of which suggested both sequestration and extrusion mechanisms. Following equilibration in 1 mM [Ca²⁺]_o, incubation with orthovanadate markedly increased [Ca²⁺]_i, results which are compatible with an active uptake of [Ca²⁺]_i by endoplasmic reticulum. In contrast, equilibration in 0 or 1 mM [Ca²⁺]_o did not influence the relatively smaller increase in [Ca²⁺]_i following incubation with oligomycin, suggesting a minor role for the mitochondrial compartment. In cells previously equilibrated in 1 mM [Ca²⁺]_o, exposure to monensin or ouabain, conditions known to decrease the [ Na⁺]_o [Na⁺]_i gradient, upon which the Na⁺ Ca²⁺ exchange pathways are dependent, markedly increased [Ca²⁺]_i. In a complementary manner, decreasing the extracellular Na⁺ gradient with Li⁺ increased [Ca²⁺]_i in a dose-dependent manner. Finally, the calcium channel blockers verapamil and isradipine inhibited the uptake of Ca²⁺ by greater than 50%, whereas diltiazem, nifedipine, and nicardipine were ineffective. The results suggest that epimastigote forms of T. cruzi maintain [Ca²⁺]_i by uptake, sequestration, and extrusion mechanisms, with properties common to eukaryotic organisms.