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 Ca2+, [Ca2+]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 Ca(2+)-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.
Oz
H S
HS
Department of Pathology, Albert Einstein College of Medicine, New York, New York 10461.
Wittner
M
M
Tanowitz
H B
HB
Bilezikian
J P
JP
Saxon
M
M
Morris
S A
SA
D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D006706:Homeostasis; D006863:Hydrogen-Ion Concentration; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D015220:Calcium Channels; D002352:Carrier Proteins; D007476:Ionophores; D019831:Sodium-Calcium Exchanger; D002110:Caffeine; D014638:Vanadates; D015759:Ionomycin; D010042:Ouabain; D012964:Sodium; D011188:Potassium; D002118:Calcium