Effect of thapsigargin on calcium homeostasis in Trypanosoma cruzi trypomastigotes and epimastigotes.
By using the fluorescent calcium indicator fura-2, it was found that the concentration of free Ca2+ in the cytoplasm of Trypanosoma cruzi trypomastigotes incubated in the presence or absence of external calcium was maintained at very low levels (10-20 nM). When trypomastigotes were incubated in the presence of succinate and ATP and permeabilized with digitonin, they lowered the medium calcium concentration to a submicromolar level. In the presence of 1 microM FCCP the initial rate of Ca2+ sequestration by these permeabilized cells was very slow. When succinate alone was present, the initial rate of Ca2+ accumulation was slower than with ATP plus succinate, and the calcium set point was about 0.6 microM. The succinate dependence and FCCP sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. High concentrations of the tumor promoter thapsigargin slightly increased cytosolic Ca2+ in the presence of extracellular Ca2+ but had no effect on the FCCP- and oligomycin/antimycin A-insensitive Ca2+ pool. In addition, when used at those concentrations (4-20 microM), thapsigargin was shown to release Ca2+ from the mitochondria and to decrease the inner mitochondrial membrane potential of trypomastigotes and epimastigotes as measured using safranine O. Despite the presence of inositol phosphates as determined by [3H]inositol incorporation, no IP3-sensitive Ca2+ release could be detected in trypomastigotes.
Docampo
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Department of Veterinary Pathobiology, University of Illinois, Urbana 61801.
Moreno
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Vercesi
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D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002463:Cell Membrane Permeability; D002851:Chromatography, High Pressure Liquid; D003593:Cytoplasm / Q000378:metabolism; D004072:Digitonin; D004533:Egtazic Acid / Q000494:pharmacology; D016257:Fura-2; D006706:Homeostasis / Q000187:drug effects; D007295:Inositol Phosphates / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000502:physiology*
D007295:Inositol Phosphates; D013729:Terpenes; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000001:Calcimycin; D004533:Egtazic Acid; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D004072:Digitonin; D002118:Calcium; D016257:Fura-2