Calcium homeostasis in Trypanosoma cruzi amastigotes: presence of inositol phosphates and lack of an inositol 1,4,5-trisphosphate-sensitive calcium pool.
The permeabilization of Trypanosoma cruzi amastigotes with digitonin allowed the study of Ca2+ fluxes between intracellular organelles in situ. In addition, fura-2 was used to determine the cytosolic Ca2+ concentration in the intact cells. When amastigotes were permeabilized in a reaction medium containing MgATP, succinate and 3.5 microM Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level, a range which correlates favorably with that detected in the intact cells with fura-2. The presence of 1 microM FCCP strongly decreased the initial rate of Ca2+ sequestration by these permeabilized cells. This FCCP-insensitive Ca2+ uptake, probably represented by the endoplasmic reticulum, was completely inhibited by 500 microM vanadate. On the other hand, when vanadate instead of FCCP was present, the initial rate of Ca2+ accumulation was decreased and the Ca2+ set point was increased to about 0.8 microM. The succinate dependence and FCCP sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. Despite the presence of inositol phosphates, as determined by [3H]inositol incorporation, and of a large extramitochondrial Ca2+ pool, no IP3-sensitive or thapsigargin-sensitive Ca2+ release could be detected in either amastigotes or epimastigotes.
Moreno
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Department of Veterinary Pathobiology, University of Illinois, Urbana 61801.
Vercesi
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Pignataro
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Docampo
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D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D004072:Digitonin / Q000494:pharmacology; D016257:Fura-2 / Q000378:metabolism; D006706:Homeostasis; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007295:Inositol Phosphates / Q000378:metabolism*; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism; D015388:Organelles / Q000378:metabolism; D010936:Plant Extracts / Q000494:pharmacology; D013053:Spectrophotometry; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D007295:Inositol Phosphates; D010936:Plant Extracts; D013729:Terpenes; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D014638:Vanadates; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D004072:Digitonin; D002118:Calcium; D016257:Fura-2