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 μM 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 μM. 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 μM), 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. © 1993.592305313Docampo, Vercesi, Ca2+ transport by coupled Trypanosoma cruzi mitochondria in situ (1989) J. Biol. Chem., 264, pp. 108-111Docampo, Vercesi, Characteristics of Ca2− transport by Trypanosoma cruzi mitochondria in situ (1989) Arch. Biochem. Biophys., 272, pp. 122-129Vercesi, Hoffmann, Bernardes, Docampo, Regulation of intracellular calcium homeostasis in Trypanosoma cruzi (1991) Effects of calmidazolium and trifluoperazine, 12, pp. 361-369. , 2nd ed., Cell CalciumVercesi, Bernardes, Hoffmann, Gadelha, Docampo, Digitonin permeabilization does not affect mitochondrial function and allows the determination of the mitochondrial membrane potential of Trypanosoma cruzi in situ (1991) J. Biol. Chem., 264, pp. 108-111Moreno, Vercesi, Pignataro, Docampo, Calcium homeostasis in Trypanosoma cruzi amastigotes: presence of inositol phosphates and lack of an inositol 1,4,5-trisphosphate-sensitive calcium pool. (1992) Mol. Biochem. Parasitol., 52, pp. 251-262Gunter, Pfeiffer, Mechanisms by which mitochondria transport calcium (1990) Am. J. Physiol., 258, pp. C755-C786Carafoli, Intracellular calcium homeostatis (1987) Annu. Rev. Biochem., 56, pp. 395-433Thastrup, Cullen, Drobak, Hanley, Dawson, Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2+--ATPase (1990) Proc. Natl Acad. Sci. USA, 87, pp. 2466-2470. , 2nd edLlopis, Chow, Kass, Gahm, Orrenius, Comparison between the effects of the microsomal Ca2+-translocase inhibitors thapsigargin and 2,5-di-(t-butyl)-1,4-benzohydroquinone on cellular calcium fluxes (1991) Biochem. J., 277, pp. 553-556Ruben, Akins, Trypanosoma brucei: the tumor promoter thapsigargin stimulates calcium release from an intracellular compartment in slender bloodstream forms (1992) Exp. Parasitol., 74, pp. 332-339Schmatz, Murray, Cultivation of Trypanosoma cruzi in irradiated muscle cells: improved synchronization and enhanced trypomastigote production (1982) Parasitology, 85, pp. 115-125Gornall, Bardawill, David, Determination of serum-proteins by means of the biuret reaction (1949) J. Biol. Chem., 177, pp. 751-766Nadal-Ginard, Commitment, fusion and biochemical differentiation of a myogenic cell line in the absence of DNA synthesis (1978) Cell, 15, pp. 855-864Docampo, Pignataro, The inositol phosphate/diacylglycerol signalling pathway in Trypanosoma cruzi (1991) Biochem. J., 275, pp. 407-411Scarpa, Measurements of cation transport with metallochromic indicators (1979) Methods Enzymol., 56, pp. 301-338Docampo, Moreno, Mason, Generation of free radical metabolites and superoxide anion by the calcium indicators arsenazo III, antipyrylazo III, and murexide in rat liver microsomes (1983) J. Biol. Chem., 258, pp. 14920-14925Moreno, Mason, Docampo, Ca2+ and Mg2+-enhanced reduction of arsenazo III to its anion free radical metabolite and generation of superoxide anion by an outer mitochondrial membrane azoreductase (1984) J. Biol. 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Ca2+/h+ Exchange In Acidic Vacuoles Of Trypanosoma Brucei

The use of digitonin to permeabilize the plasma membrane of Trypanosoma brucei procyclic and bloodstream trypomastigotes allowed the identification of a non-mitochondrial nigericin-sensitive Ca2+ compartment. The proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) was able to cause Ca2+ release from this compartment, which was also sensitive to sodium orthovanadate. Preincubation of the cells with the vacuolar H+-ATPase inhibitor bafilomycin A1 greatly reduced the nigericin-sensitive Ca2+ compartment. Bafilomycin A1 inhibited the initial rate of ATP-dependent non-mitochondrial Ca2+ uptake and stimulated the initial rate of nigericin-induced Ca2+ release by permeabilized procyclic trypomastigotes. ATP-dependent and bafilomycin A1- and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl)-sensitive Acridine Orange uptake was demonstrated in permeabilized cells. Under these conditions Acridine Orange was concentrated in abundant cytoplasmic round vacuoles by a process inhibited by bafilomycin A1, NBD-Cl, nigericin, and Ca2+. Vanadate or EGTA significantly increased Acridine Orange uptake, while Ca2+ released Acridine Orange from these preparations, thus suggesting that the dye and Ca2+ were being accumulated in the same acidic vacuole. Acridine Orange uptake was reversed by nigericin, bafilomycin A1 and NH4Cl. The results are consistent with the presence of a Ca2+/H+-ATPase system pumping Ca2+ into an acidic vacuole, that we tentatively named the acidocalcisome.304122723

Calcium Homeostasis In Procyclic And Bloodstream Forms Of Trypanosoma Brucei: Lack Of Inositol 1,4,5-trisphosphate-sensitive Ca2+ Release

When Trypanosoma brucei procyclic trypomastigotes were permeabilized with digitonin in a reaction medium containing MgATP, succinate, and 3.5 μM free Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level (0.05-0.1 μM), a range that correlates favorably with that detected in the intact cells with fura-2. The carbonyl cyanide p-trifluoromethoxyphenylhydrazone-insensitive Ca2+ uptake, certainly represented by the endoplasmic reticulum, was completely inhibited by 500 μM vanadate. When vanadate instead of carbonyl cyanide p-trifluoromethoxyphenylhydrazone was present, the Ca2+ set point was increased to 0.6-0.7 μM. The succinate dependence and carbonyl cyanide p-trifluoromethoxyphenylhydrazone sensitivity of the later Ca2+ uptake indicate that it may be exerted by the mitochondria. When bloodstream trypomastigotes were used, neither succinate nor α-glycerophosphate stimulated the mitochondrial Ca2+ uptake. The mitochondrial Ca2+ transport could be measured only in the presence of ATP and 500 μM vanadate to inhibit the endoplasmic reticulum uptake. Bloodstream trypomastigotes have a lower cytosolic Ca2+ concentration, as detected with fura-2 and a smaller extramitochondrial Ca2+ pool than procyclic trypomastigotes. Despite the presence of inositol phosphates, as determined by [3H]inositol incorporation, and the large extramitochondrial Ca2+ pool of procyclic trypomastigotes (61.7 nmol of Ca2+/mg of protein), no inositol 1,4,5-trisphosphate-sensitive Ca2+ release could be detected in these parasites.26796020602

Cytosolic-free Calcium Elevation In Trypanosoma Cruzi Is Required For Cell Invasion

To replicate, the trypomastigote form of Trypanosoma cruzi must invade host cells. Since a role for Ca2+ in the process of cell invasion by several intracellular parasites has been postulated, changes in the intracellular Ca2+ concentration in T. cruzi trypomastigotes and in tissue culture L6E9 myoblasts during their interaction were studied at the single cell level using digital imaging fluorescence microscopy or in cell suspensions by fluorescence spectrophotometry. An increase in cytosolic Ca2+ in T. cruzi trypomastigotes was detected at the single cell level after association of the parasites with the myoblasts. Ca2+ mobilization in the host cells was also detected upon contact with trypomastigotes either at the single cell level or in cells grown in coverslips and exposed to suspensions of trypomastigotes. Pretreatment of the parasites with the Ca2+ chelators quin 2 (50 μM) or bis-(o-aminophenoxy)-ethane-N,N,N',N'- tetraacetic acid (BAPTA, 50 μM) decreased the trypomastigotes' association to myoblasts by ~40 and 63%, respectively, thus indicating that an increase in intracellular Ca2+ concentration in the parasites is required for cell invasion in addition to Ca2+ mobilization in the host cells.180415351540Nogueira, N., Host and parasite factors affecting the invasion of mononuclear phagocytes by Trypanosoma cruzi (1983) CIBA Found. Symp., 99, p. 52Piras, R., Piras, M.M., Henriquez, D., Trypanosoma cruzi-fibroblastic cell interactions necessary for cellular invasion (1983) CIBA Found. Symp., 99, p. 31Snary, D., Receptors and recognition mechanisms of Trypanosoma cruzi (1985) Trans. R. Soc. Trop. Med. Hyg., 79, p. 587Zingales, B., Colli, W., Trypanosoma cruzi: Interaction with host cells (1985) Curr. Top. Microbiol. Immunol., 117, p. 129Schenkman, S., Robbins, E.S., Nussenzweig, V., Attachment of Trypanosoma cruzi to mammalian cells requires parasite energy, and invasion can be independent of the target cell cytoskeleton (1991) Infect Immun., 59, p. 645Ortega-Barria, E., Pereira, M.E.A., Entry of Trypanosoma cruzi into eukaryotic cells (1992) Infectious Agents and Disease, 1, p. 136Tardieux, I., Webster, P., Ravesloot, J., Boron, W., Lunn, J.A., Heuser, J.E., Andrews, N.W., Lysosome recruitment and fusion are early events required for trypanosome invasion of mammalian cells (1992) Cell, 71, p. 1117Hall, B.F., Webster, P., Ma, A.K., Joiner, K.A., Andrews, N.W., Desialylation of lysosomal membrane glycoproteins by Trypanosoma cruzi: A role of the surface neuraminidase in facilitating parasite entry into the host cell cytoplasm (1992) J. Exp. Med., 176, p. 313Campbell, A.K., (1983) Intracellular Calcium. Its Universal Role As Regulator, 556p. , John Wiley & Sons, Inc., New YorkTardieux, I., Nathanson, M.H., Andrews, N., Role in host cell invasion of Trypanosoma cruzi-induced cytosolic free Ca2+ transients (1994) J. Exp. Med., 179, p. 1017Morris, S.A., Tanowitz, H., Hatcher, V., Bilesikian, J.P., Wittner, M., Alterations in intracellular calcium following infection of human endothelial cell with Trypanosoma cruzi (1988) Mol. Biochem. Parasitol., 29, p. 213Osuna, A., Catanys, S., Rodriguez-Cabezas, M.N., Gamarro, F., Trypanosoma cruzi: Calcium ion movement during internalization in host cell HeLa cells (1990) Int. J. Parasitol., 20, p. 673Low, H.P., Paulin, J.J., Keith, C.H., Trypanosoma cruzi infection of BSC-1 fibroblast cells causes cytoskeletal disruption and changes in intracellular calcium levels (1992) J. 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Thapsigargin Causes Ca2+ Release And Collapse Of The Membrane Potential Of Trypanosoma Brucei Mitochondria In Situ And Of Isolated Rat Liver Mitochondria

Thapsigargin, previously reported to release Ca2+ from non-mitochondrial stores of different cell types, as well as nigericin, were found, when used at high concentrations, to release Ca2+ and collapse the membrane potential of Trypanosoma brucei bloodstream and procyclic trypomastigotes mitochondria in situ. At similarly high concentrations (>10 μM), thapsigargin was also found to release Ca2+ and collapse the membrane potential of isolated rat liver mitochondria. These results indicate that care should be taken when attributing the effects of thapsigargin in intact cells to the specific inhibition of the sarcoplasmic and endoplasmic reticulum Ca2+-ATPase family of calcium pumps. In addition, we have found no evidence for an increase in intracellular Ca2+ by release of the ion from intracellular stores by nigericin, measuring changes in cytosolic Ca2+ by dual wavelength spectrofluorometry in fura-2-loaded T. brucei bloodstream trypomastigotes or measuring Ca2+ transport in digitonin-permeabilized cells.268128564856

Acidocalcisomes In Apicomplexan Parasites

Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to man. They posses an acidic matrix that contains several cations bound to phosphates, mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. The calcium uptake occurs through a Ca2+/H+ counter transporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. 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