Reduction of the metallochromic indicators arsenazo III and antipyrylazo III to their free radical metabolites by cytoplasmic enzymes

198

202

The use of digitonin to permeabilize the plasma membrane of Trypanosoma cruzi allowed the identification of a non-mitochondrial nigericin- or bafilomycin A1-sensitive Ca(2+)-uptake mechanism. Proton uptake, as detected by ATP-dependent Acridine Orange accumulation, was also demonstrated in these permeabilized cells. Under these conditions Acridine Orange was concentrated in abundant cytoplasmic round vacuoles. This latter process was inhibited (and reversed) by bafilomycin A1, nigericin and NH4Cl in different stages of T. cruzi. Ca2+ released Acridine Orange from permeabilized cells, suggesting that the dye and Ca2+ were being accumulated in the same acidic compartment and that Ca2+ was taken up in exchange for protons. Addition of bafilomycin A1 (5 microM), nigericin (1 microM) or carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 1 microM) to fura 2-loaded epimastigotes increased their intracellular Ca2+ concentration ([Ca2+]i). Although this effect was more noticeable in the presence of extracellular Ca2+, it was also observed in its absence. Addition of NH4Cl (10-40 mM) to different stages of T. cruzi, in the nominal absence of extracellular Ca2+ to preclude Ca2+ entry, increased both [Ca2+]i in fura 2-loaded cells, and intracellular pH (pHi) in 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein acetoxymethyl ester (BCECF)-loaded cells. Treatment of the cells with the Ca2+ ionophore ionomycin under similar conditions (nominal absence of extracellular Ca2+) resulted in an increase in [Ca2+]i and a significantly higher increase in [Ca2+]i after addition of NH4Cl, nigericin or bafilomycin A1, all agents which increase the pH of acidic compartments and make ionomycin more effective as a Ca(2+)-releasing ionophore. Similar results were obtained when the order of additions was reversed. Taking into account the relative importance of the ionomycin-releasable and the ionomycin plus NH4Cl-releasable Ca2+ pools, it is apparent that most of the Ca2+ stored in different stages of T. cruzi is present in the acidic compartment thus identified. Taken together, these results are consistent with the presence of a Ca2+/H+ exchange system in an acidic vacuole, which we have named the 'acidocalcisome' and which appears to be a unique organelle present in trypanosomatids.

Section: Determination Of Ca2+ Movementsmentioning

confidence: 99%

Intracellular Ca2+ storage in acidocalcisomes of Trypanosoma cruzi

Docampo

Scott

Vercesi

et al. 1995

198

202

“…Variations in free Ca2+ concentration were monitored by measuring the changes in the absorbance spectrum of Arsenazo III [9] using an SLM Aminco DW2000 spectrophotometer at the wavelength pair 675-685 nm at 28 'C. No free radical formation from Arsenazo III occurred under the conditions used [10][11][12]. Each experiment was repeated at least three times, and the Figures show representative experiments.…”

Section: Determination Of Ca2+ Movementsmentioning

confidence: 99%

Ca2+ transport by digitonin-permeabilized Leishmania donovani. Effects of Ca2+, pentamidine and WR-6026 on mitochondrial membrane potential in situ

Vercesi

Docampo

1992

106

The use of low concentrations of digitonin allowed the quantitative determination of the mitochondrial membrane potential of Leishmania donovani promastigotes in situ using safranine O. L. donovani mitochondria were able to build up and retain a membrane potential of a value comparable with that of mammalian mitochondria. The response of promastigotes mitochondrial membrane potential to phosphate, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), valinomycin and Ca2+ indicates that these mitochondria behave similarly to vertebrate mitochondria with regard to the properties of their electrochemical proton gradient. When L. donovani promastigotes were permeabilized with digitonin in a reaction medium containing MgATP, succinate and 3.5 microM free Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level (0.05-0.1 microM). The presence of 1 microM-FCCP decreased by about 75% the initial rate of Ca2+ sequestration by these permeabilized cells. This FCCP-insensitive Ca2+ uptake, probably 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 by about 25% and the Ca2+ set point was increased to 0.7 microM. The succinate-dependence and FCCP-and Ruthenium Red-sensitivity of the Ca2+ uptake detected in the presence of vanadate indicate that this uptake is probably by the mitochondria. This interpretation was further supported by the Ruthenium Red-sensitive decrease in the mitochondrial membrane potential caused by Ca2+ addition. The anti-leishmanial cationic drugs pentamidine and WR-6026 also induced a rapid collapse of the mitochondrial inner membrane potential of L. donovani promastigotes.

“…Variations in free Ca2+ concentrations were monitored by measuring the changes in the absorbance spectrum of Arsenazo III, using the SLM Aminco DW2000 spectrophotometer at the wavelength pair 675-685 nm [30] to avoid interference by Mg2+ [31]. No free radical formation from Arsenazo III occurred under the conditions used [32][33][34].…”

Section: Determination Of Ca2+ Movementmentioning

confidence: 99%

A calmodulin-activated (Ca2+-Mg2+)-ATPase is involved in Ca2+ transport by plasma membrane vesicles from Trypanosoma cruzi

Benaím

Losada

Gadelha

et al. 1991

High-affinity Ca(2+)-activated ATPases that do not show any demonstrable dependence on Mg2+ have been reported in the plasma membranes of different trypanosomatids, and it has been suggested [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar & Bhaduri (1990) J. Biol. Chem. 265, 11345-11351] that these enzymes may have a role in Ca2+ transport by the plasma membrane and in the regulation of intracellular Ca2+ in these parasites. In this report we investigated Ca2+ transport by Trypanosoma cruzi plasma membrane vesicles using Arsenazo III as a Ca2+ indicator. These vesicles accumulated Ca2+ upon addition of ATP only when Mg2+ was present and released it in response to the Ca2+ ionophore A23187, but were insensitive to inositol 1,4,5-trisphosphate. Ca2+ transport was insensitive to antimycin A, oligomycin and carbonyl cyanide p-trifluorophenylhydrazone, ruling out any mitochondrial contamination. Staurosporine and phorbol myristate acetate had no effect on this activity, while low concentrations of vanadate (10 microM) completely inhibited it. In addition, we describe a high-affinity vanadate-sensitive (Ca(2+)-Mg2+)-ATPase in the highly enriched plasma membrane fraction of T. cruzi. Kinetic studies indicated that the apparent Km for free Ca2+ was 0.3 microM. On the other hand, Ca(2+)-ATPase activity and Ca2+ transport were both stimulated by bovine brain calmodulin and by endogenous calmodulin purified from these cells. In addition, trifluoperazine and calmidazolium, at concentrations in the range in which they normally exert anti-calmodulin effects, inhibited the calmodulin-stimulated Ca(2+)-ATPase activity. These observations support the notion that a Mg(2+)-dependent plasma membrane Ca2+ pump is present in these parasites.