Biomedical subjects

A E Vercesi

Publications and source records attributed to A E Vercesi.

At least 73 records · Page 4Linked to original sources

PubMed1996

Oxidative damage to sarcoplasmic reticulum Ca(2+)-pump induced by Fe2+/H2O2/ascorbate is not mediated by lipid peroxidation or thiol oxidation and leads to protein fragmentation.

The major protein in the sarcoplasmic reticulum (SR) membrane is the Ca2+ transporting ATPase which carries out active Ca2+ pumping at the expense of ATP hydrolysis. The aim of this work was to elucidate the mechanisms by which oxidative stress induced by Fenton's reaction (Fe(2+)+H2O2-->HO.+OH-+Fe3+) alters the function of SR. ATP hydrolysis by both SR vesicles (SRV) and purified ATPase was inhibited in a dose-dependent manner in the presence of 0-1.5 mM H2O2 plus 50 microM Fe2+ and 6 mM ascorbate. Ca2+ uptake carried out by the Ca(2+)-ATPase in SRV was also inhibited in parallel. The inhibition of hydrolysis and Ca2+ uptake was not prevented by butylhydroxytoluene (BHT) at concentrations which significantly blocked formation of thiobarbituric acid-reactive substances (TBARS), suggesting that inhibition of the ATPase was not due to lipid peroxidation of the SR membrane. In addition, dithiothreitol (DTT) did not prevent inhibition of either ATPase activity or Ca2+ uptake, suggesting that inhibition was not related to oxidation of ATPase thiols. The passive efflux of 45Ca2+ from pre-loaded SR vesicles was greatly increased by oxidative stress and this effect could be only partially prevented (ca 20%) by addition of BHT or DTT. Trifluoperazine (which specifically binds to the Ca(2+)-ATPase, causing conformational changes in the enzyme) fully protected the ATPase activity against oxidative damage. These results suggest that the alterations in function observed upon oxidation of SRV are mainly due to direct effects on the Ca(2+)-ATPase. Electrophoretic analysis of oxidized Ca(2+)-ATPase revealed a decrease in intensity of the silver-stained 110 kDa Ca(2+)-ATPase band and the appearance of low molecular weight peptides (MW < 100 kDa) and high molecular weight protein aggregates. Presence of DTT during oxidation prevented the appearance of protein aggregates and caused a simultaneous increase in the amount of low molecular weight peptides. We propose that impairment of function of the Ca(2+)-pump may be related to aminoacid oxidation and fragmentation of the protein.

Animals↗

PubMed1996

The calcium sensor ruthenium red can act as a Fenton-type reagent.

Ruthenium red (RR), an ammoniated form of tris-ruthenium(III,IV,III) oxychloride, has been widely used in the micromolar range as a strong and specific inhibitor of in vitro and in vivo Ca(2+)-mediated biochemical processes without regard for its redox properties. We show here that in the presence of tert-butyl hydroperoxide (TBHP) and an electron source, either succinate-energized rat liver mitochondria or ascorbate, RR amplifies the generation of methyl radicals. The EPR spin trapping signal of the 5,5-dimethyl-1-pyrroline-N-oxide/methyl radical (DMPO/.CH3) adduct obtained from incubations of TBHP (1.5 mM) and mitochondria (5 mg protein/ml) in an adequate medium increases upon addition of RR in a concentration-dependent fashion: sixfold at 10 microM RR. Respiring mitochondria can be replaced by ascorbate (1 mM), the characteristic EPR signal of the ascorbyl radical also being observed (aH = 0.18 mT). Spectrophotometric, cyclic voltammetric and spectroelectrochemical studies unequivocally show oxidation of RR(III,IV,III) (lambda max = 538 nm) to the ruthenium(IV,III,IV) species ("ruthenium brown," RB; lambda max = 464 nm) by TBHP, followed by its one-electron back reduction to RR by the respiratory chain or ascorbate. The calcium chelator EGTA (1 mM) strongly binds and stabilizes the RR form, slowing down its recycling by TBHP and either ascorbate or the mitochondrial electron chain. These data clearly show that Ru(III) in the RR complex can reduce TBHP via a Fenton-type reaction and thus must be considered when RR is used as a tool to study biological processes simultaneously involving Ca2+ ions and peroxides.

Animals↗

PubMed1996

Sodium-proton exchange stimulates Ca2+ release from acidocalcisomes of Trypanosoma brucei.

Acidocalcisomes are acidic vacuoles present in trypanosomatids that contain a considerable fraction of intracellular Ca2+ [Vercesi, Moreno and Docampo (1994) Biochem. J. 304, 227-233; Scott, Moreno and Docampo (1995) Biochem. J. 310, 789-794; Docampo, Scott, Vercesi and Moreno (1995) Biochem. J. 310, 1005-1012]. The data presented here indicate that Na+ stimulates Ca2+ release from the acidocalcisomes of digitonin-permeabilized Trypanosoma brucei procyclic trypomastigotes in a dose-dependent fashion, this effect being enhanced by increasing pH of the medium from 7.0 to 7.8. The hypothesis that this Na+ effect was mediated by alkalinization of the acidocalcisomes via a Na+/H+ antiporter was supported by experiments showing that Na+ promotes release of Acridine Orange previously accumulated in these vacuoles. This putative antiporter did not transport Li+ and was not sensitive to the amiloride analogue 5-(N-ethyl-N-isopropyl)amiloride. Addition of the Na+/H+ ionophore monensin to intact cells loaded with fura 2, in the nominal absence of extracellular Ca2+ to preclude Ca2+ entry, was followed by an increase in cytosolic Ca2+ concentration ([Ca2+]i), which was more accentuated in the presence of extracellular Na+. An increase in intracellular pH (pHi) of BCECF-loaded cells was detected after addition of monensin in the presence of extracellular Na+, whereas a dramatic decrease in pHi was detected in its absence, thus indicating the presence of a significant amount of releasable protons in the acidic compartments. These results are consistent with the presence of a Na+/H+ antiporter in the acidocalcisomes that could be involved in the regulation of pH1 and [Ca2+]1 in these parasites.

Animals↗

PubMed1996

Effect of inorganic phosphate concentration on the nature of inner mitochondrial membrane alterations mediated by Ca2+ ions. A proposed model for phosphate-stimulated lipid peroxidation.

Addition of high concentrations (>1 mm) of inorganic phosphate (Pi) or arsenate to Ca2+-loaded mitochondria was followed by increased rates of H2O2 production, membrane lipid peroxidation, and swelling. Mitochondrial swelling was only partially prevented either by butylhydroxytoluene, an inhibitor of lipid peroxidation, or cyclosporin A, an inhibitor of the mitochondrial permeability transition pore. This swelling was totally prevented by the simultaneous presence of these compounds. At lower Pi concentrations (1 mm), mitochondrial swelling is reversible and prevented by cyclosporin A, but not by butylhydroxytoluene. In any case (low or high phosphate concentration) exogenous catalase prevented mitochondrial swelling, suggesting that reactive oxygen species (ROS) participate in these mechanisms. Altogether, the data suggest that, at low Pi concentrations, membrane permeabilization is reversible and mediated by opening of the mitochondrial permeability transition pore, whereas at high Pi concentrations, membrane permeabilization is irreversible because lipid peroxidation also takes place. Under these conditions, lipid peroxidation is strongly inhibited by sorbate, a putative quencher of triplet carbonyl species. This suggests that high Pi or arsenate concentrations stimulate propagation of the peroxidative reactions initiated by mitochondrial-generated ROS because these anions are able to catalyze Cn-aldehyde tautomerization producing enols, which can be oxidized by hemeproteins to yield the lower Cn - 1-aldehyde in the triplet state. This proposition was also supported by experiments using a model system consisting of phosphatidylcholine/dicethylphosphate liposomes and the triplet acetone-generating system isobutanal/horseradish peroxidase, where phosphate and Ca2+ cooperate to increase the yield of thiobarbituric acid-reactive substances.

Acetates↗

PubMed1996

Opening of the mitochondrial permeability transition pore by uncoupling or inorganic phosphate in the presence of Ca2+ is dependent on mitochondrial-generated reactive oxygen species.

In this study, we show that mitochondrial membrane permeability transition in Ca(2+)-loaded mitochondria treated with carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) or inorganic phosphate (P(i)) is preceded by enhanced production of H2O2. This production is inhibited either by ethylene glycobis(b-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) or Mg2+, but not by cyclosporin A. Permeability transition is prevented either by EGTA, catalase or dithiothreitol, suggesting the involvement of Ca2+, H2O2 and oxidation of membrane protein thiols in this mechanism. When mitochondria are incubated under anaerobiosis, no permeabilization or H2O2 production occurs. Based on these results we conclude that mitochondrial permeability transition induced by P(i) or FCCP-uncoupling is dependent on mitochondrial-generated reactive oxygen species.

Animals↗

PubMed1996

The irreversibility of inner mitochondrial membrane permeabilization by Ca2+ plus prooxidants is determined by the extent of membrane protein thiol cross-linking.

We have previously shown that mitochondrial membrane potential (delta psi) drop promoted by prooxidants and Ca2+ can be reversed but not sustained by ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) unless dithiothreitol (DTT), a disulfide reductant, is also added [Valle, V. G. R., Fagian, M. M., Parentoni, L. S., Meinicke, A. R., and Vercesi, A. E. (1993). Arch. Biochem. Biophys. 307, 1-7]. In this study we show that catalase or ADP are also able to potentiate this EGTA effect. When EGTA is added long after (12 min) the completion of swelling or delta psi elimination, no membrane resealing occurs unless the EGTA addition was preceded by the inclusion of DTT, ADP, or catalase soon after delta psi was collapsed. Total delta psi recovery by EGTA is obtained only in the presence of ADP. The sensitivity of the ADP effect to carboxyatractyloside strongly supports the involvement of the ADP/ATP carrier in this mechanism. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins shows that protein aggregation due to thiol cross-linkage formed during delta psi drop continues even after delta psi is already eliminated. Titration with 5,5'-dithio-bis(2-nitrobenzoic acid) supports the data indicating that the formation of protein aggregates is paralleled by a decrease in the content of membrane protein thiols. Since the presence of ADP and EGTA prevents the progress of protein aggregation, we conclude that this process is responsible for both increased permeability to larger molecules and the irreversibility of delta psi drop. The protective effect of catalase suggests that the continuous production of protein thiol cross-linking is mediated by mitochondrial generated reactive oxygen species.

Adenosine Diphosphate↗

PubMed1996

Mechanism of tetrahydroxy-1,4-quinone cytotoxicity: involvement of CA2+ and H2O2 in the impairment of DNA replication and mitochondrial function.

In this work we investigated the toxicity of a polyphenolic p-benzoquinone derivative, the tetrahydroxy-1,4-quinone (THQ) toward V79 Chinese hamster fibroblasts and analyzed the role of H2O2 and Ca2+ in that mechanism. The exposure of exponentially growing cultures to THQ, in the presence of 1.0 mM Ca2+, caused a dose-dependent inhibition of cell growth and DNA synthesis. Complete prevention of those effects by catalase indicated that H2O2-induced damages should underlie both toxic processes. Further detection of a rise in the intracellular free Ca2+ concentration ([Ca2+]i) in cells exposed to THQ plus Ca2+, together with the partial protection conferred by the intracellular Ca(2+)-chelator fura-2 against cell growth inhibition, indicated that a disruption of Ca2+ homeostasis is a determinant event in THQ cytotoxicity. Furthermore, the intracellular accumulation of rhodizonic acid (RDZ), the primary oxidative product of THQ, indicated that THQ, or its corresponding semiquinone form, was entering the cells and undergoing further autoxidation to RDZ. It was also evidenced that mitochondria represent an important target in the development of THQ toxicity, as shown by the disruption of the transmembrane electrical potential (delta psi) of isolated rat liver mitochondria, as well as by the Ca(2+)-release by mitochondria of permeabilized V79 cells. We concluded that disruption of Ca2+ homeostasis and generation of H2O2 are critically involved in THQ-induced impairment of DNA replication and mitochondrial functions, leading ultimately to cell growth inhibition.

Animals↗

PubMed1996

Effect of organic synthetic food colours on mitochondrial respiration.

Eleven organic synthetic dyes, currently or formerly used as food colours in Brazil, were tested to determine their effect on mitochondrial respiration in mitochondria isolated from rat liver and kidney. The compounds tested were: Erythrosine, Ponceau 4R, Allura Red, Sunset yellow, Tartrazine, Amaranth, Brilliant Blue, Blue, Fast Red E, Orange GGN and Scarlet GN. All food colours tested inhibited mitochondrial respiration (State III respiration, uncoupled) supported either by alpha-ketoglutarate or succinate. This inhibition varied largely, e.g. from 100% to 16% for Erythrosine and Tartrazine respectively, at a concentration of 0.1 mg food colour per mitochondrial protein. Both rat liver and kidney mitochondria showed similar patterns of inhibition among the food colours tested. This effect was dose related and the concentration to give 50% inhibition was determined for some of the dyes. The xanthene dye Erythrosine, which showed the strongest effect, was selected for further investigation on mitochondria in vivo.

Animals↗

PubMed1995

Intracellular Ca2+ storage in acidocalcisomes of Trypanosoma cruzi.

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.

Acridine Orange↗

PubMed1995

Characteristics of Fe(II)ATP complex-induced damage to the rat liver mitochondrial membrane.

It is well established that several iron complexes can induce oxidative damage in hepatic mitochondrial membranes by catalyzing the formation of OH radicals and/or by promoting lipid peroxidation. This is a relevant process for the molecular basis of iron overload diseases. The present work demonstrates that Fe(II)ATP complexes (5-50 microM) promote an oxygen consumption burst in a suspension of isolated rat liver mitochondria (either in the absence or presence of Antimycin A), caused mainly by lipid peroxidation. Fe(II)ATP alone induced small levels of oxygen uptake but no burst. The time course of Fe(II)ATP oxidation to Fe(II)ATP in the extramitochondrial media also reveals a simultaneous 'burst phase'. The iron chelator Desferal (DFO) or the chain-break antioxidant butylated hydroxytoluene (BHT) fully prevented both lipid peroxidation (quantified as oxygen uptake burst) and mitochondrial swelling. DFO and BHT were capable of stopping the ongoing process of peroxidation at any point of their addition to the mitochondrial suspension. Conversely, DFO and BHT only halted the Fe(II)ATP-induced mitochondrial swelling at the onset of the process. Fe(II)ATP could also cause the collapse of mitochondrial potential, which was protected by BHT if added at the onset of the damaging process. These results, as well as correlation studies between peroxidation and mitochondrial swelling, suggest that a two phase process is occurring during Fe(II)ATP-induced mitochondrial damage: one dependent and another independent of lipid peroxidation. The involvement of lipid peroxidation in the overall process of mitochondrial membrane injury is discussed.

Adenosine Triphosphate↗

PubMed1995

Oxidative damage of mitochondria induced by Fe(II)citrate or t-butyl hydroperoxide in the presence of Ca2+: effect of coenzyme Q redox state.

The role of coenzyme Q on the process of mitochondrial membrane damage associated with oxidative stress was studied in a suspension of uncoupled mitochondria exposed to Ca2+ in the presence of Fe(II)citrate or t-butyl hydroperoxide. Reduction of coenzyme Q by succinate was shown to inhibit both inner membrane lipid peroxidation and permeabilization induced by Fe(II)citrate. In contrast, the inner membrane permeabilization induced by Ca2+ alone or Ca2+ plus t-butyl hydroperoxide was potentiated by the presence of succinate. These results support our previous proposition that the mitochondrial damage associated with oxidative stress generated by these pro-oxidants in the presence of Ca2+ is mediated by different mechanisms.

Animals↗

PubMed1995

Permeabilization of the inner mitochondrial membrane by Ca2+ ions is stimulated by t-butyl hydroperoxide and mediated by reactive oxygen species generated by mitochondria.

The extent of swelling undergone by deenergized mitochondria incubated in KCl/sucrose medium in the presence of Ca2+ alone or Ca2+ and t-butyl hydroperoxide decreases by decreasing molecular oxygen concentration in the reaction medium; under anaerobiosis no swelling occurs. This swelling is also inhibited by the presence of exogenous catalase or by the Fe2+ chelator o-phenanthroline in a time-dependent manner. The production of protein thiol cross-linking that leads to the formation of protein aggregates induced by Ca2+ and t-butyl hydroperoxide does not occur when mitochondria are incubated in anaerobic medium or in the presence of o-phenanthroline. In addition, it is also shown that the yield of stable methyl radical adducts, obtained from rat liver mitochondria treated with t-butyl hydroperoxide and the spin trap DMPO, is reduced by addition of EGTA and increases by addition of Ca2+ ions. These data support the hypothesis that Ca2+ ions stimulate electron leakage from the respiratory chain, increasing the mitochondrial production of reactive oxygen species.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1995

Ca(2+)-induced mitochondrial membrane permeabilization: role of coenzyme Q redox state.

Rotenone-poisoned rat liver mitochondria energized by succinate addition, after a 5-min period of preincubation in presence of 10 microM Ca2+, produce H2O2 at much faster rates, undergo extensive swelling, and are not able to retain the membrane potential and accumulated Ca2+. Similar results were obtained when a suspension of rat liver mitochondria preincubated in anaerobic medium for 5 min was reoxygenated. The addition of either ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, ruthenium red, catalase, or dithiothreitol, just before succinate or O2 addition, prevented mitochondrial swelling, indicating the involvement of Ca2+, reactive oxygen species, and oxidation of membrane protein thiols in this process of membrane permeabilization. Inhibition of mitochondrial swelling by cyclosporin A suggests that the membrane alterations observed under these experimental conditions are related to opening of the permeability transition pore. The presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, which prevents Ca2+ cycling across the membrane, did not inhibit mitochondrial swelling when Ca2+ influx into the mitochondrial matrix was driven by a high Ca2+ gradient. When rotenone plus antimycin A-poisoned mitochondria were energized by N,N,N',N'-tetramethyl-p-phenylenediamine, which reduces respiratory chain complex IV, mitochondrial swelling did not occur, unless succinate, which reduces coenzyme Q, was also added. It is concluded that reduced coenzyme Q is the electron source for oxygen radical production during Ca(2+)-stimulated oxidative damage of mitochondria.

Animals↗

PubMed1994

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.

Acridine Orange↗

PubMed1994

Oxidative damage of mitochondria induced by 5-aminolevulinic acid: role of Ca2+ and membrane protein thiols.

Reactive oxygen species (ROS) generated by metal-catalyzed 5-aminolevulinic acid (ALA) aerobic oxidation have been shown to damage the inner membrane of isolated rat liver mitochondria by a Ca(2+)-dependent mechanism. The present work describes experiments indicating that this damage can be prevented, but not completely reversed by the additions of catalase, ADP, cyclosporin A and dithiothreitol, as judged by the extent of delta psi regeneration by the injured mitochondria. In contrast, the addition of EGTA, which removes free Ca2+ and, possibly, Fe2+ present both in the intra- and extramitochondrial compartments, causes a prompt and complete regeneration of delta psi, even after long periods of mitochondrial incubations in the presence of ALA. This reversibility suggests that protein alterations such as protein thiol cross-linkings, evidenced by SDS-polyacrylamide gel electrophoresis, are the main cause of increased membrane permeability promoted by ALA oxidation. The inhibition of protein aggregation and fast regeneration of delta psi promoted by EGTA suggest that the binding of Ca2+ to some membrane proteins plays a crucial role in the mechanism of both protein polymerization (pore assembly) and pore opening. The implication of these results with the molecular pathology of acute intermittent porphyria is also discussed.

Aminolevulinic Acid↗

PubMed1994

Ca(2+)-dependent permeabilization of the inner mitochondrial membrane by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS).

We have recently shown that the permeabilization of the inner mitochondrial membrane by Ca2+ plus prooxidants is associated with oxidation of protein thiols forming cross-linked protein aggregates (Fagian, M.M., Pereira-da-Silva, L., Martins, I.S. and Vercesi, A.E. (1990) J. Biol. Chem. 265, 19955-19960). In this study we show that the incubation of rat liver mitochondria in the presence of the thiol reagent 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and Ca2+ caused production of membrane protein aggregates, mitochondrial swelling, disruption of membrane potential and Ca2+ release. The presence of DTT prevented but did not reverse the elimination of delta psi induced by DIDS. EGTA prevented delta psi elimination and decreased the amount of protein aggregates, suggesting that the binding of Ca2+ to some membrane protein may expose buried thiols to react with DIDS. Reversal of collapsed delta psi by EGTA indicates that DIDS-induced protein aggregates require the presence of Ca2+ for significant membrane permeabilization. Cyclosporin A prevented mitochondrial swelling, suggesting that DIDS-induced membrane protein polymerization mimics the condition designated as Ca(2+)-induced permeabilization transition of mitochondria. The lack of oxidation of pyridine nucleotides or significant lipid peroxidation by DIDS supports the notion that membrane permeabilization by this compound is mediated by its interaction with membrane proteins.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1994

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 microM) or bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA, 50 microM) decreased the trypomastigotes' association to myoblasts by approximately 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.

Animals↗

PubMed1994

Oxidative damage of mitochondria induced by Fe(II)citrate is potentiated by Ca2+ and includes lipid peroxidation and alterations in membrane proteins.

Isolated rat liver mitochondria exposed to Fe(II)citrate undergo lipid peroxidation and alterations in membrane proteins. These processes were associated with irreversible decrease in membrane potential and mitochondrial swelling. Lipid peroxidation was evidenced by the production of thiobarbituric acid-reactive substances and also by the reaction of these products with membrane proteins, through the formation of Schiff bases. Alterations in membrane proteins were also characterized by the loss of specific proteins that could be recovered from the mitochondrial supernatant as shown by SDS-polyacrylamide gel electrophoresis. The degree of both lipid peroxidation and alterations in membrane proteins were diminished by EGTA, ruthenium red, or dibucaine. This strongly indicates that Ca2+ potentiates the oxidative damage of mitochondria exposed to Fe(II)citrate.

Animals↗