Biomedical subjects

C Zazueta

Publications and source records attributed to C Zazueta.

At least 19 recordsLinked to original sources

PubMed2006

Ru360, a specific mitochondrial calcium uptake inhibitor, improves cardiac post-ischaemic functional recovery in rats in vivo.

BACKGROUND AND PURPOSE: The mitochondrial permeability transition pore (mPTP), an energy-dissipating channel activated by calcium, contributes to reperfusion damage by depolarizing the mitochondrial inner membrane potential. As mitochondrial Ca(2+) overload is a main inductor of mPTP opening, we examined the effect of Ru(360), a selective inhibitor of the mitochondrial calcium uptake system against myocardial damage induced by reperfusion in a rat model. EXPERIMENTAL APPROACH: Myocardial reperfusion injury was induced by a 5-min occlusion of the left anterior descending coronary artery, followed by a 5-min reperfusion in anaesthetized open-chest rats. We measured reperfusion-induced arrhythmias and functions indicative of unimpaired mitochondrial integrity to evaluate the effect of Ru(360) treatment. KEY RESULTS: Reperfusion elicited a high incidence of arrhythmias, haemodynamic dysfunction and loss of mitochondrial integrity. A bolus intravenous injection of Ru(360) (15-50 nmol kg(-1)), given 30-min before ischaemia, significantly improved the above mentioned variables in the ischaemic/reperfused myocardium. Calcium uptake in isolated mitochondria from Ru(360)-treated ventricles was partially diminished, suggesting an interaction of this compound with the calcium uniporter. CONCLUSIONS AND IMPLICATIONS: We showed that Ru(360) treatment abolishes the incidence of arrhythmias and haemodynamic dysfunction elicited by reperfusion in a whole rat model. Ru(360) administration partially inhibits calcium uptake, preventing mitochondria from depolarization by the opening of the mPTP. We conclude that myocardial damage could be a consequence of failure of the mitochondrial network to maintain the membrane potential at reperfusion. Hence, it is plausible that Ru(360) could be used in reperfusion therapy to prevent the occurrence of arrhythmia.

Aconitate Hydratase↗

PubMed2004

Different subunit location of the inhibition and transport sites in the mitochondrial calcium uniporter.

The mitochondrial calcium uniporter behaves as a cooperative mechanism, where the velocity is dependent on [Ca2+]ex. Transport kinetics follows a sigmoidal behavior with a Hill coefficient near 2.0, indicating the binding of at least two calcium molecules. Calcium transport in mitochondria is dependent on a negative inner membrane potential and is inhibited by policationic ruthenium compounds. In this study, calcium uptake activity was reconstituted into cytochrome oxidase vesicles by incorporating solubilized mitochondrial proteins. Calcium accumulation plotted against increasing Ca2+ concentrations followed a sigmoidal behavior with a Hill coefficient of 1.53. The uptake was sensitive to ruthenium policationic inhibitors, e.g. ruthenium red and Ru360. After mitochondrial proteins were separated by preparative isoelectrofocusing and incorporated into cytochrome oxidase vesicles, two peaks of calcium uptake activity were recovered. One of the activities was inhibited by Ru360, while the second activity was insensitive to Ru360 and was associated with proteins focused at very acidic isoelectric points. By using a thiol-group crosslinker and radiolabeled Ru360, we proposed a scheme of partial dissociation of the uniporter inhibitor-binding subunit under acidic conditions.

Animals↗

PubMed2003

Cardiolipin regulates the activity of the reconstituted mitochondrial calcium uniporter by modifying the structure of the liposome bilayer.

Reconstitution of mitochondrial calcium transport activity requires the incorporation of membrane proteins into a lipidic ambient. Calcium uptake has been measured previously using Cytochrome oxidase vesicles. The enrichment of these vesicles with cardiolipin, an acidic phospholipid that is found only in the inner mitochondrial membrane of eukaryotic cells, strongly inhibits calcium transport, in remarkable contrast with the activation effect that cardiolipin exerts upon other mitochondrial transporters and enzymes. The relation of the inactivation of calcium transport to the physical state of the bilayer was studied by following the polarization changes of 1,6-diphenyl-1,3,5-hexatriene (DPH) and by flow cytometry in the cardiolipin-enriched liposomes with incorporated mitochondrial solubilized proteins. Non-bilayer molecular arrangements in the cardiolipin-supplemented liposomes, detected by flow cytometry, may produce the fluidity changes observed by fluorescence polarization of DPH. Fluidity changes correlate with the abolition of calcium uptake, but have no effect on the establishment of a membrane potential in the vesicles required for calcium transport activity. Changes in the membrane structure and uniporter function are observed in the combined presence of cardiolipin and calcium leading to a modified lipid configuration.

Calcium↗

PubMed2000

Possible involvement of the adenine nucleotide translocase in the activation of the permeability transition pore induced by cadmium.

Low levels of cadmium induce a rapid calcium efflux in energized rat kidney mitochondria. This is accompanied by the collapse of the transmembrane gradient in a partial CSA-sensitive fashion. The binding of 109Cd2+ to mitochondria is a saturable function; in the presence of NEM, the binding of 2.5 nmol 109Cd2+/mg of protein suffices to induce the opening of the permeability transition pore. It was found that cadmium bound mainly to proteins of molecular weight between 30 and 50 kDa. In the presence of the monothiol reagent NEM, the label is concentrated in the 30 kDa protein. Following the addition of the reducing agent dithiothreitol, calcium is reaccumulated and the membrane potential restored. This correlates with a significant loss of label in the 30 kDa protein region. The 30 kDa protein was identified as the adenine nucleotide translocase by labelling experiments with eosin 5-maleimide and experiments of reconstitution.

Animals↗

PubMed2000

Inactivation of mitochondrial permeability transition pore by octylguanidine and octylamine.

Mitochondrial permeability transition occurs through a Ca2+-dependent opening of a transmembrane pore, whose identity has been attributed to that of the adenine nucleotide translocase (ANT). In this work, we induced permeability transition by adding 0.5 microM carboxyatractyloside. The process was evaluated analyzing Ca2+ efflux, a drop in transmembrane electric gradient, and swelling. We found that the amphiphyllic cations octylguanidine and octylamine, at the concentration of 100 microM, inhibited, almost completely, nonspecific membrane permeability. Hexylguanidine, hexylamine, as well as guanidine chloride and hydroxylamine failed to do so. The inhibition was reversed after the addition of 40 mM Li+, Na+ K+, Rb+, or Cs+; K+ was the most effective. We propose that the positive charge of the amines interact with negative charges of membrane proteins, more likely the ADP/ATP carrier, while the alkyl chain penetrates into the hydrophobic milieu of the inner membrane, fixing the reagent.

Adenosine Diphosphate↗

PubMed2000

Copper sensitizes the mitochondrial permeability transition to carboxytractyloside and oleate.

Addition of 5 microM copper to rat kidney mitochondria enhances the effect of carboxyatractyloside and oleate on pore opening, in a cyclosporin A-sensitive fashion. The effects of the pair copper-carboxyatractyloside were observed on matrix Ca2+ efflux, mitochondrial swelling and on the transmembrane electric gradient. The effect of Cu2+ emphasizes the importance of membrane thiol groups located, probably, in the ADP/ATP translocase (ANT), on permeability transition. It was also found that Cu2+ does not block the fluorescent label of ANT by eosin 5-maleimide, but abolishes the inhibition by CAT on the labeling. This suggests that the binding of Cu2+ to cysteine residues of ANT promotes a conformational change in the carrier, strengthening the effect of CAT and oleate on membrane leakage.

Animals↗

PubMed1999

Carboxyatractyloside increases the effect of oleate on mitochondrial permeability transition.

Addition of a low concentration of carboxyatractyloside (0.075 microM) renders mitochondria susceptible to the opening of the non-specific pore by 5 microM oleate, in a cyclosporin A-sensitive fashion. Matrix Ca2+ efflux as well as collapse of the transmembrane potential reveal permeability transition. The effect of oleate is reached after the titration, by carboxyatractyloside, of 38 pmol of adenine nucleotide translocase per mg mitochondrial protein. We propose that permeability transition may result from an additive action of carboxyatractyloside plus oleate on the ADP/ATP carrier.

Animals↗

PubMed1999

Inhibitory properties of ruthenium amine complexes on mitochondrial calcium uptake.

The recent finding that the inhibition of Ca2+-stimulated respiration by ruthenium red is mainly due to a binuclear ruthenium complex (Ru360) present in the commercial samples of the classical inhibitor ruthenium red (Ying et. al., 1991), showed that this complex is the more potent and specific inhibitor of the mitochondrial calcium uniporter. This work was aimed to provide insights into the mechanism by which Ru360 and other ruthenium-related compounds inhibits calcium uptake. Ruthenium red and a synthesized analog (Rrphen) were compared with Ru360. The inhibition by this binuclear complex was noncompetitive, with a Ki of 9.89 nM. The number of specific binding sites for Ru360 was 6.2 pmol/mg protein. Ruthenium red and Ru360 were mutually exclusive inhibitors. Bound La3+ was not displaced by Ru360. Rrphen was the least effective for inhibiting calcium uptake. The results support the notion of a specific binding site in the uniporter for the polycationic complexes and a negative charged region from the phospholipids in the membrane, closely associated with the uniporter inhibitor-binding site.

Amines↗

PubMed1998

Hypothyroidism renders liver mitochondria resistant to the opening of membrane permeability transition pore.

Membrane permeability was examined in liver mitochondria isolated from hypothyroid rats. It was found that such a thyroid status provides substantial protection from membrane leakiness as induced by Ca2+ loading. Thus, these mitochondria are less prone to undergoing permeability transition than mitochondria from euthyroid rats. The above conclusion was reached on the basis of the following two facts: (1) hypothyroid mitochondria are not strictly dependent on the addition of ADP to retain high matrix Ca2+ concentrations, and (2) carboxyatractyloside, antimycin A or carbonyl cyanide-m-chlorophenyl hydrazone failed to promote Ca2+ efflux. We discuss the possible relevance of the low content of membrane cardiolipin as well as the low expression of the adenine nucleotide translocase as responsible for the resistance to membrane damage.

Adenosine Diphosphate↗

PubMed1998

Mitochondrial permeability transition as induced by cross-linking of the adenine nucleotide translocase.

Mitochondrial permeability transition is caused by the opening of a transmembrane pore whose chemical nature has not been well established yet. The present work was aimed to further contribute to the knowledge of the membrane entity comprised in the formation of the non-specific channel. The increased permeability was established by analyzing the inability of rat kidney mitochondria to take up and accumulate Ca2+, as well as their failure to build up a transmembrane potential, after the cross-linking of membrane proteins by copper plus ortho-phenanthroline. To identify the cross-linked proteins, polyacrylamide gel electrophoresis was performed. The results are representative of at least three separate experiments. It is indicated that 30 microM Cu2+ induced the release of 4.3 nmol Ca2+ per mg protein. However, in the presence of 100 microM ortho-phenanthroline only 2 microM Cu2+ was required to attain the total release of the accumulated Ca2+; it should be noted that such a reaction is not inhibited by cyclosporin. The increased permeability corresponds to cross-linking of membrane proteins in which approximately 4 nmol thiol groups per mg protein appear to be involved. Such a linking process is inhibited by carboxyatractyloside. By using the fluorescent probe eosin-5-maleimide the label was found in a cross-linking 60 kDa dimer of two 30 kDa monomers. From the data presented it is concluded that copper-o-phenanthroline induces the intermolecular cross-linking of the adenine nucleotide translocase which in turn is converted to non-specific pore.

Animals↗

PubMed1998

Advances in the purification of the mitochondrial Ca2+ uniporter using the labeled inhibitor 103Ru360.

For many years the calcium uniporter has eluded attempts of purification, partly because of the difficulties inherent in the purification of low-abundance hydrophobic proteins (Reed and Bygrave, 1974). Liquid-phase preparative isoelectric focusing improved the fractionation of mitochondrial membrane proteins. A single 6-h run resulted in a 90-fold increase in specific activity of pooled active fractions over a semipurified fraction, allowing for enrichment of the calcium transport function in cytochrome oxidase vesicles. An additional powerful tool in the isolation of the uniporter was the use of the labeled inhibitor 103Ru360 as an affinity ligand; by following this procedure a protein of 18 kDa was purified in nondenatured, but rather inactive, form. The labeled protein corresponds to the protein that showed Ca2+ transport activity.

Animals↗

PubMed1998

Oxygen free-radicals mediate the damaging effect of ultraviolet light on membrane mitochondria.

This paper reports evidence that exposure of mitochondria to near-ultraviolet light inhibits electron transport, collapses the electric gradient, and increases non-specific membrane permeability to matrix solutes such as Ca2+. Membrane energization, as well as superoxide dismutase and catalase avoid membrane leakiness. Increased permeability correlates with a diminution in the titrated thiol groups. Plausibly the pore is formed through the formation of sulfhydryl bridges by the action of UV light-derived oxygen-centered free- radicals on membrane proteins.

Animals↗

PubMed1997

On the protection by inorganic phosphate of calcium-induced membrane permeability transition.

The role of inorganic phosphate as inhibitor of mitochondrial membrane permeability transition was studied. It is shown that in mitochondria containing a high phosphate concentration, i.e., 68 nmo/mg, Ca2+ did not activate the pore opening. Conversely, at lower levels of matrix phosphate, i.e., 38 nmol/mg, Ca2+ was able to induce subsequent pore opening. The inhibitory effect of phosphate was apparent in sucrose-based media, but it was not achieved in KCI media. The matrix free Ca2+ concentration and matrix pH were lowered by phosphate, but they were always higher in K+-media. In the absence of ADP, phosphate strengthened the inhibitory effect of cyclosporin A on carboxyatractyloside-induced Ca2+ efflux. Acetate was unable to replace phosphate in the induction of the aforementioned effects. It is concluded that phosphate preserves selective membrane permeability by diminishing the matrix free Ca2+ concentration.

Animals↗

PubMed1997

Membrane permeability transition as induced by dysfunction of the electron transport chain.

The results in this paper indicate that mitochondrial permeability transition is activated by dysfunction of the respiratory chain caused by anaerobiosis, exhaustion of the oxidative substrate, and antimycin A, in the presence of 100 microM Ca2+. Membrane damage coincides with the collapse of the electric gradient. The opening of the non-selective pore is prevented by cyclosporin A.

Adenosine Triphosphate↗

PubMed1996

On the mechanism by which 6-ketocholestanol protects mitochondria against uncoupling-induced Ca2+ efflux.

This work shows that 6-ketocholestanol (kCh) inhibits the effect of carbonyl cyanide-m-chlorophenyl hydrazone (CCP) on mitochondrial Ca2+ efflux. Such an effect proved to be caused by diminution of membrane fluidity, therefore, it is affected by the incubation temperature. Furthermore, kCh reversed CCP-induced Ca2+ efflux depending on the accumulation of phosphate. It is also shown that kCh enhances the effect of carboxyatractyloside on membrane permeability transition.

Animals↗

PubMed1996

Modulation of matrix Ca2+ content by the ADP/ATP carrier in brown adipose tissue mitochondria. Influence of membrane lipid composition.

The role of the adenine nucleotide translocase on Ca2+ homeostasis in mitochondria from brown adipose tissue was examined. It was found that in mitochondria incubated with 50 microM Ca2+, ADP was not needed to retain the cation, but it was required for strengthening the inhibitory effect of cyclosporin on membrane permeability transition as induced by menadione. In addition, carboxyatractyloside was unable to promote matrix Ca2+ release, even though it inhibits the ADP exchange reaction. However, when the Ca2+ concentration was increased to 150 microM carboxyatractyloside did induce Ca2+ release, and ADP favored Ca2+ retention. Determination of cardiolipin content in the inner membrane vesicles showed a greater concentration in brown adipose tissue mitochondria than that found in kidney mitochondria. It suggested that the failure of the adenine nucleotide translocase to influence membrane permeability transition depends on the lipid composition of the inner membrane.

Adenosine Diphosphate↗

PubMed1994

Characterization of Ca2+ transport in Euglena gracilis mitochondria.

The present study was designed to establish the characteristics of the Ca2+ fluxes in isolated mitochondria of the protist Euglena gracilis. Uptake of Ca2+ and Sr2+ was supported by succinate and lactate oxidation. Ca2+ influx was slightly inhibited by 5 microM Ruthenium red and completely blocked by La3+ with a half-maximal inhibition attained at 50 microM. The addition of inorganic phosphate induced a 3-fold stimulation of Ca2+ uptake. Ca2+ uptake was inhibited by Mg2+ only in the absence of phosphate. Ca2+ efflux was induced by Na+, Li+ and K+ through a diltiazem-insensitive reaction. Ca2+ release, collapse of membrane potential and swelling were induced by Hg2+ and Cd2+ but not by carboxyatractyloside; cyclosporin A did not prevent the Ca2+ release induced by the heavy metal ions. Ca2+ uptake was achieved in the presence of 3 microM antimycin or 0.1 mM cyanide; this finding indicates that the alternative respiratory chain present in Euglena mitochondria can support this energy-dependent reaction. The data obtained suggest similar pathways, but different regulatory mechanisms, for Ca2+ transport between protist and mammalian mitochondria.

Animals↗

PubMed1994

Identification of a 20-kDa protein with calcium uptake transport activity. Reconstitution in a membrane model.

This paper presents results of experiments designed to further purify the membrane system involved in mitochondrial calcium transport. A partially purified extract, which transported calcium with a specific activity of 1194 nmol 45Ca2+/mg protein/5 min, was used to obtain mouse hyperimmune serum. This serum inhibited calcium uptake both in mitoplasts and in vesicles reconstituted with mitochondrial proteins containing cytochrome oxidase. Western blot analysis of the semipurified fraction showed that the serum recognized specifically two antigens of 75 and 20 kDa. Both antibodies were purified by elution from the nitrocellulose sheets and their inhibition capacity was analyzed. The antibody that recognized the 20-kDa protein produced a higher degree of inhibition than the other one.

Animals↗