Edmundo Chávez

Evidence for the involvement of dithiol groups in mitochondrial calcium transport: studies with cadmium.

The effect of cadmium on some functions of mitochondria isolated from kidneys of rat was studied. Addition of cadmium chloride to mitochondria induced stimulation of both State 4 respiratory rate and ATPase activity, which are prevented by the addition of ruthenium red. We also show that cadmium inhibits competitively calcium translocation; this inhibitory effect of cadmium is reverted by the addition of dithiothreitol. From these results, it is proposed that, similarly to Ca2+, cadmium penetrates mitochondria and binds to a membrane dithiol group, which is essential for the translocation of the cation.

Impairment by cyclosporin A of reperfusion-induced arrhythmias

This study introduces the immunosuppressor, cyclosporin A, as a cardioprotective drug. This effect was analyzed during development of reperfusion/induced arrhythmias after 5-min period of coronary ligation in hearts of rats under anesthesia. The results indicate that cyclosporin, when given before coronary occlusion, at a dose of 20 mg/kg, effectively protects against the high incidence of arrhythmias and the fall in blood pressure induced by reperfusion. In addition, in inhibits the delivery of lactic dehydrogenase and creatine kinase enzymes to the plasma. We propose that the protective effect could be related with its well documented action to restrain Ca(2+)-induced damage of mitochondrial functions.

The mechanism of lead-induced mitochondrial Ca2+ efflux.

Addition of Pb2+ to rat kidney mitochondria is followed by induction of several reactions: inhibition of Ca2+ uptake, collapse of the transmembrane potential, oxidation of pyridine nucleotides, and a fast release of accumulated Ca2+. When the incubation media are supplemented with ruthenium red, the effect of Pb2+ on NAD(P)H oxidation, membrane ΔΨ, and Ca2+ release are not prevented if malate-glutamate are the oxidizing substrates; however, the latter two lead-induced reactions are prevented by ruthenium red if succinate is the electron donor. It is proposed that in mitochondria oxidizing NAD-dependent substrates, Pb2+ induces Ca2+ release by promoting NAD(P)H oxidation and a parallel drop in ΔΨ due to its binding to thiol groups, located in the cytosol side of the inner membrane. In addition, it is proposed that with succinate as substrate, the Ca2+-releasing effect of lead is due to the collapse of the transmembrane potential as a consequence of the uptake of Pb2+ through the calcium uniporter, since such effect is ruthenium red sensitive.

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.

In Saccharomyces cerevisiae, the phosphate carrier is a component of the mitochondrial unselective channel.

The mitochondrial permeability transition (PT) involves the opening of a mitochondrial unselective channel (MUC) resulting in membrane depolarization and increased permeability to ions. PT has been observed in many, but not all eukaryotic species. In some species, PT has been linked to cell death, although other functions, such as matrix ion detoxification or regulation of the rate of oxygen consumption have been considered. The identification of the proteins constituting MUC would help understand the biochemistry and physiology of this channel. It has been suggested that the mitochondrial phosphate carrier is a structural component of MUC and we decided to test this in yeast mitochondria. Mersalyl inhibits the phosphate carrier and it has been reported that it also triggers PT. Mersalyl induced opening of the decavanadate-sensitive Yeast Mitochondrial Unselective Channel (YMUC). In isolated yeast mitochondria from a phosphate carrier-null strain the sensitivity to both phosphate and mersalyl was lost, although the permeability transition was still evoked by ATP in a decavanadate-sensitive fashion. Polyethylene glycol (PEG)-induced mitochondrial contraction results indicated that in mitochondria lacking the phosphate carrier the YMUC is smaller: complete contraction for mitochondria from the wild type and the mutant strains was achieved with 1.45 and 1.1 kDa PEGs, respectively. Also, as expected for a smaller channel titration with 1.1 kDa PEG evidenced a higher sensitivity in mitochondria from the mutant strain. The above data suggest that the phosphate carrier is the phosphate sensor in YMUC and contributes to the structure of this channel.

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.

Triphenyltin as inductor of mitochondrial membrane permeability transition

The effect of triphenyltin on mitochondrial Ca2+ content was studied. It was found that this trialkyltin compound induces an increase in membrane permeability that leads to Ca2+ release, drop of the transmembrane potential, and efflux of matrix proteins. Interestingly, cyclosporin A was unable to inhibit triphenyltin-induced Ca2+ release. Based on these results it is proposed that the hyperpermeable state is produced by modification of 2.25 nmol of membrane thiol groups.

Intramitochondrial K+ as activator of car☐yatractyloside-induced Ca2+ release

The role of intramitochondrial K+ content on the increase in membrane permeability to Ca2+, as induced by carboxyatractyloside was studied. In mitochondria containing a high K+ concentration (83 nmol/mg), carboxyatractyloside induced a fast and extensive mitochondrial Ca2+ release, membrane de-energization, and swelling. Conversely, in K(+)-depleted mitochondria (11 nmol/mg), carboxyatractyloside was ineffective. The addition of 40 mM K+ to K(+)-depleted mitochondria restored the capability of atractyloside to induce an increase in membrane permeability to Ca2+ release. The determination of matrix free Ca2+ concentration showed that, at an external free-Ca2+ concentration of 0.8 microM, control mitochondria contained 3.9 microM of free Ca2+ whereas K(+)-depleted mitochondria contained 0.9 microM free Ca2+. It is proposed that intramitochondrial K+ affects the matrix free Ca2+ concentration required to induce a state of high membrane permeability.

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.

Copper induces permeability transition through its interaction with the adenine nucleotide translocase

In this work we examined the effect of low concentrations of Cu2+ on the opening of the mitochondrial non‐specific pore. The purpose was addressed to further contribute to the knowledge of the mechanisms that regulate the open/closed cycles of the permeability transition pore. Membrane leakage was established by measuring matrix Ca2+ efflux and mitochondrial swelling. The experimental results indicate that Cu2+ at very low concentrations promoted the release of accumulated Ca2+, as well as mitochondrial swelling, provided 1,10‐phenanthroline has been added. Carboxyatractyloside and Cu2+ exhibited additive effects on these parameters. After Cu2+ titration of membrane thiols, it might be assumed that the blockage of 5.9 nmol of SH/mg protein suffices to open the non‐specific pore. Taking into account the reinforcing effect of carboxyatractyloside, the increasing ADP concentrations, and that N‐ethylmaleimide inhibited the Cu2+‐induced Ca2+ efflux, it is proposed that the target site for Cu2+ is located in the ADP/ATP carrier.