Natalia Pavón

Bax distribution into mitochondrial detergent‐resistant microdomains is related to ceramide and cholesterol content in postischemic hearts

Diverse changes have been described in mitochondria of apoptotic cells: the phospholipid content is modified, ceramide and GD3 concentrations increase, the cristae structure is modified, and nonresident proteins are recruited into the mitochondrial membranes. In particular, Bax, a Bcl‐2 family member protein, moves from the cytosol to the mitochondria, inducing cytochrome c release. Modifications of the content and distribution of specific lipids in the mitochondrial membranes, along with the well‐known participation of the mitochondrial permeability transition pore in triggering apoptosis, led us to propose that lipid microdomains in mitochondria could coexist as structural elements with some of the mitochondrial permeability transition pore‐forming proteins and with members of the Bcl‐2 family. In this work, we demonstrated that Bax was associated preferentially with mitochondrial detergent‐resistant membranes (mDRMs) in reperfused rat hearts, a well‐known apoptotic model. Bax insertion into mDRMs correlated with cytochrome c release from such mitochondria. Bax location in mDRMs was associated with both the voltage‐dependent anion channel and the adenine nucleotide translocator, two mitochondrial permeability transition pore‐forming proteins. Interestingly, the voltage‐dependent anion channel was more abundant in the mDRM fraction than in the Triton X‐100‐soluble fraction. Ceramide and cholesterol contents were higher in mDRMs from reperfused hearts. Our results suggest that membrane microenvironments enriched in cholesterol and ceramide in mitochondria favor Bax translocation to this organelle, fostering propagation of the apoptotic cascade.

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.

Sexual hormones: Effects on cardiac and mitochondrial activity after ischemia-reperfusion in adult rats. Gender difference

In this work we studied the influence of sex hormones on heart and mitochondrial functions, from adult castrated female and male, and intact rats. Castration was performed at their third week of life and on the fourth month animals were subjected to heart ischemia and reperfusion. Electrocardiogram and blood pressure recordings were made, cytokines levels were measured, histopathological studies were performed and thiobarbituric acid reactive species were determined. At the mitochondrial level respiratory control, transmembranal potential and calcium management were determined; Western blot of some mitochondrial components was also performed. Alterations in cardiac function were worst in intact males and castrated females as compared with those found in intact females and castrated males, cytokine levels were modulated also by hormonal status. Regarding mitochondria, in those obtained from hearts from castrated females without ischemia-reperfusion, all evaluated parameters were similar to those observed in mitochondria after ischemia-reperfusion. The results show hormonal influences on the heart at functional and mitochondrial levels.

Titration of cardiolipin by either 10-N-nonyl acridine orange or acridine orange sensitizes the adenine nucleotide carrier to permeability transition.

Under the action of carboxyatractyloside or fatty acids, adenine nucleotide translocase switches its function from nucleotide carrier to modulator of the opening of a non-specific pore. In addition to the effect of these agents, this modification in activity is, in some way, dependent on the influence of the lipid milieu of the membrane. Cardiolipin is, among other membrane phospholipids, the one that interacts the most with the translocase. This work shows that 10-N-nonyl acridine orange and acridine orange, probes for this phospholipid, modify the sensitivity of the translocase to carboxyatractyloside, oleate, and palmitate to induce permeability transition. The results also show that these probes stimulate the release of mitochondrial cytochrome c, and increase labeling of the carrier by eosin 5-maleimide. Based on the aforementioned it is proposed that the increase in sensitivity is due to a conformational change in the translocase, induced by the binding of the probe to cardiolipin.

Cyclosporin a is unable to inhibit carboxyatractyloside-induced permeability transition in aged mitochondria

We studied the effect of mitochondrial ageing on membrane permeability transition. The results obtained indicate that aged mitochondria are neither able to retain Ca2+ nor to maintain a high transmembrane electric gradient. In addition, aged mitochondria undergo a large amplitude swelling. These dysfunctions were circumvented by the addition of cyclosporin A. Furthermore, it is shown that ageing-induced permeability transition causes oxidative damage on the matrix enzyme aconitase. The observed damage in aged mitochondria requires Ca2+ addition; therefore, it was not seen when Sr2+ replaced Ca2+. Two important findings in this work were the fact that despite of the presence of cyclosporin A, carboxyatractyloside was still able to induce permeability transition, and that ageing induced mitochondrial DNA disruption and release of cytochrome c. It is likely that the membranes increased permeability is due to the effect of fatty acids, since bovine serum albumin makes mitochondria able to retain Ca2+. However, the possibility that the damage might be the result of oxidative stress cannot be discarded.

The Effect of N -Ethylmaleimide on Permeability Transition as Induced by Carboxyatractyloside, Agaric Acid, and Oleate

In this work, we studied the effect of N-ethylmaleimide on permeability transition. The findings indicate that the amine inhibited the effects of carboxyatractyloside and agaric acid. It is known that these reagents interact with the adenine nucleotide carrier through the cytosolic side. When oleate, which interacts through the matrix side, was used it was found that the amine amplified the effects of oleate on permeability transition. The results also show that N-ethylmaleimide strengthened the inhibition induced by carboxyatractyloside, agaric acid, and oleate on ADP exchange. Furthermore, it was also found that oleate improved the binding of eosin-5-maleimide on the adenine nucleotide translocase.

Induction of Mitochondrial Permeability Transition by the DNA-intercalating Cationic Dye Ethidium Bromide

This work shows that the DNA cationic probe, ethidium bromide (EtBr), induces the transition from selective to non-selective mitochondrial permeability. This statement is based on the findings, indicating: (i) EtBr induced the release of accumulated Ca(2+) through a mechanism sensitive to cyclosporin A and octylguanidine; (ii) EtBr induced the release of cytochrome c and (iii) EtBr induced mitochondrial swelling. Interestingly, mersalyl inhibited, in a non-competitive fashion, EtBr uptake, which would indicate that the uptake may be carried out through a protein membrane system. This work also shows that the effect of the dye on permeability transition was stimulated by carboxyatractyloside. Taking into account the facts that EtBr inhibited the ADP exchange reaction and increased the binding of the fluorescent probe eosin-5-maleimide to adenine nucleotide translocase, it is tempting to assume a possible interaction between EtBr and the ADP/ATP carrier.

On the Opening of an Insensitive Cyclosporin A Non-specific Pore by Phenylarsine Plus Mersalyl

The purpose of this work was addressed to provide new information on the effect of thiol reagents on mitochondrial non-specific pore opening, and its response to cyclosporin A (CSA). To meet this proposal phenylarsine oxide (PHA) and mersalyl were employed as tools to induce permeability transition and CSA to inhibit it. PHA-induced mitochondrial dysfunction, characterized by Ca2+ efflux, swelling, and membrane de-energization, was inhibited by N-ethylmaleimide and CSA. Conversely, mersalyl failed to inhibit the inducing effect of phenylarsine oxide, it rather strengthened it. In addition, the effect of mersalyl was associated with cross-linking of membrane proteins. The content of membrane thiol groups accessible to react with PHA, mersalyl, and PHA plus mersalyl was determined. In all situations, permeability transition was accompanied by a significant decrease in the whole free membrane thiol content. Interestingly, it is also shown that mersalyl hinders the protective effect of cyclosporin A on PHA-induced matrix Ca2+ efflux.

Cardioprotective properties of citicoline against hyperthyroidism-induced reperfusion damage in rat hearts.

Hyperthyroidism represents an increased risk factor for cardiovascular morbidity, especially when the heart is subjected to an ischemia/reperfusion process. The aim of this study was to explore the possible protective effect of the nucleotide citicoline on the susceptibility of hyperthyroid rat hearts to undergo reperfusion-induced damage, which is associated with mitochondrial dysfunction. Hence, we analyzed the protective effect of citicoline on the electrical behavior and on the mitochondrial function in rat hearts. Hyperthyroidism was established after a daily i.p. injection of triiodothyronine (at 2 mg/kg of body weight) during 5 days. Thereafter, citicoline was administered i.p. (at 125 mg/kg of body weight) for 5 days. In hyperthyroid rat hearts, citicoline protected against reperfusion-induced ventricular arrhythmias. Moreover, citicoline maintained the accumulation of mitochondrial Ca(2+), allowing mitochondria to reach a high transmembrane electric gradient that protected against the release of cytochrome c. It also preserved the activity of the enzyme aconitase that inhibited the release of cytokines. The protection also included the inhibition of oxidative stress-induced mDNA disruption. We conclude that citicoline protects against the reperfusion damage that is found in the hyperthyroid myocardium. This effect might be due to its inhibitory action on the permeability transition in mitochondria.

Citicoline (CDP-choline) protects myocardium from ischemia/ reperfusion injury via inhibiting mitochondrial permeability transition

AIMS Oxidative stress emerges after reperfusion of an organ following an ischemic period and results in tissue damage. In the heart, an amplified generation of reactive oxygen species and a significant Ca(2+) accumulation cause ventricular arrhythmias and mitochondrial dysfunction. This occurs in consequence of increased non-specific permeability. A number of works have shown that permeability transition is a common substrate that underlies the reperfusion-induced heart injury. The aim of this work was to explore the possibility that CDP-choline may circumvent heart damage and mitochondrial permeability transition. MAIN METHODS Rats were injected i.p. with CDP-choline at 20 mg/kg body weight. Heart electric behavior was followed during a closure/opening cycle of the left coronary descendent artery. Heart mitochondria were isolated from rats treated with CDP-choline, and their function was evaluated by analyzing Ca(2+) movements, achievement of a high level of the transmembrane potential, and respiratory control. Oxidative stress was estimated following the activity of the enzymes cis-aconitase and superoxide dismutase, as well as the disruption of mitochondrial DNA. KEY FINDINGS This study shows that CDP-choline avoided ventricular arrhythmias and drop of blood pressure. Results also show that mitochondria, isolated from CDP-choline-treated rats, maintained selective permeability, retained accumulated Ca(2+), an elevated value of transmembrane potential, and a high ratio of respiratory control. Furthermore, activity of cis-aconitase enzyme and mDNA structure were preserved. SIGNIFICANCE This work introduces CDP-choline as a useful tool to preserve heart function from reperfusion damage by inhibiting mitochondrial permeability transition.