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Mitochondrial permeability transition is characterized by the opening of a transmembranal pore that switches membrane permeability from specific to nonspecific. This structure allows the free traffic of ions, metabolites, and water across the mitochondrial inner membrane. The opening of the permeability transition pore is triggered by oxidative stress along with calcium overload. In this work, we explored if oxidative stress is a consequence, rather than an effector of the pore opening, by evaluating the interaction of agaric acid with the adenine nucleotide translocase, a structural component of the permeability transition pore. We found that agaric acid induces transition pore opening, increases the generation of oxygen-derived reactive species, augments the oxidation of unsaturated fatty acids in the membrane, and promotes the detachment of cytochrome c from the inner membrane. The effect of agaric acid was inhibited by the antioxidant tamoxifen in association with decreased binding of the thiol reagent eosin-3 maleimide to the adenine nucleotide translocase. We conclude that agaric acid promotes the opening of the pore, increasing ROS production that exerts oxidative modification of critical thiols in the adenine nucleotide translocase.

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In the kidney, the accumulation of heavy metals such as Cd2+ produces mitochondrial dysfunctions, i.e., uncoupling of the oxidative phosphorylation, inhibition of the electron transport through the respiratory chain, and collapse of the transmembrane electrical gradient. This derangement may be due to the fact that Cd2+ induces the transition of membrane permeability from selective to nonselective via the opening of a transmembrane pore. In fact, Cd2+ produces this injury through the stimulation of oxygen-derived radical generation, inducing oxidative stress. Several molecules have been used to avoid or even reverse Cd2+-induced mitochondrial injury, for instance, cyclosporin A, resveratrol, dithiocarbamates, and even EDTA. The aim of this study was to explore the possibility that the antioxidant tamoxifen could protect mitochondria from the deleterious effects of Cd2+. Our results indicate that the addition of 1 µmol/L Cd2+ to mitochondria collapsed the transmembrane electrical gradient, induced the release of cytochrome c, and increased both the generation of H2O2 and the oxidative damage to mitochondrial DNA (among other measured parameters). Of interest, these mitochondrial dysfunctions were ameliorated after the addition of tamoxifen.

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Several studies have demonstrated that the mitochondrial membrane switches from selective to non-selective permeability because of its improved matrix Ca2+ accumulation and oxidative stress. This process, known as permeability transition, evokes severe dysfunction in mitochondria through the opening of a non-specific pore, whose chemical nature is still under discussion. There are some proposals regarding the components of the pore structure, e.g., the adenine nucleotide translocase and dimers of the F1 Fo-ATP synthase. Our results reveal that Ca2+ induces oxidative stress, which not only increases lipid peroxidation and ROS generation but also brings about both the collapse of the transmembrane potential and the membrane release of cytochrome c. Additionally, it is shown that Ca2+ increases the binding of the probe eosin-5-maleimide to adenine nucleotide translocase. Interestingly, these effects are diminished after the addition of ADP. It is suggested that pore opening is caused by the binding of Ca2+ to the adenine nucleotide translocase.

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Heavy metal ions are known to produce harmful alterations on kidney function. Specifically, the accumulation of Hg2+ in kidney tissue may induce renal failure. In this work, the protective effect of CDP-choline against the deleterious effects induced by Hg2+ on renal function was studied. CDP-choline administered ip at a dose of 125 mg/kg body weight prevented the damage induced by Hg2+ administration at a dose of 3 mg/kg body weight. The findings indicate that CDP-choline guards mitochondria against Hg2+ -toxicity by preserving their ability to retain matrix content, such as accumulated Ca2+ . This nucleotide also protected mitochondria from Hg2+ -induced loss of the transmembrane electric gradient and from the generation of hydrogen peroxide and membrane TBARS. In addition, CDP-choline avoided the oxidative damage of mtDNA and inhibited the release of the interleukins IL-1 and IL6, recognized as markers of acute inflammatory reaction. After the administration of Hg2+ and CDP, CDP-choline maintained nearly normal levels of renal function and creatinine clearance, as well as blood urea nitrogen (BUN) and serum creatinine.

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In this work, we studied the protective effects of tamoxifen (TAM) on disulfiram (Dis)-induced mitochondrial membrane insult. The results indicate that TAM circumvents the inner membrane leakiness manifested as Ca2+ release, mitochondrial swelling, and collapse of the transmembrane electric gradient. Furthermore, it was found that TAM prevents inactivation of the mitochondrial enzyme aconitase and detachment of cytochrome c from the inner membrane. Interestingly, TAM also inhibited Dis-promoted generation of hydrogen peroxide. Given that TAM is an antioxidant molecule, it is plausible that its protection may be due to the inhibition of Dis-induced oxidative stress.

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In this work, we studied the effect of tamoxifen and cyclosporin A on mitochondrial permeability transition caused by addition of the thiol-oxidizing pair Cu2+ -orthophenanthroline. The findings indicate that tamoxifen and cyclosporin A circumvent the oxidative membrane damage manifested by matrix Ca2+ release, mitochondrial swelling, and transmembrane electrical gradient collapse. Furthermore, it was found that tamoxifen and cyclosporin A prevent the generation of TBARs promoted by Cu2+ -orthophenanthroline, as well as the inactivation of the mitochondrial enzyme aconitase and disruption of mDNA. Electrophoretic analysis was unable to demonstrate a cross-linking reaction between membrane proteins. Yet, it was found that Cu2+ -orthophenanthroline induced the generation of reactive oxygen species. It is thus plausible that membrane leakiness is due to an oxidative stress injury.

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AIMS: Mitochondrial permeability transition is a process established through massive Ca(2+) load in addition to an inducer reagent. Ebselen (Ebs), an antioxidant seleno compound, has been introduced as a reagent which inhibits mitochondrial dysfunction induced by permeability transition. Paradoxically enough, it has been shown that Ebs may also be able to induce the opening of the mitochondrial non-selective pores. This study was performed with the purpose of establishing the membrane system involved in Ebs-induced pore opening. MAIN METHODS: Permeability transition was appraised by analyzing the following: i) matrix Ca(2+) release, and mitochondrial swelling, ii) efflux of cytochrome c, and iii) the inhibition of superoxide dismutase. All of these adverse reactions were inhibited by N-ethylmaleimide and cyclosporin A. KEY FINDINGS: At concentrations from 5 to 20 µM, we found that Ebs induces non-specific membrane permeability. Remarkably, Ebs blocks the binding of the fluorescent reagent eosin-5-maleimide to the thiol groups of the adenine nucleotide translocase. SIGNIFICANCE: Based on the above, it is tempting to hypothesize that Ebs induces pore opening through its binding to the ADP/ATP carrier.

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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.

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Hyperthyroidism, known to have deleterious effects on heart function, and is associated with an enhanced metabolic state, implying an increased production of reactive oxygen species. Tamoxifen is a selective antagonist of estrogen receptors. These receptors make the hyperthyroid heart more susceptible to ischemia/reperfusion. Tamoxifen is also well-known as an antioxidant. The aim of the present study was to explore the possible protective effect of tamoxifen on heart function in hyperthyroid rats. Rats were injected daily with 3,5,3'-triiodothyronine at 2mg/kg body weight during 5 days to induce hyperthyroidism. One group was treated with 10mg/kg tamoxifen and another was not. The protective effect of the drug on heart rhythm was analyzed after 5 min of coronary occlusion followed by 5 min reperfusion. In hyperthyroid rats not treated with tamoxifen, ECG tracings showed post-reperfusion arrhythmias, and heart mitochondria isolated from the ventricular free wall lost the ability to accumulate and retain matrix Ca(2+) and to form a high electric gradient. Both of these adverse effects were avoided with tamoxifen treatment. Hyperthyroidism-induced oxidative stress caused inhibition of cis-aconitase and disruption of mitochondrial DNA, effects which were also avoided by tamoxifen treatment. The current results support the idea that tamoxifen inhibits the hypersensitivity of hyperthyroid rat myocardium to reperfusion damage, probably because its antioxidant activity inhibits the mitochondrial permeability transition.

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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.

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Chemical modification of primary amino groups of mitochondrial membrane proteins by the fluorescent probe fluorescamine induces non-specific membrane permeabilisation. Titration of the lysine ϵ-amino group promoted efflux of accumulated Ca(2+), collapse of transmembrane potential and mitochondrial swelling. Ca(2+) release was inhibited by cyclosporin A. Considering the latter, we assumed that fluorescamine induces permeability transition. Carboxyatractyloside also inhibited the reaction. Using a polyclonal antibody for adenine nucleotide translocase, Western blot analysis showed that the carrier appeared labelled with the fluorescent probe. The results point out the importance of the ϵ-amino group of lysine residues, located in the adenine nucleotide carrier, on the modulation of membrane permeability, since its blockage suffices to promote opening of the non-specific nanopore.

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Cecropin 3 (Ccrp3) is an antimicrobial peptide from Anopheles albimanus, which is expressed during Plasmodium berghei infection. Here, we report that synthetic Ccrp3, aside from antibacterial activity, also shows cardio regulatory functions. In rats, Ccrp3 significantly diminishes blood pressure as well as the heartbeat frequency at nanomolar concentration. Ccrp3 affect the rat cardiac muscle mitochondria, inducing uncoupling of oxidative phosphorylation, oxygen consumption and transport of Ca(2). Ccrp3 treatment of the mitochondria causes mitochondrial damage promoting oxidative stress, causing overproduction of reactive oxygen species (ROS) and inhibition of superoxide dismutase. At nM concentration, Ccrp3 inhibits superoxide dismutase activity through direct interaction, diminishing by its enzymatic activity. Ccrp3 induces the release of the pro-apoptotic marker Bax from the mitochondria. Altogether, these results suggest that Ccrp3 pro-oxidative activity on cardiac muscle mitochondria could be responsible for triggering the heartbeat frequency and blood pressure lowering observed the Ccrp3 injected rats.

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Castrated rats of either sex were used in this work, and sex hormones of their own gender or cross-sex hormones were administered for 4 months. Animals were then put through 5 min of myocardial ischemia followed by a 5-min reperfusion injury. Electrocardiographic recordings were made and serum was obtained. Sex hormone levels were measured. Cardiac frequency was calculated, arterial pressure was determined, and the levels of lactate dehydrogenase (LDH), creatinine kinase (CK), and thiobarbituric acid reactive species (TBARs) were analyzed. Proinflammatory cytokine levels were measured in homogenized hearts; besides this, five hearts of each experimental group were obtained and fixed for histopathologic analysis. In male rats with estradiol replacement, the incidence of tachyarrhythmias and CK levels were higher when compared to the rest of the animals. Their cytokine levels were also elevated when compared to the group that received testosterone. Estradiol replacement protected female rats from variations in all of the parameters evaluated, whereas testosterone did not show a protective effect. In the presence of testosterone, the incidence of tachyarrhythmia was higher and TBARs, cytokines, CK, and LDH levels were also elevated. The results shown reinforce the idea that cross-sex hormone administration can damage the cardiovascular system.

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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.

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Permeability transition was examined in heart mitochondria isolated from neonate rats. We found that these mitochondria were more susceptible to Ca(2+)-induced membrane leakiness than mitochondria from adult rats. In K(+) containing medium, at 25 °C, mitochondria were unable to accumulate Ca(2+). Conversely, in Na(+) containing medium, mitochondria accumulated effectively Ca(2+). At 15 °C mitochondria accumulated Ca(2+) regardless of the presence of K(+). Kinetics of Ca(2+) accumulation showed a similar Vmax as that of adult mitochondria. Lipid milieu of inner membrane contained more unsaturated fatty acids than adult mitochondria. Aconitase inhibition and high thiobarbituric acid-reactive substances (TBARS) indicate that oxidative stress caused mitochondrial damage. In addition, proteomics analysis showed that there is a considerable diminution of succinate dehydrogenase C and subunit 4 of cytochrome oxidase in neonate mitochondria. Our proposal is that dysfunction of the respiratory chain makes neonate mitochondria more susceptible to damage by oxidative stress.

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Heavy metals are known to induce functional alterations in kidney mitochondria, this damage plays a central role in the mercury-induced acute renal failure. In fact, mercury causes rapid and dramatic changes in the membrane's ionic permeability in such a way that a supra load of mitochondrial Ca(2+) occurs. As a consequence, the phenomenon of permeability transition takes place. In this work we studied in vitro and in vivo the protective effect of the selective estrogen receptor modulator tamoxifen on the deleterious action of mercury-induced nonselective permeability in kidney mitochondria. Added in vitro tamoxifen inhibited membrane nonspecific pore opening, brought about by Hg(2+), as well as the oxidative damage of the enzyme cis-aconitase. In vivo the administration of tamoxifen prevented Hg(2+)-induced poisoning on mitochondrial energy-dependent functions. Permeability transition was analyzed by measuring matrix Ca(2+) retention, mitochondrial swelling, and the build up and maintenance of a transmembrane electric gradient. The pharmacologic action of tamoxifen on mercury poisoning could be ascribed to its cyclosporin-like action.

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Hypothyroidism induces several metabolic changes that allow understanding some physiopathological mechanisms. Under experimental hypothyroid conditions in rats, heart and kidney are protected against oxidative damage induced by ischemia reperfusion. An increased resistance to opening of the permeability transition pore seems to be at the basis of such protection. Moreover, glomerular filtration rate of hypothyroid kidney is low as a result of adenosine receptors-induced renal vasoconstriction. The vascular tone of aorta is also regulated by adenosine in hypothyroid conditions. In other context, thyroid hormones regulate lipoprotein metabolism. High plasma level of LDL cholesterol is a common feature in hypothyroidism, due to a low expression of the hepatic LDL receptor. In contrast, HDL-cholesterol plasma levels are variable in hypothyroidism; several proteins involved in HDL metabolism and structure are expressed at lower levels in experimental hypothyroidism. Based on the positive influence of thyroid hormones on lipoprotein metabolism, thyromimetic drugs are promising for the treatment of dyslipidemias. In summary, hypothyroid status has been useful to understand molecular mechanisms involved in ischemia reperfusion, regulation of vascular function and intravascular metabolism of lipoproteins.

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AIMS: Mitochondrial permeability transition is established after massive Ca(2+) accumulation inside the matrix, in addition to an inducer. The closure of the pore can be accomplished by adenosine diphosphate and the immunosuppressant cyclosporin A. Recently, the estrogen antagonist, tamoxifen, has been introduced as an inhibitor of the opening of the permeability transition pore. However, the mechanism by which this drug inhibits pore opening is still under discussion. This work was performed with the purpose of establishing the membrane system involved in tamoxifen-induced pore closure. For this purpose, permeability transition was induced after the addition of carboxyatractyloside, which is a specific reagent that interacts with the adenine nucleotide translocase. MAIN METHODS: Permeability transition was assessed by analyzing matrix Ca(2+) release, transmembrane electric gradient, and mitochondrial swelling in aged, as well as in freshly prepared mitochondria. Also, cytochrome c content was analyzed in membrane mitochondria as well as in the supernatant. KEY FINDINGS: In freshly prepared mitochondria, tamoxifen, at the concentration of 10 µM, totally inhibited nonspecific membrane permeability induced by 1 µM carboxyatractyloside. In addition, tamoxifen inhibited non-specific permeability in aged mitochondria and diminished membrane fluidity. SIGNIFICANCE: Plausibly, the inhibitory effect of tamoxifen on nonspecific membrane permeability, as induced by carboxyatractyloside, should be ascribed to a diminution, of membrane fluidity by this drug.

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Mercurials are known to induce morphological and functional modifications in kidney. The protective effect of octylguanidine on the injury induced by Hg(2+) on renal functions was studied. Octylguanidine administered at a dose of 10 mg/kg body weight prevented the damage induced by Hg(2+) administration at a dose of 3 mg/kg body weight. The findings indicate that octylguanidine spared mitochondria from Hg(2+)-poisoning by preserving their ability to retain matrix content, such as accumulated Ca(2+) and pyridine nucleotides. The hydrophobic amine also protected mitochondria from the Hg(2+)-induced loss of the transmembrane potential, and from the oxidative injury of mitochondrial DNA. In addition, octylguanidine maintained renal functions, such as normal values of creatinine clearance and blood urea nitrogen (BUN), and serum creatinine after Hg(2+) administration. It is proposed that octylguanidine protects kidney by inhibiting Hg(2+) uptake to kidney tissue, and in consequence its binding to mitochondrial membrane through a screening phenomenon, in addition to its known action as inhibitor of permeability transition.

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Ca²+ loading in mitochondria promotes the opening of a non-selective transmembrane pathway. Permeability transition is also associated with the interaction of cyclophilin D at the internal surface of the non-specific transmembrane pore. This interaction is circumvented by cyclosporin A and ADP. Our results show that, in the absence of ADP, liver mitochondria were unable to retain Ca²+, they underwent a fast and large amplitude swelling, as well as a rapid collapse of the transmembrane potential. In contrast, in the absence of ADP, kidney mitochondria retained Ca²+, swelling did not occur, and the collapse of the membrane potential was delayed. Ca²+ efflux was reversed by the addition of ADP and cyclosporin A. Our findings indicate that the differences between liver and kidney mitochondria are due to the low association of cyclophilin D to the ADP/ATP carrier found in kidney mitochondria as compared to liver mitochondria.

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