RESUMO

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.

RESUMO

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.

Assuntos

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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.

Assuntos

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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.

Assuntos

RESUMO

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.

Assuntos

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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.

Assuntos

RESUMO

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.

Assuntos

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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.

Assuntos

RESUMO

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.

RESUMO

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.

Assuntos