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1.
Arch Biochem Biophys ; 726: 109231, 2022 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-35660298

RESUMO

Complex I (NADH-ubiquinone reductase) and Complex III (ubiquinol-cytochrome c reductase) supplemented with NADH generated O2-at maximum rates of 9.8 and 6.5 nmol/min/mg of protein, respectively, while, in the presence of superoxide dismutase, the same systems generated H2O2 at maximum rates of 5.1 and 4.2 nmol/min/mg of protein, respectively. H2O2 was essentially produced by disproportionation of O2-, which constitutes the precursor of H2O2. The effectiveness of the generation of oxygen intermediates by Complex I in the absence of other specific electron acceptors was 0.95 mol of O2- and 0.63 mol of H2O2/mol of NADH. A reduced form of ubiquinone appeared to be responsible for the reduction of O2 to O2-, since (a) ubiquinone constituted the sole common major component of Complexes I and III, (b) H202 generation by Complex I was inhibited by rotenone, and (c) supplementation of Complex I with exogenous ubiquinones increased the rate of H2O2 generation. The efficiency of added quinones as peroxide generators decreased in the order Q1 > Q0 > Q2 > Q6 = Q10, in agreement with the quinone capacity of acting as electron acceptor for Complex I. In the supplemented systems, the exogenous quinone was reduced by Complex I and oxidized nonenzymati- cally by molecular oxygen. Additional evidence for the role of ubiquinone as peroxide generator is provided by the generation of O2- and H2O2 during autoxidation of quinols. In oxygenated buffers, ubiquinol (Q0H2), benzoquinol, duroquinol and menadiol generated O2-with k3 values of 0.1 to 1.4 M-1 s-1 and H2O2 with k4 values of 0.009 to 4.3 m-1·s-1.


Assuntos
Complexo I de Transporte de Elétrons , Superóxidos , Animais , Bovinos , Complexo I de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Peróxido de Hidrogênio/metabolismo , Mitocôndrias Cardíacas/metabolismo , NAD/metabolismo , Oxigênio/metabolismo , Quinonas , Superóxidos/metabolismo , Ubiquinona/metabolismo
2.
Food Funct ; 10(5): 2528-2537, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-30993288

RESUMO

In order to study the in vitro effect of flavan-3-ol (+)-catechin on the enzymatic activities of mitochondrial complex I and nitric oxide synthase (mtNOS), as well as the consequences on the membrane potential and H2O2 production rate, isolated mitochondria from rat heart were exposed to 3 nM to 100 µM (+)-catechin. NADH-Q1 reductase (complex I) and mtNOS activities were inhibited 25% and 50%, respectively, by the addition of 10 nM (+)-catechin to the reaction medium. Moreover, in the nM range, (+)-catechin decreased state 4 mitochondrial membrane potential by about 10 mV, but failed to change the membrane potential measured in the presence of ADP. (+)-Catechin (10 nM) inhibited not only complex I activity, but also the H2O2 production rate (35%) sustained by malate-glutamate, in accordance with the decrease observed in mitochondrial membrane potential. Considering (+)-catechin concentrations lower than 10 nM, linear and positive correlations were obtained between mitochondrial complex I activity and either NO (r2 = 0.973) or H2O2 production rates (r2 = 0.958), suggesting a functional association among these parameters. Altogether, the results indicate that (+)-catechin, at nM concentrations, inhibits mitochondrial complex I activity, leading to membrane potential decline and consequently to reduction in H2O2 and NO production rates. The decrease in mtNOS activity could also be a consequence of the direct action of (+)-catechin on the NOS structure, this effect being in accordance with the functional interaction between complex I and mtNOS, as previously reported.


Assuntos
Catequina/farmacologia , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Coração/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Animais , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Feminino , Cinética , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Miocárdio/química , Miocárdio/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase/metabolismo , Ratos , Ratos Sprague-Dawley
3.
PLoS One ; 13(2): e0193022, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29489891

RESUMO

Activation of ERK1/2 implies the phosphorylation of tyrosine (pTyr) and threonine (pThr) by MEK1/2; both reactions were thought to be cytoplasmic, promoting ERK to reach the nucleus where it activates several transcription factors. In addition, H2O2 concentrations are known to modulate ERK intracellular translocation, which impacts on cellular proliferation. In this context, the objective of this work was to study the sequence of ERK phosphorylation under two redox conditions and to analyze a putative mitochondrial contribution to this process, in LP07 murine lung cells. A time-course of H2O2 administration was used and ERK phosphorylation was analyzed in cytosol, mitochondria and nuclei. At 1µM H2O2, a proliferative redox stimulus, immunoblot revealed a fast and transient increase in cytosol pTyr and a sustained increase in mitochondrial pTyr content. The detection for pThr/pTyrERK (2pERK) showed in cytosol a marked increase at 5 minutes with a fast dephosphorylation after that time, for both H2O2 concentrations. However, at 50 µM H2O2, an anti-proliferative condition, 2pERK was gradually retained in mitochondria. Interestingly, these results were confirmed by in vivo experiments using mice treated with a highly oxidizing agent [H2O2]. By the use of two ERK2 mutant constructions, where Tyr and Thr were replaced by alanine, we confirmed that 2pERK relied almost completely on pThr183. Confocal microscopy confirmed ERK subcellular distribution dependence on the incidence of cytosolic pTyr and mitochondrial pThr at 1µM H2O2. This work shows for the first time, both in vitro and in vivo, an ERK cycle involving a cross-talk between cytosol and mitochondria phosphorylation events, which may play a significant role in cell cycle progression, proliferation or differentiation under two different redox conditions.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Pulmão/metabolismo , Treonina/metabolismo , Tirosina/metabolismo , Animais , Linhagem Celular , Fator de Crescimento Epidérmico/farmacologia , Peróxido de Hidrogênio/metabolismo , Pulmão/citologia , Masculino , Camundongos , Mitocôndrias/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Oxirredução , Fosforilação , Frações Subcelulares/metabolismo
4.
Free Radic Biol Med ; 112: 267-276, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28756312

RESUMO

This study, in an experimental model of type I Diabetes Mellitus in rats, deals with the mitochondrial production rates and steady-state concentrations of H2O2 and NO, and ATP levels as part of a network of signaling molecules involved in heart mitochondrial biogenesis. Sustained hyperglycemia leads to a cardiac compromise against a work overload, in the absence of changes in resting cardiac performance and of heart hypertrophy. Diabetes was induced in male Wistar rats by a single dose of Streptozotocin (STZ, 60mg × kg-1, ip.). After 28 days of STZ-injection, rats were sacrificed and hearts were isolated. The mitochondrial mass (mg mitochondrial protein × g heart-1), determined through cytochrome oxidase activity ratio, was 47% higher in heart from diabetic than from control animals. Stereological analysis of cardiac tissue microphotographs showed an increase in the cytosolic volume occupied by mitochondria (30%) and in the number of mitochondria per unit area (52%), and a decrease in the mean area of each mitochondrion (23%) in diabetic respect to control rats. Additionally, an enhancement (76%) in PGC-1α expression was observed in cardiac tissue of diabetic animals. Moreover, heart mitochondrial H2O2 (127%) and NO (23%) productions and mtNOS expression (132%) were higher, while mitochondrial ATP production rate was lower (~ 40%), concomitantly with a partial-mitochondrial depolarization, in diabetic than in control rats. Changes in mitochondrial H2O2 and NO steady-state concentrations and an imbalance between cellular energy demand and mitochondrial energy transduction could be involved in the signaling pathways that lead to the novo synthesis of mitochondria. However, this compensatory mechanism triggered to restore the mitochondrial and tissue normal activities, did not lead to competent mitochondria capable of supplying the energetic demands in diabetic pathological conditions.


Assuntos
Trifosfato de Adenosina/metabolismo , Diabetes Mellitus Experimental/metabolismo , Peróxido de Hidrogênio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miocárdio/metabolismo , Óxido Nítrico/metabolismo , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Expressão Gênica , Masculino , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias Cardíacas/patologia , Miocárdio/patologia , Biogênese de Organelas , Tamanho das Organelas , Estresse Oxidativo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Ratos , Ratos Wistar , Estreptozocina
5.
J Inorg Biochem ; 172: 94-99, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28445841

RESUMO

Rat liver mitochondria (1.5-2.1mg protein·mL-1) supplemented with either 25 and 100µM Cu2+ or 100 and 500µM Fe3+ show inhibition of active respiration (O2 consumption in state 3) and increased phospholipid peroxidation . Liver mitochondria were supplemented with the antioxidants reduced glutathione, N-acetylcysteine or butylated hydroxitoluene, to evaluate their effects on the above-mentioned alterations. Although the mitochondrial dysfunction is clearly associated to phospholipid peroxidation, the different responses to antioxidant supplementation indicate that the metal ions have differences in their mechanisms of toxicity. Mitochondrial phospholipid peroxidation through the formation of hydroxyl radical by a Fenton/Haber-Weiss mechanism seems to precede the respiratory inhibition and to be the main fact in Fe-induced mitochondrial dysfunction. In the case of Cu2+, it seems that the ion oxidizes glutathione, and low molecular weight protein thiol groups in a direct reaction, as part of its intracellular redox cycling. The processes involving phospholipid peroxidation, protein oxidation and mitochondrial respiratory inhibition characterize a redox dyshomeostatic situation that ultimately leads to cell death. However, Cu2+ exposure involves an additional, yet unidentified, toxic event as previous reduction of the metal with N-acetylcysteine has only a minor effect in preventing the mitochondrial damage.


Assuntos
Antioxidantes/farmacologia , Respiração Celular/efeitos dos fármacos , Cobre/farmacologia , Ferro/fisiologia , Peroxidação de Lipídeos/efeitos dos fármacos , Mitocôndrias Hepáticas/efeitos dos fármacos , Animais , Cobre/química , Radicais Livres/metabolismo , Íons/farmacologia , Ferro/química , Masculino , Modelos Biológicos , Fosfolipídeos/metabolismo , Ratos
6.
Artigo em Inglês | MEDLINE | ID: mdl-28347745

RESUMO

Several oxidative stress markers and liver oxygen consumption were measured in different tissues of the marine fish Trichiurus lepturus in late summer and late winter, as well as in juveniles and adult females. Oxygen consumption in liver, superoxide dismutase (SOD) and catalase (CAT) activity in liver, red cells, lens and roe, vitamin E, ubiquinol10, ß-carotene in liver, red cells, and roe, as well as contents of reduced glutathione (GSH) and lipoperoxidation (TBARS) in red cells were evaluated. Regarding ontogeny, compared to adult fish, juveniles showed significant higher SOD activity in liver and lens, as well as higher liver contents of vitamin E. In contrast, adult females showed higher contents of vitamin E in roe, ubiquinol10 in liver and roe, and higher GSH levels in red cells, while the other markers remained unchanged. Regarding seasonal changes, no differences were detected in adult females for liver CAT and ubiquinol10, CAT in roe, vitamin E in roe and in red cells, liver and red cell ubiquinol10, and in GSH in red cells. However, and coinciding with the spawning period of late summer, liver oxygen consumption, SOD and CAT activity and ubiquinol10 contents in roe and SOD activity in red cells, and red cell TBARS contents were higher compared to late winter. These temporal antioxidant adjustments of Trichiurus lepturus seem to be parallel to the higher oxygen consumption typical of juvenile forms and also to the intense spawning and foraging activities of adult females in late summer.


Assuntos
Proteínas de Peixes/metabolismo , Peixes/fisiologia , Peroxidação de Lipídeos , Fígado/metabolismo , Morfogênese , Estresse Oxidativo , Oxirredutases/metabolismo , Animais , Ilhas Atlânticas , Oceano Atlântico , Comportamento Animal , Biomarcadores/sangue , Biomarcadores/metabolismo , Brasil , Eritrócitos/enzimologia , Eritrócitos/metabolismo , Comportamento Alimentar , Feminino , Peixes/sangue , Peixes/crescimento & desenvolvimento , Glutationa/sangue , Fígado/enzimologia , Fígado/crescimento & desenvolvimento , Óvulo/enzimologia , Óvulo/metabolismo , Oxirredutases/sangue , Consumo de Oxigênio , Reprodução , Estações do Ano , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo
7.
Mol Cell Biochem ; 432(1-2): 169-178, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28316061

RESUMO

Transition from compensated to decompensated left ventricular hypertrophy (LVH) is accompanied by functional and structural changes. Here, the aim was to evaluate dystrophin expression in murine models and human subjects with LVH by transverse aortic constriction (TAC) and aortic stenosis (AS), respectively. We determined whether doxycycline (Doxy) prevented dystrophin expression and myocardial stiffness in mice. Additionally, ventricular function recovery was evaluated in patients 1 year after surgery. Mice were subjected to TAC and monitored for 3 weeks. A second group received Doxy treatment after TAC. Patients with AS were stratified by normal left ventricular end-diastolic wall stress (LVEDWS) and high LVEDWS, and groups were compared. In mice, LVH decreased inotropism and increased myocardial stiffness associated with a dystrophin breakdown and a decreased mitochondrial O2 uptake (MitoMVO2). These alterations were attenuated by Doxy. Patients with high LVEDWS showed similar results to those observed in mice. A correlation between dystrophin and myocardial stiffness was observed in both mice and humans. Systolic function at 1 year post-surgery was only recovered in the normal-LVEDWS group. In summary, mice and humans present diastolic dysfunction associated with dystrophin degradation. The recovery of ventricular function was observed only in patients with normal LVEDWS and without dystrophin degradation. In mice, Doxy improved MitoMVO2. Based on our results it is concluded that the LVH with high LVEDWS is associated to a degradation of dystrophin and increase of myocardial stiffness. At least in a murine model these alterations were attenuated after the administration of a matrix metalloprotease inhibitor.


Assuntos
Distrofina/deficiência , Hipertrofia Ventricular Esquerda/metabolismo , Mitocôndrias Cardíacas/metabolismo , Miocárdio/metabolismo , Proteólise , Animais , Modelos Animais de Doenças , Doxiciclina/efeitos adversos , Doxiciclina/farmacologia , Humanos , Hipertrofia Ventricular Esquerda/induzido quimicamente , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/patologia , Masculino , Camundongos , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/patologia , Miocárdio/patologia
8.
Stem Cell Rev Rep ; 13(4): 491-498, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28214945

RESUMO

Intercellular communication is one of the most important events in cell population behavior. In the last decade, tunneling nanotubes (TNTs) have been recognized as a new form of long distance intercellular connection. TNT function is to allow molecular and subcellular structure exchange between neighboring cells via the transfer of molecules and organelles such as calcium ions, prions, viral and bacterial pathogens, small lysosomes and mitochondria. New findings support the concept that mesenchymal stem cells (MSCs) can affect cell microenvironment by the release of soluble factors or the transfer of cellular components to neighboring cells, in a way which significantly contributes to cell regulation and tissue repair, although the underlying mechanisms remain poorly understood. MSCs have many advantages for their implementation in regenerative medicine. The TNTs in these cell types are heterogeneous in both structure and function, probably due to their highly dynamic behavior. In this work we report an extensive and detailed description of types, structure, components, dynamics and functionality of the TNTs bridging neighboring human umbilical cord MSCs obtained from Wharton"s jelly. Characterization studies were carried out through phase contrast, fluorescence, electron microscopy and time lapse images with the aim of describing cells suitable for an eventual regenerative medicine.


Assuntos
Comunicação Celular , Células-Tronco Mesenquimais/metabolismo , Nanotubos/química , Humanos , Células-Tronco Mesenquimais/citologia
9.
J Inorg Biochem ; 166: 5-11, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27815982

RESUMO

Increased copper (Cu) and iron (Fe) levels in liver and brain are associated to oxidative stress and damage with increased phospholipid oxidation process. The aim of this work was to assess the toxic effects of Cu2+ and Fe3+ addition to rat liver mitochondria by determining mitochondrial respiration in states 3 (active respiration) and 4 (resting respiration), and phospholipid peroxidation. Both, Cu2+ and Fe3+ produced decreases in O2 consumption in a concentration-dependent manner in active state 3: both ions by 42% with malate-glutamate as complex I substrate (concentration for half maximal response (C50) 60µM Cu2+ and 1.25mM Fe3+), and with succinate as complex II substrate: 64-69% with C50 of 50µM Cu2+ and with C50 of 1.25mM of Fe3+. Respiratory control decreased with Cu2+ (C50 50µM) and Fe3+ (C50 1.25-1-75mM) with both substrates. Cu2+ produced a 2-fold increase and Fe3+ a 5-fold increase of thiobarbituric acid-reactive substances (TBARS) content from 25µM Cu2+ (C50 40µM) and from 100µM Fe3+ (C50 1.75mM). Supplementations with Cu2+ and Fe3+ ions induce mitochondrial dysfunction with phospholipid peroxidation in rat liver mitochondria. Although is proved that a Fenton/Haber Weiss mechanism of oxidative damage occurs in metal-ion induced mitochondrial toxicity, slightly different responses to the metal ions suggest some differences in the mechanism of intracellular toxicity. The decreased rates of mitochondrial respiration and the alteration of mitochondrial function by phospholipid and protein oxidations lead to mitochondrial dysfunction, cellular dyshomeostasis and cell death.


Assuntos
Cobre/farmacologia , Ferro/farmacologia , Peroxidação de Lipídeos/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Fosfolipídeos/metabolismo , Animais , Complexo I de Transporte de Elétrons/metabolismo , Masculino , Mitocôndrias Hepáticas/patologia , Proteínas Mitocondriais/metabolismo , Ratos , Ratos Sprague-Dawley
10.
Int J Biochem Cell Biol ; 81(Pt B): 335-345, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27682517

RESUMO

Diabetes is a chronic disease associated to a cardiac contractile dysfunction that is not attributable to underlying coronary artery disease or hypertension, and could be consequence of a progressive deterioration of mitochondrial function. We hypothesized that impaired mitochondrial function precedes Diabetic Cardiomyopathy. Thus, the aim of this work was to study the cardiac performance and heart mitochondrial function of diabetic rats, using an experimental model of type I Diabetes. Rats were sacrificed after 28days of Streptozotocin injection (STZ, 60mgkg-1, ip.). Heart O2 consumption was declined, mainly due to the impairment of mitochondrial O2 uptake. The mitochondrial dysfunction observed in diabetic animals included the reduction of state 3 respiration (22%), the decline of ADP/O ratio (∼15%) and the decrease of the respiratory complexes activities (22-26%). An enhancement in mitochondrial H2O2 (127%) and NO (23%) production rates and in tyrosine nitration (58%) were observed in heart of diabetic rats, with a decrease in Mn-SOD activity (∼50%). Moreover, a decrease in contractile response (38%), inotropic (37%) and lusitropic (58%) reserves were observed in diabetic rats only after a ß-adrenergic stimulus. Therefore, in conditions of sustained hyperglycemia, heart mitochondrial O2 consumption and oxidative phosphorylation efficiency are decreased, and H2O2 and NO productions are increased, leading to a cardiac compromise against a work overload. This mitochondrial impairment was detected in the absence of heart hypertrophy and of resting cardiac performance changes, suggesting that mitochondrial dysfunction could precede the onset of diabetic cardiac failure, being H2O2, NO and ATP the molecules probably involved in mitochondrion-cytosol signalling.


Assuntos
Diabetes Mellitus Experimental/complicações , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/fisiopatologia , Mitocôndrias Cardíacas/patologia , Trifosfato de Adenosina/metabolismo , Animais , Peróxido de Hidrogênio/metabolismo , Óxido Nítrico/metabolismo , Ratos
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