RESUMO
Limited uncoupling of oxidative phosphorylation is known to be beneficial in various laboratory models of diseases. The search for cationic uncouplers is promising as their protonophorous effect is self-limiting because these uncouplers lower membrane potential which is the driving force for their accumulation in mitochondria. In this work, the penetrating cation Rhodamine 19 butyl ester (C4R1) was found to decrease membrane potential and to stimulate respiration of mitochondria, appearing to be a stronger uncoupler than its more hydrophobic analog Rhodamine 19 dodecyl ester (C12R1). Surprisingly, C12R1 increased H(+) conductance of artificial bilayer lipid membranes or induced mitochondria swelling in potassium acetate with valinomycin at concentrations lower than C4R1. This paradox might be explained by involvement of mitochondrial proteins in the uncoupling action of C4R1. In experiments with HeLa cells, C4R1 rapidly and selectively accumulated in mitochondria and stimulated oligomycin-sensitive respiration as a mild uncoupler. C4R1 was effective in preventing oxidative stress induced by brain ischemia and reperfusion in rats: it suppressed stroke-induced brain swelling and prevented the decline in neurological status more effectively than C12R1. Thus, C4R1 seems to be a promising example of a mild uncoupler efficient in treatment of brain pathologies related to oxidative stress.
Assuntos
Mitocôndrias Hepáticas/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Rodaminas/farmacologia , Desacopladores/farmacologia , Animais , Células HeLa , Humanos , Bicamadas Lipídicas , Ratos , Rodaminas/químicaRESUMO
Lipid peroxidation plays an important role in various pathologies and aging, at least partially mediated by ferroptosis. The role of mitochondrial lipid peroxidation during ferroptosis remains poorly understood. We show that supplementation of exogenous iron in the form of ferric ammonium citrate at submillimolar doses induces production of reactive oxygen species (ROS) and lipid peroxidation in mitochondria that precede ferroptosis in H9c2 cardiomyocytes. The mitochondria-targeted antioxidant SkQ1 and the redox mediator methylene blue, which inhibits the production of ROS in complex I of the mitochondrial electron transport chain, prevent both mitochondrial lipid peroxidation and ferroptosis. SkQ1 and methylene blue also prevented accumulation of lipofuscin observed after 24 h incubation of cardiomyocytes with ferric ammonium citrate. Using isolated cardiac mitochondria as an in vitro ferroptosis model, it was shown that rotenone (complex I inhibitor) in the presence of ferrous iron stimulates lipid peroxidation and lipofuscin accumulation. Our data indicate that ROS generated in complex I stimulate mitochondrial lipid peroxidation, lipofuscin accumulation, and ferroptosis induced by exogenous iron.
Assuntos
Ferroptose , Ferro , Peroxidação de Lipídeos , Lipofuscina , Miócitos Cardíacos , Espécies Reativas de Oxigênio , Peroxidação de Lipídeos/efeitos dos fármacos , Ferroptose/efeitos dos fármacos , Lipofuscina/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Animais , Ratos , Espécies Reativas de Oxigênio/metabolismo , Ferro/metabolismo , Linhagem Celular , Compostos de Amônio Quaternário/farmacologia , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Azul de Metileno/farmacologia , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/efeitos dos fármacos , Compostos Férricos , Plastoquinona/análogos & derivadosRESUMO
Ferroptosis induced by erastin (an inhibitor of cystine transport) and butionine sulfoximine (an inhibitor of glutathione biosynthesis) was prevented by the mitochondria-targeted antioxidants SkQ1 and MitoTEMPO. These effects correlate with the prevention of mitochondrial lipid peroxidation, which precedes cell death. Methylene blue, a redox agent that inhibits the production of reactive oxygen species (ROS) in complex I of the mitochondrial electron transport chain, also inhibits ferroptosis and mitochondrial lipid peroxidation. Activation of ROS production in complex I with rotenone in the presence of ferrous iron stimulates lipid peroxidation in isolated mitochondria, while ROS produced by complex III are ineffective. SkQ1 and methylene blue inhibit lipid peroxidation. We suggest that ROS formed in complex I promote mitochondrial lipid peroxidation and ferroptosis.
Assuntos
Ferroptose , Peroxidação de Lipídeos , Espécies Reativas de Oxigênio/metabolismo , Azul de Metileno/metabolismo , Mitocôndrias/metabolismoRESUMO
Ferroptosis is a regulated form of necrotic cell death reliant on iron-catalyzed lipid peroxidation. Although the precise involvement of mitochondria in ferroptosis remains incompletely elucidated, recent research indicates that mitochondrial oxidative events wield a pivotal influence in this mechanism. This article centers on the most recent discoveries, spotlighting the significance of mitochondrial lipid peroxidation in the occurrence of ferroptosis. Modern investigative tools, such as mitochondria-specific dyes responsive to lipid peroxidation and antioxidants targeting mitochondria, have been employed to delve into this phenomenon. The authors' recent empirical evidence demonstrates that mitochondrial lipid peroxidation, quantified using the innovative fluorescent ratiometric probe MitoCLox, takes place prior to the onset of ferroptotic cell death. The mitochondria-targeted antioxidant SkQ1 hinders mitochondrial lipid peroxidation and thwarts ferroptosis, all while leaving unaffected the buildup of reactive oxygen species within the cytoplasm, an antecedent to mitochondrial lipid peroxidation. Similarly, the redox agent methylene blue, impeding the genesis of reactive oxygen species in complex I of the electron transport chain, also imparts a comparable protective effect. These findings collectively imply that reactive oxygen species originating from complex I might hold particular significance in fomenting mitochondrial lipid peroxidation, a pivotal trigger of ferroptosis.
RESUMO
A limited decrease in mitochondrial membrane potential can be beneficial for cells, especially under some pathological conditions, suggesting that mild uncouplers (protonophores) causing such an effect are promising candidates for therapeutic uses. The great majority of protonophores are weak acids capable of permeating across membranes in their neutral and anionic forms. In the present study, protonophorous activity of a series of derivatives of cationic rhodamine 19, including dodecylrhodamine (C(12)R1) and its conjugate with plastoquinone (SkQR1), was revealed using a variety of assays. Derivatives of rhodamine B, lacking dissociable protons, showed no protonophorous properties. In planar bilayer lipid membranes, separating two compartments differing in pH, diffusion potential of H(+) ions was generated in the presence of C(12)R1 and SkQR1. These compounds induced pH equilibration in liposomes loaded with the pH probe pyranine. C(12)R1 and SkQR1 partially stimulated respiration of rat liver mitochondria in State 4 and decreased their membrane potential. Also, C(12)R1 partially stimulated respiration of yeast cells but, unlike the anionic protonophore FCCP, did not suppress their growth. Loss of function of mitochondrial DNA in yeast (grande-petite transformation) is known to cause a major decrease in the mitochondrial membrane potential. We found that petite yeast cells are relatively more sensitive to the anionic uncouplers than to C(12)R1 compared with grande cells. Together, our data suggest that rhodamine 19-based cationic protonophores are self-limiting; their uncoupling activity is maximal at high membrane potential, but the activity decreases membrane potentials, which causes partial efflux of the uncouplers from mitochondria and, hence, prevents further membrane potential decrease.
Assuntos
Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Rodaminas , Saccharomyces cerevisiae/metabolismo , Desacopladores , Animais , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Concentração de Íons de Hidrogênio , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Ratos , Rodaminas/química , Rodaminas/farmacologia , Desacopladores/química , Desacopladores/farmacologiaRESUMO
Receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of Alzheimer's disease. We have previously revealed that RAGE fragment sequence (60-76) and its shortened analogs sequence (60-70) and (60-65) under intranasal insertion were able to restore memory and improve morphological and biochemical state of neurons in the brain of bulbectomized mice developing major AD features. In the current study, we have investigated the ability of RAGE peptide (60-76) and five shortened analogs to bind beta-amyloid (Aß) 1-40 in an fluorescent titration test and show that all the RAGE fragments apart from one [sequence (65-76)] were able to bind Aß in vitro. Moreover, we show that all RAGE fragments apart from the shortest one (60-62), were able to protect neuronal primary cultures from amyloid toxicity, by preventing the caspase 3 activation induced by Aß 1-42. We have compared the data obtained in the present research with the previously published data in the animal model of AD, and offer a probable mechanism of neuroprotection of the RAGE peptide.
RESUMO
The release of cytochrome c from the intermembrane space of mitochondria into the cytosol is one of the critical events in apoptotic cell death. In the present study, it is shown that release of cytochrome c and apoptosis induced by tumor necrosis factor alpha (TNF) in HeLa cells can be inhibited by (i) overexpression of an oncoprotein Bcl-2, (ii) Cyclosporin A, an inhibitor of the mitochondrial permeability transition pore (PTP) or (iii) oligomycin, an inhibitor of H+- ATP-synthase. Staurosporine-induced apoptosis is sensitive to Bcl-2 but insensitive to Cyclosporin A and oligomycin. The effect of oligomycin is not due to changes in mitochondrial membrane potential or to inhibition of ATP synthesis/hydrolysis since (a) uncouplers (CCCP, DNP) which discharge the membrane potential fail to abolish the protective action of oligomycin and (b) aurovertin B (another inhibitor of H+-ATP-synthase, affecting its F1 component) do not affect apoptosis. A role of oligomycin-sensitive F0 component of H+-ATP-synthase in the TNF-induced PTP opening and apoptosis is suggested.
Assuntos
Grupo dos Citocromos c/metabolismo , Inibidores Enzimáticos/farmacologia , Mitocôndrias/efeitos dos fármacos , Oligomicinas/farmacologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Aurovertinas/farmacologia , Ciclosporina/farmacologia , Citosol/enzimologia , Desoxiglucose/farmacologia , Emetina/farmacologia , Genes bcl-2 , Células HeLa/efeitos dos fármacos , Células HeLa/enzimologia , Humanos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/enzimologia , Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade Mitocondrial , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , ATPases Translocadoras de Prótons/fisiologia , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Recombinantes/farmacologia , Estaurosporina/farmacologia , Transfecção , Fator de Necrose Tumoral alfa/farmacologia , Desacopladores/farmacologiaRESUMO
In HeLa cells, complete inhibition of oxidative phosphorylation by oligomycin, myxothiazol or FCCP combined with partial inhibition of glycolysis by DOG resulted in a steady threefold decrease in the intracellular ATP level. The ATP level recovers when the DOG-containing medium was replaced by that with high glucose. In 48 h after a transient (3 h) [ATP] lowering followed by recovery of the ATP level, the majority of the cells commits suicide by means of apoptosis. The cell death does not occur if DOG or an oxidative phosphorylation inhibitor was added separately, treatments resulting in 10-35% lowering of [ATP]. Apoptosis is accompanied by Bax translocation to mitochondria, cytochrome c release into cytosol, caspase activation, reactive oxygen species (ROS) generation, and reorganization and decomposition of chromatin. Apoptosis appears to be sensitive to oncoprotein Bcl-2 and a pancaspase inhibitor zVADfmk. In the latter case, necrosis is shown to develop instead of apoptosis. The cell suicide is resistant to cyclosporine A, a phospholipase inhibitor trifluoroperazine, the JNK and p38 kinase inhibitors, oligomycin, N-acetyl cysteine and mitoQ, differing in these respects from the tumor necrosis factor (TNF)- and H(2)O(2)-induced apoptoses. It is suggested that the ATP concentration in the cell is monitored by intracellular "ATP-meter(s)" generating a cell suicide signal when ATP decreases, even temporarily, below some critical level (around 1 mM).
Assuntos
Trifosfato de Adenosina/metabolismo , Apoptose/fisiologia , Espaço Intracelular/metabolismo , Trifosfato de Adenosina/análise , Trifosfato de Adenosina/deficiência , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Caspases/metabolismo , Citocromos c/metabolismo , Citosol/metabolismo , Desoxiglucose/farmacologia , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Metacrilatos , Mitocôndrias/metabolismo , Oligomicinas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tiazóis/farmacologia , Proteína X Associada a bcl-2RESUMO
The effects of specific inhibitors of respiratory chain, F(o)F(1)ATP synthase and uncouplers of oxidative phosphorylation on survival of carcinoma HeLa cells and on the structure of mitochondria in the cells were studied. The inhibitors of respiration (piericidin, antimycin, myxothiazol), the F(1)-component of ATP synthase (aurovertin) and uncouplers (DNP, FCCP) did not affect viability of HeLa cells, apoptosis induced by TNF or staurosporin and the anti-apoptotic action of Bcl-2. Apoptosis was induced by combined action of respiratory inhibitors and uncouplers indicating possible pro-apoptotic action of reactive oxygen species (ROS) generated by mitochondria. Short-term incubation of HeLa cells with the mitochondrial inhibitors and 2-deoxyglucose followed by 24-48 h recovery resulted in massive apoptosis. Apoptosis correlated to transient (3-4 h) and limited (60-70%) depletion of ATP. More prolonged or more complete transient ATP depletion induced pronounced necrosis. The inhibitors of respiration and uncouplers caused fragmentation of tubular mitochondria and formation of small round bodies followed by swelling. These transitions were not accompanied with release of cytochrome c into the cytosol and were fully reversible. The combined effect of respiratory inhibitors and uncouplers developed more rapidly indicating possible involvement of ROS generated by mitochondria. More prolonged (48-72 h) incubation with this combination of inhibitors caused clustering and degradation of mitochondria.
Assuntos
Antimicina A/análogos & derivados , Apoptose , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Trifosfato de Adenosina/metabolismo , Animais , Antibacterianos/farmacologia , Antifúngicos/farmacologia , Antimicina A/farmacologia , Aurovertinas/farmacologia , Linhagem Celular , Glucose/metabolismo , Células HeLa , Humanos , Metacrilatos , Oxigênio/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Piridinas/farmacologia , Espécies Reativas de Oxigênio , Tiazóis/farmacologia , Fatores de Tempo , Desacopladores/farmacologiaRESUMO
A conjugate of plastoquinone with decylrhodamine 19 (SkQR1) selectively accumulates in mitochondria of normal and tumor cells. SkQR1 protected the cellular pool of reduced glutathione under oxidative stress. Overexpression of P-glycoprotein (Pgp 170) multidrug resistance pump strongly suppresses accumulation of SkQR1. The inhibitors of Pgp 170 stimulate accumulation of SkQR1 in various cell lines indicating that SkQR1 is a substrate of Pgp 170. The protective effect of SkQR1 against oxidative stress is diminished in the cells overexpressing Pgp 170. It is suggested that mitochondria-targeted antioxidants could selectively protect normal (Pgp 170-negative) cells against the toxic effect of anti-cancer treatments related to oxidative stress.