RESUMEN
Measuring respiration rate can be a powerful way to assess energetic function in isolated mitochondria. Current, plate-based methods have several advantages over older, suspension-based systems, including greater throughput and the requirement of only µg quantities of material. In this chapter, we describe a plate-based method for measuring oxygen consumption by isolated adherent mitochondria.
Asunto(s)
Respiración de la Célula , Fluorometría/métodos , Mitocondrias Musculares/metabolismo , Consumo de Oxígeno , Animales , Fluorometría/instrumentación , Ratas , Ratas WistarRESUMEN
S-Adenosyl-L-methionine (SAM) is an enzyme cofactor used in methyl transfer reactions and polyamine biosynthesis. The biosynthesis of SAM from ATP and L-methionine is performed by the methionine adenosyltransferase enzyme family (Mat; EC 2.5.1.6). Human methionine adenosyltransferase 2A (Mat2A), the extrahepatic isoform, is often deregulated in cancer. We identified a Mat2A inhibitor, PF-9366, that binds an allosteric site on Mat2A that overlaps with the binding site for the Mat2A regulator, Mat2B. Studies exploiting PF-9366 suggested a general mode of Mat2A allosteric regulation. Allosteric binding of PF-9366 or Mat2B altered the Mat2A active site, resulting in increased substrate affinity and decreased enzyme turnover. These data support a model whereby Mat2B functions as an inhibitor of Mat2A activity when methionine or SAM levels are high, yet functions as an activator of Mat2A when methionine or SAM levels are low. The ramification of Mat2A activity modulation in cancer cells is also described.
Asunto(s)
Metionina Adenosiltransferasa/antagonistas & inhibidores , Quinolinas/farmacología , S-Adenosilmetionina/metabolismo , Triazoles/farmacología , Sitio Alostérico/efectos de los fármacos , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Metionina Adenosiltransferasa/aislamiento & purificación , Metionina Adenosiltransferasa/metabolismo , Quinolinas/química , Relación Estructura-Actividad , Triazoles/químicaRESUMEN
Brown adipose tissue (BAT) possesses the inherent ability to dissipate metabolic energy as heat through uncoupled mitochondrial respiration. An essential component of the mitochondrial electron transport chain is coenzyme Q (CoQ). While cells synthesize CoQ mostly endogenously, exogenous supplementation with CoQ has been successful as a therapy for patients with CoQ deficiency. However, which tissues depend on exogenous CoQ uptake as well as the mechanism by which CoQ is taken up by cells and the role of this process in BAT function are not well understood. Here, we report that the scavenger receptor CD36 drives the uptake of CoQ by BAT and is required for normal BAT function. BAT from mice lacking CD36 displays CoQ deficiency, impaired CoQ uptake, hypertrophy, altered lipid metabolism, mitochondrial dysfunction, and defective nonshivering thermogenesis. Together, these data reveal an important new role for the systemic transport of CoQ to BAT and its function in thermogenesis.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Antígenos CD36/metabolismo , Ubiquinona/metabolismo , Animales , Ataxia/genética , Ataxia/metabolismo , Antígenos CD36/genética , Cromatografía Líquida de Alta Presión , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Debilidad Muscular/genética , Debilidad Muscular/metabolismo , Oxidación-Reducción , Ácido Palmítico/metabolismo , Termogénesis/genética , Termogénesis/fisiología , Ubiquinona/deficiencia , Ubiquinona/genéticaRESUMEN
The sites and rates of mitochondrial production of superoxide and H2O2 in vivo are not yet defined. At least 10 different mitochondrial sites can generate these species. Each site has a different maximum capacity (e.g. the outer quinol site in complex III (site IIIQo) has a very high capacity in rat skeletal muscle mitochondria, whereas the flavin site in complex I (site IF) has a very low capacity). The maximum capacities can greatly exceed the actual rates observed in the absence of electron transport chain inhibitors, so maximum capacities are a poor guide to actual rates. Here, we use new approaches to measure the rates at which different mitochondrial sites produce superoxide/H2O2 using isolated muscle mitochondria incubated in media mimicking the cytoplasmic substrate and effector mix of skeletal muscle during rest and exercise. We find that four or five sites dominate during rest in this ex vivo system. Remarkably, the quinol site in complex I (site IQ) and the flavin site in complex II (site IIF) each account for about a quarter of the total measured rate of H2O2 production. Site IF, site IIIQo, and perhaps site EF in the ß-oxidation pathway account for most of the remainder. Under conditions mimicking mild and intense aerobic exercise, total production is much less, and the low capacity site IF dominates. These results give novel insights into which mitochondrial sites may produce superoxide/H2O2 in vivo.
Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Peróxido de Hidrógeno/metabolismo , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Superóxidos/metabolismo , Animales , Citocromos b/metabolismo , Complejo II de Transporte de Electrones/metabolismo , Femenino , Malatos/metabolismo , Mitocondrias Musculares/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Oligomicinas/farmacología , Consumo de Oxígeno/fisiología , Condicionamiento Físico Animal/fisiología , Ratas , Ratas Wistar , Descanso/fisiología , Ácido Succínico/metabolismo , Técnicas de Cultivo de Tejidos , Desacopladores/farmacologíaRESUMEN
p53 Inducible gene 6 (PIG6) encodes mitochondrial proline dehydrogenase (PRODH) and is up-regulated several fold upon p53 activation. Proline dehydrogenase is proposed to generate radicals that contribute to cancer cell apoptosis. However, there are at least 10 mitochondrial sites that can produce superoxide and/or H2O2, and it is unclear whether proline dehydrogenase generates these species directly, or instead drives production by other sites. Amongst six cancer cell lines, ZR75-30 human breast cancer cells had the highest basal proline dehydrogenase levels, and mitochondria isolated from ZR75-30 cells consumed oxygen and produced H2O2 with proline as sole substrate. Insects use proline oxidation to fuel flight, and mitochondria isolated from Drosophila melanogaster were even more active with proline as sole substrate than ZR75-30 mitochondria. Using mitochondria from these two models we identified the sites involved in formation of superoxide/H2O2 during proline oxidation. In mitochondria from Drosophila the main sites were respiratory complexes I and II. In mitochondria from ZR75-30 breast cancer cells the main sites were complex I and the oxoglutarate dehydrogenase complex. Even with combinations of substrates and respiratory chain inhibitors designed to minimize the contributions of other sites and maximize any superoxide/H2O2 production from proline dehydrogenase itself, there was no significant direct contribution of proline dehydrogenase to the observed H2O2 production. Thus proline oxidation by proline dehydrogenase drives superoxide/H2O2 production, but it does so mainly or exclusively by providing anaplerotic carbon for other mitochondrial dehydrogenases and not by producing superoxide/H2O2 directly.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Mitocondrias/metabolismo , Prolina/metabolismo , Superóxidos/metabolismo , Animales , Línea Celular , Drosophila melanogaster , Humanos , Ratones , Mitocondrias/química , Oxidación-ReducciónRESUMEN
Several flavin-dependent enzymes of the mitochondrial matrix utilize NAD(+) or NADH at about the same operating redox potential as the NADH/NAD(+) pool and comprise the NADH/NAD(+) isopotential enzyme group. Complex I (specifically the flavin, site IF) is often regarded as the major source of matrix superoxide/H2O2 production at this redox potential. However, the 2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-oxoacid dehydrogenase (BCKDH), and pyruvate dehydrogenase (PDH) complexes are also capable of considerable superoxide/H2O2 production. To differentiate the superoxide/H2O2-producing capacities of these different mitochondrial sites in situ, we compared the observed rates of H2O2 production over a range of different NAD(P)H reduction levels in isolated skeletal muscle mitochondria under conditions that favored superoxide/H2O2 production from complex I, the OGDH complex, the BCKDH complex, or the PDH complex. The rates from all four complexes increased at higher NAD(P)H/NAD(P)(+) ratios, although the 2-oxoacid dehydrogenase complexes produced superoxide/H2O2 at high rates only when oxidizing their specific 2-oxoacid substrates and not in the reverse reaction from NADH. At optimal conditions for each system, superoxide/H2O2 was produced by the OGDH complex at about twice the rate from the PDH complex, four times the rate from the BCKDH complex, and eight times the rate from site IF of complex I. Depending on the substrates present, the dominant sites of superoxide/H2O2 production at the level of NADH may be the OGDH and PDH complexes, but these activities may often be misattributed to complex I.
Asunto(s)
Peróxido de Hidrógeno/metabolismo , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Mitocondrias Musculares/metabolismo , Superóxidos/metabolismo , Animales , Femenino , Mitocondrias Musculares/enzimología , Músculo Esquelético/enzimología , Músculo Esquelético/metabolismo , NAD/metabolismo , Oxidación-Reducción , Complejo Piruvato Deshidrogenasa/metabolismo , Ratas , Ratas WistarRESUMEN
Mitochondrial radical production is important in redox signaling, aging and disease, but the relative contributions of different production sites are poorly understood. We analyzed the rates of superoxide/H2O2 production from different defined sites in rat skeletal muscle mitochondria oxidizing a variety of conventional substrates in the absence of added inhibitors: succinate; glycerol 3-phosphate; palmitoylcarnitine plus carnitine; or glutamate plus malate. In all cases, the sum of the estimated rates accounted fully for the measured overall rates. There were two striking results. First, the overall rates differed by an order of magnitude between substrates. Second, the relative contribution of each site was very different with different substrates. During succinate oxidation, most of the superoxide production was from the site of quinone reduction in complex I (site IQ), with small contributions from the flavin site in complex I (site IF) and the quinol oxidation site in complex III (site IIIQo). However, with glutamate plus malate as substrate, site IQ made little or no contribution, and production was shared between site IF, site IIIQo and 2-oxoglutarate dehydrogenase. With palmitoylcarnitine as substrate, the flavin site in complex II (site IIF) was a major contributor (together with sites IF and IIIQo), and with glycerol 3-phosphate as substrate, five different sites all contributed, including glycerol 3-phosphate dehydrogenase. Thus, the relative and absolute contributions of specific sites to the production of reactive oxygen species in isolated mitochondria depend very strongly on the substrates being oxidized, and the same is likely true in cells and in vivo.
Asunto(s)
Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Superóxidos/metabolismo , Animales , Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/metabolismo , Complejo III de Transporte de Electrones/química , Complejo III de Transporte de Electrones/metabolismo , Femenino , Glicerofosfatos/metabolismo , Malatos/metabolismo , Palmitoilcarnitina/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Ácido Succínico/metabolismoRESUMEN
Mitochondrial reactive oxygen species (ROS) are widely implicated in physiological and pathological pathways. We propose that it is critical to understand the specific sites of mitochondrial ROS production and their mechanisms of action. Mitochondria possess at least eight distinct sites of ROS production in the electron transport chain and matrix compartment. In this chapter, we describe the nature of the mitochondrial ROS-producing machinery and the relative capacities of each site. We provide detailed methods for the measurement of H2O2 release and the conditions under which maximal rates from each site can be achieved in intact skeletal muscle mitochondria.
Asunto(s)
Peróxido de Hidrógeno/análisis , Mitocondrias Musculares/metabolismo , Especies Reactivas de Oxígeno/análisis , Animales , Bioquímica/métodos , Complejo I de Transporte de Electrón/metabolismo , Complejo II de Transporte de Electrones/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Glicerolfosfato Deshidrogenasa/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Músculo Esquelético/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
A high membrane potential across the mitochondrial inner membrane leads to the production of the reactive oxygen species (ROS) implicated in aging and age-related diseases. A prototypical drug for the correction of this type of mitochondrial dysfunction is presented. MitoDNP-SUM accumulates in mitochondria in response to the membrane potential due to its mitochondria-targeting alkyltriphenylphosphonium (TPP) cation and is uncaged by endogenous hydrogen peroxide to release the mitochondrial uncoupler, 2,4-dinitrophenol (DNP). DNP is known to reduce the high membrane potential responsible for the production of ROS. The approach potentially represents a general method for the delivery of drugs to the mitochondrial matrix through mitochondria targeting and H(2)O(2)-induced uncaging.
Asunto(s)
2,4-Dinitrofenol/farmacología , Antioxidantes/farmacología , Peróxido de Hidrógeno/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Profármacos/farmacología , 2,4-Dinitrofenol/química , 2,4-Dinitrofenol/metabolismo , Animales , Antioxidantes/química , Antioxidantes/metabolismo , Femenino , Mitocondrias/metabolismo , Compuestos Organofosforados/química , Compuestos Organofosforados/metabolismo , Compuestos Organofosforados/farmacología , Estrés Oxidativo/efectos de los fármacos , Profármacos/química , Profármacos/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismoRESUMEN
H2O2 production by skeletal muscle mitochondria oxidizing palmitoylcarnitine was examined under two conditions: the absence of respiratory chain inhibitors and the presence of myxothiazol to inhibit complex III. Without inhibitors, respiration and H2O2 production were low unless carnitine or malate was added to limit acetyl-CoA accumulation. With palmitoylcarnitine alone, H2O2 production was dominated by complex II (44% from site IIF in the forward reaction); the remainder was mostly from complex I (34%, superoxide from site IF). With added carnitine, H2O2 production was about equally shared between complexes I, II, and III. With added malate, it was 75% from complex III (superoxide from site IIIQo) and 25% from site IF. Thus complex II (site IIF in the forward reaction) is a major source of H2O2 production during oxidation of palmitoylcarnitine ± carnitine. Under the second condition (myxothiazol present to keep ubiquinone reduced), the rates of H2O2 production were highest in the presence of palmitoylcarnitine ± carnitine and were dominated by complex II (site IIF in the reverse reaction). About half the rest was from site IF, but a significant portion, â¼40pmol H2O2·min(-1)·mg protein(-1), was not from complex I, II, or III and was attributed to the proteins of ß-oxidation (electron-transferring flavoprotein (ETF) and ETF-ubiquinone oxidoreductase). The maximum rate from the ETF system was â¼200pmol H2O2·min(-1)·mg protein(-1) under conditions of compromised antioxidant defense and reduced ubiquinone pool. Thus complex II and the ETF system both contribute to H2O2 productionduring fatty acid oxidation under appropriate conditions.
Asunto(s)
Ácidos Grasos/metabolismo , Peróxido de Hidrógeno/metabolismo , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Superóxidos/metabolismo , Animales , Complejo II de Transporte de Electrones/fisiología , Femenino , Oxidación-Reducción , Consumo de Oxígeno , Palmitoilcarnitina/metabolismo , Ratas , Ratas WistarRESUMEN
The oxidation of sn-glycerol 3-phosphate by mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a major pathway for transfer of cytosolic reducing equivalents to the mitochondrial electron transport chain. It is known to generate H(2)O(2) at a range of rates and from multiple sites within the chain. The rates and sites depend upon tissue source, concentrations of glycerol 3-phosphate and calcium, and the presence of different electron transport chain inhibitors. We report a detailed examination of H(2)O(2) production during glycerol 3-phosphate oxidation by skeletal muscle, brown fat, brain, and heart mitochondria with an emphasis on conditions under which mGPDH itself is the source of superoxide and H(2)O(2). Importantly, we demonstrate that a substantial portion of H(2)O(2) production commonly attributed to mGPDH originates instead from electron flow through the ubiquinone pool into complex II. When complex II is inhibited and mGPDH is the sole superoxide producer, the rate of superoxide production depends on the concentrations of glycerol 3-phosphate and calcium and correlates positively with the predicted reduction state of the ubiquinone pool. mGPDH-specific superoxide production plateaus at a rate comparable with the other major sites of superoxide production in mitochondria, the superoxide-producing center shows no sign of being overreducible, and the maximum superoxide production rate correlates with mGPDH activity in four different tissues. mGPDH produces superoxide approximately equally toward each side of the mitochondrial inner membrane, suggesting that the Q-binding pocket of mGPDH is the major site of superoxide generation. These results clarify the maximum rate and mechanism of superoxide production by mGPDH.
Asunto(s)
Glicerolfosfato Deshidrogenasa/metabolismo , Mitocondrias/enzimología , Superóxidos/metabolismo , Animales , Grupo Citocromo b/metabolismo , Proteínas del Complejo de Cadena de Transporte de Electrón/antagonistas & inhibidores , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Femenino , Glicerofosfatos/metabolismo , Peróxido de Hidrógeno/metabolismo , Membranas Mitocondriales/metabolismo , Especificidad de Órganos , Oxidación-Reducción , Ratas , Ratas WistarRESUMEN
A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and α-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigor.
Asunto(s)
S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo/genética , Ácidos Grasos/metabolismo , Técnicas de Inactivación de Genes , Lipoilación , Mitocondrias/metabolismo , Proteínas Mitocondriales/genética , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo/metabolismo , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/ultraestructura , Anemia/genética , Animales , Respiración de la Célula , Ácidos Grasos/genética , Femenino , Cuerpos Cetónicos/sangre , Ácido Láctico/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mitocondrias/genética , Proteínas Mitocondriales/metabolismo , Miocardio/metabolismo , Prolapso Rectal/genética , Transducción de SeñalRESUMEN
Individual sites of superoxide production in the mitochondrial respiratory chain have previously been defined and partially characterized using specific inhibitors, but the native contribution of each site to total superoxide production in the absence of inhibitors is unknown. We estimated rates of superoxide production (measured as H(2)O(2)) at various sites in rat muscle mitochondria using specific endogenous reporters. The rate of superoxide production by the complex I flavin (site I(F)) was calibrated to the reduction state of endogenous NAD(P)H. Similarly, the rate of superoxide production by the complex III site of quinol oxidation (site III(Qo)) was calibrated to the reduction state of endogenous cytochrome b(566). We then measured the endogenous reporters in mitochondria oxidizing NADH-generating substrates, without added respiratory inhibitors, with and without ATP synthesis. We used the calibrated reporters to calculate the rates of superoxide production from sites I(F) and III(Qo). The calculated rates of superoxide production accounted for much of the measured overall rates. During ATP synthesis, site I(F) was the dominant superoxide producer. Under nonphosphorylating conditions, overall rates were higher, and sites I(F) and III(Qo) and unidentified sites (perhaps the complex I site of quinone reduction, site I(Q)) all made substantial contributions to measured H(2)O(2) production.
Asunto(s)
Citocromos b/metabolismo , Transporte de Electrón , Mitocondrias Musculares/metabolismo , NADP/metabolismo , Superóxidos/metabolismo , Animales , Calibración , Femenino , Ácido Glutámico/metabolismo , Peróxido de Hidrógeno/metabolismo , Cinética , Malatos/metabolismo , Oxidación-Reducción , Ratas , Ratas WistarRESUMEN
Respiratory complex II oxidizes succinate to fumarate as part of the Krebs cycle and reduces ubiquinone in the electron transport chain. Previous experimental evidence suggested that complex II is not a significant contributor to the production of reactive oxygen species (ROS) in isolated mitochondria or intact cells unless mutated. However, we find that when complex I and complex III are inhibited and succinate concentration is low, complex II in rat skeletal muscle mitochondria can generate superoxide or H(2)O(2) at high rates. These rates approach or exceed the maximum rates achieved by complex I or complex III. Complex II generates these ROS in both the forward reaction, with electrons supplied by succinate, and the reverse reaction, with electrons supplied from the reduced ubiquinone pool. ROS production in the reverse reaction is prevented by inhibition of complex II at either the ubiquinone-binding site (by atpenin A5) or the flavin (by malonate), whereas ROS production in the forward reaction is prevented by malonate but not by atpenin A5, showing that the ROS from complex II arises only from the flavin site (site II(F)). We propose a mechanism for ROS production by complex II that relies upon the occupancy of the substrate oxidation site and the reduction state of the enzyme. We suggest that complex II may be an important contributor to physiological and pathological ROS production.
Asunto(s)
Complejo II de Transporte de Electrones/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Peróxido de Hidrógeno/metabolismoRESUMEN
Oxidative phosphorylation is an important energy-conserving mechanism coupling mitochondrial electron transfer to ATP synthesis. Coupling between respiration and phosphorylation is not fully efficient due to proton and electron leaks. In this chapter, methods are presented to measure proton and electron leak activities in isolated mitochondria. The relative strength of a modular kinetic approach to probe oxidative phosphorylation is emphasised.
Asunto(s)
Electrones , Mitocondrias Musculares/metabolismo , Protones , Animales , Cinética , Potencial de la Membrana Mitocondrial , Fosforilación Oxidativa , Consumo de Oxígeno , Ratas , Ratas WistarRESUMEN
Complex I (NADH-ubiquinone oxidoreductase) can form superoxide during forward electron flow (NADH-oxidizing) or, at sufficiently high protonmotive force, during reverse electron transport from the ubiquinone (Q) pool (NAD(+)-reducing). We designed an assay system to allow titration of the redox state of the superoxide-generating site during reverse electron transport in rat skeletal muscle mitochondria: a protonmotive force generated by ATP hydrolysis, succinate:malonate to alter electron supply and modulate the redox state of the Q pool, and inhibition of complex III to prevent QH(2) oxidation via the Q cycle. Stepwise oxidation of the QH(2)/Q pool by increasing malonate concentration slowed the rates of both reverse electron transport and rotenone-sensitive superoxide production by complex I. However, the superoxide production rate was not uniquely related to the resultant potential of the NADH/NAD(+) redox couple. Thus, there is a superoxide producer during reverse electron transport at complex I that responds to Q pool redox state and is not in equilibrium with the NAD reduction state. In contrast, superoxide production during forward electron transport in the presence of rotenone was uniquely related to NAD redox state. These results support a two-site model of complex I superoxide production; one site in equilibrium with the NAD pool, presumably the flavin of the FMN moiety (site I(F)) and the other dependent not only on NAD redox state, but also on protonmotive force and the reduction state of the Q pool, presumably a semiquinone in the Q-binding site (site I(Q)).
Asunto(s)
Complejo I de Transporte de Electrón/metabolismo , Mitocondrias/enzimología , Superóxidos/metabolismo , Animales , Transporte de Electrón , Femenino , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Ratas , Ratas WistarRESUMEN
Superoxide production from antimycin-inhibited complex III in isolated mitochondria first increased to a maximum then decreased as substrate supply was modulated in three different ways. In each case, superoxide production had a similar bell-shaped relationship to the reduction state of cytochrome b(566), suggesting that superoxide production peaks at intermediate Q-reduction state because it comes from a semiquinone in the outer quinone-binding site in complex III (Q(o)). Imposition of a membrane potential changed the relationships between superoxide production and b(566) reduction and between b(562) and b(566) redox states, suggesting that b(562) reduction also affects semiquinone concentration and superoxide production. To assess whether this behavior was consistent with the Q-cycle mechanism of complex III, we generated a kinetic model of the antimycin-inhibited Q(o) site. Using published rate constants (determined without antimycin), with unknown rate constants allowed to vary, the model failed to fit the data. However, when we allowed the rate constant for quinol oxidation to decrease 1000-fold and the rate constant for semiquinone oxidation by b(566) to depend on the b(562) redox state, the model fit the energized and de-energized data well. In such fits, quinol oxidation was much slower than literature values and slowed further when b(566) was reduced, and reduction of b(562) stabilized the semiquinone when b(566) was oxidized. Thus, superoxide production at Q(o) depends on the reduction states of b(566) and b(562) and fits the Q-cycle only if particular rate constants are altered when b oxidation is prevented by antimycin. These mechanisms limit superoxide production and short circuiting of the Q-cycle when electron transfer slows.
Asunto(s)
Complejo III de Transporte de Electrones/metabolismo , Mitocondrias/metabolismo , Quinonas/metabolismo , Superóxidos/metabolismo , Animales , Antibacterianos , Antimicina A/análogos & derivados , Antimicina A/farmacología , Grupo Citocromo b/metabolismo , Transporte de Electrón , Proteínas de Escherichia coli/metabolismo , Femenino , Cinética , Mitocondrias/efectos de los fármacos , Oxidación-Reducción , Ratas , Ratas WistarRESUMEN
The production of H(2)O(2) by isolated mitochondria is frequently used as a measure of mitochondrial superoxide formation. Matrix superoxide dismutase quantitatively converts matrix superoxide to H(2)O(2). However, matrix enzymes such as the glutathione peroxidases can consume H(2)O(2) and compete with efflux of H(2)O(2), causing an underestimation of superoxide production. To assess this underestimate, we depleted matrix glutathione in rat skeletal muscle mitochondria by more than 90% as a consequence of pretreatment with 1-chloro-2,4-dintrobenzene (CDNB). The pretreatment protocol strongly diminished the mitochondrial capacity to consume exogenous H(2)O(2), consistent with decreased peroxidase capacity, but avoided direct stimulation of superoxide production from complex I. It elevated the observed rates of H(2)O(2) formation from matrix-directed superoxide by up to two-fold from several sites of production, as defined by substrates and electron transport inhibitors, over a wide range of control rates, from 0.2-2.5 nmol H(2)O(2) min(-1) mg protein(-1). Similar results were obtained when glutathione was depleted using monochlorobimane or when soluble matrix peroxidase activity was removed by preparation of submitochondrial particles. The data indicate that the increased H(2)O(2) efflux observed with CDNB pretreatment was a result of glutathione depletion and compromised peroxidase activity. A hyperbolic correction curve was constructed, making H(2)O(2) efflux a more quantitative measure of matrix superoxide production. For rat muscle mitochondria, the correction equation was: CDNB-pretreated rate = control rate + [1.43 x (control rate)]/(0.55 + control rate). These results have significant ramifications for the rates and topology of superoxide production by isolated mitochondria.
Asunto(s)
Glutatión/deficiencia , Peróxido de Hidrógeno/metabolismo , Mitocondrias Musculares/metabolismo , Superóxidos/metabolismo , Animales , Femenino , Glutatión/metabolismo , Mitocondrias Musculares/química , Mitocondrias Musculares/enzimología , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismoRESUMEN
Mitochondria are central players in the pathophysiology of ischemia-reperfusion. Activation of plasma membrane G-coupled receptors or the Na,K-ATPase triggers cytosolic signaling pathways that result in cardioprotection. Our working hypothesis is that the occupied receptors migrate to caveolae, where signaling enzymes are scaffolded into signalosomes that bud off the plasma membrane and migrate to mitochondria. The signalosome-mitochondria interaction then initiates intramitochondrial signaling by opening the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)). MitoK(ATP) opening causes an increase in ROS production, which activates mitochondrial protein kinase C epsilon (PKCvarepsilon), which inhibits the mitochondrial permeability transition (MPT), thus decreasing cell death. We review the experimental findings that bear on these hypotheses and other modes of protection involving mitochondria.
Asunto(s)
Mitocondrias Cardíacas/fisiología , Transducción de Señal/fisiología , Animales , Humanos , Canales KATP/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteína Quinasa C-epsilon/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Perfusion of the heart with bradykinin triggers cellular signaling events that ultimately cause opening of mitochondrial ATP-sensitive K+ (mitoKATP) channels, increased H2O2 production, inhibition of the mitochondrial permeability transition (MPT), and cardioprotection. We hypothesized that the interaction of bradykinin with its receptor induces the assembly of a caveolar signaling platform (signalosome) that contains the enzymes of the signaling pathway and that migrates to mitochondria to induce mitoKATP channel opening. We developed a novel method for isolating and purifying signalosomes from Langendorff-perfused rat hearts treated with bradykinin. Fractions containing the signalosomes were found to open mitoKATP channels in mitochondria isolated from untreated hearts via the activation of mitochondrial PKC-epsilon. mitoKATP channel opening required signalosome-dependent phosphorylation of an outer membrane protein. Immunodetection analysis revealed the presence of the bradykinin B2 receptor only in the fraction isolated from bradykinin-treated hearts. Immunodetection and immunogold labeling of caveolin-3, as well as sensitivity to cholesterol depletion and resistance to Triton X-100, attested to the caveolar nature of the signalosomes. Ischemic preconditioning, ischemic postconditioning, and perfusion with ouabain also led to active signalosome fractions that opened mitoKATP channels in mitochondria from untreated hearts. These results provide initial support for a novel mechanism for signal transmission from a plasma membrane receptor to mitoKATP channels.