RESUMO
The study aimed to explore the role of age-associated elevated cytosolic Ca2+ in changes of brain mitochondria energetic processes. Two groups of rats, young adults (4 months) and advanced old (24 months), were evaluated for potential alterations of mitochondrial parameters, the oxidative phosphorylation (OxPhos), membrane potential, calcium retention capacity, activity of glutamate/aspartate carrier (aralar), and ROS formation. We demonstrated that the brain mitochondria of older animals have a lower resistance to Ca2+ stress with resulting consequences. The suppressed complex I OxPhos and decreased membrane potential were accompanied by reduction of the Ca2+ threshold required for induction of mPTP. The Ca2+ binding sites of mitochondrial aralar mediated a lower activity of old brain mitochondria. The altered interaction between aralar and mPTP may underlie mitochondrial dysregulation leading to energetic depression during aging. At the advanced stages of aging, the declined metabolism is accompanied by the diminished oxidative background.
Assuntos
Mitocôndrias , Proteínas de Transporte da Membrana Mitocondrial , Ratos , Animais , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Encéfalo/metabolismo , Fosforilação Oxidativa , Cálcio/metabolismoRESUMO
Mitochondrial defects have been shown to be associated with the pathogenesis of Parkinson's disease (PD). Yet, experience in PD research linking mitochondrial dysfunction, e.g., deregulation of oxidative phosphorylation, with neuronal degeneration and behavioral changes is rather limited. Using the 6-hydroxydopamine (6-OHDA) rat model of PD, we have investigated the potential role of mitochondria in dopaminergic neuronal cell death in the substantia nigra pars compacta by high-resolution respirometry. Mitochondrial function was correlated with the time course of disease-related motor behavior asymmetry and dopaminergic neuronal cell loss, respectively. Unilateral 6-OHDA injections (>2.5 µg/2 µl) into the median forebrain bundle induced an impairment of oxidative phosphorylation due to a decrease in complex I activity. This was indicated by increased flux control coefficient. During the period of days 2-21, a progressive decrease in respiratory control ratio of up to -58 % was observed in the lesioned compared to the non-lesioned substantia nigra of the same animals. This decrease was associated with a marked uncoupling of oxidative phosphorylation. Mitochondrial dysfunction, motor behavior asymmetry, and dopaminergic neuronal cell loss correlated with dosage (1.25-5 µg/2 µl). We conclude that high-resolution respirometry may allow the detection of distinct mitochondrial dysfunction as a suitable surrogate marker for the preclinical assessment of potential neuroprotective strategies in the 6-OHDA model of PD.
Assuntos
Neurônios Dopaminérgicos/efeitos dos fármacos , Feixe Prosencefálico Mediano/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Oxidopamina/toxicidade , Transtornos Parkinsonianos/fisiopatologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Neurônios Dopaminérgicos/patologia , Neurônios Dopaminérgicos/fisiologia , Relação Dose-Resposta a Droga , Lateralidade Funcional , Imuno-Histoquímica , Masculino , Feixe Prosencefálico Mediano/patologia , Feixe Prosencefálico Mediano/fisiopatologia , Mitocôndrias/fisiologia , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Fosforilação Oxidativa/efeitos dos fármacos , Ratos Sprague-Dawley , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Existing literature on estradiol indicates that it affects mitochondrial functions at low micromolar concentrations. Particularly blockade of the permeability transition pore (PTP) or modulation of the enzymatic activity of one or more complexes of the respiratory chain were suspicious. We prepared mitoplasts from rat liver mitochondria (RLM) to study by single-channel patch-clamp techniques the PTP, and from rat astrocytes to study the potassium BK-channel said to modulate the PTP. Additionally, we measured respiration of intact RLM. After application of 17ß-estradiol (ßE) our single-channel results reveal a transient increase of activity of both, the BK-channel and the PTP followed by their powerful inhibition. Respiration measurements demonstrate inhibition of the Ca(2+)-induced permeability transition, as well, though only at higher concentrations (≥30µM). At lower concentrations, we observed an increase of endogenous- and state 2-respiration. Furthermore, we show that ßE diminishes the phosphorylating respiration supported by complex I-substrates (glutamate/malate) or by the complex II-substrate succinate. Taken together the results suggest that ßE affects mitochondria by several modes, including partial inhibition of the activities of ion channels of the inner membrane and of respiration. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).
Assuntos
Estradiol/farmacologia , Estrogênios/farmacologia , Mitocôndrias Hepáticas/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Animais , Cálcio/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Poro de Transição de Permeabilidade Mitocondrial , Fosforilação/efeitos dos fármacos , RatosRESUMO
This review focuses on problems of the intracellular regulation of mitochondrial function in the brain via the (i) supply of mitochondria with ADP by means of ADP shuttles and channels and (ii) the Ca(2+) control of mitochondrial substrate supply. The permeability of the mitochondrial outer membrane for adenine nucleotides is low. Therefore rate dependent concentration gradients exist between the mitochondrial intermembrane space and the cytosol. The existence of dynamic ADP gradients is an important precondition for the functioning of ADP shuttles, for example CrP-shuttle. Cr at mM concentrations instead of ADP diffuses from the cytosol through the porin pores into the intermembrane space. The CrP-shuttle isoenzymes work in different directions which requires different metabolite concentrations mainly caused by dynamic ADP compartmentation. The ADP shuttle mechanisms alone cannot explain the load dependent changes in mitochondrial energization, and a complete model of mitochondrial regulation have to account the Ca(2+) -dependent substrate supply too. According to the old paradigmatic view, Ca(2+) (cyt) taken up by the mitochondrial Ca(2+) uniporter activates dehydrogenases within the matrix. However, recently it was found that Ca(2+) (cyt) at low nM concentrations exclusively activates the state 3 respiration via aralar, the mitochondrial glutamate/aspartate carrier. At higher Ca(2+) (cyt) (> 500 nM), brain mitochondria take up Ca(2+) for activation of substrate oxidation rates. Since brain mitochondrial pyruvate oxidation is only slightly influenced by Ca(2+) (cyt) , it was proposed that the cytosolic formation of pyruvate from its precursors is tightly controlled by the Ca(2+) dependent malate/aspartate shuttle. At low (50-100 nM) Ca(2+) (cyt) the pyruvate formation is suppressed, providing a substrate limitation control in neurons. This so called "gas pedal" mechanism explains why the energy metabolism of neurons in the nucleus suprachiasmaticus could be down-regulated at night but activated at day as a basis for the circadian changes in Ca(2+) (cyt) . It also could explain the energetic disadvantages caused by altered Ca(2+) (cyt) at mitochondrial diseases and neurodegeneration.
Assuntos
Cálcio/metabolismo , Retroalimentação Fisiológica , Mitocôndrias/metabolismo , Núcleo Supraquiasmático/metabolismo , Difosfato de Adenosina/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Antiporters/metabolismo , Ácido Aspártico/metabolismo , Ritmo Circadiano/fisiologia , Citosol/metabolismo , Metabolismo Energético , Humanos , Membranas Intracelulares/metabolismo , Malatos/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Neurônios/metabolismo , Fosforilação Oxidativa , Ácido Pirúvico/metabolismoRESUMO
The glutamate-dependent respiration of isolated BM (brain mitochondria) is regulated by Ca2+(cyt) (cytosolic Ca2+) (S0.5=225±22 nM) through its effects on aralar. We now also demonstrate that the α-glycerophosphate-dependent respiration is controlled by Ca2+(cyt) (S0.5=60±10 nM). At higher Ca2+(cyt) (>600 nM), BM accumulate Ca2+ which enhances the rate of intramitochondrial dehydrogenases. The Ca2+-induced increments of state 3 respiration decrease with substrate in the order glutamate>α-oxoglutarate>isocitrate>α-glycerophosphate>pyruvate. Whereas the oxidation of pyruvate is only slightly influenced by Ca2+(cyt), we show that the formation of pyruvate is tightly controlled by Ca2+(cyt). Through its common substrate couple NADH/NAD+, the formation of pyruvate by LDH (lactate dehydrogenase) is linked to the MAS (malate-aspartate shuttle) with aralar as a central component. A rise in Ca2+(cyt) in a reconstituted system consisting of BM, cytosolic enzymes of MAS and LDH causes an up to 5-fold enhancement of OXPHOS (oxidative phosphorylation) rates that is due to an increased substrate supply, acting in a manner similar to a 'gas pedal'. In contrast, Ca2+(mit) (intramitochondrial Ca2+) regulates the oxidation rates of substrates which are present within the mitochondrial matrix. We postulate that Ca2+(cyt) is a key factor in adjusting the mitochondrial energization to the requirements of intact neurons.
Assuntos
Ácido Aspártico/metabolismo , Encéfalo/metabolismo , Cálcio/metabolismo , Citosol/metabolismo , Malatos/metabolismo , Mitocôndrias/metabolismo , Ácido Pirúvico/metabolismo , Animais , Cinética , Camundongos , Fosforilação OxidativaRESUMO
Carboxyatractylate (CAT) and atractylate inhibit the mitochondrial adenine nucleotide translocator (ANT) and stimulate the opening of permeability transition pore (PTP). Following pretreatment of mouse liver mitochondria with 5 microM CAT and 75 microM Ca2+, the activity of PTP increased, but addition of 2 mM ADP inhibited the swelling of mitochondria. Extramitochondrial Ca2+ concentration measured with Calcium-Green 5N evidenced that 2 mM ADP did not remarkably decrease the free Ca2+ but the release of Ca2+ from loaded mitochondria was stopped effectively after addition of 2 mM ADP. CAT caused a remarkable decrease of the maximum amount of calcium ions, which can be accumulated by mitochondria. Addition of 2 mM ADP after 5 microM CAT did not change the respiration, but increased the mitochondrial capacity for Ca2+ at more than five times. Bongkrekic acid (BA) had a biphasic effect on PT. In the first minutes 5 microM BA increased the stability of mitochondrial membrane followed by a pronounced opening of PTP too. BA abolished the action about of 1 mM ADP, but was not able to induce swelling of mitochondria in the presence of 2 mM ADP. We conclude that the outer side of inner mitochondrial membrane has a low affinity sensor for ADP, modifying the activity of PTP. The pathophysiological importance of this process could be an endogenous prevention of PT at conditions of energetic depression.
Assuntos
Difosfato de Adenosina/farmacologia , Atractilosídeo/análogos & derivados , Membranas Intracelulares/metabolismo , Fígado/metabolismo , Mitocôndrias/metabolismo , Porinas/metabolismo , Translocador 3 do Nucleotídeo Adenina/antagonistas & inibidores , Difosfato de Adenosina/metabolismo , Animais , Atractilosídeo/farmacologia , Transporte Biológico/efeitos dos fármacos , Ácido Bongcréquico/farmacologia , Cálcio/metabolismo , Camundongos , Consumo de Oxigênio/fisiologia , Permeabilidade/efeitos dos fármacos , Espectrometria de FluorescênciaRESUMO
l-Aminocarnitine (l-AC) has been shown to inhibit carnitine palmitoyltransferases (CPT) in rat muscle and in rat liver. However, there are no reports on interactions of l-AC with CPT II and CPT I of human muscle. Therefore, the aim of the present work was to characterize the inhibition of human muscle CPT I and CPT II by l-AC in muscle mitochondria, skinned fibers and muscle homogenates in comparison to the established action of malonyl-CoA. Both isoenzymes were inhibited by l-AC, but sensitivity was different (CPT I, K(d)=3.8 mM l-AC; CPT II, K(d)=21.3 microM l-AC). A mixed inhibition type in respect to carnitine was detected (K(i)=3.5 microM l-AC). At 0.5 mM l-AC, CPT II was completely inhibited without affection of CPT I. In contrast, CPT I was completely inhibited by 0.4 mM malonyl-CoA (K(d)=0.5 microM), whereas CPT II was nearly not affected by this inhibitor. Using these inhibitors in muscle homogenates, activities of CPT II and CPT I were detected to be 38+/-10% and 63+/-10% of total, respectively (n=21). In intact mitochondria and different fractions of muscle homogenates after selective solubilization of CPT II by Tween 20, the extent of specific CPT inhibition changed in relation to the accessible isoenzyme pattern. Palmitoyl-carnitine-dependent respiration in skinned fibers was inhibited by high concentrations of l-AC, indicating that the inhibitor can be transported via the acyl-carnitine transporter, too. The combined use of both inhibitors (l-AC and malonyl-CoA) allows the kinetic characterization of CPT I and CPT II in human muscle homogenates. In addition, it has been shown that l-AC can be used for the study of metabolic consequences of CPT II deficiency on function of intact mitochondria.
Assuntos
Betaína/análogos & derivados , Betaína/metabolismo , Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Carnitina , Músculo Esquelético/metabolismo , Betaína/farmacologia , Biópsia , Inibidores Enzimáticos/farmacologia , Humanos , Malonil Coenzima A/farmacologia , Mitocôndrias Musculares/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Consumo de Oxigênio , Rabdomiólise/metabolismoRESUMO
We have previously provided evidence that diffusion of metabolites across the porin pores of mitochondrial outer membrane is hindered. A functional consequence of this diffusion limitation is the dynamic compartmentation of ADP in the intermembrane space. These earlier studies were done on isolated mitochondria suspended in isotonic media without macromolecules, in which intermembrane space of mitochondria is enlarged. The present study was undertaken to assess the diffusion limitation of outer membrane in the presence of 10% (w/v) dextran M20, in order to mimic the action of cytosolic macromolecules on mitochondria. Under these conditions, mitochondria have a more native, condensed configuration.Flux-dependent concentration gradients of ADP were estimated by measuring the ADP diffusion fluxes across the porin pores of isolated rat heart mitochondria incubated together with pyruvate kinase (PK), both of which compete for ADP regenerated by mitochondrial creatine kinase (mtCK) within the intermembrane space or by yeast hexokinase (HK) extramitochondrially. From diffusion fluxes and bulk phase concentrations of ADP, its concentrations in the intermembrane space were calculated using Fick's law of diffusion. Flux-dependent gradients up to 23 microM ADP (for a diffusion rate of J(Dif)=1.9 micromol ADP/min/mg mitochondrial protein) were observed. These gradients are about twice those estimated in the absence of dextran and in the same order of magnitude as the cytosolic ADP concentration (30 microM), but they are negligibly low for cytosolic ATP (5 mM). Therefore, it is concluded that the dynamic ADP compartmentation is of biological importance for intact heart cells. If mtCK generates ADP within the intermembrane space, the local ADP concentration can be clearly higher than in the cytosol resulting in higher extramitochondrial phosphorylation potentials. In this way, mtCK contributes to ensure optimal kinetic conditions for ATP-splitting reactions in the extramitochondrial compartment.
Assuntos
Difosfato de Adenosina/metabolismo , Membranas Intracelulares/metabolismo , Mitocôndrias Cardíacas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Creatina Quinase/metabolismo , Dextranos , Difusão , Substâncias Macromoleculares , Modelos Biológicos , Fosforilação Oxidativa , Piruvato Quinase/metabolismo , RatosRESUMO
To clarify the importance of deleted protein and tRNA genes on the impairment of mitochondrial function, we performed a quantitative analysis of biochemical, genetic and morphological findings in skeletal muscles of 16 patients with single deletions and 5 patients with multiple deletions of mtDNA. Clinically, all patients showed chronic progressive external ophthalmoplegia (CPEO). The size of deletions varied between 2.5 and 9 kb, and heteroplasmy between 31% and 94%. In patients with single deletions, the citrate synthase (CS) activity was nearly doubled. Decreased ratios of pyruvate- and succinate-dependent respiration were detected in fibers of all patients in comparison to controls. Inverse and linear correlations without thresholds were established between heteroplasmy and (i) CS referenced activities of the complexes of respiratory chain, (ii) CS referenced maximal respiratory rates, (iii) and cytochrome-c-oxidase (COX) negative fibers. In patients with single and multiple deletions, all respiratory chain complexes as well as the respiratory rates were decreased to a similar extent. All changes detected in patients with single deletions were independent of deletion size. In one patient, only genes of ND5, ND4L as well as tRNA(Leu(CUN)), tRNA(Ser(AGY)), and tRNA(His) were deleted. The pronounced decrease in COX activity in this patient points to the high pathological impact of these missing tRNA genes. The activity of nuclear encoded SDH was also significantly decreased in patients, but to a lesser extent. This is an indication of secondary disturbances of mitochondria at CPEO. In conclusion, we have shown that different deletions cause mitochondrial impairments of the same phenotype correlating with heteroplasmy. The missing threshold at the level of mitochondrial function seems to be characteristic for large-scale deletions were tRNA and protein genes are deleted.
Assuntos
DNA Mitocondrial/química , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais/biossíntese , Oftalmoplegia Externa Progressiva Crônica/metabolismo , Citrato (si)-Sintase/metabolismo , DNA Mitocondrial/genética , Transporte de Elétrons , Complexo I de Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Deleção de Genes , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias Musculares/química , Proteínas Mitocondriais/genética , Músculo Esquelético/metabolismo , NADH NADPH Oxirredutases/metabolismo , Oftalmoplegia Externa Progressiva Crônica/genética , Consumo de Oxigênio , Análise de Regressão , Succinato Desidrogenase/metabolismoRESUMO
Due to the large importance of mitochondrial function for numerous diseases the detection of respiratory chain defects for diagnostic purposes is an important task of mitochondrial medicine. For comparing the methods, standard mitochondrial homogenate prepared from bovine skeletal muscle was sent on dry ice to 14 labs of 8 countries. Activities of complexes I, I + III, II, II + III, IV (cytochrome c oxidase) and V (F0F1ATPase) as well as of citrate synthase were measured. For all enzymes the results differed at more than one order of magnitude. From eight labs we were able to compare the results with their control values for human skeletal muscle. Four labs found normal activity of cytochrome-c-oxidase whereas three labs found higher and one lab found lower activities compared to the own controls. Since all labs used different temperatures (25, 30 and 37 degrees C) in one lab the temperature dependencies were measured experimentally. The temperature correction did not much reduce the divergence of the results. It is concluded that differences in the lab protocols are the reason for the large variation of results. Since the experimental results strongly depend on the used method a strict standardization is necessary.
RESUMO
Mitochondria not only provide most of the ATP needed for cell work and numerous specific anabolic and catabolic functions, they also contribute to Ca++ signalling and play a key role in the pathway to cell death. Impairment of mitochondrial functions caused by mutations of the mt-genome or by acute processes, is responsible for numerous diseases. Decreased concentrations of adenine nucleotides, leaky outer and inner mitochondrial membranes, and decreased activities of respiratory chain enzymes contribute to depression of cellular energy metabolism, one of the most important consequences of mitochondrial impairment as characterized by decreased cytosolic phosphorylation potentials.
Assuntos
Mitocôndrias/metabolismo , Nucleotídeos de Adenina/metabolismo , Animais , Sinalização do Cálcio , Transporte de Elétrons , Metabolismo Energético , Humanos , Membranas Intracelulares/metabolismo , Doenças Mitocondriais/metabolismo , Sepse/metabolismoRESUMO
Mitochondria, that provide most of the ATP needed for cell work, and that play numerous specific functions in biosyntheses and degradations, as well as contributing to Ca2+ signaling, also play a key role in the pathway to cell death. Impairment of mitochondrial functions caused by mutations of mt-genome, and by acute processes, are responsible for numerous diseases. The involvement of impaired mitochondria in the pathogenesis of sepsis is discussed. By means of the skinned fiber technique and high resolution respirometry, we have detected significantly reduced rates of mitochondrial respiration in heart and skeletal muscle of endotoxaemic rabbits. Mitochondria from heart were more affected than those from skeletal muscle. Decreased respiration rates were accompanied by reduced activities of complex I + III of the respiratory chain. Endotoxin-caused impairment was also detectable at the level of the Langendorff perfused heart, where the coronary vascular resistance was significantly increased. For an investigation of the influence of bacteraemia on the mitochondrial respiratory chain, baboons were made septic by infusion of high and low amounts of E. coli. For complex I + III and II + III, a clear dose-dependent decrease was detectable and in animals which died in septic shock, a further decrease of enzyme activities in comparison to the controls were found. These results are discussed in the light of current knowledge on the role of mitochondria in cell pathology in respect to sepsis. In conclusion, we present evidence that mitochondrial function is disturbed during sepsis. Besides ischaemic and poison-induced disturbances of mitochondrial function, sepsis is a further example of an acute disease where impaired mitochondria have to be taken into account.
Assuntos
Bacteriemia/fisiopatologia , Endotoxemia/fisiopatologia , Doenças Mitocondriais/fisiopatologia , Sepse/fisiopatologia , Animais , Bacteriemia/metabolismo , Endotoxemia/metabolismo , Doenças Mitocondriais/metabolismo , Papio , Coelhos , Sepse/metabolismoRESUMO
We have introduced a sensitive method for studying oxygen/glucose deprivation (OGD)-induced mitochondrial alterations in homogenates of organotypic hippocampal slice cultures (slices) by high-resolution respirometry. Using this approach, we tested the neuroprotective potential of the novel non-immunosuppressive cyclosporin (CsA) derivative Cs9 in comparison with CsA, the immunosuppressive CsA analog [D-Ser](8)CsA, and MK 801, a N-methyl-d-aspartate (NMDA) receptor antagonist. OGD/reperfusion reduced the glutamate/malate dependent (and protein-related) state 3 respiration to 30% of its value under control conditions. All of the above drugs reversed this effect, with an increase to >88% of the value for control slices not exposed to OGD. We conclude that Cs9, [D-Ser](8)CsA, and MK 801, despite their different modes of action, protect mitochondria from OGD-induced damage.
Assuntos
Respiração Celular , Ciclosporinas/metabolismo , Maleato de Dizocilpina/metabolismo , Glucose/metabolismo , Hipocampo/fisiologia , Fármacos Neuroprotetores/metabolismo , Oxigênio/metabolismo , Animais , Hipocampo/efeitos dos fármacos , Técnicas In Vitro , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ratos , Ratos WistarRESUMO
We present unexpected and novel results revealing that glutamate-dependent oxidative phosphorylation (OXPHOS) of brain mitochondria is exclusively and efficiently activated by extramitochondrial Ca(2+) in physiological concentration ranges (S(0.5) = 360 nM Ca(2+)). This regulation was not affected by RR, an inhibitor of the mitochondrial Ca(2+) uniporter. Active respiration is regulated by glutamate supply to mitochondria via aralar, a mitochondrial glutamate/aspartate carrier with regulatory Ca(2+)-binding sites in the mitochondrial intermembrane space providing full access to cytosolic Ca(2+). At micromolar concentrations, Ca(2+) can also enter the intramitochondrial matrix and activate specific dehydrogenases. However, the latter mechanism is less efficient than extramitochondrial Ca(2+) regulation of respiration/OXPHOS via aralar. These results imply a new mode of glutamate-dependent OXPHOS regulation as a demand-driven regulation of mitochondrial function. This regulation involves the mitochondrial glutamate/aspartate carrier aralar which controls mitochondrial substrate supply according to the level of extramitochondrial Ca(2+).
Assuntos
Cálcio/farmacologia , Ácido Glutâmico/metabolismo , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Difosfato de Adenosina/farmacologia , Animais , Respiração Celular/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Ratos , Ratos Wistar , Rutênio Vermelho/metabolismoRESUMO
The aim of the study was to determinate mitochondrial oxidative phosphorylation (OXPHOS) functions in rat rhabdomyosarcoma R1H (R1H) and rat skeletal muscles. For that purpose skinned fiber technique and multiple substrate inhibitor titration were adapted to tumor samples. In our animal tumor model (R1H) functional abnormalities of OXPHOS were found compared to skeletal muscles. In R1H the state 3 respiration of pyruvate + malate was decreased: 0.56 +/- 0.28 nmol O(2)/mg/min versus 2.32 +/- 1.19 nmol O(2)/mg/min, P < 0.001, whereas the state 3 respiration of succinate + rotenone was increased: 36 +/- 14% versus 19 +/- 11%, P < 0.001. In R1H the rotenone-insensitive respiration reached higher levels than the antimycin A-insensitive respiration, whereas in normal muscles the converse was observed. Additionally, the obvious difference between the CAT- and the antimycin A-independent respiration indicates an increased part of leak respiration in R1H. By now, the high feasibility of these techniques is appreciated for the investigation of muscles and prospectively for tumors, too.
Assuntos
Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Rabdomiossarcoma/metabolismo , Animais , Antimicina A/farmacologia , Linhagem Celular Tumoral , Transporte de Elétrons/efeitos dos fármacos , Feminino , Mitocôndrias Musculares/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ácido Pirúvico/metabolismo , Ratos , Ratos Endogâmicos , Rotenona/farmacologia , Succinatos/metabolismo , Desacopladores/farmacologiaRESUMO
Mitochondria, providing most of ATP needed for cell work, realizing numerous specific functions as biosyntheses or degradations, contributing to Ca2+ signalling also play a key role in the pathways to cell death. Impairment of mitochondrial functions caused by mutations of mt-genome and by acute processes are responsible for numerous diseases. The relations between changes on the level of molecules and the clinical state are rather complex, and the prediction of thresholds is difficult. Therefore investigations on different levels of an organismus (genome, metabolites, enzymes, mitochondrial function in vivo and in vitro) are necessary (multi level approach). Metabolic control theory is a valuable tool for understanding the different effects of mutations on the level of enzyme activities and mitochondrial function. Decreased concentrations of adenine nucleotides, leaky outer and inner mitochondrial membranes, decreased rates of mitochondrial linked pathways and decreased activities of respiratory chain enzymes contribute to depression of cellular energy metabolism characterized by decreased cytosolic phosphorylation potentials as one of the most important consequences of mitochondrial impairments. This review regards classical bioenergetic mechanisms of mitochondrial impairment which contribute to energetic depression.