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PubMed2004

Mitochondrial dysfunction and ultrastructural damage in the hippocampus during kainic acid-induced status epilepticus in the rat.

PURPOSE: Prolonged and continuous epileptic seizure (status epilepticus) results in cellular changes that lead to neuronal damage. We investigated whether these cellular changes entail mitochondrial dysfunction and ultrastructural damage in the hippocampus, by using a kainic acid (KA)-induced experimental status epilepticus model. METHODS: In Sprague-Dawley rats maintained under chloral hydrate anesthesia, KA (0.5 nmol) was microinjected unilaterally into the CA3 subfield of the hippocampus to induce seizure-like hippocampal EEG activity. The activity of key mitochondrial respiratory chain enzymes in the dentate gyrus (DG), or CA1 or CA3 subfield of the hippocampus was measured 30 or 180 min after application of KA. Ultrastructure of mitochondria in those three hippocampal subfields during KA-induced status epilepticus also was examined with electron microscopy. RESULTS: Microinjection of KA into the CA3 subfield of the hippocampus elicited progressive build-up of seizure-like hippocampal EEG activity. Enzyme assay revealed significant depression of the activity of nicotinamide adenine dinucleotide cytochrome c reductase (marker for Complexes I+III) in the DG, or CA1 or CA3 subfields 180 min after KA-elicited temporal lobe status epilepticus. Conversely, the activities of succinate cytochrome c reductase (marker for Complexes II+III) and cytochrome c oxidase (marker for Complex IV) remained unaltered. Discernible mitochondrial ultrastructural damage, varying from swelling to disruption of membrane integrity, also was observed in the hippocampus 180 min after hippocampal application of KA. CONCLUSIONS: Our results demonstrated that dysfunction of Complex I respiratory chain enzyme and mitochondrial ultrastructural damage in the hippocampus are associated with prolonged seizure during experimental temporal lobe status epilepticus.

Animals↗
PubMed2006

Short communication metabolic and mitochondrial effects of switching antiretroviral-experienced patients to enfuvirtide, tenofovir and saquinavir/ritonavir.

OBJECTIVE: Investigate the metabolic and mitochondrial effects of switching a highly active antiretroviral therapy (HAART) regimen with a high mitochondrial toxicity profile to a HAART with a theoretically low mitochondrial toxicity. PATIENTS AND METHODS: Six consecutive HAART-experienced patients receiving at least one dideoxy-nucleoside reverse transcriptase inhibitor (NRTI) switched to enfuvirtide plus tenofovir plus saquinavir/ritonavir (T20+TDF+SQV/r). Blood samples were collected at baseline, 12 and 24 weeks after the switch, and viral load (VL) and lymphocyte CD4+ T-cell count were determined. Metabolic parameters consisted of fasting serum triglycerides, cholesterol (total and fractions), glucose, insulin, C-peptide and lactate. Mitochondrial assessment consisted on mitochondrial DNA (mtDNA) quantification, COX-II mitochondrial protein expression rate, mitochondrial respiratory chain complex III and IV activities, and oxygen consumption in peripheral blood mononuclear cells. For baseline mitochondrial comparisons, we included six HIV-infected patients naive for ART. RESULTS: Switched patients exhibited a mean increase of 26 CD4+ T-cells/mm3 and a mean decrease of 1.1 log in VL (P = NS for both). Lactate, lipids and glycaemia remained stable during the study; only insulin levels increased significantly (P < 0.05). Switched patients exhibited, at baseline, low mitochondrial measurements, being significant only for complex III and IV activities with respect to naive patients (P < 0.05 for both). MtDNA content did not rise significantly during the study. However, we observed increases in COX-II mitochondrial protein synthesis (124%, P < 0.05), complex III activity (127%, P < 0.05), complex IV activity (86%, P = 0.37) and oxygen consumption (194%, P < 0.05). CONCLUSION: Switching a HAART-containing dideoxy-NRTI to T20+TDF+SQV/r minimally alters metabolic parameters and exerts beneficial effects on mitochondrial function at 24 weeks. Mitochondrial improvement should be considered as an additional advantage of this rescue therapy.

Adenine↗
PubMed1996

Threshold effects and control of oxidative phosphorylation in nonsynaptic rat brain mitochondria.

The amount of control exerted by respiratory chain complexes in isolated nonsynaptic mitochondria prepared from rat brain on the rate of oxygen consumption was assessed using inhibitor titrations. Rotenone, myxothiazol, and KCN were used to titrate the activities of NADH:ubiquinone oxidoreductase (EC 1.6.5.3; complex I), ubiquinol:ferrocytochrome c oxidoreductase (EC 1.10.2.2; complex III), and cytochrome c oxidase (EC 1.9.3.1; complex IV ), respectively. Complexes I, III, and IV shared some of the control of the rate of oxygen consumption in nonsynaptic mitochondria, having flux control coefficients of 0.14, 0.15, and 0.24, respectively. Threshold effects in the control of oxidative phosphorylation were demonstrated for complexes I, III, and IV. It was found that complex I activity could be decreased by approximately 72% before major changes in mitochondrial respiration and ATP synthesis took place. Similarly, complex III and IV activities could be decreased by approximately 70 and 60%, respectively, before major changes in mitochondrial respiration and ATP synthesis occurred. These results indicate that previously observed decreases in respiratory chain complex activities in some neurological disorders need to be reassessed as these decreases might not affect the overall capability of nonsynaptic mitochondria to maintain energy homeostasis unless a certain threshold of decreased complex activity has been reached. Possible implications for synaptic mitochondria and neurodegenerative disorders are also discussed.

Adenosine Triphosphate↗
PubMed2003

Essential role of complex II of the respiratory chain in hypoxia-induced ROS generation in the pulmonary vasculature.

In the pulmonary vasculature, the mechanisms responsible for oxygen sensing and the initiation of hypoxia-induced vasoconstriction and vascular remodeling are still unclear. Nitric oxide (NO) and reactive oxygen species (ROS) are discussed as early mediators of the hypoxic response. Here, we describe a quantitative analysis of NO- and ROS-producing cells within the vascular walls of murine lung sections cultured at normoxia or hypoxia. Whereas the number of NO-producing cells was not changed by hypoxia, the number of ROS-generating cells was significantly increased. Addition of specific inhibitors revealed that mitochondria were the source of ROS. The participation of the individual mitochondrial complexes differed in normoxic and hypoxic ROS generation. Whereas normoxic ROS production required complexes I and III, hypoxic ROS generation additionally demanded complex II. Histochemically demonstrable succinate dehydrogenase activity of complex II in the arterial wall decreased during hypoxia. Inhibition of the reversed enzymatic reaction, i.e., fumarate reductase, by application of succinate, specifically abolished hypoxic, but not normoxic, ROS generation. Thus complex II plays an essential role in hypoxic ROS production. Presumably, its catalytic activity switches from succinate dehydrogenase to fumarate reductase at reduced oxygen tension, thereby modulating the directionality of the electron flow.

Animals↗
PubMedDate not supplied

Nitric-oxide-induced inhibition of mitochondrial complexes following aglycaemic hypoxia in neonatal cortical rat brain slices.

The effect of aglycaemic hypoxia (AH) on the activity of the mitochondrial respiratory chain complexes was measured in superfused neonatal cortical brain slices. After 30 min AH, there were no significant changes in the activities of complex I, II-III and IV or citrate synthase compared to controls. Following 30 min AH and a 30-min reperfusion period (with oxygen and glucose), the activities of complex II-III and complex IV were significantly reduced (by 25 and 17%, respectively). These reductions in enzyme activity were not abrogated by removing external calcium prior to and throughout AH, but could be reversed by the presence of the nitric oxide (NO) synthase inhibitor N omega-nitro-L-arginine during these periods. These data suggest that NO or an NO-derived species is involved in the decreases in mitochondrial enzyme activities observed after AH

Animals↗
PubMed1994

Cortical cytochrome oxidase activity is reduced in Alzheimer's disease.

A defect in energy metabolism may play a role in the pathogenesis of neurodegenerative diseases, such as Alzheimer's disease. In the present study, we examined the activities of the enzymes that catalyze oxidative phosphorylation in frontal, temporal, parietal, and occipital cortex from Alzheimer's disease patients and age-matched controls. Complex I and complex II-III activities showed a small decrease in occipital cortex, but were unaffected in the other cortical areas. The most consistent change was a significant decrease of cytochrome oxidase (complex IV) activity of 25-30% in the four cortical regions examined. These results provide further evidence of a cytochrome oxidase defect in Alzheimer's disease postmortem brain tissue. A deficiency in this key energy-metabolizing enzyme could lead to a reduction in energy stores and thereby contribute to the neurodegenerative process.

Alzheimer Disease↗
PubMed1991

Decreased activities of ubiquinol:ferricytochrome c oxidoreductase (complex III) and ferrocytochrome c:oxygen oxidoreductase (complex IV) in liver mitochondria from rats with hydroxycobalamin[c-lactam]-induced methylmalonic aciduria.

Rats treated with hydroxycobalamin[c-lactam] (HCCL), a cobalamin analogue that induces methylmalonic aciduria, have increased hepatic mitochondrial content and increased oxidative metabolism of pyruvate and palmitate per hepatocyte. The present studies were undertaken to characterize oxidative metabolism in isolated liver mitochondria from rats treated with HCCL. After 5-6 weeks, state 3 oxidation rates for diverse substrates are reduced in mitochondria from HCCL-treated rats. Similar reductions of mitochondrial oxidation rates are obtained with dinitrophenol-uncoupled mitochondria excluding defective phosphorylation as a cause for the observed decrease in mitochondrial oxidation. The activities of mitochondrial oxidases are reduced in HCCL-treated rats and demonstrate a defect in complex IV. Investigation of the complexes of the respiratory chain reveals a 32% decrease of ubiquinol:ferricytochrome c oxidoreductase (complex III) activity and a 72% decrease of ferrocytochrome c:oxygen oxidoreductase (complex IV) activity in mitochondria from 5-6-week HCCL-treated rats as compared with controls. Liver mitochondria from HCCL-treated rats also demonstrate decreased cytochrome content per mg of mitochondrial protein (25% decrease of cytochrome b and 52% decrease of cytochrome a + a3 as compared with control rats). The HCCL-treated rat represents an animal model for the study of the consequences of respiratory chain defects in liver mitochondria.

Animals↗
PubMed1993

Electron transfer complexes I and IV of platelets are abnormal in Parkinson's disease but normal in Parkinson-plus syndromes.

Using a technique which requires only 100 ml blood we investigated the electron transfer complexes (ETC) I, III and IV in platelet mitochondria of 44 control subjects, 27 patients with idiopathic Parkinson's disease and eight patients with Parkinson-plus syndromes due to multiple system atrophy. In both control subjects and patients, ETC measurements were repeated at intervals of several months. The activities varied considerably among normal subjects, but intra-individual variation of ETC activities were low at repetitive measurements. In normal subjects there was no correlation between enzyme activities and age or training state. There was no difference in enzyme activities between smokers and non-smokers in the control group. Complex I activity was lower in Parkinson's disease patients than in controls (14 versus 29 nmol/min/mg platelet protein; P < 0.001). Furthermore, the group difference in complex IV activity also reached statistical significance (83 versus 58 nmol/min/mg platelet protein; P < 0.001). Additionally, in some Parkinson's disease patients, activities of complex III were low and lay outside the control range, but the group difference did not reach significance. There was no correlation between complex I activity and disease duration or severity as well as the daily L-dopa dose in Parkinson's disease patients. Repeated measurements in five Parkinson's disease patients in the earliest stages of their illness demonstrated that the decrease in complex I and IV activities can develop rapidly within 1 year. In Parkinson-plus patients suffering from multiple system atrophy the ETC activities were normal.

Aged↗
PubMed2006

Protections of pinocembrin on brain mitochondria contribute to cognitive improvement in chronic cerebral hypoperfused rats.

To study effects of pinocembrin, a natural compound extracted from propolis, on cognitive ability impaired by chronic cerebral hypoperfusion in rats, and if it did so, to investigate its effects on brain mitochondria. Rat chronic cerebral hypoperfusion was achieved by permanent bilateral common carotid arteries ligation, with regional cerebral blood flow evaluated. Cognitive ability was tested by Morris water maze task. Production of reactive oxygen species and origin targets including mitochondria membrane potential, respiratory chain complex I, complex III activities and mitochondria swelling degree were evaluated. Cytochrome oxidase was determined on its expression level by western blotting. Pinocembrin alleviated cognitive impairments in Morris water maze and decreased mitochondria reactive oxygen species production, in accordance with its improvements on complex I activity, membrane potential level, mitochondria swelling degree and cytochrome oxidase deficits. Pinocembrin could improve rat cognitive impairments induced by chronic cerebral hypoperfusion, contributed to its protections on brain mitochondria structure and function.

Animals↗
PubMed2000

Mitochondrial function in flying honeybees (Apis mellifera): respiratory chain enzymes and electron flow from complex III to oxygen.

The biochemical bases for the high mass-specific metabolic rates of flying insects remain poorly understood. To gain insights into mitochondrial function during flight, metabolic rates of individual flying honeybees were measured using respirometry, and their thoracic muscles were fixed for electron microscopy. Mitochondrial volume densities and cristae surface densities, combined with biochemical data concerning cytochrome content per unit mass, were used to estimate respiratory chain enzyme densities per unit cristae surface area. Despite the high content of respiratory enzymes per unit muscle mass, these are accommodated by abundant mitochondria and high cristae surface densities such that enzyme densities per unit cristae surface area are similar to those found in mammalian muscle and liver. These results support the idea that a unit area of mitochondrial inner membrane constitutes an invariant structural unit. Rates of O(2) consumption per unit cristae surface area are much higher than those estimated in mammals as a consequence of higher enzyme turnover rates (electron transfer rates per enzyme molecule) during flight. Cytochrome c oxidase, in particular, operates close to its maximum catalytic capacity (k(cat)). Thus, high flux rates are achieved via (i) high respiratory enzyme content per unit muscle mass and (ii) the operation of these enzymes at high fractional velocities.

Animals↗
PubMed1996

Mitochondrial dysfunction after fetal alcohol exposure.

Specific mitochondrial enzyme activities and mRNA levels were measured in the heart, brain, and liver tissues of a group of 1-day-old neonatal rats whose mothers were alcohol-fed during pregnancy and compared with a control group. The results show a significant decrease in mitochondrial ATP synthase activity in both the brain and liver, as well as a decrease in complex III activity in the liver of rats exposed to alcohol. Other mitochondrial enzymes activities (e.g., citrate synthase, cytochrome c oxidase, and complex I), as well as specific mitochondrial transcript levels, were not significantly affected. Heart mitochondrial enzyme activities were not significantly affected. These data reveal that a tissue-specific response occurs after fetal exposure to alcohol and may explain some of the cellular events occurring in fetal alcohol syndrome resulting in abnormal growth and neurological development.

ATP Synthetase Complexes↗
PubMed1984

Cytochrome c-mediated electron transfer between ubiquinol-cytochrome c reductase and cytochrome c oxidase. Kinetic evidence for a mobile cytochrome c pool.

Ubiquinol oxidase has been reconstituted from ubiquinol-cytochrome c reductase (Complex III), cytochrome c and cytochrome c oxidase (Complex IV). The steady-state level of reduction of cytochrome c by ubiquinol-2 varies with the molar ratios of the complexes and with the presence of antimycin in a way that can be quantitatively accounted for by a model in which cytochrome c acts as a freely diffusible pool on the membrane. This model was based on that of Kröger & Klingenberg [(1973) Eur. J. Biochem. 34, 358-368] for ubiquinone-pool behaviour. Further confirmation of the pool model was provided by analysis of ubiquinol oxidase activity as a function of the molar ratio of the complexes and prediction of the degree of inhibition by antimycin.

Antimycin A↗
PubMed1998

Mutations in the membrane anchor of yeast cytochrome c1 compensate for the absence of Oxa1p and generate carbonate-extractable forms of cytochrome c1.

Oxa1p is a mitochondrial inner membrane protein that is mainly required for the insertion/assembly of complex IV and ATP synthase and is functionally conserved in yeasts, humans, and plants. We have isolated several independent suppressors that compensate for the absence of Oxa1p. Molecular cloning and sequencing reveal that the suppressor mutations (CYT1-1 to -6) correspond to amino acid substitutions that are all located in the membrane anchor of cytochrome c1 and decrease the hydrophobicity of this anchor. Cytochrome c1 is a catalytic subunit of complex III, but the CYT1-1 mutation does not seem to affect the electron transfer activity. The double-mutant cyt1-1,164, which has a drastically reduced electron transfer activity, still retains the suppressor activity. Altogether, these results suggest that the suppressor function of cytochrome c1 is independent of its electron transfer activity. In addition to the membrane-bound cytochrome c1, carbonate-extractable forms accumulate in all the suppressor strains. We propose that these carbonate-extractable forms of cytochrome c1 are responsible for the suppressor function by preventing the degradation of the respiratory complex subunits that occur in the absence of Oxa1p.

Adenosine Triphosphatases↗
PubMed2006

Differential inhibitory effects of methylmalonic acid on respiratory chain complex activities in rat tissues.

Methylmalonic acidemia is an inherited metabolic disorder biochemically characterized by tissue accumulation of methylmalonic acid (MMA) and clinically by progressive neurological deterioration and kidney failure, whose pathophysiology is so far poorly established. Previous studies have shown that MMA inhibits complex II of the respiratory chain in rat cerebral cortex, although no inhibition of complexes I-V was found in bovine heart. Therefore, in the present study we investigated the in vitro effect of 2.5mM MMA on the activity of complexes I-III, II, II-III and IV in striatum, hippocampus, heart, liver and kidney homogenates from young rats. We observed that MMA caused a significant inhibition of complex II activity in striatum and hippocampus (15-20%) at low concentrations of succinate in the medium, but not in the peripheral tissues. We also verified that the inhibitory property of MMA only occurred after exposing brain homogenates for at least 10 min with the acid, suggesting that this inhibition was mediated by indirect mechanisms. Simultaneous preincubation with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME) and catalase (CAT) plus superoxide dismutase (SOD) did not prevent MMA-induced inhibition of complex II, suggesting that common reactive oxygen (superoxide, hydrogen peroxide and hydroxyl radical) and nitric (nitric oxide) species were not involved in this effect. In addition, complex II-III (20-35%) was also inhibited by MMA in all tissues tested, and complex I-III only in the kidney (53%) and liver (38%). In contrast, complex IV activity was not changed by MMA in all tissues studied. These results indicate that MMA differentially affects the activity of the respiratory chain pending on the tissues studied, being striatum and hippocampus more vulnerable to its effect. In case our in vitro data are confirmed in vivo in tissues from methylmalonic acidemic patients, it is feasible that that the present findings may be related to the pathophysiology of the tissue damage characteristic of these patients.

Animals↗
PubMed1994

Mitochondrial respiratory failure in skeletal muscle from patients with Parkinson's disease and multiple system atrophy.

We studied mitochondrial respiratory chain function in skeletal muscle taken from 27 patients with idiopathic Parkinson's disease (PD; 21 Dopa-treated PD patients and 6 de novo patients), 5 patients with multiple system atrophy (MSA) and from 43 age-matched controls in order to determine the occurrence of mitochondrial respiratory chain abnormalities in parkinsonian syndromes. In our control subjects, we found a significant age-related decrease in the activity of respiratory chain complex I. As compared to carefully age-matched control subjects, activity of complex (NADH:ubiquinone reductase) was significantly lower in muscle mitochondria from patients with PD and MSA and a mean remaining activity < 30% of controls was observed. Mean activities of complexes III (ubiquinol:cytochrome c reductase) and IV (cytochrome c oxidase) were also lower in PD patients than controls, but a low activity (remaining activity < 30% of controls) was observed in only 5 PD patients for complex I and III or I and IV. No deficit in complex II activity (succinate:ubiquinone reductase) was observed. Our results support the hypothesis of a wide-spread mitochondrial complex I deficiency in PD and MSA as compared to age-matched controls, who showed age-related deficiency. This deficit can be found in de novo PD patients as well as in treated patients. The observed respiratory enzyme chain deficiency could not be explained by the dose and duration of L-Dopa or dopaminergic agonist treatment, the severity of the disease, anxiety or depression since no significant correlation was found between these parameters and enzyme complexes activities.

Adult↗
PubMed2006

Physiological diversity of mitochondrial oxidative phosphorylation.

To investigate the physiological diversity in the regulation and control of mitochondrial oxidative phosphorylation, we determined the composition and functional features of the respiratory chain in muscle, heart, liver, kidney, and brain. First, we observed important variations in mitochondrial content and infrastructure via electron micrographs of the different tissue sections. Analyses of respiratory chain enzyme content by Western blot also showed large differences between tissues, in good correlation with the expression level of mitochondrial transcription factor A and the activity of citrate synthase. On the isolated mitochondria, we observed a conserved molar ratio between the respiratory chain complexes and a variable stoichiometry for coenzyme Q and cytochrome c, with typical values of [1-1.5]:[30-135]:[3]:[9-35]:[6.5-7.5] for complex II:coenzyme Q:complex III:cytochrome c:complex IV in the different tissues. The functional analysis revealed important differences in maximal velocities of respiratory chain complexes, with higher values in heart. However, calculation of the catalytic constants showed that brain contained the more active enzyme complexes. Hence, our study demonstrates that, in tissues, oxidative phosphorylation capacity is highly variable and diverse, as determined by different combinations of 1) the mitochondrial content, 2) the amount of respiratory chain complexes, and 3) their intrinsic activity. In all tissues, there was a large excess of enzyme capacity and intermediate substrate concentration, compared with what is required for state 3 respiration. To conclude, we submitted our data to a principal component analysis that revealed three groups of tissues: muscle and heart, brain, and liver and kidney.

Animals↗
PubMed2004

Focused proteomics: monoclonal antibody-based isolation of the oxidative phosphorylation machinery and detection of phosphoproteins using a fluorescent phosphoprotein gel stain.

We have raised monoclonal antibodies capable of immunocapturing all five complexes involved in oxidative phosphorylation for evaluating their post-translational modifications. Complex I (NADH dehydrogenase), complex II (succinate dehydrogenase), complex III (cytochrome c reductase), complex IV (cytochrome c oxidase), and complex V (F1F0 ATP synthase) from bovine heart mitochondria were obtained in good yield from small amounts of tissue in more than 90% purity in one step. The composition and purity of the complexes was evaluated by Western blotting using monoclonal antibodies against individual subunits of the five complexes. In this first study, the phosphorylation state of the proteins without inducing phosphorylation or dephosphorylation was identified by using the novel Pro-Q Diamond phosphoprotein gel stain. The major phosphorylated components were the same as described before in sucrose gradient enriched complexes. In addition a few additional potential phosphoproteins were observed. Since the described monoclonal antibodies show cross reactivity to human proteins, this procedure will be a fast and efficient way of studying post-translational modifications in control and patient samples using only small amounts of tissue.

Animals↗
PubMed2003

Polarographic evaluation of mitochondrial enzymes activity in isolated mitochondria and in permeabilized human muscle cells with inherited mitochondrial defects.

Inherited disturbances of the mitochondrial energy generating system represent a heterogeneous group of disorders associated with a broad spectrum of metabolic abnormalities and clinical symptoms. We used the polarographic and spectrophotometric method for detection of mitochondrial disorders, because these two techniques provide a different insight into mitochondrial function. In six patients suspected of mitochondrial disease we found defects of complex I (two patients), complex III (one patient), complex IV (two patients) and a combination of defect of complex III and IV (one patient). Citrate synthase activity, used as the reference enzyme, was not changed. A comparison of the two methods showed several differences in evaluation of mitochondrial enzymes activity due to the fact that both methods used different conditions for enzyme activity measurements. In contrast to oxygen consumption measurements, where the function of the whole-integrated respiratory chain is characterized, spectrophotometric measurements characterize activities of isolated complexes in disintegrated membranes. However, it may be concluded from our experiments that both methods provide useful and complementary data about mitochondrial energetic functions. Whereas spectrophotometric data are suitable for evaluation of maximal enzyme activities of mitochondrial enzyme complexes, polarographic data provide better information about enzyme activities in cells with mitochondrial defects under in situ conditions.

Adolescent↗