Effect of astrocytic nitric oxide production on neuronal mitochondrial activity.
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Determining and understanding the control of flux. An illustration in submitochondrial particles of how to validate schemes of metabolic control.
Two complementary methods were used to determine how the rate of respiration and that of ATP hydrolysis were controlled in rat liver submitochondrial particles. In the first, 'direct control analysis' method, respiration was titrated with malonate, antimycin or cyanide at 20, 30 and 37 degrees C, to determine the flux control exerted by succinate dehydrogenase, cytochrome bc1 complex and cytochrome c oxidase, respectively. Together, the three respiratory complexes only controlled the flux by about 50%, leaving the other 50% of flux control to the H+ leak. In the second, 'elasticity based' method, the elasticity coefficients of the respiratory chain or the H+-ATPase and the H+ leak towards the H+ gradient were determined. Then, the flux control coefficients were calculated using the connectivity and summation laws of metabolic control theory. The correspondence between the flux control coefficients determined in the two ways validated the two methods. This allowed us to use the second method to analyse what was the kinetic origin of the observed distribution of control. Control of ATP hydrolysis by the ATPase decreased with increasing ATPase activity; hence, the control exerted by the H+ leak increased with increasing ATPase activity, due to a diminishing elasticity towards the H+ gradient. Reverse electron transport was mainly controlled by the ATPase; the sum of flux control coefficients of succinate dehydrogenase, NADH-CoQ oxidoreductase, and H+-ATPase yielded a value greater than one, indicating that the H+ leak exerted a significant negative control on this pathway.
Adenosine Triphosphatases↗
Astrocyte-derived nitric oxide causes both reversible and irreversible damage to the neuronal mitochondrial respiratory chain.
Cytokine-stimulated astrocytes produce nitric oxide (NO), which, along with its metabolite peroxynitrite (ONOO(-)), can inhibit components of the mitochondrial respiratory chain. We used astrocytes as a source of NO/ONOO(-) and monitored the effects on neurons in coculture. We previously demonstrated that astrocytic NO/ONOO(-) causes significant damage to the activities of complexes II/III and IV of neighbouring neurons after a 24-h coculture. Under these conditions, no neuronal death was observed. Using polytetrafluoroethane filters, which are permeable to gases such as NO but impermeable to NO derivatives, we have now demonstrated that astrocyte-derived NO is responsible for the damage observed in our coculture system. Expanding on these observations, we have now shown that 24 h after removal of NO-producing astrocytes, neurons exhibit complete recovery of complex II/III and IV activities. Furthermore, extending the period of exposure of neurons to NO-producing astrocytes does not cause further damage to the neuronal mitochondrial respiratory chain. However, whereas the activity of complex II/III recovers with time, the damage to complex IV caused by a 48-h coculture with NO-producing astrocytes is irreversible. Therefore, it appears that neurons can recover from short-term damage to mitochondrial complex II/III and IV, whereas exposure to astrocytic-derived NO for longer periods causes permanent damage to neuronal complex IV.
Animals↗
Mitochondrial respiration and triiodothyronine concentration in liver from postpubertal and adult rats.
The purpose of this study was to investigate the decline in rat liver mitochondria respiration found in adult rats compared to younger ones, and to find a link between this respiratory impairment and a tissue hypothyroidism state. To this end, hepatic concentration and serum levels of triiodothyronine were measured in postpubertal rats (60 days old) and adult rats (180 days old). In addition, in these rats we measured oxidative phosphorylation in homogenate together with coupled and uncoupled respiration in isolated mitochondria using succinate or durohydroquinone as substrate. We found that mitochondria from adult rats consumed less oxygen compared to younger rats due to lower electron transport chain and phosphorylating system activity. In addition, we found that in state 4 condition, mitochondria from adult rats consumed less oxygen than mitochondria from young rats. Finally, we found a decrease in liver triiodothyronine concentration in adult rats. In conclusion, the results of this study show that hepatic mitochondria in adult rats have a decreased ATP synthesis capacity and proton permeability, both consistent with the tissue hypothyroidism found in the liver of adult rats.
Adenosine Triphosphate↗
Coordinated patterns of gene expression for substrate and energy metabolism in skeletal muscle of diabetic mice.
Metabolic abnormalities underlying diabetes are primarily the result of the lack of adequate insulin action and the associated changes in protein phosphorylation and gene expression. To define the full set of alterations in gene expression in skeletal muscle caused by diabetes and the loss of insulin action, we have used Affymetrix oligonucleotide microarrays and streptozotocin-diabetic mice. Of the genes studied, 235 were identified as changed in diabetes, with 129 genes up-regulated and 106 down-regulated. Analysis revealed a coordinated regulation at key steps in glucose and lipid metabolism, mitochondrial electron transport, transcriptional regulation, and protein trafficking. mRNAs for all of the enzymes of the fatty acid beta-oxidation pathway were increased, whereas those for GLUT4, hexokinase II, the E1 component of the pyruvate dehydrogenase complex, and subunits of all four complexes of the mitochondrial electron transport chain were all coordinately down-regulated. Only about half of the alterations in gene expression in diabetic mice could be corrected toward normal after 3 days of insulin treatment and euglycemia. These data point to as of yet undefined mechanisms for highly coordinated regulation of gene expression by insulin and potential new targets for therapy of diabetes mellitus.
Adenosine Triphosphatases↗
Role of positively charged transmembrane segments in the insertion and assembly of mitochondrial inner-membrane proteins.
The biogenesis of membrane oligomeric complexes is an intricate process that requires the insertion and assembly of transmembrane (TM) domains into the lipid bilayer. The Oxa1p family plays a key role in this process in organelles and bacteria. Hell et al. (2001, EMBO J., 20, 1281-1288) recently have proposed that Oxa1p could act as part of a general membrane insertion machinery for mitochondrial respiratory complex subunits. We have previously shown that mutations in the TM domain of Cyt1p can partially compensate for the absence of Oxa1p. Here, we demonstrate that a single amino acid substitution in the TM domain of Qcr9p can bypass Oxa1p in yeast. Qcr9p and Cyt1p are two subunits of the respiratory complex bc1 and their relative roles in the assembly of other respiratory complexes have been investigated. The mutations we have isolated in Cyt1p or Qcr9p introduce positively charged amino acids, and we show that the mutant TM domain of Cyt1p mediates the restoration of complex assembly. We propose that the positive charges introduced in Cyt1p and Qcr9p TM domains promote interactions with negatively charged TM domains of other respiratory complex subunits, allowing the coinsertion of both domains into the membrane, in the absence of Oxa1p. This model argues in favor of a role of Oxa1p in the insertion and the lateral exit of less hydrophobic TM domains from the translocation site into the lipid bilayer.
Amino Acid Substitution↗
Oxidation process of bovine heart ubiquinol-cytochrome c reductase as studied by stopped-flow rapid-scan spectrophotometry and simulations based on the mechanistic Q cycle model.
Stopped-flow rapid-scan spectrophotometry was employed to study complicated oxidation processes of ubiquinol-cytochrome c reductase (QCR) that was purified from bovine heart mitochondria and maximally contained 0.36 mol of ubiquinone-10/mol of heme c1. When fully reduced QCR was allowed to react with dioxygen in the presence of cytochrome c plus cytochrome c oxidase, the oxidation of b-type hemes accompanied an initial lag, apparently low potential heme bL was oxidized first, followed by high potential heme bH. Antimycin A inhibited the oxidation of both b-type hemes. The oxidation of heme c1 was triphasic and became biphasic in the presence of antimycin A. On the other hand, starting from partially reduced QCR that was poised at a higher redox potential with succinate and succinate-cytochrome c reductase, the b-type hemes were oxidized immediately without a lag. When the ubiquinone content in QCR was as low as 0.1 mol/mol heme c1 the oxidation of the b-type hemes was almost suppressed. As the Q-deficient QCR was supplemented with ubiquinol-2, the rapid oxidation of b-type hemes was restored to some extent. These results indicate that a limited amount of ubiquinone-10 found in purified preparations of QCR is obligatory for electron transfer from the b-type hemes to iron-sulfur protein (ISP) and heme c1. The characteristic oxidation profiles of heme bL, heme bH, and heme c1 were simulated successfully based on a mechanistic Q cycle model. According to the simulations the two-electron oxidation of ubiquinol-10 via the ISP and heme c1 pathway, which is more favorable thermodynamically than the bifurcation of electron flow into both ISP and heme bL, does really occur as long as heme bL is in the reduced state and provides ubiquinone-10 at center i. Mechanistically this process takes time, thus explaining the initial lag in the oxidation of the b-type hemes. With the partially reduced QCR, inherent ubisemiquinone at center i immediately oxidizes reduced heme bH thus eliminating the lag. The mechanistic Q cycle model consists of 56 reaction species, which are interconnected by the reaction paths specified with microscopic rate constants. The simulations further indicate that the rate constants for electron transfer between the redox centers can be from 10(5) to 10(3) s-1 and are rarely rate-limiting. On the other hand, a shuttle of ubiquinone or ubiquinol between center o and center i and the oxidation of heme c1 can be rate-limiting. The interplay of the microscopic rate constants determines the actual reaction pathway that is shown schematically by the "reaction map." Most significantly, the simulations support the consecutive oxidation of ubiquinol in center o as long as both heme bL and heme bH are in the reduced state. Only when heme bL is oxidized and ISP is reduced can SQo donate an electron to heme bL. Thus, we propose that a kinetic control mechanism, or "a kinetic switch," is significant for the bifurcation of electron flow.
Animals↗
Threshold effect and tissue specificity. Implication for mitochondrial cytopathies.
Mitochondrial cytopathies present a tissue specificity characterized by the fact that even if a mitochondrial DNA mutation is present in all tissues, only some will be affected and induce a pathology. Several mechanisms have been proposed to explain this phenomenon such as the appearance of a sporadic mutation in a given stem cell during embryogenesis or mitotic segregation, giving different degrees of heteroplasmy in tissues. However, these mechanisms cannot be the only ones involved in tissue specificity. In this paper, we propose an additional mechanism contributing to tissue specificity. It is based on the metabolic expression of the defect in oxidative phosphorylation (OXPHOS) complexes that can present a biochemical threshold. The value of this threshold for a given OXPHOS complex can vary according to the tissue; thus different tissues will display different sensitivities to a defect in an OXPHOS complex. To verify this hypothesis and to illustrate the pathological consequences of the variation in biochemical thresholds, we studied their values for seven OXPHOS complexes in mitochondria isolated from five different rat tissues. Two types of behavior in the threshold curves can be distinguished corresponding to two modes of OXPHOS response to a deficiency. We propose a classification of tissues according to their type of OXPHOS response to a complex deficiency and therefore to their threshold values.
Animals↗
The cytochrome bc1 and cytochrome c oxidase complexes associate to form a single supracomplex in yeast mitochondria.
The mitochondrial electron transport chain complexes are large multisubunit complexes embedded in the inner membrane. We report here that in the yeast Saccharomyces cerevisiae, the cytochrome bc(1) and cytochrome c oxidase complexes co-exist as a larger complex of approximately 1000 kDa in the mitochondrial membrane. Following solubilization with a mild detergent, the cytochrome bc(1)-cytochrome c oxidase complex remains stable. It was analyzed using the techniques of gel filtration and blue native-polyacrylamide gel electrophoresis. Direct physical association of subunits of the cytochrome bc(1) complex with those of the cytochrome c oxidase complex was verified by co-immunoprecipitation analysis. Our data indicate that the cytochrome bc(1) complex is exclusively in association with the cytochrome c oxidase complex in yeast mitochondria. We term this complex the cytochrome bc(1)-cytochrome c oxidase supracomplex.
Electron Transport↗
Purification of a cytochrome bc-aa3 supercomplex with quinol oxidase activity from Corynebacterium glutamicum. Identification of a fourth subunity of cytochrome aa3 oxidase and mutational analysis of diheme cytochrome c1.
The aerobic respiratory chain of the Gram-positive Corynebacterium glutamicum involves a bc(1) complex with a diheme cytochrome c(1) and a cytochrome aa(3) oxidase but no additional c-type cytochromes. Here we show that the two enzymes form a supercomplex, because affinity chromatography of either strep-tagged cytochrome b (QcrB) or strep-tagged subunit I (CtaD) of cytochrome aa(3) always resulted in the copurification of the subunits of the bc(1) complex (QcrA, QcrB, QcrC) and the aa(3) complex (CtaD, CtaC, CtaE). The isolated bc(1)-aa(3) supercomplexes had quinol oxidase activity, indicating functional electron transfer between cytochrome c(1) and the Cu(A) center of cytochrome aa(3). Besides the known bc(1) and aa(3) subunits, few additional proteins were copurified, one of which (CtaF) was identified as a fourth subunit of cytochrome aa(3). If either of the two CXXCH motifs for covalent heme attachment in cytochrome c(1) was changed to SXXSH, the resulting mutants showed severe growth defects, had no detectable c-type cytochrome, and their cytochrome b level was strongly reduced. This indicates that the attachment of both heme groups to apo-cytochrome c(1) is not only required for the activity but also for the assembly and/or stability of the bc(1) complex.
Amino Acid Sequence↗
The iron-sulfur cluster of the Rieske iron-sulfur protein functions as a proton-exiting gate in the cytochrome bc(1) complex.
The destruction of the Rieske iron-sulfur cluster ([2Fe-2S]) in the bc(1) complex by hematoporphyrin-promoted photoinactivation resulted in the complex becoming proton-permeable. To study further the role of this [2Fe-2S] cluster in proton translocation of the bc(1) complex, Rhodobacter sphaeroides mutants expressing His-tagged cytochrome bc(1) complexes with mutations at the histidine ligands of the [2Fe-2S] cluster were generated and characterized. These mutants lacked the [2Fe-2S] cluster and possessed no bc(1) activity. When the mutant complex was co-inlaid in phospholipid vesicles with intact bovine mitochondrial bc(1) complex or cytochrome c oxidase, the proton ejection, normally observed in intact reductase or oxidase vesicles during the oxidation of their corresponding substrates, disappeared. This indicated the creation of a proton-leaking channel in the mutant complex, whose [2Fe-2S] cluster was lacking. Insertion of the bc(1) complex lacking the head domain of the Rieske iron-sulfur protein, removed by thermolysin digestion, into PL vesicles together with mitochondrial bc(1) complex also rendered the vesicles proton-permeable. Addition of the excess purified head domain of the Rieske iron-sulfur protein partially restored the proton-pumping activity. These results indicated that elimination of the [2Fe-2S] cluster in mutant bc(1) complexes opened up an otherwise closed proton channel within the bc(1) complex. It was speculated that in the normal catalytic cycle of the bc(1) complex, the [2Fe-2S] cluster may function as a proton-exiting gate.
Animals↗
The effect of dietary lipid changes on the fatty acid composition and function of liver, heart and brain mitochondria in the rat at different ages.
A correlation between dietary lipids and cellular enzyme activities is a problem that has only been partially addressed by nutritionists. Therefore, changes in the fatty acid composition and the activities of some key metabolic enzymes (ubiquinol-2-cytochrome c reductase (EC 1.10.2.2), cytochrome oxidase (EC 1.9.3.1) and ATPase (EC 3.6.1.3)) in the mitochondria of liver, heart and brain of rats fed on diets differing extensively in their polyunsaturated fatty acid compositions have been investigated. The results showed that fatty acid compositional changes brought about by the dietary differences were associated with extensive changes in the activities of these key enzymes in the mitochondria. The extent of the influence differed considerably with the period over which the diets were fed. The role of dietary lipids to effect changes through the preservation of membrane structural integrity is discussed.
Adenosine Triphosphatases↗
Characterization of mitochondrial respiratory chain energetics in the vestibular nucleus complex.
CONCLUSIONS: Despite having very high neuronal firing rates, the VNC does not have unusually high mitochondrial activity in vitro. This study is the first in which functionally active mitochondria from the hindbrain have been isolated and characterized. OBJECTIVE: Neurons in the vestibular nucleus complex (VNC) have exceptionally high spontaneous firing rates. Neuronal mitochondria generate adenosine triphosphate critical for maintaining the membrane potentials required for axon firing. We therefore hypothesized a high rate of mitochondrial activity in the VNC. MATERIAL AND METHODS: To test this hypothesis, we compared mitochondrial activity in the VNC with mitochondrial activity from another area of the hindbrain, the cerebellum. Mitochondrial respiratory activity was assessed by measuring oxidative phosphorylation and mitochondrial respiratory enzyme complex activity. RESULTS: Assay results were not significantly different in the VNC compared to those obtained with the cerebellum or with rat brain mitochondria in previous studies.
Adenosine Triphosphate↗
Mitochondrial complex I, II/III, and IV activities in familial and sporadic Parkinson's disease.
A possible role of mitochondrial respiratory chain dysfunction in the pathogenesis of sporadic Parkinson's disease (PD) has been described. There are only a few reports concerning mitochondrial involvement in familial Parkinson's disease. The present study investigated mitochondrial complex I-IV activity in patients with sporadic and familial PD, compared to controls. Platelets were isolated from venous blood and platelet mitochondria were obtained through sonication and differential centrifugation. Complex I, II/III, and IV activities were measured in 17 patients with family history of Parkinson's disease (PDF), 15 patients with sporadic Parkinson disease (PDS), and 17 age-matched, healthy controls. The mitochondrial enzyme activities did not differ significantly between patient groups and controls. In addition, there was no correlation between mitochondrial complex activities and age, severity of disease, or age at onset of disease in the patient groups. In this study, the data indicate no significant differences in mitochondrial complex I-IV activities in PDF and PDS.
Aged↗
Absence of mitochondrial dysfunction in polymyalgia rheumatica. Evidence based on a simultaneous molecular and biochemical approach.
OBJECTIVE: To investigate the molecular and biochemical profile of skeletal muscle mitochondria of patients with isolated polymyalgia rheumatica (PMR). PATIENTS AND METHODS: We included patients with a recent diagnosis of PMR and as control healthy individuals submitted to orthopedic surgery. Skeletal muscle was obtained from quadriceps, thus was mitochondria immediately isolated. Long polymerase chain reaction and Southern blot transference were performed to detect deleted mtDNA molecules. Mitochondrial oxidative activity using different substrates and individual enzyme activity of respiratory chain complexes were assessed to search for any biochemical dysfunction. RESULTS: Fifty-one individuals (PMR=25, controls=26) were included. Mean age was 72 (11) years; 45% were females. We found no significant increase of deleted mtDNA molecules in PMR patients compared to controls. Both groups differed neither on oxygen consumption (p=NS for all substrates) nor enzymatic activity (p=NS for all complexes). CONCLUSIONS: Skeletal muscle mitochondria are molecularly and biochemically unaffected in PMR.
Adenosine Triphosphatases↗
Effects of ubiquinone enriched diet on deterioration of mitochondrial respiratory function caused by fried beef derived mutagenic factor in rats.
2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP), a potent mutagenic agent produced during thermal processing of meats, shows detrimental effects on mitochondrial respiratory function. Effects of dietary ubiquinone-10 was examined on PhIP-induced mitochondrial respiratory dysfunction in rats. Rats fed with 0.2% ubiquinone-10 diet mitigated the PhIP-induced decrease in mitochondrial complex I activities observed in rat heart, diaphragm, and psoai major. Therefore, dietary anti-oxidants such as ubiquinone-10 could cancel out the unfavorable effects of PhIP and contribute to the preservation of mitochondrial function.
Animals↗
A high rate (20%-30%) of parental consanguinity in cytochrome-oxidase deficiency.
By studying a large series of 157 patients, we found that complex I (33%), complex IV (28%), and complex I+IV (28%) deficiencies were the most common causes of respiratory chain (RC) defects in childhood. Truncal hypotonia (36%), antenatal (20%) and postnatal (31%) growth retardation, cardiomyopathy (24%), encephalopathy (20%), and liver failure (20%) were the main clinical features in our series. No correlation between the type of RC defect and the clinical presentation was noted, but complex I and complex I+IV deficiencies were significantly more frequent in cases of cardiomyopathy (P<.01) and hepatic failure (P<.05), respectively. The sex ratio (male/female) in our entire series was mostly balanced but was skewed toward males being affected with complex I deficiency (sex ratio R=1.68). Interestingly, a high rate of parental consanguinity was observed in complex IV (20%) and complex I+IV (28%) deficiencies. When parental consanguinity was related to geographic origin, an even higher rate of inbreeding was observed in North African families (76%, P<.01). This study gives strong support to the view that an autosomal recessive mode of inheritance is involved in most cases of mitochondrial disorders in childhood, a feature that is particularly relevant to genetic counseling for this devastating condition.
Abnormalities, Multiple↗
Robust cluster analysis of microarray gene expression data with the number of clusters determined biologically.
MOTIVATION: The success of each method of cluster analysis depends on how well its underlying model describes the patterns of expression. Outlier-resistant and distribution-insensitive clustering of genes are robust against violations of model assumptions. RESULTS: A measure of dissimilarity that combines advantages of the Euclidean distance and the correlation coefficient is introduced. The measure can be made robust using a rank order correlation coefficient. A robust graphical method of summarizing the results of cluster analysis and a biological method of determining the number of clusters are also presented. These methods are applied to a public data set, showing that rank-based methods perform better than log-based methods. AVAILABILITY: Software is available from http://www.davidbickel.com.
Algorithms↗