Component Of Mitochondrial Respiratory Chain Research Articles

Newborn neurons acquire high levels of reactive oxygen species and increased mitochondrial proteins upon differentiation from progenitors

A population of embryonic rat cortical cells cultured in the presence of FGF2 and having neuronal morphology expressed higher levels of reactive oxygen species (ROS) than did progenitor cells, astrocytes, and several cell lines of neuronal and non-neuronal origin. ROS were assessed using 5-(and-6)-chlormethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H 2DCF-DA), and high levels persisted in the presence of antioxidants or lowered levels of ambient oxygen. Greater than 95% of high ROS-producing cells, isolated by fluorescence-activated cell sorting, expressed the neuronal marker β III tubulin. These cells did not incorporate BrdU or express nestin, unlike low ROS-producing cells, 99% of which exhibited both of these characteristics. Upon growth factor removal, low ROS-expressing cells differentiated into neurons and astrocytes and these neurons expressed high levels of ROS, indicating that ROS accumulation accompanies the differentiation of progenitors into neurons. ROS levels were decreased by added superoxide dismutase and catalase, suggesting that both superoxide and hydrogen peroxide contribute to the ROS signal. High ROS-expressing cells also contained higher levels of several mitochondrial respiratory chain components. Although ROS have been associated with conditions that lead to cell death, our results and recent studies on the role of ROS as regulators of signal pathways are consistent with the possibility that ROS play a role in the development of the neuronal phenotype. Moreover, the differential production of ROS provides a useful method to isolate from mixed populations cells that are highly enriched for either progenitor cells or neurons.

Mitochondrial complex I mutations in Caenorhabditis elegans produce cytochrome c oxidase deficiency, oxidative stress and vitamin-responsive lactic acidosis.

Mitochondrial dysfunction, with an estimated incidence of 1 in 10 000 live births, is among the most common genetically determined conditions. Missense mutations in the human NDUFV1 gene, which encodes the 51 kDa active site subunit of the NADH-ubiquinone oxidoreductase or complex I, can lead to severe neurological disorders. Owing to the rare and often sporadic nature of mitochondrial disorders, the mechanisms of pathogenesis of most mutations remain poorly understood. We have generated transgenic strains of Caenorhabditis elegans that express disease-causing mutations in the nuo-1 gene, the C. elegans homolog of the NDUFV1 gene. The transgenic strains demonstrate hallmark features of complex I dysfunction such as lactic acidosis and decreased NADH-dependent mitochondrial respiration. They are also hypersensitive to exogenous oxidative stress, suggesting that cellular defense mechanisms against reactive oxygen species are already taxed by an endogenous stress. The lactic acidosis induced by the NDUFV1 mutations could be partially corrected with the vitamins riboflavin and thiamine or with sodium dichloroacetate, an activator of the pyruvate dehydrogenase complex, resulting in significant increases in animal fitness. Surprisingly, cytochrome c oxidase activity and protein levels were reduced, establishing a connection between complexes I and IV. Our results indicate that complex I mutations exert their pathogenic effects in multiple ways: by impeding the metabolism of NADH, by increasing the production of reactive oxygen species, and by interfering with the function or assembly of other mitochondrial respiratory chain components.

Feeding fried oil changes antioxidant and fatty acid pattern of rat and affects rat liver mitochondrial respiratory chain components.

Fat frying is a popular food preparation method but several components like antioxidant vitamins could be lost due to oxidation and some others with toxic effects could appear. Because of such large consumption of frying oils, the effect of high temperatures on the oils is of major concern both for product quality and nutrition, taking into account that dietary fat source deeply influences several biochemical parameters, especially of mitochondrial membranes. Virgin olive oil possesses specific features for modulating the damages occurred by endogenous and exogenous oxidative stress being particularly rich in antioxidant molecules. We evaluated the extent of modifications suffered by virgin olive oil following a short-time deep fat frying procedure: vitamin E and phenolic compound as well as total antioxidant capacity (measured by ESR) decreased, while polar compounds increased. The intake of such an altered oil mainly affected the hydroperoxide and TBARS contents of mitochondrial membranes which were enhanced after the dietary treatments. Also, several mitochondrial respiratory chain components (Coenzyme Q, cytochrome b, c + c1, and a + a3) were affected.

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Ethanol, oxidative stress, reactive aldehydes, and the fetus.

The fetotoxic effects of maternal ethanol (E) consumption have been documented for over two decades, yet the mechanisms underlying this devastating phenomenon remain uncertain. The wide variety of cellular/biochemical effects of E on fetal tissues is itself a puzzle and strongly suggests that fetotoxic responses to E reflect a multifactorial setting. Many of these responses can be conceptually connected to effects on membrane structure and function. Representative of this, are studies in our laboratory documenting E effects on fetal cell replication, membrane transport systems, membrane fluidity, Na+-K+ pump expression, and EGF receptor expression. Recent studies have provided evidence that oxidative stress may be one mechanism by which E produces these membrane-related events. We initially observed E-induced oxidative stress in cultured fetal rat hepatocytes, the latter exhibiting morphological and biochemical signs of mitochondrial damage. E increased H2O2, O2-, lipid peroxidation products, along with signs of membrane damage. Supplementation with antioxidants or agents that enhance glutathione stores reversed these effects. E was found to inhibit activities of mitochondrial respiratory chain components (a potential source of the enhanced levels of H2O2, and O2-) and this could be reversed by antioxidant treatment. Subsequent studies have documented oxidative stress and membrane lipid peroxidation in fetal brain and liver (gestation day 19) following a two day maternal E consumption and in gestation day 14 and 17 "embryos" immediately following a single dose of E to the pregnant dam. The means by which E can induce oxidative stress in fetal cells is under investigation. We have examined effects of E on activities of key antioxidant enzymes and found no depressant responses. However, the low levels of antioxidants in fetal tissues and an exaggerated response of fetal mitochondria to prooxidant stimulation in vitro, suggest that fetal cells are strongly predisposed to oxidative stress. Additionally, recent studies have suggested that fetal tissues are likewise prone to the formation and subsequent accumulation of at least one toxic lipid peroxidation product, 4-hydroxynonenal. We conclude that maternal E consumption induces oxidative stress in fetal tissues and that this is responsible for some toxic responses to E. Additionally, the low antioxidant defenses in fetal tissues and accumulation of toxic aldehyde products of lipid peroxidation predispose the fetus to oxidative damage.

Changes in Mitochondrial Respiratory Chain Components of Petunia Cells during Culture in the Presence of Antimycin A.

When petunia (Petunia hybrida Vilm, cv Rosy Morn) cells are cultured in the presence of 2 [mu]M antimycin A (AA), respiration proceeds mainly via the cyanide-resistant pathway. Cyanide-resistant respiratory rates were higher in mitochondria from AA cells than in control mitochondria. Compared with control cells, an increase in alternative oxidase protein was observed in AA cells, as well as an increase in ubiquinone (UQ) content. A change in the kinetics of succinate dehydrogenase was observed: there was a much higher activity at high UQ reduction in mitochondria from AA cells compared with control mitochondria. No changes were found for external NADH dehydrogenase kinetics. In AA cells in vivo, UQ reduction was only slightly higher than in control cells, indicating that increased electron transport via the alternative pathway can prevent high UQ reduction levels. Moreover, O2 consumption continues at a similar rate as in control cells, preventing O2 danger. These adaptations to stress conditions, in which the cytochrome pathway is restricted, apparently require, in addition to an increase in alternative oxidase protein, a new setup of the relative amounts and/or kinetic parameters of all of the separate components of the respiratory network.

Nitric oxide-mediated inhibition of the mitochondrial respiratory chain in cultured astrocytes.

The Ca(2+)-independent form of nitric oxide synthase was induced in rat neonatal astrocytes in primary culture by incubation with lipopolysaccharide (1 microgram/ml) plus interferon-gamma (100 U/ml), and the activities of the mitochondrial respiratory chain components were assessed. Incubation for 18 h produced 25% inhibition of cytochrome c oxidase activity. NADH-ubiquinone-1 reductase (complex I) and succinate-cytochrome c reductase (complex II-III) activities were not affected. Prolonged incubation for 36 h gave rise to a 56% reduction of cytochrome c oxidase activity and a 35% reduction in succinate-cytochrome c reductase activity, but NADH-ubiquinone-1 reductase activity was unchanged. Citrate synthase activity was not affected by any of these conditions. The inhibition of the activities of these mitochondrial respiratory chain complexes was prevented by incubation in the presence of the specific nitric oxide synthase inhibitor NG-monomethyl-L-arginine. The lipopolysaccharide/interferon-gamma treatment of the astrocytes produced an increase in glycolysis and lactate formation. These results suggest that inhibition of the mitochondrial respiratory chain after induction of astrocytic nitric oxide synthase may represent a mechanism for nitric oxide-mediated neurotoxicity.

A computer-assisted rapid-scanning spectrophotometer with applications to tissues in vitro and in vivo

A flexible approach is described to acquire, store, retrieve, and manipulate the large volume of data provided by a rapid-scanning spectrophotometer designed for biological preparations. Data can be displayed in spectral and kinetic formats. This spectrophotometer provides a means to simultaneously measure the concentrations of components of light scattering preparations and to record chemical reactions as they occur. Although application of this instrument for the study of reduction/oxidation shifts of mitochondrial respiratory chain components is emphasized, the desing is suitable for many spectrophotometric applications.

Oxygen insufficiency during hypoxic hypoxia in rat brain cortex

To relate cerebral oxygen sufficiency and insufficiency with arterial tension, reduction/oxidation responses of the initial and terminal members of the mitochondrial respiratory chain (NADH and cytochrome c oxidase) were recorded in situ by optical techniques when increased cerebral energy use was provoked by direct electrical stimulation. Small decreases in p aO 2, produced by forced ventilation of hypoxic gas mixtures, resulted in decreased amplitude of the characteristic negative shift in extracellular potential which accompanies such stimulation and smaller oxidative response of NADH and cytochrome oxidase. When p aO 2 fell below 40–50 Torr, stimulation produced reductive rather than oxidative shifts of the mitochondrial respiratory chain components. The data suggest that hen arterial oxygen tension is slightly decreased, compensating mechanisms allow brain function to continue with minimal changes in cortical metabolites and high energy phosphate compounds. When arterial oxygen falls below 40–50 Torr, however, compensation can no longer prevent tissue hypoxia during times of increased energy demand. Thus, hypoxemia is not necessarily synonymous with oxygen insufficiency, but oxygen sufficiency must be defined with due regard to the degree of energy demand.

The cerebral oxidative metabolic response to acute ethanol administration in rats and cats

Ethanol effects on mitochondrial respiratory chain components in intact cerebral cortex of cats and rats are reported. Changes in redox levels were monitored by non-invasive optical techniques (microfluorometry for intramitochondrial NADH: dual wavelength reflection spectrophotometry for cytochrome a, a 3). Acute ethanol injections were accompanied by progressive increases in the level of reduced cytochrome a, a 3, a response consistent with the cytochrome response to other depressants such as phenobarbital and chlorpromazine. Low ethanol doses (below approx. 1 g/kg) were accompanied by increased levels of reduced NAD and it appeared that the ethanol effect in vivo is due to a decrease in tissue activity common to central depressants. Higher ethanol doses (1–3 g/kg) produced increased levels of oxidized NAD whereas phenobarbital and chlorpromazine each resulted in increased levels of reduced NAD. Since many of the effects of ethanol have been attributed to the activity of its primary metabolic product, acetaldehyde. the latter's effects on energy metabolism were also considered. Acetaldehyde at all effective doses produced similar changes in NAD (i.e. oxidation) and cytochrome a, a 3 (reduction) to those of the higher ethanol doses. The oxidation of NAD at higher ethanol doses and at all doses of acetaldehyde was interpreted as a unique effect of ethanol and due to acetaldehyde.