Glycerophosphate-dependent hydrogen peroxide production by rat liver mitochondria

We studied the extent to which hormonally-induced mitochondrial glycerophosphate dehydrogenase (mGPDH) activity contributes to the supply of reducing equivalents to the mitochondrial respiratory chain in the rat liver. The activity of glycerophosphate oxidase was compared with those of NADH oxidase and/or succinate oxidase. It was found that triiodothyronine-activated mGPDH represents almost the same capacity for the saturation of the respiratory chain as Complex II. Furthermore, the increase of mGPDH activity induced by triiodothyronine correlated with an increase of capacity for glycerophosphate-dependent hydrogen peroxide production. As a result of hormonal treatment, a 3-fold increase in glycerophosphate-dependent hydrogen peroxide production by liver mitochondria was detected by polarographic and luminometric measurements.     

Glycerophosphate-dependent hydrogen peroxide production by brown adipose tissue mitochondria and its activation by ferricyanide

Oxidation of glycerophosphate (GP) by brown adipose tissue mitochondria in the presence of antimycin A was found to be accompanied by significant production of hydrogen peroxide. GP-dependent hydrogen peroxide production could be detected by p-hydroxyphenylacetate fluorescence changes or as an antimycin A-insensitive oxygen consumption. One-electron acceptor, potassium ferricyanide, highly stimulated the rate of GP-dependent antimycin A-insensitive oxygen uptake, which was prevented by inhibitors of mitochondrial GP dehydrogenase (mGPDH) or by coenzyme Q(CoQ). GP-dependent ferricyanide-induced peroxide production was also determined luminometrically, using mitochondria or partially purified mGPDH. Ferricyanide-induced peroxide production was negligible, when succinate or NADH was used as a substrate. These results indicate that hydrogen peroxide is produced directly by mGPDH and reflect the differences in the transport of reducing equivalents from mGPDH and succinate dehydrogenase to the CoQ pool. The data suggest that more intensive production of reactive oxygen species may be present in mammalian cells with active mGPDH.     

Inhibition of lipid peroxidation by a novel compound (CV-2619) in brain mitochondria and mode of action of the inhibition

Lipid peroxidation in rat brain mitochondria was induced by NADH in the presence of ADP and FeCl3. CV-2619 inhibited the lipid peroxidation in a concentration-dependent manner; the concentration giving 50% inhibition (IC50) was 84 microM. In addition, the inhibitory effect of CV-2619 was strongly enhanced by adding substrates of mitochondrial respiration; when succinate, glutamate, or succinate plus glutamate was added, the IC50 of CV-2619 was changed to 1.1, 10, or 0.5 microM, respectively. Metabolites of CV-2619 also inhibited the lipid peroxidation. The inhibitory effect of CV-2619 on mitochondrial lipid peroxidation disappeared when TTFA, an inhibitor of complex II in mitochondrial respiratory chain, was added. The results indicate that in mitochondria CV-2619 is changed to its reduced form which inhibits lipid peroxidation.     

Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid

Propionic and methylmalonic acidemic patients have severe neurologic symptoms whose etiopathogeny is still obscure. Since increase of lactic acid is detected in the urine of these patients, especially during metabolic decompensation when high concentrations of methylmalonate (MMA) and propionate (PA) are produced, it is possible that cellular respiration may be impaired in these individuals. Therefore, we investigated the effects of MMA and PA (1, 2.5 and 5 mM), the principal metabolites which accumulate in these conditions, on the mitochondrial respiratory chain complex activities succinate: 2,6-dichloroindophenol (DCIP) oxireductase (complex II); succinate: cytochrome c oxireductase (complexII+CoQ+III); NADH: cytochrome c oxireductase (complex I+CoQ+complex III); and cytochrome c oxidase (COX) (complex IV) from cerebral cortex homogenates of young rats. The effect of MMA on ubiquinol: cytochrome c oxireductase (complex III) and NADH: ubiquinone oxireductase (complex I) activities was also tested. Control groups did not contain MMA and PA in the incubation medium. MMA significantly inhibited complex I+III (32–46%), complex I (61–72%), and complex II+III (15–26%), without affecting significantly the activities of complexes II, III and IV. However, by using 1 mM succinate in the assay instead of the usual 16 mM concentration, MMA was able to significantly inhibit complex II activity in the brain homogenates. In contrast, PA did not affect any of these mitochondrial enzyme activities. The effect of MMA and PA on succinate: phenazine oxireductase (soluble succinate dehydrogenase (SDH)) was also measured in mitochondrial preparations. The results showed significant inhibition of the soluble SDH activity by MMA (11–27%) in purified mitochondrial fractions. Thus, if the in vitro inhibition of the oxidative phosphorylation system is also expressed under in vivo conditions, a deficit of brain energy production might explain some of the neurological abnormalities found in patients with methylmalonic acidemia (MMAemia) and be responsible for the lactic acidemia/aciduria identified in some of them.     

Respiration of wheat roots during inhibition of phospholipase A2 by 4-bromphenacylbromide

Dependence of oxygen consumption by wheat root cells on the activity of phospholipase A2 (PLA2) was studied. The treatment of excised roots with 4-bromophenacile bromide (BPB), a specific inhibitor of PLA2, caused a decrease in the content of free fatty acids (FFA) and in oxygen consumption of root cells. The latter was prevented by exogenous application of a mixture of FFA. A similar inhibitory effect was caused by BPB after the activation of root respiration by 2,4-dinitrophenol (DNP). These data suggest that FFA may be involved in the regulation of respiration through the formation of succinate. This is supported by the fact of reduction of DNP-induced stimulation of oxygen consumption by malonate, known to be an inhibitor of succinate dehydrogenase, and by stimulation of respiration by exogenous application of succinate.     

The effect of substrate, ADP and uncoupler on the respiration of tomato pollen during incubation in vitro at moderately high temperature

Pollen of tomato cv. Supermarmande was collected from greenhouse-grown plants at various intervals throughout the year and arbitrarily classified as of high, medium or low respiratory activity on the basis of CO₂ production during 8 h incubation in vitro at 30°C, a temperature that is considered to be moderately high for tomato fruit set. After an initial burst of respiration during the first stage of hydration at 30°C (>1 h), the respiration rate of pollen of all three categories declined, the decrease being greater in the lots with a low or medium respiratory activity than in the high category. During hydration (10 min after the start of incubation), the addition of succinate or reduced β-nicotinamide adenine dinucleotide (NADH) to the substrate increased the respiratory rate of slowly-respiring pollen more than that of fast-respiring pollen, but carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and adenosine 5′-diphosphate (ADP) had less effect. After 1–4 h incubation, the respiration rate of the slow- or medium-respiring pollen lots had decreased, but was stimulated by succinate or NADH, and to a lesser degree by ADP. By 7 h, the respiration rate of all pollen lots had declined and was stimulated less by substrate, ADP or CCCP. The oxidation of NADH by tomato pollen contrasts with the failure of other pollen species to utilize this substrate; moreover, a synergistic effect of NADH and succinate was consistently observed. We conclude that the decline in respiration during incubation for up to 4 h at 30°C may reflect a lack of respiratory substrate. After 7 h, however, the decreased response to substrate indicates a loss of mitochondrial integrity or an accumulation of metabolic inhibitors. It is concluded that at 30°C (a moderately high temperature for tomato pollen), the initially high rate of respiration leads to exhaustion of the endogenous respiratory substrates (particularly in pollen with low to medium respiratory activity), but subsequently to ageing and a loss of mitochondrial activity.     

Metabolic consequences of the cytochrome c oxidase deficiency in brain of copper-deficient Mo(vbr) mice

Biochemical micromethods were used for the investigation of changes in mitochondrial oxidative phosphorylation associated with cytochrome c oxidase deficiency in brain cortex from Mo(vbr) (mottled viable brindled) mice, an animal model of Menkes' copper deficiency syndrome. Enzymatic analysis of cortex homogenates from Mo(vbr) mice showed an approximately twofold decrease in cytochrome c oxidase and a 1.4-fold decrease in NADH:cytochrome c reductase activities as compared with controls. Assessment of mitochondrial respiratory function was performed using digitonin-treated homogenates of the cortex, which exhibited the main characteristics of isolated brain mitochondria. Despite the substantial changes in respiratory chain enzyme activities, no significant differences were found in maximal pyruvate or succinate oxidation rates of brain cortex homogenates from Mo(vbr) and control mice. Inhibitor titrations were used to determine flux control coefficients of NADH:CoQ oxidoreductase and cytochrome c oxidase on the rate of mitochondrial respiration. Application of amobarbital to titrate the activity of NADH:CoQ oxidoreductase showed very similar flux control coefficients for control and mutant animals. Alternately, titration of respiration with azide revealed for Mo(vbr) mice significantly sharper inhibition curves than for controls, indicating a more than twofold elevated flux control coefficient of cytochrome c oxidase. Owing to the reserve capacity of respiratory chain enzymes, the reported changes in activities do not seem to affect whole-brain high-energy phosphates, as observed in a previous study using 31P NMR.     

The role of succinate in the respiratory chain of Trypanosoma brucei procyclic trypomastigotes.

Trypanosoma brucei procyclic trypomastigotes were made permeable by using digitonin (0-70 micrograms/mg of protein). This procedure allowed exposure of coupled mitochondria to different substrates. Only succinate and glycerol phosphate (but not NADH-dependent substrates) were capable of stimulating oxygen consumption. Fluorescence studies on intact cells indicated that addition of succinate stimulates NAD(P)H oxidation, contrary to what happens in mammalian mitochondria. Addition of malonate, an inhibitor of succinate dehydrogenase, stimulated NAD(P)H reduction. Malonate also inhibited intact-cell respiration and motility, both of which were restored by further addition of succinate. Experiments carried out with isolated mitochondrial membranes showed that, although the electron transfer from succinate to cytochrome c was inhibitable by antimycin, NADH-cytochrome c reductase was antimycin-insensitive. We postulate that the NADH-ubiquinone segment of the respiratory chain is replaced by NADH-fumarate reductase, which reoxidizes the mitochondrial NADH and in turn generates succinate for the respiratory chain. This hypothesis is further supported by the inhibitory effect on cell growth and respiration of 3-methoxyphenylacetic acid, an inhibitor of the NADH-fumarate reductase of T. brucei.     

Studies of the mitochondria from Eimeria tenella and inhibition of the electron transport by quinolone coccidiostats.

Intact but fragile mitochondria were isolated from unsporulated oocysts of Eimeria tenella. The mitochondria respired in response to succinate, malate plus pyruvate, and L-ascorbate at rates of 1.00, 0.40, and 0.25 mu1 O2/min/mg protein, respectively. Spectrophotometric analyses of the cytochromes in mitochondria and whole oocysts revealed b-type and o-type cytochromes, at roughly similar levels, but no cytochrome c could be detected. The mitochondrial respiration was inhibited by cyanide, azide, carbon monoxide, antimycin A, and 2-heptyl-4-hydroxyquinoline-N-oxide, but was relatively resistant to rotenone and amytal. The quinolone coccidiostats buquinolate, amquinate, methyl benzoquate, and decoquinate were identified as very powerful inhibitiors of succinate and malate plus pyruvate supported respiration in E. tenella mitochondria. None of these four drugs exhibited any inhibitory effect on chicken liver mitochondria. Only 3 pmol of the quinolones per mg mitochondrial protein was needed to achieve 50% inhibition. The inhibition could not be reversed by coenzymes Q6 or Q10. Since the quinolones did not affect L-ascorbate-supported respiration or the activities of submitochondrial succinate dehydrogenase and NADH dehydrogenase, the site of action of the quinolone coccidiostats was tentatively identified as probably near cytochrome b in E. tenella mitochondria. Mitochondria isolated from an E. tenella amquinate-resistant mutant were much less susceptible to quinolone coccidiostats; 50% inhibition was attained by 300 pmol of the drugs/mg mitochondrial protein. The results suggest that the mechanisms of action of quinolone coccidiostats is by inhibiting the cytochrome-mediated electron transport in the mitochondria of coccidia. 2-Hydroxynaphthoquinone coccidiostats were identified as inhibitors of mitochondrial respiration of both E. tenella and chicken liver. They inhibited submitochondrial succinate dehydrogenase and NADH dehydrogenase of E. tenella, and remained equally active against the mitochondrial function of E. tenella amquinolate-resistant mutant.     

Interaction of 3-[3H]-2-n-nonyl-4-hydroxy-quinoline-N-oxide with submitochondrial particles of beef heart. I. Inhibition of the respiratory activity

The inhibitory effect of 3-3H-2-n-nonyl-4-hydroxy-quinoline-N-oxide (3H-NQNO) on the respiration induced by NADH or by succinate was studied in submitochondrial particles from beef heart. Polarographic experiments showed that oxygen uptake by submitochondrial particles was inhibited by 3H-NQNO. Similar results were obtained with the unlabelled compound NQNO. The inhibitory activity of 3H-NQNO on the respiration was 2 or 3 times better than that of the heptyl derivatives (HQNO) and additive with that shown by antimycin.     

Saturation kinetics of coenzyme Q in NADH and succinate oxidation in beef heart mitochondria.

The saturation kinetics of NADH and succinate oxidation for Coenzyme Q (CoQ) has been re-investigated in pentane-extracted lyophilized beef heart mitochondria reconstituted with exogenous CoQ10. The apparent 'Km' for CoQ10 was one order of magnitude lower in succinate cytochrome c reductase than in NADH cytochrome c reductase. The Km value in NADH oxidation approaches the natural CoQ content of beef heart mitochondria, whereas that in succinate oxidation is close to the content of respiratory chain enzymes.     

Antimycin A- and hydroxamate-insensitive respiration in yeasts

In this paper evidence is presented for the mitochondrial localization of the antimycin A (AA) + salicylhydroxamate (SHAM)-insensitive respiration of the yeasts Kluyveromyces lactis, Endomycopsis capsularis and Hansenula saturnus. Such a respiration, which can be sustained by NADH and NADPH but not by succinate, is inhibited by high concentrations of azide. AA + SHAM-insensitive respiration is not phosphorylating and its postulated physiological role is to oxidize NADH.The research has been supported by a grant of Ministero della Publica Istruzione and of C.N.R.     

Coenzyme Q10 Protects Astrocytes from ROS-Induced Damage through Inhibition of Mitochondria-Mediated Cell Death Pathway

Coenzyme Q10 (CoQ10) acts by scavenging reactive oxygen species to protect neuronal cells against oxidative stress in neurodegenerative diseases. The present study was designed to examine whether CoQ10 was capable of protecting astrocytes from reactive oxygen species (ROS) mediated damage. For this purpose, ultraviolet B (UVB) irradiation was used as a tool to induce ROS stress to cultured astrocytes. The cells were treated with 10 and 25 μg/ml of CoQ10 for 3 or 24 h prior to the cells being exposed to UVB irradiation and maintained for 24 h post UVB exposure. Cell viability was assessed by MTT conversion assay. Mitochondrial respiration was assessed by respirometer. While superoxide production and mitochondrial membrane potential were measured using fluorescent probes, levels of cytochrome C (cyto-c), cleaved caspase-9, and caspase-8 were detected using Western blotting and/or immunocytochemistry. The results showed that UVB irradiation decreased cell viability and this damaging effect was associated with superoxide accumulation, mitochondrial membrane potential hyperpolarization, mitochondrial respiration suppression, cyto-c release, and the activation of both caspase-9 and -8. Treatment with CoQ10 at two different concentrations started 24 h before UVB exposure significantly increased the cell viability. The protective effect of CoQ10 was associated with reduction in superoxide, normalization of mitochondrial membrane potential, improvement of mitochondrial respiration, inhibition of cyto-c release, suppression of caspase-9. Furthermore, CoQ10 enhanced mitochondrial biogenesis. It is concluded that CoQ10 may protect astrocytes through suppression of oxidative stress, prevention of mitochondrial dysfunction, blockade of mitochondria-mediated cell death pathway, and enhancement of mitochondrial biogenesis.     

Action of halothane upon mitochondrial respiration

The inhibitory action of halothane upon respiration was studied with rat liver mitochondria (RLM³), beef heart mitochondria (HBHM), and electron-transport particles (ETP). With intact mitochondrial preparations the oxidation of NADH-linked substrates but not of succinate was markedly suppressed by low concentrations of halothane (<2 mm as determined by gas-liquid chromatography). This inhibitory action of halothane was completely reversible. In contrast, a number of other mitochondrial processes were found to be sensitive in an irreversible manner at higher concentrations of the anesthetic. Likewise, the oxidation of added NADH by HBHM, ETP, and detergent-disrupted RLM was found to be sensitive in a reversible manner to low concentrations of halothane. The energy-dependent transfer of electrons from succinate to NAD by ETP_H was also sensitive to halothane. On the other hand, the NADH-ferricyanide reductase and the succinic oxidase activities of ETP and the NADH-cytochrome c reductase activity of microsomes were all insensitive to halothane. The site of inhibition by halothane appears to be in the vicinity of the rotenone-sensitive site of complex I (NADH-CoQ reductase). A number of other general anesthetics inhibited respiration at or near the same site as halothane.     

Inhibition of mitochondrial respiration by phosphoenolpyruvate

The cytosolic factors that influence mitochondrial oxidative phosphorylation rates are relatively unknown. In this report, we examine the effects of phosphoenolpyruvate (PEP), a glycolytic intermediate, on mitochondrial function. It is reported here that in rat heart mitochondria, PEP delays the onset of state 3 respiration in mitochondria supplied with either NADH-linked substrates or succinate. However, the maximal rate of state 3 respiration is only inhibited when oxidative phosphorylation is supported by NADH-linked substrates. The capacity of PEP to delay and/or inhibit state 3 respiration is dependent upon the presence or absence of ATP. Inhibition of state 3 is exacerbated in uncoupled mitochondria, with a 40% decrease in respiration seen with 0.1mM PEP. In contrast, ATP added exogenously or produced by oxidative phosphorylation completely prevents PEP-mediated inhibition. Mechanistically, the results support the conclusion that the main effects of PEP are to impede ADP uptake and inhibit NADH oxidation. By altering the NADH/NAD(+) status of mitochondria, it is demonstrated that PEP enhances succinate dehydrogenase activity and increase free radical production. The results of this study indicate PEP may be an important modulator of mitochondrial function under conditions of decreased ATP.     

Loss of Sensitivity to Helminthosporium maydis Race T Toxin during Aging of Mitochondria Isolated from Texas Cytoplasm Corn

Helminthosporium maydis Race T toxin caused the expected changes in freshly isolated mitochondria from T cytoplasm corn, namely complete uncoupling of oxidative phosphorylation, pronounced stimulation of succinate and NADH respiration, complete inhibition of malate respiration, and increased mitochondrial swelling. In contrast, identical toxin treatments of the mitochondria after 12 hours aging on ice resulted in partial uncoupling, much lower stimulation of succinate and NADH respiration, no inhibition of malate respiration, and no mitochondrial swelling. Almost all of the toxin sensitivity was lost by 6 hours aging. At this stage, the mitochondria were 208× and 66× less sensitive to toxin-induced changes in coupling of malate respiration and state 4 malate respiration rates, respectively. Loss of toxin sensitivity did not occur when the mitochondria were aged under nitrogen or in the presence of 5 millimolar dithiothreitol. This suggested that the aging effect was due to oxidation, possibly of sulfhydryl groups in one or more mitochondrial membrane proteins.     

Alternative Oxidase Activity and the Ubiquinone Redox Level in Soybean Cotyledon and Arum Spadix Mitochondria during NADH and Succinate Oxidation

In Arum and soybean (Glycine max L.) mitochondria, the dependence of the alternative oxidase activity on the redox level of ubiquinone, with NADH and succinate as substrates, was studied, using a voltametric procedure to measure the ubiquinone redox poise in the mitochondrial membrane. The results showed that when the enzyme was activated by pyruvate the relationship between the alternative oxidase rate and the redox state of the ubiquinone pool was the same for both NADH and succinate oxidations. In the absence of pyruvate the alternative oxidase had an apparent lower affinity for ubiquinol. This was more marked with NADH than with succinate and was possibly due to pyruvate production during succinate oxidation or to an activation of the alternative oxidase by succinate itself. In Arum spadix (unlike soybean cotyledon) mitochondria, succinate oxidation via the alternative oxidase maintained the ubiquinone pool in a partially reduced state (60%), whereas NADH oxidation kept it almost completely reduced. Previous data comparing mitochondria from thermogenic and nonthermogenic tissues have not examined the full range of ubiquinone redox levels in both tissues, leading to the suggestion that the activity of alternative oxidase for Arum was different from nonthermogenic tissues. When the complete range of redox states of ubiquinone is used and the oxidase is fully activated, the alternative oxidase from thermogenic tissue (Arum) behaves similarly to that of nonthermogenic tissue (soybean).     

Relations Between Tocopherol Depletion and Coenzyme Q During Lipid Peroxidation in rat Liver Mitochondria

In order to evaluate different mitochondrial antioxidant systems, the depletion of alpha-tocopherol and the levels of the reduced and oxidized forms of CoQ were measured in rat liver mitochondria during Fe⁺⁺/ascorbate and NADPH/ADP/Fe⁺⁺ induced lipid peroxidation. During the induction phase of malondialdehyde formation, alpha-tocopherol declined moderately to about 80% of initial contents, whereas the total CoQ pool remained nearly unchanged, but reduced CoQ9 continuously declined. At the start of massive malondialdehyde formation, CoQ9 reaches its fully oxidized state. At the same time alpha-tocopherol starts to decline steeply, but never becomes fully exhausted in both experimental systems. Evidently the oxidation of the CoQ9 pool constitutes a prerequisite for the onset of massive lipid peroxidation in mitochondria and for the subsequent depletion of alpha-tocopherol. Trapping of the GSH by addition of dinitrochlorbenzene (a substrate of the GSH transferase), results in a moderate acceleration of lipid peroxidation, but alpha-tocopherol and ubiquinol levels remained unchanged when compared with the controls. Addition of succinate to GSH depleted mitochondria effectively suppressed MDA formation as well as alpha-tocopherol and ubiquinol depletion. The data support the assumption that the protective effect of respiratory substrates against lipid peroxidation in the absence of mitochondrial GSH is mediated by the regeneration of the lipid soluble antioxidants CoQ and alpha-tocopherol.     

MDH2 produced OAA is a metabolic switch rewiring the fuelling of respiratory chain and TCA cycle

The mitochondrial respiratory chain (RC) enables many metabolic processes by regenerating both mitochondrial and cytosolic NAD⁺ and ATP. The oxidation by the RC of the NADH metabolically produced in the cytosol involves redox shuttles as the malate-aspartate shuttle (MAS) and is of paramount importance for cell fate. However, the specific metabolic regulations allowing mitochondrial respiration to prioritize NADH oxidation in response to high NADH/NAD⁺ redox stress have not been elucidated. The recent discovery that complex I (NADH dehydrogenase), and not complex II (Succinate dehydrogenase), can assemble with other respiratory chain complexes to form functional entities called respirasomes, led to the assumption that this supramolecular organization would favour NADH oxidation. Unexpectedly, characterization of heart and liver mitochondria demonstrates that the RC systematically favours electrons provided by the ‘respirasome free’ complex II. Our results demonstrate that the preferential succinate driven respiration is tightly controlled by OAA levels, and that OAA feedback inhibition of complex II rewires RC fuelling increasing NADH oxidation capacity. This new regulatory mechanism synergistically increases RC's NADH oxidative capacity and rewires MDH2 driven anaplerosis of the TCA, preventing malate production from succinate to favour oxidation of cytosolic malate. This regulatory mechanism synergistically adjusts RC and TCA fuelling in response to extramitochondrial malate produced by the MAS.     

Skeletal muscle mitochondrial free-fatty-acid content and membrane potential sensitivity in different thyroid states: involvement of uncoupling protein-3 and adenine nucleotide translocase

The effect of triiodothyronine (T3) on mitochondrial efficiency could be related to an increase in the concentrations of some proteins, such as uncoupling proteins (UCPs). Free fatty acids (FFA) seem to be a cofactor essential for the uncoupling activity of UCP3. In this paper, we report that the hypothyroidism-hyperthyroidism transition is accompanied by increases: (i) in the endogenous levels of mitochondrial FFA and (ii) in the sensitivity to FFA shown by the mitochondrial respiration rate and membrane potential, which correlated with the level of UCP3 protein. The level of the mRNA for adenine-nucleotide translocase-1 (ANT) was not affected by the thyroid state, while the ANT contribution to FFA-induced changes in mitochondrial uncoupling was low in the hypothyroid and euthyroid states but became more relevant in the hyperthyroid state at the highest concentration of FFA.