Heart and breast muscle mitochondrial dysfunction in pulmonary hypertension syndrome in broilers (Gallus domesticus)

This study was conducted to determine function and defects in electron transport in muscle mitochondria of meat chickens (broilers) with pulmonary hypertension syndrome (PHS). The respiratory control ratio (RCR, indicative of respiratory chain coupling) was higher in the control than in PHS breast and heart muscle mitochondria, but there were no differences in the ADP/O (an index of oxidative phosphorylation). Sequential additions of ADP improved the RCR in the control breast muscle mitochondria and the ADP/O in PHS breast and heart muscle mitochondria. Basal hydrogen peroxide production, (an indicator of electron leak), was higher in PHS breast and heart muscle mitochondria than in controls and differences in electron leak in PHS mitochondria were magnified by inhibiting electron transport at Complex I and III (cyt b(562)). Complex I activity was lower in PHS heart mitochondria but there was no difference in Complex II activity. Thus, compared to controls, PHS mitochondria exhibited site-specific defects in electron transport within Complex I and III that could contribute to lower respiratory chain coupling. Additionally, it appears that healthy broilers may exhibit higher basal levels of electron leak compared to other avian species. Together, these findings provide insight into inefficient cellular use of oxygen that may contribute to the development of PHS in broilers.     

Dietary inorganic nitrate improves mitochondrial efficiency in humans

Nitrate, an inorganic anion abundant in vegetables, is converted in vivo to bioactive nitrogen oxides including NO. We recently demonstrated that dietary nitrate reduces oxygen cost during physical exercise, but the mechanism remains unknown. In a double-blind crossover trial we studied the effects of a dietary intervention with inorganic nitrate on basal mitochondrial function and whole-body oxygen consumption in healthy volunteers. Skeletal muscle mitochondria harvested after nitrate supplementation displayed an improvement in oxidative phosphorylation efficiency (P/O ratio) and a decrease in state 4 respiration with and without atractyloside and respiration without adenylates. The improved mitochondrial P/O ratio correlated to the reduction in oxygen cost during exercise. Mechanistically, nitrate reduced the expression of ATP/ADP translocase, a protein involved in proton conductance. We conclude that dietary nitrate has profound effects on basal mitochondrial function. These findings may have implications for exercise physiology- and lifestyle-related disorders that involve dysfunctional mitochondria.     

Delta sleep inducing peptide (DSIP): effect on respiration activity in rat brain mitochondria and stress protective potency under experimental hypoxia

Neuromodulatory delta sleep inducing peptide (DSIP) seems to be implicated in the attenuation of stress-induced pathological metabolic disturbances in various animal species and human beings. Mitochondria, as cell organelles, are considered especially sensitive to stress conditions. In this work, the influence of DSIP and Deltaran((R))-a recently developed product based upon DSIP-on processes of oxidative phosphorylation and ATP production in rat brain mitochondria and rat brain homogenates was studied. A polarographic measurement of oxygen consumption was applied to evaluate the impact of DSIP on maximal rates of mitochondrial respiration and coupling of respiration to ATP production. We provide evidence that DSIP affected the efficiency of oxidative phosphorylation on isolated rat brain mitochondria. This peptide significantly increased the rate of phosphorylated respiration V3, while the rate of uncoupled respiration V(DNP) remaining unchanged. It enhanced the respiratory control ratio RCR and the rate of ADP phosphorylation. DSIP and Deltaran exhibited the same action in rat brain homogenates. We also examined the influence of DSIP under hypoxia when mitochondrial respiratory activity is altered. In rats subjected to hypoxia, we detected a significant stress-mediated reduction of V3 and ADP/t values. Pretreatment of rats with DSIP at the dose of 120 microgram/kg (i.p.) prior to their subjection to hypoxia completely inhibited hypoxia-induced reduction of mitochondrial respiratory activity. The revealed capacity of DSIP to enhance the efficiency of oxidative phosphorylation found in vitro experiments could contribute to understanding pronounced stress protective and antioxidant action of this peptide in vivo.     

Effects of paraquat on mitochondria of rat skeletal muscle

The effect of the herbicide paraquat (N,N'-dimethyl 4,4'-bipyridium), known to damage the lipid cellular membrane by peroxidation with superoxide radicals and a singlet oxygen, was investigated on skeletal muscle mitochondria. Minced rat gastrocnemius muscles were incubated in 8 mM paraquat solution. Mitochondrial fractions prepared from the incubated muscles were examined with respect to respiratory function and the enzyme activity of cytochrome c oxidase and succinate-cytochrome c reductase in the electron transport chain. The ADP/O ratio, RCR, and state 3 rates (= oxygen consumption in state 3) decreased gradually. State 4 rates (= oxygen consumption in state 4) increased in the initial stages and decreased after longer incubations. Enzyme activities gradually increased. These results suggested that paraquat damaged the mitochondrial membrane and disrupted oxidative phosphorylation in the early stage of incubation. Also, the electron transport chain was accelerated in the earlier stage and broken following a longer incubation. The inhibitory modality of paraquat on mitochondrial respiration was shown to be different from that of other known inhibitors.     

Effects of 1,4-dihydropyridine derivatives (cerebrocrast, gammapyrone, glutapyrone, and diethone) on mitochondrial bioenergetics and oxidative stress: a comparative study

The potential protective action of 1,4-dihydropyridine derivatives (cerebrocrast, gammapyrone, glutapyrone, and diethone) against oxidative stress was assessed on mitochondrial bioenergetics, inner membrane anion channel (IMAC), Ca2+-induced opening of the permeability transition pore (PTP), and oxidative damage induced by the oxidant pair adenosine diphosphate (ADP)/Fe2+ (lipid peroxidation) of mitochondria isolated from rat liver. By using succinate as the respiratory substrate, respiratory control ratio (RCR), ADP to oxygen ratio (ADP/O), state 3, state 4, and uncoupled respiration rates were not significantly affected by gammapyrone, glutapyrone, and diethone concentrations up to 100 microM. Cerebrocrast at concentrations higher than 25 microM depressed RCR, ADP/O, state 3, and uncoupled respiration rates, but increased three times state 4 respiration rate. The transmembrane potential (deltapsi) and the phosphate carrier rate were also decreased. At concentrations lower than 25 microM, cerebrocrast inhibited the mitochondrial IMAC and partially prevented Ca2+-induced opening of the mitochondrial PTP, whereas gammapyrone, glutapyrone, and diethone were without effect. Cerebrocrast, gammapyrone, and glutapyrone concentrations up to 100 microM did not affect ADP/Fe2+-induced lipid peroxidation of rat liver mitochondria, while very low diethone concentrations (up to 5 microM) inhibited it in a dose-dependent manner, as measured by oxygen consumption and thiobarbituric acid reactive substances formation. Diethone also prevented deltapsi dissipation due to lipid peroxidation initiated by ADP/Fe2+. It can be concluded that: none of the compounds interfere with mitochondrial bioenergetics at concentrations lower than 25 microM; cerebrocrast was the only compound that affected mitochondrial bioenergetics, but only for concentrations higher than 25 microM; at concentrations that did not affect mitochondrial bioenergetics (< or = 25 microM), only cerebrocrast inhibited the IMAC and partially prevented Ca2+-induced opening of the PTP; diethone was the only compound that expressed antioxidant activity at very low concentrations (< or = 5 microM). Cerebrocrast acting as an inhibitor of the IMAC and diethone acting as an antioxidant could provide effective protective roles in preventing mitochondria from oxidative damage, favoring their therapeutic interest in the treatment of several pathological situations known to be associated with cellular oxidative stress.     

Age-associated deficit of mitochondrial oxidative phosphorylation in skeletal muscle: Role of carnitine and lipoic acid

Mitochondrial damage has implicated a major contributor for ageing process. In the present study, we measured mitochondrial membrane swelling, mitochondrial respiration (state 3 and 4) by using oxygen electrode in skeletal muscle of young (3–4 months old) and aged rats (above 24 months old) with supplementation of l-carnitine and dl-α-lipoic acid. Our results shows that the mitochondrial membrane swelling and state 4 respiration were increased more in skeletal muscle mitochondria of aged rats than in young control rats, whereas the state 3 respiration, respiratory control ratio (RCR) and ADP:O ratio decreased more in aged rats than in young rats. After supplementation of carnitine and lipoic acid to aged rats for 30 days, the state 3 respiration and RCR were increased, whereas the state 4 and mitochondrial membrane swelling were decreased to near normal rats. From our results, we conclude that combined supplementation of carnitine and lipoic acids to aged rats increases the skeletal muscle mitochondrial respiration, thereby increasing the level of ATP. (Mol Cell Biochem xxx: 83–89, 2005)     

The effect of low concentrations of sodium nitrites and nitrates on respiration and oxidative phosphorylation in rat liver mitochondria]

Nitrite incubation in mitochondria and nitrate intoxication of rats have been studied for their effect on aerobic energetic processes in the liver. Sodium nitrite in concentration of 2 mg/l causes an inhibition of ADP-stimulated respiration and provides uncoupling processes of oxidative phosphorylation and respiration in mitochondria, when adding succinate as a substrate. Low doses of nitrate in vivo promote oxygen economization in mitochondria. Intoxication of rats with nitrate in a dose of 50 mg/l for 30 days induces a decrease of the respiration rate after ADP-phosphorylation and an increase of the coefficient of oxidative phosphorylation efficiency (ADP/O). Intraperitoneal administration of adrenalin in a dose of 25 micrograms/100 g to rats after 30-day nitrate intoxication in a concentration of 10 mg/l induces no typical increase of ADP-stimulated respiration and rate of oxidative phosphorylation and succinate oxidation.     

Control of skeletal muscle mitochondria respiration by adenine nucleotides: differential effect of ADP and ATP according to muscle contractile type in pigs

Skeletal muscle exhibits considerable variation in mitochondrial content among fiber types, but it is less clear whether mitochondria from different fiber types also present specific functional and regulatory properties. The present experiment was undertaken on ten 170-day-old pigs to compare functional properties and control of respiration by adenine nucleotides in mitochondria isolated from predominantly slow-twitch (Rhomboideus (RM)) and fast-twitch (Longissimus (LM)) muscles. Mitochondrial ATP synthesis, respiratory control ratio (RCR) and ADP-stimulated respiration with either complex I or II substrates were significantly higher (25-30%, P<0.05) in RM than in LM mitochondria, whereas no difference was observed for basal respiration. Based on mitochondrial enzyme activities (cytochrome c oxidase [COX], F0F1-ATPase, mitochondrial creatine kinase [mi-CK]), the higher ADP-stimulated respiration rate of RM mitochondria appeared mainly related to a higher maximal oxidative capacity, without any difference in the maximal phosphorylation potential. Mitochondrial K(m) for ADP was similar in RM (4.4+/-0.9 microM) and LM (5.9+/-1.2 microM) muscles (P>0.05) but the inhibitory effect of ATP was more marked in LM (P<0.01). These findings demonstrate that the regulation of mitochondrial respiration by ATP differs according to muscle contractile type and that absolute muscle oxidative capacity not only relies on mitochondrial density but also on mitochondrial functioning per se.     

Altered Mitochondrial Function in Canine Ceroid-Lipofuscinosis

The neuronal ceroid-lipofuscinoses (NCL) are a group of autosomal recessively inherited neurodegenerative disorders characterized by progressive dementia, neuronal atrophy, and premature death. The late infantile and juvenile types of NCL show massive accumulation of mitochondrial ATP synthase subunit c protein in both mitochondria and lysosomes. The specific accumulation of this mitochondrial protein suggests that mitochondrial function may be impaired in the NCL diseases. Therefore, a study was conducted to determine whether oxidative phosphorylation is altered in liver mitochondria from English setters with NCL, an animal model in which there is also massive accumulation of the subunit c protein. The ADP/O ratios were significantly depressed in affected and carrier dogs, suggesting that the disease mutation led to a partial uncoupling of oxidative phosphorylation. On the other hand, ADP-stimulated respiration rates were higher than normal in both carriers and affected dogs. The increased respiration rates were highest in the carriers, and may reflect a compensatory response to the reduced efficiency of oxidative phosphorylation. Accompanying the increased respiration rates were elevations in mitochondrial ADP content with the elevation being greater in the carriers than in the affected dogs. This suggests that the increased respiration rates may be due, at least in part, to enhanced ADP uptake by the mitochondria. In the carriers, the enhanced respiration rate may be sufficient to offset the reduced efficiency of oxidative phosphorylation. In the affected animals, which had lower respiration rates than the carriers, the enhanced respiration rates may not be sufficient to offset the reduced efficiency of oxidative phosphorylation. Impaired mitochondrial function may therefore contribute to the disease pathology.     

An assessment of the role of proton leaks in the mechanistic stoichiometry of oxidative phosphorylation.

Rat liver mitochondria were incubated in the presence of varying concentrations of ATP, followed by ADP to initiate phosphorylation. Analysis of phosphorylation to oxygen ratios (P/O) was carried out with varied initial phosphorylation potentials (or ATP/ADP ratios). Rates of phosphorylation and respiration and magnitude of membrane potential (delta psi) were measured. The results are discussed in the framework of P/total O and P/"extra" O ratios in determination of the mechanistic P/O ratio. It is concluded that the former underestimates, and the latter overestimates the mechanistic P/O ratio.     

Effect of Low Temperature lmbibition on Mito chondrium Respiration and Phosphorylation of PEG Primed Soybean Seed

Cotyledon mitochondrium respiration and oxidative phosphorylation activity of PEG primed and unprimed (control) soybean seeds which have been exposed to low temperature imbibition before germination are studied. The ADP stimulated respiration rates of control mitochondria are evidently higher than state Ⅲ respiration rates of mitochondria from primed seed when L-Mal, α-Kg and Succ are used as substrates respectively. The mitochondria from the unprimed do not possess respiratory control (RC.) On the contrary, mitochondria from the primed, even after seeds being exposed to 2–3 ℃ imbibition for 24 h, phosphorylate normally. The ADP/O and RC values are consistent with those of theoretical expectation. When NADH is used as substrate, unprimed seed mitochondria still possess oxidative phosphorylation activity, while ADP/O and RC values are obviously lower than those of mitochondria from the primed. The emerging sequence of the activity of the diverse phosphorylation sites during germination is also studied. When a different substrate is used, the emerging sequence of the primed is as follows: 1. NADH (12 h), 2.α-Kg (24 h), 3. L-Mal and Succ (48 h). This corresponds to occurrence sequence of ADP stimulated respiration in control mitochondria. The above results show that low temperature imbibition has an irreversible destructive effect on oxidative phosphorylation activity of control mitochondria, and PEG priming has a protective effect on structure and function of the mitochondria under low temperature imbibition stress. The mechanism of soybean imbibitional chilling injury and protective effect of PEG priming are discussed.     

Nature of enhanced mitochondrial oxidative metabolism by a calf blood extract

The effect of calf blood extract (Solcoseryl, SS) on mitochondrial oxidative function in various states was studied polarographically in vitro. 1) Mitochondrial respiration in all 4 conventional study states (Estabrook, 1967) was enhanced by the addition of SS, including states 1 and 2 (endogenous substrates only). 2) The effect of SS on mitochondrial oxygen consumption was concentration dependent, while ADP/O ratio remained constant. The effect of added respiratory substrates varied with the particular substrate at optimally active concentrations. With suboptimal substrate levels, ADP/O ratios were concentration dependent, in contrast to the SS effect. Under oligomycin ATPase inhibition, SS was no longer active, in contrast to DNP, which remained active. 3) In states 3 (added ADP) and 4 (ADP exhausted), oxygen consumption and oxidative phosphorylation were enhanced by SS in the presence or absence of citrate, glutamate, pyruvate, lactate, or ascorbate. However, in the presence of succinate, SS had no effect. 4) ADP/O ratio was decreased by SS in the presence of added substrate, suggesting that SS activation of H(+)-ATPase enhances ATP hydrolysis as well as oxidative phosphorylation and ATP synthesis. 5) The enhancing effect of SS on mitochondrial function is due to hydrophilic components of SS. The lipidic components obtained by Folch fraction of SS have no effect. It is concluded that the effects of SS respiratory substrates and uncouplers on mitochondrial function are essentially different. SS enhances both ATP synthesis and oxygen consumption by mitochondria.     

Oxidative phosphorylation by pyridoxine-deficient rat brain mitochondria

Abstract— Oxygen uptake, ADP/O ratios and respiratory control ratios (RCR) were studied by the oxygen electrode technique in mitochondria prepared from adult and neonatal brains from normal and pyridoxine-deficient rats. The mitochondria from neonatal brain exhibited decreased rates of substrate oxidation, ADP/O ratios and respiratory control ratios in comparison to those obtained with mitochondria from the respective adult brains. The cytochrome contents of the neonatal brains were also less than those of the adults. Within the neonatal or adult groups, there were no differences in any of the parameters tested between the normal and pyridoxine-deficient rats.     

The effects of palmitic acid on skeletal muscle mitochondria of cold and warm acclimated rats.

The effects of palmitic acid on skeletal muscle mitochondria isolated from the hind limb muscle of cold and warm acclimated rats were studied. At higher concentrations of the fatty acid, a greater depression of both ADP/O and RCR (respiratory control ratio) was observed in the cold acclimated group. Initial ADP/O and RCC however, were higher in the cold acclimated group. The enhanced sensitivity of skeletal muscle mitochondria of the cold acclimated rat is discussed.     

Effect of exhaustive exercise on liver mitochondrial function in the rat

The oxidative and phosphorylative function of rat liver mitochondria after exhaustive exercise was investigated. The stimulation of state 4 respiration (without ADP) with NADH and FADH2 dependent substrates was demonstrated. The reduction in RCR ratio (the rate of oxidation in state 3/the rate of oxidation in state 4) and enhanced activity of oligomycin sensitive ATP-ase was also found. The results suggest an inhibition of liver mitochondrial phosphorylative activity in rats exercised till exhaustion.     

Respiratory control and ADP:O coupling ratios of isolated chick heart mitochondria

Heart mitochondria isolated from 14- to 21-day-old chicks are highly coupled and often have respiratory control ratio (RCR) values exceeding 100. This paper presents data from a study of some of the properties of these mitochondria. The studies show that: (a) The ADP:O ratios and the state 4 rates of respiration are highly dependent upon the concentration of mitochondria at which these parameters are measured. (b) The mitochondrial isolate is contaminated with at least two divalent cation-stimulated ATPase, of which one is the F1F0-ATPase of broken mitochondria. (c) The oligomycin-sensitive component of state 4 respiration is completely inhibited by ethylene glycol bis(beta-amino-ethylether) N,N'-tetraacetic acid (EGTA). This inhibition is biphasic and attributable to the differential affinity of EGTA for Ca(II) and Mg(II). (d) Ca(II) and Mg(II) stimulate state 4 respiration, thereby depressing RCR values. These cations also decrease ADP:O ratios from greater than or equal to 3.25 to 3.0 for some NAD-linked substrates. (e) Uncoupled (i.e., oligomycin-insensitive) state 4 respiration can be abolished by treating the mitochondria with Nagarse and by preincubating mitochondria with exogenous substrate. (f) The ADP:O ratios obtained when these heart mitochondria oxidize pyruvate/malate, alpha-ketoglutarate, and beta-hydroxybutyrate are fractional and significantly greater than 3.0.     

Identification and characterization of uncoupling protein in heart and muscle mitochondria of canary birds

An uncoupling protein (cUCP) was identified in heart and skeletal muscle mitochondria of canary birds. cUCP was immunodetected using polyclonal antibodies raised against murine UCP2. Its molecular mass was similar to those of mammalian UCPs (32 kDa). The activity of cUCP was stimulated by palmitic acid (PA) and inhibited by GTP mainly in state 3 respiration. Additions of PA augmented state 4 respiration and lowered the ADP/O ratio. Thus, the activity of cUCP diverted energy from oxidative phosphorylation in state 3 respiration. cUCP in heart and skeletal muscles of canary birds might have implications in thermogenesis as well as protection against free radical production.     

Simulation of state 4 --> state 3 transition in isolated mitochondria

The mathematical dynamic model of oxidative phosphorylation developed previously and in the accompanying paper was modified to involve isolated mitochondria conditions; it was also used to simulate state 4 --> state 3 transition in rat liver mitochondria incubated with succinate as respiratory substrate and glucose-hexokinase as an ADP-regenerating system. Changes in the respiration rate, protonmotive force and reduction level of ubiquinone and cytochrome c as well as the internal (i) and external (e) ATP/ADP ratio between state 4 and state 3 were calculated and compared with the experimental data. Flux control coefficients with respect to oxygen consumption flux for different reactions and processes of oxidative phosphorylation were simulated for different values of the respiration rate (state 4, state 3 and intermediate states). Flux control coefficients for the oxidation, phosphorylation and proton leak subsystems with respect to the oxidation, phosphorylation and proton leak fluxes for different values of the respiration rate were also calculated. These theoretical data were compared with the experimental results obtained in the frame of metabolic control analysis and the 'top-down' approach to this analysis. A good agreement was obtained. Simulated time courses of the respiration rate, the protonmotive force (Deltap) and other parameters after addition of a small amount of ADP to mitochondria in state 4 mimicked at least semiquantitatively the experimentally measured time courses of these parameters. It was concluded, therefore, that in the present stage, the model is able to reflect different properties of the oxidative phosphorylation system in a broad range of conditions fairly well, allows deeper insight into the mechanisms responsible for control and regulation of this process, and can be used for simulation of new experiments, thus inspiring experimental verification of the theoretical predictions.     

Proton leak induced by reactive oxygen species produced during in vitro anoxia/reoxygenation in rat skeletal muscle mitochondria

Superoxide anion generation and the impairment of oxidative phosphorylation yield were studied in rat skeletal muscle mitochondria submitted to anoxia/reoxygenationk in vitro. Production of superoxide anion was detected after several cycles of anoxia/reoxygenationk. Concomitantly, a decrease of state 3 respiration and phosphorylation yield (ADP/O) were observed. The latter resulted from a proton leak. The presence of palmitic acid during anoxia/reoxygenationk cycles led to a dose-dependent inhibition of superoxide anion production together with a partial protection of the ADP/O ratio measured after anoxia/reoxygenationk. The ADP/O decrease was shown to be due to a permeability transition pore-sustained proton leak, as it was suppressed by cyclosporine A. The permeability transition pore activation was induced during anoxia/reoxygenationk by superoxide anion, as it was cancelled by the spin trap (POBN), which scavenges superoxide anion and by palmitic acid, which induces mitochondrial uncoupling. It can be proposed that the palmitic acid-induced proton leak cancels the production of superoxide anion by mitochondria during anoxia/reoxygenationk and therefore prevents the occurrence of the superoxide anion-induced permeability transition pore-mediated proton leak after anoxia/reoxygenationk.     

Studies on the respiratory properties of mitochondria isolated from developing winter wheat seedlings

Mitochondria isolated from shoots of 2 days, light- and dark-grown winter wheat (Triticum aestivum L. cv. Rideau) seedlings oxidize alpha-ketoglutarate and l-malate with good respiratory control and ADP: O ratios. The efficiency of oxidative phosphorylation, and respiratory control are both reduced significantly when succinate or NADH is employed as substrate. Respiratory control values and ADP: O ratios show a general decline in mitochondria from seedlings of increasing age, whether grown in light or dark. In light-grown seedlings, the decrease in respiratory control with aging is due principally to a decrease in the rate of state 3 respiration, while in dark-grown material, the decrease appears to be due mainly to an increased rate of state 4 respiration. In both light- and dark-grown seedlings, oxygen consumption during state 3 respiration is severely inhibited by oligomycin. During state 4 respiration, 2,4-dinitrophenol stimulates oxygen uptake to a level approximately two-thirds the normal ADP-stimulated rate.