Changes of mitochondrial oxidation and phosphorylation in the testes of white rats under excessive supply of sodium nitrate into the organism

The functional state of mitochondria, obtained from the testicles, after chronic intoxication by sodium nitrate (in a dose of 200 mg/kg during 14; 30 and 90 days) was investigated in experiment on white rats. It was revealed, that in dynamics of changes of mitochondrial oxidation and phosphorylation in the testicles under the excessive supply of sodium nitrate into the organism the determined phases should be marked: on the 14th day of intoxication this is the ascending of rate of respiration, on the 30th and 90th day--separation of the mitochondrial oxidation and ADP phosphorylation. It is supposed, that in pathogenesis of these changes the essential role belongs to nitric oxide production.     

Rat liver mitochondria impairments under acute carbon tetrachloride-induced intoxication. Effects of melatonin

The aim of the present work was to investigate the mechanisms of oxidative damage of rat liver mitochondria in vitro, under hypochlorous acid (HOCl)-induced oxidative stress, and in vivo, under acute carbon tetrachloride-induced intoxication in rats. Hypochlorous acid (50–300 μM), the main inflammatory agent, inhibited liver mitochondria respiratory activity and caused uncoupling in the respiratory and phos-porylation processes. The toxic damage of rat liver after 24 h of acute carbon tetrachloride-induced intoxication (4 g/kg, intragastrically) was accompanied by a significant reduction in succinate- and glutamate-dependent respiration rate in state 3 (by 65%, p < 0.001, and by 50%, p < 0.01, respectively). The respiration control ratio approached 1, reflecting the loss of respiration control. The phosphorylation coefficient significantly decreased due to uncoupling of the oxidation and phosphorylation processes. The mitochondrial alterations were associated with oxidation of intramitochondrial GSH by 25% (p < 0.05), the marked inhibition of succinate dehydrogenase (complex II) by 35% (p < 0.05), and the rise of blood plasma nitric oxide level by 45% (p < 0.05). The impairment of mitochondrial respiratory function may result from the inhibition of enzymatic activities in the respiratory chain and the damage of mitochondrial membrane during intoxication and plays a key role in the development of the CCl₄-induced hepatotoxicity. Melatonin administration under CCl₄-induced intoxication (three times at a dose of 10 mg/kg) increased the rate of succinate oxidation in state 3 by 30% (p < 0.05) and reversed the increase in glutathione peroxidase activity. Melatonin prevented an elevation of nitric oxide level in the blood plasma of intoxicated animals but did not protect mitochondrial functions under acute intoxication.     

Effects of peginol C-2000 on respiration and oxidative phosphorylation in liver mitochondria of rats poisoned with butylcaptax]

The data on the effect of synthetic antioxidant Paginol C-2000 on respiration and oxidative phosphorylation in liver mitochondria of rats primed with butylcaptax are given. It is shown that intoxication of organism with butylcaptax in the dose of 1/10 LD50 during 5 days resulted in suppression of respiration and oxidative phosphorylation in rat liver mitochondria. It is found, that administration of Raginol C-2000 into organisms of primed animals leads to the reduction of succinate pathway of oxidation, that, in its turn, results in the normalization of functioning level of ATP-synthesizing system of mitochondria.     

Effect of L-arginine and N(omega)-nitro-L-arginine on oxidative phosphorylation and lipid peroxidation in rats with various tolerance to hypoxia under stressful conditions

The influence of L-arginine (600 mg/kg) and NO-synthase blocator N omega-nitro-L-arginine L-NNA (35 mg/kg) on processes of ADP-stimulated respiration (under using 0.35 mM succinate, 1 mM alpha-ketoglutarate, 2 mM pyruvate, 2 mM glutamate, 2 Mm malate and succinate dehydrohenase blocator--2 mM malonate as substrates of oxidation), lipid peroxidation (concentration of DK and MDA), activities of succinate dehydrohenase and aminotransferases in rats tissues with different resistance to hypoxia under stress conditions have been investigated. It have been shown that the energy metabolism indices (respiration rate and efficiency of phosphorilation ADP/O) are higher in high resistent (HR) animals in the control group. Stress causes the increase of ADP-stimulated respiration in low resistent (LR) under succinate oxidation and decrease of NADPH-dependent utilization, indicative of more effort of energy system in LR animals. Stress conditions are connected with the increase of lipid peroxidation products in blood both in LR and in HR animals, though in hepar their concentration change unimportantly. Injection of L-arginine decreases aerobic component of energy metabolism on the background decreasing aminotransferases ways of oxidation and succinate dehydrohenase activity. L-arginine causes decrease of lipid peroxidation products in LR, in HR the same effect reaches by L-NNA injection. The has been made conclusion about tight correlation between energy metabolism, processes of lipid peroxidation with resistance to hypoxia and functioning of nitric oxide cycle under stress conditions.     

Corrections by melatonin of liver mitochondrial disorders under diabetes and acute intoxication in rats

The aim of the present work was to investigate the mechanisms of oxidative damage of the liver mitochondria under diabetes and intoxication in rats as well as to evaluate the possibility of corrections of mitochondrial disorders by pharmacological doses of melatonin. The experimental (30 days) streptozotocin‐induced diabetes mellitus caused a significant damage of the respiratory activity in rat liver mitochondria. In the case of succinate as a respiratory substrate, the ADP‐stimulated respiration rate V₃ considerably decreased (by 25%, p < 0·05) as well as the acceptor control ratio (ACR) V₃/V₂ markedly diminished (by 25%, p < 0·01). We observed a decrease of the ADP‐stimulated respiration rate V₃ by 35% (p < 0·05), with glutamate as substrate. In this case, ACR also decreased (by 20%, p < 0·05). Surprisingly, the phosphorylation coefficient ADP/O did not change under diabetic liver damage. Acute rat carbon tetrachloride‐induced intoxication resulted in considerable decrease of the phosphorylation coefficient because of uncoupling of the oxidation and phosphorylation processes in the liver mitochondria. The melatonin administration during diabetes (10 mg·kg^‐1 body weight, 30 days, daily) showed a considerable protective effect on the liver mitochondrial function, reversing the decreased respiration rate V₃ and the diminished ACR to the control values both for succinate‐dependent respiration and for glutamate‐dependent respiration. The melatonin administration to intoxicated animals (10 mg·kg⁻¹ body weight, three times) partially increased the rate of succinate‐dependent respiration coupled with phosphorylation. The impairment of mitochondrial respiratory plays a key role in the development of liver injury under diabetes and intoxication. Melatonin might be considered as an effector that regulates the mitochondrial function under diabetes. Copyright © 2011 John Wiley & Sons, Ltd.     

Respiratory activity of isolated hepatocytes

Oligomycin and uncoupler of oxidative phosphorylation have been studied for their effect on the respiration activity of hepatocytes in rats. The respiration rate in the presence of oligomycin and uncoupler is higher than it is with the respiration uncoupled in the absence of oligomycin. Exogenic succinate makes endogenic respiration of hepatocytes in the presence of digitonin 5 times more intensive. The obtained results evidence for the fact that the uncoupled respiration is limited by the concentration of substrates able to be oxidized in the respiration chain of mitochondria. Oligomycin induces accumulation of substrates and following addition of the disconnector evokes their fast oxidation.     

Effect of Ca ions on the transmembrane electric potential, synthesis and hydrolysis of ATP in brain mitochondria

The transmembrane potential (delta psi) of rabbit brain mitochondria was measured with the fluorescent dye dis--C2--5. During oxidative phosphorylation a fall in delta psi in the order of 20% was observed. In the presence of inhibitors of ATP synthesis, there was a good correlation between the fall in delta psi and the ADP-stimulated increase in respiration rate. The influence of endogenous calcium on the energetic metabolism of mitochondria was studied by measuring the changes of delta psi. An amount of 12 nmol Ca2+/mg protein cause half-inhibition of the ATP synthesis rate; 50 nmol/mg completely inhibits oxidative phosphorylation. The effect of the Ca2+ load on the ATPase activity of intact mitochondria was studied. It was found that endogenous calcium inhibits in a similar degree synthesis and hydrolysis of ATP. It was shown that both Ca ATP and Mg ATP can serve as a substrate for the mitochondrial ATPase.     

Combined effect of propofol and GSNO on oxidative phosphorylation of isolated rat liver mitochondria.

Isolated rat liver mitochondria have been treated with the general anaesthetic propofol (2,6-diisopropylphenol, 200 microM) and the physiological NO donor nitrosoglutathione (GSNO, 200 or 250 microM). The efficiency of the oxidative phosphorylation has been evaluated by measuring the respiration and ATP synthesis rates and the behavior of transmembrane electrical potential. In mitochondria energized by succinate, the simultaneous presence of both propofol and GSNO gives rise to a synergic action in affecting the resting and the ADP-stimulated respiration, the respiratory control ratio, the ATP synthesis, and the formation and utilization of the electrochemical transmembrane potential.     

Studies of energy-linked reactions. Inhibition of oxidative phosphorylation by DL-8-methyldihydrolipoate.

1. DL-8-Methyldihydrolipoate was shown to be a potent inhibitor of mitochondrial oxidative phosphorylation and ATP-driven energy-linked reactions. 2. ADP-stimulated respiration utilizing pyruvate + malate and succinate in both ox heart and rat liver mitochondria is inhibited; oxidative phosphorylation using pyruvate + malate, succinate and ascorbate + NNN'N'-tetramethyl-p-phenylenediamine as substrates is also inhibited; uncoupler-stimulated respiration is unaffected regardless of the substrate used. 3. Mitochondrial oligomycin-sensitive adenosine triphosphatase is inhibited in both the membrane-bound form and the purified detergent-dispersed preparation. 4. ATP-driven transhydrogenase and the ATP-driven energy-linked reduction of NAD+ by succinate in ox heart submitochondrial particles are inhibited, whereas the respiratory-chain-driven transhydrogenase is unaffected. 5. DL-8-Methyl-lipoate has no immediate effect on the above reactions, demonstrating the requirement for the reduced form for inhibition. 6. The inhibitory properties of DL-8-methyldihydrolipoate are analogous to those of oligomycin and provide further evidence of a role for lipoic acid in oxidative phosphorylation.     

The possible role of palmitoyl-CoA in the regulation of the adenine nucleotides transport in mitochondria under different metabolic states. I. Comparison of liver mitochondria from starved and fed rats.

It has been shown that KM values for ADP when rat liver mitochondria oxidized succinate were strictly dependent on the values of the respiratory control ratios. The Ki values for palmitoyl-CoA inhibition of the ADP-stimulated succinate oxidation and the inhibition of the uncoupler-stimulated ATPase activity were equal to 0.5 muM. Mitochondria from livers of starved rats showed 30% inhibition of the state 3 respiratory rate (compared to the uncoupled respiratory rate) which was abolished by addition of carnitine. It was supposed that this inhibition was due to the influence of acyl-CoAs bound to the inner mitochondrial membrane on the adeninenucleotide translocase. Mitochondria from livers of fed rats showed a strong inhibition of succinate oxidation both in state 4 and state 3, although the rate of uncoupled respiration was normal. It was assumed that in this case the changes in mitochondrial behaviour was caused by the decrease in the concentration of ADP and ATP in the matrix space of mitochondria.     

Intermittent hypoxic training and L-arginine as corrective agents for myocardial energy supply under acute hypoxia

It NO has been shown play to the primary role in several mitochondrial functions. Our aim for this study was to investigate whether exogenous NO (L-arginine) or NO blocker L-NNA modulated the adaptive reactions of rat myocardial tissue respiration on intermittent hypoxic training (IHT). In the control rats an acute hypoxic test (inhalation of 7% O2, 30 min) provoked sharp augmentation of ADP-stimulated tissue respiration with the increase of respiratory coefficient and phosphorylation rate, the decrease of O2 uptake efficacy and switching the energy supply to succinate oxidation pathway. The same hypoxic test but following 14 days of IHT (11% O2, 15-min sessions with 15 min rest intervals, 5 times daily) produced a stimulation of oxidative phosphorylation with primary activation of NAD-dependent pathway, the marked increase of ADP/O ratio. The combination of IHT with L-arginine treatment (600 mg/kg intraperitoneally, daily before IHT sessions) provoked the decrease of tissue oxygen consumption in comparison with untrained animals. L-arginine effects abolished by the NO-synthase blocker L-NNA. Its effects on mitochondrial function deals with succinic acid inhibition utilizatin (increasing level ADP/O) and activation NADH-dependent oxidation. We conclude that the combination of IHT with NO-precursor treatment was capable to increase significantly the tolerance to episodes of acute hypoxia.     

Protective effect of ursodeoxycholic acid on liver mitochondrial function in rats with alloxan-induced diabetes: link with oxidative stress

We investigated the effects of ursodeoxycholic acid (UDCA) on mitochondrial functions and oxidative stress and evaluated their relationships in the livers of rats with alloxan-induced diabetes. Diabetes was induced in male Wistar rats by a single alloxan injection (150 mg kg^− 1 b.w., i.p.). UDCA (40 mg kg^− 1 b.w., i.g., 30 days) was administered from the 5th day after the alloxan treatment. Mitochondrial functions were evaluated by oxygen consumption with Clark oxygen electrode using succinate, pyruvate + malate or palmitoyl carnitine as substrates and by determination of succinate dehydrogenase and NADH dehydrogenase activities. Liver mitochondria were used to measure chemiluminiscence enhanced by luminol and lucigenin, reduced liver glutathione and the end-products of lipid peroxidation. The activities of both NADH dehydrogenase and succinate dehydrogenase as well as the respiratory control (RC) value with all the substrates and the ADP/O ratio with pyruvate + malate and succinate as substrates were significantly decreased in diabetic rats. UDCA developed the beneficial effect on the mitochondrial respiration and oxidative phosphorylation parameters in alloxan-treated rats, whereas the activities of mitochondrial enzymes were increased insignificantly after the administration of UDCA. The contents of polar carbonyls and MDA as well as the chemiluminescence with luminol were elevated in liver mitochondria of diabetic rats. The treatment with UDCA normalized all the above parameters measured except the MDA content. UDCA administration prevents mitochondrial dysfunction in rats treated with alloxan and this process is closely connected with inhibition of oxidative stress by this compound.     

Effect of tricarboxylic acid cycle intermediates on nitric oxide system during acute hypoxia

Effects Crebs Cycle of exogenous intermediates sodium succinate (50 mg/kg) and sodium alpha-ketoglutarate (200 mg/kg) on processes of mitochondrial ADP-stimulated respiration (using as substrates of oxidation 0.35 mM succinate, 1 mM alpha-ketoglutarate), production of nitric oxide under NO2-, NO3-, as well as carbamide, putrescyne content and processes of lipid peroxidation in the rats liver under acute hypoxia (7% O2 in N2, 30 min) have been studied. It was shown, that the exogenous sodium alpha-ketoglutarate increases nitric oxide content, aminotransferase activation, inhibition of succinatedehydrogenase simultaneously more than exogenous sodium succinate. It correlates with decreasing of processes lipid peroxidation in liver.     

Effect of ethanol intake on rat liver mitochondrial respiration and oxidative phosphorylation

The effect of ethanol intake on liver mitochondrial functions was investigated by feeding rats with a liquid isocaloric diet containing various concentrations of ethanol. We found that after feeding the liquid diet for 2 to 3 months, the body weight of rats did not show a significant difference between treated and control groups. However, the mitochondrial respiration rate decreased significantly with the increase of ethanol concentration in the diet. We found that when the rats were fed on 10.8% ethanol, the average succinate-supported State 3 respiration rate decreased from 54.5 to 44.8 nmol O2/min/mg and the glutamate-malate-supported State 3 respiration rate decreased from 38.8 to 23.6 nmol O2/min/mg as compared with the control. Interestingly, we noted that ethanol intake caused a more drastic effect on State 3 respiration than on State 4 respiration, irrespective of the substrate utilized by the mitochondria. In addition, the respiratory control and ADP/O ratios were found to decrease concomitantly with the increase of ethanol level in the diet. Moreover, we found that the effect of ethanol on both respiratory control and ADP/O ratios of liver mitochondria was more pronounced in glutamate-malate-supported respiration than succinate-supported respiration. These results clearly demonstrate that ethanol intake by the rat can cause impairment of liver mitochondrial respiration and oxidative phosphorylation, and that these effects are exerted through damage to mitochondrial membranes.     

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.     

Changes in the stimulation of mitochondrial respiration by adrenaline depending upon the dose

Effects of low and high 25 and 100 micrograms per 100 g of body weight doses of adrenaline on respiration and oxidative phosphorylation in rat liver mitochondria are compared. The high dose of adrenaline is shown to decrease activation of respiration and phosphorylation typical of the low doses. This decrease is caused by inhibition of succinate dehydrogenase and is accompanied by uncoupling of respiration and phosphorylation in mitochondria.     

Inhibition of oxidative phosphorylation by organotin thiocarbamates

A series of triphenyl-, tricyclohexyl- and tribenzyltin compounds have been synthesized and examined as inhibitors of mitochondrial oxidative phosphorylation. All compounds tested inhibit oxidative phosphorylation linked to succinate oxidation by potato tuber mitochondria. All of the organotin compounds inhibit ADP-stimulated O2 uptake linked to succinate oxidation with concentrations for 50% inhibition in the range 2-50 microM. This inhibition is not due to inhibition of electron transport from succinate to O2 per se: none of the organotin compounds at 50 microM substantially inhibit the rate of succinate oxidation in the presence of 2,4-dinitrophenol. Representative organotin compounds at 0.5-50 microM do not act as uncouplers of succinate oxidation. It is concluded that the organotin compounds act as energy transfer inhibitors to inhibit oxidative phosphorylation in potato tuber mitochondria. A similar mode of action of representative organotin compounds was found with rat liver mitochondria. These organotin compounds inhibit a hydrophobic Ca2+-dependent plant protein kinase in the absence but not in the presence of thiols.     

The respiration and oxidative phosphorylation of the brain mitochondria in rats with different types of behavior]

Respiration and oxidative phosphorylation of brain mitochondria were studied in outbred rats with different types of behaviour in open-field test and in forced swimming test. It was found that in rats with "active" type of behaviour (with high locomotor activity and low level of depressiveness) the brain mitochondria have at succinate oxidation a higher rate of respiration in active metabolic state and in presence of uncoupler 2,4-DNP, and a higher rate of ADP phosphorylation than in rats with "passive" type of behaviour (with low locomotor activity and high and moderate levels of depressiveness). Thus rats with active type of behaviour have a higher succinate oxidase activity than rats with passive type of behaviour. It is supposed that revealed differences in brain energy metabolism may affect the dominance of certain type of behaviour.     

Effects of chronic ethanol treatment of mitochondrial functions damage to coupling site I

Chronic ethanol feeding to rats produces changes in hepatic mitochondria which persist in the absence of ethanol metabolism. The integrity of isolated mitochondria is well preserved, as evidenced by unchanged activities of latent, Mg²⁺- and dinitrophenol-stimulated ATPase activity, and unaltered permeability to NADH. With succinate or ascorbate as substrates, oxygen uptake by mitochondria from ethanol-fed rats was decreased compared to pair-fed controls. The decrease was comparable under state 4 or state 3 conditions, or in the presence of an uncoupler. However, with the NAD⁺-dependent substrates, ADP-stimulated oxygen consumption (state 3) was decreased to a greater extent than state 4 or uncoupler-stimulated oxygen consumption in mitochondria from ethanol-fed rats. This suggests that the decrease in energy-dependent oxygen consumption at site I may be superimposed upon damage to the respiratory chain. Using NAD⁺-dependent substrates (glutamate, α-ketoglutarate or β-hydroxybutyrate) the respiratory control ratio and the $\text{P}\text{O}$ ratio of oxidative phosphorylation were significantly decreased in mitochondria isolated from the livers of rats fed ethanol. By contrast, when succinate or ascorbate served as the electron donor these functions were unchanged. The rate of phosphorylation is decreased 70% with the NAD⁺-dependent substrates because of a decreased flux of electrons, as well as a lower efficiency of oxidative phosphorylation. With succinate and ascorbate as substrates, the rate of phosphorylation is decreased 20–30%, owing to a decreased flux of electrons. These data suggest the possibility that, in addition to effects on the respiratory chain, energy-coupling site I may be damaged by ethanol feeding. Energy-dependent Ca²⁺ uptake, supported by either substrate oxidation or ATP hydrolysis, was inhibited by chronic ethanol feeding.Concentrations of acetaldehyde (1–3 mm) which inhibited phosphorylation associated with the oxidation of NAD⁺-dependent substrates had no effect on that of succinate or ascorbate. Many of the effects of chronic ethanol feeding on mitochondrial functions are similar to those produced by acetaldehyde in vitro.     

Molecular mechanism for the selective impairment of cancer mitochondrial function by a mitochondrially targeted vitamin E analogue

The effects of α-tocopheryl succinate (α-TOS), α-tocopheryl acetyl ether (α-TEA) and triphenylphosphonium-tagged vitamin E succinate (mitochondrially targeted vitamin E succinate; MitoVES) on energy-related mitochondrial functions were determined in mitochondria isolated from AS-30D hepatoma and rat liver, bovine heart sub-mitochondrial particles (SMPs), and in rodent and human carcinoma cell lines and rat hepatocytes. In isolated mitochondria, MitoVES stimulated basal respiration and ATP hydrolysis, but inhibited net state 3 (ADP-stimulated) respiration and Ca(2+) uptake, by collapsing the membrane potential at low doses (1-10μM). Uncoupled mitochondrial respiration and basal respiration of SMPs were inhibited by the three drugs at concentrations at least one order of magnitude higher and with different efficacy: MitoVES>α-TEA>α-TOS. At high doses (>10μM), the respiratory complex II (CII) was the most sensitive MitoVES target. Acting as an uncoupler at low doses, this agent stimulated total O(2) uptake, collapsed ?ψ(m), inhibited oxidative phosphorylation and induced ATP depletion in rodent and human cancer cells more potently than in normal rat hepatocytes. These findings revealed that in situ tumor mitochondria are preferred targets of the drug, indicating its clinical relevance.