Effect of morphine in vitro on the oxidative phosphorylation in rat liver mitochondria]

The rates of respiration in the presence of ADP and of phosphorylation as an ATP-ase activity of rat liver mitochondria was inhibited was in vitro by morphine with Ki=6.5 mM. The uncoupler-stimulated respiration of the mitochondria and the activity of ATP-ase and synthesis of ATP in the submitochondrial particles were not altered in the presence of morphine. It is suggested that morphine inhibited the adenine nucleotide transport through the mitochondrial membrane     

Effect of insulin on oxidative phosphorylation in the liver and heart mitochondria of adrenalectomized rats

The effect of insulin was studied as applied to the inhibited under conditions of adrenalectomy process of oxidative phosphorylation in the rat liver and heart mitochondria. It is established that adrenalectomy does not change oxidative activity of mitochondria but inhibits the process of phosphorylation, which results in the decreased values of the ADP/O coefficient and the respiratory control. Insulin administered to the adrenalectomized rats 3h before the experiments reduces the disturbed oxidative phosphorylation in mitochondria of the liver and heart by intensifying the degree of ADP phosphorylation.     

Chemical modification of mitochondria. II. Effect of labeling on oxidative phosphorylation

The labeling of rat liver mitochondria (RLM) by the uncoupler 2,4-dinitro-5-(bromoacetoxyethoxy)phenol (DNBP) was studied and related to the effect of this molecule on oxidative phosphorylation. Alkylation of the cysteine residues was measured both with respect to incubation time of RLM with DNBP and with increasing DNBP concentration. At 3.3 × 10^?5m DNBP, the amount of S-carboxymethyl-cysteine formed was found to level off after about 3 min. The rate of ATP synthesis in RLM is reduced by increasing concentrations of DNBP and falls to zero, with either hydroxybutyrate or succinate as substrate, at 2 × 10^?4m DNBP. To characterize the effect of labeling on oxidative phosphorylation, the $\text{P}\text{O}$ ratio were measured after incubating RLM with DNBP for various times between 10 and 300 sec. The $\text{P}\text{O}$ ratio increases and tends to level off as the incubation time increases. No increase in $\text{P}\text{O}$ ratio was noted when RLM were similarly incubated with the nonlabeling uncoupler 2,4-dinitro-5-(acetoxyethoxy) phenol. Further, the effect of labeling on oxidative phosphorylation was determined with RLM which had been treated with DNBP and then washed free of the excess unreacted uncoupler. DNBP produces specific labeling in RLM which, when related to the effects of this uncoupler on oxidative phosphorylation, suggests that the labeled proteins may be involved in the primary energy transduction process.     

Effect of alpha-tocopherol and its derivatives on ATPase activity and oxidative phosphorylation in rat liver mitochondria

The effects of alpha-tocopherol and its derivatives on the energy metabolism in mitochondria were studied. It was shown that alpha-tocopherol derivatives with short hydrocarbon chain produce uncoupling effects, while alpha-tocopherol itself as well as its long-chain derivatives (tocopheryl acetate and tocopheryl stearate) exert no effect. All the derivatives studied produced an inhibitory effect on DNP-stimulated mitochondrial ATPase. As far as these compounds did not influence the activity of mitochondrial F1 factor, it was concluded that the effects of the compounds were due to membrane modification.     

The effects of protein synthesis inhibitors on oxidative phosphorylation by plant mitochondria

Inhibitors can be successfully used if they are specific for only one process. Published data suggest that some inhibitors of protein synthesis may also inhibit respiration or oxidative phosphorylation. The effect of a range of protein synthesis inhibitors on respiration and phosphorylation has been studied, using tightly coupled mitochondria from several plant species including turnips (Brassica napus).Puromycin, actinomycin D, lincomycin, mitomycin C and d-serine did not uncouple or inhibit respiration. Cycloheximide caused a partial inhibition (maximum 22% at 3 mM) of malate but not succinate-driven respiration. Chloramphenicol was a potent inhibitor of electron transport, but not of phosphorylation. The activity of the isomers of chloramphenicol varied in the order l-threo >d-threo >l-erythro >d-erythro. From evidence presented it is concluded that chloramphenicol has three sites of action, the flavoprotein level being most sensitive, the second site of variable sensitivity lies between cytochromes b and c and the third site at the cytochrome a level is only slightly affected by the inhibitor.     

Effect of primary aliphatic amines on oxidative phosphorylation in rat liver mitochondria

The dynamics of primary aliphatic amines (ethylamine, propylamine) effects on the processes of oxidative phosphorylation in rat liver mitochondria was estimated. The inhibiting action of ethylamine and propylamine on the oxidative phosphorylation processes in the rat liver mitochondria was revealed.     

Functioning of oxidative phosphorylation in liver mitochondria of high-fat diet fed rats

We proposed that inhibition of mitochondrial adenine nucleotide translocator (ANT) by long chain acyl-CoA (LCAC) underlies the mechanism associating obesity and type 2 diabetes. Here we test that after long-term exposure to a high-fat diet (HFD): (i) there is no adaptation of the mitochondrial compartment that would hinder such ANT inhibition, and (ii) ANT has significant control of the relevant aspects of oxidative phosphorylation. After 7 weeks, HFD induced a 24+/-6% increase in hepatic LCAC concentration and accumulation of the oxidative stress marker N(epsilon)-(carboxymethyl)lysine. HFD did not significantly affect mitochondrial copy number, oxygen uptake, membrane potential (Deltapsi), ADP/O ratio, and the content of coenzyme Q(9), cytochromes b and a+a(3). Modular kinetic analysis showed that the kinetics of substrate oxidation, phosphorylation, proton leak, ATP-production and ATP-consumption were not influenced significantly. After HFD-feeding ANT exerted considerable control over oxygen uptake (control coefficient C=0.14) and phosphorylation fluxes (C=0.15), extra- (C=0.23) and intramitochondrial (C=-0.56) ATP/ADP ratios, and Deltapsi (C=-0.11). We conclude that although HFD induces accumulation of LCAC and N(epsilon)-(carboxymethyl)lysine, oxidative phosphorylation does not adapt to these metabolic challenges. Furthermore, ANT retains control of fluxes and intermediates, making inhibition of this enzyme a more probable link between obesity and type 2 diabetes.     

The effect of hypoxia on oxidative phosphorylation and lipid peroxidation in rat liver mitochondria upon lung inflammation

Hypoxic trainings of rats (maintenance in the test chamber at the "altitude" 4 km above the sea level for 7 hours a day for two weeks) prevent pneumonia-induced activation of peroxidation for lipids of the liver mitochondria. This increases the phosphorylating respiration rate when lipemic serum is used as an oxidation substrate (not succinate). In these experiments the efficiency of oxidative phosphorylation (delta ADP/delta O) when either succinate and glutamate or glutamate and malate have been oxidated corresponded to the values typical of intact animals and was higher when lipemic serum was used. It is supposed that rearrangement of energy reactions of mitochondria is connected with intensification of utilization of lipids and conjugation between their oxidation and phosphorylation. This rearrangement is apparently aimed to prevent the energy deficiency in the organism which arises in patients with pneumonia.     

Regulation of oxidative phosphorylation by liver mitochondria receptors after adaptation by rats to periodic normal pressure and acute hypoxia

It is known that NO-dependent mechanisms are involved in mitochondrial adaptive reactions to different factors. The object of this study was to investigate the role of cholino- and adrenoreceptors in NO-dependent reactions of rat liver mitochondria to acute hypoxia (AH) and intermittent hypoxic training (IHT). Eight groups of Wistar male rats participated in the study. Animals of Gr. I underwent daily i.p. saline injections during 14 days. Gr. II was examined after a single AH test (inhalation of 7% O2, 30 min) with the same saline treatment. Another six groups were exposed to IHT (11% O2, 15-min sessions with 15 min rest intervals, 5 times daily during 14-days), at that 15 min before every IHT session animals underwent i.p. treatment: Gr. III and IV--saline, Gr. V--L-arginine, Gr. VI--NO synthase blocker L-NNA, Gr. VII--L-arginine with alpha-, beta-adrenoblockers phentolamine and obzidane, Gr. VIII--L-arginine with M- and N-cholinoreceptor blockers athropine and benzohexonium. After IHT Gr. IV-VIII were exposed to a single AH test and decapitated just after that. In control rats AH provoked: 1) in the presence of succinate, a 33% augmentation of ADP-stimulated mitochondrial respiration (V3) with a 18% decrease of O2 uptake efficiency (ADP/O ratio); 2) in the presence of alpha-ketoglutarate, a NAD-dependent substrate, no changes in V3 were observed, also 21% augmentation of ADP/O ratio registered. These events were accompanied by 36% increase in succinate dehydrogenase (SDG) activity, two-fold augmentation of malon dialdehyde (MDA) content and 43% increase in diene conjugates (DK). IHT caused reorganization of mitochondrial energy metabolism improving the protection against acute hypoxia. A decrease by 40% in activation of mitochondrial respiration in the presence of succinate (V3--by 40% and V4--by 34%), a reduction of MDA and DK content (by 32% and 20%, respectively), an increase in SGD activity by 31% was observed in Gr. IY compared to Gr. II. Extra exogenous NO (Gr.Y) did not influence V3 and V4 in the presence of succinate, but in the presence of alpha-ketoglutarate decreased them by 9% and 29%, respectively, as well as ADP/O ratio by 28% on the background of SDG inhibition by 24% and the decrease of MDA content by 34%, that is reduced aerobic energy supply and reactive oxygen species production. L-arginine effects were abolished by L-NNA. Effects of cholinoreceptor blockers over L-arginine (Gr. VIII) resembled effects of AH: considerable activation of succinate and alpha-ketoglutarate oxidation in stage V3 by 44% and 75%, respectively, was observed which was accompanied by a decrease in ADP/O by 21% and 31%, and V3/V4 by 15% and 28%, respectively, in comparison to Gr.Y. It indicates that effects of L-arginine are mediated mainly by cholinoreceptors. The effects of adrenoreceptors blockade strengthened the combined effects of IHT with L-arginine treatment, confirming primary role of cholinoreceptors in NO-dependent mitochondrial reactions to IHT. Thus, oxygen uptake and its effective usage depend on dynamic status of adreno- and cholinoreceptors. We conclude that protective effects of the combination of IHT with NO-donor treatment under acute hypoxia are mainly realized through cholinoreceptors.     

Effect of ascorbic acid and oat polyphenols on respiration and oxidative phosphorylation in liver mitochondria in alloxan diabetic rats

30 mg of ascorbic acid and 80 mg of dry oats extracts were administered to rats with alloxan diabetes during a day per 1 kg of live weight. Administration of these preparations during 6, 12 and 24 days prevents the uncoupling action of respiration and oxidative phosphorylation, that was observed in the rats with alloxan diabetes which were not given ascorbic acid and oats polyphenols. The P/O coefficient on the alloxan diabetes rats on the 6, 12 and 24 days was 1.32 +/- 0.027; 1.26 +/- 0.013; 1.22 +/- 0.18, respectively; in the rats which were given ascorbic acid and oats polyphenols to P/O coefficient was 1.85 +/- 0,026, 1.80 +/- 0.024 and 1.75 +/- 0.028, respectively.     

Anandamide inhibits oxidative phosphorylation in isolated liver mitochondria

A study on the effect of anandamide (AEA) in energy coupling of rat liver mitochondria is presented. Micromolar concentrations of AEA, while almost ineffective on substrate supported oxygen consumption rate and on uncoupler stimulated respiration, strongly inhibited the respiratory state III. AEA did not change the rate and the extent of substrate generated membrane potential, but markedly delayed rebuilding by respiration of the potential collapsed by ADP addition. Overall, these data suggest that anandamide inhibits the oxidative phosphorylation process. Direct measurement of the F_oF₁ ATP synthase activity showed that the oligomycin sensitive ATP synthesis was inhibited by AEA, (IC₅₀, 2.5 μM), while the ATP hydrolase activity was unaffected. Consistently, AEA did not change the membrane potential generated by ATP hydrolysis.     

Heart mitochondria in rats submitted to chronic hypoxia.

The effect of prolonged exposure to normobaric hypoxia on the mitochondria of myocard of rats exposed for several weeks to 8 and 7% O2 has been morphometrically evaluated. Twelve male Wistar rats housed in Nalgene cages (2 per cage) with a batch of six cages placed in plexiglass chambers were maintained in air/N2 mixtures containing different concentrations of O2. Six animals kept in similar cages under normoxia served as controls. When at day 60 the FIO2 was reduced to 8%, the weight increase stagnated and after the 81st test day, on which the hypoxic animals were subdivided into 8% and 7% groups the weight curve showed a decrease in the mean body weight for both groups. The arrest and the following loss of weight beyond the 85th day may be interpreted as the expression of a limit reached in the compensation capacity. In the 8%-group the shape of the mitochondria varied more markedly often with budding and furrowing of the surface. In the 7%-group bizarre shapes and wide variations in size with a decided shift towards larger mitochondria were noteworthy. While rats kept under 8% oxygen exhibited a numerical increase in myocardial mitochondria compared to controls, the mitochondria of the 7%-group were numerically reduced. The results suggest that hypoxia of 8% oxygen is compensatable, if only to some extent, by an increasing surface of mitochondrial membranes, and that further reduction of oxygen causes compensation mechanisms to fail as seen by the severe alterations of the mitochondrial population of the cardiomyocyte in the 7%-group.     

Effect of ethanol on the palmitate-induced uncoupling of oxidative phosphorylation in liver mitochondria

The effect of ethanol on the uncoupling activity of palmitate and recoupling activities of carboxyatractylate and glutamate was studied in liver mitochondria at various Mg²⁺ concentrations and medium pH values (7.0, 7.4, and 7.8). Ethanol taken at concentration of 0.25 M had no effect on the uncoupling activity of palmitic acid in the presence of 2 mM MgCl₂ and decreased the recoupling effects of carboxyatractylate and glutamate added to mitochondria either just before or after the fatty acid. However, ethanol did not modify the overall recoupling effect of carboxyatractylate and glutamate taken in combination. The effect of ethanol decreased as medium pH was decreased to 7.0. Elevated concentration of Mg²⁺ (up to 8 mM) inhibits the uncoupling effect of palmitate. Ethanol eliminates substantially the recoupling effect of Mg²⁺ under these conditions, but does not influence the recoupling effects of carboxyatractylate and glutamate. It is inferred that ADP/ATP and aspartate/glutamate antiporters are involved in uncoupling function as single uncoupling complex with the common fatty acid pool. Fatty acid molecules gain the ability to migrate under the action of ethanol: from ADP/ATP antiporter to aspartate/glutamate antiporter on addition of carboxyatractylate and in opposite direction on addition of glutamate. Possible mechanisms of fatty acid translocation from one transporter to another are discussed.     

Studies on the effects of copper deficiency on rat liver mitochondria. II. Effects on oxidative phosphorylation

Effects of dietary copper deficiency in rats on respiratory enzymes of isolated rat liver mitochondria have been studied. After 2 weeks of Cu-depletion, cytochrome c oxidase (EC 1.9.3.1) activity had declined by 42% and between 4 and 8 weeks exhibited between 20 and 25% of the activity of control mitochondria. Activities of NADH cytochrome c reductase (EC 1.6.99.3) and succinate cytochrome c reductase (EC 1.3.99.1), were unaffected initially but declined by 32 and 46%, respectively, after 8 weeks of Cu-depletion. After 4 weeks there was a significant (34%) decline in succinate supported state 3 respiration with only a modest (18%) decline in state 4 respiration. The ADP:O ratio was unaffected by Cu-depletion after 6 and 8 weeks of dietary Cu-restriction. State 3 respiration was significantly reduced after 6 weeks when glutamate/malate or beta-hydroxybutyrate were used as substrates, whereas state 4 respiration and ADP:O ratios were unaffected. The fall in state 3 respiration was of sufficient magnitude at 8 weeks to cause a significant decline in the respiratory control ratio with all substrates. Comparisons between the relative activities of cytochrome c oxidase and reductase activities in Cu-deficient preparations, the relatively specific effect of the deficiency on state 3 respiration with all substrates tested and the ability to increase significantly oxygen consumption in excess of maximal state 3 respiration by the uncoupler 2,4-dinitrophenol suggest that the defect in Cu-deficient mitochondria cannot be attributed solely to the decreased activity of cytochrome c oxidase.