ascorbic-acid and antimycin

Here we investigate the possible involvement of the sympathetic nervous system in the respiratory properties of intermyofibrillar and subsarcolemmal mitochondrial populations from heart and gastrocnemius muscles. Mitochondrial oxidative phosphorylation was assessed polarographically by using succinate (plus rotenone), and ascorbate plus N,N,N',N'-tetramethyl-p-phenyl-enediamine (plus antimycin) as respiratory substrates. We report that chronic chemical sympathectomy with guanethidine (150 mg/kg, daily for 3 weeks) induced a marked decrease in whole body metabolic and heart rates, in plasma metabolites (fatty acids and glucose) and norepinephrine levels. Guanethidine treatment decreased mainly the oxidative phosphorylation capacity of subsarcolemmal mitochondria in heart, irrespective of the substrate used. In contrast, both mitochondrial populations were affected by the treatment in skeletal muscle. This suggests that sympathetic nervous system activity can alter the energetic status of muscle cells, and to some extent play a thermogenic role in birds.

Topics: Animals; Antimycin A; Ascorbic Acid; Body Weight; Catecholamines; Ducks; Fatty Acids; Glucose; Guanethidine; Heart Rate; Male; Mitochondria; Oxidative Phosphorylation; Oxygen; Rotenone; Sarcolemma; Succinic Acid; Tetramethylphenylenediamine; Uncoupling Agents

Senescence marker protein-30 (SMP30) decreases with aging. Mice with SMP30 deficiency, a model of aging, have a short lifespan with increased oxidant stress. To elucidate SMP30's effect on coronary circulation derived from myocytes, we measured the changes in the diameter of isolated coronary arterioles in wild-type (WT) mice exposed to supernatant collected from isolated paced cardiac myocytes from SMP30 KO or WT mice. Pacing increased hydrogen peroxide in myocytes, and hydrogen peroxide was greater in SMP30 KO myocytes compared to WT myocytes. Antimycin enhanced and FCCP (oxidative phosphorylation uncoupler in mitochondria) decreased superoxide production in both groups. Addition of supernatant from stimulated myocytes, either SMP30 KO or WT, caused vasodilation. The degree of the vasodilation response to supernatant was smaller in SMP30 KO mice compared to WT mice. Administration of catalase to arterioles eliminated vasodilation in myocyte supernatant of WT mice and converted vasodilation to vasoconstriction in myocyte supernatant of SMP30 KO mice. This vasoconstriction was eliminated by olmesartan, an angiotensin II receptor antagonist. Thus, SMP30 deficiency combined with oxidant stress increases angiotensin and hydrogen peroxide release from cardiac myocytes. SMP30 plays an important role in the regulation of coronary vascular tone by myocardium.

Topics: Animals; Antimycin A; Arterioles; Ascorbic Acid; Calcium-Binding Proteins; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Catalase; Cell Survival; Coronary Vessels; Electric Stimulation; Hydrogen Peroxide; Intracellular Signaling Peptides and Proteins; Male; Mice, Inbred C57BL; Mice, Knockout; Myocardium; Myocytes, Cardiac; NADPH Oxidases; Reactive Oxygen Species; Staining and Labeling; Subcellular Fractions; Superoxides; Vasoconstriction; Vasodilation

Cytochemical reactions for mitochondrial NADH-dependent dehydrogenases (diaphorase/NADH which is related to flavoprotein), NAD-dependent dehydrogenases (isocitrate, malate) and succinate dehydrogenase were carried out in rat spermatozoa. In addition to a morphological evaluation, the intensity of the reactions was assessed using a computer image analysing system (Quantimet 600 S). The intensity of the reactions was examined in sperm midpieces by measuring integrated optical density (IOD) and mean optical density (MOD). The activity of mitochondrial respiratory chain complexes was also analysed using the polarographic method. In the population of spermatozoa studied, all whole spermatozoa midpieces were completely filled with formazans, the product of the cytochemical reaction. These morphological findings corresponded to the values obtained for IOD and MOD for the given enzymes. In the oxygraphic studies, the spermatozoa demonstrated consumption of oxygen in the presence of substrates for I, II and IV complexes and their mitochondria revealed normal integrity and sensitivity to the substrates and inhibitors. However, the oxygraphic studies revealed differences between the sperm and somatic cells. These differences concerned the stimulation of pyruvate oxidation by malate, the lack of an effect of malonic acid on phenazine methosulphate (an acceptor of electrons) oxidation and the lack of an effect of cytochrome c on ascorbate oxidation. The cytochemical method, together with densitometric measurements, enables: (1) the reaction intensity to be determined objectively; (2) subtle and dramatic differences in reaction intensity to be revealed between spermatozoa that do not differ under morphological evaluation of the intensity; (3) possible defects within the mitochondrial sheath to be located and assessed in a large number of spermatozoa. This method can be used as a screening method alongside the routine morphological examination of spermatozoa. On the other hand, the oxygraphic method in the inner membrane of mitochondria can reveal functional changes which are related to the action of respiratory chain complexes and display characteristic features of mitochondria energy metabolism. The methods used are complementary and allow the complex evaluation of mitochondria in spermatozoa. Both methods can be used in experimental and clinical studies.

Topics: Animals; Antimycin A; Ascorbic Acid; Dihydrolipoamide Dehydrogenase; Intracellular Membranes; Isocitrate Dehydrogenase; Malate Dehydrogenase; Male; Microscopy, Electron; Mitochondria; Online Systems; Oxygen Consumption; Polarography; Rats; Rats, Wistar; Rotenone; Spermatozoa; Succinate Dehydrogenase

Two sets of studies have been reported on the electron transfer pathway of complex III in bovine heart submitochondrial particles (SMP). 1) In the presence of myxothiazol, MOA-stilbene, stigmatellin, or of antimycin added to SMP pretreated with ascorbate and KCN to reduce the high potential components (iron-sulfur protein (ISP) and cytochrome c(1)) of complex III, addition of succinate reduced heme b(H) followed by a slow and partial reduction of heme b(L). Similar results were obtained when SMP were treated only with KCN or NaN(3), reagents that inhibit cytochrome oxidase, not complex III. The average initial rate of b(H) reduction under these conditions was about 25-30% of the rate of b reduction by succinate in antimycin-treated SMP, where both b(H) and b(L) were concomitantly reduced. These results have been discussed in relation to the Q-cycle hypothesis and the effect of the redox state of ISP/c(1) on cytochrome b reduction by succinate. 2) Reverse electron transfer from ISP reduced with ascorbate plus phenazine methosulfate to cytochrome b was studied in SMP, ubiquinone (Q)-depleted SMP containing

Topics: Animals; Anti-Bacterial Agents; Antimycin A; Ascorbic Acid; Cattle; Cyanates; Cytochrome b Group; Cytochrome c Group; Electron Transport Complex III; Electrons; Enzyme Inhibitors; Heme; Methylphenazonium Methosulfate; Mitochondria; Models, Biological; Myocardium; Oxidation-Reduction; Succinic Acid; Time Factors; Ubiquinone

To examine the combined effects of 2-week endurance training and 3-week feeding with beta-guanidinopropionic acid (GPA) on regional adaptability of skeletal muscle mitochondria, intermyofibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) were isolated from quadriceps muscles of sedentary control, trained control, sedentary GPA-fed and trained GPA-fed rats. Mitochondrial oxidative phosphorylation was assessed polarographically by using pyruvate plus malate, succinate (plus rotenone), and ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) (plus antimycin) as respiratory substrates. Assays of cytochrome c oxidase and F(1)-ATPase activities were also performed. In sedentary control rats, IFM exhibited a higher oxidative capacity than SSM, whereas F(1)-ATPase activities were similar. Training increased the oxidative phosphorylation capacity of mitochondria with both pyruvate plus malate and ascorbate plus TMPD as substrates, with no differences between IFM and SSM. In contrast, the GPA diet mainly improved the overall SSM oxidative phosphorylation capacity, irrespective of the substrate used. Finally, the superimposition of training to feeding with GPA strongly increased both oxidase and enzymic activities in SSM, whereas no cumulative effects were found in IFM mitochondria. It therefore seems that endurance training and feeding with GPA, which are both known to alter the energetic status of the muscle cell, might mediate distinct biochemical adaptations in regional skeletal muscle mitochondria.

Topics: Animals; Antimycin A; Ascorbic Acid; Creatine; Diet; Electron Transport Complex IV; Guanidines; Humans; Malates; Male; Mitochondria; Muscle, Skeletal; Oxidative Phosphorylation; Oxygen Consumption; Physical Conditioning, Animal; Propionates; Proton-Translocating ATPases; Pyruvic Acid; Rats; Rats, Wistar; Rotenone; Sarcolemma; Succinic Acid; Tetramethylphenylenediamine; Time Factors

The redox reactions of the bis-heme cytochrome b of the ubiquinol:cytochrome c oxidoreductase complex (complex III, bc1 complex) were studied in bovine heart submitochondrial particles (SMP). It was shown that (i) when SMP were treated with the complex III inhibitor myxothiazol (or MOA-stilbene or stigmatellin) or with KCN and ascorbate to reduce the high potential centers of complex III (iron-sulfur protein and cytochromes c + c1), NADH or succinate reduced heme bL slowly and incompletely. In contrast, heme bH was reduced by these substrates completely and much more rapidly. Only when the complex III inhibitor was antimycin, and the high potential centers were in the oxidized state, NADH or succinate was able to reduce both bH and bL rapidly and completely. (ii) When NADH or succinate was added to SMP inhibited at complex III by antimycin and energized by ATP, the bis-heme cytochrome b was reduced only partially. Prereduction of the high potential centers was not necessary for this partial b reduction, but slowed down the reduction rate. Deenergization of SMP by uncoupling (or addition of oligomycin to inhibit ATP hydrolysis) resulted in further b reduction. Addition of ATP after b was reduced by substrate resulted in partial b oxidation, and the heme remaining reduced appeared to be mainly bL. Other experiments suggested that the redox changes of cytochrome b effected by energization and deenergization of SMP occurred via electronic communication with the ubiquinone pool. These results have been discussed in relation to current concepts regarding the mechanism of electron transfer by complex III.

Topics: Animals; Antifungal Agents; Antimycin A; Ascorbic Acid; Cattle; Cytochrome b Group; Electron Transport Complex III; Ferricyanides; Methacrylates; Models, Chemical; NAD; Oxidation-Reduction; Potassium Cyanide; Spectrophotometry, Atomic; Submitochondrial Particles; Succinates; Succinic Acid; Thiazoles

This article is a study of the relationship between lipid peroxidation and protein modification in beef heart submitochondrial particles, and the protective effect of endogenous ubiquinol (reduced coenzyme Q) against these effects. ADP-Fe3+ and ascorbate were used to initiate lipid peroxidation and protein modification, which were monitored by measuring TBARS and protein carbonylation, respectively. Endogenous ubiquinone was reduced by the addition of succinate and antimycin. The parameters investigated included extraction and reincorporation of ubiquinone, and comparison of the effect of ubiquinol with those of various antioxidant compounds and enzymes, as well as the iron chelator EDTA. Under all conditions employed there was a close correlation between lipid peroxidation and protein carbonylation, and the inhibition of these effects by endogenous ubiquinol. SDS-PAGE analysis revealed a differential effect on individual protein components and its prevention by ubiquinol. Conceivable mechanisms behind the observed oxidative modifications of membrane phospholipids and proteins and of the role of ubiquinol in preventing these effects are considered.

Topics: Animals; Antimycin A; Antioxidants; Ascorbic Acid; Cattle; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Kinetics; Lipid Peroxidation; Mitochondria, Heart; Proteins; Submitochondrial Particles; Succinates; Succinic Acid; Thiobarbituric Acid Reactive Substances; Ubiquinone

Cytochrome b-561 in chromaffin granules interacts with antimycin and its alpha-peak shifts 1 nm towards red. When chromaffin granules were treated with Triton X-100 antimycin no effect was observed. Cytochrome b-561 is located in the plasma membrane isolated from the chromaffin cells. The plasma membrane b-561 does not seem to interact with antimycin. A number of NADH or NADPH (acceptor) oxidoreductase activity has been observed in isolated plasma membrane providing clues to the origin of plasma membrane dehydrogenase. The possible role of cytochrome b-561 in secretory granules other than its accredited energy conserving electron transport property is projected.

Topics: Adrenal Medulla; Animals; Antimycin A; Ascorbic Acid; Cattle; Cell Membrane; Chromaffin Granules; Chromaffin System; Cytochrome b Group; Dithionite; Kinetics; Oxidation-Reduction; Spectrophotometry

1. Oxygen-pulse experiments on antimycin-treated liver mitochondria from young rats in the absence of added substrates indicate the presence of an endogenous hydrogenated reductant on the oxygen side of the antimycin inhibition site. This reductant has been estimated to be present in a 20--50-fold molar excess over cytochrome aa3. Oxidation of the reductant by small bursts of electron flow to oxygen gives a ratio H+ translocated:O atoms taken up of 2.1 +/- 0.2. 2. The endogenous hydrogenated reductant competes with added ferrocyanide and with ascorbate/NNN'N'-tetramethyl-p-phenylenediamine as a source of reducing equivalents to oxygen. 3. When the contribution of endogenous substrate to H+ production is taken into account, there still remains some respiration-dependent H+ translocation consistent with a H+ pumping activity of cytochrome oxidase. The stoichiometry of this 'extra' H+ translocation is variable and depends on the rate of electron flow.

Topics: Animals; Antimycin A; Ascorbic Acid; Ferricyanides; Ferrocyanides; Hydrogen-Ion Concentration; In Vitro Techniques; Mitochondria, Liver; Oxidation-Reduction; Oxygen; Rats