Interaction of a synthetic polyanion with rat liver mitochondria

The effect of a polyanion (a copolymer of methacrylate, malaete and styrene in a 1:2:3 proportion with an average molecular weight of 10 000) on respiration, ATPase activity and ADP/ATP exchange activity of rat liver mitochondria and submitochondrial particles has been studied.The polyanion (at 17–150 μg/ml concentration, 100 μg polyanion corresponding to 0.83 μequiv. of carboxylic groups) inhibits the oxidation of succinate and NAD-linked substrates in state 3 in a concentration-dependent manner. The extent of this inhibition can be decreased by elevating the concentration of ADP. State 4 respiration is not affected by the polyanion. It has also a slight inhibitory effect on the oxidation of the above mentioned substrates in the uncoupled state (a maximum inhibition of 37% at 166 μg/ml polyanion concentration), which is unaffected by ADP. The strong inhibition of state 3 respiration can be relieved by 2,4-dinitrophenol to the low level observed in the uncoupled state. Ascorbate+TMPD oxidation is slightly inhibited in state 3, while it is not inhibited at all in the uncoupled state.The polyanion, depending on its concentration, strongly inhibits also the DNP-activated ATPase activity of mitochondria (50% inhibition at 40 μg/ml polyanion concentration).The ATPase activity of sonic submitochondrial particles is also inhibited. However, this inhibition is incomplete (reaching a maximum of 65%) and higher concentrations of the polyanion are required than to inhibit the ATPase activity of intact mitochondria.The polyanion inhibits the ADP/ATP translocator activity of mitochondria, measured by the “back exchange” of [2-³H]ADP. After a short preincubation of the mitochondria with the polyanion, the concentration dependence of the inhibition by the polyanion corresponds to that of the DNP-activated ATPase activity of intact mitochondria.It is concluded that, in intact mitochondria, the polyanion has at least a dual effect, i.e. it partially inhibits the respiratory chain between cytochrome b and cytochrome c, and strongly oxidative phosphorylation by blocking the ADP/ATP translocator.     

Oxidative phosphorylation in mitochondria isolated from small intestinal epithelium of thiamphenicol-treated rats and control rats

Treatment of rats with thiamphenicol in a dose of 125 mg/kg per day for 60–64 h causes specific inhibition of mitochondrial protein synthesis, leading to a drastic decrease of the cytochrome c oxidase activity in intestinal epithelium. At the same time the mitochondrial ATPase activity becomes resistant to inhibition by oligomycin. Experiments with isolated intestinal mitochondria revealed that respiration in state 3 is diminished by 55% with succinate (5 mM) and by 40% with pyruvate (10 mM) plus L-malate (2 mM) as the substrates, both as compared to intestinal mitochondria isolated from control rats. P : O ratios as well as respiratory control indices are comparable in the two groups of animals. Uncoupled respiration is inhibited by 35% with succinate as the substrate, while the succinate cytochrome c reductase activity remains unaltered. No inhibition of uncoupled respiration with pyruvate plus L-malate as the substrates was observed. The ATP-P_i exchange activity in the mitochondria from the treated animals is diminished by about 75%. It is concluded that in the mitochondria of the treated animals the inhibition of the coupled respiration (state 3) is caused by the limitation of the ATP-generating capacity and that electron transport is rate limiting only with the rapidly oxidized substrates such as succinate, if respiration is uncoupled.     

The metabolic effects and uptake of ethidium bromide by rat liver mitochondria.

The uptake of ethidium bromide by rat liver mitochondria and its effect on mitochondria, submitochondrial particles, and F₁ were studied. Ethidium bromide inhibited the State 4-State 3 transition with glutamate or succinate as substrates. With glutamate, ethidium bromide did not affect State 4 respiration, but with succinate it induced maximal release of respiration. These effects appear to depend on the uptake and concentration of the dye within the mitochondrion. In submitochondrial particles, the aerobic oxidation of NADH is much more sensitive to ethidium bromide than that of succinate. Ethidium bromide partially inhibited the ATPase activity of submitochondrial particles and of a soluble F₁ preparation. Ethidium bromide behaves as a lipophilic cation which is concentrated through an energy-dependent process within the mitochondria, producing its effects at different levels of mitochondrial function. The ability of mitochondria to concentrate ethidium bromide may be involved in the selectivity of the dye as a mitochondrial mutagen.     

Effects of equisetin on rat liver mitochondria: Evidence for inhibition of substrate anion carriers of the inner membrane

The effect of equisetin, an antibiotic produced byFusarium equiseti, has been studied on mitochondrial functions (respiration, ATPase, ion transport). Equisetin inhibits the DNP-stimulated ATPase activity of rat liver mitochondria and mitoplasts in a concentration-dependent manner; 50% inhibition is caused by about 8 nmol equisetin/mg protein. The antibiotic is without effect either on the ATPase activity of submitochondrial particles or on the purified F₁-ATPase. It inhibits both the ADP- or DNP-activated oxygen uptake by mitochondria in the presence of glutamate + malate or succinate as substrates, but only the ADP-stimulated respiration is inhibited if the electron donors are TMPD + ascorbate. It does not affect the NADH or succinate oxidation of submitochondrial particles. Equisetin inhibits in a concentration-dependent manner the active Ca²⁺-uptake of mitochondria energized both by ATP or succinate without affecting the Ca²⁺-uniporter itself. The antibiotic inhibits the ATP-uptake by mitochondria (50% inhibition at about 8 nmol equisetin/mg protein) and the P_i and dicarboxylate carrier. It does not lower the membrane potential at least up to 200 nmol/mg protein concentration. The data presented in this paper indicate that equisetin specifically inhibits the substrate anion carriers of the mitochondrial inner membrane.Abbreviations EGTA ethyleneglycol bis/-aminoethylether/-N, N-tetraacetic acid - DNP 2, 4-dinitrophenol - TMPD N,N,N,N,tetramethyl-p-phenylenediamine - CCP carbonylcyanide-m-chlorophenyl hydrazone - TPP tetraphenyl-phosphonium - Hepes /4,(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid/     

Effect of rapid freezing on the functional state of rat heart mitochondria

As evidenced by respiration, oxidative phosphorylation, ATPase and NADH-oxidase activities, mitochondria composing heart tissue slices are more damaged by freezing-thawing than isolated mitochondria. A change in the functional activity of mitochondria is manifested in an increased respiratory rate in the second metabolic state and decreased respiratory rate in the third metabolic state upon oxidation of succinate and alpha-ketoglutarate; the ability of mitochondria to synthetize ATP (inhibition of the respiratory control) varied and the ATPase and NADH-oxidase activities increased. These changes in the functional state of mitochondria appeared to be due to a rise of the proton conductivity of the inner mitochondrial membrane by freezing-thawing.     

Control of state 3 respiration in liver mitochondria from rats subjected to chronic ethanol consumption

Male Sprague-Dawley rats were pair-fed a liquid diet containing 36% of calories as ethanol for at least 31 days. Mitochondria were isolated from the livers and assayed for state 3, state 4 and uncoupled respiration at all three coupling sites. Assay conditions were established that maximized state 3 respiration with each substrate while maintaining a high respiratory control ratio. In mitochondria from ethanol-fed animals, state 3 respiratory rates were decreased at all three coupling sites. The decreased state 3 rate observed at site III was still significantly higher than the state 3 rates observed at site II in mitochondria from either ethanol-fed or control animals. Moreover, the maximal (FCCP-uncoupled) rates with succinate and -ketoglutarate were the same in mitochondria from ethanol-fed and control animals, whereas with glutamate-malate as substrate it was lowered 23% by chronic ethanol consumption. To investigate the role of cytochrome oxidase in modulating the respiratory rate with site I and site II substrates, the effects of cyanide on state 3 and FCCP-uncoupled respiration were determined. When the mitochondria were uncoupled there was no decrease in the rate of succinate oxidation until the rates of ascorbate and succinate oxidation became equivalent. Conversely, parallel inhibition of ascorbate, succinate and glutamate-malate state 3 respiratory rates were observed at all concentrations (1–50 μM) of cyanide utilized. These observations suggest strongly that in coupled mitochondria ethanol-elicited decreases in cytochrome oxidase activity depress the state 3 respiratory rates with site I and II substrates.     

ATP Accelerates Respiration of Mitochondria From Rat Lung and Suppresses Their Release of Hydrogen Peroxide

Lung mitochondria were isolated by differential centrifugation from pentobarbital-anesthetized male rats. One to three millimolar Mg²⁺-ATP increased the consumption of oxygen of lung mitochondria oxidizing 10 mM succinate > fourfold (P < 0.01) whereas ATP increased the respiration of liver mitochondria by < 35%. ATP also hyperpolarized partially uncoupled lung mitochondria in the presence of the mitochondria-specific antagonist, oligomycin. However, only 20% of the ATPase activity in the lung mitochondria was blocked by oligomycin compared to a blockade of 91% for liver mitochondria. We investigated the effect of reducing the non-mitochondrial ATPase activity in the lung preparation. A purer suspension of lung mitochondria from a Percoll gradient was inhibited 95% by oligomycin. The volume fraction identified as mitochondria by electron microscopy in this suspension (73.6± 3.5%) did not differ from that for liver mitochondria (69.1± 4.9%). ATP reduced the mean area of the mitochondrial profiles in this Percoll fraction by 15% (P <0.01) and increased its state 3 respiration with succinate as substrate by 1.5-fold (P < 0.01) with no change in the state 4 respiration measured after carboxyatractyloside. Hence, ATP increased the respiratory control ratio (state 3/state 4, P <0.01). In contrast, state 3 respiration with the complex 1-selective substrates, glutamate and malate, did not change with addition of ATP. The acceleration of respiration by ATP was accompanied by decreased production of H₂O₂. Thus ATP-dependent processes that increase respiration appear to improve lung mitochondrial function while minimizing the release of reactive oxygen species.     

Control of state 3 respiration in liver mitochondria from rats subjected to chronic ethanol consumption

Male Sprague-Dawley rats were pair-fed a liquid diet containing 36% of calories as ethanol for at least 31 days. Mitochondria were isolated from the livers and assayed for state 3, state 4 and uncoupled respiration at all three coupling sites. Assay conditions were established that maximized state 3 respiration with each substrate while maintaining a high respiratory control ratio. In mitochondria from ethanol-fed animals, state 3 respiratory rates were decreased at all three coupling sites. The decreased state 3 rate observed at site III was still significantly higher than the state 3 rates observed at site II in mitochondria from either ethanol-fed or control animals. Moreover, the maximal (FCCP-uncoupled) rates with succinate and alpha-ketoglutarate were the same in mitochondria from ethanol-fed and control animals, whereas with glutamate-malate as substrate it was lowered 23% by chronic ethanol consumption. To investigate the role of cytochrome oxidase in modulating the respiratory rate with site I and site II substrates, the effects of cyanide on state 3 and FCCP-uncoupled respiration were determined. When the mitochondria were uncoupled there was no decrease in the rate of succinate oxidation until the rates of ascorbate and succinate oxidation became equivalent. Conversely, parallel inhibition of ascorbate, succinate and glutamate-malate state 3 respiratory rates were observed at all concentrations (1-50 microM) of cyanide utilized. These observations suggest strongly that in coupled mitochondria ethanol-elicited decreases in cytochrome oxidase activity depress the state 3 respiratory rates with site I and II substrates.     

Effects of 5,5'-diphenyl-2-thiohydantoin on respiration and oxidative phosphorylation of rat liver mitochondria.

5,5'-Diphenyl-2-thiohydantoin (DPTH) administered in vitro, inhibited state 3 oxidation, stimulated state 4 oxidation and decreased ADP:O ratio when 3-hydroxybutyrate and succinate were used as substrates. Considerably lower DPTH concentrations were required for the inhibition of 3-hydroxybutyrate oxidation (50% inhibition occurred at approximately 0.17 mumoles DPTH/mg protein) than were needed for inhibition of succinate oxidation (50% inhibition occurred at about 0.62 mumoles DPTH/mg protein). DPTH showed no inhibitory effects when ascorbate plus tetramethylphenylenediamine (TMPD) served as the substrate. The inhibition of state 3 respiration was not reversed by 2,4-dinitrophenol (DNP), although there was a slight increase in the DNP rate:state 3 rate suggesting the presence of a weak DPTH inhibotory site located within the Site I energy transport chain. Uncoupling, in the presence of DPTH, was observed with all substrates. In experiments utilizing sonicated mitochondria, DPTH inhibited NADH-linked oxidation, but did not inhibit succinate or ascorbate plus TMPD oxidation. The effects of DPTH were reversed by dilution and by addition of albumin. DPTH concentrations which produced inhibition of state 3 respiration in vitro were reached, in vivo, in the livers of rats receiving a single oral dose of 40 mg/kg of DPTH.     

Effect of catecholamine depletion on oxidative energy metabolism in rat liver, brain and heart mitochondria; use of reserpine

Regulation of mitochondrial functions in vivo by catecholamines was examined indirectly by depleting the catecholamines stores by reserpine treatments of the experimental animals. Reserpine treatment resulted in decreased respiratory activity in liver and brain mitochondria with the two NAD+-linked substrates: glutamate and pyruvate + malate with succinate ATP synthesis rate decreased in liver mitochondria only. With ascorbate + TMPD system, the ADP/O ratio and ADP phosphorylation rate decreased in brain mitochondria. For the heart mitochondria, state 3 respiration rates decreased for all substrates. In the liver mitochondria basal ATPase activity decreased by 51%, but in the presence of Mg2+ and/or DNP increased significantly. In the brain and heart mitochondria ATPase activities were unchanged. The energy of activation in high temperature range increased liver mitochondrial ATPase while in brain mitochondria reserpine treatment resulted in abolishment in phase transition. Total phospholipid (TPL) content of the brain mitochondria increased by 22%. For the heart mitochondria TPL content decreased by 19% and CHL content decreased by 34%. Tissue specific differential effects were observed for the mitochondrial phospholipid composition. Liver mitochondrial membranes were more fluidized in the reserpine-treated group. The epinephrine and norepinephrine contents in the adrenals decreased by 68 and 77% after reserpine treatment.     

Effect of bedaquiline on the functions of rat liver mitochondria

The paper considers the effects of bedaquiline (BDQ), an antituberculous preparation of the new generation, on rat liver mitochondria. It was shown that 50?μM BDQ inhibited mitochondrial respiration measured with substrates of complexes I and II (glutamate/malate and succinate/rotenone systems respectively) in the states V₃ and V_DNP. At the same time, at concentrations below 50?μM, BDQ slightly stimulated respiration with substrates of complex I in the state V₂. BDQ was also found to suppress, in a dose-dependent manner, the activity of complex II and the total activity of complexes II?+?III of the mitochondrial transport chain. It was discovered that at concentrations up to 10?μM, BDQ inhibited H₂O₂ production in mitochondria. BDQ (10–50?μM) suppressed the opening of Ca²⁺-dependent CsA-sensitive mitochondrial permeability transition pore. The latter was revealed experimentally as the inhibition of Ca²⁺/P_i-dependent swelling of mitochondria, suppression of cytochrome c release, and an increase in the Ca²⁺ capacity of the organelles. BDQ also decreased the rate of mitochondrial energy-dependent K⁺ transport, which was evaluated by the energy-dependent swelling of mitochondria in a K⁺ buffer and DNP-induced K⁺ efflux from the organelles. The possible mechanisms of BDQ effect of rat liver mitochondria are discussed.     

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.     

On the mechanism of action of alkylguanidines on oxidative phosphorylation in mitochondria.

The effect of octylguanidine and oligomycin on the oxygen uptake of rat liver mitochondria and on the ATPase activity of "sonic" submitochondrial particles has been studied. 1. Octylguanidine inhibits state 3 respiration with glutamate-malate and succinate as substrates, but much lower concentrations are required to inhibit oxygen uptake with the former substrates. State 4 respiration is unaffected by octylguanidine. 2. The titration-curve for the octylguanidine inhibition of glutamate-malate oxidation is hyperbolic and apparently biphasic, half-maximal inhibition is obtained at 30 muM octylguanidine. The octylguanidine-curve for inhibition of succinate oxidation is sigmoid with half-maximal inhibition at about 250 muM. 3. Octylguanidine and oligomycin show additive inhibitory action on state 3 respiration with both glutamate plus malage and succinate as respiratory substrates. 4. Concentrations of oligomycin or octylguanidine, which added separately are ineffective on state 3 respiration, become inhibitory when the two inhibitors are added together. 5. Octylguanidine inhibits the ATPase activity of sonic submitochondrial particles with a hyperbolic titration-curve analogous to that obtained for oligomycin inhibition. The inhibitory actions of octylguanidine and oligomycin on the ATPase activity are additive. 6. It is concluded that octylguanidine acts directly on the ATPase complex and that its binding at the action site is mutually exclusive with the binding of oligomycin. A kinetic explanation is given for the reported higher sensitivity of site I phosphorylation to octylguanidine.     

Effects of Fluoride on Mitochondrial Activity in Higher Plants

The effects of fluoride on respiration of plant tissue and mitochondria were investigated. Fumigation of young soybean plants (Glycine max Merr. cv. Hawkeye) with 9–12 μg × m^?3 HF caused a stimulation of respiration at about 2 days of treatment followed by inhibition 2 days later. Mitochondria isolated from the stimulated tissue had higher respiration rates, greater ATPase activity, and lower P/O ratios, while in mitochondria from inhibited tissue, all three were reduced. Treatment of etiolated soybean hypocotyl sections in Hoagland's solution containing KF for 3 to 10 h only resulted in inhibition of respiration. Mitochondria isolated from this tissue elicited increased respiration rates with malate as substrate and inhibited respiration with succinate. With both substrates respiratory control and ADP/O ratios were decreased. Direct treatment of mitochondria from the etiolated soybean hypocotyl tissue with fluoride resulted in inhibition of state 3 respiration and lower ADP/O ratios with the substrates succinate, malate, and NADH. Fluoride was also found to increase the amount of osmotically induced swelling and cause a more rapid leakage of protein with mitochondria isolated from etiolated corn shoots (Zea mays L. cv. Golden Cross Bantam). The results are discussed with respect to possible effects of fluoride on mitochondrial membranes.     

Effects of piperine on enzyme activities and bioenergetic functions in isolated rat liver mitochondria and hepatocytes

The influence of piperine on the enzymes and bioenergetic functions in isolated rat liver mitochondria and hepatocytes was studied. Piperine at lower concentrations (<50 μM) did not affect the RCR and ADP:O ratios, state 4 and 3 respirations supported by site-specific substrates, viz. glutamate + malate, succinate, and ascorbate + TMPD. The site-specific effects became significantly apparent only at higher concentrations. Only the state 3 respiration supported by NAD-linked substrates was impaired equipotently in mitochondria and permeabilized hepatocytes; the effect appeared to be localized at energy-coupling site 1. In hypotonic treated mitochondria, respiration supported by three kinds of substrates was not affected. Among the respiratory chain-linked enzymes, the activity of NADH-dehydrogenase registered a significant decrease of about 25, 42, and 53% at 100, 150, and 180 μM piperine, respectively. The activity of Mg⁺⁺-ATPase, however, was stimulated at concentrations above 150 μM. Among the matrix enzymes, only malate and succinate dehydrogen-ases were studied. Malate dehydrogenase only showed a strong concentration-related inhibition in both the forward and backward directions. Enzyme kinetics indicated noncompetitive inhibition with a very low Ki of 10 μM. The presence of unsaturated double bonds in the side chain of piperine appeared essential for producing this strong inhibition. The studies suggested that piperine produces concentration related site-specific effects on mitochondrial bioenergetics and enzymes of energy metabolism.     

ATPase complex and oxidative phosphorylation in chloramphenicol-induced megamitochondria from mouse liver.

1. Functional properties of the ATPase complex are investigated in megamitochondria isolated from livers of weanling mice fed a diet containing 2% chloramphenicol, as an inhibitor of mitochondrial protein synthesis. 2. Whereas the specific activity of ATPase remains unchanged in chloramphenicol-induced megamitochondria, about 40% of the enyzme activity is resistant to inhibition by oligomycin, triethyltin or venturicidin. It is concluded that the ATPase complex lacks one or more components whose synthesis or accumulation is dependent on mitochondrial translation. The inhibitor-resistant ATPase portion appears tightly bound to the mitochondrial membrane. 3. Respiratory chain phosphorylation is tightly coupled in isolated megamitochondria. ATP synthesis and ATP-Pi exchange are diminished by 40%, as compared to control mitochondria, but both processes are sensitive to oligomycin, triethyltin or venturicidin. 4. The decrease in ATP synthesis and ATP-Pi exchange in megamitochondria correlates quite well with the emergence of inhibitor-resistant ATPase. 5. The following electron transport activities in the megmitochondria are reduced: NADH-cytochrome c reductase, by 60%, cytochrome oxidase, by 80%; the amount of antimycin required to gain complete inhibition of the bc1-segment is diminished by more than 50%. On the other hand succinate dehydrogenase activity is increased by 50%. 6. Chloramphenicol-induced megamitochondria appear to be a useful system for studying the role of mitochondrial translation in the assembly of mammalian mitochondria.     

In Phosphorylating <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> Mitochondria the Sensitivity of Uncoupling Protein Activity to GTP Depends on the Redox State of Quinone

In isolated Acanthamoeba castellanii mitochondria respiring in state 3 with external NADH or succinate, the linoleic acid-induced purine nucleotide-sensitive uncoupling protein activity is able to uncouple oxidative phosphorylation. The linoleic acid-induced uncoupling can be inhibited by a purine nucleotide (GTP) when quinone (Q) is sufficiently oxidized, indicating that in A. castellanii mitochondria respiring in state 3, the sensitivity of uncoupling protein activity to GTP depends on the redox state of the membranous Q. Namely, the inhibition of the linoleic acid-induced uncoupling by GTP is not observed in uninhibited state 3 respiration as well as in state 3 respiration progressively inhibited by complex III inhibitors, i.e., when the rate of quinol (QH₂)-oxidizing pathway is decreased. On the contrary, the progressive decrease of state 3 respiration by declining respiratory substrate availability (by succinate uptake limitation or by decreasing external NADH concentration), i.e., when the rate of Q-reducing pathways is decreased, progressively leads to a full inhibitory effect of GTP. Moreover, in A. castellanii mitochondria isolated from cold-treated cells, where a higher uncoupling protein activity is observed, the inhibition of the linoleic acid-induced proton leak by GTP is revealed for the same low values of the Q reduction level.     

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.     

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.     

Action of the antitumor and antispermatogenic agent lonidamine on electron transport in ehrlich ascites tumor mitochondria

The effect of lonidamine, an antispermatogenic and antitumor drug, on the oxygen consumption, ATPase activity, and redox state of the electron carriers of Ehrlich ascites tumor mitochondria has been studied. Lonidamine inhibits ADP- and uncoupler-stimulated respiration on various NAD- and FAD-linked substrates, but does not affect state 4 respiration. Experiments to determine its site of action showed that lonidamine does not significantly inhibit electron flow through cytochrome oxidase. Electron flow through site 2, the ubiquinone-cytochrome b-cytochrome c₁ complex, also was unaffected by lonidamine, which failed to inhibit the oxidation of duroquinol. Moreover, inhibition of electron flow through site 2 was also excluded because of the inability of the N,N,N′,N′-tetramethyl-p-phenylenediamine bypass to relieve the lonidamine inhibition of the oxidation of pyruvate + malate. The F₀F₁ATPase activity and vectorial H⁺ ejection are also unaffected by lonidamine. The inhibition of succinate oxidation by lonidamine was found to take place at a point between succinate and iron-sulfur center S3. Spectroscopic experiments demonstrated that lonidamine inhibits the reduction of mitochondrial NAD⁺ by pyruvate + malate and other NAD-linked substrates in the transition from state 1 to state 4. However, lonidamine does not inhibit reduction of added NAD⁺ by submitochondrial vesicles or by soluble purified NAD-linked dehydrogenases. These observations, together with other evidence, suggest that electron transport in tumor mitochondria is inhibited by lonidamine at the dehydrogenase-coenzyme level, particularly when the electron carriers are in a relatively oxidized state and/or when the inner membrane-matrix compartment is in the condensed state. The action of lonidamine in several respects resembles the selective inhibition of electron transport in tumor cells produced by cytotoxic macrophages.