Rate Of Succinate Oxidation Research Articles

“Ca2+-dependent regulation of the proton permeability of the inner membrane of lupine cotyledon mitochondria”

change in volume, and proton permeability of the inner membrane of mitochondria isolated from cotyledons of etiolated seedlings of narrow-leaved lupine (Lupinus angustifolius L.) was studied. The mitochondria used in the work were characterized by a strong coupling of oxidation and phosphorylation processes. The high functional activity of mitochondria was confirmed by their ability to generate a transmembrane gradient of protons on the inner membrane (membrane potential or ∆Ψ) during the oxidation of succinate and also sustainably maintain it for a long time , both due to the operation of the electron transport chain and due to ATP hydrolysis under conditions of anaerobiosis. It was shown that the presence of 60–120 µM CaCl2 in the mitochondrial incubation medium had no significant effect on the rate of succinate oxidation and the parameters of oxidative phosphorylation; however, it induced ∆Ψ dissipation under conditions of oxygen depletion in the incubation medium. The most complete removal Ca2+ from the medium in the presence of chelators (EGTA, EDTA) prevented the membrane potential dissipation. Ca2+-dependent depolarization of the inner membrane was inhibited by dithiothreitol, suggesting involvement in this process of reactive oxygen species. The reset of the membrane potential was not accompanied by swelling of mitochondria and was not sensitive to cyclosporine A. Using metallochromic Ca2+-indicator arsenazo III, it was shown that the mitochondria of lupine cotyledons are able to actively absorb exogenous Ca2+ and store it in the matrix. Ca2+-induced dissipation of ∆Ψ under conditions of anaerobiosis was accompanied by the release of Ca2+ from mitochondria, the rate of which sharply increased in the presence of calcium ionophore A23 (A23187). It is assumed that the accumulation of Ca2+ and an increase in the level of reactive oxygen species in the matrix induces reversible permeabilization of the inner mitochondrial membrane of lupine cotyledons under conditions of anaerobiosis, which is due to the opening of a pore of nonspecific permeability in a state of low conductivity permeable to protons and, possibly, to other small cations (Na+, K+, Ca2+).

Mitochondrial plasticity in the cerebellum of two anoxia-tolerant sharks: contrasting responses to anoxia/re-oxygenation.

Exposure to anoxia leads to rapid ATP depletion, alters metabolic pathways and exacerbates succinate accumulation. Upon re-oxygenation, the preferential oxidation of accumulated succinate most often impairs mitochondrial function. Few species can survive prolonged periods of hypoxia and anoxia at tropical temperatures and those that do may rely on mitochondria plasticity in response to disruptions to oxygen availability. Two carpet sharks, the epaulette shark (Hemiscyllium ocellatum) and the grey carpet shark (Chiloscyllium punctatum) display different adaptive responses to prolonged anoxia: while H. ocellatum enters energy-conserving metabolic depression, C. punctatum temporarily elevates its haematocrit, prolonging oxygen delivery. High-resolution respirometry was used to investigate mitochondrial function in the cerebellum, a highly metabolically active organ that is oxygen sensitive and vulnerable to injury after anoxia/re-oxygenation (AR). Succinate was titrated into cerebellar preparations in vitro, with or without pre-exposure to AR, then the activity of mitochondrial complexes was examined. As in most vertebrates, C. punctatum mitochondria significantly increased succinate oxidation rates, with impaired complex I function post-AR. In contrast, H. ocellatum mitochondria inhibited succinate oxidation rates and both complex I and II capacities were conserved, resulting in preservation of oxidative phosphorylation capacity post-AR. Divergent mitochondrial plasticity elicited by elevated succinate post-AR parallels the inherently divergent physiological adaptations of these animals to prolonged anoxia, namely the absence (C. punctatum) and presence (H. ocellatum) of metabolic depression. As anoxia tolerance in these species also occurs at temperatures close to that for humans, examining their mitochondrial responses to AR could provide insights for novel interventions in clinical settings.

Medium-chain TAG improve energy metabolism and mitochondrial biogenesis in the liver of intra-uterine growth-retarded and normal-birth-weight weanling piglets.

We previously reported that medium-chain TAG (MCT) could alleviate hepatic oxidative damage in weanling piglets with intra-uterine growth retardation (IUGR). There is a relationship between oxidative status and energy metabolism, a process involved in substrate availability and glucose flux. Therefore, the aim of this study was to investigate the effects of IUGR and MCT on hepatic energy metabolism and mitochondrial function in weanling piglets. Twenty-four IUGR piglets and twenty-four normal-birth-weight (NBW) piglets were fed a diet of either soyabean oil (SO) or MCT from 21 d of postnatal age to 49 d of postnatal age. Then, the piglets' biochemical parameters and gene expressions related to energy metabolism and mitochondrial function were determined (n 4). Compared with NBW, IUGR decreased the ATP contents and succinate oxidation rates in the liver of piglets, and reduced hepatic mitochondrial citrate synthase (CS) activity (P<0·05). IUGR piglets exhibited reductions in hepatic mitochondrial DNA (mtDNA) contents and gene expressions related to mitochondrial biogenesis compared with NBW piglets (P<0·05). The MCT diet increased plasma ghrelin concentration and hepatic CS and succinate dehydrogenase activities, but decreased hepatic pyruvate kinase activity compared with the SO diet (P<0·05). The MCT-fed piglets showed improved mtDNA contents and PPARγ coactivator-1α expression in the liver (P<0·05). The MCT diet alleviated decreased mRNA abundance of the hepatic PPARα induced by IUGR (P<0·05). It can therefore be postulated that MCT may have beneficial effects in improving energy metabolism and mitochondrial function in weanling piglets.

In vitro Toxicological Study of Metal Oxides Nanoparticles on Oxidation of Succinate in Krebs Cycle and Their Resultant Effect in Metabolic Pathways

AbstractIn vitro study to evaluate toxic effects of metal oxides nanoparticles on energy producing cycle was conducted. In this study oxidation of sodium succinate in aqueous solution was investigated by using potassium ferricyanide as an oxidizing agent. Kinetic measurements were carried out on kinetic mode of spectrophotometer at λmax 420 nm. Effect of different metal oxides (MgO and CaO) nanoparticles was observed on the oxidation of succinate at 25 ± 0.5 °C. These metal oxides nanoparticles were prepared by hydrothermal method and their characterization were done by using FTIR, TGA, SEM‐EDX, TEM and XRD. Kinetic results indicated that these nanoparticles inhibit the conversion of succinate into fumerate. The inhibition is directly dependent on particle size of the nanoparticles and it was observed that particle size is inversely related to the oxidation rate of succinate. It was concluded at the end that elevated concentrations of metal oxides nanoparticles can severely affect the Krebs cycle by inhibiting succinate oxidation and lead to various metabolic disorders.

Characteristics of functioning of succinate dehydrogenase from flight muscles of the bumblebee Bombus terrestris (L.)

It was found that the succinate oxidation rate in mitochondria of flight muscles of Bombus terrestris L. in- creased by a factor of 2.15 after flying for 1 h. An electrophoretically homogenous preparation of succinate dehydrogenase with a specific activity of 7.14 U/mg protein and 81.55-fold purity was isolated from B. terrestris flight muscles. It is shown that this enzyme is represented in the muscle tissue by only one isoform with R,f = 0.24. The molecular weight of the native molecule and its subunits A and B was determined. The kinetic characteristics ofsuccinate dehydrogenase (Km = 0.33 mM) and the optimal concentration of hydrogen ions (pH 7.8) were established, and the effect of salts on the enzyme activity was studied. The role of succinate as a respiratory substrate in stress and the structural and functional characteristics of the succinate dehydrogenase system in the flight muscles of insects are discussed.

Hydroxynonenal-stimulated activity of the uncoupling protein in Acanthamoeba castellanii mitochondria under phosphorylating conditions

The influence of 4-hydroxy-2-nonenal (HNE), a lipid peroxidation end product, on the activity of the amoeba Acanthamoeba castellanii uncoupling protein (AcUCP) in isolated phosphorylating mitochondria was studied. Under phosphorylating conditions, exogenously added HNE induced GTP-sensitive AcUCP-mediated mitochondrial uncoupling. The HNE-induced proton leak decreased the yield of oxidative phosphorylation in an HNE concentration-dependent manner. The present study describes how the contributions of ATP synthase and HNE-induced AcUCP in phosphorylating respiration vary when the rate of succinate oxidation is decreased by limiting succinate uptake or inhibiting complex III activity within the range of a constant membrane potential. In phosphorylating mitochondria, at a given HNE concentration (100 μM), the efficiency of AcUCP in mitochondrial uncoupling increased as the respiratory rate decreased because the AcUCP contribution remained constant while the ATP synthase contribution decreased with the respiratory rate. HNE-induced uncoupling can be inhibited by GTP only when ubiquinone is sufficiently oxidized, indicating that in phosphorylating A. castellanii mitochondria, the sensitivity of AcUCP activity to GTP depends on the redox state of the membranous ubiquinone.

C04 Reproductive parameters and mitochondrial function in spermatozoa of F1 and F2 minipig boars transgenic for n-terminal part of the human mutated huntingtin

Testicular degeneration of Huntington9s disease involves the reduction of germ cells and wall thickening of seminiferous tubule. Since changes in ejaculates of patients and mammalians models has not yet been tested, the aim of our study was to analyse motility, amount, fertilising capacity and mitochondrial function of spermatozoa from two HD minipig boars from F1 generation and two WT control animals from 12 to 24 month of age. Random analysis of HD boars from F2 generation at the age of 8 to 12 month was also performed. After semen collections of transgenic and control boars, spermatozoa were prepared for in vitro penetration test. The HD F1 boars showed significantly decreasing tendency in motility, total sperm number and fertilising capacity staring at about 13 month of age in comparison with control boars. The slight depression in sperm quality and quantity was observed in two of four tested HD F2 boars. Evaluating the sperm quantity, motility and fertilising capacity, we can conclude the age related depression in all observed parameters in HD F1 transgenic boars. Mitochondrial energy generating system capacity was analysed in homogenates of minipig boar spermatozoa by measuring oxidation rates and oxygen consumption was determined by high resolution polarography. Decreased oxidation rates were found in spermatozoa of the transgenic F1 minipig boars at the age of 18, 21 and 24 months in comparison with controls. Oxidation rate of succinate and decreased oxygen consumption after addition of succinate indicate impaired function of complex II. Our results demonstrate disturbed bioenergetic function of mitochondria in spermatozoa of transgenic boars. Funding Supported by PRVOUK P24/LF1/3 and TACR Alfa: TA0101146.

A new organosilicon adsorbent for air-gas mixture purification

Natural zeolites (klinoptiolits) were treated with Onkosorb adsorbent (N,N′-bis(3-triethoxysilyl-propyl-thiocarbamide)) to attain greater nitrosamine (NA) and polycyclic aromatic hydrocarbon (PAH) adsorption capacity. The efficiency of volatile NA recovery rose from 30 to 77%; that of PAH recovery, from 20 to 63.7 %. The increased adsorption efficiency of the toxicants from an air-gas mixture was also reflected in biological indicators. Prolonged exposure of rats to the air-gas mixture (similar in composition to the air in industrial zones) led to a 25% reduction in the maximum rates of succinate oxidation and the efficiency of oxidative phosphorylation in liver mitochondria. The changes in the energetics of mitochondria were apparently associated with the action of the toxicants, as is indicated by a 22 % increase of P-450 cytochrome content in the liver tissue. An air-gas mixture passed through an Onkosorb adsorbent layer on zeolite did not produce any of the above changes in the mitochondrion energetics or the effect on P-450 cytochrome content in the liver tissue. We assume a high level of efficiency for Onkosorb adsorbent on zeolite in PAH and NA absorption.

Effect of Bovine Serum Albumin on Mitochondrial Respiration in the Brain and Liver of Mice and Rats

We studied the effect of BSA (in the isolation medium) on the oxidation rate of succinate, glutamate, pyruvate, and α-ketoglutarate by mitochondria of the brain and liver from C57Bl/6g mice and Taconic Sprague Dawley rats. BSA had no effect on liver mitochondrial respiration, but increased oxidation of substrates (particularly of succinate) in brain mitochondria. Therefore, the major effect of BSA on brain mitochondria is manifested in activation of SDH. The improvement of mitochondrial properties in the brain after treatment with BSA is associated with antioxidant activity of this agent. Our results confirm the hypothesis that inhibition of SDH in brain mitochondria is not the artifact. This process serves as a mechanism protecting neurons from free oxygen radicals during succinate oxidation.

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.

Energization of Bacillus subtilis membrane vesicles increases catalytic activity of succinate: Menaquinone oxidoreductase

In this work, high DeltamicroH+-dependent succinate oxidase activity has been demonstrated for the first time with membrane vesicles isolated from Bacillus subtilis. The maximal specific rate of succinate oxidation by coupled inside-out membrane vesicles isolated from a B. subtilis strain overproducing succinate:menaquinone oxidoreductase approaches the specific rate observed with the intact cells. Deenergization of the membrane vesicles with ionophores or alamethicin brings about an almost complete inhibition of succinate oxidation. An apparent K(m) for succinate during the energy-dependent succinate oxidase activity of the vesicles (2.2 mM) is higher by an order of magnitude than the K(m) value measured for the energy-independent reduction of 2,6-dichlorophenol indophenol. The data reveal critical importance of DeltamicroH+ for maintaining active electron transfer by succinate:menaquinone oxidoreductase. The role of DeltamicroH+ might consist in providing energy for thermodynamically unfavorable menaquinone reduction by succinate by virtue of transmembrane electron transport within the enzyme down the electric field; alternatively, DeltamicroH+ could play a regulatory role by maintaining the electroneutrally operating enzyme in a catalytically active conformation.

Characteristics of the succinate transport into Saccharomices cerevisiae cellsafter prolonged cold preincubation

A nonconventional approach to the measurement of succinate transport through plasmalemma is proposed. It is based on the conditions in which the succinate oxidation rate is limited by transport through plasmalemma. Impermeable specific inhibitor of plasmalemma dicarboxylate transporter was employed as a tool to optimize conditions for the transport activity assay. For this purpose yeast culture was grown in synthetic medium under selected conditions. After aerobic preincubation of S. cerevisiae cells at 0 degree C (instead of incubation at 15 degrees C), the rate of endogenous respiration decreased substantially and was stabilized during measurements at a level that was five times lower than oxidation rates in the presence of exogenous substrate. This approach allowed for the reproducible determination of K(M) for the dicarboxylate transporter (7.3 +/- 2.1 mM) within a half-hour period. The advantages and drawbacks of this fast, but indirect, assay of slow substrates transport into the cell are compared with conventional methods.

The Neuromediator Glutamate, through Specific Substrate Interactions, Enhances Mitochondrial ATP Production and Reactive Oxygen Species Generation in Nonsynaptic Brain Mitochondria

The finding that upon neuronal activation glutamate is transported postsynaptically from synaptic clefts and increased lactate availability for neurons suggest that brain mitochondria (BM) utilize a mixture of substrates, namely pyruvate, glutamate, and the tricarboxylic acid cycle metabolites. We studied how glutamate affected oxidative phosphorylation and reactive oxygen species (ROS) production in rat BM oxidizing pyruvate + malate or succinate. Simultaneous oxidation of glutamate + pyruvate + malate increased state 3 and uncoupled respiration by 52 and 71%, respectively. The state 4 ROS generation increased 100% over BM oxidizing pyruvate + malate and 900% over that of BM oxidizing glutamate + malate. Up to 70% of ROS generation was associated with reverse electron transport. These effects of pyruvate + glutamate + malate were observed only with BM and not with liver or heart mitochondria. The effects of glutamate + pyruvate on succinate-supported respiration and ROS generation were not organ-specific and depended only on whether mitochondria were isolated with or without bovine serum albumin. With the non-bovine serum albumin brain and heart mitochondria oxidizing succinate, the addition of pyruvate and glutamate abrogated inhibition of Complex II by oxaloacetate. We conclude that (i) during neuronal activation, simultaneous oxidation of glutamate + pyruvate temporarily enhances neuronal mitochondrial ATP production, and (ii) intrinsic inhibition of Complex II by oxaloacetate is an inherent mechanism that protects against ROS generation during reverse electron transport.

Respiration, hydrogen peroxide levels and antioxidant enzyme activities during cold storage of zucchini squash fruit

Steady-state levels of hydrogen peroxide and activities of catalase and peroxidase were measured in the peel and pulp of zucchini squash ( Cucurbita pepo L., cv., Giambo) during storage at 10 °C or 0 °C for two weeks. No visible damage occurred during this storage time; epidermal brown pits become visible later, at day 20 in fruit stored at 10 °C and at day 15 in those stored at 0 °C. In order to analyze the early effects of the chilling-induced oxidative stress during zucchini storage, rates of succinate oxidation, alternative oxidase activity, membrane fluidity and phospholipid composition were also measured in mitochondria isolated from the zucchini pulp. A decrease in hydrogen peroxide levels, an increase in the activity of detoxifying enzymes, a recovery of chilling-induced mitochondrial membrane fluidity and an increase in alternative oxidase (AOX) activity were detected in the early stages of zucchini storage at 10 °C. The peroxidase and the AOX activities of the pulp of zucchini stored at 0 °C were also found to increase but to a lesser extent and later during storage, suggesting that these fruit can also activate these ROS regulatory systems, possibly preventing the occurrence of early visible damage in the peel but not the occurrence of cold stress.

Plasmalemma dicarboxylate transporter of Saccharomyces cerevisiae is involved in citrate and succinate influx and is modulated by pH and cations

Succinate and citrate transport into yeast (Saccharomyces cerevisiae) cells was studied by measuring substrate oxidation rates in the presence and in the absence of effective impermeable oxidation inhibitors O-palmitoyl-L-malate and 2-undecyl malonate. Linearity of the Dixon plot for 2-undecyl malonate suggests that this inhibitor blocked the rate-limiting step upon oxidation of both substrates, which was, most probably, transport of these substrates across the plasma membrane (due to inability of the inhibitor to penetrate into the membrane). This approach allowed fast (within 30–40 min) measurement of kinetic parameters of the transporter in individual samples without losing control over limiting conditions. In case of succinate transport, the limiting rate of succinate oxidation (V max) depended on pH and increased monotonously from near-zero at pH 4.5 to the maximum level at pH 7.5. At pH 5.5, succinate and citrate transport was insensitive to the protonophore FCCP, being activated by Na+ ions and competitively inhibited by 2-undecyl malonate and K+ ions. Values of K i for 2-undecyl malonate were similar for both substrates. These data suggest that citrate and succinate influx is mediated by a common plasma membrane transporter. This is not typical of fungi. At pH 6.5, Tris+, K+ and Na+ had no effect on succinate oxidation. In monosodium media pH increase was accompanied by a decrease of succinate K m due to higher proportion of the dianionic form of the substrate. Atypical substrate specificity and mechanisms of functional activity of the dicarboxylate transporter in plasma membrane of S. cerevisiae are discussed.

Properties of yeast Saccharomyces cerevisiae plasma membrane dicarboxylate transporter

Transport of succinate into Saccharomyces cerevisiae cells was determined using the endogenous coupled mitochondrial succinate oxidase system. The dependence of succinate oxidation rate on the substrate concentration was a curve with saturation. At neutral pH the K(m) value of the mitochondrial "succinate oxidase" was fivefold less than that of the cellular "succinate oxidase". O-Palmitoyl-L-malate, not penetrating across the plasma membrane, completely inhibited cell respiration in the presence of succinate but not glucose or pyruvate. The linear inhibition in Dixon plots indicates that the rate of succinate oxidation is limited by its transport across the plasmalemma. O-Palmitoyl-L-malate and L-malate were competitive inhibitors (the K(i) values were 6.6 +/- 1.3 microM and 17.5 +/- 1.1 mM, respectively). The rate of succinate transport was also competitively inhibited by the malonate derivative 2-undecyl malonate (K(i) = 7.8 +/- 1.2 microM) but not phosphate. Succinate transport across the plasma membrane of S. cerevisiae is not coupled with proton transport, but sodium ions are necessary. The plasma membrane of S. cerevisiae is established to have a carrier catalyzing the transport of dicarboxylates (succinate and possibly L-malate and malonate).

Changes in mitochondrial electron transport chain activity during insect metamorphosis

The midgut of the tobacco hornworm (Manduca sexta) is a highly aerobic tissue that is destroyed by programmed cell death during larval-pupal metamorphosis. The death of the epithelium begins after commitment to pupation, and the oxygen consumption of isolated midgut mitochondria decreases soon after commitment. To assess the role of the electron transport chain in this decline in mitochondrial function, the maximal activities of complexes I-IV of the respiratory chain were measured in isolated midgut mitochondria. Whereas there were no developmental changes in the activity of complex I or III, activities of complexes II and IV [cytochrome c oxidase (COX)] were higher in mitochondria from precommitment than postcommitment larvae. This finding is consistent with a higher rate of succinate oxidation in mitochondria isolated from precommitment larvae and reveals that the metamorphic decline in mitochondrial respiration is due to the targeted destruction or inactivation of specific sites within the mitochondria, rather than the indiscriminate destruction of the organelles. The COX turnover number (e- x s(-1) x cytochrome aa3(-1)) was greater for the enzyme from precommitment than postcommitment larvae, indicating a change in the enzyme structure and/or its lipid environment during the early stages of metamorphosis. The turnover number of COX in the intact mitochondria (in organello COX) was also lower in postcommitment larvae. In addition to changes in the protein or membrane phospholipids, the metamorphic decline in this rate constant may be a result of the observed loss of endogenous cytochrome c.

Contribution of ATP synthase to stimulation of respiration by Ca2+ in heart mitochondria

A variety of experimental conditions were applied with the aim to estimate the correlation between the contribution of ATP synthase to the respiratory flux control and the calcium-induced activation of succinate oxidation in heart mitochondria isolated from rat, rabbit and guinea pig. The sensitivity of respiration in heart mitochondria to the decrease in temperature from 37 degrees C to 28 degrees C decreases in the order rabbit > guinea pig > rat. Ca2+ effect on succinate oxidation rate in state 3 respiration was species- and temperature-dependent and ranged from 0 (rat, 37 degrees C) to +44% (rabbit, 28 degrees C). For mitochondria from all experimental animals, the increase of Ca2+ in physiological range of concentration did not change state 2 respiration rate, and the stimulatory effect of Ca2+ on state 3 respiration was more pronounced at 28 degrees C than at 37 degrees C. The respiratory subsystem was sensitive to Ca2+ ions only in rabbit heart mitochondria. A high positive correlation between Ca2+ ability to stimulate succinate oxidation in state 3 and the control exerted by ATP synthase over the respiratory flux provides argument confirming stimulation of ATP synthase by Ca2+ ions.

Protective Effect of Bacoside A on Cigarette Smoking-Induced Brain Mitochondrial Dysfunction in Rats

Chronic exposure to cigarette smoke affects the structure and function of mitochondria, which may account for the pathogenesis of smoking-related diseases. Bacopa monniera Linn., used in traditional Indian medicine for various neurological disorders, was shown to possess mitrochondrial membrane-stabilizing properties in the rat brain during exposure to morphine. We investigated the protective effect of bacoside A, the active principle of Bacopa monniera, against mitochondrial dysfunction in rat brain induced by cigarette smoke. Male Wistar albino rats were exposed to cigarette smoke and administered bacoside A for a period of 12 weeks. The mitochondrial damage in the brain was assessed by examining the levels of lipid peroxides, cholesterol, phospholipid, cholesterol/phospholipid (C/P) ratio, and the activities of isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, NADH dehydrogenase, and cytochrome C oxidase. The oxidative phosphorylation (rate of succinate oxidation, respiratory control ratio and ADP/O ratio, and the levels of ATP) was evaluated for the assessment of mitochondrial functional capacity. We found significantly elevated levels of lipid peroxides, cholesterol, and C/P ratio, and decreased levels of phospholipids and mitochondrial enzymes in the rats exposed to cigarette smoke. Measurement of oxidative phosphorylation revealed a marked depletion in all the variables studied. Administration of bacoside A prevented the structural and functional impairment of mitochondria upon exposure to cigarette smoke. From the results, we suggest that chronic cigarette smoke exposure induces damage to the mitochondria and that bacoside A protects the brain from this damage by maintaining the structural and functional integrity of the mitochondrial membrane.

Cyclosporin A-sensitive cytochrome c release and activation of external pathway of NADH oxidation in liver mitochondria due to pore opening by acidification of phosphate-containing incubation medium.

Acidification of a high phosphate incubation medium from pH 7.4 to 6.5 promotes increase in rates of succinate oxidation and exogenous NADH oxidation via external (rotenone-and myxothiazol-resistant) pathway by factors 2 and 2.3 respectively. Cyclosporin A prevents these effects. To measure the cytochrome c release, mitochondrial cytochrome c concentration was calculated from absorption spectrum of alpha-band of cytochromes c + c1. The cytochrome c release is shown to be equal to 27 +/- 4%, 40 +/- 12%, 70 +/- 5% at pH 7.4, 7.0, 6.5, respectively, the last value being reduced by cyclosporin A to 10 +/- 3%. Immunoblot method gives the similar results. It is concluded that acidification of the high phosphate medium induces release of a large part of the cytochrome c pool from liver mitochondria due to opening the Ca(2+)-dependent cyclosporin A-sensitive permeability transition pore and subsequent high amplitude swelling.