Development and mechanism of barbiturate tolerance in glial cell cultures

The effects of exposure of glial cells in primary culture and in continuous line (clone NN) to pentobarbital over various periods of time on cellular respiration and activities of enzymes involved in carbohydrate metabolism were studied. The results obtained in glial cells in primary culture were qualitatively identical to those obtained in glial cells in clonal line (NN). Both types of glial cells were shown to develop biochemical tolerance to pentobarbital as defined by an attenuated response to the depressant effects of a challenging dose of pentobarbital on cellular respiration in barbiturate-cultivated cells compared to those grown in drug-free medium. The biochemical tolerance was evident in the presence of glucose and succinate but not malate as substrate. This tolerance to pentobarbital was accompanied by increased activities of hexokinase, glucose-6-phosphate dehydrogenase, succinate dehydrogenase, and glutamate dehydrogenase and by a marked increase in the number of glial cell mitochondria as observed in electron micrographs. The results are interpreted to indicate a compensation of glial cells to the continuous presence of PB by an accelerated glucose uptake and metabolism, an accelerated metabolism of succinate, and an increased mitochondrial activity.     

Kinetics of substrate oxidation by whole cells and cell membranes of Helicobacter pylori

Abstract Oxygen uptake by Helicobacter pylori cells and membranes was determined. Cells from stirred broth cultures or agar plates, suspended in buffer, possessed a variable and apparently endogenous respiration which could be sustained for several hours. In contrast, oxygen consumption by cells from statically incubated broth cultures, in the absence of added substrate, was transient or undetectable. These latter cells, however, oxidised ethanol, fumarate, glucose, d-lactate, pyruvate and succinate, though glucose-oxidising ability declined rapidly. The K _m s for d-lactate, pyruvate and succinate metabolism were low (≤20 μM) and oxygen uptake was approximately 1.5, 2 and 2 mol per mol substrate respectively, indicating metabolism beyond acetate plus CO₂ and implying the presence of tricarboxylic acid cycle activity. Cell membranes oxidised fumarate, d-lactate, NADH, NADPH and succinate. NADPH oxidation was six times more rapid than that of NADH. Rates of oxygen uptake by cells suspended in buffer with metabolisable substrate were < 20% of those for cells suspended in a brain heart infusion medium. Uninoculated medium consumed significant quantities of oxygen.     

Investigation of mitochondrial dysfunction by sequential microplate-based respiration measurements from intact and permeabilized neurons

Mitochondrial dysfunction is a component of many neurodegenerative conditions. Measurement of oxygen consumption from intact neurons enables evaluation of mitochondrial bioenergetics under conditions that are more physiologically realistic compared to isolated mitochondria. However, mechanistic analysis of mitochondrial function in cells is complicated by changing energy demands and lack of substrate control. Here we describe a technique for sequentially measuring respiration from intact and saponin-permeabilized cortical neurons on single microplates. This technique allows control of substrates to individual electron transport chain complexes following permeabilization, as well as side-by-side comparisons to intact cells. To illustrate the utility of the technique, we demonstrate that inhibition of respiration by the drug KB-R7943 in intact neurons is relieved by delivery of the complex II substrate succinate, but not by complex I substrates, via acute saponin permeabilization. In contrast, methyl succinate, a putative cell permeable complex II substrate, failed to rescue respiration in intact neurons and was a poor complex II substrate in permeabilized cells. Sequential measurements of intact and permeabilized cell respiration should be particularly useful for evaluating indirect mitochondrial toxicity due to drugs or cellular signaling events which cannot be readily studied using isolated mitochondria.     

Preparation of enriched fractions from cerebral cortex containing isolated, metabolically active neuronal and glial cells

1. A procedure has been developed for the separation of intact metabolically active neuronal and glial cells in bulk from rat cerebral cortex. Separation depended on dispersion of the tissue in a Ficoll medium followed by centrifugation on a discontinuous Ficoll gradient. Up to 1.5x10(7) neuronal cells could be collected from 12 brains within 3hr. The morphological appearance of these cells seemed good, and the fraction was 8.5-fold purified in terms of dry weight. Average dry weight per neuron was 2300mumug. Maximum glial contamination of the neuronal fraction was 11% as determined by carbonic anhydrase measurements. The glial fraction was free from neurons but contained various subcellular contaminants. 2. Concentrations of nucleic acids, phospholipid, protein and phosphoprotein were determined in the separated fractions. The neuronal fraction was richer than the glial in all except phospholipid. Succinate dehydrogenase was equally distributed between neurons and glia but the neuronal fraction was 1.8-fold enriched in cytochrome oxidase. 3. Measurement of respiration by the cells showed an endogenous uptake of 117mmumoles of oxygen/mg./hr. in neurons, and 173mmumoles of oxygen/mg./hr. in glia. Addition of substrate at 10mm stimulated uptake to similar values in both fractions. With glucose it was 390, with pyruvate 355, and with glutamate 215mmumoles of oxygen/mg./hr. This represented a larger stimulation of neuronal than of glial respiration compared with the basal level. 4. Respiration in cell suspensions was 70-80% of that of slices, whereas fractionated tissue homogenates had respiratory rates of only one-third those of the cell suspensions. Lactate dehydrogenase content of cell suspensions was maintained during gradient centrifugation and washing. 5. The possible uses of isolated cell preparations are discussed.     

Regulation of Alternative Oxidase Activity by Pyruvate in Soybean Mitochondria

The regulation of alternative oxidase activity by the effector pyruvate was investigated in soybean (Glycine max L.) mitochondria using developmental changes in roots and cotyledons to vary the respiratory capacity of the mitochondria. Rates of cyanide-insensitive oxygen uptake by soybean root mitochondria declined with seedling age. Immunologically detectable protein levels increased slightly with age, and mitochondria from younger, more active roots had less of the protein in the reduced form. Addition of pyruvate stimulated cyanide-insensitive respiration in root mitochondria, up to the same rate, regardless of seedling age. This stimulation was reversed rapidly upon removal of pyruvate, either by pelleting mitochondria (with succinate as substrate) or by adding lactate dehydrogenase with NADH as substrate. In mitochondria from cotyledons of the same seedlings, cyanide-insensitive NADH oxidation was less dependent on added pyruvate, partly due to intramitochondrial generation of pyruvate from endogenous substrates. Cyanide-insensitive oxygen uptake with succinate as substrate was greater than that with NADH, in both root and cotyledon mitochondria, but this difference became much less when an increase in external pH was used to inhibit intramitochondrial pyruvate production via malic enzyme. Malic enzyme activity in root mitochondria declined with seedling age. The results indicate that the activity of the alternative oxidase in soybean mitochondria is very dependent on the presence of pyruvate: differences in the generation of intramitochondrial pyruvate can explain differences in alternative oxidase activity between tissues and substrates, and some of the changes that occur during seedling development.     

Brown adipose tissue mitochondria: modulation by GDP and fatty acids depends on the respiratory substrates

The UCP1 [first UCP (uncoupling protein)] that is found in the mitochondria of brown adipocytes [BAT (brown adipose tissue)] regulates the heat production, a process linked to non-shivering thermogenesis. The activity of UCP1 is modulated by GDP and fatty acids. In this report, we demonstrate that respiration and heat released by BAT mitochondria vary depending on the respiratory substrate utilized and the coupling state of the mitochondria. It has already been established that, in the presence of pyruvate/malate, BAT mitochondria are coupled by faf-BSA (fatty-acid-free BSA) and GDP, leading to an increase in ATP synthesis and mitochondrial membrane potential along with simultaneous decreases in both the rates of respiration and heat production. Oleate restores the uncoupled state, inhibiting ATP synthesis and increasing the rates of both respiration and heat production. We now show that in the presence of succinate: (i) the rates of uncoupled mitochondria respiration and heat production are five times slower than in the presence of pyruvate/malate; (ii) faf-BSA and GDP accelerate heat and respiration as a result and, in coupled mitochondria, these two rates are accelerated compared with pyruvate/malate; (iii) in spite of the differences in respiration and heat production noted with the two substrates, the membrane potential and the ATP synthesized were the same; and (iv) oleate promoted a decrease in heat production and respiration in coupled mitochondria, an effect different from that observed using pyruvate/malate. These effects are not related to the production of ROS (reactive oxygen species). We suggest that succinate could stimulate a new route to heat production in BAT mitochondria.     

Flavoprotein-linked substrate oxidation in preparations of hamster brown adipocytes. A discrimination between internally and externally oxidized substrates

Noradrenaline-stimulated oxidative metabolism in isolated hamster brown fat cells is very reproducible between different cell preparations, 565 +/- 81 (S.D.) nmol O/min per 10(6) cells (n = 25). In contrast, the oxygen consumption rate induced by the addition of succinate or sn-glycerol 3-phosphate strongly varies between different cell preparation, although these substances have been reported to be potent substrates for isolated hamster brown fat cells. By filtration and by successive washings we demonstrate that the flavoprotein-linked substrate oxidation is mainly dependent on extracellular succinate and sn-glycerol 3-phosphate-oxidizing enzymes. These enzymes originate from damaged and broken cells and are present in different amounts in different cell preparations. In discriminating between intra- and extracellular succinate oxidation 5,5'- dithiobis(2-nitrobenzoate) is used as an inhibitor of the extracellular portion. This application of 5,5'-dithiobis(2-nitrobenzoate) ought to be useful also in other cell or tissue preparations. Added succinate can, however, be oxidized by the intact brown adipocyte but at very low rate, probably as a result of a limited transport rate over the membrane(s). In the presence of noradrenaline, added succinate can potentiate the noradrenaline-inducible oxygen consumption by catalytically increasing the oxidative capacity of the citric acid cycle. Our conclusions is that the only effectors which significantly increase oxidative metabolism in intact isolated hamster brown fat cells are catecholamines and free fatty acids. Provided the cells are uncoupled, also pyruvate can function as substrate for these cells.     

Use of β-Methylene-D,L-Aspartate to Assess the Role of Aspartate Aminotransferase in Cerebral Oxidative Metabolism

Several inhibitors of aspartate aminotransferase, a key enzyme of the malate-aspartate shuttle, were investigated for their effects on cerebral oxidative metabolism in vitro. beta-Methylene-D,L-aspartate (2 mM), aminooxyacetate (0.1 mM), and D,L-vinylglycine (20 mM) all significantly reduced the activity of aspartate aminotransferase and the rate of oxygen consumption of rat cerebral cortex slices respiring on glucose. In the presence of beta-methyleneaspartate, a one-to-one correlation was found between the degree of inhibition of tissue respiration and the degree of inhibition of transaminase activity. Slices of rat liver incubated in the presence of glucose and beta-methyleneaspartate showed a similar one-to-one relationship between inhibition of oxygen comsumption and inhibition of aspartate aminotransferase activity, whereas with rat kidney cortex slices, the inhibition of aspartate aminotransferase activity was greater than the inhibition of oxygen consumption. Structural analogs of beta-methyleneaspartate (D,L-beta-methyl-D,L-aspartate, gamma-methyl-D,L-glutamate, and alpha-methyl-D,L-didehydroglutamate) that did not inhibit the activity of aspartate aminotransferase similarly did not inhibit the rate of oxygen consumption by cerebral cortex slices. In the presence of beta-methyleneaspartate, pyruvate oxidation by cerebral cortex slices was inhibited to almost the same extent as was glucose oxidation, and the oxidation of succinate was decreased by approximately 20%. The artificial electron acceptor phenazine methosulfate (0.1 mM) only partially overcame the beta-methyleneaspartate-mediated inhibition of respiration with glucose as substrate. The content of ATP and phosphocreatine declined steadily in slices incubated with glucose and beta-methyleneaspartate. At 1 h the concentration of lactate and the lactate/pyruvate ratio, an indicator of the cytoplasmic redox state, increased threefold, whereas the concentrations of malate, citrate, and aspartate decreased. The findings are interpreted in the context of the hypothesis that enzymes common to the malate-aspartate shuttle and the tricarboxylic acid cycle are physically complexed in brain, so that inhibition of aspartate aminotransferase, a component of the complex, impedes the flow of carbon through both metabolic pathways.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of various respiration substrates and growth marker proteins on oxygen consumption in rabbit heart mitochondria

Oxygen uptake by myocardium mitochondria of healthy, carcinomatous and new-born rabbits in the presence of different substrates was studied under the effect of immunoglobulin G and beta-globulin peculiar to the normal and malignant growth. It is stated that the growth marker proteins representing a subfraction of immunoglobulin G of tumour patients blood serum and beta-globulin of new-born rabbits blood serum in the presence of pyruvate, alpha-ketoglutarate and glutamate inhibit the oxygen uptake by healthy rabbits heart mitochondria. Studies conducted on mitochondria of new-born and carcinomatous rabbits show that the action of the proteins under study depends on the respiration substrate. In the presence of succinate the proteins under study activate the oxygen uptake and pyruvate, alpha-ketoglutarate and glutamate inhibit this process. A conclusion is drawn that the effect of proteins peculiar to the growth process on the biological oxidation depends both on the substrate and structural state of mitochondria.     

An implication of novel methodology to study pancreatic acinar mitochondria under in situ conditions

Mitochondria maintain numerous energy‐consuming processes in pancreatic acinar cells, yet characteristics of pancreatic mitochondrial oxidative phosphorylation in native conditions are poorly studied. Besides, it is not known which type of solution is most adequate to preserve functions of pancreatic mitochondria in situ. Here we propose a novel experimental protocol suitable for in situ analysis of pancreatic mitochondria metabolic states. Isolated rat pancreatic acini were permeabilized with low doses of digitonin. Different metabolic states of mitochondria were examined in KCl‐ and sucrose‐based solutions using Clark oxygen electrode. Respiration of digitonin‐treated, unlike of intact, acini was substantially intensified by succinate or mixture of pyruvate plus malate. Substrate‐stimulated respiration rate did not depend on solution composition. In sucrose‐based solution, oligomycin inhibited State 3 respiration at succinate oxidation by 65.4% and at pyruvate plus malate oxidation by 60.2%, whereas in KCl‐based solution, by 32.0% and 36.1%, respectively. Apparent respiratory control indices were considerably higher in sucrose‐based solution. Rotenone or thenoyltrifluoroacetone severely inhibited respiration, stimulated by pyruvate plus malate or succinate, respectively. This revealed low levels of non‐mitochondrial oxygen consumption of permeabilized acinar cells. These results suggest a stronger coupling between respiration and oxidative phosphorylation in sucrose‐based solution. Copyright © 2012 John Wiley & Sons, Ltd.     

Oxidation of acetoacetate and palmitylcarnitine by brain and liver mitochondria from suckling and adult rats

Experiments in which we investigated the possible oxidative utilization of lipoid substrates by brain and liver mitochondria were carried out with rats aged 5 and 90 days, kept under completely standardized conditions. Brain mitochondria were isolated on a Ficoll gradient after Clark and Nicklas (1970). Respiratory activity (or the respiratory control index-R.C.) was determined in the manner described in an earlier paper (Dobesová and Mourek 1980). Na succinate or Na malate was used as the testing substrate; palmityl carnitine, acetyl carnitine and acetoacetate were used as lipoid substrates. Oxygen consumption was measured with a Clark's oxygen electrode and respiration was expressed in nAt oxygen per min per mg protein, which was measured by the method of Lowry et al. (1951). When using succinate or malate, in agreement with our previous results we did not find any development changes in the respiratory activity of the brain mitochondria. The oxidation of acetoacetate by the brain mitochondria of 5-day-old rats was about five times greater, and of acetyl carnitine over two times greater, compared with the CNS mitochondria of adult rats. The oxidative utilization of lipoid substrates by the liver mitochondria of 5-day-old rats was significantly greater than their utilization by CNS mitochondria (in the case of palmityl carnitine three times greater, for example) and was always significantly greater than in the liver mitochondria of adult rats. We demonstrated that mitochondria isolated from the brain of 5-day-old rats are equipped with an enzymatic apparatus which allows them to utilize lipoid substrates on a significantly greater scale than in adulthood.     

K+--induced changes of oxygen uptake by neuronal enriched and glial enriched fractions from mouse brain cortex.

Neuronal and glial enriched fractions were incubated in a medium with 10mM pyruvate, 5mM fumarate and 0.9mM 5'-AMP and the effect of increased external K+ concentrations was studied upon oxygen uptake. A concentration of 65 mM K+ had a different effect on the oxygen consumption of glial and neuronal perikarya. The rate of oxygen uptake by glia was stimulated by 52.81% whilst an insignificant decrease of 15.79% occurred in the neurones. The highest rate of oxygen uptake by incubated cells was estimated in the presence of the substrate system containing pyruvate, fumarate and 5'-AMP. The significance of components in the substrate system for a high rate of oxygen uptake by cells was also tested with 6.2 mM K+ and 65 mM K+.     

Relationship of energy production to gluconeogenesis in renal cortical tubules.

Isolated tubules prepared by collagenase treatment of rat renal cortex retained their ultrastructural integrity and responded to added lactate and succinate with an increase in gluconeogenesis and respiration. Inhibition of the mitochondrial respiratory chain with rotenone, or energy conservation sites with oligomycin caused a marked reduction in respiration and ATP content thereby completely inhibiting net gluconeogenesis. Dissociation of gluconeogenesis from respiration was accomplished with quinolinic acid and hydrazine, inhibitors of gluconeogenesis. At 5 times 10(-3) M quinolinic acid, gluconeogenesis from succinate was inhibited approximately 50% and from lactate nearly 100%. This concentration of quinolinic acid did not affect oxygen uptake or the ATP content of tubules in the presence or absence of substrate. Hydrazine at 10(-3) M resulted in approximately 75% inhibition of glucose formation from succinate and complete inhibition from lactate without interfering with respiration or ATP content. The increased mitochondrial energy generation, as manifested by accelerated respiration was independent of gluconeogenesis. The unchanging cell ATP concentration with a higher respiratory rate upon addition of exogenous substrate bespeaks increased ATP turnover. ATP utilization for the substrate-induced enhancement of gluconeogenesis could not account for the increment in ATP hydrolysis.     

Flavoprotein-linked substrate oxidation in preparations of hamster brown adipocytes: A discrimination between internally and externally oxidized substrates

Noradrenaline-stimulated oxidative metabolism in isolated hamster brown fat cells is very reproducible between different cell preparations, 565 ± 81 (S.D.) nmol O/min per 10⁶ cells (n = 25).In contrast, the oxygen consumption rate induced by the addition of succinate or sn-glycerol 3-phosphate strongly varies between different cell preparation, although these substances have been reported to be potent substrates for isolated hamster brown fat cells.By filtration and by successive washings we demonstrate that the flavoprotein-linked substrate oxidation is mainly dependent on extracellular succinate and sn-glycerol 3-phosphate-oxidizing enzymes. These enzymes originate from damaged and broken cells and are present in different amounts in different cell preparations.In discriminating between intra- and extracellular succinate oxidation 5,5′-dithiobis(2-nitrobenzoate) is used as an inhibitor of the extracellular portion. This application of 5,5′-dithiobis(2-nitrobenzoate) ougth to be useful also in other cell or tissue preparations.Added succinate can, however, be oxidized by the intact brown adipocyte but a very low rate, probably as a result of a limited transport rate over the membrane(s). In the presence of noradrenaline, added succinated can potentiate the noradrenaline-inducible oxygen consumption by catalytically increasing the oxidative capacity of the citric acid cycle.Our conclusions is that the only effectors which significantly increase oxidative metabolism in intact isolated hamster brown fat cells are catecholamines and free fatty acids. Provided the cells are uncoupled, also pyruvate can function as substrate for these cells.     

Synaptosomal bioenergetics. The role of glycolysis, pyruvate oxidation and responses to hypoglycaemia

The bioenergetic interaction between glycolysis and oxidative phosphorylation in isolated nerve terminals (synaptosomes) from guinea-pig cerebral cortex is characterized. Essentially all synaptosomes contain functioning mitochondria. There is a tight coupling between glycolytic rate and respiration: uncoupler causes a tenfold increase in glycolysis and a sixfold increase in respiration. Synaptosomes contain little endogenous glycolytic substrate and glycolysis is dependent on external glucose. In glucose-free media, or following addition of iodoacetate, synaptosomes continue to respire and to maintain high ATP/ADP ratios. In contrast to glucose, the endogenous substrate can neither maintain high respiration in the presence of uncoupler nor generate ATP in the presence of cyanide. Pyruvate, but not succinate, is an excellent substrate for intact synaptosomes. The in-situ mitochondrial membrane potential (delta psi m) is highly dependent upon the availability of glycolytic or exogenous pyruvate; glucose deprivation causes a 20-mV depolarization, while added pyruvate causes a 6-mV hyperpolarization even in the presence of glucose. Inhibition of pyruvate dehydrogenase by arsenite or pyruvate transport by alpha-cyano-4-hydroxycinnamate has little effect on ATP/ADP ratios; however respiratory capacity is severely restricted. It is concluded that synaptosomes are valuable models for studying the control of mitochondrial substrate supply in situ.     

Intermediary carbon metabolism of Azospirillum brasilense.

Azospirillum brasilense Sp 7 grew rapidly in AZO medium containing reduced nitrogen and succinate as an energy source, with a doubling time of 43 min. No growth was measured with glucose as the sole carbon source. In contrast, Azospirillum lipoferum Sp 59b could grow in media containing either succinate or glucose with a doubling time of 69 min and 223 min, respectively. Warburg-Barcroft respirometry showed that the rate of oxygen consumption by A. brasilense Sp 7 on glucose medium (0.034 mumol of O2 min-1 mg-1 of cell protein) was only one-quarter of that on succinate medium (0.14 mumol of O2 min-1 mg-1). Radioisotopic labeling showed that very little glucose was assimilated by A. brasilense Sp 7 as compared to succinate. High respiration rates were measured on A. lipoferum Sp 59b with either succinate (0.15 mumol of O2 min-1 mg-1) or glucose (0.13 mumol of O2 min-1 mg-1) as the sole carbon source. The pattern of CO2 evolution from differentially labeled succinate indicated that A. brasilense Sp 7 had a complete tricarboxylic acid cycle. Assimilation of most of the radioactivity from labeled succinate, pyruvate, and acetate into lipids suggested a strong anabolic metabolism and the presence of an active malic enzyme of phosphoenolpyruvate carboxykinase. The distribution of radioactivity from differentially labeled pyruvate showed that gluconeogenesis competed with pyruvate dehydrogenase. Uptake and incorporation of labeled acetate also indicated the presence of a glyoxylate cycle in A. brasilense Sp 7.     

Bacterial nutritional approach to mechanisms of oxygen toxicity

Gottlieb, Sheldon F. (Union Carbide Corp., Tonawanda, N.Y.). Bacterial nutritional approach to mechanisms of oxygen toxicity. J. Bacteriol. 92:1021-1027. 1966.-Inhibition by oxygen of growth of the bacterium Achromobacter P6 was reversed by amino acid supplements. The reversal of oxygen-induced growth inhibition was not due to the presence of reducing substances in the growth medium. Oxygen primarily exerts a bacteriostatic effect. The oxygen inhibition of growth occurred over a wide pH range. Oxygen inhibition of growth was observed when 1-amino-2-propanol, acetate, lactate, citrate, or glucose was used as the sole source of carbon and energy. No inhibition of growth was obtained when succinate, fumarate, malate, or glutamate was used as the source of carbon and energy. Oxygen markedly depressed the respiration of P6 when 1-amino-2-propanol was the substrate. There was no depression of respiration under oxygen with succinate as substrate. P6 grown in the presence of high oxygen tensions had a higher rate of respiration under oxygen than similar air-grown cells. Chloramphenicol did not affect the rate of oxygen consumption or cause a further depression of the respiratory rate in the presence of oxygen. It is suggested that microbes may serve as a model system for studying the cellular and subcellular mechanisms of oxygen toxicity.     

Intermediary metabolism in Legionella pneumophila: utilization of amino acids and other compounds as energy sources.

The utilization of amino acids and other compounds as carbon and energy sources by Legionella pneumophila was examined. Based on the stimulation of oxygen consumption in washed-cell suspensions, glutamate, serine, threonine, and tyrosine were the only amino acids which were utilized as energy sources. Other stimulators of oxygen uptake were lactate, pyruvate, acetate, fumarate, and succinate. Citrate was a good stimulator only when the bacteria were grown in the presence of the substrate. Radiolabeling studies showed that [14C]glutamate was rapidly metabolized, with the label distributed evenly in all cell fractions. [14C]pyruvate and [14C]acetate were incorporated into the lipid-containing cell fraction, whereas glucose and glycerol were found in both the lipid- and polysaccharide-containing cell fractions. Radiorespirometry of differentially labeled [14C]glucose indicated that this compound was metabolized primarily by the pentose phosphate and Entner-Doudoroff pathways rather than by the glycolytic pathway.     

Nature of enhanced mitochondrial oxidative metabolism by a calf blood extract

The effect of calf blood extract (Solcoseryl, SS) on mitochondrial oxidative function in various states was studied polarographically in vitro. 1) Mitochondrial respiration in all 4 conventional study states (Estabrook, 1967) was enhanced by the addition of SS, including states 1 and 2 (endogenous substrates only). 2) The effect of SS on mitochondrial oxygen consumption was concentration dependent, while ADP/O ratio remained constant. The effect of added respiratory substrates varied with the particular substrate at optimally active concentrations. With suboptimal substrate levels, ADP/O ratios were concentration dependent, in contrast to the SS effect. Under oligomycin ATPase inhibition, SS was no longer active, in contrast to DNP, which remained active. 3) In states 3 (added ADP) and 4 (ADP exhausted), oxygen consumption and oxidative phosphorylation were enhanced by SS in the presence or absence of citrate, glutamate, pyruvate, lactate, or ascorbate. However, in the presence of succinate, SS had no effect. 4) ADP/O ratio was decreased by SS in the presence of added substrate, suggesting that SS activation of H(+)-ATPase enhances ATP hydrolysis as well as oxidative phosphorylation and ATP synthesis. 5) The enhancing effect of SS on mitochondrial function is due to hydrophilic components of SS. The lipidic components obtained by Folch fraction of SS have no effect. It is concluded that the effects of SS respiratory substrates and uncouplers on mitochondrial function are essentially different. SS enhances both ATP synthesis and oxygen consumption by mitochondria.     

Inhibition of energy-producing pathways of HepG2 cells by 3-bromopyruvate

3-BrPA (3-bromopyruvate) is an alkylating agent with anti-tumoral activity on hepatocellular carcinoma. This compound inhibits cellular ATP production owing to its action on glycolysis and oxidative phosphorylation; however, the specific metabolic steps and mechanisms of 3-BrPA action in human hepatocellular carcinomas, particularly its effects on mitochondrial energetics, are poorly understood. In the present study it was found that incubation of HepG2 cells with a low concentration of 3-BrPA for a short period (150 microM for 30 min) significantly affected both glycolysis and mitochondrial respiratory functions. The activity of mitochondrial hexokinase was not inhibited by 150 microM 3-BrPA, but this concentration caused more than 70% inhibition of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 3-phosphoglycerate kinase activities. Additionally, 3-BrPA treatment significantly impaired lactate production by HepG2 cells, even when glucose was withdrawn from the incubation medium. Oxygen consumption of HepG2 cells supported by either pyruvate/malate or succinate was inhibited when cells were pre-incubated with 3-BrPA in glucose-free medium. On the other hand, when cells were pre-incubated in glucose-supplemented medium, oxygen consumption was affected only when succinate was used as the oxidizable substrate. An increase in oligomycin-independent respiration was observed in HepG2 cells treated with 3-BrPA only when incubated in glucose-supplemented medium, indicating that 3-BrPA induces mitochondrial proton leakage as well as blocking the electron transport system. The activity of succinate dehydrogenase was inhibited by 70% by 3-BrPA treatment. These results suggest that the combined action of 3-BrPA on succinate dehydrogenase and on glycolysis, inhibiting steps downstream of the phosphorylation of glucose, play an important role in HepG2 cell death.