Mechanism of Superoxide Anion Generation in Intact Mitochondria in the Presence of Lucigenin and Cyanide

In the presence of cyanide and various respiratory substrates (succinate or pyruvate + malate) addition of high concentrations of lucigenin (400 microM; Luc2+) to rat liver mitochondria can induce a short-term flash of high amplitude lucigenin-dependent chemiluminescence (LDCL). Under conditions of cytochrome oxidase inhibition by cyanide the lucigenin-induced cyanide-resistant respiration (with succinate as substrate) was not inhibited by uncouplers (FCCP) and oligomycin. Increase in transmembrane potential (Deltaphi) value by stimulating F0F1-ATPase functioning (induced by addition of MgATP to the incubation medium) caused potent stimulation of the rate of cyanide-resistant respiration. At high Deltaphi values (in the presence of MgATP) cyanide resistant respiration of mitochondria in the presence of succinate or malate with pyruvate was insensitive to tenoyltrifluoroacetone (TTFA) or rotenone, respectively. However, in both cases respiration was effectively inhibited by myxothiazol or antimycin A. Mechanisms responsible for induction of LDCL and cyanide resistant mitochondrial respiration differ. In contrast to cyanide-resistant respiration, generation of LDCL signal, that was suppressed only by combined addition of Complex III inhibitors, antimycin A and myxothiazol, is a strictly potential-dependent process. It is observed only under conditions of high Deltaphi value generated by F0F1-ATPase functioning. The data suggest lucigenin-induced intensive generation of superoxide anion in mitochondria. Based on results of inhibitor analysis of cyanide-resistant respiration and LDCL, a two-stage mechanism of autooxidizable lucigenin cation-radical (Luc*+) formation in the respiratory chain is proposed. The first stage involves two-electron Luc2+ reduction by Complexes I and II. The second stage includes one-electron oxidation of reduced lucigenin (Luc(2e)). Reactions of Luc(2e) oxidation involve coenzyme Q-binding sites of Complex III. This results in formation of autooxidizable Luc*+ and superoxide anion generation. A new scheme for lucigenin-dependent electron pathways is proposed. It includes formation of fully reduced form of lucigenin and two-electron-transferring shunts of the respiratory chain. Lucigenin-induced activation of superoxide anion formation in mitochondria is accompanied by increase in ion permeability of the inner mitochondrial membrane.     

Hepatocyte mitochondrion respiratory chain in rats with experimental toxic hepatitis

The purpose of this study was to examine hepatocyte mitochondrion respiratory chain in rats subjected to ethanol and CCl4 administration within 4 weeks to induce an experimental hepatitis. Oxygen consumption was determined as a measure of mitochondrion respiration chain function. The development of liver pathology was accompanied by fat accumulation, fibrosis, triglycerides and lipid peroxidation increase. Respiratory chain characteristics damage was found. Endogenous oxygen consumption by hepatocytes isolated from pathological liver was found 34% higher compared to control. Exogenous malate and pyruvate substrates delivery didn't stimulate cell respiration. Rotenone (the inhibitor of the I complex) decreased 27% oxygen consumption by pathological hepatocytes while dinitrophenol produced 37% cell respiration increase. States 3 (V3) and 4 (V4) mitochondrial respiration with malate + glutamate as substrates were found to be 70 and 56% higher accordingly compared to control level. V3 and Vd (dinitrophenol respiration) for mitochondria from pathological liver didn't differ from control when being tested with malate + glutamate or succinate as substrates. Cytochrome c oxidase activity increased (+ 80%) as compared to control. Administration of hypolipidemic agent simvastatin simultaneously with ethanol and CC14 resulted in decrease liver fat accumulation, fibrosis and peroxidation products. Simvastatin administration caused hepatocyte endogenous respiration decrease while malate + pyruvate, dinitrophenol or rotenone delivery produced oxygen consumption alterations similar to control. However, when isolated mitochondria from liver of simvastatin treated animals being tested the decrease of oxidative phosphorylation coupling for substrates malate + glutamate was found. While simvastatin did not cause changes in cytochrome c oxidase activity. We propose the hypothesis that the NCCR complex in rat mitochondria with experimental toxic hepatitis works extensively on superoxydanion production. Alterations of SCCR, Coenzyme Q-cytochrome c-reductase, cytochrome c oxidase and ATP-synthase activities have an adaptive nature to compensate for impaired NCCR function.     

Substrate-dependent differential regulation of mitochondrial bioenergetics in the heart and kidney cortex and outer medulla

The kinetics and efficiency of mitochondrial oxidative phosphorylation (OxPhos) can depend on the choice of respiratory substrates. Furthermore, potential differences in this substrate dependency among different tissues are not well-understood. Here, we determined the effects of different substrates on the kinetics and efficiency of OxPhos in isolated mitochondria from the heart and kidney cortex and outer medulla (OM) of Sprague-Dawley rats. The substrates were pyruvate+malate, glutamate+malate, palmitoyl-carnitine+malate, alpha-ketoglutarate+malate, and succinate±rotenone at saturating concentrations. The kinetics of OxPhos were interrogated by measuring mitochondrial bioenergetics under different ADP perturbations. Results show that the kinetics and efficiency of OxPhos are highly dependent on the substrates used, and this dependency is distinctly different between heart and kidney. Heart mitochondria showed higher respiratory rates and OxPhos efficiencies for all substrates in comparison to kidney mitochondria. Cortex mitochondria respiratory rates were higher than OM mitochondria, but OM mitochondria OxPhos efficiencies were higher than cortex mitochondria. State 3 respiration was low in heart mitochondria with succinate but increased significantly in the presence of rotenone, unlike kidney mitochondria. Similar differences were observed in mitochondrial membrane potential. Differences in H₂O₂ emission in the presence of succinate±rotenone were observed in heart mitochondria and to a lesser extent in OM mitochondria, but not in cortex mitochondria. Bioenergetics and H₂O₂ emission data with succinate±rotenone indicate that oxaloacetate accumulation and reverse electron transfer may play a more prominent regulatory role in heart mitochondria than kidney mitochondria. These studies provide novel quantitative data demonstrating that the choice of respiratory substrates affects mitochondrial responses in a tissue-specific manner.     

Glucose,Lactate and Glutamine but not Glutamate Support Depolarization-Induced Increased Respiration in Isolated Nerve Terminals

Synaptosomes prepared from various aged and gene modified experimental animals constitute a valuable model system to study pre-synaptic mechanisms. Synaptosomes were isolated from whole brain and the XFe96 extracellular flux analyzer (Seahorse Bioscience) was used to study mitochondrial respiration and glycolytic rate in presence of different substrates. Mitochondrial function was tested by sequentially exposure of the synaptosomes to the ATP synthase inhibitor, oligomycin, the uncoupler FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone) and the electron transport chain inhibitors rotenone and antimycin A. The synaptosomes exhibited intense respiratory activity using glucose as substrate. The FCCP-dependent respiration was significantly higher with 10 mM glucose compared to 1 mM glucose. Synaptosomes also readily used pyruvate as substrate, which elevated basal respiration, activity-dependent respiration induced by veratridine and the respiratory response to uncoupling compared to that obtained with glucose as substrate. Also lactate was used as substrate by synaptosomes but in contrast to pyruvate, mitochondrial lactate mediated respiration was comparable to respiration using glucose as substrate. Synaptosomal respiration using glutamate and glutamine as substrates was significantly higher compared to basal respiration, whereas oligomycin-dependent and FCCP-induced respiration was lower compared to the responses obtained in the presence of glucose as substrate. We provide evidence that synaptosomes are able to use besides glucose and pyruvate also the substrates lactate, glutamate and glutamine to support their basal respiration. Veratridine was found to increase respiration supported by glucose, pyruvate, lactate and glutamine and FCCP was found to increase respiration supported by glucose, pyruvate and lactate. This was not the case when glutamate was the only energy substrate.     

Relation between glutamate and adenine nucleotide levels of heart mitochondria during hypoxia

The effect of acute respiratory hypoxia in rats on mitochondrial respiration, adenine nucleotides and some amino acids of the heart was studied. The decrease in the total (ATP + ADP + AMP) and exchangeable (ATP + ADP) adenine nucleotide pool of the mitochondria was accompanied by a pronounced loss of state 3 respiration with glutamate plus malate and a slight decrease with succinate plus rothenone. The uncoupled respiration of mitochondria with glutamate and malate was decreased in the same degree as in the absence of 2,4-dinitrophenol. State 4 respiration with substrates of both types was unaffected by hypoxia. These data point to a hypoxia-induced impairment of complex I of the respiratory chain. The decrease of tissue and mitochondrial glutamate was accompanied by the elevation of alanine content in the heart and an increase in intramitochondrial aspartate. The ADP-stimulated respiration of mitochondria was correlated with mitochondrial glutamate and ATP as well as with exchangeable adenine nucleotide pools during hypoxia. The experimental results suggest that mitochondrial dysfunction induced by hypoxia may also be attributed to the low level of mitochondrial glutamate.     

State of liver mitochondrial respiratory chain in rats with experimental toxic hepatitis

Polarographical determination of oxygen concentration has shown that in rats with experimental hepatitis induced by combined ethanol and CCl₄ administration for 4 weeks, the functioning of the hepatocyte mitochondrial respiratory chain is impaired. Development of liver pathology was accompanied by adipose dystrophy, fibrosis, and an increase of triglycerides and lipid peroxidation products in the liver tissue. The endogenous respiration rate in hepatocytes isolated from the pathologically altered liver was 34% higher than in the control. Cell respiration was not stimulated by the addition of the substrates malate and pyruvate with digitonine. An uncoupler of oxidation and phosphorylation, 2,4-dinitrophenol, increased the hepatocyte oxygen consumption rate by 37%, while addition of the inhibitor of the I complex, rotenone, decreased cell respiration in pathologically altered hepatocytes by 27%. The states 3 (V₃) and 4 (V₄) of mitochondrial respiration with malate + glutamate as substrates were found to be higher by 70% and 56%, respectively, as compared with the control level. When using malate + glutamate or succinate as substrates, V₃ and Vd (dinitrophenol respiration) in the toxic hepatitis hepatocyte mitochondria did not differ from the control, which indicates no uncoupling occurred of the oxidation and phosphorylation processes. Cytochrome c oxidase activity was elevated (+80%) as compared with the control. Administration of the hypolipidemic agent symvastatin simultaneously with ethanol and CCl₄ resulted in a reduction of the degree of liver adipose dystrophy, prevented activation of lipid peroxidation, and decreased the hepatocyte endogenous respiration rate. Addition of malate + pyruvate, dinitrophenol or rotenone produced oxygen consumption changes similar to those in the control. However, in mitochondria isolated from the pathologically altered liver, symvastatin induced an uncoupling effect on the respiratory chain in the presence of the substrates malate + glutamate, but did not change the cytochrome c oxidase activity. We suggest that functioning of the NCCR complex in the hepatocyte mitochondria of animals with experimental toxic hepatitis is impaired, which leads to an intensive superoxide anion production at the level of this complex. Under these conditions, the defect of the NADH-coenzyme Q-oxidoreductase is compensated by functioning of other complexes of the respiratory chain (SCCR, coenzyme Q-cytochrome c-reductase, cytochrome c oxidase, and ATP-synthase activities).     

Hepatocyte mitochondrion electron-transport chain alterations in CCl<Subscript>4</Subscript> and alcohol induced hepatitis in rats and their correction with simvastatin

The goal of this study was to examine the state of hepatocyte mitochondrial respiratory chain of rats with toxic hepatitis induced by CCl₄ and ethanol. Oxygen consumption by hepatocytes and mitochondria was determined. Endogenous oxygen consumption by pathological hepatocytes was 1.3-fold higher compared with control. Rotenone resulted in 27% suppression of respiration by pathological hepatocytes whereas 2,4-dinitrophenol produced a 1.4-fold increase of respiration. States 3 and 4 of mitochondrial respiration with malate and glutamate were found to be higher as compared to control. State dinitrophenol and state 3 respirations were similar within every group of animals when being tested with malate and glutamate or succinate. Cytochrome c oxidase activity in hepatitis was 1.8-fold higher compared with control. Simvastatin administration resulted in a decrease in hepatocyte endogenous respiration in hepatitis. The presented data lead to the assumption that the increased oxygen consumption by the respiratory chain of pathological mitochondria to be linked mainly with the altered function of complex I.     

Adenine nucleotides, glutamate and respiratory function of heart mitochondria during acute hypoxia

The effect of acute hypoxia on adenine nucleotides, glutamate, aspartate, alanine and respiration of heart mitochondria was studied in rats. The losses of intramitochondrial adenine nucleotides (ATP+ADP+AMP) during hypoxia were related to depression of state 3 respiration supported by glutamate and malate, as well as decrease in uncoupled respiration. Hypoxia had less prominent effect on succinate-dependent state 3 respiration. Non-phosphorylating (state 4) respiratory rates and ADP/O ratios were slightly affected by oxygen deprivation. Glutamate fall in tissue and mitochondria of hypoxic hearts was concomitant with significant increase in tissue alanine and mitochondrial aspartate. The losses of intramitochondrial ATP and respiratory activity with NAD-dependent substrates during hypoxia were related to a decrease in mitochondrial glutamate. The results suggest that hypoxia-induced impairment of complex I of respiratory chain and a loss of glutamate from the matrix may limit energy-producing capacity of heart mitochondria.     

Rotenone-like action of the branched-chain phytanic acid induces oxidative stress in mitochondria

Phytanic acid (Phyt) increase is associated with the hereditary neurodegenerative Refsum disease. To elucidate the still unclear toxicity of Phyt, mitochondria from brain and heart of adult rats were exposed to free Phyt. Phyt at low micromolar concentrations (maximally: 100 nmol/mg of protein) enhances superoxide (O(2)(.))(2) generation. Phyt induces O(2)(.) in state 3 (phosphorylating), as well as in state 4 (resting). Phyt stimulates O(2)(.) generation when the respiratory chain is fed with electrons derived from oxidation of glutamate/malate, pyruvate/malate, or succinate in the presence of rotenone. With succinate alone, Phyt suppresses O(2)(.) generation caused by reverse electron transport from succinate to complex I. The enhanced O(2)(.) generation by Phyt in state 4 is in contrast to the mild uncoupling concept. In this concept uncoupling by nonesterified fatty acids should abolish O(2)(.) generation. Stimulation of O(2)(.) generation by Phyt is paralleled by inhibition of the electron transport within the respiratory chain or electron leakage from the respiratory chain. The interference of Phyt with the electron transport was demonstrated by inhibition of state 3- and p-trifluoromethoxyphenylhydrazone (FCCP)-dependent respiration, inactivation of the NADH-ubiquinone oxidoreductase complex in permeabilized mitochondria, decrease in reduction of the synthetic electron acceptor 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in state 4, and increase of the mitochondrial NAD(P)H level in FCCP-uncoupled mitochondria. Thus, we suggest that complex I is the main site of Phyt-stimulated O(2)(.) generation. Furthermore, inactivation of aconitase and oxidation of the mitochondrial glutathione pool show that enhanced O(2)(.) generation with chronic exposure to Phyt causes oxidative damage.     

Hyperglycemia Preserves Brain Mitochondrial Respiration During Anoxia

We examined brain mitochondrial function in normo- (5 mM) and hyperglycemic (50 mM) cats after 8 min of anoxia. In anoxic normoglycemic cats, mitochondrial state 3 respiration with NAD-linked substrates glutamate or pyruvate (both plus malate) was inhibited 30-50%. The uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) maximally stimulated respiration, indicating that inhibition of phosphorylation, not impairment of electron transport, substrate transport, or oxidation was present. State 3 respiration with succinate (plus rotenone) was unaffected. Mitochondrial respiratory control ratios trended toward reductions whereas ADP/O ratios remained unchanged. In contrast, brain mitochondria from anoxic hyperglycemic cats showed no such inhibition of state 3 respiration and no differences in function from normo- and hyperglycemic control animals except for trends toward loose coupling. Significantly higher brain tissue glucose concentrations were present in hyperglycemic controls as the only metabolite difference compared to normoglycemic controls. At the end of anoxia, hyperglycemic cats exhibited significantly higher cortical lactate and glucose levels but similarly reduced high-energy phosphate concentrations compared to normoglycemic cats. These results demonstrate that increased availability of glucose to gray matter as a consequence of hyperglycemia maintains normal mitochondrial state 3 respiration during exposure to anoxia. Previous survival studies have shown that lower serum glucose concentrations during anoxia are relatively brain protective. This result indicates that the presently described alterations in mitochondrial respiration must be fully reversible.     

On the Function of Mitochondrial Metabolism during Photosynthesis in Spinach (Spinacia oleracea L.) Leaves (Partitioning between Respiration and Export of Redox Equivalents and Precursors for Nitrate Assimilation Products)

The functioning of isolated spinach (Spinacia oleracea L.) leaf mitochondria has been studied in the presence of metabolite concentrations similar to those that occur in the cytosol in vivo. From measurements of the concentration dependence of the oxidation of the main substrates, glycine and malate, we have concluded that the state 3 oxidation rate of these substrates in vivo is less than half of the maximal rates due to substrate limitation. Analogously, we conclude that under steady-state conditions of photosynthesis, the oxidation of cytosolic NADH by the mitochondria does not contribute to mitochondrial respiration. Measurements of mitochondrial respiration with glycine and malate as substrates and in the presence of a defined malate:oxaloacetate ratio indicated that about 25% of the NADH formed in vivo during the oxidation of these metabolites inside the mitochondria is oxidized by a malate-oxaloacetate shuttle to serve extramitochondrial processes, e.g. reduction of nitrate in the cytosol or of hydroxypyruvate in the peroxisomes. The analysis of the products of the oxidation of malate indicates that in the steady state of photosynthesis the activity of the tricarboxylic acid cycle is very low. Therefore, we have concluded that the mitochondrial oxidation of malate in illuminated leaves produces mainly citrate, which is converted via cytosolic aconitase and NADP-isocitrate dehydrogenase to yield 2-oxoglutarate as the precursor for the formation of glutamate and glutamine, which are the main products of photosynthetic nitrate assimilation.     

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.     

Wy-14,643 and fenofibrate inhibit mitochondrial respiration in isolated rat cardiac mitochondria

We investigated the direct effects of two selective PPARalpha ligands, fenofibrate and Wy-14,643, on mitochondrial respiratory function using isolated rat cardiac mitochondria. Isolated left ventricular mitochondria were incubated with increasing concentrations of fenofibrate or Wy-14,643 (10, 100, and 500 microM) and mitochondrial respiration determined using: malate/glutamate (complex I), succinate (complex II) and palmitoyl-l-carnitine as oxidative substrates. Our data show that acute exposure to Wy-14,643 and fenofibrate differentially perturb cardiac mitochondrial respiration i.e., fenofibrate more potently inhibited mitochondrial respiration and bioenergetic capacity compared to Wy-14,643. Moreover, we found that both agents increased uncoupling of mitochondrial oxidative phosphorylation.     

Similar nature of inhibition of mitochondrial respiration of heart tissue and malignant cells by methylglyoxal. A vital clue to understand the biochemical basis of malignancy

The effect of methylglyoxal on the oxygen consumption of mitochondria of heart and of several other organs of normal animals of different species has been tested. The results indicate that methylglyoxal (3.5 mM) strongly inhibits ADP-stimulated -oxoglutarate and malate plus pyruvate-dependent respiration of exclusively heart mitochondria of normal animals of different species. Whereas, with the same substrates, but at a higher concentration of methylglyoxal (7.5 mM), the respiration of mitochondria of other organs of normal animals is not inhibited. Methylglyoxal also inhibits the respiration of slices of rat and toad hearts. But this inhibition is less pronounced. However, methylglyoxal (15 mM) fails to have any effect on perfused toad heart. Using rat heart mitochondria as a model, the effect of methylglyoxal on the oxygen consumption was also tested with different respiratory substrates, electron donors at different segments of the mitochondrial respiratory chain and site-spe inhibitors to identify the specific respiratory complex which might be involved in the inhibitory effect of methylglyoxal. The results strongly suggest that methylglyoxal inhibits the electron flow through complex I of rat heart mitochondrial respiratory chain. Moreover, lactaldehyde (0.6 mM), a catabolite of methylglyoxal, can exert a protective effect on the inhibition of rat heart mitochondrial respiration by methylglyoxal (2.5 mM). The effect of methylglyoxal on heart mitochondria as described in the present paper is strikingly similar to the results of our previous work with mitochondria of Ehrlich ascites carcinoma cells and leukemic leukocytes. We have recently proposed a new hypothesis on cancer which suggests that excessive ATP formation in cells may lead to malignancy. The above mentioned similarity apparently provides a solid experimental foundation for the proposed hypothesis which has been discussed.     

Does the respiratory rate in sea urchin embryos increase during early development without proliferation of mitochondria?

During early development of the sea urchin, the respiratory rate, enhanced upon fertilization, is maintained up to hatching (pre-hatching period) and then gradually increases to a maximum at the gastrula stage (post-gastrula period). Except for a short duration after fertilization, respiration in embryos is strongly inhibited by CN⁻ and antimycin A. During the whole span of early development, the amounts of proteins, cytochromes and the specific activities of cytochrome c oxidase and reduced nicotinamide adenine dinucleotide (NADH) cytochrome c reductase in mitochondria are practically the same as in unfertilized eggs. A marked augmentation of mitochondrial respiration after hatching probably occurs without net increase in whole mitochondrial intrinsic capacities. Carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) or tetramethyl p-phenylenediamine (TMPD) enhances the respiratory rate in the pre-hatching period but hardly augments the respiration in the post-gastrula period. In the presence of both FCCP and TMPD, the respiratory rate in the pre-hatching period was as high as in the post-gastrula period. Probably, electron transport in the mitochondrial respiratory chain is regulated by acceptor control and limitation of cytochrome c reduction in the pre-hatching period and released from those regulations in the post-gastrula period. Acceptor control of respiration is experimentally reproduced in isolated mitochondria by making adenine nucleotide levels as those levels in the pre-hatching period.     

Involvement of the energy-dissipating systems in modulating the energetic efficiency of respiration in mitochondria from etiolated winter wheat seedlings

Effects of cyanide-resistant alternative oxidase (AOX) and modulators of plant uncoupling mitochondrial proteins (PUMP) on respiration rate and generation of transmembrane electric potential (ΔΨ) were investigated during oxidation of various substrates by isolated mitochondria from etiolated coleoptiles of winter wheat (Triticum aestivum L.). Oxidative phosphorylation in wheat mitochondria during malate and succinate oxidation was quite effective (it was characterized by high respiratory control ratio as defined by Chance, high ADP/O ratio, and rapid ATP synthesis). Nevertheless, the effectiveness of oxidative phosphorylation was substantially modulated by operation of energy-dissipating systems. The application of safranin dye revealed the partial dissipation of ΔΨ during inhibition of cytochrome-mediated malate oxidation by cyanide and antimycin A and demonstrated the operation of AOX-dependent compensatory mechanism for ΔΨ generation. The complex I of mitochondrial electron transport chain was shown to play the dominant role in ΔΨ generation and ATP synthesis during AOX functioning upon inhibition of electron transport through the cytochrome pathway. Effects of linoleic acid (PUMP activator) at physiologically low concentrations (4–10 μM) on respiration and ΔΨ generation in mitochondria were examined. The uncoupling effect of linoleic acid was shown in activation of the State 4 respiration, as well as in ΔΨ dissipation; this effect was eliminated in the presence of BSA but was insensitive to purine nucleotides. The uncoupling effect of linoleic acid was accompanied by reversible inhibition of AOX activity. The results are discussed with regard to possible physiological role of mitochondrial energy-dissipating systems in regulation of energy transduction in plant cells under stress conditions.     

Isolation and properties of flight muscle mitochondria of the bumblebee Bombus terrestris (L.)

This report describes the isolation procedure and properties of tightly coupled flight muscle mitochondria of the bumblebee Bombus terrestris (L.). The highest respiratory control index was observed upon oxidation of pyruvate, whereas the highest respiration rates were registered upon oxidation of a combination of the following substrates: pyruvate + malate, pyruvate + proline, or pyruvate + glutamate. The respiration rates upon oxidation of malate, glutamate, glutamate + malate, or succinate were very low. At variance with flight muscle mitochondria of a number of other insects reported earlier, B. terrestris mitochondria did not show high rates of respiration supported by oxidation of proline. The maximal respiration rates were observed upon oxidation of α-glycerophosphate. Bumblebee mitochondria are capable of maintaining high membrane potential in the absence of added respiratory substrates, which was completely dissipated by the addition of rotenone, suggesting high amount of intramitochondrial NAD-linked oxidative substrates. Pyruvate and α-glycerophosphate appear to be the optimal oxidative substrates for maintaining the high rates of oxidative metabolism of the bumblebee mitochondria.     

The effects of ischemia and experimental conditions on the respiration rate of cardiac fibers

The mitochondrial respiratory parameters were measured in situ, i.e. in saponin-skinned rabbit cardiac fibers and in fibers treated with saponin + collagenase. It was found that the decrease of maximal ADP-stimulated respiration rate of saponin-skinned fibers with pyruvate + malate under the conditions of total ischemia (0.5–1.5 h) is less pronounced as compared to isolated mitochondria. Maximal succinate oxidation rate (+ADP), however, was not different from control (1 h ischemia) but it exceeded the control level when measured in the medium supplemented with cytochrome c. It was also demonstrated that treatment of fibers with collagenase alone or in combination with saponin significantly (almost 2 fold) enhanced the maximal ADP-stimulated respiration rate if compared with saponin-skinned fibers. The data obtained suggest that mitochondrial respiration in saponin-skinned rabit cardiac fibers is not completely revealed, most probably, due to insufficient permeabilization of sarcolemma by saponin and, thus, inadequate accessibility of mitochondria to exogenous substrates, ADP in particular. These parameters can be improved by pre-treatment of fibers with collagenase. (Mol Cell Biochem 174: 87–90, 1997)     

Cytochrome c Effect on Respiration of Heart Mitochondria: Influence of Various Factors

The effect of exogenous cytochrome c on respiration rate of the rat and human heart mitochondria was assessed in situ, using permeabilized fibers. It was (i) much more pronounced in State 2 and 4 than in State 3 with all the respiratory substrates (pyruvate+malate, succinate, palmitoyl-CoA+carnitine and octanoyl-L-carnitine), (ii) different with different substrates, (iii) much higher after ischemia in both metabolic states, particularly in the case of succinate oxidation compared to pyruvate+malate, (iv) the highest in State 4 with succinate as a substrate. Similar results were obtained with the isolated rat and rabbit heart mitochondria. The differences in the degree of stimulation of mitochondrial respiration by cytochrome c and, thus, sensitivity of cytochrome c test in evaluation of the intactness/injury of outer mitochondrial membrane are probably determined by the differences in the cytochrome c role in the control of mitochondrial respiration in the above-described conditions.     

The effect of cold exposure on rat liver mitochondrial respiratory capacity.

1. The effect of cold exposure on the respiratory capacity of rat liver mitochondria has been studied using succinate as the substrate. 2. The mitochondria obtained in this study were well coupled, as shown by the RCR and ADP/O ratios. 3. In addition, durohydroquinone was used to eliminate the regulation of substrate supply. Likewise, we measured uncoupled respiration to evaluate the maximal electron flow through the respiratory chain. 4. We found that oxygen consumption using succinate or durohydroquinone + FCCP as substrates, as well as ATP production were not affected by cold exposure. 5. Our results also show that, when succinate is used, the maximal capacity of the respiratory chain is measured. 6. The data obtained do not support a role of the electron transport chain as a target of cold action.