Effect of thyroxine on the NADH cytochrome c reductase activity of microsomes and outer mitochondrial membrane of rat liver depending on age

The rotenone-insensitive NADH-cytochrome c reductase activity and cytochrome b5 content in mitochondria and microsomes of liver of 1-, 3-, 12- and 24-month-old rats were studied. Thyroxine injection caused a considerable decrease in the microsomal activity in all age groups under study; in mitochondria this effect was increased with age. The decrease of the cytochrome b5 content was maximal in the microsomes and mitochondria of 12- and 24-month-old animals and was insignificant in 1-month-old rats. It was assumed that the age variations in regulation of the outer mitochondrial membrane function can be due changes in their population.     

Cytochrome b5-like hemoprotein/cytochrome b5 reductase complex in rat liver mitochondria has NADH-linked aquacobalamin reductase activity

Rat liver mitochondrial NADH-linked aquacobalamin reductase was characterized to clarify its enzymological properties. Most of the enzyme was solubilized with 10 g/L Triton X-100 from rat liver mitochondrial membranes. The elution behavior of the solubilized enzyme was identical to that of NADH-cytochrome c reductase (b-type cytochromes/cytochrome b5 reductase complex) during DEAE-Sepharose Fast Flow column chromatography. By mixing both purified cytochrome b5-like hemoprotein (outer membrane-cytochrome b) and cytochrome b5 reductase, cob(II)alamin was formed from aquacobalamin and NADH. These results provide evidence that the outer membrane-cytochrome b/cytochrome b5 reductase complex has the activity of the NADH-linked aquacobalamin reductase in rat liver mitochondria. Some properties of the NADH-linked aquacobalamin reductase were studied using the function of rat liver mitochondrial membranes. The specific activity (109.5 +/- 14.3 nmol.min-1.mg protein-1) of the enzyme was shown under physiological conditions (pH 7.1 at 40 degrees C). The optimal pH and temperature for activity were 7.1 and 40 degrees C, respectively. The apparent Km values were 41.9 mumol/L for aquacobalamin in the presence of 0.2 mmol/L NADH and 14.4 mumol/L for NADH in the presence of 0.1 mmol/L aquacobalamin. The enzyme was specific for aquacobalamin, because cyanocobalamin could not be reduced by the enzyme.     

Membrane potential generation coupled to oxidation of external NADH in liver mitochondria

Oxidation of added NADH by rat liver mitochondria has been studied. It is found that exogenous NADH, when oxidized by rat liver mitochondria in sucrose hypotonic medium supplemented with Mg2+ and EGTA, generates a membrane potential (delta psi) even in the absence of added cytochrome c. ADP and phosphate decrease delta psi, the effect being reversed by oligomycin. Rotenone and myxothiazol do not inhibit delta psi generated by oxidation of exogenous NADH. Added cytochrome c increases the rate of the exogenous NADH oxidation and coupled delta psi formation. In sucrose isotonic medium, or in hypotonic medium without Mg2+, exogenous NADH fails to stimulate respiration and to form a membrane potential. In the presence of Mg2+, exogenous NADH appears to be effective in delta psi generation in isotonic sucrose medium if mitochondria were treated with digitonin. In isotonic KCl without Mg2+, oxidation of exogenous NADH is coupled to the delta psi formation and MgCl2 addition before mitochondria prevents this effect. In hypotonic (but not in isotonic) sucrose medium, Mg2+ makes a portion of the cytochrome c pool reducible by exogenous NADH or ascorbate. It is assumed that (i) hypotonic treatment or digitonin causes disruption of the outer mitochondrial membrane, and, as a consequence, desorption of the membrane-bound cytochrome c in a Mg2+-dependent fashion; (ii) incubation in isotonic KCI without Mg2+ results in swelling of mitochondrial matrix, disruption of the outer membrane and cytochrome c desorption whereas Mg2+ lowers the K+ permeability of the inner membrane and, hence, prevents swelling; (iii) desorbed cytochrome c is reduced by added NADH via NADH-cytochrome b5 reductase and cytochrome b5 or by ascorbate and is oxidized by cytochrome oxidase. The role of desorbed cytochrome c in oxidation of superoxide and cytoplasmic NADH as well as possible relations of these events to apoptosis are discussed.     

t-Butylhydroperoxide and gliotoxin stimulate Ca2+ release from rat skeletal muscle mitochondria

Rat liver mitochondria have a specific Ca2+ release pathway which operates when NAD+ is hydrolysed to nicotinamide and ADPribose. NAD+ hydrolysis is Ca(2+)-dependent and inhibited by cyclosporine A (CSA). Mitochondrial Ca2+ release can be activated by the prooxidant t-butylhydroperoxide (tbh) or by gliotoxin (GT), a fungal metabolite of the epipolythiodioxopiperazine group. Tbh oxidizes NADH to NAD+ through an enzyme cascade consisting of glutathione peroxidase, glutathione reductase, and the energy linked transhydrogenase, whereas GT oxidizes some vicinal thiols to the disulfide form, a prerequisite for NAD+ hydrolysis. We report now that rat skeletal muscle mitochondria also contain a specific Ca2+ release pathway activated by both tbh and GT. Ca2+ release increases with the mitochondrial Ca2+ load, is completely inhibited in the presence of CSA, and is paralleled by pyridine nucleotide oxidation. In the presence of tbh and GT, mitochondria do not lose their membrane potential and do not swell, provided continuous release and re-uptake of Ca2+ ('Ca2+ cycling') is prevented. These data support the notion that both tbh- and GT-induced Ca2+ release are not the consequence of an unspecific increase of the inner membrane permeability ('pore' formation). Tbh induces Ca2+ release from rat skeletal muscle less efficiently than from liver mitochondria indicating that the coupling between tbh and NADH oxidation is much weaker in skeletal muscle mitochondria. This conclusion is corroborated by a much lower glutathione peroxidase activity in skeletal muscle than in liver mitochondria. The prooxidant-dependent pathway promotes, under drastic conditions (high mitochondrial Ca2+ loads and high tbh concentrations), Ca2+ release to about the same extent and rate as the Na+/Ca2+ exchanger. This renders the prooxidant-dependent pathway relevant in the pathophysiology of mitochondrial myopathies where its activation by an increased generation of reactive oxygen species probably results in excessive Ca2+ cycling and damage to mitochondria.     

Analysis of the sorting signals directing NADH-cytochrome b5 reductase to two locations within yeast mitochondria

Mitochondrial NADH-cytochrome b5 reductase (Mcr1p) is encoded by a single nuclear gene and imported into two different submitochondrial compartments: the outer membrane and the intermembrane space. We now show that the amino-terminal 47 amino acids suffice to target the Mcr1 protein to both destinations. The first 12 residues of this sequence function as a weak matrix-targeting signal; the remaining residues are mostly hydrophobic and serve as an intramitochondrial sorting signal for the outer membrane and the intermembrane space. A double point mutation within the hydrophobic region of the targeting sequence virtually abolishes the ability of the precursor to be inserted into the outer membrane but increases the efficiency of transport into the intermembrane space. Import of Mcr1p into the intermembrane space requires an electrochemical potential across the inner membrane, as well as ATP in the matrix, and is strongly impaired in mitochondria lacking Tom7p or Tim11p, two components of the translocation machineries in the outer and inner mitochondrial membranes, respectively. These results indicate that intramitochondrial sorting of the Mcr1 protein is mediated by specific interactions between the bipartite targeting sequence and components of both mitochondrial translocation systems.     

Glutathione peroxidase and catalase modulate the genotoxicity of arsenite

In the present study, the endogenous formation of reactive oxygen species was localized in rat liver and small intestine. The 3,3'-diaminobenzidine (DAB)-Mn2+ technique in which cobalt ions were included in the incubation medium was applied to unfixed cryostat sections of intact tissues. Addition of manganese ions to the DAB-Co(2+)-containing medium greatly increased the amounts of final reaction product formed compared with incubations with only DAB and cobalt ions. In liver, a blue final reaction product was deposited, particularly in hepatocytes surrounding portal tracts. In the small intestine, the DAB-cobalt complex was mainly found at the basal side of enterocytes. Goblet cells remained unstained. Electron microscopical images revealed that an electron-dense reaction product was exclusively present at both inner and outer membranes and at the intermembrane space in mitochondria of liver parenchymal cells and duodenal enterocytes. It was shown that the spontaneous formation of final reaction product was enzymatic and dependent on the presence of oxygen in the medium. Sulphide decreased the reaction, which may indicate that cytochrome c oxidase was partially involved. Benzoquinone and histidine, which are scavengers of superoxide anions and singlet oxygen respectively, reduced the amount of final reaction product considerably. Furthermore, the formation of final reaction product was sensitive to specific inhibitors of NADH:coenzyme Q reductase and aldehyde oxidase, indicating that these enzymes were at least partly responsible for the generation of superoxide anions and singlet oxygen and for the formation of the DAB-cobalt complex.     

Acute and chronic ethanol increases reactive oxygen species generation and decreases viability in fresh, isolated rat hepatocytes

Although reactive oxygen species (ROS) have been implicated in the etiology of alcohol-induced liver disease, neither their relative contribution to cell death nor the cellular mechanisms mediating their formation are known. The purpose of this study was to test the hypothesis that acute and chronic ethanol exposure enhances the mitochondrial generation of ROS in fresh, isolated hepatocytes. Acute ethanol exposure stimulated ROS production, increased the cellular NADH/NAD+ ratio, and decreased hepatocyte viability slightly, which was prevented by pretreatment with 4-methylpyrazole (4-MP), an inhibitor of alcohol dehydrogenase. Similarly, xylitol, an NADH-generating compound, enhanced hepatocyte ROS production and decreased viability. Incubation with pyruvate, an NADH-oxidizing compound, and cyanamide, an inhibitor of aldehyde dehydrogenase, significantly decreased ROS levels in acute ethanol-treated hepatocytes. Chronic ethanol consumption produced a sixfold increase in hepatocyte ROS production compared with levels measured in controls. Hepatocytes from ethanol-fed rats were less viable compared with controls, e.g., viability was 68% +/- 2% (ethanol) versus 83% +/- 1% (control) after 60 minutes of incubation. Antimycin A increased ROS production and decreased cell viability; however, the toxic effect of antimycin A was more pronounced in ethanol-fed hepatocytes. These results suggest that acute and chronic ethanol exposure exacerbates mitochondrial ROS production, contributing to cell death.     

Mitochondrial aldehyde reductase: identification and characterization in rat liver and kidney cortex

Aldehyde reductase (EC 1.1.1.2) has been regarded so far as an exclusively cytosolic enzyme. The present investigation shows that mitochondria of rat liver, kidney cortex and, tentatively, heart also contain an enzyme catalyzing oxidation of NADPH by aldehydes, p-nitrobenzaldehyde, methylglyoxal and glyceraldehyde. Activity of the mitochondrial enzyme can only be measured after the organelles are disrupted by sonication or solubilized with nonionic detergents. Mitochondrial aldehyde reductase activity contributed to about 4.6% and 2.5% of the total cellular activity in liver and kidney cortex, respectively. However, the specific activity in liver mitochondria was about one third and in kidney cortex mitochondria one tenth of that in the cytosol of the corresponding organ. The mitochondrial enzyme resembled the cytosolic one by its absolute specificity towards NADPH as the electron donor, a similar profile of aldehydic electron acceptors and identical Km values. Mitochondrial aldehyde reductase differed from the cytosolic enzyme by low sensitivity to known inhibitors of cytosolic aldehyde reductase, AL-1576, AL-4114 and ONO-2235. In liver, about 60% of the mitochondrial activity was tightly bound to the membranes whereas about 40% was present in the mitochondrial matrix. The membrane-bound activity was inactivated by digestion of mitoplasts with trypsin, alpha-chymotrypsin or papain, thus pointing to exposition of the substrate-binding site at the external surface of the inner membrane. On the other hand, latency of the enzyme in intact mitochondria indicates that the NADPH-binding site is located at the inner surface. These data provide the first direct evidence for the existence of aldehyde reductase in mitochondria of some rat tissues.     

Human endothelial cell storage granules: a novel intracellular site for isoforms of the endothelin-converting enzyme

The participation of oxidative mechanisms in major histocompatibility complex (MHC) class II-restricted antigen presentation was studied in vitro. In general, antigen processing is inhibited when peritoneal macrophages (MO) are incubated with scavengers of reactive oxygen intermediates (ROI): mannitol (an.OH scavenger), dimethylurea (DMTU, which reacts with H2O2 and HOCl) and NCO-700 (an epoxysuccinic acid derivative which inhibits oxidant production by activated phagocytes and can scavenge reactive oxygen species in both NaOCl and hypoxanthine (XOD) systems). However, neither rotenone and antimycins (inhibitors of O-2 production at the NADH dehydrogenase and ubiquinone-cytochrome b regions, respectively) nor aminoguanidine (an inducible nitric oxide synthase inhibitor) impaired antigen presentation, thus indirectly discarding the participation of mitochondrial oxidation and reactive nitrogen intermediates (RNI) in antigen processing. ROI scavengers do not inhibit the MHC class II-restricted presentation of antigens that need processing but have their disulphide bonds reduced. It can be shown that oxidation of protein antigens (either by chlorination or performic acid treatment) allow protein unfolding and enhance both processing and exposure of immunogenic epitopes to specific T cells.     

Ursodeoxycholate protects against ethanol-induced liver mitochondrial injury

The purpose of this work was to examine whether ursodeoxycholate (UDC), a hydrophilic bile salt, could reduce mitochondrial liver injury from chronic ethanol consumption in rats. Animals were pair-fed liquid diets containing 36% of calories as ethanol or isocaloric carbohydrates. They were randomly assigned into 4 groups of 7 rats each and received a specific treatment for 5 weeks: control diet, ethanol diet, control diet + UDC, and ethanol diet + UDC. Respiratory rates of isolated liver mitochondria were measured using a Clark oxygen electrode with sodium succinate as substrate. Mitochondria from rats chronically fed ethanol demonstrated an impaired ability to produce energy. At the fatty liver stage, the ADP-stimulated respiration (V3) was depressed by 33%, the respiratory control ratio (RC) by 25% and the P/O ratio by 15%. In ethanol-fed rats supplemented with UDC, both the rate and efficiency of ATP synthesis via the oxidative phosphorylation were improved: V3 was increased by 35%, P/O by 8%. All the respiratory parameters were similar in control group and control + UDC group. On the other hand, the number and size of mitochondria were assessed by electron microscopy and computer-assisted quantitative analysis. The number of mitochondria from ethanol-treated rats was decreased by 29%, and they were enlarged by 74%. Both parameters were normalized to control values by UDC treatment. These studies demonstrate that UDC has a protective effect against ethanol-induced mitochondrial injury by improving ATP synthesis and preserving liver mitochondrial morphology. These UDC positive effects may contribute to the observed decrease in fat accumulation and may delay the progression of alcoholic injury to more advanced stages.     

Defects at center P underlie diabetes-associated mitochondrial dysfunction

Detailed respiration studies on isolated liver mitochondria from streptozotocin-induced diabetic Sprague-Dawley rats revealed a disease-associated decrease in the ADP/O ratio, a marker for mitochondrial ability to couple the consumption of oxygen to the phosphorylation of ADP. This decrease was observed following induction of respiration with glutamate/malate, succinate, or duroquinol, which enter the electron transport chain selectively at complexes I (NADH dehydrogenase), II (succinate dehydrogenase), or III (cytochrome bc1 complex), respectively. These data, coupled with studies using respiratory inhibitors (most importantly antimycin A and myxothiazol), localize at least a portion of this defect to a single site within the electron transport chain (center P in the Q-cycle portion of complex III). These results suggest that liver mitochondria from diabetic animals may generate increased levels of reactive oxygen species at the portion of the electron transport chain already established as the major site of mitochondrial free radical generation. The reduction in the ADP/O ratio occurred in mitochondria that do not have overt defects in the respiratory control ratio or in State 3 and State 4 respiration. The data in this paper suggest that defects in center P of the electron transport chain likely increase mitochondrial exposure to oxidants in the diabetic. This data may partially explain the evidence of altered exposure and/or response to reactive species in mitochondria from diabetics. This work thus provides further clues to the interaction between oxidative stress and diabetes-associated mitochondrial dysfunction.     

Changes in hepatic enzyme activities related to ethanol metabolism in mice following chronic ethanol administration

Male mice of three strains, C57BL, DBA and C3H/He, were fed on commercial food with 10% (v/v) ethanol solution as drinking liquid ad libitum for eighty days, and the changes in the activities of enzymes in the metabolic pathway of ethanol in the liver were examined. C57BL and C3H/He mice showed a preference for drinking the 10% (v/v) ethanol solution, while DBA mice did not. The ethanol intake g/g of body weight of C3H/He mice showed the highest value among all three strains and that of C57BL mice tended to show higher value than that of DBA mice. The liver weights of C57BL and C3H/He mice increased significantly following chronic ethanol administration, but that of DBA did not. The cytosolic enzyme alcohol dehydrogenase (ADH) showed no changes in any of the strains following chronic ethanol administration. The microsomal ethanol-oxidizing system (MEOS) of C57BL mice exhibited approximately 2-fold higher activity compared to that of DBA and C3H/He mice but did not increase in any strain following chronic ethanol administration. However, the microsomal aniline hydroxylase activity in the liver increased significantly in C57BL and C3H/He mice following chronic administration of ethanol. The microsomal cytochrome P-450 content also tended to slightly increase in the same strains of mice. It seemed that cytochrome P-450IIE1 was induced in the liver microsomes of these strains. Total aldehyde dehydrogenase (ALDH) activities together with high-Km ALDH activity increased markedly in the microsomes of C57BL mice and tended to increase in C3H/He mice, while it did not change in DBA mice following chronic ethanol administration. In the mitochondria of C57BL, total ALDH activities increased slightly and high-Km ALDH activities tended to increase. These mitochondrial ALDH activities of C3H/He and DBA mice tended to increase following chronic ethanol administration. The cytosolic ALDH activity showed no changes in any strain of mice following chronic ethanol administration. It seemed that in the microsomes, the activities of enzymes related to oxidation of ethanol increased in C57BL and C3H/He mice, which tended to consume a large amount of ethanol, and did not in DBA mice which tended to consume a small amount of it. It seemed that the increases in activities of enzymes related to oxidation of acetaldehyde in the microsomes and in the mitochondria were responsible for the strain difference.     

Effect of S-adenosyl-L-methionine and dilinoleoylphosphatidylcholine on liver lipid composition and ethanol hepatotoxicity in isolated perfused rat liver

We investigated whether S-adenosyl-L-methionine (SAMe), dilinoleoylphosphatidylcholine (DLPC), or SAMe + DLPC influence liver lipid composition as well as acute ethanol hepatotoxicity in the isolated perfused rat liver (IPRL). SAMe (25 mg/kg intramuscularly three times a day) was administered for five consecutive days, while DLPC was administered intraperitoneally for five days. The liver was then isolated, perfused with taurocholate to stabilize bile secretion, and exposed to 0.5% ethanol for 70 min. SAMe, without changing total phospholipid (PL) content, induced an increase in the phosphatidylcholine/phosphatidylethanolamine (PC/PE) molar ratio in both liver homogenate and microsomes and a significant enrichment of 16:0-20:4 and 18:0-20:4 PC molecular species. DLPC induced a significant enrichment of PL in liver homogenate and microsomes due to a contemporary increase in PC and PE. The PC enrichment specifically involved 16:0-20:4 and 18:0-20:4 PC molecular species besides the HPLC peak containing the administered 18:2-18:2 PC species. DLPC + SAMe increased the concentration of PC in liver homogenate and microsomes due to a specific enrichment of 16:0-22:6, 16:0-20:4, and 18:0-20:4 PC molecular species, and the HPLC peak containing the administered 18:2-18:2 PC species. Ethanol acute exposure in the control IPRLs for 70 min induced a depletion of cholesterol in both liver homogenate and microsomes without significant changes in the composition of PL classes and PC molecular species. SAMe, DLPC, or SAMe + DLPC counteracted the cholesterol depletion induced by ethanol, indicating that phospholipid changes promoted by these treatments all induce a major resistance of liver membranes to the effect of ethanol. Ethanol administration in control IPRLs induced a fivefold increase of AST and LDH release in the perfusate, depletion of glutathione in homogenates and mitochondria, decreased oxygen liver consumption, and inhibition of bile flow. These effects of ethanol were significantly antagonized by SAMe. In contrast, DLPC alone only minimally attenuated enzyme release in the perfusate and the inhibitory effect of ethanol on bile flow, but it failed to influence the depletion of total and mitochondrial glutathione or the depressed oxygen consumption induced by ethanol. DLPC, administered together with SAMe, added nothing to the protective effect of SAMe against ethanol hepatotoxicity and cholestasis. In conclusion, this study demonstrates that both SAMe and DLPC induced marked modifications in the lipid composition of liver membranes with a similar enrichment of polyunsaturated PC molecular species. Only SAMe, however, significantly protected against the hepatotoxic and cholestatic effect of acute ethanol administration, an effect associated with maintained normal glutathione mitochondrial levels and oxygen liver consumption. This indicates that the protective effect of SAMe against ethanol toxicity is linked to multiple mechanisms, the maintenance of glutathione levels probably being one of the most important.     

Prevention of mitochondrial injury by manganese superoxide dismutase reveals a primary mechanism for alkaline-induced cell death

Alkalosis is a clinical complication resulting from various pathological and physiological conditions. Although it is well established that reducing the cellular proton concentration is lethal, the mechanism leading to cell death is unknown. Mitochondrial respiration generates a proton gradient and superoxide radicals, suggesting a possible link between oxidative stress, mitochondrial integrity, and alkaline-induced cell death. Manganese superoxide dismutase removes superoxide radicals in mitochondria, and thus protects mitochondria from oxidative injury. Cells cultured under alkaline conditions were found to exhibit elevated levels of mitochondrial membrane potential, reactive oxygen species, and calcium which was accompanied by mitochondrial damage, DNA fragmentation, and cell death. Overexpression of manganese superoxide dismutase reduced the levels of intracellular reactive oxygen species and calcium, restored mitochondrial transmembrane potential, and prevented cell death. The results suggest that mitochondria are the primary target for alkaline-induced cell death and that free radical generation is an important and early event conveying cell death signals under alkaline conditions.     

Characterization of NAD(P)H-dependent ubiquinone reductase activities in rat liver microsomes

Exogenous ubiquinone-10 was efficiently reduced by rat liver microsomes in the presence of NADH and NADPH under anaerobic conditions. Ubiquinone-10 reduced under anaerobic conditions was rapidly re-oxidized by the re-aeration. The reduction and re-oxidation were not observed when the reactions were carried out with the boiled microsomes or without microsomes, suggesting that the reactions were enzymatically catalyzed by the electron transport system(s) from NAD(P)H to O2 through the ubiquinone. The Km and Vmax of the reductase activity for NADH were 0.4 mM and 1.7 nmol/min per mg of protein, and those for NADPH were 19 microM and 2.1 nmol/min per mg of protein, respectively. The NADH-dependent oxidoreduction system was different from the NADPH-dependent system because of the following observations; (1) rotenone inhibited only the NADH-dependent ubiquinone-10 reductase, (2) dicoumarol inhibited the NADPH-dependent ubiquinone-10 reduction more potently than the NADH-dependent reduction and (3) the activity oxidizing the reduced ubiquinone-10 in the presence of NADH was less than that in the presence of NADPH. Endogenous ubiquinone-9 was also reduced and re-oxidized in essentially the same manner as exogenous ubiquinone-10. Thus, ubiquinone-10 oxidoreductase in rat liver microsomes acts on endogenous ubiquinone-9.     

Asymptomatic hypertension in the ED

(R)-(+)-Menthofuran is a potent, mechanism-based inactivator of human liver cytochrome P450 (CYP or P450) 2A6. Menthofuran caused a time- and concentration-dependent loss of CYP2A6 activity. The inactivation of CYP2A6 was characterized by a Ki of 2.5 microM and a kinact of 0.22 min-1 for human liver microsomes and a Ki of 0.84 microM and a kinact of 0.25 min-1 for purified expressed CYP2A6. Addition of various nucleophiles, a chelator of iron, or scavengers of reactive oxygen species or extensive dialysis failed to protect CYP2A6 from inactivation. An antibody to metallothionein conjugates of a suspected reactive metabolite of menthofuran was used to detect reactive menthofuran metabolite adducts with CYP2A6. These adducts were formed only in the presence of NADPH-P450 reductase and NADPH. Glutathione, methoxylamine, and semicarbazide did not prevent adduction of reactive menthofuran metabolites to CYP2A6, however. The menthofuran metabolite formation/CYP2A6 inactivation partition ratio was determined to be 3.5 +/- 0.6 nmol/nmol of P450. Menthofuran was unable to inactivate CYP1A2, CYP2D6, CYP2E1, or CYP3A4 in a time- and concentration-dependent manner.     

Genetic-demographic characteristics of farming regions and small cities of the Tomsk district

The effect of the herbicide 4,6-dinitro-o-cresol (DNOC), a structural analogue of the classical protonophore 2,4-dinitrophenol, on the bioenergetics and inner membrane permeability of isolated rat liver mitochondria was studied. We observed that DNOC (10-50 microM) acts as a classical uncoupler of oxidative phosphorylation in rat liver mitochondria, promoting both an increase in succinate-supported mitochondrial respiration in the presence or absence of ADP and a decrease in transmembrane potential. The protonophoric activity of DNOC was evidenced by the induction of mitochondrial swelling in hyposmotic K(+)-acetate medium, in the presence of valinomycin. At higher concentrations (> 50 microM), DNOC also induces an inhibition of succinate-supported respiration, and a decrease in the activity of the succinate dehydrogenase can be observed. The addition of uncoupling concentrations of DNOC to Ca(2+)-loaded mitochondria treated with Ruthenium Red results in non-specific membrane permeabilization, as evidenced by mitochondrial swelling in isosmotic sucrose medium. Cyclosporin A, which inhibits mitochondrial permeability transition, prevented DNOC-induced mitochondrial swelling in the presence of Ca2+, which was accompanied by a decrease in mitochondrial membrane protein thiol content, owing to protein thiol oxidation. Catalase partially inhibits mitochondrial swelling and protein thiol oxidation, indicating the participation of mitochondrial-generated reactive oxygen species in this process. It is concluded that DNOC is a potent potent protonophore acting as a classical uncoupler of oxidative phosphorylation in rat liver mitochondria by dissipating the proton electrochemical gradient. Treatment of Ca(2+)-loaded mitochondria with uncoupling concentrations of DNOC results in mitochondrial permeability transition, associated with membrane protein thiol oxidation by reactive oxygen species.     

Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria

Mitochondrial physiology is disrupted in either apoptosis or necrosis. Here, we report that a wide variety of apoptotic and necrotic stimuli induce progressive mitochondrial swelling and outer mitochondrial membrane rupture. Discontinuity of the outer mitochondrial membrane results in cytochrome c redistribution from the intermembrane space to the cytosol followed by subsequent inner mitochondrial membrane depolarization. The mitochondrial membrane protein Bcl-xL can inhibit these changes in cells treated with apoptotic stimuli. In addition, Bcl-xL-expressing cells adapt to growth factor withdrawal or staurosporine treatment by maintaining a decreased mitochondrial membrane potential. Bcl-xL expression also prevents mitochondrial swelling in response to agents that inhibit oxidative phosphorylation. These data suggest that Bcl-xL promotes cell survival by regulating the electrical and osmotic homeostasis of mitochondria.     

Mitochondrial complex I defects in aging

According to the 'mitochondrial theory of aging' it is expected that the activity of NADH Coenzyme Q reductase (Complex I) would be most severely affected among mitochondrial enzymes, since mitochondrial DNA encodes for 7 subunits of this enzyme. Being these subunits the site of binding of the acceptor substrate (Coenzyme Q) and of most inhibitors of the enzyme, it is also expected that subtle kinetic changes of quinone affinity and enzyme inhibition could develop in aging before an overall loss of activity would be observed. The overall activity of Complex I was decreased in several tissues from aged rats, nevertheless it was found that direct assay of Complex I using artificial quinone acceptors may underevaluate the enzyme activity. The most acceptable results could be obtained by applying the 'pool equation' to calculate Complex I activity from aerobic NADH oxidation; using this method it was found that the decrease in Complex I activity in mitochondria from old animals was greater than the activity calculated by direct assay of NADH Coenzyme Q reductase. A decrease of NADH oxidation and its rotenone sensitivity was observed in nonsynaptic mitochondria, but not in synaptic 'light' and 'heavy' mitochondria of brain cortex from aged rats. In a study of Complex I activity in human platelet membranes we found that the enzyme activity was unchanged but the titre for half-inhibition by rotenone was significantly increased in aged individuals and proposed this change as a suitable biomarker of aging and age-related diseases.     

Nature of inhibition of mitochondrial respiratory complex I by 6-Hydroxydopamine

The catecholaminergic neurotoxin 6-hydroxydopamine causes parkinsonian symptoms in animals and it has been proposed that reactive oxygen species and oxidative stress, enhanced by iron, may play a key role in its toxicity. The present results demonstrate that 6-hydroxydopamine reversibly inhibits complex I (NADH dehydrogenase) of brain mitochondrial respiratory chain in isolated mitochondria. 6-Hydroxydopamine itself, rather than its oxidative products, was responsible for the inhibition. Iron (III) did not enhance inhibition but decreased it by stimulating the nonenzyme oxidation of 6-hydroxydopamine. Inhibition was potentiated to some extent by calcium ion. Desferrioxamine protected complex I activity against the inhibition, but it was not due to its chelator or antioxidative properties. Desferrioxamine was also shown to activate NADH dehydrogenase in the absence of 6-hydroxydopamine. Activation of mitochondrial respiration by desferrioxamine may contribute to the enhanced neuron survival in the presence of desferrioxamine in some neurodegenerative conditions.