Oxidative phosphorylation by in situ synaptosomal mitochondria from whole brain of young and old rats

Synaptosomes, isolated from the whole brain of young (3 months) and old (24 months) rats were used to study the major bioenergetic systems of neuronal mitochondria in situ, within the synaptosome. Approximately 85% of the resting oxygen consumption of synaptosomes from both young and old rats was a result of proton leak (and possibly other ion cycling) across the mitochondrial inner membrane. There were no significant differences between synaptosomes from the young and old rats in the kinetic responses of the substrate oxidation system, the mitochondrial proton leak and the phosphorylation system to changes in the proton electrochemical gradient. Flux control coefficients of 0.71, 0.27 and 0.02 were calculated for substrate oxidation system, phosphorylation system and the proton leak, respectively, at maximal ATP producing capacity in synaptosomes from young animals. The corresponding values calculated for synaptosomes from old animals were 0.53, 0.43 and 0.05. Thus substrate oxidation had greatest control over oxygen consumption at maximal phosphorylating capacity for synaptosomes from whole brain, with proton leak, having little control under maximal ATP producing capacity. The uncoupled rate of oxygen consumption, in the presence of the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), was significantly lower (p = 0.0124) in synaptosomes from old rats (6.08 +/- 0.42, n = 11) when compared with those from the young rats (7.87 +/- 0.48, n = 8). Thus, there is an impaired flux through the substrate oxidation system is synaptosomes from old rats, as compared to synaptosomes from the young animals. These in situ results may have important implications for the interpretation of theories that age-dependent impairment of mitochondrial energy production may result in increased susceptibility to neurodegeneration.     

Membrane potential and H2O2 production in duodenal mitochondria from broiler chickens (Gallus gallus domesticus) with low and high feed efficiency

Increased hydrogen peroxide (H2O2) production was observed in duodenal mitochondria obtained from broiler chickens with low feed efficiency (FE). As a decrease in mitochondrial membrane potential (Deltapsi(m)) due to mild uncoupling of oxidative phosphorylation reduces reactive oxygen species production, this study was conducted to evaluate the effect of uncoupling on Deltapsi(m) and H2O2 production in duodenal mitochondria isolated from broilers with low (0.48+/-0.02) and high (0.68+/-0.01) FE. Membrane potential and H2O2 production were measured fluorometrically and in the presence of different levels of an uncoupler, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP). The Deltapsi(m) was higher (P相似文献   

Comparative effects of herbicide dicamba and related compound on plant mitochondrial bioenergetics

The herbicide dicamba (3,6-dichloro-2-methoxybenzoic acid) was evaluated for its effects on bioenergetic activities of potato tuber mitochondria to elucidate putative mechanisms of action and to compare its toxicity with 2-chlorobenzoic acid. Dicamba (4 micro mol/mg mitochondrial protein) induces a limited stimulation of state 4 respiration of ca. 10%, and the above concentrations significantly inhibit respiration, whereas 2-chlorobenzoic acid maximally stimulates state 4 respiration (ca. 50%) at about 25 micro mol/mg mitochondrial protein. As opposed to these limited effects on state 4 respiration, transmembrane electrical potential is strongly decreased by dicamba and 2-chlorobenzoic acid. Dicamba (25 micro mol/mg mitochondrial protein) collapses, almost completely, Deltapsi; similar concentrations of 2-chlorobenzoic acid promote Deltapsi drops of about 50%. Proton permeabilization partially contributes to Deltapsi collapse since swelling in K-acetate medium is stimulated, with dicamba promoting a stronger stimulation. The Deltapsi decrease induced by dicamba is not exclusively the result of a stimulation on the proton leak through the mitochondrial inner membrane, since there was no correspondence between the Deltapsi decrease and the change on the O(2) consumption on state 4 respiration; on the contrary, for concentrations above 8 micro mol/mg mitochondrial protein a strong inhibition was observed. Both compounds inhibit the activity of respiratory complexes II and III but complex IV is not significantly affected. Complex I seems to be sensitive to these xenobiotics. In conclusion, dicamba is a stronger mitochondrial respiratory chain inhibitor and uncoupler as compared to 2-chlorobenzoic acid. Apparently, the differences in the lipophilicity are related to the different activities on mitochondrial bioenergetics.     

Carvedilol inhibits mitochondrial complex I and induces resistance to H2O2 -mediated oxidative insult in H9C2 myocardial cells

Carvedilol, a beta-adrenoreceptor antagonist with strong antioxidant activity, produces a high degree of cardioprotection in a variety of experimental models of ischemic cardiac injury. Although growing evidences suggest specific effects on mitochondrial metabolism, how carvedilol would exert its overall activity has not been completely disclosed. In the present work we have investigated the impact of carvedilol-treatment on mitochondrial bioenergetic functions and ROS metabolism in H9C2 cells. This analysis has revealed a dose-dependent decrease in respiratory fluxes by NAD-dependent substrates associated with a consistent decline of mitochondrial complex I activity. These changes were associated with an increase in mitochondrial H(2)O(2) production, total glutathione and protein thiols content. To evaluate the antioxidant activity of carvedilol, the effect of the exposure of control and carvedilol-pretreated H9C2 cells to H(2)O(2) were investigated. The H(2)O(2)-mediated oxidative insult resulted in a significant decrease of mitochondrial respiration, glutathione and protein thiol content and in an increased level of GSSG. These changes were prevented by carvedilol-pretreatment. A similar protective effect on mitochondrial respiration could be obtained by pre-treatment of the cells with a sub-saturating amount of rotenone, a complex I inhibitor. We therefore suggest that carvedilol exerts its protective antioxidant action both by a direct antioxidant effect and by a preconditioning-like mechanism, via inhibition of mitochondrial complex I.     

Regulation of mitochondrial uncoupling respiration during exercise in rat heart: role of reactive oxygen species (ROS) and uncoupling protein 2

The physiological significance of cardiac mitochondrial uncoupling protein 2 (UCP2)-mediated uncoupling respiration in exercise is unknown. In the current study, mitochondrial respiratory function, UCP2 mRNA level, UCP2-mediated respiration (UCR), and reactive oxygen species (ROS) generation, as well as manganese superoxide dismutase (MnSOD) activity were determined in rat heart with or without endurance training after an acute bout of exercise of different duration. In the untrained rats, state 4 respiration and UCR-independent respiration rates were progressively increased with exercise time and were 64 and 70% higher, respectively, than resting rate at 150 min, whereas UCR was elevated by 86% with no significant change in state 3 respiration. UCP2 mRNA level showed a 5- and 4-fold increase, respectively, after 45 and 90 min of exercise, but returned to resting level at 120 and 150 min. Mitochondrial ROS production and membrane potential (Deltapsi) increased progressively until 120 min, followed by a decrease to the resting level at 150 min. MnSOD mRNA abundance showed a 2-fold increase at 120 min but MnSOD activity did not change with exercise. Training significantly increased mitochondrial ATP synthetase activity, ADP to oxygen consumption (P/O) ratio, respiratory control ratio, and MnSOD activity, whereas exercise-induced state 4 respiration, UCR, ROS production, and Deltapsi were attenuated in the trained rats. We conclude that (1) UCP2 mRNA expression and activity in rat heart can be upregulated during prolonged exercise, which may reduce cross-membrane Deltapsi and thus ROS production; and (2) endurance training can blunt exercise-induced UCP2 and UCR, and improve mitochondrial efficiency of oxidative phosphorylation due to increased removal of ROS.     

Changes in the calcium transport systems of rat brain synaptosomes and their possible role in the pathophysiology of aging

Studies were undertaken on the age-associated peculiarities of the Ca2+ transport systems of the rat brain synaptosomes. It has been found that 45Ca2+ uptake reduced with ageing. The above reduction was not linked with the changes in the permeability of potential-dependent synaptosomal membrane Ca2+ depending upon the membrane potential. The distribution of calcium across the mitochondrial membrane changed with ageing, shifting towards higher extramitochondrial calcium levels in old rats, both in isolated and in synaptosomal mitochondria. While studying calcium efflux from mitochondria, it was found that, at equivalent calcium loads, the calcium efflux rates were slower in old rats as compared to adult animals. As observed, both resting [Ca2+]i and that obtained after K-depolarization drastically increased in old animals. The possible pathogenic mechanisms in neuronal injury, conditioned by this increase, are discussed.     

Effect of voluntary exercise on H2O2 release by subsarcolemmal and intermyofibrillar mitochondria

Previous data have demonstrated that, to handle the oxidative stress encountered with training at high intensity, skeletal muscle relies on an increase in mitochondrial biogenesis, a reduced H(2)O(2) production, and an enhancement of antioxidant enzymes. In the present study, we evaluated the influence of voluntary running on mitochondrial O(2) consumption and H(2)O(2) production by intermyofibrillar mitochondria (IFM) and subsarcolemmal mitochondria (SSM) isolated from oxidative muscles in conjunction with the determination of antioxidant capacities. When mitochondria are incubated with succinate as substrate, both maximal (state 3) and resting (state 4) O(2) consumption were significantly lower in SSM than in IFM populations. Mitochondrial H(2)O(2) release per unit of O(2) consumed was 2-fold higher in SSM than in IFM. Inhibition of H(2)O(2) formation by rotenone suggests that complex I of the electron transport chain is likely the major physiological H(2)O(2)-generating system. In Lou/C rats (an inbred strain of rats of Wistar origin), neither O(2) consumption nor H(2)O(2) release by IFM and SSM were affected by long-term, voluntary wheel training. In contrast, glutathione peroxidase and catalase activity were significantly increased despite no change in oxidative capacities with long-term, voluntary exercise. Furthermore, chronic exercise enhanced heat shock protein 72 accumulation within skeletal muscle. It is concluded that the antioxidant status of muscle can be significantly improved by prolonged wheel exercise without necessitating an increase in mitochondrial oxidative capacities.     

Influence of mitochondrial membrane fatty acid composition on proton leak and H2O2 production in liver

Mitochondrial membrane fatty acid composition has been proposed to play a role in determining mitochondrial proton leak rate. The purpose of this study was to determine if feeding rats diets with different fatty acid sources produces changes in liver proton leak and H(2)O(2) production. Six-month-old male FBNF(1) rats were fed diets with a primary fat source of either corn or fish oil for a 6-month period. As expected, diet manipulations produced substantial differences in mitochondrial fatty acid composition. These changes were most striking for 20:4n6 and 22:6n3. However, proton leak and phosphorylation kinetics as well as lipid and protein oxidative damage were not different (P > 0.10) between fish and corn oil groups. Metabolic control analysis, however, did show that control of both substrate oxidation and phosphorylation was shifted away from substrate oxidation reactions to increased control by phosphorylation reactions in fish versus corn oil groups. Increased mitochondrial H(2)O(2) production was observed in corn versus fish oil-fed rats when mitochondria were respiring on succinate alone or on either succinate or pyruvate/malate in the presence of antimycin A. These results show that mitochondrial H(2)O(2) production and the regulation of oxidative phosphorylation are altered in liver mitochondria from rats consuming diets with either fish or corn oil as the primary lipid source.     

T cell activation-induced mitochondrial hyperpolarization is mediated by Ca2+- and redox-dependent production of nitric oxide

Activation, proliferation, or programmed cell death of T lymphocytes is regulated by the mitochondrial transmembrane potential (Deltapsi(m)) through controlling ATP synthesis, production of reactive oxygen intermediates (ROI), and release of cell death-inducing factors. Elevation of Deltapsi(m) or mitochondrial hyperpolarization is an early and reversible event associated with both T cell activation and apoptosis. In the present study, T cell activation signals leading to mitochondrial hyperpolarization were investigated. CD3/CD28 costimulation of human PBL elevated cytoplasmic and mitochondrial Ca(2+) levels, ROI production, and NO production, and elicited mitochondrial hyperpolarization. Although T cell activation-induced Ca(2+) release, ROI levels, and NO production were diminished by inositol 1,4,5-triphosphate receptor antagonist 2-aminoethoxydiphenyl borane, superoxide dismutase mimic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride, spin trap 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide, and NO chelator carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, mitochondrial hyperpolarization was selectively inhibited by carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (-85.0 +/- 10.0%; p = 0.008) and, to a lesser extent, by 2-aminoethoxydiphenyl borane. Moreover, NO precursor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate diethylenetriamine elicited NO and ROI production, Ca(2+) release, transient ATP depletion, and robust mitochondrial hyperpolarization (3.5 +/- 0.8-fold; p = 0.002). Western blot analysis revealed expression of Ca-dependent endothelial NO synthase and neuronal NO synthase isoforms and absence of Ca-independent inducible NO synthase in PBL. CD3/CD28 costimulation or H(2)O(2) elicited severalfold elevations of endothelial NO synthase and neuronal NO synthase expression, as compared with beta-actin. H(2)O(2) also led to moderate mitochondrial hyperpolarization; however, Ca(2+) influx by ionomycin or Ca(2+) release from intracellular stores by thapsigargin alone failed to induce NO synthase expression, NO production, or Deltapsi(m) elevation. The results suggest that T cell activation-induced mitochondrial hyperpolarization is mediated by ROI- and Ca(2+)-dependent NO production.     

Increase of sodium delivery stimulates the mitochondrial respiratory chain H2O2 production in rat renal medullary thick ascending limb

The mitochondria-rich epithelial cells of the renal medullary thick ascending limb (mTAL) reabsorb nearly 25% of filtered sodium (Na(+)) and are a major source of cellular reactive oxygen species. Although we have shown that delivery of Na(+) to the mTAL of rats increases superoxide (O(2)(·-)) production in mTAL, little is known about H(2)O(2) production, given the lack of robust and selective fluorescent indicators for determining changes within the whole cell, specifically in the mitochondria. The present study determined the effect of increased tubular flow and Na(+) delivery to mTAL on the production of mitochondrial H(2)O(2) in mTAL. H(2)O(2) responses were determined in isolated, perfused mTAL of Sprague-Dawley rats using a novel mitochondrial selective fluorescent H(2)O(2) indicator, mitochondria peroxy yellow 1, and a novel, highly sensitive and stable cytosolic-localized H(2)O(2) indicator, peroxyfluor-6 acetoxymethyl ester. The results showed that mitochondrial H(2)O(2) and cellular fluorescent signals increased progressively over a period of 30 min following increased tubular perfusion (5-20 nl/min), reaching levels of statistical significance at ～10-12 min. Responses were inhibited with rotenone or antimycin A (inhibitors of the electron-transport chain), polyethylene glycol-catalase and by reducing Na(+) transport with furosemide or ouabain. Inhibition of membrane NADPH-oxidase with apocynin had no effect on mitochondrial H(2)O(2) production. Cytoplasmic H(2)O(2) (peroxyfluor-6 acetoxymethyl ester) increased in parallel with mitochondrial H(2)O(2) (mitochondria peroxy yellow 1) and was partially attenuated (～65%) by rotenone and completely inhibited by apocynin. The present data provide clear evidence that H(2)O(2) is produced in the mitochondria in response to increased flow and delivery of Na(+) to the mTAL, and that whole cell H(2)O(2) levels are triggered by the mitochondrial reactive oxygen species production. The mitochondrial production of H(2)O(2) may represent an important target for development of more effective antioxidant therapies.     

Catalase-dependent measurement of H2O2 in intact mitochondria

Mitochondria generate potentially damaging amounts of superoxide and H2O2 during oxidative metabolism. Although many assays are available to measure mitochondrial H2O2 generation, most detect H2O2 that has escaped the organelle. To measure H2O2 within mitochondria that contain catalase, we have developed an assay based on the ability of H2O2 to inhibit catalase in the presence of 3-amino-1,2,4-triazole. The assay is simple to perform, does not require expensive instrumentation, and is specific for H2O2. Results from this assay show that H2O2 generation in rat heart mitochondria reflects the activity of the electron transport chain. Further, liver mitochondria prepared from selenium-deficient rats have increased succinate-stimulated rates of H2O2 generation. This indicates that mitochondrial selenoenzymes are important for H2O2 removal. It also demonstrates the utility of this assay in measuring H2O2 release from mitochondria that do not contain catalase. The assay should be useful for study of both superoxide-dependent H2O2 generation in situ, and the role of endogenous mitochondrial catalase in H2O2 removal.     

Xanthine oxidase and mitochondria contribute to vascular superoxide anion generation in DOCA-salt hypertensive rats

Vascular superoxide anion (O(2)(*-)) levels are increased in DOCA-salt hypertensive rats. We hypothesized that the endothelin (ET)-1-induced generation of ROS in the aorta and resistance arteries of DOCA-salt rats originates partly from xanthine oxidase (XO) and mitochondria. Accordingly, we blocked XO and the mitochondrial oxidative phosphorylation chain to investigate their contribution to ROS production in mesenteric resistance arteries and the aorta from DOCA-salt rats. Systolic blood pressure rose in DOCA-salt rats and was reduced after 3 wk by apocynin [NAD(P)H oxidase inhibitor and/or radical scavenger], allopurinol (XO inhibitor), bosentan (ET(A/B) receptor antagonist), BMS-182874 (BMS; ET(A) receptor antagonist), and hydralazine. Plasma uric acid levels in DOCA-salt rats were similar to control unilaterally nephrectomized (UniNx) rats, reduced with allopurinol and bosentan, and increased with BMS. Levels of thiobarbituric acid-reacting substances were increased in DOCA-salt rats versus UniNx rats, and BMS, bosentan, and hydralazine prevented their increase. Dihydroethidium staining showed reduced O(2)(*-) production in mesenteric arteries and the aorta from BMS- and bosentan-treated DOCA-salt rats compared with untreated DOCA-salt rats. Increased O(2)(*-) derived from XO was reduced or prevented by all treatments in mesenteric arteries, whereas bosentan and BMS had no effect on aortas from DOCA-salt rats. O(2)(*-) generation decreased with in situ treatment by tenoyltrifluoroacetone and CCCP, inhibitors of mitochondrial electron transport complexes II and IV, respectively, whereas rotenone (mitochondrial complex I inhibitor) had no effect. Our findings demonstrate the involvement of ET(A) receptor-modulated O(2)(*-) derived from XO and from mitochondrial oxidative enzymes in arteries from DOCA-salt rats.     

High-sucrose diet increases ROS generation, FFA accumulation, UCP2 level, and proton leak in liver mitochondria

Obesity, a risk factor for insulin resistance, contributes to the development of type 2 diabetes and cardiovascular diseases. The relationship between increased levels of free fatty acids in the liver mitochondria, mitochondrial function, and ROS generation in rat model of obesity induced by a high-sucrose diet was not sufficiently established. We determined how the bioenergetic functions and ROS generation of the mitochondria respond to a hyperlipidemic environment. Mitochondria from sucrose-fed rats generated H(2)O(2) at a higher rate than the control mitochondria. Adding fatty acid-free bovine serum albumin to mitochondria from sucrose-fed rats significantly reduced the rate of H(2)O(2) generation. In contrast, adding exogenous oleic or linoleic acid exacerbated the rate of H(2)O(2) generation in both sucrose-fed and control mitochondria, and the mitochondria from sucrose-fed rats were more sensitive than the control mitochondria. The increased rate of H(2)O(2) generation in sucrose-fed mitochondria corresponded to decreased levels of reduced GSH and vitamin E and increased levels of Cu/Zn-SOD in the intermembrane space. There was no difference between the levels of lipid peroxidation and protein carbonylation in the two types of mitochondria. In addition to the normal activity of Mn-SOD, GPX and catalase detected an increased activity of complex II, and upregulation of UCP2 was observed in mitochondria from sucrose-fed rats, all of which may accelerate respiration rates and reduce generation of ROS. In turn, these effects may protect the mitochondria of sucrose-fed rats from oxidative stress and preserve their function and integrity. However, in whole liver these adaptive mechanisms of the mitochondria were inefficient at counteracting redox imbalances and inhibiting oxidative stress outside of the mitochondria.     

Leishmania donovani: intracellular ATP level regulates apoptosis-like death in luteolin induced dyskinetoplastid cells

Leishmaniasis presents a spectrum of diseases ranging from benign cutaneous lesions to the often-fatal visceralizing form. Luteolin, a dietary flavone induces apoptosis-like death in both promastigote and amastigote forms of Leishmania, the causative agent of the diseases. Here, we have elucidated the mechanism of action of luteolin by analyzing the mitochondrial and cytosolic changes associated with apoptosis-like death of leishmanial cells. In Leishmania donovani, treatment with luteolin induces the loss of both maxicircles and minicircles which resulted in the formation of dyskinetoplastid cells. The loss of mitochondrial DNA causes reduction in the activities of complex I, II, III, and IV of electron transport chain. However, the mitochondrial ATPase activity of complex V remains almost unaltered during treatment with luteolin but the sensitivity to oligomycin is lost. The inactivation of ETC complex is associated with decrease in mitochondrial as well as glycolytic ATP production, which is responsible for depolarization of Deltapsi(m) and alteration in mitochondrial structure. This event is followed by the release of cytochrome c from mitochondria in mt-DNA depleted leishmanial cells and causes an activation of caspase like proteases. Collectively our results provide the first insight into the mechanistic pathway of apoptosis-like death where inhibition of glycolytic ATP production is an essential event responsible for depolarization of Deltapsi(m) in mt-DNA depleted cells to propagate apoptosis-like death in leishmanial cells.     

Modulation of brain mitochondrial function by deprenyl

The present study shows that deprenyl, a known inhibitor of monoamine oxidase B (MAO B), may generate changes in mitochondrial function. Brain submitochondrial membranes (SMP), synaptosomes and cytosolic fractions were incubated with different deprenyl concentrations and nitric oxide synthase (NOS) activity was measured. The effect of deprenyl on oxygen consumption, calcium-induced permeability transition and hydrogen peroxide (H(2)O(2)) production rates was studied in intact mitochondria. Respiratory complexes and monoamine oxidase activities were also measured in submitochondrial membranes. Incubation of brain submitochondrial membranes with deprenyl 10, 25 and 50 microM inhibited nitric oxide synthase activity in a concentration-dependent manner. The same effect was observed in cytosolic fractions and synaptosomes. Monoamine oxidase activity was inhibited at lower deprenyl concentrations (from 0.5 microM). Cytochrome oxidase (complex IV) activity was found 42% increased in the presence of 25 microM deprenyl in a condition of maximal nitric oxide synthase activity. Incubation of brain mitochondria with deprenyl 25 microM produced a 60% increase in oxygen uptake in state 3, but no significant changes were observed in state 4. Pre-incubation of brain mitochondria with deprenyl 0.5 and 1 microM inhibited calcium-induced mitochondrial permeability transition and decreased hydrogen peroxide production rates. Our results suggest that in vitro effects of deprenyl on mitochondrial function can occur through two different mechanisms, involving nitric oxide synthase inhibition and decreased hydrogen peroxide production.     

Effect of training on H(2)O(2) release by mitochondria from rat skeletal muscle

In this study, oxygen consumption and H(2)O(2) release rate by succinate or pyruvate/malate supplemented mitochondria isolated from skeletal muscle of trained and untrained rats were investigated. The overall mitochondrial antioxidant capacity and the effect of preincubation of mitochondria with GDP, an inhibitor of uncoupling proteins UCP1 and UCP2, on both succinate-supported H(2)O(2) release and membrane potential were also determined. The results indicate that training does not affect mitochondrial oxygen consumption with both complex-I- and complex II-linked substrates. Succinate-supported H(2)O(2) release was lower in trained than in untrained rats both in State 4 and State 3. Even the antimycin A-stimulated release was lower in trained rats. When pyruvate/malate were used as substrates, H(2)O(2) release rate was lower in trained rats only in the presence of antimycin A. The increase of mitochondrial protein content (determined by the ratio between cytochrome oxidase activities in homogenates and mitochondria) in trained muscle was such that the succinate-supported H(2)O(2) release per g of tissue was not significantly different in trained and untrained rats, while that supported by pyruvate/malate was higher in trained than in untrained animals. The lack of training-induced changes in overall antioxidant capacity of mitochondria indicates that the decrease in mitochondrial H(2)O(2) release cannot be attributed to a greater capacity of mitochondria to scavenge the reactive oxygen intermediates derived from univalent O(2) reduction by respiratory chain components. In contrast, the above decrease seems to depend on the drop induced by training in mitochondrial membrane potential. These training effects are not due to an increased level of mitochondrial uncoupling protein, because in the presence of GDP the increase in both membrane potential and H(2)O(2) release was greater in untrained than in trained rats.     

Aromatase activity in microsomes from rat ventral prostate and Dunning R3327H rat prostatic adenocarcinoma

We have measured aromatase activity in microsomes obtained from rat ventral prostate, using the 3H2O release method as described by Weisz. Production of 3H2O from 1 beta-[3H]androstenedione correlated with estrogen production measured by RIA and by TLC. The assay was optimized for incubation time and protein concentration, and used to determine the aromatase activity of ventral prostate microsomes from rats of varying age. Aromatase activity per mg microsomal protein increased from an average of 4 pmol/mg protein X h in 3-month old rats to 68 pmol/mg protein X h in 8-month old rats. Aromatase activity was also measured in microsomes from the Dunning R3327H rat prostatic adenocarcinoma, and was increased in tumors removed 225 days after implantation compared to tumors removed 141 days after implantation. Tumors removed 225 days after implantation from rats which had been treated with DES for 14 days displayed increased aromatase activity compared to untreated tumors. The presence of aromatase activity in the rat ventral prostate and rat prostatic adenocarcinoma would allow regulation of estrogen levels independent of circulating estrogen. Thus, in situ changes in estrogen production with age may contribute to the development of prostatic disease.     

Lower oxidative DNA damage despite greater ROS production in muscles from rats selectively bred for high running capacity

Artificial selection in rat has yielded high-capacity runners (HCR) and low-capacity runners (LCR) that differ in intrinsic (untrained) aerobic exercise ability and metabolic disease risk. To gain insight into how oxygen metabolism may have been affected by selection, we compared mitochondrial function, oxidative DNA damage (8-dihydroxy-guanosine; 8dOHG), and antioxidant enzyme activities in soleus muscle (Sol) and gastrocnemius muscle (Gas) of adult and aged LCR vs. HCR rats. In Sol of adult HCR rats, maximal ADP-stimulated respiration was 37% greater, whereas in Gas of adult HCR rats, there was a 23% greater complex IV-driven respiratory capacity and 54% greater leak as a fraction of electron transport capacity (suggesting looser mitochondrial coupling) vs. LCR rats. H(2)O(2) emission per gram of muscle was 24-26% greater for both muscles in adult HCR rats vs. LCR, although H(2)O(2) emission in Gas was 17% lower in HCR, after normalizing for citrate synthase activity (marker of mitochondrial content). Despite greater H(2)O(2) emission, 8dOHG levels were 62-78% lower in HCR rats due to 62-96% higher superoxide dismutase activity in both muscles and 47% higher catalase activity in Sol muscle in adult HCR rats, with no evidence for higher 8 oxoguanine glycosylase (OGG1; DNA repair enzyme) protein expression. We conclude that genetic segregation for high running capacity has generated a molecular network of cellular adaptations, facilitating a superior response to oxidative stress.     

Mitochondrial bound hexokinase activity as a preventive antioxidant defense: steady-state ADP formation as a regulatory mechanism of membrane potential and reactive oxygen species generation in mitochondria

Brain hexokinase is associated with the outer membrane of mitochondria, and its activity has been implicated in the regulation of ATP synthesis and apoptosis. Reactive oxygen species (ROS) are by-products of the electron transport chain in mitochondria. Here we show that the ADP produced by hexokinase activity in rat brain mitochondria (mt-hexokinase) controls both membrane potential (Deltapsi(m)) and ROS generation. Exposing control mitochondria to glucose increased the rate of oxygen consumption and reduced the rate of hydrogen peroxide generation. Mitochondrial associated hexokinase activity also regulated Deltapsi(m), because glucose stabilized low Deltapsi(m) values in state 3. Interestingly, the addition of glucose 6-phosphate significantly reduced the time of state 3 persistence, leading to an increase in the Deltapsi(m) and in H(2)O(2) generation. The glucose analogue 2-deoxyglucose completely impaired H(2)O(2) formation in state 3-state 4 transition. In sharp contrast, the mt-hexokinase-depleted mitochondria were, in all the above mentioned experiments, insensitive to glucose addition, indicating that the mt-hexokinase activity is pivotal in the homeostasis of the physiological functions of mitochondria. When mt-hexokinase-depleted mitochondria were incubated with exogenous yeast hexokinase, which is not able to bind to mitochondria, the rate of H(2)O(2) generation reached levels similar to those exhibited by control mitochondria only when an excess of 10-fold more enzyme activity was supplemented. Hyperglycemia induced in embryonic rat brain cortical neurons increased ROS production due to a rise in the intracellular glucose 6-phosphate levels, which were decreased by the inclusion of 2-deoxyglucose, N-acetyl cysteine, or carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Taken together, the results presented here indicate for the first time that mt-hexokinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.