Mitochondrial involvement in memory impairment induced by scopolamine in rats.

OBJECTIVE: Scopolamine (SCO) administration to rats induces molecular features of AD and other dementias, including impaired cognition, increased oxidative stress, and imbalanced cholinergic transmission. Although mitochondrial dysfunction is involved in different types of dementias, its role in cognitive impairment induced by SCO has not been well elucidated. The aim of this work was to evaluate the in vivo effect of SCO on different brain mitochondrial parameters in rats to explore its neurotoxic mechanisms of action. METHODS: Saline (Control) or SCO (1 mg/kg) was administered intraperitoneally 30 min prior to neurobehavioral and biochemical evaluations. Novel object recognition and Y-maze paradigms were used to evaluate the impact on memory, while redox profiles in different brain regions and the acetylcholinesterase (AChE) activity of the whole brain were assessed to elucidate the amnesic mechanism of SCO. Finally, the effects of SCO on brain mitochondria were evaluated both ex vivo and in vitro, the latter to determine whether SCO could directly interfere with mitochondrial function. RESULTS: SCO administration induced memory deficit, increased oxidative stress, and increased AChE activities in the hippocampus and prefrontal cortex. Isolated brain mitochondria from rats administered with SCO were more vulnerable to mitochondrial swelling, membrane potential dissipation, H2O2 generation and calcium efflux, all likely resulting from oxidative damage. The in vitro mitochondrial assays suggest that SCO did not affect the organelle function directly. CONCLUSION: In conclusion, the present results indicate that SCO induced cognitive dysfunction and oxidative stress may involve brain mitochondrial impairment, an important target for new neuroprotective compounds against AD and other dementias.

Cobalt oxide nanoparticles aggravate DNA damage and cell death in eggplant via mitochondrial swelling and NO signaling pathway.

BACKGROUND: Despite manifold benefits of nanoparticles (NPs), less information on the risks of NPs to human health and environment has been studied. Cobalt oxide nanoparticles (Co3O4-NPs) have been reported to cause toxicity in several organisms. In this study, we have investigated the role of Co3O4-NPs in inducing phytotoxicity, cellular DNA damage and apoptosis in eggplant (Solanum melongena L. cv. Violetta lunga 2). To the best of our knowledge, this is the first report on Co3O4-NPs showing phytotoxicity in eggplant. RESULTS: The data revealed that eggplant seeds treated with Co3O4-NPs for 2 h at a concentration of 1.0 mg/ml retarded root length by 81.5 % upon 7 days incubation in a moist chamber. Ultrastructural analysis by transmission electron microscopy (TEM) demonstrated the uptake and translocation of Co3O4-NPs into the cytoplasm. Intracellular presence of Co3O4-NPs triggered subcellular changes such as degeneration of mitochondrial cristae, abundance of peroxisomes and excessive vacuolization. Flow cytometric analysis of Co3O4-NPs (1.0 mg/ml) treated root protoplasts revealed 157, 282 and 178 % increase in reactive oxygen species (ROS), membrane potential (ΔΨm) and nitric oxide (NO), respectively. Besides, the esterase activity in treated protoplasts was also found compromised. About 2.4-fold greater level of DNA damage, as compared to untreated control was observed in Comet assay, and 73.2 % of Co3O4-NPs treated cells appeared apoptotic in flow cytometry based cell cycle analysis. CONCLUSION: This study demonstrate the phytotoxic potential of Co3O4-NPs in terms of reduction in seed germination, root growth, greater level of DNA and mitochondrial damage, oxidative stress and cell death in eggplant. The data generated from this study will provide a strong background to draw attention on Co3O4-NPs environmental hazards to vegetable crops.

Cobalt oxide nanoparticles aggravate DNA damage and cell death in eggplant via mitochondrial swelling and NO signaling pathway

BACKGROUND: Despite manifold benefits of nanoparticles (NPs), less information on the risks of NPs to human health and environment has been studied. Cobalt oxide nanoparticles (Co3O4-NPs) have been reported to cause toxicity in several organisms. In this study, we have investigated the role of Co3O4-NPs in inducing phytotoxicity, cellular DNA damage and apoptosis in eggplant (Solanum melongena L. cv. Violetta lunga 2). To the best of our knowledge, this is the first report on Co3O4-NPs showing phytotoxicity in eggplant. RESULTS: The data revealed that eggplant seeds treated with Co3O4-NPs for 2 h at a concentration of 1.0 mg/ml retarded root length by 81.5 % upon 7 days incubation in a moist chamber. Ultrastructural analysis by transmission electron microscopy (TEM) demonstrated the uptake and translocation of Co3O4-NPs into the cytoplasm. Intracellular presence of Co3O4-NPs triggered subcellular changes such as degeneration of mitochondrial cristae, abundance of peroxisomes and excessive vacuolization. Flow cytometric analysis of Co3O4-NPs (1.0 mg/ml) treated root protoplasts revealed 157, 282 and 178 % increase in reactive oxygen species (ROS), membrane potential (APm) and nitric oxide (NO), respectively. Besides, the esterase activity in treated protoplasts was also found compromised. About 2.4-fold greater level of DNA damage, as compared to untreated control was observed in Comet assay, and 73.2 % of Co3O4-NPs treated cells appeared apoptotic in flow cytometry based cell cycle analysis. CONCLUSION: This study demonstrate the phytotoxic potential of Co3O4-NPs in terms of reduction in seed germination, root growth, greater level of DNA and mitochondrial damage, oxidative stress and cell death in eggplant. The data generated from this study will provide a strong background to draw attention on Co3O4-NPs environmental hazards to vegetable crops.

Tissue and sex specificities in Ca2+ handling by isolated mitochondria in conditions avoiding the permeability transition.

NEW FINDINGS: What is the central question of this study? The assessment of Ca(2+) handling by isolated mitochondria can be biased by dysfunctions secondary to Ca(2+) -induced mitochondrial permeability transition (MPT). As a result of this uncertainty and the differing experimental conditions between studies, the tissue and sex diversities in mitochondrial Ca(2+) transport are still unsettled questions. What is the main finding and its importance? If MPT is not prevented during Ca(2+) transport assays, some measured variables are biased. Accounting for the implied importance of preventing MPT, we observed substantial tissue specificities in the mitochondrial Ca(2+) handling, particularly in the Ca(2+) efflux pathways. The characteristics of mitochondria, including their Ca(2+) transport functions, may exhibit tissue specificity and sexual dimorphism. Given that measurements of Ca(2+) handling by isolated mitochondria may be biased by dysfunction secondary to Ca(2+) -induced mitochondrial permeability transition (MPT) pore opening, this study evaluated the extent to which MPT inhibition by ciclosporin affected the measurement of Ca(2+) transport in isolated rat liver mitochondria. The results indicate that the steady-state levels of external Ca(2+) and the rates of mitochondrial Ca(2+) efflux through the selective pathways can be overestimated by up to fourfold if MPT pore opening is not prevented. We analysed Ca(2+) transport in isolated mitochondria from the liver, skeletal muscle, heart and brain of male and female rats in incubation conditions containing MPT inhibitors, NAD-linked substrates and relevant levels of free Ca(2+), Mg(2+) and Na(+). The Ca(2+) influx rates were similar among the samples, except that the liver mitochondria displayed values fourfold higher. In contrast, the Ca(2+) efflux rates exhibited more tissue diversity, especially in the presence of Na(+). Interestingly, the Na(+)-independent Ca(2+) efflux was highest in the heart mitochondria (∼ 4 nmol mg(-1) min(-1)), thus challenging the view that cardiac mitochondrial Ca(2+) efflux relies almost exclusively on a Na(+)-dependent pathway. Sex specificity was observed in only two kinetic indexes of heart mitochondrial Ca(2+) homeostasis and in the ADP-stimulated respiration of liver mitochondria (∼ 20% higher in females). The present study shows the methodological importance of preventing MPT when measuring the properties and the physiological variability of the Ca(2+) handling by isolated mitochondria.

Ethylmalonic acid induces permeability transition in isolated brain mitochondria.

Predominant accumulation of ethylmalonic acid (EMA) in tissues and biological fluids is a characteristic of patients affected by short chain acyl-CoA dehydrogenase deficiency and ethylmalonic encephalopathy. Neurological abnormalities are frequently found in these disorders, but the mechanisms underlying the brain injury are still obscure. Since hyperlacticacidemia is also found in many affected patients indicating a mitochondrial dysfunction; in the present work, we evaluated the in vitro and ex vivo effects of EMA plus Ca(2+) on mitochondrial integrity and redox balance in succinate-supported brain organelles. We verified that the evaluated parameters were disturbed only when EMA was associated with exogenous micromolar Ca(2+) concentrations. Thus, we found that this short chain organic acid plus Ca(2+) dissipated the membrane potential and provoked mitochondrial swelling, as well as impaired the mitochondrial Ca(2+) retention capacity, resulting in a rapid Ca(2+) release and decreased NAD(P)H matrix content. In contrast, EMA was not able to stimulate mitochondrial hydrogen peroxide generation. We also observed that all these effects were prevented by the mitochondrial Ca(2+) uptake inhibitor ruthenium red and the mitochondrial permeability transition (MPT) inhibitors cyclosporin A (CsA) and ADP. Furthermore, mitochondria isolated from rat brains after in vivo intrastriatal administration of EMA was more susceptible to Ca(2+)-induced swelling, which was fully prevented by CsA and ADP. Finally, EMA significantly decreased striatal slice viability, which was attenuated by CsA. The data strongly indicate that EMA reduced the mitochondrial threshold for Ca(2+)-induced MPT reinforcing the role of this cation in EMA-induced disruption of mitochondrial bioenergetics. It is, therefore, presumed that EMA acting synergistically with Ca(2+) compromises mitochondrial energy homeostasis in the central nervous system that may explain at least in part the neurologic alterations presented by patients with abnormal levels of this organic acid.

Titration of lysine residues on adenine nucleotide translocase by fluorescamine induces permeability transition.

Chemical modification of primary amino groups of mitochondrial membrane proteins by the fluorescent probe fluorescamine induces non-specific membrane permeabilisation. Titration of the lysine ϵ-amino group promoted efflux of accumulated Ca(2+), collapse of transmembrane potential and mitochondrial swelling. Ca(2+) release was inhibited by cyclosporin A. Considering the latter, we assumed that fluorescamine induces permeability transition. Carboxyatractyloside also inhibited the reaction. Using a polyclonal antibody for adenine nucleotide translocase, Western blot analysis showed that the carrier appeared labelled with the fluorescent probe. The results point out the importance of the ϵ-amino group of lysine residues, located in the adenine nucleotide carrier, on the modulation of membrane permeability, since its blockage suffices to promote opening of the non-specific nanopore.

Hyperbaric oxygen therapy and ischemia and reperfusion: a valuable association to attenuate ischemic lesion and hepatic reperfusion.

PURPOSE: To investigate the consequences of the association between hyperbaric oxygen therapy and hepatic ischemia / reperfusion. METHODS: Wistar rats were divided into three groups: SHAM, rats submitted to surgical stress and anesthetic but not hepatic ischemia or reperfusion, I / R, rats submitted to total hepatic pedicle ischemia for 30 min, followed by 5 min of reperfusion; HBO120, rats submitted to 120 min of hyperbaric oxygen therapy at two absolute atmospheres and immediately after submitted to the experimental protocol of ischemia and reperfusion. The preservation of the hepatic function was evaluated by determining mitochondrial swelling and malondialdehyde tissue level, as well as alanine aminotransferase and aspartate aminotranferase serum levels. The results were analyzed using the Mann-Whitney test and differences were considered significant for p<0.05. RESULTS: There were significant differences in values: mitochondrial swelling of the I / R group compared to SHAM and HBO120; malondialdehyde between SHAM vs. I / R, SHAM vs HBO120, and I / R vs HBO120, alanine aminotransferase between SHAM vs. I / R . There was no significant difference between groups in aspartate aminotransferase serum levels. CONCLUSION: The association between hyperbaric oxygen therapy and hepatic ischemia and reperfusion process was positive.

Hyperbaric oxygen therapy and ischemia and reperfusion: a valuable association to attenuate ischemic lesion and hepatic reperfusion

PURPOSE: To investigate the consequences of the association between hyperbaric oxygen therapy and hepatic ischemia / reperfusion. METHODS: Wistar rats were divided into three groups: SHAM, rats submitted to surgical stress and anesthetic but not hepatic ischemia or reperfusion, I / R, rats submitted to total hepatic pedicle ischemia for 30 min, followed by 5 min of reperfusion; HBO120, rats submitted to 120 min of hyperbaric oxygen therapy at two absolute atmospheres and immediately after submitted to the experimental protocol of ischemia and reperfusion. The preservation of the hepatic function was evaluated by determining mitochondrial swelling and malondialdehyde tissue level, as well as alanine aminotransferase and aspartate aminotranferase serum levels. The results were analyzed using the Mann-Whitney test and differences were considered significant for p<0.05. RESULTS: There were significant differences in values: mitochondrial swelling of the I / R group compared to SHAM and HBO120; malondialdehyde between SHAM vs. I / R, SHAM vs HBO120, and I / R vs HBO120, alanine aminotransferase between SHAM vs. I / R . There was no significant difference between groups in aspartate aminotransferase serum levels. CONCLUSION: The association between hyperbaric oxygen therapy and hepatic ischemia and reperfusion process was positive.

Mitochondrial swelling and incipient outer membrane rupture in preapoptotic and apoptotic cells.

Outer mitochondrial membrane (OMM) rupture was first noted in isolated mitochondria in which the inner mitochondrial membrane (IMM) had lost its selective permeability. This phenomenon referred to as mitochondrial permeability transition (MPT) refers to a permeabilized inner membrane that originates a large swelling in the mitochondrial matrix, which distends the outer membrane until it ruptures. Here, we have expanded previous electron microscopic observations that in apoptotic cells, OMM rupture is not caused by a membrane stretching promoted by a markedly swollen matrix. It is shown that the widths of the ruptured regions of the OMM vary from 6 to 250 nm. Independent of the perforation size, herniation of the mitochondrial matrix appeared to have resulted in pushing the IMM through the perforation. A large, long focal herniation of the mitochondrial matrix, covered with the IMM, was associated with a rupture of the OMM that was as small as 6 nm. Contextually, the collapse of the selective permeability of the IMM may precede or follow the release of the mitochondrial proteins of the intermembrane space into the cytoplasm. When the MPT is a late event, exit of the intermembrane space proteins to the cytoplasm is unimpeded and occurs through channels that transverse the outer membrane, because so far, the inner membrane is impermeable. No channel within the outer membrane can expose to the cytoplasm a permeable inner membrane, because it would serve as a conduit for local herniation of the mitochondrial matrix.

Acute brain damage induced by acetaminophen in mice: effect of diphenyl diselenide on oxidative stress and mitochondrial dysfunction.

Organoselenium compounds exhibit antioxidant activity, as well as a variety of biological activities, with potential pharmacological and therapeutic applications. The aim of this study was to investigate the effect of diphenyl diselenide (PhSe)(2) in reversing oxidative brain damage and mitochondrial dysfunction caused by administration of acetaminophen (APAP) in mice. Mice received a toxic dose of APAP, followed by a dose of (PhSe)(2) 1 h later. Four hours after the administration of APAP, plasma was withdrawn from the mice and used for biochemical assays of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) as markers of hepatotoxicity. Brain homogenate was examined to determine oxidative stress. Isolated brain mitochondria were examined to quantify mitochondrial transmembrane's electrical potential and mitochondrial swelling and to estimate reactive oxygen species (ROS) production. APAP administration caused an increase in plasma ALT and AST activities. APAP administration also caused a significant increase in the levels of thiobarbituric acid reactive substances (TBARS) and dichlorofluorescein oxidation in brain homogenate. Similarly, mitochondrial swelling and ROS production increased after APAP administration. APAP treatment also caused a decrease in Na(+), K(+)- ATPase activity and in mitochondrial membrane potential. These alterations observed in the brain of APAP-treated mice were restored by (PhSe)(2). Glutathione levels were decreased by APAP, but (PhSe)(2) did not reverse this change. Treatment with (PhSe)(2) after APAP administration can reverse the neurotoxicity caused by a single toxic dose of APAP. The neuroprotective effect of (PhSe)(2) is likely associated with its antioxidant properties.

Interaction of 1,3,4-thiadiazolium mesoionic derivatives with mitochondrial membrane and scavenging activity: Involvement of their effects on mitochondrial energy-linked functions.

The aim of this work was to assess the significance of the interaction of the 1,3,4-thiadiazolium derivatives MI-J, MI-4F and MI-2,4diF with mitochondrial membrane and their effects on energy-linked functions. Mitochondrial swelling in the absence of substrate was inhibited by all derivatives; however, the fluorine derivatives were most effective. MI-4F decreased swelling by ~32% even at the lowest concentration (65 nmol mg(-1) protein), reaching ~67% at the concentration of 130 nmol mg(-1) protein. Swelling of mitochondria in the presence of oxidizable substrates was also strongly decreased by all derivatives. This effect was more pronounced when using glutamate plus malate, and also fluorine derivatives, which promoted complete inhibition at all concentrations (6.5-130 nmol mg(-1) protein). Swelling occurred when succinate was the substrate in the presence of MI-J (6.5-65 nmol mg(-1) protein); however, the shrinkage rate was strongly decreased. MI-4F and MI-2,4diF also inhibited swelling, with total inhibition occurring at a concentration of 65 nmol mg(-1) protein. Lipid peroxidation induced by Fe(3+)-ADP/2-oxoglutarate in isolated mitochondria was inhibited time- and dose-dependently by the derivatives, reaching complete inhibition at the highest concentration (80 nmol mg(-1) protein). However, when lipid peroxidation was initiated by peroxyl radicals generated from AAPH, the inhibition was less intense, reaching ~50%, ~40% and ~58% with MI-J, MI-4F and MI-2,4diF (80 nmol mg(-1) protein), respectively. The mesoionic compounds also showed superoxide radical scavenging ability of ~22%, ~32% and ~40% (80 nmol mg(-1) protein), respectively. Fluorescence polarization experiments showed that the derivatives are able to enter the bilayer, decreasing its fluidity in the hydrophobic DMPC membrane region and ordering the fluid phase. Our results suggest that MI-J, MI-4F and MI-2,4diF interact significantly, albeit in different modes, with mitochondrial membrane, and that fluorine derivatives seem to alter the membrane's properties more markedly.

Mitochondrial function and nitric oxide production in hippocampus and cerebral cortex of rats exposed to enriched environment.

Male rats (21days) were assigned to enriched environment (EE) or to standard environment (SE) for 1year. Oxygen consumption and the sensitivity to calcium induced mitochondrial permeability transition (MPT), through mitochondrial membrane potential (DeltaPsi(m)) and swelling, were determined in isolated hippocampal and cerebral cortex mitochondria. Mitochondrial H(2)O(2) production rate, and NOS activity and expression associated with mitochondrial membranes were also assayed. Results showed that state 3 respiratory rate was increased by 80% in cerebral cortex mitochondria from EE rats and no changes were observed in hippocampal mitochondria after EE exposure. Calcium induced-swelling was 40% and 53% lower in hippocampal and cerebral cortex mitochondria from EE rats, as compared with SE rats. Calcium loading induced membrane depolarization in cerebral cortex mitochondria from EE rats but did not affect mitochondrial DeltaPsi(m) in hippocampal mitochondria from EE animals, probably due to decreased H(2)O(2) formation. NO production associated to mitochondrial membranes was increased by 195% in cerebral cortex mitochondria but decreased by 47% in hippocampal mitochondria from EE rats, as compared with SE rats. Western blot analysis from nNOS protein expression associated to mitochondrial samples revealed a similar pattern. Our results suggest that in hippocampus and cerebral cortex, EE exposure protects mitochondria against calcium-induced MPT maintaining a convenient membrane potential, which assures a continuous energy supply.

Ischemic preconditioning protects the pig liver by preserving the mitochondrial structure and downregulating caspase-3 activity.

BACKGROUND DATA: The beneficial effects of ischemic preconditioning (IPC) on hepatic ischemia-reperfusion injury (I/RI) have been described. However, the way in which IPC causes the changes in mitochondrial ultrastructure seen in hepatic I/RI is not well understood. OBJECTIVE: The objective of the present study was to determine whether IPC protects the liver from changes in mitochondrial structure and caspase 3 activity in the early phase of post-ischemic injury. METHODS: A pig model consisting of 90 min of hepatic ischemia and 180 min of reperfusion was employed. Eighteen female pigs were randomly divided into three groups: sham-operated, non-preconditioned, and ischemic preconditioned (10 min ischemia followed by 10 min reperfusion). Serum concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and thiobarbituric acid reactive substances (TBARS), as well as bile flow, were measured. Liver biopsies were taken after reperfusion for histological, immunohistochemical (anti-caspase 3), and ultrastructural examinations. RESULTS: The IPC procedure increased bile flow (p < 0.01), reduced serum AST level (p < 0.01), and reduced serum concentration of TBARS at 180 min of reperfusion (p = 0.05). Ischemic-preconditioned liver cells had less caspase 3 activity than the non-preconditioning group (p < 0.01), and changes in mitochondrial ultrastructure were reduced (p < 0.01). CONCLUSION: IPC exerts a powerful protective effect against hepatic I/RI in the early phase of reperfusion, which may be mediated by preservation of mitochondrial structure and inhibition of caspase-3 activity.

Redox properties of the adenoside triphosphate-sensitive K+ channel in brain mitochondria.

Brain mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) opening by diazoxide protects against ischemic damage and excitotoxic cell death. Here we studied the redox properties of brain mitoK(ATP) . MitoK(ATP) activation during excitotoxicity in cultured cerebellar granule neurons prevented the accumulation of reactive oxygen species (ROS) and cell death. Furthermore, mitoK(ATP) activation in isolated brain mitochondria significantly prevented H2O2 release by these organelles but did not change Ca2+ accumulation capacity. Interestingly, the activity of mitoK(ATP) was highly dependent on redox state. The thiol reductant mercaptopropionylglycine prevented mitoK(ATP) activity, whereas exogenous ROS activated the channel. In addition, the use of mitochondrial substrates that led to higher levels of endogenous mitochondrial ROS release closely correlated with enhanced K+ transport activity through mitoK(ATP). Altogether, our results indicate that brain mitoK(ATP) is a redox-sensitive channel that controls mitochondrial ROS release.

1,3-diene probes for detection of triplet carbonyls in biological systems.

Electronically excited triplet carbonyls are formed during the oxidative degradation of polyunsaturated fatty acids, amino acids, and beta-dicarbonyl metabolites. Due to their long lifetime and high alkoxyl radical-like reactivity, triplet carbonyls may initiate deleterious reactions in biological systems. Here we study the quenching properties of conjugated dienes, specifically 2,4-hexadienoate (sorbate) and its alkyl ester, on triplet acetone generated chemically (thermolysis of tetramethyl-1,2-dioxetane) or enzymatically (horseradish peroxidase-catalyzed aerobic oxidation of isobutanal). Triplet acetone quenching rates were near diffusion control ( k q = 10 (8)-10 (9) M (-1) s (-1)) and accompanied by diene cis-trans isomerization. None of the dienes displays antioxidant activity in classical systems known to generate reactive oxygen species: superoxide anion radical, hydroxyl radical, alkoxyl and alkylperoxyl radicals, or singlet oxygen. Experiments with model systems used widely to study lipid peroxidation showed that sorbate can inhibit mitochondrial swelling induced by enzymically formed triplet benzophenone and quench the chemiluminescence of microsome preparations challenged with iron and ascorbate. Altogether, our data indicate that conjugated dienes can be used as specific quenchers of triplet carbonyls formed in biological systems during oxidative stress. Moreover, they suggest that the well-known food preservative properties of sorbate may be due to its triplet carbonyl quenching activity.

Mitochondrial permeability transition relevance for apoptotic triggering in the post-ischemic heart.

Dysfunction of mitochondrial calcium homeostasis transforms this cation from a key regulator of mitochondrial function, into a death effector during post-ischemic reperfusion. High intramitochondrial calcium and prevailing cellular conditions favor the opening of the mitochondrial permeability transition pore (mPTP), that induces mitochondrial swelling and provides a mechanism for cytochrome c release, a hallmark signal protein of the mitochondrial apoptosis pathway; indeed, a second mechanism induced by pro-apoptotic BAX protein, could account for cytochrome c leak in the post-ischemic heart. The present study was undertaken to determine which one of these mechanisms triggers the mitochondrial apoptosis pathway in the reperfused heart. To accomplish this goal we prevented the opening of the mPTP in such hearts, by diminishing calcium overload with Ru360, a specific mitochondrial calcium uniporter inhibitor. We found that mPTP opening in reperfused hearts increased along with reperfusion time and concurs with cytochrome c release from mitochondria. Maximal cytochrome c release correlated with mitochondrial dysfunction and complete NAD+ deletion. Fully inserted BAX was detected early after reperfusion and remained unchanged during the evaluated reperfusion times. Remarkably, heart perfusion with Ru360, inhibited mPTP opening and BAX docking into the mitochondrial membranes, suggesting a mPTP upstream role on BAX migration/insertion.

Hyperlipidemic mice present enhanced catabolism and higher mitochondrial ATP-sensitive K+ channel activity.

BACKGROUND & AIMS: Changes in mitochondrial energy metabolism promoted by uncoupling proteins (UCPs) are often found in metabolic disorders. We have recently shown that hypertriglyceridemic (HTG) mice present higher mitochondrial resting respiration unrelated to UCPs. Here, we disclose the underlying mechanism and consequences, in tissue and whole body metabolism, of this mitochondrial response to hyperlipidemia. METHODS: Oxidative metabolism and its response to mitochondrial adenosine triphosphate (ATP)-sensitive K+ channel (mitoK(ATP)) agonists and antagonists were measured in isolated mitochondria, livers, and mice. RESULTS: Mitochondria isolated from the livers of HTG mice presented enhanced respiratory rates compared with those from wild-type mice. Changes in oxygen consumption were sensitive to adenosine triphosphate (ATP), diazoxide, and 5-hydroxydecanoate, indicating they are attributable to mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) activity. Indeed, mitochondria from HTG mice presented enhanced swelling in the presence of K+ ions, sensitive to mitoK(ATP) agonists and antagonists. Furthermore, mitochondrial binding to fluorescent glibenclamide indicates that HTG mice expressed higher quantities of mitoK(ATP). The higher content and activity of liver mitoK(ATP) resulted in a faster metabolic state, as evidenced by increased liver oxygen consumption and higher body CO(2) release and temperature in these mice. In agreement with higher metabolic rates, food ingestion was significantly larger in HTG mice, without enhanced weight gain. CONCLUSIONS: These results show that primary hyperlipidemia leads to an elevation in liver mitoK(ATP) activity, which may represent a regulated adaptation to oxidize excess fatty acids in HTG mice. Furthermore, our data indicate that mitoK(ATP), in addition to UCPs, may be involved in the control of energy metabolism and body weight.

Iron complexing activity of mangiferin, a naturally occurring glucosylxanthone, inhibits mitochondrial lipid peroxidation induced by Fe2+-citrate.

Mangiferin, a naturally occurring glucosylxanthone, has been described as having antidiabetic, antiproliferative, immunomodulatory and antioxidant activities. In this study we report for the first time the iron-complexing ability of mangiferin as a primary mechanism for protection of rat liver mitochondria against Fe(2+)-citrate induced lipid peroxidation. Thiobarbituric acid reactive substances and antimycin A-insensitive oxygen consumption were used as quantitative measures of lipid peroxidation. Mangiferin at 10 microM induced near-full protection against 50 microM Fe(2+)-citrate-induced mitochondrial swelling and loss of mitochondrial transmembrane potential (DeltaPsi). The IC(50) value for mangiferin protection against Fe(2+)-citrate-induced mitochondrial thiobarbituric acid reactive substance formation (9.02+/-1.12 microM) was around 10 times lower than that for tert-butylhydroperoxide mitochondrial induction of thiobarbituric acid reactive substance formation. The xanthone derivative also inhibited the iron citrate induction of mitochondrial antimycin A-insensitive oxygen consumption, stimulated oxygen consumption due to Fe(2+) autoxidation and prevented Fe(3+) ascorbate reduction. Absorption spectra of mangiferin-Fe(2+)/Fe(3+) complexes also suggest the formation of a transient charge transfer complex between Fe(2+) and mangiferin, accelerating Fe(2+) oxidation and the formation of a more stable Fe(3+)-mangiferin complex unable to participate in Fenton-type reaction and lipid peroxidation propagation phase. In conclusion, these results show that in vitro antioxidant activity of mangiferin is related to its iron-chelating properties and not merely due to the scavenging activity of free radicals. These results are of pharmacological relevance since mangiferin and its naturally contained extracts could be potential candidates for chelation therapy in diseases related to abnormal intracellular iron distribution or iron overload.

ATP-sensitive K+ channels in renal mitochondria.

Isolated kidney mitochondria swell when incubated in hyposmotic solutions containing K+ salts in a manner inhibited by ATP, ADP, 5-hydroxydecanoate, and glibenclamide and stimulated by GTP and diazoxide. These results suggest the existence of ATP-sensitive K+ channels in these mitochondria, similar to those previously described in heart, liver, and brain. Renal mitochondrial ATP-sensitive K+ uptake rates are approximately 140 nmol.min-1.mg protein-1. This K+ transport results in a slight increase in respiration and decrease in the inner membrane potential. In addition, the activation of ATP-inhibited K+ uptake using diazoxide leads to a decrease of ATP hydrolysis through the reverse activity of the F0F1 ATP synthase when respiration is inhibited. In conclusion, we characterize an ATP-sensitive K+ transport pathway in kidney mitochondria that affects volume, respiration, and membrane potential and may have a role in the prevention of mitochondrial ATP hydrolysis.

Ischemic preconditioning inhibits mitochondrial respiration, increases H2O2 release, and enhances K+ transport.

Ischemic preconditioning, or the protective effect of short ischemic episodes on a longer, potentially injurious, ischemic period, is prevented by antagonists of mitochondrial ATP-sensitive K+ channels (mitoKATP) and involves changes in mitochondrial energy metabolism and reactive oxygen release after ischemia. However, the effects of ischemic preconditioning itself on mitochondria are still poorly understood. We determined the effects of ischemic preconditioning on isolated heart mitochondria and found that two brief (5 min) ischemic episodes are sufficient to induce a small but significant decrease ( approximately 25%) in mitochondrial NADH-supported respiration. Preconditioning also increased mitochondrial H2O2 release, an effect related to respiratory inhibition, because it is not observed in the presence of succinate plus rotenone and can be mimicked by chemically inhibiting complex I in the presence of NADH-linked substrates. In addition, preconditioned mitochondria presented more substantial ATP-sensitive K+ transport, indicative of higher mitoKATP activity. Thus we directly demonstrate that preconditioning leads to mitochondrial respiratory inhibition in the presence of NADH-linked substrates, increased reactive oxygen release, and activation of mitoKATP.