Mitochondrial dysfunction due to in vitro exposure to atrazine and its metabolite in striatum.

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) has been described as a potential toxic for dopaminergic metabolism both in vivo and in vitro. Its main metabolite diamino-chloro triazine (DACT) has been shown to achieve higher levels in brain tissue than atrazine. The aim of this study was to evaluate the in vitro effects of atrazine and DACT on striatal mitochondrial function, active oxygen species generation, and nitric oxide (NO) content. Incubation of mitochondria with atrazine (10 µM) was not able to modify oxygen consumption. However, a 50% increase in malate-glutamate state 4 respiratory rates was observed after DACT treatment (100 µM) without changes in respiratory state 3. Atrazine was able to inhibit complex I-III activity by 30% and DACT induced a tendency to decrease by 17% in the striatum. Regarding reactive oxygen species (ROS), DACT increased H2 O2 production by 43%. Also, superoxide anion levels were higher (14%) after atrazine exposure than in control mitochondria. Incubation of striatal mitochondria with atrazine and DACT induced membrane depolarization by 15% and 19%, respectively. Also, atrazine increased NO content by 10% but no significant changes were observed after exposure of mitochondria to DACT. Glutathione peroxidase activity was inhibited (56%) by DACT and atrazine inhibited superoxide dismutase activity by 60%. Also, cardiolipin oxidation (15%) was observed after atrazine treatment. Summing up, the obtained results suggest that in vitro atrazine and DACT induce ROS production affecting striatal mitochondrial function. The atrazine effects would be attributed to a direct effect on the mitochondrial respiratory chain and superoxide dismutase activity while DACT appears to disturb glutathione-related enzyme system.

Alcohol hangover induces nitric oxide metabolism changes by impairing NMDA receptor-PSD95-nNOS pathway.

Alcohol hangover is defined as the combination of mental and physical symptoms experienced the day after a single episode of heavy drinking, starting when blood alcohol concentration approaches zero. We previously evidenced increments in free radical generation and an imbalance in antioxidant defences in non-synaptic mitochondria and synaptosomes during hangover. It is widely known that acute alcohol exposure induces changes in nitric oxide (NO) production and blocks the binding of glutamate to NMDAR in central nervous system. Our aim was to evaluate the residual effect of acute ethanol exposure (hangover) on NO metabolism and the role of NMDA receptor-PSD95-nNOS pathway in non-synaptic mitochondria and synaptosomes from mouse brain cortex. Results obtained for the synaptosomes fraction showed a 37% decrease in NO total content, a 36% decrease in NOS activity and a 19% decrease in nNOS protein expression. The in vitro addition of glutamate to synaptosomes produced a concentration-dependent enhancement of NO production which was significantly lower in samples from hangover mice than in controls for all the glutamate concentrations tested. A similar patter of response was observed for nNOS activity being decreased both in basal conditions and after glutamate addition. In addition, synaptosomes exhibited a 64% and 15% reduction in NMDA receptor subunit GluN2B and PSD-95 protein expression, respectively. Together with this, glutamate-induced calcium entry was significant decreased in synaptosomes from alcohol-treated mice. On the other hand, in non-synaptic mitochondria, no significant differences were observed in NO content, NOS activity or nNOS protein expression. The expression of iNOS remained unaltered in synaptosomes and non-synaptic mitochondria. Here we demonstrated that hangover effects on NO metabolism are strongly evidenced in synaptosomes probably due to a disruption in NMDAR/PSD-95/nNOS pathway.

Ketamine induced cell death can be mediated by voltage dependent calcium channels in PC12 cells.

Ketamine is widely used both as anesthetic and abuse drug. In this study, we investigated the effects of a wide range of ketamine concentrations (100-500-1000 µM) on calcium mobilization and the induction of cell death in undifferentiated PC12 cells, 24 h after treatment. Calcium mobilization was measured as the percentage of fluorescence one minute after depolarization by flow cytometry. For the kinetic changes in [Ca2+]c, fluorescence microscopy with Live Imaging was used with a resolution time of 0.87 s (exposure time: 20 ms). Fluo-4 AM was used for both methods. Flow cytometry using TMRE, NAO, and Annexin V-FITC/PI probes were employed for the evaluation of mitochondrial membrane potential (ΔΨm), cardiolipin content and type of cell death respectively. Fluorescence microscopy was used for the evaluation of DNA fragmentation by TUNEL assay with dUTP-conjugated FITC. Results obtained by flow cytometry showed a clear increment in cell response to depolarization after addition of 50 mM and 70 mM KCl in PC12 cells. Simultaneously, cells treated with 100 µM and 500 µM ketamine during 24 h, induced a decreased response to depolarization as compared with control cells. In addition, 1000 µM ketamine induced a similar increase in Fluo4AM fluorescence either after addition of 50 or 70 mM KCl. The kinetic assays showed that after 100 mM KCl, cells pre-treated with ketamine showed a marked decrease in [Ca2+]c as compared with control cells. In the case of 1000 µM ketamine treatment, an increased and sustained [Ca2+]c was observed along the whole assay, indicating a cell disability to maintain calcium homeostasis. Associated with these cytosolic calcium alterations, mitochondrial depolarization, cardiolipin depletion and alteration in Bax protein expression were observed after ketamine treatment. Our data demonstrate that ketamine action in these cells seems to be independent from NMDAR, as observed by the absence of glutamate­calcium response. Acute disturbance in [Ca2+]c could be mediated by the inhibition of VDCCs as part of the molecular mechanism of ketamine cytotoxicity leading to mitochondrial dysfunction and cell death by apoptosis and necrosis.

Brain cortex mitochondrial bioenergetics in synaptosomes and non-synaptic mitochondria during aging.

Alterations in mitochondrial bioenergetics have been associated with brain aging. In order to evaluate the susceptibility of brain cortex synaptosomes and non-synaptic mitochondria to aging-dependent dysfunction, male Swiss mice of 3 or 17 months old were used. Mitochondrial function was evaluated by oxygen consumption, mitochondrial membrane potential and respiratory complexes activity, together with UCP-2 protein expression. Basal respiration and respiration driving proton leak were decreased by 26 and 33 % in synaptosomes from 17-months old mice, but spare respiratory capacity was not modified by aging. Succinate supported state 3 respiratory rate was decreased by 45 % in brain cortex non-synaptic mitochondria from 17-month-old mice, as compared with young animals, but respiratory control was not affected. Synaptosomal mitochondria would be susceptible to undergo calcium-induced depolarization in 17 months-old mice, while non-synaptic mitochondria would not be affected by calcium overload. UCP-2 was significantly up-regulated in both synaptosomal and submitochondrial membranes from 17-months old mice, compared to young animals. UCP-2 upregulation seems to be a possible mechanism by which mitochondria would be resistant to suffer oxidative damage during aging.

Mitochondrial dysfunction in brain cortex mitochondria of STZ-diabetic rats: effect of l-Arginine.

Mitochondrial dysfunction has been implicated in many diseases, including diabetes. It is well known that oxygen free radical species are produced endogenously by mitochondria, and also nitric oxide (NO) by nitric oxide synthases (NOS) associated to mitochondrial membranes, in consequence these organelles constitute main targets for oxidative damage. The aim of this study was to analyze mitochondrial physiology and NO production in brain cortex mitochondria of streptozotocin (STZ) diabetic rats in an early stage of diabetes and the potential effect of L-arginine administration. The diabetic condition was characterized by a clear hyperglycaemic state with loose of body weight after 4 days of STZ injection. This hyperglycaemic state was associated with mitochondrial dysfunction that was evident by an impairment of the respiratory activity, increased production of superoxide anion and a clear mitochondrial depolarization. In addition, the alteration in mitochondrial physiology was associated with a significant decrease in both NO production and nitric oxide synthase type I (NOS I) expression associated to the mitochondrial membranes. An increased level of thiobarbituric acid-reactive substances (TBARS) in brain cortex homogenates from STZ-diabetic rats indicated the presence of lipid peroxidation. L-arginine treatment to diabetic rats did not change blood glucose levels but significantly ameliorated the oxidative stress evidenced by lower TBARS and a lower level of superoxide anion. This effect was paralleled by improvement of mitochondrial respiratory function and a partial mitochondrial repolarization.In addition, the administration of L-arginine to diabetic rats prevented the decrease in NO production and NOSI expression. These results could indicate that exogenously administered L-arginine may have beneficial effects on mitochondrial function, oxidative stress and NO production in brain cortex mitochondria of STZ-diabetic rats.

Mitochondrial bioenergetics and cytometric characterization of a synaptosomal preparation from mouse brain cortex.

Mitochondrial function at synapses can be assessed in isolated nerve terminals. Synaptosomes are structures obtained in vitro by detaching the nerve endings from neuronal bodies under controlled homogenization conditions. Several protocols have been described for the preparation of intact synaptosomal fractions. Herein a fast and economical method to obtain synaptosomes with optimal intrasynaptic mitochondria functionality was described. Synaptosomal fractions were obtained from mouse brain cortex by differential centrifugation followed by centrifugation in a Ficoll gradient. The characteristics of the subcellular particles obtained were analyzed by flow cytometry employing specific tools. Integrity and specificity of the obtained organelles were evaluated by calcein and SNAP-25 probes. The proportion of positive events of the synaptosomal preparation was 75 ± 2 % and 48 ± 7% for calcein and Synaptosomal-Associated Protein of 25 kDa (SNAP-25), respectively. Mitochondrial integrity was evaluated by flow cytometric analysis of cardiolipin content, which indicated that 73 ± 1% of the total events were 10 N-nonylacridine orange (NAO)-positive. Oxygen consumption, ATP production and mitochondrial membrane potential determinations showed that mitochondria inside synaptosomes remained functional after the isolation procedure. Mitochondrial and synaptosomal enrichment were determined by measuring synaptosomes/ homogenate ratio of specific markers. Functionality of synaptosomes was verified by nitric oxide detection after glutamate addition. As compared with other methods, the present protocol can be performed briefly, does not imply high economic costs, and provides an useful tool for the isolation of a synaptosomal preparation with high mitochondrial respiratory capacity and an adequate integrity and function of intraterminal mitochondria.

Lactic Acid Transport Mediated by Aquaporin-9: Implications on the Pathophysiology of Preeclampsia.

Aquaporin-9 (AQP9) expression is significantly increased in preeclamptic placentas. Since feto-maternal water transfer is not altered in preeclampsia, the main role of AQP9 in human placenta is unclear. Given that AQP9 is also a metabolite channel, we aimed to evaluate the participation of AQP9 in lactate transfer across the human placenta. Explants from normal term placentas were cultured in low glucose medium with or without L-lactic acid and in the presence and absence of AQP9 blockers (0.3 mM HgCl2 or 0.5 mM Phloretin). Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and lactate dehydrogenase release. Apoptotic indexes were analyzed by Bax/Bcl-2 ratio and Terminal Deoxynucleotidyltransferase-Mediated dUTP Nick-End Labeling assay. Heavy/large and light/small mitochondrial subpopulations were obtained by differential centrifugation, and AQP9 expression was detected by Western blot. We found that apoptosis was induced when placental explants were cultured in low glucose medium while the addition of L-lactic acid prevented cell death. In this condition, AQP9 blocking increased the apoptotic indexes. We also confirmed the presence of two mitochondrial subpopulations which exhibit different morphologic and metabolic states. Western blot revealed AQP9 expression only in the heavy/large mitochondrial subpopulation. This is the first report that shows that AQP9 is expressed in the heavy/large mitochondrial subpopulation of trophoblasts. Thus, AQP9 may mediate not only the lactic acid entrance into the cytosol but also into the mitochondria. Consequently, its lack of functionality in preeclamptic placentas may impair lactic acid utilization by the placenta, adversely affecting the survival of the trophoblast cells and enhancing the systemic endothelial dysfunction.

Protective effects of the synthetic cannabinoids CP55,940 and JWH-015 on rat brain mitochondria upon paraquat exposure.

The effects of cannabinoids in mitochondria after acute oxidative stress insult are not fully established. We investigated the ability of CP55,940 and JWH-015 to scavenge reactive oxygen species and their effect on mitochondria permeability transition (MPT) in either a mitochondria-free superoxide anion generation system, intact rat brain mitochondria or in sub-mitochondrial particles (SMP) treated with paraquat (PQ). Oxygen consumption, mitochondrial membrane potential (Deltapsi(m)) and MPT were determined as parameters of mitochondrial function. It is found that both cannabinoids effectively attenuate mitochondrial damage against PQ-induced oxidative stress by scavenging anion superoxide radical (O(2)(*-)) and hydrogen peroxide (H(2)O(2)), maintaining Deltapsi(m) and by avoiding Ca(2+)-induced mitochondrial swelling. Understanding the mechanistic action of cannabinoids on mitochondria might provide new insights into more effective therapeutic approaches for oxidative stress related disorders.

Dopamine modifies oxygen consumption and mitochondrial membrane potential in striatal mitochondria.

Dopamine is a neurotransmitter that has been related to mitochondrial dysfunction. In this study, striatal intact mitochondria and submitochondrial membranes were incubated with different dopamine concentrations, and changes on mitochondrial function, hydrogen peroxide, and nitric oxide production were evaluated. A 35% decrease in state 3 oxygen uptake (active respiration state) was found after 1 mM dopamine incubation. In addition, mitochondrial respiratory control significantly decreased, indicating mitochondrial dysfunction. High dopamine concentrations induced mitochondrial depolarization. Also, evaluation of hydrogen peroxide production by intact striatal mitochondria showed a significant increase after 0.5 and 1 mM dopamine incubation. Incubation with 0.5 and 1 mM dopamine increased nitric oxide production in submitochondrial membranes by 28 and 49%, respectively, as compared with control values. This study provides evidence that high dopamine concentrations induce striatal mitochondrial dysfunction through a decrease in mitochondrial respiratory control and loss of membrane potential, probably mediated by free radical production.

Ketamine treatment affects hippocampal but not cortical mitochondrial function in prepubertal rats.

Previous reports have shown that ketamine triggered apoptosis in immature developing brain involving mitochondrial-mediated pathways. However, no data for ketamine effects on hippocampal and cortical mitochondrial function are available in prepubertal rats. Twenty-one-day-old Sprague-Dawley rats received ketamine (40 mg/kg i.p.) for 3 days and were killed 24 hr after the last injection. Hippocampal mitochondria from ketamine-treated rats showed decreased malate-glutamate state 4 and 3 respiratory rates and an inhibition in complex I and IV activities. Hippocampal mitochondrial membrane depolarization and mitochondrial permeability transition induction were observed. This was not reflected in an increment of H2 O2 production probably due to increased Mn-SOD and catalase activities, 24 hr after treatment. Interestingly, increased H2 O2 production rates and cardiolipin oxidation were found in hippocampal mitochondria shortly after ketamine treatment (45 min). Unlike the hippocampus, ketamine did not affect mitochondrial parameters in the brain cortex, being the area less vulnerable to suffer ketamine-induced oxidative damage. Results provide evidences that exposure of prepubertal rats to ketamine leads to an induction of mitochondrial ROS generation at early stages of treatment that was normalized by the triggering of antioxidant systems. Although hippocampal mitochondria from prepubertal rats were capable of responding to the oxidative stress, they remain partially dysfunctional.

Alcohol hangover effects on brain cortex non-synaptic mitochondria and synaptosomes bioenergetics.

Alcohol hangover (AH) has been associated with oxidative stress and mitochondrial dysfunction. We herein postulate that AH-induced mitochondrial alterations can be due to a different pattern of response in synaptosomes and non-synaptic (NS) mitochondria. Mice received intraperitoneal (i.p.) injections of ethanol (3.8 g/kg) or saline and were sacrificed 6 h afterward. Brain cortex NS mitochondria and synaptosomes were isolated by Ficoll gradient. Oxygen consumption rates were measured in NS mitochondria and synaptosomes by high-resolution respirometry. Results showed that NS-synaptic mitochondria from AH animals presented a 26% decrease in malate-glutamate state 3 respiration, a 64% reduction in ATP content, 28-37% decrements in ATP production rates (malate-glutamate or succinate-dependent, respectively), and 44% inhibition in complex IV activity. No changes were observed in mitochondrial transmembrane potential (ΔΨ) or in UCP-2 expression in NS-mitochondria. Synaptosome respiration driving proton leak (in the presence of oligomycin), and spare respiratory capacity (percentage ratio between maximum and basal respiration) were 30% and 15% increased in hangover condition, respectively. Synaptosomal ATP content was 26% decreased, and ATP production rates were 40-55% decreased (malate-glutamate or succinate-dependent, respectively) in AH mice. In addition, a 24% decrease in ΔΨ and a 21% increase in UCP-2 protein expression were observed in synaptosomes from AH mice. Moreover, mitochondrial respiratory complexes I-III, II-III, and IV activities measured in synaptosomes from AH mice were decreased by 18%, 34%, and 50%, respectively. Results of this study reveal that alterations in bioenergetics status during AH could be mainly due to changes in mitochondrial function at the level of synapses.

Dopamine enhances mtNOS activity: implications in mitochondrial function.

Dopamine and nitric oxide systems can interact in different processes in the central nervous system. Dopamine and oxidation products have been related to mitochondrial dysfunction. In the present study, intact mitochondria and submitochondrial membranes were incubated with different DA concentrations for 5 min. Dopamine (1 mM) increased nitric oxide production in submitochondrial membranes and this effect was partially prevented in the presence of both DA and NOS inhibitor N(omega)-nitro-L-arginine (L-NNA). A 46% decrease in state 3 oxygen uptake (active respiration state) was found after 15 mM dopamine incubation. When mitochondria were incubated with 15 mM dopamine in the presence of L-NNA, state 3 respiratory rate was decreased by only 17% showing the involvement of NO. As shown for O(2) consumption, the inhibition of cytochrome oxidase by 1 mM DA was mediated by NO. Hydrogen peroxide production significantly increased after 15 mM DA incubation, being mainly due to its metabolism by MAO. Also, DA-induced depolarization was prevented by the addition of L-NNA showing the involvement of nitric oxide in this process too. This work provides evidence that in the studied conditions, dopamine modifies mitochondrial function by a nitric oxide-dependent pathway.

Time course of regression of the protection conferred by simulated high altitude to rat myocardium: correlation with mtNOS.

During acclimatization to sustained hypobaric hypoxia, retardation of age-associated decline in left ventricle mechanical activity and improved posthypoxic recovery were accompanied by upregulation of mitochondrial nitric oxide synthase (mtNOS). To evaluate the time course of regression of these effects on deacclimatization, rats exposed to 53.8 kPa in a hypopressure chamber for 5 mo were returned to 101.3 kPa, whereas controls remained at 101.3 kPa throughout the study. At three time points, contractile function in response to calcium and to hypoxia-reoxygenation (H/R) were determined in papillary muscle, and NOS activity and expression were determined in mitochondria isolated from left ventricle. Developed tension was, before H/R, 65, 58, and 40%, and, after H/R, 129, 107, and 71% higher than in controls at 0.4, 2, and 5 mo of normoxia, respectively. Maximal rates of contraction and relaxation followed a similar pattern. All three parameters showed a linear decline during deacclimatization, with mean half-time (t(1/2)) of 5.9 mo for basal mechanical activity and 5.3 mo for posthypoxic recovery. Left ventricle mtNOS activity was 42, 27, and 20% higher than in controls at 0.4, 2, and 5 mo, respectively (t(1/2) = 5.0 mo). The expression of mtNOS showed similar behavior. The correlation of mtNOS activity with muscle contractility sustained a biphasic modulation, suggesting an optimal mtNOS activity. This experimental model would provide the most persistent effect known at present on preservation of myocardial mechanical activity and improved tolerance to O(2) deprivation. Results support the putative role of mtNOS in the mechanism involved.

Mitochondrial dysfunction and cellular stress progression after ultraviolet B irradiation in human keratinocytes.

BACKGROUND: Ultraviolet (UV) radiation is the major environmental harmful factor that affects human skin. UVB radiation is known to be a potent inducer of reactive oxygen species (ROS) production and has also been associated with the generation of nitric oxide (NO), all of which have been implicated in various skin disorders. It is well known that mitochondria can also be affected by UVB, leading to alterations in their membrane structure and permeabilization with cytochrome c release, which consequently affects the cell function. However, the loss of keratinocyte mitochondrial function generated by UVB, as well as its kinetics, has not been characterized completely. METHODS: We evaluated the effect of UVB irradiation on HaCat cells' mitochondrial function, assessed by membrane potential loss and superoxide anion (O(2)(*-)) production, correlating with apoptosis, p53 expression, ROS levels and NO production, 0, 6, 12, 24 and 48 h post-irradiation. RESULTS: HaCat cells progressed toward apoptotic cell death as the time post-irradiation increased, with the highest levels found 48 h after irradiation. Increased levels of ROS were observed 6 h after irradiation while high O(2)(*-) levels and mitochondrial membrane depolarization were detected 12 h post-UVB. Nevertheless, NO production was not significantly increased at any of the evaluated times. CONCLUSIONS: The kinetics of mitochondrial dysfunction after UVB irradiation in human keratinocytes progressed in a time post-irradiation-dependent manner, and they are closely related to cell death. However, there are certain levels of apoptosis, although low, in the absence of mitochondrial alterations. In addition, our data suggest that ROS play a greater role in keratinocyte UVB damage than reactive nitrogen species.

Mitochondrial metabolic states regulate nitric oxide and hydrogen peroxide diffusion to the cytosol.

Mitochondria isolated from rat heart, liver, kidney and brain (respiratory control 4.0-6.5) release NO and H2O2 at rates that depend on the mitochondrial metabolic state: releases are higher in state 4, about 1.7-2.0 times for NO and 4-16 times for H2O2, than in state 3. NO release in rat liver mitochondria showed an exponential dependence on membrane potential in the range 55 to 180 mV, as determined by Rh-123 fluorescence. A similar behavior was reported for mitochondrial H2O2 production by [S.S. Korshunov, V.P. Skulachev, A.A. Starkov, High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15_18.]. Transition from state 4 to state 3 of brain cortex mitochondria was associated to a decrease in NO release (50%) and in membrane potential (24-53%), this latter determined by flow cytometry and DiOC6 and JC-1 fluorescence. The fraction of cytosolic NO provided by diffusion from mitochondria was 61% in heart, 47% in liver, 30% in kidney, and 18% in brain. The data supports the speculation that NO and H2O2 report a high mitochondrial energy charge to the cytosol. Regulation of mtNOS activity by membrane potential makes mtNOS a regulable enzyme that in turn regulates mitochondrial O2 uptake and H2O2 production.

Brain nitric oxide synthases and mitochondrial function.

Nitric oxide is a small signaling molecule, which may act as a neurotransmitter and neuromodulator, exerting a regulatory effect on neuronal function. It can diffuse from its site of synthesis to different intra and extracellular compartments, being therefore present in the pre-synaptic, synaptic and post-synaptic spaces. Recently, a NOS located in the mitochondria (mtNOS) has been observed in different brain regions, responsible for the production of NO in these organelles and identified as nNOS. A regulatory effect of NO on mitochondrial function was described in brain mitochondria, where NO acts mainly by inhibiting cytochrome oxidase activity. Hippocampal mitochondrial dysfunction and decreased mtNOS activity and expression were reported in association with ultrastructural damage in an experimental model of hepatic encephalopathy. Enriched environment exposure preserved the aged animals from spatial cognition impairment; also environment and training modulated neuronal plasticity in pre-pubertal rats through NO-dependent mechanisms. In addition, brain cortical mitochondrial respiration and mtNOS activity and expression were analyzed as function of age. Mitochondrial NO production showed a decreasing tendency as a function of age. These results are in accordance with the protein expression analyzed by Western Blot of mitochondrial fractions which was 6.5 times higher in 1 month aged rats as compared with 14 old animals. Concomitant with these results, a clear increasing oxygen uptake tendency in state 3 respiration was observed, meanwhile only a slight increase was observed in state 4. All these results seems to be clearly related with the reversible and concentration-dependent attenuation of the respiratory chain by NO.

Free radical production and antioxidant status in brain cortex non-synaptic mitochondria and synaptosomes at alcohol hangover onset.

Alcohol hangover (AH) is the pathophysiological state after a binge-like drinking. We have previously demonstrated that AH induced bioenergetics impairments in a total fresh mitochondrial fraction in brain cortex and cerebellum. The aim of this work was to determine free radical production and antioxidant systems in non-synaptic mitochondria and synaptosomes in control and hangover animals. Superoxide production was not modified in non-synaptic mitochondria while a 17.5% increase was observed in synaptosomes. A similar response was observed for cardiolipin content as no changes were evidenced in non-synaptic mitochondria while a 55% decrease in cardiolipin content was found in synaptosomes. Hydrogen peroxide production was 3-fold increased in non-synaptic mitochondria and 4-fold increased in synaptosomes. In the presence of deprenyl, synaptosomal H2O2 production was 67% decreased in the AH condition. Hydrogen peroxide generation was not affected by deprenyl addition in non-synaptic mitochondria from AH mice. MAO activity was 57% increased in non-synaptic mitochondria and 3-fold increased in synaptosomes. Catalase activity was 40% and 50% decreased in non-synaptic mitochondria and synaptosomes, respectively. Superoxide dismutase was 60% decreased in non-synaptic mitochondria and 80% increased in synaptosomal fractions. On the other hand, GSH (glutathione) content was 43% and 17% decreased in synaptosomes and cytosol. GSH-related enzymes were mostly affected in synaptosomes fractions by AH condition. Acetylcholinesterase activity in synaptosomes was 11% increased due to AH. The present work reveals that AH provokes an imbalance in the cellular redox homeostasis mainly affecting mitochondria present in synaptic terminals.

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.

Arsenic stimulates release of cytochrome c from isolated mitochondria via induction of mitochondrial permeability transition.

Arsenic trioxide, As(III), is a known environmental toxicant, co-carcinogen, and potent chemotherapeutic agent. In model experiments with isolated rat liver mitochondria, As(III) stimulated a dose-dependent, cyclosporin A-sensitive release of cytochrome c via induction of mitochondrial permeability transition and subsequent swelling of mitochondria. Mitochondrial GSH does not seem to be a target for As(III) which, however, appears to cause oxidative modification of thiol groups of pore forming proteins, notably adenine nucleotide translocase. In mouse embryonic fibroblasts, 10 microM As(III) stimulated cytochrome c release and apoptosis via a Bax/Bak-dependent mechanism. At high concentrations (125 microM and higher), cells died by Bax/Bak-independent necrosis; at this concentration range As(III) targets mitochondria directly, particularly complex I of the mitochondrial respiratory chain. Since pyruvate, a substrate of complex I, is a predominant mitochondrial substrate in the cell, inhibition of complex I will cause mitochondrial instability and a decrease of Delta psi that facilitates permeability transition and necrotic cell death.

Hippocampal mitochondrial dysfunction with decreased mtNOS activity in prehepatic portal hypertensive rats.

Portal hypertension is a major complication of human cirrhosis that frequently leads to central nervous system dysfunction. In our study, rats with prehepatic portal hypertension developed hippocampal mitochondrial dysfunction as indicated by decreased respiratory rates, respiratory control and mitochondrial nitric oxide synthase (mtNOS) activity in mitochondria isolated from the whole hippocampus. Succinate-dependent respiratory rates decreased by 29% in controlled state 4 and by 42% in active state 3, and respiratory control diminished by 20%. Portal hypertensive rats showed a decreased mtNOS activity of 46%. Hippocampal mitochondrial dysfunction was associated with ultrastructural damage in the mitochondria of hippocampal astrocytes and endothelial cells. Swollen mitochondria, loss of cristae and rupture of outer and inner membrane was observed in astrocytes and endothelial cells of the blood-brain barrier in parallel with the ammonia gradient. It is concluded that the moderate increase in plasma ammonia that followed portal hypertension was the potential primary cause of the observed alterations.