Clark-type Oxygen Electrode Research Articles

Refined method to study the posttranslational regulation of alternative oxidases from Arabidopsis thaliana in vitro.

In isolated membranes, posttranslational regulation of quinol oxidase activities can only be determined simultaneously for all oxidases-quinol oxidases as well as cytochrome c oxidases-because of their identical localization. In this study, a refined method to determine the specific activity of a single quinol oxidase is exemplarily described for the alternative oxidase (AOX) isoform AOX1A from Arabidopsis thaliana and its corresponding mutants, using the respiratory chain of an Escherichia coli cytochrome bo and bd-I oxidase double mutant as a source to provide electrons necessary for O2 reduction via quinol oxidases. A highly sensitive and reproducible experimental set-up with prolonged linear time intervals of up to 60 s is presented, which enables the determination of constant activity rates in E. coli membrane vesicles enriched in the quinol oxidase of interest by heterologous expression, using a Clark-type oxygen electrode to continuously follow O2 consumption. For the calculation of specific quinol oxidase activity, activity rates were correlated with quantitative signal intensity determinations of AOX1A present in a membrane-bound state by immunoblot analyses, simultaneously enabling normalization of specific activities between different AOX proteins. In summary, the method presented is a powerful tool to study specific activities of individual quinol oxidases, like the different AOX isoforms, and their corresponding mutants upon modification by addition of effectors/inhibitors, and thus to characterize their individual mode of posttranslational regulation in a membranous environment.

REDUCED CITRATE SYNTHASE ACTIVITY EFFECT ON OXYGEN CONSUMPTION RATES IN ISOLATED MITOCHONDRIA FROM MICE LIVER AND MUSCLES

Background. Liver and skeletal muscles play the major role in metabolism. Mitochondria are of particular importance in functioning of these organs. We tested the hypothesis that reduced citrate synthase (CS) activity could induce improved fatty substrate and carbohydrate oxidation in mitochondria extracted from liver and hind limb muscles of mice. Methods. Eight mice each of 12-week-old control C57B6/J (B6) and congenic B6.A-(rs3676616-D10Utsw1)/ Kjn (B6.A) mice were studied. The mitochondria were isolated by differential centrifugation method followed by assessment of mitochondrial respiration and citrate synthase (CS) activity. Mitochondrial respiration was measured as oxygen consumption with Clark-type oxygen electrode by using polarography system. CS enzyme activity was measured spectrophotometrically. Results. The activity of CS was by ~32% lower for mitochondria for B6.A compared to B6 mice (603.9 ± 135.6 U/g and 894.2 ± 193.2 U/g, respectively). Mitochondrial respiration did not differ significantly between the strains. Conclusions. 30% reduction in citrate synthase activity does not impair mitochondrial respiration.

Advanced and High-Throughput Method for Mitochondrial Bioenergetics Evaluation in Neurotrauma.

Mitochondrial dysfunction is one of the key posttraumatic neuropathological events observed in various experimental models of traumatic brain injury (TBI). The extent of mitochondrial dysfunction has been associated with the severity and time course of secondary injury following brain trauma. Critically, several mitochondrial targeting preclinical drugs used in experimental TBI models have shown improved mitochondrial bioenergetics, together with cortical tissue sparing and cognitive behavioral outcome. Mitochondria, being a central regulator of cellular metabolic pathways and energy producer of cells, are of a great interest for researchers aiming to adopt cutting-edge methodology for mitochondrial bioenergetics assessment. The traditional way of mitochondrial bioenergetics analysis utilizing a Clark-type oxygen electrode (aka. oxytherm) is time-consuming and labor-intensive. In the present chapter, we describe an advanced and high-throughput method for mitochondrial bioenergetics assessments utilizing the Seahorse Biosciences XF(e)24 Flux Analyzer. This allows for simultaneous measurement of multiple samples with higher efficiency than the oxytherm procedure. This chapter provides helpful guidelines for conducting mitochondrial isolation and studying mitochondrial bioenergetics in brain tissue homogenates following experimental TBI.

Mechanism for the antibacterial activity of a mixture of 3,4,5-trihydroxy benzoic acid and magnesium oxide

In this paper, the mechanism for the antibacterial activity of a mixture of 3,4,5-trihydroxy benzoic acid (GA) and magnesium oxide (MgO) for Escherichia coli (JCM1649, E coli) is investigated. Antibacterial activity was assayed by the liquid dilution method against E coli. E coli in the logarithmic phase and the culture medium with pepton, yeast extract, NaCl, glucose, and MgSO4 7H2O were used in all experiments. The logarithmic phase culture of E coli (1x10 5 CFU cm -3 ) was added to the culture medium with GA and/or MgO. A Clark-type oxygen electrode with 1.1 cm -3 volume cell was used for the measurements of the oxygen consumption profile of a mixture of GA and MgO. The concentration of hydrogen peroxide (H 2O2) in the mixture was estimated by using porphyrinato-titanium complex. Efficient antibacterial activity was observed on a mixture of 2 mg cm -3 GA and 2 mg cm -3 MgO. The addition of 110 units cm -3 catalase, which is an enzyme of H2O2 decomposition, in a mixture of 2 mg cm -3 GA and 2 mg cm -3 MgO led to the decrease of the antibacterial activity as same as the case of 2 mg cm -3 MgO. Moreover, the production of H2O2 in the mixture was confirmed by the oxygen consumption profile under the addition of 500 units cm -3 catalase. The accumulation of 3-4 mmol dm -3 H2O2 was confirmed in a mixture of 2 mg cm -3 GA and 2 mg cm -3 MgO. The increase in the concentration of H2O2 from 3 mmol dm -3 to 4 mmol dm -3 led to a drastic increase in the antibacterial activity. Based on the results of this study, a possible mechanism for the antibacterial activity of a mixture of GA and MgO is discussed, concerning a synergy effect on the antibacterial action of MgO and that of H2O2.

Mediatorless electron transfer in glucose dehydrogenase/laccase system adsorbed on carbon nanotubes

A mediatorless electron transfer in the chain of glucose dehydrogenase (GDH) and laccase (LAC) catalysing the oxidation of glucose by molecular oxygen was studied. To demonstrate mediatorless processes, the GDH from Ewingella americana was adsorbed on single-walled carbon nanotubes (SWCNT). The effective mediatorless oxidation of glucose proceeded at 0.2–0.4V vs. SCE. The electrode was most active at pH 6.1, and generated 0.8mAcm−2 biocatalytic current in the presence of 50mM glucose. The electrode showed a bell-shaped pH dependence with pKa values of 4.1 and 7.5. LAC from Trichaptum abietinum adsorbed on SWCNT exhibited mediatorless oxygen reduction at electrode potential less than 0.65V. The electrode was most active at pH 3.0–4.0 and generated 1.1mAcm−2 biocatalytic current in the presence of 0.254mM oxygen, with an apparent pKa of 1.0 and 5.4. The electrodes prepared by simultaneous adsorption of GDH and LAC on SWCNT exhibited glucose oxidation at a potential higher than 0.25V.The oxygen consumption in the chain was demonstrated using a Clark-type oxygen electrode. The dependence of oxygen consumption on glucose and lactose concentrations as well as activity of the system on pH were measured. A model of the pH dependence as well as mediatorless consecutive glucose oxidation with oxygen catalysed by LAC/GDH system is presented. This work provides a novel approach towards the synthesis of artificial multi enzyme systems by wiring oxidoreductases with SWCNT, and offers a better understanding of natural electron transfer networks and chains.

Catalytic activity of human indoleamine 2,3-dioxygenase (hIDO1) at low oxygen

A cytokine-inducible extrahepatic human indoleamine 2,3-dioxygenase (hIDO1) catalyzes the first step of the kynurenine pathway. Immunosuppressive activity of hIDO1 in tumor cells weakens host T-cell immunity, contributing to the progression of cancer. Here we report on enzyme kinetics and catalytic mechanism of hIDO1, studied at varied levels of dioxygen (O2) and l-tryptophan (l-Trp). Using a cytochrome b5-based activating system, we measured the initial rates of O2 decay with a Clark-type oxygen electrode at physiologically-relevant levels of both substrates. Kinetics was also studied in the presence of two substrate analogs: 1-methyl-l-tryptophan and norharmane. Quantitative analysis supports a steady-state rather than a rapid equilibrium kinetic mechanism, where the rates of individual pathways, leading to a ternary complex, are significantly different, and the overall rate of catalysis depends on contributions of both routes. One path, where O2 binds to ferrous hIDO1 first, is faster than the second route, which starts with the binding of l-Trp. However, l-Trp complexation with free ferrous hIDO1 is more rapid than that of O2. As the level of l-Trp increases, the slower route becomes a significant contributor to the overall rate, resulting in observed substrate inhibition.

Characterization of mitochondrial bioenergetics in neonatal anoxic model of rats.

Neonatal anoxia at the time of birth can lead to mitochondrial dysfunction and further neurodevelopmental abnormalities. The present study investigated the mitochondrial bioenergetics and associated sensorimotor changes in the anoxic neonatal rats. Rat pups after 30h to birth (2days) were subjected to anoxia of two episodes (10min in each) at a time interval of 24h by passing 100%N2 into an enclosed chamber. Brain mitochondrial respiration was measured using clark type oxygen electrode. A significant decrease in brain respiratory control ratio (RCR; State III/IV respiration) at all-time points, complex I (24h) and complex II (30min, 6 and 24h) enzyme activities indicated loss of mitochondrial integrity and function A significant increase in levels of nitric oxide was observed after second anoxic episode at all-time points. A significant change in sensorimotor activity in terms of increased reflex latency was observed 24h after second episode in this model, which is an indication of loss of subcortical maturation. All the above changes were observed after second but not after the first anoxic exposure. Therefore, this anoxic model shows significant changes in mitochondrial bioenergetics, nitric oxide levels and sensorimotor effects after second episode of anoxia. This model may be helpful to evaluate mitochondrial targeted pharmacological intervention for the treatment of anoxia.

Abstract 14641: Deficiency in the E3 Ubiquitin Ligase Parkin Exacerbates Alcoholic Cardiomyopathy: Role of Mitophagy

Background: Long-term heavy alcohol consumption has been shown to promote mitochondrial injury, unfavorable geometric and contractile changes in the heart. Parkin, a cytosolic E3 ubiquitin ligase encoded by PARK2 gene, plays an important role in the regulation of selective mitophagy. This study was designed to examine the role of Parkin in alcohol-induced myocardial injury (aka alcoholic cardiomyopathy) and the underlying mechanism with a focus on mitophagy. Methods: Adult male wild-type C57 and PARKIN2 knockout (Parkin-/-) mice were placed on alcohol (4%) or control diet for 4 weeks. Echocardiographic and cardiomyocyte mechanical properties were assessed. Mitochondrial morphology, function and mitophagy were examined using transmission electronic microscopy, Clark-type oxygen electrode, and Western blot, respectively. Results: Our results revealed that chronic alcohol consumption triggered unfavorable geometric and contractile changes [decreased fractional shortening (FS) and ejection fraction (EF), with enlarged left ventricular chamber; decreased peak shortening (PS) and velocity of shortening +dL/dt, increased time-to-90% relengthening TR90], the effects of which were exacerbated by Parkin deficiency. In addition, our data showed that chronic alcohol intake promoted myocardial mitochondrial swelling with cristae disarrangement, induced myocardial mitochondrial depolarization and respiration inhibition, which were exacerbated by Parkin knockout. Furthermore, chronic alcohol consumption promoted mitophagy activation, as evidenced by accumulation of Parkin and LC3BII in mitochondria and mitochondrial ubiquitination level in the heart, the effect of which was nullified by Parkin knockout. Conclusion: These data suggest that chronic alcohol consumption triggered mitophagy by stimulating Parkin translocation to the mitochondria, which may be an adaptive response in the heart. Our findings implicated the therapeutic potential of mitophagy as a target in the management of alcoholic cardiomyopathy.

Neuroprotective effects of gallic acid against hypoxia/reoxygenation-induced mitochondrial dysfunctions in vitro and cerebral ischemia/reperfusion injury in vivo

Oxidative stress and mitochondrial dysfunction are frequently implicated in the pathology of secondary neuronal damage following cerebral ischemia/reperfusion. Recent evidence suggests that gallic acid (GA) reverses oxidative stress in rat model of streptozotocin-induced dementia, but the roles and mechanisms of GA on cerebral ischemia/reperfusion injury remain unknown. Here we investigated the potential roles and mechanisms of GA in hypoxia/reoxygenation induced by sodium hydrosulfite (Na2S2O4) in vitro and cerebral ischemia/reperfusion induced by middle cerebral artery occlusion (MCAO) in vivo. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazol carbocyanine iodide (JC-1), Dichlorofluorescin diacetate (DCF-DA) and MitoSOX fluorescent assay, Clark-type oxygen electrode, firefly luciferase assay, and calcium-induced mitochondrial swelling were conducted to detect cell death, mitochondrial membrane potential (MMP), intracellular and mitochondrial reactive oxygen species (ROS), oxygen consumption, ATP level, and mitochondrial permeability transition pore (MPTP) viability. We firstly find that modulation of the mitochondrial dysfunction is an important mechanism by GA attenuating hypoxia/reoxygenation insult. To further assess the effects of GA on cerebral ischemia/reperfusion injury, 2, 3, 5-triphenyl-tetrazolium chloride (TTC) staining, dUTP nick-end labeling (TUNEL) assay, and Cytochrome C (Cyt C) release were performed in MCAO rats. The results support that GA is useful against cerebral ischemia/reperfusion injury as a potential protective agent.

The flavonols quercetin, myricetin, kaempferol, and galangin inhibit the net oxygen consumption by immune complex-stimulated human and rabbit neutrophils.

Stimulated human neutrophils exhibit increased net oxygen consumption (NOC) due to the conversion of O2 into the superoxide anion by the NADPH oxidase enzymatic complex during the respiratory burst. In several inflammatory diseases, overproduction of these oxidants causes tissue damage. The present study aims to: (a) optimize the experimental conditions used to measure the NOC in serum-opsonized zymosan (OZ)- and insoluble immune complex (i-IC)-stimulated human and rabbit neutrophils; and (b) compare the effect of four flavonols (quercetin, myricetin, kaempferol, and galangin) on this activity. We used a Clark-type oxygen electrode to measure the NOC of stimulated neutrophils. Eliciting the neutrophil respiratory burst with OZ and i-IC yielded similar maximum O2 uptake levels within the same species, but the human neutrophil NOC was almost four times higher than the rabbit neutrophil NOC. The optimal experimental conditions established for both cell types were 4 x 10(6) neutrophils mL(-1), 2 mg mL(-1) OZ, and 240 microg mL(-1) i-IC. Upon stimulation with OZ or i-IC, the tested flavonols reduced the human and rabbit neutrophil NOC in the same order of potency--quercetin and galangin were the most and the least potent, respectively. These compounds were around four times more effective in inhibiting the rabbit as compared to the human neutrophil NOC, respectively. The four flavonols were not toxic to human or rabbit neutrophils. The experimental conditions used are suitable for both the determination of human and rabbit neutrophil NOC and for the assessment of the modulatory effects of natural compounds on these activities. The relationship between the level of NOC and the inhibitory potency of the flavonols suggests that rabbit neutrophils can be useful experimental models to predict the effect of drugs on immune complex-stimulated human neutrophils.

The effect of Leonurus cardiaca herb extract and some of its flavonoids on mitochondrial oxidative phosphorylation in the heart.

Motherwort (Leonurus cardiaca) possesses antibacterial, antioxidant, anti-inflammatory, and analgesic activities, and is used as a complementary remedy to improve heart function and blood circulation. Since cardiovascular diseases are often associated with an alteration of mitochondria, the main producers of ATP in cardiac muscle cells, the aim of our work was to determine bioactive constituents present in motherwort aerial parts extract in ethanol and investigate their effects on the functions of cardiac mitochondria. Quantitative determination of polyphenols in L.cardiaca herb extract was performed by HPLC. Mitochondrial respiration rates were evaluated using a Clark-type oxygen electrode. Mitochondrial ROS generation was determined fluorimetrically with Amplex Red and horseradish peroxidase. The results showed that constituents (chlorogenic acid, orientin, quercetin, hyperoside, and rutin) of L.cardiaca herb extract uncouple (by 20-90 %) mitochondrial oxidation from phosphorylation, partially inhibit (by ~ 40 %) the mitochondrial respiratory chain in cases of pyruvate and malate as well as succinate oxidation, and effectively attenuate the generation of free radicals in mitochondria. Since partial uncoupling of mitochondria, respiratory inhibition, and decreased ROS production are proposed as possible mechanisms of cardioprotection, our results imply that L. cardiaca herb extract could be a useful remedy to protect cardiac muscles from the effects of pathogenic processes.

Essential Roles of Neutral Ceramidase and Sphingosine in Mitochondrial Dysfunction Due to Traumatic Brain Injury

In addition to immediate brain damage, traumatic brain injury (TBI) initiates a cascade of pathophysiological events producing secondary injury. The biochemical and cellular mechanisms that comprise secondary injury are not entirely understood. Herein, we report a substantial deregulation of cerebral sphingolipid metabolism in a mouse model of TBI. Sphingolipid profile analysis demonstrated increases in sphingomyelin species and sphingosine concurrently with up-regulation of intermediates of de novo sphingolipid biosynthesis in the brain. Investigation of intracellular sites of sphingosine accumulation revealed an elevation of sphingosine in mitochondria due to the activation of neutral ceramidase (NCDase) and the reduced activity of sphingosine kinase 2 (SphK2). The lack of change in gene expression suggested that post-translational mechanisms are responsible for the shift in the activities of both enzymes. Immunoprecipitation studies revealed that SphK2 is complexed with NCDase and cytochrome oxidase (COX) subunit 1 in mitochondria and that brain injury hindered SphK2 association with the complex. Functional studies showed that sphingosine accumulation resulted in a decreased activity of COX, a rate-limiting enzyme of the mitochondrial electron transport chain. Knocking down NCDase reduced sphingosine accumulation in mitochondria and preserved COX activity after the brain injury. Also, NCDase knockdown improved brain function recovery and lessened brain contusion volume after trauma. These studies highlight a novel mechanism of secondary TBI involving a disturbance of sphingolipid-metabolizing enzymes in mitochondria and suggest a critical role for mitochondrial sphingosine in promoting brain injury after trauma.

Renal hypoxia due to increased oxygen metabolism is an independent pathway to nephropathy (890.6)

The proposed pathways for the development of nephropathy are many and likely to be complex and overlapping. Recent literature strongly supports a role of renal tissue hypoxia as a pathway to nephropathy but the evidence remains inconclusive as the role of hypoxia cannot be separated from confounding factors such as hyperglycemia, hypertension and oxidative stress. By increasing kidney oxygen consumption (QO2) using triodothyronine (T3) we aimed to investigate the role of renal hypoxia per se for the development of nephropathy.Normoglycemic Sprague‐Dawley rats were treated with T3 (10 mg/kg bw/day) for 56 days. Thereafter, glomerular filtration rate (GFR), renal blood flow (RBF), total kidney QO2, kidney oxygen tension (pO2, Clark‐type oxygen electrodes) proteinuria, oxidative stress and histological alterations were evaluated and compared to corresponding controls.T3 increased kidney QO2 (18±2 vs. 11±1 µmol/min/kidney, p<0.05) and decreased kidney pO2 (27±1 vs. 44±1 mmHg, p<0.05). Furthermore, T3 resulted in proteinuria (74±6 vs. 54±5 µg/min/kidney, p<0.05) and four of eight T3‐treated rats, but none of the controls, presented with focal tubulointerstitial fibrosis. GFR (1.4±0.2 vs. 1.5±0.2 ml/min), RBF (12.7±1.6 vs. 11.1±0.8 ml/min), TBARS (15±2 vs. 16±3 fmol/min/kidney) and blood glucose (4.8±0.2 vs. 5.1±0.2 mM) were unaffected by T3. The detected mitochondrial uncoupling and increased ATP synthesis provide a mechanistic explanation for the increased kidney QO2.In conclusion, the present study adds further evidence in support of renal tissue hypoxia as an independent pathway to nephropathy.

Epicatechin stimulates mitochondrial activity and selectively sensitizes cancer cells to radiation.

Radiotherapy is the treatment of choice for solid tumors including pancreatic cancer, but the effectiveness of treatment is limited by radiation resistance. Resistance to chemotherapy or radiotherapy is associated with reduced mitochondrial respiration and drugs that stimulate mitochondrial respiration may decrease radiation resistance. The objectives of this study were to evaluate the potential of (-)-epicatechin to stimulate mitochondrial respiration in cancer cells and to selectively sensitize cancer cells to radiation. We investigated the natural compound (-)-epicatechin for effects on mitochondrial respiration and radiation resistance of pancreatic and glioblastoma cancer cells using a Clark type oxygen electrode, clonogenic survival assays, and Western blot analyses. (-)-Epicatechin stimulated mitochondrial respiration and oxygen consumption in Panc-1 cells. Human normal fibroblasts were not affected. (-)-Epicatechin sensitized Panc-1, U87, and MIA PaCa-2 cells with an average radiation enhancement factor (REF) of 1.7, 1.5, and 1.2, respectively. (-)-Epicatechin did not sensitize normal fibroblast cells to ionizing radiation with a REF of 0.9, suggesting cancer cell selectivity. (-)-Epicatechin enhanced Chk2 phosphorylation and p21 induction when combined with radiation in cancer, but not normal, cells. Taken together, (-)-epicatechin radiosensitized cancer cells, but not normal cells, and may be a promising candidate for pancreatic cancer treatment when combined with radiation.

Electron Transport Activity in Embryonic Hearts Requires the Formation of Supercomplexes

The heart is the first functional organ in a vertebrate embryo. Mitochondria appear to be important to heart development and closing the permeability transition pore (PTP) between embryonic day (E) 9.5 and E11.5 drives the maturation of mitochondrial structure and regulates myocyte differentiation. These data also suggested that closure of the PTP is associated with changes of individual complexes (Cx) of the electron transport chain (ETC), and the objective here was to further define these changes.We assessed the activity of the Cx- 1, Cx-2 and Cx-3 of the ETC of C57BL/6N (wildtype (WT)) and cyclophilin D knockout (CypD KO) mice during embryonic development from E9.5 to E13.5 with a Clark-type oxygen electrode and enzymatic assays. Our results show that at E9.5 mitochondrial oxygen consumption appears uncoupled and there is no difference between state 2, 3 and 4 respiration. In contrast, oxygen consumption in E9.5 CypD KO embryos shows distinct state 2, 3 and 4 respiration, which can be mimicked in E9.5 WT hearts by incubation with 200 nM CsA. By E11.5 mitochondrial oxygen consumption in WT hearts is not significantly different when compared to E11.5 CypD KO mice or E13.5 hearts.In addition, supercomplexes were not detectable in WT hearts of E9.5 and E11.5 embryos by native electrophoresis or immunocapturing. This may explain why NADH-cytochrome c reductase activity is not detectable in WT embryos at E9.5 but in E11.5 or E13.5 WT mice. Furthermore, Cx-1 is in its de-active form at E9.5 and becomes activated around E11.5.Our data suggests that at E11.5 the PTP is closed and Cx-1, Cx- 2 and Cx-3 of the ETC are present, but are not yet arranged in supercomplexes and a coordinated electron transfer is still developing.

Phenol determination by an amperométrico biosensor based on lyophilized mushroom (Agaricus bisporus) tissue

A simple and inexpensive biosensor based on lyophilized mushroom tissue (Agaricus bisporus) was developed for amperometric determination of phenol. This fungi tissue contains tyrosinase (EC 1.14.18.1) enzyme that catalysis two sequential oxidation reactions with phenolic substrates. Both reactions involve molecular oxygen; therefore, the commercial Clark-type oxygen electrode was selected as a transducer. The lyophilized biocomponent was tested in two different forms: cubes (at two positions in the biosensor system) or powder. In characterization studies of the biosensor, some parameters such as time reaction, linear range and repeatability were investigated. For the best biosensor configuration, a linear response was observed from 0.1 to 10.0 mg L−1 phenol; variation coefficient and standard deviation were calculated as 0.02% and±0.11 mg L−1, respectively.

Intermittent hypoxia protects cerebral mitochondrial function from calcium overload

Hypoxia leads to Ca(2+) overload and results in mitochondrial uncoupling, decreased ATP synthesis, and neuronal death. Inhibition of mitochondrial Ca(2+) overload protects mitochondrial function after hypoxia. The present study was aimed to investigate the effect of intermittent hypoxia on mitochondrial function and mitochondrial tolerance to Ca(2+) overload. Wistar rats were divided into control and intermittent hypoxia (IH) groups. The IH group was subject to hypoxia for 4 h daily in a hypobaric cabin (5,000 m) for 7 days. Brain mitochondria were isolated on day 7 following hypoxia. The baseline mitochondrial functions, such as ST3, ST4, and respiratory control ratio (RCR = ST3/ST4), were measured using a Clark-type oxygen electrode. Mitochondrial adenine nucleotide concentrations were measured by HPLC. Mitochondrial membrane potential was determined by measuring rhodamine 123 (Rh-123) fluorescence in the absence and presence of high Ca(2+) concentration (0.1 M), which simulates Ca(2+) overload. Our results revealed that IH did not affect mitochondrial respiratory functions, but led to a reduction in AMP and an increase in ADP concentrations in mitochondria. Both control and IH groups demonstrated decreased mitochondrial membrane potential in the presence of high Ca(2+) (0.1 M), while the IH group showed a relative higher mitochondrial membrane potential. These results indicated that the neuroprotective effect of intermittent hypoxia was resulted partly from preserving mitochondrial membrane potential, and increasing mitochondrial tolerance to high calcium levels. The increased ADP and decreased AMP in mitochondria following intermittent hypoxia may be a mechanism underlying this protection.

Inhibition of cytochrome P450s enhances (+)‐usnic acid cytotoxicity in primary cultured rat hepatocytes

(+)-Usnic acid (UA) is consumed as a dietary supplement to promote weight loss; however, dietary supplements containing UA have been associated with clinical cases of severe liver injury. UA has been shown to be hepatotoxic in rats and is extensively metabolized by hepatic cytochrome P450s (CYPs); therefore, we examined if UA metabolism results in the formation of cytotoxic metabolites or if metabolism is a detoxification process in primary rat hepatocytes. When CYP activity was suppressed by the non-isoenzyme-selective inhibitor SKF-525A (20 μM), or the CYP1A inhibitor alpha-naphthoflavone (10 μM), or the CYP3A inhibitor ketoconazole (25 μM), the cytotoxicity of UA at 3~6 μM after 3~20 h of exposure was significantly increased as measured by lactate dehydrogenase (LDH) leakage. At 2 h after UA exposure, an earlier time point prior to LDH release, these CYP inhibitors potentiated UA-induced inhibition of cellular respiration as determined by the Clark type oxygen electrode. Cellular adenosine triphosphate (ATP) depletion by UA was also exacerbated by these CYP inhibitors. The CYP2B/2C inhibitor, ticlopidine at 20 μM, showed no effects in parallel experiments. These data demonstrate that UA is bio-transformed to less toxic metabolites in rat primary hepatocytes, probably mainly by CYP1A and 3A, but not 2B/2C. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

Hepatic mitochondrial efficiency in lines of mice differing in feed intake12

Selection was practiced for high (MH) and low (ML) heat loss using direct calorimetry to create lines of mice differing in feed intake per unit average BW (FI/BW). A total of 25 generations of selection was practiced during maintenance of an unselected control (MC). As a percentage of MC, mice of MH and ML lines differed in heat loss and FI/BW by 56 and 34%, respectively, when selection ceased. The purpose of this study was to determine hepatic mitochondrial efficiency in mice at maintenance across the genetic lines. After measurement of feed intakeand BW over a 2-wk period, mice were euthanized, and their livers were extracted. Livers were homogenized and mitochondria were isolated in buffer for measurement of oxygen consumption and, hence, mitochondrial activity using a Clark-type oxygen electrode. Mitochondria of ML mice expressed greater (P < 0.03) respiratory control ratio by 22% than that for MH mice, revealing greater degree of uncoupling during electron transport in MH mice. Difference in FI/BW (P < 0.001) was large with MH mice consuming 34% more than ML mice. The respiratory control ratio differed between mice of MH and ML lines as expected; however, regression analysis revealed that it explained essentially no variation in feed intake per BW.

Abstract 19619: A Novel Role of the Valosin-Containing Protein (VCP) In Cardiac Protection via Regulation of Mitochondrial Protein Nitrosylation

Introduction: We have previously shown that over expression of the valosin-containing protein (VCP) in a cardiac specific transgenic (TG) mouse conferred a cardiac protection against ischemia as well as a dose dependent induction of inducible isoform of nitric oxide synthase (iNOS). However, the link between iNOS and the VCP-conferred cardiac protection remains unclear. Hypothesis: Our hypothesis is that VCP elicits cardiac protection by the induction of inducible isoform of nitric oxide synthase (iNOS), leading to the nitrosylation of mitochondrial proteins, resulting in the stimulation of mitochondrial respiration. Methods and Results . Biotin switch was used to identify S-nitrosylated proteins in the VCP TG mouse heart, which a majority of them were found to be mitochondrial proteins involved in the regulation of mitochondrial metabolism and respiration, including succinate semialdehyde dehydrogenase, mitochondrial heat shock protein 60, cytochrome c1. One of the most upregulated nitrosylated proteins was Malic enzyme 3 (Me3), an enzyme which catalyzes the oxidative decarboxylation of malate to pyruvate, was significantly increased by 2.5 fold (p&lt;0.01) in TG mouse compared to WT, although the total protein level was unchanged. Reciprocally, we found a significant decrease by 50% of S-nitrosylated complex I subunit, (NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 9 (NDUFA9)), in VCP TG heart compared to WT (p&lt;0.05). Since S-nitrosylation serves to inhibit Complex I activity, this reduction indicates a reversal of this inhibition by VCP. Basal cardiac mitochondrial respiration was measured using a Clark type oxygen electrode on VCP TG mice (n = 5) and wild-type (WT) mice (n = 6). Mitochondrial State 3 respiration was significantly (p &lt; 0.01) elevated in the VCP TG (72 ±2 nmol O2/min/mg prot) mice compared to WT (45.9 ±6 nmol O2/min/mg prot) with no difference in State 4 or State 2, indicating that VCP over expression enhances the capacity for ATP production without a change in basal levels of respiration. Conclusion: In conclusion, our data reveals a critical and novel role of VCP in mediating cardioprotection through the stimulation of mitochondrial function by inducing nitrosylation of mitochondrial proteins via increased expression of iNOS.