Reduction In Mitochondrial Respiration Research Articles

Superoxide dismutase 1 mutation in a cellular model of amyotrophic lateral sclerosis shifts energy generation from oxidative phosphorylation to glycolysis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder involving the progressive degeneration of motor neurons in the brain and spinal cord. Mitochondrial dysfunction plays a key role in ALS disease progression and has been observed in several ALS cellular and animal models. Here, we show that fibroblasts isolated from ALS cases with a Cu/Zn superoxide dismutase (SOD1) I113T mutation recapitulate these mitochondrial defects. Using a novel technique, which measures mitochondrial respiration and glycolytic flux simultaneously in living cells, we have shown that SOD1 mutation causes a reduction in mitochondrial respiration and an increase in glycolytic flux. This causes a reduction in adenosine triphosphate produced by oxidative phosphorylation and an increase in adenosine triphosphate produced by glycolysis. Switching the energy source from glucose to galactose caused uncoupling of mitochondria with increased proton leak in SOD1I113T fibroblasts. Assessment of the contribution of fatty acid oxidation to total respiration, suggested that fatty acid oxidation is reduced in SOD1 ALS fibroblasts, an effect which can be mimicked by starving the control cells of glucose. These results highlight the importance of understanding the interplay between the major metabolic pathways, which has the potential to lead to strategies to correct the metabolic dysregulation observed in ALS cases.

Regulation of mitochondrial function by PINK1, Parkin and DJ-1

Loss-of-function mutations in PTEN-induced kinase 1 (PINK1), Parkin and DJ-1 genes have been linked to recessively inherited forms of Parkinsonism. PINK1 is a putative serine-threonine kinase containing a mitochondrial localization signal, whereas Parkin and DJ-1 function as E3 ubiquitin-protein ligase and scavenger for reactive oxygen species (ROS), respectively. Interestingly, all three of them have been implicated in the control of the mitochondrial function. In this study using primary mouse embryonic fibroblasts (MEFs), primary neurons and mice brain, we investigated how the mitochondrial respiration is affected by the loss of PINK1, Parkin and DJ-1. We found that intact mitochondria in PINK1-/- and Parkin-/- cells recapitulate all the defects in mitochondrial respiration observed previously in isolated mitochondria from PINK1-/- and Parkin-/- mouse brains, suggesting that these cells are a valid experimental system to study the underlying mechanisms. Our data showed that these defects are not induced by changes in expression levels and activities of all individual complexes composing the electron transport system in PINK1-/- and Parkin-/-. In parallel, we found that loss of DJ-1 does not affect mitochondrial respiration in intact and permeabilized DJ-1-/- MEFs and in isolated mitochondria from the cerebral cortex of DJ-1-/- mice at 3 months or 2 years of age. Interestingly, the mitochondrial transmembrane potential in MEFs cells is reduced in absence of PINK1, Parkin or DJ-1. We first discovered that this reduction is linked to an increase of the opening of the mitochondrial permeability transition pore in PINK1-/-, Parkin-/- and DJ-1-/- cells. Consistent with earlier reports, production of ROS species is increased in DJ-1-/- MEFs. The accumulation of ROS seems linked to the decreased mitochondrial transmembrane potential and to the increased mitochondrial permeability transition pore opening in DJ-17-MEFs, as antioxidant treatment can restore them, whereas oxidative stress inducers have the opposite effects. Interestingly, the specific inhibition of mitochondrial permeability transition pore opening was able to rescue the transmembrane potential and respiration defects in PINK1-/- and Parkin-/- cells. According to our earlier findings in mouse brains, mitochondrial morphology is similar between PINK1-/- and wild-type cells, indicating that the observed mitochondrial functional defects are not due to morphological changes. The intra-mitochondrial calcium concentration measured after FCCP treatment is increased in PINK1-/- and Parkin-/- but not in DJ-1-/- cells. Together, our study suggests that the excessive mitochondrial permeability transition pore opening may underlie the selective reduction of mitochondrial respiration and transmembrane potential as well as an elevation of mitochondrial calcium observed in PINK1-/- and Parkin-/- cells, while increased ROS production seems to be the origin of mitochondrial functional defects observed DJ-1-/-. Supported by NINDS (R01 NS041779)

Activity of the mTOR inhibitor RAD001, the dual mTOR and PI3‐kinase inhibitor BEZ235 and the PI3‐kinase inhibitor BKM120 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide with only few therapeutic options for patients with advanced disease. There is growing evidence indicating that activation of the PI3K/Akt/mTOR pathway plays an important role in HCC and therefore represents a promising target for novel therapeutic approaches. The aim of this study was to evaluate and compare the antitumour activity of the mTOR inhibitor RAD001, the dual mTOR and PI3-kinase inhibitor BEZ235 and the PI3-kinase inhibitor BKM120 in vitro and in vivo. The antitumour effects of RAD001, BEZ235 and BKM120 were analysed in seven hepatoma cell lines as mono and combination therapy with Doxorubicin, Cisplatin, Irinotecan or 5-Flourouracil in vitro and in xenografts. Cell-cycle progression, apoptosis, and autophagy were analysed. Furthermore, effects on mitochondrial respiration and glycolysis were assessed. Treatment with RAD001, BEZ235 and BKM120 markedly reduced tumour cell viability. Combination of PI3K inhibitors with chemotherapy was most effective. RAD001, BEZ235 and BKM120 reduced tumour growth mainly by inhibiting cell-cycle progression rather than by inducing apoptosis. Interestingly, the antitumour effects were strongly associated with a reduction of mitochondrial respiration. BKM120, which exhibited the strongest antiproliferative effect, most strongly impaired oxidative phosphorylation compared with the other drugs. In this study, BKM120 showed the strongest antitumour activity. Our findings suggest impairment of mitochondrial function as a relevant mechanism of BKM120. Moreover, combination of PI3K and mTOR inhibitors with cytotoxic agents could be promising option for non-cirrhotic HCC patients.

Oncosecretomics coupled to bioenergetics identifies α-amino adipic acid, isoleucine and GABA as potential biomarkers of cancer: Differential expression of c-Myc, Oct1 and KLF4 coordinates metabolic changes

Bioenergetic profiling of tumors is a new challenge of cancer research and medicine as therapies are currently being developed. Meanwhile, methodological means must be proposed to gather information on tumor metabolism in order to adapt these potential therapies to the bioenergetic specificities of tumors. Studies performed on tumors and cancer cell lines have shown that cancer cells bioenergetics is highly variable. This profile changes with microenvironmental conditions (eg. substrate availability), the oncogenes activated (and the tumor suppressors inactivated) and the interaction with the stroma (i.e. reverse Warburg effect). Here, we assessed the power of metabolic footprinting (MFP) to unravel the bioenergetics and associated anabolic changes induced by three oncogenes, c-Myc, KLF4 and Oct1. The MFP approach provides a quantitative analysis of the metabolites secreted and consumed by cancer cells. We used ultra performance liquid chromatography for quantifying the amino acid uptake and secretion. To investigate the potential oncogene-mediated alterations in mitochondrial metabolism, we measured oxygen consumption rate and ATP production as well as the glucose uptake and lactate release. Our findings show that c-Myc deficiency initiates the Warburg effect along with a reduction of mitochondrial respiration. KLF4 deficiency also stimulated glycolysis, albeit without cellular respiration impairment. In contrast, Oct1 deficiency reduced glycolysis and enhanced oxidative phosphorylation efficiency. MFP revealed that c-Myc, KLF4 and Oct1 altered amino acid metabolism with specific patterns. We identified isoleucine, α-aminoadipic acid and GABA (γ-aminoisobutyric acid) as biomarkers related. Our findings establish the impact of Oct1, KLF4 and c-Myc on cancer bioenergetics and evidence a link between oncosecretomics and cellular bioenergetics profile.

Age-Dependent Reductions in Mitochondrial Respiration are Exacerbated by Calcium in the Female Rat Heart

Cardiovascular disease mortality increases rapidly after menopause by poorly defined mechanisms. Because mitochondrial function and Ca(2+) sensitivity are important regulators of cell death after myocardial ischemia, we sought to determine whether aging and/or estrogen deficiency (ovariectomy) increased mitochondrial Ca(2+) sensitivity. Mitochondrial respiration was measured in ventricular mitochondria isolated from adult (6 months; n = 26) and aged (24 months; n = 25), intact or ovariectomized female rats using the substrates α-ketoglutarate/malate (complex I); succinate/rotenone (complex II); ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/antimycin (complex IV). State 2 and 3 respiration was initiated by sequential addition of mitochondria and adenosine diphosphate. Ca(2+) sensitivity was assessed by Ca(2+)-induced swelling of de-energized mitochondria and reduction in state 3 respiration. Propylpyrazole triol (PPT) was administered intraperitoneally 45 minutes before euthanasia to assess mitochondrial protective effects through estrogen receptor (ER) α activation. Aging decreased the respiratory control index (RCI; state 3/state 2) for complexes I and II by 12% and 8%, respectively, independent of ovary status (P < 0.05). Of interest, Ca(2+) induced a greater decrease (18%-30%; P < 0.05) in complex I state 3 respiration in aged and ovariectomized animals, and mitochondrial swelling occurred twice as quickly in aged (vs adult) female rats (P < 0.05). Pretreatment with PPT increased RCI by 8% and 7% at complexes I and II, respectively (P < 0.05) but surprisingly increased Ca(2+) sensitivity. Age-dependent decreases in RCI and sensitization to Ca(2+) may explain in part the age-associated reductions in female ischemic tolerance; however, protection afforded by ER agonism involves more complex mechanisms.

Regulation of life span by mitochondrial respiration: the HIF-1 and ROS connection

A mild reduction in mitochondrial respiration extends the life span of many species, including C. elegans. We recently showed that hypoxia-inducible factor 1 (HIF-1) is required for the acquisition of a long life span by mutants with reduced respiration in C. elegans. We suggested that increased levels of reactive oxygen species (ROS) produced in the respiration mutants increase HIF-1 activity and lead to this longevity. In this research perspective, we discuss our findings and recent advances regarding the roles of ROS and HIF-1 in aging, focusing on the longevity caused by reduced respiration.

Alterations of respiratory chain complexes in sporadic pheochromocytoma

Succinate dehydrogenase (SDH) has been associated with carcinogenesis in hereditary pheochromocytoma (PC) and paraganglioma. We investigated if a similar association applies to sporadic pheochromocytoma. No genetic alteration was found in the SDHB, SDHC or SDHD genes of sporadic PC. However, in eight of nine sporadic PCs the SDH activity was, on average, reduced by 40%; moreover, the activities of the other oxidative phosphorylation (OXPHOS) complexes and citrate synthase were significantly lower compared to normal kidney tissue. Furthermore, immunohistochemical staining revealed a significant down-regulation of respiratory chain complexes. Since no pathogenic mutations were detected in the von Hippel-Lindau (VHL) gene, we can rule out that VHL deficiency is causing the general reduction of OXPHOS enzymes observed in the PCs investigated. In contrast to the single enzyme defects found in a subset hereditary PCs, a more generalized reduction of mitochondrial respiration seems to be present in most sporadic PCs. Strikingly, one of the nine PCs showed specific loss of complex I and a compensatory up-regulation of complexes II-V, which is a phenotype usually characteristic of oncocytic tumors.

Lycopene prevents 3-nitropropionic acid-induced mitochondrial oxidative stress and dysfunctions in nervous system

3-Nitropropionic acid (3-NP), an irreversible inhibitor of succinic acid dehydrogenase (SDH), induces neurodegeneration similar to that observed in Huntington's disease (HD). The present study was designed to investigate neuroprotective effect of lycopene on 3-NP induced mitochondrial dysfunctions and oxidative stress. Rats administered with 3-NP (25mg/kg, intraperitoneally) for four consecutive days exhibited deficits in cognitive and motor functions on day 15, whereas, lycopene (10mg/kg, orally) administration for 15 days ameliorated 3-NP-induced neurobehavioral deficits. The activities of mitochondrial Complexes-II, IV and V were found to be significantly lowered in striatum along with the reduction in mitochondrial respiration. However, no significant change in Complex-I activity was observed in 3-NP treated animals. 3-NP administration increased the rate of reactive oxygen species (ROS) and nitrite production which was accompanied by increase in lipid peroxidation in mitochondria. Thiol content and superoxide dismutase activity were depressed in 3-NP treated brain. 3-NP treatment induced mitochondrial swelling with increased cytochrome c release. Expression of p53 and active caspase-3 were increased in 3-NP treated animals. On the other hand, lycopene administration exhibited protective effect on 3-NP induced mitochondrial dysfunctions and oxidative stress. The results of the present study provide evidence for effectiveness of lycopene in preventing mitochondrial dysfunctions in 3-NP-induced HD.

Melatonin reduces striatal dopaminergic and mitochondrial deficits associated with chronic Parkinson's disorder

Neuronal oxidative stress and mitochondrial dysfunction have been implicated in Parkinson's disease (PD). We have recently correlated striatal dopaminergic (DA) neurodegeneration with mitochondrial deficit in a chronic mouse model of PD (MPD) induced by 10 injections of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine and probenecid over 5 weeks. Melatonin is a natural antioxidant and free radical scavenger, which has been shown to reduce cellular oxidative stress, protect mitochondria, and slow aging. In this study, we examined the effect of long‐term melatonin treatment (5 mg/kg/day, i.p., 5 days/week for 18 weeks) on mitochondrial and DA neuronal functions in the striatum of the chronic MPD. Melatonin alone did not alter the normal neuronal, mitochondrial and motor functions as seen in the saline‐treated control mice. The chronic MPD exhibited marked loss of DA, reduction of mitochondrial respiration and ATP level in the striatum, and impaired motor performance on the challenging beam. Interestingly, melatonin treatment for 18 weeks significantly reduced DA loss and motor impairment and prevented mitochondrial deficit as detected in the chronic MPD. This study demonstrates that long‐term melatonin is not only mitochondria‐protective but also neuroprotective in the chronic MPD supporting the hypothesis that melatonin may slow the progression of PD neurodegeneration. Supported by NINDS R01 NS47920.

Isolated Cytochrome c Oxidase Deficiency in G93A SOD1 Mice Overexpressing CCS Protein

G93A SOD1 transgenic mice overexpressing CCS protein develop an accelerated disease course that is associated with enhanced mitochondrial pathology and increased mitochondrial localization of mutant SOD1. Because these results suggest an effect of mutant SOD1 on mitochondrial function, we assessed the enzymatic activities of mitochondrial respiratory chain complexes in the spinal cords of CCS/G93A SOD1 and control mice. CCS/G93A SOD1 mouse spinal cord demonstrates a 55% loss of complex IV (cytochrome c oxidase) activity compared with spinal cord from age-matched non-transgenic or G93A SOD1 mice. In contrast, CCS/G93A SOD1 spinal cord shows no reduction in the activities of complex I, II, or III. Blue native gel analysis further demonstrates a marked reduction in the levels of complex IV but not of complex I, II, III, or V in spinal cords of CCS/G93A SOD1 mice compared with non-transgenic, G93A SOD1, or CCS/WT SOD1 controls. With SDS-PAGE analysis, spinal cords from CCS/G93A SOD1 mice showed significant decreases in the levels of two structural subunits of cytochrome c oxidase, COX1 and COX5b, relative to controls. In contrast, CCS/G93A SOD1 mouse spinal cord showed no reduction in levels of selected subunits from complexes I, II, III, or V. Heme A analyses of spinal cord further support the existence of cytochrome c oxidase deficiency in CCS/G93A SOD1 mice. Collectively, these results establish that CCS/G93A SOD1 mice manifest an isolated complex IV deficiency which may underlie a substantial part of mutant SOD1-induced mitochondrial cytopathy.

Endotoxin challenge reduces aconitase activity in myocardial tissue

Sepsis impairs mitochondrial respiration but the mechanisms responsible are incompletely understood. We propose that Krebs cycle enzymes are inhibited in sepsis, contributing to reduced rates of oxidative phosphorylation. Hypothesis. The activities of Krebs cycle enzymes are decreased in endotoxemia and contribute to reduced rates of oxidative phosphorylation. Methods. Adult male rats received an intraperitoneal injection of either endotoxin or saline. Cardiac mitochondria were subsequently isolated and measures of mitochondrial respiration and enzyme activities performed. Main results. By 24 h post endotoxin administration, there was a 28% reduction in mitochondrial respiration ( P = 0.0005) and a 24% reduction in aconitase activity ( P = 0.001). Functional activity of the electron transport chain was unaffected. Conclusion. Our data demonstrate that in the heart, the administration of endotoxin significantly and selectively decreased aconitase activity in association with reduced rates of oxidative phosphorylation. We conclude that decreased activity of aconitase contributes to the endotoxin-stimulated reduction in mitochondrial respiration.

INVESTIGATION OF A HEPATOTOXICITY SCREENING SYSTEM IN PRIMARY CELL CULTURES-"WHAT BIOMARKERS WOULD NEED TO BE ADDRESSED TO ESTIMATE TOXICITY IN CONVENTIONAL AND NEW APPROACHES?"-

High throughput toxicological estimation is required for safety evaluation in the early stage of drug discovery. In this context, establishment of an in vitro screening system reflecting in vivo toxicity is demanded for earlier safety assessment. We investigated LDH release and mitochondrial respiration (WST-1 reduction assay; WST-1) to detect cytotoxicity, morphological evaluation, and proteomics for estimating the reliable and sensitive biomarkers by using rat primary hepatocytes exposed to the compounds (acetaminophen, amiodarone, tetracycline and carbon tetrachloride) that are known to induce hepatotoxicity. In LDH release, no significant difference was detected between the control and compound exposed cells after exposure for 3 or 6 hr, but a dose-dependent increase was observed after exposure for 24 hr. Regarding the WST-1 assay, a dose-dependent reduction was detected after exposure for 6 and 24 hr to all of the compounds evaluated. In the proteomics analysis, 31 candidate proteins were identified from among the 103 demonstrating altered expression spots after exposure to acetaminophen. It was concluded that the cytotoxicity was detected earlier by measuring WST-1 than by measuring LDH release because the reduction of mitochondrial respiration is an expressions of earlier toxicity for cellular function, while the measured increase in the LDH release occurs after the failure of the cell membrane. Mitochondrial respiration ability was a useful parameter for cytotoxicity in in vitro hepato-toxicity screening, as cytotoxicity can be detected during the early stage of exposure. In addition to the conventional biomarkers, several protein biomarkers which relate to oxidative stress and metabolism-regulation were detected. Further comprehensive analysis of defined proteins would be necessary to estimate the more sensitive toxicology biomarker.

Microvesicular Steatosis Induced by a Short Chain Fatty Acid: Effects on Mitochondrial Function and Correlation with Gene Expression

Hepatotoxicity characterized by microvesicular steatosis (MVS) is characterized by an abnormal accumulation of numerous small cytoplasmic lipid droplets in hepatocytes. Fulminant or progressive cases of microvesicular steatosis may lead to liver failure and death. Experimentally, short-chain carboxylic acids are known to induce microvesicular steatosis. The identification of gene changes that correlate with MVS concomitant with biochemical and histological indices could provide a better understanding of how this toxicity occurs as well as biomarkers that could be used to avoid this toxicity in the future. Sprague-Dawley rats were dosed days with cyclopropane carboxylic acid (CPCA) a short-chain fatty acid that can induce microvesicular steatosis, and with butyrate, a short chain fatty acid that served as a negative control. CPCA initiated microvesicular steatosis while butyrate did not. In addition, CPCA inhibited beta-oxidation in a concentration-dependent manner in vitro and caused a reduction in mitochondrial respiration ex vivo; no inhibition was evident with butyrate. Microarray results showed that gene expression changes with CPCA resulted in regulation of genes involved in beta-oxidation, as well as other genes associated with mitochondrial function. Overall, these results support altered hepatic mitochondrial function as a mechanism of the toxicity induced by a short-chain fatty acid and may provide potential biomarkers for this toxicity.

Methionine 35 oxidation reduces toxic and pro-apoptotic effects of the amyloid β-protein fragment (31–35) on isolated brain mitochondria

Amyloid β-peptide (AβP), the central constituent of senile plaques in Alzheimer's disease (AD) brain, has been shown to be a source of free radical oxidative stress that may lead to neurodegeneration. In particular, it is well known that oxidation of methionine 35, is strongly related to the pathogenesis of AD, since it represents the residue in AβP most susceptible to oxidation in vivo. In the present study, we used the fragment 31–35 of AβP, which has a single methionine at residue 35, in order to investigate the influence of the oxidation state of methionine-35 on the toxic and pro-apoptotic effects induced by Aβ(31–35) on isolated brain mitochondria. The obtained results show that exposure of isolated mitochondria from rat brain to AβP(31–35) determines (i) a large release of cytochrome c (ii) a significant reduction in mitochondrial respiration and (iii) a slight drop in the mitochondrial membrane potential (ΔΨ). In contrast, the amplitude of these events resulted attenuated or completely abrogated in isolated brain mitochondria exposed to the AβP(31–35)Met35 OX, in which methionine 35 was oxidized to methionine sulfoxide. We have further characterized the action of AβP(31–35) and Aβ(31–35)Met35 OX peptide on PC12 cells. Although these two peptides, compromised mitochondrial function at a different extent as assessed by MTT reduction, neither one of them decreased cell viability as measured by Trypan Blue exclusion assay. The results obtained in this study support the hypothesis that the oxidative state of Met-35 may play a critical role in the mechanisms responsible of neurotoxicity exerted by this peptide.

Methamphetamine induces apoptosis in an immortalized rat striatal cell line by activating the mitochondrial cell death pathway

Methamphetamine is a neurotoxic drug of abuse known to cause cell death both in vitro and in vivo. Nevertheless, the molecular and cellular mechanisms involved in this process remain to be clarified. Herein, we show that methamphetamine-induced apoptosis is associated with early (2 h) overexpression of bax, decreases of mitochondrial membrane potential and oxygen consumption as well as release of cytochrome c from mitochondria. In addition, activated caspase-9 was detected at 4 h post-METH exposure. Cell death was detectable by annexin V and propidium iodide staining after 8 h of methamphetamine exposure. At that time, the majority of the cells were stained by annexin V alone, with some cells being stained for both annexin V and propidium iodide. Moreover, cleavage of caspase-3, poly (ADP-ribose) polymerase and DNA fragmentation-related factor 45 was detected at 8 h post drug treatment. These results indicate that methamphetamine-induced apoptotic cell death results from early overexpression of bax, reduction of mitochondrial respiration and membrane potential and release of mitochondrial cytochrome c with subsequent activation of the caspase cascade.

5-Aminoisoquinolinone, a potent inhibitor of poly (adenosine 5′-diphosphate ribose) polymerase, reduces myocardial infarct size

This study investigates the effects of a novel, water-soluble inhibitor of the activity of poly (adenosine 5′-diphosphate ribose) polymerase, 5-aminoisoquinolinone [5-aminoisoquinolin-1(2 H)-one], on (i) poly (adenosine 5′-diphosphate ribose) polymerase activity in rat cardiac myoblasts and (ii) the infarct size caused by regional myocardial ischaemia and reperfusion in the rat. Exposure of H9c2 cells to hydrogen peroxide (H 2O 2, 1 mM) caused a significant increase in poly (adenosine 5′-diphosphate ribose) polymerase activity and an 80–90% reduction in mitochondrial respiration (cellular injury). Pretreatment of these cells with 5-aminoisoquinolinone (0.003–1 mM) caused a concentration-dependent inhibition of poly (adenosine 5′-diphosphate ribose) polymerase activity (IC 50: ∼4.5 μM, n=6–9) and cell injury (EC 50: ∼4.45 μM, n=9). In a rat model of myocardial infarction, left anterior descending coronary artery occlusion (25 min) and reperfusion (2 h) resulted in an infarct size of 50±3%. Administration (1 min before reperfusion) of 5-aminoisoquinolinone reduced myocardial infarct size in a dose-related fashion. Thus, 5-aminoisoquinolinone is a potent inhibitor of poly (adenosine 5′-diphosphate ribose) polymerase activity in cardiac myoblasts and reduces myocardial infarct size in vivo.

Zn2+ Inhibits α-Ketoglutarate-stimulated Mitochondrial Respiration and the Isolated α-Ketoglutarate Dehydrogenase Complex

Intracellular free Zn(2+) is elevated in a variety of pathological conditions, including ischemia-reperfusion injury and Alzheimer's disease. Impairment of mitochondrial respiration is also associated with these pathological conditions. To test whether elevated Zn(2+) and impaired respiration might be linked, respiration of isolated rat liver mitochondria was measured after addition of Zn(2+). Zn(2+) inhibition (K(i)(app) = approximately 1 micrometer) was observed for respiration stimulated by alpha-ketoglutarate at concentrations well within the range of intracellular Zn(2+) reported for cultured hepatocytes. The bc(1) complex is inhibited by Zn(2+) (Link, T. A., and von Jagow, G. (1995) J. Biol. Chem. 270, 25001-25006). However, respiration stimulated by succinate (K(i)(app) = approximately 6 micrometer) was less sensitive to Zn(2+), indicating the existence of a mitochondrial target for Zn(2+) upstream from bc(1) complex. Purified pig heart alpha-ketoglutarate dehydrogenase complex was strongly inhibited by Zn(2+) (K(i)(app) = 0.37 +/- 0.05 micrometer). Glutamate dehydrogenase was more resistant (K(i)(app) = 6 micrometer), malate dehydrogenase was unaffected, and succinate dehydrogenase was stimulated by Zn(2+). Zn(2+) inhibition of alpha-ketoglutarate dehydrogenase complex required enzyme cycling and was reversed by EDTA. Reversibility was inversely related to the duration of exposure and the concentration of Zn(2+). Physiological free Zn(2+) may modulate hepatic mitochondrial respiration by reversible inhibition of the alpha-ketoglutarate dehydrogenase complex. In contrast, extreme or chronic elevation of intracellular Zn(2+) could contribute to persistent reductions in mitochondrial respiration that have been observed in Zn(2+)-rich diseased tissues.

Does elevated atmospheric CO2 concentration inhibit mitochondrial respiration in green plants?

ATP, adenosine triphosphate Km, Michaelis‐Menton coefficient Ca, concentration of CO2 in the air (μmol mol–1) NAD, oxidized nicotin adenine dinucleotide NADH, reduced nicotin adenine dinucleotide NADP, oxidized nicotin adenine phosphate dinucleotide NADPH, reduced nicotine adenine phosphate dinucleotide R, rate of respiration per unit DW [μmol g DW–1], Rubisco, ribulose‐1,5‐bisphosphate carboxylase/oxygenase Vc,max, maximum in vivo rate of carboxylation at Rubisco (μmol m–2 s–1) There is abundant evidence that a reduction in mitochondrial respiration of plants occurs when atmospheric CO2 (Ca) is increased. Recent reviews suggest that doubling the present Ca will reduce the respiration rate [per unit dry weight (DW)] by 15 to 18%. The effect has two components: an immediate, reversible effect observed in leaves, stems, and roots of plants as well as soil microbes, and an irreversible effect which occurs as a consequence of growth in elevated Ca and appears to be specific to C3 species. The direct effect has been correlated with inhibition of certain respiratory enzymes, namely cytochrome‐c‐oxidase and succinate dehydrogenase, and the indirect or acclimation effect may be related to changes in tissue composition. Although no satisfactory mechanisms to explain these effects have been demonstrated, plausible mechanisms have been proposed and await experimental testing. These are carbamylation of proteins and direct inhibition of enzymes of respiration. A reduction of foliar respiration of 15% by doubling present ambient Ca would represent 3 Gt of carbon per annum in the global carbon budget.

Inhibitors of the activity of poly (ADP-ribose) synthetase reduce the cell death caused by hydrogen peroxide in human cardiac myoblasts.

Poly (ADP-ribose) synthetase (PARS) is a nuclear enzyme activated by strand breaks in DNA which are caused by reactive oxygen species (ROS). Inhibitors of PARS activity reduce the degree of reperfusion injury of the heart in vivo and in vitro. Here we investigate the role of PARS in the cell death of human cardiac myoblasts caused by hydrogen peroxide. Exposure of human cardiac myoblasts to hydrogen peroxide caused a time- and concentration-dependent reduction in mitochondrial respiration (cell injury), an increase in cell death (LDH release), as well as an increase in PARS activity. The PARS inhibitors 3-aminobenzamide (3 mM), 1,5-dehydroxyisoquinoline (300 microM) or nicotinamide (3 mM) attenuated the cell injury and death as well as the increase in PARS activity caused by hydrogen peroxide (3 mM; 4 h for cell injury/death, 60 min for PARS activity) in human cardiac myoblasts. In contrast, the inactive analogues 3-aminobenzoic acid (3 mM) or nicotinic acid (3 mM) were without effect. The iron chelator deferoxamine (1-10 mM) caused a concentration-dependent reduction in the cell injury and death caused by hydrogen peroxide in these human cardiac myoblasts. Thus, the cell injury/death caused by hydrogen peroxide in human cardiac myoblasts is secondary to the formation of hydroxyl radicals and due to an increase in PARS activity. We therefore propose that activation of PARS contributes to the cell injury/cell death associated with oxidant stress in the heart.

Enterocyte respiration rates in feline small intestine exposed to graded ischemia

The purpose of our study was to investigate the changes in enterocyte cellular and mitochondrial respiration rates subsequent to ischemia of graded duration. The small intestine of anesthetized adult cats was assigned to one of five treatment regimens: control or ischemia of 15-, 30-, 60-, or 90-min duration. Cellular and mitochondrial respiration was measured using a Clark-type O2 electrode at 0 and 4 h postharvest. Ischemia of increasing duration caused a progressive decrease in cellular and mitochondrial respiration in enterocytes at 0 h postharvest. By 4 h postharvest, cellular and mitochondrial respiration rates for the 15-, 30-, and 60-min ischemic groups had recovered to near control levels, whereas the 90-min group showed minimal recovery. These data suggest that ischemia suppresses cellular and mitochondrial respiration of intestinal epithelial cells, the magnitude of which is related to the ischemic duration. The ischemia-induced suppression in cellular respiration primarily reflects a reduction in mitochondrial respiration.