A novel high-throughput assay for respiration in isolated brain microvessels reveals impaired mitochondrial function in the aged mice

Cerebral blood flow (CBF) is uniquely regulated by the anatomical design of the cerebral vasculature as well as through neurovascular coupling. The process of directing the CBF to meet the energy demands of neuronal activity is referred to as neurovascular coupling. Microvasculature in the brain constitutes the critical component of the neurovascular coupling. Mitochondria provide the majority of ATP to meet the high-energy demand of the brain. Impairment of mitochondrial function plays a central role in several age-related diseases such as hypertension, ischemic brain injury, Alzheimer’s disease, and Parkinson disease. Interestingly, microvessels and small arteries of the brain have been the focus of the studies implicating the vascular mechanisms in several age-related neurological diseases. However, the role of microvascular mitochondrial dysfunction in age-related diseases remains unexplored. To date, high-throughput assay for measuring mitochondrial respiration in microvessels is lacking. The current study presents a novel method to measure mitochondrial respiratory parameters in freshly isolated microvessels from mouse brain ex vivo using Seahorse XFe24 Analyzer. We validated the method by demonstrating impairments of mitochondrial respiration in cerebral microvessels isolated from old mice compared to the young mice. Thus, application of mitochondrial respiration studies in microvessels will help identify novel vascular mechanisms underlying a variety of age-related neurological diseases.     

High dose clopidogrel decreases mice liver mitochondrial respiration function in vitro

The effects of clopidogrel on mitochondrial respiratory function have not been previously investigated. We show in vitro that isolated mice liver mitochondria treated with very high doses of clopidogrel 10 microg/ml significantly reduces pre-treatment mitochondrial respiratory state 3 (P<0.05) and state 4 respiration (P<0.01), while oxygen consumption in State 3 is prolonged. This suggests a compromise to mitochondrial oxidative phosphorylation following the addition of high dose clopidogrel. Because clopidogrel at human therapeutic doses 40 ng/ml did not affect isolated mitochondrial respiration, it is thus unlikely, in the absence of cellular bioaccumulation, that clinical doses of clopidogrel would affect mitochondrial bioenergetics in vivo.     

Myoglobin-mediated oxygen delivery to mitochondria of isolated cardiac myocytes.

Myoglobin-mediated oxygen delivery to intracellular mitochondria is demonstrated in cardiac myocytes isolated from the hearts of mature rats. Myocytes are held at high ambient oxygen pressure, 40-340 torr (5-45 kPa); sarcoplasmic myoglobin is fully oxygenated. In this condition oxygen availability does not limit respiratory rate; myoglobin-facilitated diffusion contributes no additional oxygen flux and, since oxygen consumption is measured in steady states, the storage function of myoglobin vanishes. Carbon monoxide, introduced stepwise, displaces oxygen from intracellular oxymyoglobin without altering the optical spectrum of the largely oxidized intracellular mitochondria. A large part, about one-third, of the steady-state oxygen uptake is abolished by carbon monoxide blockade of myoglobin oxygenation. The myoglobin-dependent component of the oxygen uptake decreases linearly with decreasing fraction of intracellular oxymyoglobin, with a slope near unity. Studies using inhibitors of mitochondrial electron transport indicate that myoglobin-delivered oxygen uptake depends on electron flow through the mitochondrial electron transport chain. We conclude that cardiac mitochondria accept two additive simultaneous flows of oxygen: a flow of dissolved oxygen to cytochrome oxidase and a flow of myoglobin-bound oxygen to a mitochondrial terminus. Myoglobin-mediated oxygen delivery supports ATP generation by heart cells at physiological ambient oxygen pressure.     

A high fat diet increases mitochondrial fatty acid oxidation and uncoupling to decrease efficiency in rat heart

Elevated levels of cardiac mitochondrial uncoupling protein 3 (UCP3) and decreased cardiac efficiency (hydraulic power/oxygen consumption) with abnormal cardiac function occur in obese, diabetic mice. To determine whether cardiac mitochondrial uncoupling occurs in non-genetic obesity, we fed rats a high fat diet (55% kcal from fat) or standard laboratory chow (7% kcal from fat) for 3 weeks, after which we measured cardiac function in vivo using cine MRI, efficiency in isolated working hearts and respiration rates and ADP/O ratios in isolated interfibrillar mitochondria; also, measured were medium chain acyl-CoA dehydrogenase (MCAD) and citrate synthase activities plus uncoupling protein 3 (UCP3), mitochondrial thioesterase 1 (MTE-1), adenine nucleotide translocase (ANT) and ATP synthase protein levels. We found that in vivo cardiac function was the same for all rats, yet oxygen consumption was 19% higher in high fat-fed rat hearts, therefore, efficiency was 21% lower than in controls. We found that mitochondrial fatty acid oxidation rates were 25% higher, and MCAD activity was 23% higher, in hearts from rats fed the high fat diet when compared with controls. Mitochondria from high fat-fed rat hearts had lower ADP/O ratios than controls, indicating increased respiratory uncoupling, which was ameliorated by GDP, a UCP3 inhibitor. Mitochondrial UCP3 and MTE-1 levels were both increased by 20% in high fat-fed rat hearts when compared with controls, with no significant change in ATP synthase or ANT levels, or citrate synthase activity. We conclude that increased cardiac oxygen utilisation, and thereby decreased cardiac efficiency, occurs in non-genetic obesity, which is associated with increased mitochondrial uncoupling due to elevated UCP3 and MTE-1 levels.     

Changes of hepatic fatty acid metabolism produced by chronic thioacetamide administration in rats.

Hepatic mitochondrial functions related to fatty acid metabolism, including the respiratory control ratio, fatty acid oxidative capacity and carnitine palmitoyltransferase I activity, were studied in vitro with mitochondria isolated from rats treated with thioacetamide for up to 12 wk. The levels of ketone bodies, carnitine, carnitine esters and malonyl-coenzyme A were also determined in liver extracts. Polarography of mitochondrial respiration from succinate or glutamate plus malate showed a lower respiratory control ratio in thioacetamide-treated rats, whereas uncoupled oxygen consumption was not altered. This suggests that the mitochondrial respiratory chain capacity remained intact in the thioacetamide-treated rats. The oxygen consumption associated with palmitoyl-coenzyme A and palmitoyl-L-carnitine oxidation by isolated liver mitochondria was increased by thioacetamide treatment on both a per-mitochondrial protein and a per-total liver basis. The carnitine palmitoyl-transferase I activity; the tissue levels of ketone bodies, carnitine and carnitine esters; and the beta-hydroxybutyrate/acetoacetate ratio were all higher in the livers of thioacetamide-treated animals than in control livers, whereas the hepatic malonyl-coenzyme A level was decreased by thioacetamide. These results indicate the increased diversion of cytosolic long-chain acyl-coenzyme As into the mitochondria for beta-oxidation rather than their esterification and use in lipogenesis. These intrahepatic metabolic changes induced by chronic thioacetamide administration may reflect the whole-body catabolic state and can be seen as adaptive for maintaining energy homeostasis under conditions of impaired glucose tolerance.     

Idebenone induces oxygen consumption rate modifications in aged rat brain mitochondria

Idebenone effects on oxygen consumption in brain mitochondria, obtained from young and aged rats, were evaluated. Sixty rats (3 and 20 months of age) were treated for 3 months with 30 mg/kg of idebenone and compared to a placebo group. Brain mitochondria oxygen consumption rate was measured by polarographic techniques in basal (State 4), ADP-stimulated (State 3) and uncoupled conditions. When Complex I substrates (pyruvate + malate) were used, aged non-treated rats showed a significant increase in State 4 (175%) and uncoupled (152%) O(2)-uptake rate; no difference was found in State 3 respiration and in ADP/O(2) ratio. Idebenone was able to reverse these age-related effects probably acting on lipid peroxidation and the mitochondrial respiratory chain. No differences were found in mitochondrial enzymatic activities.     

Oxidative capacity of muscle and mitochondria: correlation of physiological, biochemical, and morphometric characteristics.

The oxidative capacity of cat skeletal muscles (soleus, gracilis, and gracilis chronically stimulated for 28 days) was derived from the total mitochondrial content in the muscle, the surface area of mitochondrial inner membranes, and respiratory activities of isolated mitochondria. Mitochondrial content was estimated by standard morphometry. The surface area of mitochondrial inner membranes per unit volume of mitochondria was estimated by a stereological method. The respiratory activities of isolated mitochondria were measured biochemically, using pyruvate/malate, glutamate/malate, succinate, or cytochrome c as substrate. Structurally and functionally, mitochondria from the three muscle types showed nearly identical characteristics. Oxidative activity was dependent on substrate; with succinate, 5.8 ml of O2 per min per ml of mitochondria was the rate most likely to represent physiological conditions. Oxidative activities of 3.1 ml.min-1.ml-1 with pyruvate/malate and 14.5 ml.min-1.ml-1 with cytochrome c as substrates were theoretical lower and upper bounds. The oxidative capacity of each of the three muscles was thus in direct proportion to the total volume of mitochondria in the muscle. The respiratory capacity of isolated mitochondria was very near to the maximal oxygen uptake rate of mitochondria that is commonly estimated in intact muscles of a wide variety of animals.     

Impact of imidapril on cardiac mitochondrial function in an ex-vivo animal model of global myocardial ischemia.

Imidapril is an angiotensin I converting enzyme inhibitor, a class of drugs with known cardioprotective activity. It is now known that this is due not only to their antihypertensive activity, but also to the fact that they decrease cellular and tissue levels of angiotensin II, a potent vasoconstrictor and inducer of myocardial fibrosis. These mechanisms may explain the good clinical results of this class of drugs in the treatment of coronary artery disease and heart failure, two diseases whose etiopathogenesis is closely related to the activation of the renin-angiotensin-aldosterone system. However, the impact of this class of drugs on cardiac mitochondrial function during acute myocardial ischemia is still largely unknown. With the aim of studying the effect of imidapril on cardiac mitochondrial function during acute ischemia, we used an ex-vivo animal model, perfused in a Langerdorff system and then subjected to ischemia in the presence or absence of imidapril. We evaluated mitochondrial membrane electrical potential, respiratory chain O2 consumption, and rate and amplitude of mitochondrial swelling. We conclude that imidapril did not significantly change oxygen consumption by cardiac mitochondria, as assessed by the rate of respiratory state 3 (the state that corresponds to the active phosphorylation phase). However, imidapril significantly increased transmembrane electrical potential and, in ischemic cardiac mitochondria, was able to prevent the calcium-induced increase in the rate and amplitude of mitochondrial swelling, thus enabling better preservation of mitochondrial membrane structure, with consequent improvement of electrical potential after the phosphorylation cycle. These findings enabled a better understanding of the mechanisms behind the cytoprotection provided by imidapril during ischemic cardiomyopathy, clearly highlighting, at a cellular biology level, the importance of pharmacological modulation of cardiac mitochondrial function during acute ischemia.     

Chronic alcohol consumption increases the sensitivity of rat liver mitochondrial respiration to inhibition by nitric oxide

Chronic alcohol consumption is a well-known risk factor for hepatic injury, and mitochondrial damage plays a significant role in this process. Nitric oxide (NO) is an important modulator of mitochondrial function and is known to inhibit mitochondrial respiration. However, the impact of chronic alcohol consumption on NO-dependent control of liver mitochondrial function is unknown. This study examines the effect of alcohol exposure on liver mitochondria in a rat model and explores the interaction of NO and mitochondrial respiration in this context. Mitochondria were isolated from the liver of both control and ethanol-fed rats after 5 to 6 weeks of alcohol consumption. Mitochondria isolated from ethanol-treated rats showed a significant decrease in state 3 respiration and respiratory control ratio that was accompanied by an increased sensitivity to NO-dependent inhibition of respiration. In conclusion, we show that chronic alcohol consumption leads to increased sensitivity to the inhibition of respiration by NO. We propose that this results in a greater vulnerability to hypoxia and the development of alcohol-induced hepatotoxicity.     

Incorporation of marine lipids into mitochondrial membranes increases susceptibility to damage by calcium and reactive oxygen species: evidence for enhanced activation of phospholipase A2 in mitochondria enriched with n-3 fatty acids.

Experiments were designed to evaluate the susceptibility of mitochondrial membranes enriched with n-3 fatty acids to damage by Ca2+ and reactive oxygen species. Fatty acid content and respiratory function were assessed in renal cortical mitochondria isolated from fish-oil- and beef-tallow-fed rats. Dietary fish oils were readily incorporated into mitochondrial membranes. After exposure to Ca2+ and reactive oxygen species, mitochondria enriched in n-3 fatty acids, and using pyruvate and malate as substrates, had significantly greater changes in state 3 and uncoupled respirations, when compared with mitochondria from rats fed beef tallow. Mitochondrial site 1 (NADH coenzyme Q reductase) activity was reduced to 45 and 85% of control values in fish-oil- and beef-tallow-fed groups, respectively. Exposure to Ca2+ and reactive oxygen species enhance the release of polyunsaturated fatty acids enriched at the sn-2 position of phospholipids from mitochondria of fish-oil-fed rats when compared with similarly treated mitochondria of beef-tallow-fed rats. This release of fatty acids was partially inhibited by dibucaine, the phospholipase A2 inhibitor, which we have previously shown to protect mitochondria against damage associated with Ca2+ and reactive oxygen species. The results indicate that phospholipase A2 is activated in mitochondria exposed to Ca2+ and reactive oxygen species and is responsible, at least in part, for the impairment of respiratory function. Phospholipase A2 activity and mitochondrial damage are enhanced when mitochondrial membranes are enriched with n-3 fatty acids.     

The effect of pancreatitis associated ascitic fluid on some functions of rat liver mitochondria. A possible mechanism of the damage to the liver in acute pancreatitis

The damage to the liver during acute pancreatitis (AP) could be partly dependent on depressive action of pancreatitis associated ascitic fluid (PAAF) on the energy metabolism of hepatocytes. The aim of the study was to assess the effect of PAAF from dogs with acute experimental pancreatitis (AEP) and from humans with AP on the respiratory function of isolated rat liver mitochondria (RLM). The mitochondrial oxygen consumption rate in state 3 respiration (with ADP) and in state 4 (without ADP) using sodium succinate as substrate and oxygen Clark's electrode was estimated. Respiratory control ratio (RCR) and P/O ratio were calculated. PAAF was collected after 6 h of AEP induced by Elliott's method in 8 dogs, and from 4 patients with AP, intraoperatively. Both animal and human PAAFs increase the oxygen consumption rate by RLM in state 4 dose dependently (by 65% with 50 microL to 150% with 200 microL of canine PAAF). This uncoupling effect of human PAAF was twice more potent than the canine. Dialysis of PAAF reduced this effect almost completely. The mitochondrial ATPase activity in RLM treated with PAAF was stimulated and this effect was also reduced by dialysis. The conclusion was that the damage to the liver in AEP could be partly dependent on the toxicity of dializable component(s) of PAAF on the energy metabolism of mitochondria. These findings may partly explain the beneficial effects of peritoneal lavage in acute pancreatitis.     

Lack of effect of caloric restriction on bioenergetics and reactive oxygen species production in intact rat hepatocytes

To investigate the hypothesis that caloric restriction alters mitochondrial function in situ, intact hepatocytes were isolated from fully fed and calorie-restricted (55% of control food intake, 4 months duration) male Brown-Norway rats at 6 months of age, and various parameters were determined. Overall, the production of reactive oxygen species was not affected by caloric restriction, neither were the mitochondrial membrane potential, oxygen consumption driving proton leak, or oxygen consumption driving ATP turnover. It is concluded that while isolated mitochondria from liver tissue of calorie-restricted animals display a reduction in the generation of reactive oxygen species, it was not possible to confirm this effect in isolated hepatocytes. Further work is required to establish what effect, if any, caloric restriction has on the rate of generation of reactive oxygen species in intact cells and tissues and importantly at the whole-animal level.     

Melatonin treatment restores mitochondrial function in Alzheimer's mice: a mitochondrial protective role of melatonin membrane receptor signaling

Mitochondrial dysfunction is a hallmark of Alzheimer's disease (AD) and is observed in mutant amyloid precursor protein (APP) transgenic mouse models of familial AD. Melatonin is a potent antioxidant, can prevent toxic aggregation of Alzheimer's beta-amyloid (Aβ) peptide and, when taken long term, can protect against cognitive deficits in APP transgenic mice. To study the effects of melatonin on brain mitochondrial function in an AD model, APP/PS1 transgenic mice were treated for 1 month with melatonin. Analysis of isolated brain mitochondria from mice indicated that melatonin treatment decreased mitochondrial Aβ levels by two- to fourfold in different brain regions. This was accompanied by a near complete restoration of mitochondrial respiratory rates, membrane potential, and ATP levels in isolated mitochondria from the hippocampus, cortex, or striatum. When isolated mitochondria from untreated young mice were given melatonin, a slight increase in respiratory rate was observed. No such effect was observed in mitochondria from aged mice. In APP-expressing neuroblastoma cells in culture, mitochondrial function was restored by melatonin or by the structurally related compounds indole-3-propionic acid or N(1)-acetyl-N(2)-formyl-5-methoxykynuramine. This restoration was partially blocked by melatonin receptor antagonists indicating melatonin receptor signaling is required for the full effect. Therefore, treatments that stimulate melatonin receptor signaling may be beneficial for restoring mitochondrial function in AD, and preservation of mitochondrial function may an important mechanism by which long term melatonin treatment delays cognitive dysfunction in AD mice.     

Mitochondrial function after global cardiac ischemia and reperfusion: Influences of organelle isolation protocols

Summary Dog hearts were made globally ischemic for 1 hr at normothermia, at 28°C, or at normothermia after perfusion with a hyperkalemic cardioplegia solution. After 1 hr of reperfusion mitochondria were isolated from each heart using three protocols involving: processing (homogenization and centrifugation) exclusively in KCl, Tris-EDTA plus albumin (KEA) solution; homogenizing in KEA but washing mitochondria in EDTA-depleted media (KA); or processing exclusively in EDTA-free medium.Postreperfusion contractile measurements indicated that only hypothermic hearts had function not significantly different from nonischemic controls, but significantly better than that of normothermic, nontreated ischemic hearts. Mitochondrial studies indicated no differences between ischemic-hypothermic, potassium-arrested or nonischemic groups. Mitochondrial function paralleled contractile function only in the severely damaged hearts made ischemic at 37°C without perfusion with cardioplegic solution. These comparisons were not dependent upon whether mitochondrial function was assessed in terms of respiratory rates, respiratory control (R.C.) or ADP: O ratios, or the oxidative phosphorylation rate (O.P.R.; State 3 oxygen consumption rate X ADP: O ratio).Adding EDTA to organelles isolated by gradually removing EDTA after homogenization in KEA had differing effects. In the ischemic nontreated group, adding EDTA increased State 3 rate, decreased State 4 rate, and increased both R.C. ratios and the O.P.R. Adding EDTA to mitochondria from nonischemic hearts or hearts which were hypothermic or potassium-arrested decreased State 4 rates, increasing the R.C. ratio, but did not affect State 3 rates or the O.P.R. Initial homogenization of tissue in EDTA-free medium gave poorly-functioning mitochondria in all groups, and readministration of EDTA was without benefit.We concluded that hypothermia was superior to normothermic cardioplegia in terms of contractile function, but not based on mitochondrial function. This suggests that in some hearts subjected to ischemic damage, defects of ATP utilization, rather than of ATP synthesis, may predominate.We also concluded that for studies of cardiac mitochondrial function, EDTA or a similar chelator must be present in homogenizing media to provide meaningful data, but the chelator can be removed and readministered as desired during subsequent processing. However, the qualitative and quantitative effects of EDTA readministration depend upon the severity of ischemic damage to the myocardium from which the organelles are isolated.This study was supported in part by Grants-in-Aid from the Michigan Heart Association, the American Heart Association, and the National Heart, Lung and Blood Institute (HL-19782-03).Preliminary data were presented at the 1979 Annual Meeting of the Federation of American Societies for Experimental Biology.     

Biochemical and morphological changes in myocardium during coronary occlusion and reperfusion in canine hearts: effects of propranolol on myocardial damage

To clarify the mechanism of irreversible myocardial damage, we studied the relationship between ischaemic mitochondrial dysfunction and leakage of lysosomal enzymes, and the effects of propranolol on myocardial damage. Open chest anaesthetised dogs were divided into six groups: 30 min occlusion of the left anterior coronary artery (LAD); 2 h LAD occlusion; 2 h LAD occlusion after premedication with 0.3 mg.kg-1 propranolol; 30 min LAD occlusion/l h reperfusion; 2 h LAD occlusion/l h reperfusion; and 2 h LAD occlusion/l h reperfusion after propranolol premedication. After occlusion or reperfusion, heart mitochondria were prepared from normal and occluded or reperfused areas, and mitochondrial function (rate of oxygen consumption in State III, and respiratory control index) was measured polarographically. Myocardial tissue was fractionated and activities of lysosomal enzymes (N-acetyl-beta-glucosaminidase and beta-glucuronidase) were measured. Electron microscopic studies were performed. Thirty min occlusion induced mitochondrial dysfunction without leakage of lysosomal enzymes. Reperfusion for 1 h reversed these changes. However occlusion for 2 h induced mitochondrial dysfunction associated with the leakage of lysosomal enzymes, and mitochondrial dysfunction was not reversed by 1 h reperfusion. Propranolol reduced mitochondrial dysfunction after 2 h occlusion and prevented leakage of lysosomal enzymes. Mitochondrial function was fairly well maintained after 1 h reperfusion in dogs premedicated with propranolol. Structural changes in mitochondria were observed in the 2 h occlusion/l h reperfusion group, and were reduced by premedication with propranolol. These results suggest that irreversible injury of ischaemic mitochondria is closely linked with instability of lysosomal membranes, and that propranolol prevented irreversible myocardial mitochondrial dysfunction.     

Progesterone and estrogen regulate oxidative metabolism in brain mitochondria

The ovarian hormones progesterone and estrogen have well-established neurotrophic and neuroprotective effects supporting both reproductive function and cognitive health. More recently, it has been recognized that these steroids also regulate metabolic functions sustaining the energetic demands of this neuronal activation. Underlying this metabolic control is an interpretation of signals from diverse environmental sources integrated by receptor-mediated responses converging upon mitochondrial function. In this study, to determine the effects of progesterone (P4) and 17beta-estradiol (E2) on metabolic control via mitochondrial function, ovariectomized rats were treated with P4, E2, or E2 plus P4, and whole-brain mitochondria were isolated for functional assessment. Brain mitochondria from hormone-treated rats displayed enhanced functional efficiency and increased metabolic rates. The hormone-treated mitochondria exhibited increased respiratory function coupled to increased expression and activity of the electron transport chain complex IV (cytochrome c oxidase). This increased respiratory activity was coupled with a decreased rate of reactive oxygen leak and reduced lipid peroxidation representing a systematic enhancement of brain mitochondrial efficiency. As such, ovarian hormone replacement induces mitochondrial alterations in the central nervous system supporting efficient and balanced bioenergetics reducing oxidative stress and attenuating endogenous oxidative damage.     

Decrease in mitochondrial complex I activity in ischemic/reperfused rat heart: involvement of reactive oxygen species and cardiolipin

Reactive oxygen species (ROS) are considered an important factor in ischemia/reperfusion injury to cardiac myocytes. Mitochondrial respiration is an important source of ROS production and hence a potential contributor to cardiac reperfusion injury. In this study, we have examined the effect of ischemia and ischemia followed by reperfusion of rat hearts on various parameters related to mitochondrial function, such as complex I activity, oxygen consumption, ROS production, and cardiolipin content. The activity of complex I was reduced by 25% and 48% in mitochondria isolated from ischemic and reperfused rat heart, respectively, compared with the controls. These changes in complex I activity were associated with parallel changes in state 3 respiration. The capacity of mitochondria to produce H2O2 increased on reperfusion. The mitochondrial content of cardiolipin, which is required for optimal activity of complex I, decreased by 28% and 50% as function of ischemia and reperfusion, respectively. The lower complex I activity in mitochondria from reperfused rat heart could be completely restored to the level of normal heart by exogenous added cardiolipin. This effect of cardiolipin could not be replaced by other phospholipids nor by peroxidized cardiolipin. It is proposed that the defect in complex I activity in ischemic/reperfused rat heart could be ascribed to a ROS-induced cardiolipin damage. These findings may provide an explanation for some of the factors responsible for myocardial reperfusion injury.     

Curcumin Protects from Cardiac Reperfusion Damage by Attenuation of Oxidant Stress and Mitochondrial Dysfunction

This study was designed to investigate whether the pretreatment with curcumin, a yellow pigment from turmeric (Curcuma longa) known for its potent antioxidant capacity, was able to protect against the oxidant damage and mitochondrial dysfunction induced by reperfusion injury in isolated hearts. Rats were treated with a daily intragastric dose of curcumin (200 mg/kg) for 7 days prior to experimental ischemia (30 min) and reperfusion (60 min) (I/R). Cardiac mechanical work was measured during periods of stabilization, ischemia, and reperfusion. Oxidant stress and activity of antioxidant enzymes were measured in both homogenates of cardiac tissue and in isolated mitochondria. In addition, oxygen consumption was measured in isolated mitochondria. It was found that curcumin pretreatment attenuates the I/R injury as evidenced by (a) loss of cardiac mechanical work, (b) oxidant stress (increase in lipid peroxidation and decrease in reduced glutathione content) and (c) decrease in the activity of the antioxidant enzymes superoxide dismutase and glutathione reductase in both cardiac tissue and isolated mitochondria, and (d) decrease in mitochondrial respiratory capacity. In conclusion, the protective effect of curcumin was associated with the attenuation of oxidant stress and mitochondrial dysfunction secondary to I/R injury.     

Mitochondrial oxygen consumption in asexual and sexual blood stages of the human malarial parasite, Plasmodium falciparum

The two developmental stages of human malarial parasite Plasmodium falciparum, asexual and sexual blood stages, were continuously cultivated in vitro. Both asexual and sexual stages of the parasites were assayed for mitochondrial oxygen consumption by using a polarographic assay. The rate of oxygen consumption by both stages was found to be relatively low, and was not much different. Furthermore, the mitochondrial oxygen consumption by both stages was inhibited to various degrees by mammalian mitochondrial inhibitors that targeted each component of complexes I- IV of the respiratory system. The oxygen consumption by both stages was also affected by 5-fluoroorotate, a known inhibitor of enzyme dihydroorotate dehydrogenase of the pyrimidine pathway and by an antimalarial drug atovaquone that acted specifically on mitochondrial complex III of the parasite. Moreover, antimalarials primaquine and artemisinin had inhibitory effects on the oxygen consumption by both stages of the parasites. Our results suggest that P. falciparum in both developmental stages have functional mitochondria that operate a classical electron transport system, containing complexes I-IV, and linked to the pyrimidine biosynthetic pathway.