Expression of Classical Mitochondrial Respiratory Responses in Homogenates of Rat Forebrain

Respiratory studies of brain mitochondria have, in general, been limited to purified preparations. Conventional procedures for mitochondrial isolation yield relatively small and potentially selected subfractions of mitochondria. Examination of respiratory responses of homogenates of rat forebrain indicated that key respiratory properties of mitochondria are fully expressed in these preparations. In a high K+ buffer, comparable to those commonly used for purified mitochondria, forebrain homogenates exhibited many of the characteristics of oxygen uptake by "free" mitochondria: requirement for both pyruvate and malate for maximal respiration, stimulation (over threefold) by ADP, stimulation by uncoupling agent [carbonyl cyanide m-chlorophenylhydrazone (CCCP)], but little effect of digitonin. In a modified Krebs-Ringer phosphate buffer (a physiological buffer), respiratory responses were primarily due to mitochondria enclosed in synaptosomes: respiration with glucose was markedly stimulated by CCCP, further stimulated by pyruvate, and extensively inhibited by digitonin (which disrupts the cholesterol-rich synaptosomal membranes). Studies with purified mitochondria and synaptosomes supported the specificity of these responses. These data indicate that classical mitochondrial responses are expressed in whole brain homogenates and, under appropriate conditions, provide functional measures of the total pools of free and synaptosomal mitochondria.     

Altered Mitochondrial Respiration in Selectively Vulnerable Brain Subregions Following Transient Forebrain Ischemia in the Rat

Mitochondrial respiratory function, assessed from the rate of oxygen uptake by homogenates of rat brain subregions, was examined after 30 min of forebrain ischemia and at recirculation periods of up to 48 h. Ischemia-sensitive regions which develop extensive neuronal loss during the recirculation period (dorsal-lateral striatum, CA1 hippocampus) were compared with ischemia-resistant areas (paramedian neocortex, CA3 plus CA4 hippocampus). All areas showed reductions (to 53-69% of control) during ischemia for oxygen uptake rates determined in the presence of ADP or an uncoupling agent, which then recovered within 1 h of cerebral recirculation. In the ischemia-resistant regions, oxygen uptake rates remained similar to control values for at least 48 h of recirculation. After 3 h of recirculation, a significant decrease in respiratory activity (measured in the presence of ADP or uncoupling agent) was observed in the dorsal-lateral striatum which progressed to reductions of greater than 65% of the initial activity by 24 h. In the CA1 hippocampus, oxygen uptake rates were unchanged for 24 h, but were significantly reduced (by 30% in the presence of uncoupling agent) at 48 h. These alterations parallel the development of histological evidence of ischemic cell change determined previously and apparently precede the appearance of differential changes between sensitive and resistant regions in the content of high-energy phosphate compounds. These results suggest that alterations of mitochondrial activity are a relatively early change in the development of ischemic cell death and provide a sensitive biochemical marker for this process.     

Selective Impairment of Respiration in Mitochondria Isolated from Brain Subregions Following Transient Forebrain Ischemia in the Rat

Using Percoll density gradient centrifugation, free (nonsynaptosomal) mitochondria were isolated from the dorsal-lateral striatum and paramedian neocortex of rats during complete forebrain ischemia and reperfusion. Mitochondria prepared from either region after 30 min of ischemia showed decreased state 3 (ADP and substrate present) and uncoupled respiration rates (19-45% reductions) with pyruvate plus malate as substrates, whereas state 4 respiration (no ADP present) was preserved. At 6 h of recirculation, state 3 and uncoupled respiration rates for mitochondria from the paramedian neocortex (a region resistant to ischemic damage) were similar to or even increased compared with control values. By contrast, in mitochondria from the dorsal-lateral striatum (a region containing neurons susceptible to global ischemia), decreases in state 3 and uncoupled respiration rates (25 and 30% less than control values) were again observed after 6 h of recirculation. With succinate as respiratory substrate, however, no significant differences from control values were found in either region at this time point. By 24 h of recirculation, respiratory activity with either pyruvate plus malate or succinate was greatly reduced in samples from the dorsal-lateral striatum, probably reflecting complete loss of function in some organelles. In contrast with these marked changes in free mitochondria, the respiratory properties of synaptosomal mitochondria, assessed from measurements in unfractionated homogenates, were unchanged from controls in the dorsal-lateral striatum at each of the time points studied, but showed reductions (19-22%) during ischemia and after 24 h of recirculation in the paramedian neocortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subsynaptosomal Calcium Distribution During Hypoxia and 3,4-Diaminopyridine Treatment

Previous results demonstrate that hypoxia (low oxygen) diminishes calcium uptake by synaptosomes. The present studies examined the effects of low oxygen on calcium homeostasis in the digitonin-resistant (mitochondrial) and the digitonin-labile (nonmitochondrial) compartments of intact synaptosomes and their relation to altered membrane potentials. A 10-min hypoxic incubation in low-potassium media reduced total (-38.3%), mitochondrial (-43.3%), and nonmitochondrial (-27.8%) calcium uptake. In high-potassium media, low oxygen reduced mitochondrial (-41.2%) and total (-34.4%) uptake whereas nonmitochondrial (+ 6%) calcium uptake was essentially unaffected. A temporal analysis of nonmitochondrial calcium uptake revealed an initial depression (0-5 min) followed by a stimulation (5-10 min). Hypoxic-induced alterations in the subsynaptosomal distribution of calcium resembled those produced by uncouplers [FCCP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone) or rotenone plus oligomycin]. 3,4-Diaminopyridine partially ameliorated the hypoxic- and FCCP-induced decreases in synaptosomal calcium uptake. Low oxygen reduced the total synaptosomal membrane potential (i.e., plasma plus mitochondrial membrane potential) as measured by an increased efflux of tetraphenylphosphonium ion. This hypoxic-induced efflux of tetraphenylphosphonium was slowed by pretreatment with 3,4-diaminopyridine. Thus, both drug and membrane potential studies suggest that hypoxic-induced alterations in the subcellular distribution of calcium may be due to an uncoupling mechanism and a collapse of the synaptosomal mitochondrial membrane potential.     

The Effect of Acute Hypoxia on Synaptosomes from Rat Brain

Abstract: Synaptosomes have been isolated from the brains of nonanesthetized and nembutal-anesthetized rats subjected to 30 min hypoxia induced by breathing 7% oxygen in nitrogen. The respiratory rate was depressed in synaptosomes from starved hypoxic animals but was not significantly different from the respective control values in preparations from fed hypoxic animals, anesthetized animals, and hypoxic nonanesthetized animals allowed to recover from the hypoxic episode by 60 min of normoxic conditions. Observations are also reported concerning the levels of various metabolites in the synaptosomes isolated from the brains of the same groups of animals. It is suggested that hypoxia results in damage to the synaptosomal and/or mitochondrial membrane, which modifies substrate oxidation in the mitochondria and decreases availability of reducing equivalents for the respiratory chain. Results obtained on afflicted and recovered animals indicate that synaptosomal preparations provide a useful model for the study of hypoxic damage.     

Rapid Isolation of Metabolically Active Mitochondria from Rat Brain and Subregions Using Percoll Density Gradient Centrifugation

Two procedures are described for isolating free (nonsynaptosomal) mitochondria from rat brain. Both procedures employ a discontinuous Percoll gradient and yield well coupled mitochondria which exhibit high rates of respiratory activity and contain little residual contamination by synaptosomes or myelin. The procedures are considerably more rapid than methods described previously for the isolation of brain mitochondria and do not require an ultracentrifuge or swing-out rotor. The first method separates mitochondria by gradient centrifugation from a P2 (crude mitochondrial) fraction and is likely to be widely applicable for studies in which at least 500 mg of tissue are available as starting material. In the second method, the unfractionated homogenate is subjected directly to gradient centrifugation. This method requires the preparation of more gradients (per gram of tissue) than the first method and yields a subcellular fraction with slightly more synaptosomal contamination. However, this second procedure is more rapid, requires less manipulation of the tissue, and is suitable for obtaining mitochondria with well preserved metabolic characteristics from subregions of single rat brains.     

Energy transduction in intact synaptosomes. Influence of plasma-membrane depolarization on the respiration and membrane potential of internal mitochondria determined in situ.

A method is described, based on the differential accumulation of Rb+ and methyltriphenylphosphonium, for the simultaneous estimation of the membrane potentials across the plasma membrane of isolated nerve endings (synaptosomes), and across the inner membrane of mitochondria within the synaptosomal cytoplasm. These determinations, together with measurements of respiratory rates, and ATP and phosphocreatine concentrations, are used to define the bioenergetic behaviour of isolated synaptosomes under a variety of conditions. Under control conditions, in the presence of glucose, the plasma and mitochondrial membrane potentials are respectively 45 and 148mV. Addition of a proton translocator induces a 5-fold increase in respiration, and abolishes the mitochondrial membrane potential. The addition of rotenone to inhibit respiration does not affect the plasma membrane potential, and only lowers the mitochondrial membrane potential to 128mV. Evidence is presented that ATP synthesis by anaerobic glycolysis is sufficient under these conditions to maintain ATP-dependent processes, including the reversal of the mitochondrial ATP synthetase. Addition of oligomycin under non-respiring conditions leads to a complete collapse of the mitochondrial potential. Even under control conditions the plasma membrane (Na+ + K+)-dependent ATPase is responsible for a significant proportion of the synaptosomal ATP turnover. Veratridine greatly increases respiration, and depolarizes the plasma membrane, but only slightly lowers the mitochondrial membrane potential. High K+ and ouabain also lower the plasma membrane potential without decreasing the mitochondrial membrane potential. In non-respiring synaptosomes, anaerobic glycolysis is incapable of maintaining cytosolic ATP during the increased turnover induced by veratridine, and the mitochondrial membrane potential collapses. It is concluded that the internal mitochondria must be considered in any study of synaptosomal transport.     

Analysis of polyadenylated RNA from brain synaptosomes and mitochondria

We have isolated RNA from sheep brain synaptosomes and mitochondria separated by an aqueous two-phase system composed of dextran and poly(ethylene glycol). RNA was fractionated through oligo(dT)-cellulose columns and analyzed by electrophoresis through agarose slab gels containing methylmercuric hydroxide and stained with ethidium bromide. The electrophoretic patterns of the poly(A)-containing RNA fraction from synaptosomes and mitochondria are very similar although some high molecular weight RNA species, clearly visible in the synaptosomal fraction, are scarcely detected in the mitochondrial preparations. The electrophoretic analysis of a cleaner RNA preparation from digitonin-treated free mitochondria (mitoplasts) showed that all the poly (A)-RNA species of the synaptosomal preparation are also present in mitoplast. These results strongly suggest that all the discrete poly(A)-RNA species identified in brain synaptosomes are of mitochondrial origin.     

Comparison of respiration in rat, guinea pig and muskrat heart mitochondria

1. Subsarcolemmal and interfibrillar mitochondria were isolated from the hearts of the diving muskrat and non-diving guinea pig and rat. Respiration rates, respiratory control ratio (RCR) and phosphorous to oxygen (P:O) ratios determined. 2. There was no significant difference in these values among the three species or between mitochondrial populations. 3. Mitochondrial yield as measured by citrate synthase of whole heart homogenates was greatest in the rat, intermediate in the muskrat and lowest in the guinea pig. 4. Muskrat heart mitochondria do not differ from rat and guinea pig heart mitochondria in the ability to use pyruvate as a substrate. 5. Differences in heart mitochondrial function between diving and non-diving rodents were not found and thus do not appear to be adaptations for the hypoxia of diving.     

Dissociation of cytochrome c from the inner mitochondrial membrane during cardiac ischemia

Mitochondria isolated from ischemic cardiac tissue exhibit diminished rates of respiration and ATP synthesis. The present study was undertaken to determine whether cytochrome c release was responsible for ischemia-induced loss in mitochondrial function. Rat hearts were perfused in Langendorff fashion for 60 min (control) or for 30 min followed by 30 min of no flow ischemia. Mitochondria isolated from ischemic hearts in a buffer containing KCl exhibited depressed rates of maximum respiration and a lower cytochrome c content relative to control mitochondria. The addition of cytochrome c restored maximum rates of respiration, indicating that the release of cytochrome c is responsible for observed declines in function. However, mitochondria isolated in a mannitol/sucrose buffer exhibited no ischemia-induced loss in cytochrome c content, indicating that ischemia does not on its own cause the release of cytochrome c. Nevertheless, state 3 respiratory rates remained depressed, and cytochrome c release was enhanced when mitochondria from ischemic relative to perfused tissue were subsequently placed in a high ionic strength buffer, hypotonic solution, or detergent. Thus, events that occur during ischemia favor detachment of cytochrome c from the inner membrane increasing the pool of cytochrome c available for release. These results provide insight into the sequence of events that leads to release of cytochrome c and loss of mitochondrial respiratory activity during cardiac ischemia/reperfusion.     

Hormonal effects on mitochondrial respiration: potential role of endogenous lipolytic activities

Hormonal effects on heart mitochondrial metabolism are investigated by comparing respiratory rates, Ca2+ uptake capacity, and lipolytic activities of mitochondria isolated from control rats to those of mitochondria isolated from thyroparathyroidectomized animals. Two biochemically and morphologically distinct populations of heart mitochondria are prepared--one derived from the region of the cell directly beneath the sarcolemma (subsarcolemmal mitochondria), the other originally between the myofibrils (interfibrillar mitochondria). Subsarcolemmal mitochondria isolated from normal rat cardiac tissue have both lower respiratory rates and Ca2+ uptake capacity than do interfibrillar mitochondria. However, when these mitochondrial populations are isolated from hearts from thyroparathyroidectomized rats, there is a selective increase in the maximal ability of the subsarcolemmal mitochondria to accumulate Ca2+, which is accompanied by a proportionate increase in their maximal respiratory rates. Neither Ca2+ uptake capacity nor respiratory rates are similarly increased in the interfibrillar mitochondria. Cytochrome contents and mitochondrial protein recoveries are not significantly changed in either of these mitochondrial preparations. The relationship between these selective increases in respiratory properties of the subsarcolemmal mitochondria to endogenous lipolytic activities is also investigated. It was previously demonstrated that, in the absence of Ca2+, both the rate and extent of formation of free fatty acids from endogenous phospholipids is greater in subsarcolemmal than interfibrillar mitochondria (J. W. Palmer et al. (1981) Arch. Biochem. Biophys. 211, 674-682). In this study it is shown that lipolysis is also more sustained in the subsarcolemmal mitochondria when Ca2+ is added. In the subsarcolemmal mitochondria isolated from thyroparathyroidectomized rats, however, the rates of release of stearic acid and oleic acid are reduced in both the presence and absence of Ca2+. In the presence of added Ca2+, the rate of release of arachidonic acid is also decreased compared to control subsarcolemmal mitochondria, suggesting that the expressed activity of Ca2+-activated phospholipase A2 is lower in those mitochondria isolated from the thyroparathyroidectomized animals, in which respiratory rates and Ca2+ uptake capacity are increased.     

Synaptosomes from Rat Brain: Morphology, Compartmentation, and Transmembrane pH and Electrical Gradients

Abstract: Morphological studies of synaptosomes isolated from rat brains show that approximately 68% of the synaptosomes in these preparations contain synaptic vesicles (range, 62–72.5%). Approximately 30% of the synaptosomes contain mitochondria, and only less than 20% of the total mitochondria in good preparations are free and not enclosed in synaptic structures. The mitochondrial volume percent calculated on the basis of the measured cytochrome c content is 5% for synaptosomes isolated from anesthetized animals and 11% for synaptosomes isolated from unanesthetized animals. These numbers bracket the value of 8.7% obtained from electron micrographs. The volume percent of intrasynaptic vesicles is 1.5% as calculated from electron micrographs. The pH gradient between the extracellular pH and the mean intracellular pH is -0.45, as measured by equilibrium distributions of methylamine and dimethylamine, and -0.05, as determined by equilibrium distributions of 5,5-dimethyloxazolidine-2,4-dione and trimethylacetic acid. Analysis of these data shows that there cannot be a large pH gradient (alkaline inside) across the mitochondria, nor can the synaptic vesicle compartment be very large (<1.85%). Equilibrium distribution of [³H]triphenylmethyl-phosphonium ion in synaptosomal preparations gives a calculated apparent potential of -85 mV, in agreement with our previous value. Analysis of these data using the measured volumes of mitochondrial and intrasynaptic vesicular compartments (8.7 and 1.5%, respectively) gives a maximum possible trans mitochondrial membrane potential of -59 mV.     

Factors influencing the establishment of the normal values of the respiratory activities of human liver mitochondria.

Some factors that influence the values of respiratory activities of liver mitochondria isolated from surgical biopsy specimens have been studied. By sedimentating of mitochondria at a lower centrifugal force (5,500 g) than usually used for rat liver mitochondria, and washing the mitochondrial pellet twice, the contamination with lysosomes and microsomes was lowered. At 37 degrees C, and in the presence of hexokinase and glucose, the oxygen uptake was greater than at 25 degrees C and in their absence. The respiratory control was good and the respiratory activities were rather stable during the first 3-4 h after isolation. The respiratory activities of mitochondria isolated from patients with duodenal or gastric ulcers, biliary diseases, and subjects with no digestive diseases (all having normal liver) were compared. Differences in oxygen uptake and acceptor control index values with some substrates were noted. The conditions for selection of controls in studies on subcellular fractions of human liver include: absence of any hepatic antecedents; no clinical evidence of liver involvement; no abnormality in routine liver function tests; a histologic aspect free of pathological conditions, and a normal aspect of the tissue during the homogenization and the fractionation procedure (absence of steatosis or fibrosis). These data provide a basis for the standardization of methods in establishing the reference values of mitochondrial activities for the modifications in a variety of diseases.     

Monoamine Oxidase-Catalyzed Metabolism of 3,4-Dihydroxyphenylethylamine in the Dopaminergic Synaptosomes from Rat Corpus Striatum

Abstract: This communication describes conditions under which the monoamine oxidase-catalyzed metabolism of 3,4-dihydroxyphenylethylamine can be assayed in dopaminergic synaptosomes from the rat striatum. In contrast to the activity of the isolated enzyme or that of free mitochondria, the synaptosomal reaction exhibits sigmoidal kinetics with respect to substrate concentration. This is consistent with a kinetic mechanism in which intrasynaptosomal substrate partitions between reaction and saturable storage in synaptic vesicles. The reaction is inhibited at moderately decreased oxygen tension, where catecholamine uptake is unaffected. The specificity of this effect suggests that it reflects limited availability of oxygen as an enzyme substrate.     

The Calmodulin Antagonists, Trifluoperazine and R24571, Depolarize the Mitochondria Within Guinea Pig Cerebral Cortical Synaptosomes

Abstract: The effects of trifluoperazine and l-[bis( p -chlorophenyl)methyl] - 3 - [2,4 - dichloro - 3 - (2,4 - dichloroben-zyloxy)phenethyl]imidazolium chloride (R24571) upon synaptosomal calcium transport, plasma membrane potential, in situ mitrochondrial membrane potential, and ATP levels are investigated in order to assess the suitability of these calmodulin antagonists for investigating calmodulin-dependent processes in the nerve terminal. Both agents appear to act selectively at the mitochondrial membrane, causing extensive depolarization at concentrations in excess of 10 μ M (trifluoperazine) or 0.5 μ M (R24571). The extent of Ca uptake into the synaptosomes is decreased, consistent with the loss of the mitochondrial compartment. There is no inhibition of the efflux of Ca from the synaptosomes. Depolarization-dependent Ca uptake is not prevented by R24571. Synaptosomal ATP levels decrease to an extent consistent with the collapse of the mitochondrial potential. It is concluded that the uncoupling effect of these agents on the in situ mitochondria prevents their being used to investigate the role of calmodulin in intact synaptosomes.     

Mitochondria from human term placenta. I. Isolation and assay conditions for oxidative phosphorylation.

Human term placental mitochondria were resolved by differential centrifugation into three fractions, heavy mitochondria, light mitochondria and a third, less dense fraction. Approximately equal amounts of mitochondrial protein were found in the three fractions. These mitochondrial preparations differed in physical properties. ATPase and "ADPase" content and oxidative capacities. Assay conditions were developed which permitted the polarographic measurement of respiration and coupled phosphorylation carried out by all three mitocondrial preparations despite the variable nucleotide-phosphate phosphatase activities present. With heavy mitochondria, rates of respiration were consistently higher than those previously reported for unfractionated placental mitochondria. Respiratory control ratios were comparable to those of mitochondria from other steroid hormone-producing endocrine tissues and ADP/O ratios approaching the theoretical maxima were obtained. Both lighter placental mitochondrial fractions displayed somewhat lower respiration rates and respiratory control but their primary defect was a selective uncoupling of the third site of energy conservation. Modification of isolation procedures were evaluated in terms of quantitative yield and functional activity of the three fractions.     

Changes in the calcium transport systems of rat brain synaptosomes and their possible role in the pathophysiology of aging

Studies were undertaken on the age-associated peculiarities of the Ca2+ transport systems of the rat brain synaptosomes. It has been found that 45Ca2+ uptake reduced with ageing. The above reduction was not linked with the changes in the permeability of potential-dependent synaptosomal membrane Ca2+ depending upon the membrane potential. The distribution of calcium across the mitochondrial membrane changed with ageing, shifting towards higher extramitochondrial calcium levels in old rats, both in isolated and in synaptosomal mitochondria. While studying calcium efflux from mitochondria, it was found that, at equivalent calcium loads, the calcium efflux rates were slower in old rats as compared to adult animals. As observed, both resting [Ca2+]i and that obtained after K-depolarization drastically increased in old animals. The possible pathogenic mechanisms in neuronal injury, conditioned by this increase, are discussed.     

Metabolic disturbances of synaptosomes isolated from ischemic gerbil brain

The effects of cerebral ischemia, induced for 10 min by bilateral common carotid ligation in the Mongolian gerbil, on the brain and synaptosomal content of phospholipids and free fatty acids were measured. Moreover, the incorporation of arachidonic acid and oleoyl-CoA into phospholipids, as well as the respiration and the accumulation of⁴⁵Ca, norepinephrine, dopamine, choline, glutamate, and -aminobutyrate in the ischemic brain synaptosomal fraction were studied. Analyses of lipids showed a drop in phospholipids content with concomitant increase of lysocompounds and free fatty acids in ischemic cerebral cortex. Disturbances in lipid metabolism including rapid phospholipids hydrolysis and changes in the incorporation of arachidonic acid into inositol and choline phosphoglycerides were also shown in the synaptosomal fraction of ischemic brain. The uptake of neurotransmitter substances, expressed as a percent of control value, was reduced 21% for norepinephrine, 40% for dopamine, 20% for choline, 24% for glutamate and 13% for -aminobutyrate in ischemic synaptosomes. There was no significant effect of ischemia on synaptosomal respiration and⁴⁵Ca uptake in both control and high potassium media. the inhibition of neurotransmitter uptake in ischemic brain synaptosomes may be caused by the disturbance of fatty acid metabolism.     

Free radicals alter maximal diaphragmatic mitochondrial oxygen consumption in endotoxin-induced sepsis

Recent studies indicate that sepsis is associated with enhanced generation of several free radical species (nitric oxide, superoxide, hydrogen peroxide) in skeletal muscle. While studies suggest that free radical generation causes uncoupling of oxidative phosphorylation in sepsis, no previous report has examined the role of free radicals in modulating skeletal muscle oxygen consumption during State 3 respiration or inhibiting the electron transport chain in sepsis. The purpose of the present study was to examine the effects of endotoxin-induced sepsis on State 3 diaphragm mitochondrial oxygen utilization and to determine if inhibitors/scavengers of various free radical species would protect against these effects. We also examined mitochondrial protein electrophoretic patterns to determine if observed endotoxin-related physiological derangements were accompanied by overt alterations in protein composition. Studies were performed on: (a) control animals, (b) endotoxin-treated animals, (c) animals given endotoxin plus PEG-SOD, a superoxide scavenger, (d) animals given endotoxin plus L-NAME, a nitric oxide synthase inhibitor, (e) animals given only PEG-SOD or L-NAME, (f) animals given endotoxin plus D-NAME, and (g) animals given endotoxin plus denatured PEG-SOD. We found: (a) no alteration in maximal State 3 mitochondrial oxygen consumption rate at 24 h after endotoxin administration, but (b) a significant reduction in oxygen consumption rate at 48 h after endotoxin, (c) no effect of endotoxin to induce uncoupling of oxidative phosphorylation, (d) either PEG-SOD or L-NAME (but neither denatured PEG-SOD nor D-NAME) prevented endotoxin-mediated reductions in State 3 respiration rates, (e) some mitochondrial proteins underwent tyrosine nitrosylation at 24 h after endotoxin administration, and (f) SDS-page electrophoresis of mitochondria from endotoxin-treated animals revealed a selective depletion of several proteins at 48 h after endotoxin administration (but not at 24 h); (g) administration of L-NAME or PEG-SOD prevented this protein depletion. These data provide the first evidence that endotoxin-induced reductions in State 3 mitochondrial oxygen consumption are free radical-mediated.     

Lipid peroxidation and alterations to oxidative metabolism in mitochondria isolated from rat heart subjected to ischemia and reperfusion.

Ischemia-reperfusion injury to cardiac myocytes involves membrane damage mediated by oxygen free radicals. Lipid peroxidation is considered a major mechanism of oxygen free radical toxicity in reperfused heart. Mitochondrial respiration is an important source of these reactive oxygen species and hence a potential contributor to reperfusion injury. We have examined the effects of ischemia (30 min) and ischemia followed by reperfusion (15 min) of rat hearts, on the kinetic parameters of cytochrome c oxidase, on the respiratory activities and on the phospholipid composition in isolated mitochondria. Mitochondrial content of malonyldialdheyde (MDA), an index of lipid peroxidation, was also measured. Reperfusion was accompanied by a significant increase in MDA production. Mitochondrial preparations from control, ischemic and reperfused rat heart had equivalent Km values for cytochrome c, although the maximal activity of the oxidase was 25 and 51% less in ischemic and reperfused mitochondria than that of controls. These changes in the cytochrome c oxidase activity were associated to parallel changes in state 3 mitochondrial respiration. The cytochrome aa3 content was practically the same in these three types of mitochondria. Alterations were found in the mitochondrial content of the major phospholipid classes, the most pronounced change occurring in the cardiolipin, the level that decreased by 28 and by 50% as function of ischemia and reperfusion, respectively. The lower cytochrome c oxidase activity in mitochondria from reperfused rat hearts could be almost completely restored to the level of control hearts by exogenously added cardiolipin, but not by other phospholipids nor by peroxidized cardiolipin. It is proposed that the reperfusion-induced decline in the mitochondrial cytochrome c oxidase activity can be ascribed, at least in part, to a loss of cardiolipin content, due to peroxidative attack of its unsaturated fatty acids by oxygen free radicals. These findings may provide an explanation for some of the factors that lead to myocardial reperfusion injury.