Chromium(VI) interaction with plant and animal mitochondrial bioenergetics: a comparative study

The mechanism of Cr(VI)-induced toxicity in plants and animals has been assessed for mitochondrial bioenergetics and membrane damage in turnip root and rat liver mitochondria. By using succinate as the respiratory substrate, ADP/O and respiratory control ratio (RCR) were depressed as a function of Cr(VI) concentration. State 3 and uncoupled respiration were also depressed by Cr(VI). Rat mitochondria revealed a higher sensitivity to Cr(VI), as compared to turnip mitochondria. Rat mitochondrial state 4 respiration rate triplicated in contrast to negligible stimulation of turnip state 4 respiration. Chromium(VI) inhibited the activity of the NADH-ubiquinone oxidoreductase (complex I) from rat liver mitochondria and succinate-dehydrogenases (complex II) from plant and animal mitochondria. In rat liver mitochondria, complex I was more sensitive to Cr(VI) than complex II. The activity of cytochrome c oxidase (complex IV) was not sensitive to Cr(VI). Unique for plant mitochondria, exogenous NADH uncoupled respiration was unaffected by Cr(VI), indicating that the NADH dehydrogenase of the outer leaflet of the plant inner membrane, in addition to complexes III and IV, were insensitive to Cr(VI). The ATPase activity (complex V) was stimulated in rat liver mitochondria, but inhibited in turnip root mitochondria. In both, turnip and rat mitochondria, Cr(VI) depressed mitochondrial succinate-dependent transmembrane potential (Deltapsi) and phosphorylation efficiency, but it neither affected mitochondrial membrane permeabilization to protons (H+) nor induced membrane lipid peroxidation. However, Cr(VI) induced mitochondrial membrane permeabilization to K+, an effect that was more pronounced in turnip root than in rat liver mitochondria. In conclusion, Cr(VI)-induced perturbations of mitochondrial bioenergetics compromises energy-dependent biochemical processes and, therefore, may contribute to the basal mechanism underlying its toxic effects in plant and animal cells.     

Mode of Action of the Antibiotic Siccanin on Intact Cells and Mitochondria of Trichophyton mentagrophytes

Siccanin at 3 mug/ml completely inhibited the growth of Trichophyton mentagrophytes. The primary site of action of siccanin on T. mentagrophytes is succinate dehydrogenase in the terminal electron transport system. At a concentration of siccanin giving 50% inhibition of growth (0.3 mug/ml), respiration of intact cells was inhibited more strongly than any other cellular functions tested, including the syntheses of cellular ribonucleic acid, deoxyribonucleic acid, phospholipid, protein, and cell wall fractions. In addition, at the same concentration siccanin did not cause any detectable damage in the permeability of the cells. Furthermore, the oxidation of succinate in mitochondrial preparation is more sensitive to the antibiotic than respiration in intact cells. Oxidation of other substrates tested was less sensitive to siccanin than that of succinate. The antibiotic inhibited both phosphorylation and oxidation, without causing changes in the P:O ratio. Siccanin at 0.03 mug/ml, which caused 50% inhibition of succinate oxidation in mitochondria, had effect neither on the exchange reaction between inorganic phosphate (P(i)) and adenosine triphosphate (ATP) nor on that between adenosine diphosphate and ATP. An ATP phosphohydrolase activity was also insensitive to the antibiotic. At very high concentrations, however, the antibiotic slightly inhibited the P(i)-ATP exchange reaction. From those results, it was concluded that siccanin inhibits fungal growth by inhibiting the respiratory electron transport system.     

The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1. II. Cyanide-insensitive respiration. Function and regulation.

1. The cyanide-insensitive respiration in Paramecium tetraurelia was found to be located in mitochondria. 2. Sensitivity of the mitochondrial respiration to cyanide depended on growth conditions. Under standard conditions of growth, 15--20% of respiration was insensitive to 1 mM cyanide. Full resistance to 1 mM cyanide was observed by growing cells in the presence of erythromycin (100--400 microgram/ml) 0.2 mM cyanide. The mitochondrial respiration of the mutant Cl1 harvested during the exponential phase of growth was largely insensitive to cyanide (more than 80%). 3. Pyruvate was oxidized at the same rate by wild type mitochondria and mitochondria of the mutant Cl1. In contrast, succinate oxidation was 2--3 times faster in mitochondria of the mutant Cl1 than in wild type mitochondria. 4. The cyanide-insensitive respiration was inhibited by 1 mM salicylhydroxamic acid to nearly 100%. Other efficient respiratory inhibitors included amytal and heptylhydroxyquinoline. Antimycin was not inhibitory even at concentrations as high as 5 microgram/mg protein, a finding consistent with the lack of antimycin binding sites.     

The effect of heating on the respiration of MC7 and D23 tumour tissue

The sensitivity of the tissue respiration of two tumours and liver slices to heat has been studied. The tumour tissue is sensitive to in vitro preincubation at temperatures above 43°C, whereas liver slices were less temperature sensitive. The inclusion of 1 mM tetracaine during preincubation sensitizes the tumour tissue to heating. In vivo heating of the tumour tissue at 44°C for 1h was not inhibitory of respiration when subsequently measured at 37°C. Mitochondria isolated from the D23 hepatoma tissue showed coupled respiration, however mitochondria isolated from in vivo heated tumour did not show coupled respiration. It contrast to mitochondria isolated from unheated tissue, these mitochondria lacked cristae and contained electron-dense granules, indicators of damage. The lack of effect of an in vivo heat-dose, known to cause tumour regression, on respiration and the reports that ATP levels are unaffected by such heating, suggests that cell respiration is not a primary lesion in cellular heat injury. This implies that the observed impairment of mitochondrial function following in vivo heating is best explained if the heating sensitized the mitochondria to subsequent damage during isolation.     

Assessment of ram sperm mitochondrial function by quantitative determination of sperm rhodamine 123 accumulation

A simple procedure is described for determining the functional state of ram sperm mitochondria by quantitative measurement of sperm rhodamine 123 (R 123) accumulation. Sperm were incubated with 1 μg/ml R 123, and the accumulated R 123 was measured fluorimetrically after release from washed sperm by detergent lysis. Ram sperm R 123 uptake was maximal after 30 min of incubation and responded to changes in both sperm (P < 0.01) and R 123 (P < 0.01) concentration. There was a linear relationship (r = 0.98) between R 123 uptake and the proportion of cold-shocked sperm present in a sperm sample. R 123 uptake was unaffected by 20 mM 2-deoxyglucose or by 10 mM malonate (the latter being sufficient to reduce O₂ uptake; P < 0.01). R 123 accumulation in ram sperm was reduced by 6 mg/ml sodium pentobarbitone (P 0.05), by 1 μM 2,4-dinitrophenol (P < 0.01), and by 0.05% Triton X-100 (P < 0.01). It is concluded that quantitative estimation of R 123 uptake complements oxygen uptake in detecting mitochondrial dysfunction in ram sperm. While it is largely unaffected by inhibition of glycolysis, and is less sensitive than oxygen uptake to trichloroacetic acid cycle inhibition, R 123 uptake is sensitive to factors directly reducing the mitochondrial membrane potential of ram sperm. It may therefore be useful in the evaluation of the effects of such membrane-mediated injuries as cold shock and freezing damage on ram sperm mitochondria. © 1993 Wiley-Liss, Inc.     

Mechanism of toxicity of precocene II in rat hepatocyte cultures

Precocene II was more toxic in 24 hour cultures than in 72 hour cultures of rat hepatocytes. In 24 hour cultures, there was no observable toxicity at 75 μM precocene II after exposure for 6 hours, but after 24 hours, 65% of the cells were dead. In contrast, although 794 μM killed 50% of the cells in the 72 hour cultures after a 24 hour exposure, 1 mM killed 96% of the cells within 6 hours. In both 24 and 72 hour cultures, cell death was preceded by a rapid, early loss of mitochondrial membrane potential, followed by decreases in glutathione, reduced pyridine nucleotide status, and plasma membrane Na⁺/K⁺-ATPase activity. There was also a rapid loss of ATP in the 72 hour cultures but not in the 24 hour cultures; therefore, onset of cell death may be closely linked to loss of ATP. Inhibition of cytochrome P-450 prevented the toxicity, and partially protected against the loss of membrane potential and glutathione, in 24 hour cultures but was ineffective in 72 hour cultures. Therefore, in addition to depletion of glutathione, precocene II appears to damage mitochondria and plasma membrane functions and can do so by more than one pathway. © 1996 John Wiley & Sons, Inc.     

Potential Use of Surface-active Agents for Controlling Mycoplasma Contamination in Animal Cell Cultures

Seven nonionic detergents, which were determined to be relatively nontoxic to selected animal cell cultures, were tested for their lethal effect on the GDL strain of Mycoplasma hyorhinis. Of the seven detergents tested, five were found to cause complete lysis of the organism in vitro within 24 hr at 37 C. These detergents included Triton WR-1339 and Tweens 20, 40, 60, and 80. When different concentrations of the detergents were tested, Tween 80 was found to be the most effective and Triton WR-1339 the least effective in lysing the mycoplasmata. These same five detergents were used to treat a rat nephroma cell line which was chronically infected with the GDL strain. The mycoplasmata were eliminated from those cultures treated with Triton but they persisted in cultures exposed to the Tween compounds. The Triton-treated cells remained free from infection over a 7-month period, as determined both by cultural methods and fluorescent-antibody staining. The "cure" was effected by treating the cells for either 48 hr with maintenance media containing 1 mg of Triton per ml or for 96 hr with a concentration of 500 mug/ml. Triton was also effective in eliminating the GDL, strain from experimentally infected rat embryo cells after a 48-hr treatment with a concentration of 1 mg/ml. Four other species of Mycoplasma, which were completely lysed by Triton in vitro, were not eliminated from experimentally infected cells by a single treatment with Triton, although the severity of the infection was apparently reduced.     

Differential effects of osmotic pressure on mitochondrial respiratory chain and indices of oxidative phosphorylation

Oxidative phosphorylation was critically evaluated in terms of activities which are sensitive and insensitive to variations in external osmotic pressure in mitochondria. Integrity of mitochondria was determined in terms of a variety of parameters, including the latency of the occluded enzymes, by careful titrations as a function of external osmotic pressure as well as detergent concentrations. The evidence indicated that the rate-limiting step in respiratory states 2 and 4 would be osmotically insensitive, as opposed to the osmotically sensitive respiration of states 1 and 3 and uncoupler-stimulated respiration with glutamate + malate and succinate. Cytochrome oxidase activity in mitochondria as well as in purified reconstituted systems exhibited osmotic insensitivity but marked sensitivity to ionic strength, offering an interesting model to study the osmotically insensitive respiration. Cytochrome oxidase activity led to permeation of mannitol across the mitochondrial inner membrane. Stimulation of cytochrome oxidase activity by uncouplers did not require an intact membrane.     

Illumination with blue light reactivates respiratory activity of mitochondria inhibited by nitric oxide, but not by glycerol trinitrate

Nitric oxide (NO) is known to inhibit mitochondrial respiration reversibly. This study aimed at clarifying whether low level illumination at specific wavelengths recovers mitochondrial respiration inhibited by NO and glycerol-trinitrate (GTN), a clinically used NO mimetic. NO fully inhibited respiration of liver mitochondria at concentrations occurring under septic shock. The respiration was completely restored by illumination at the wavelength of 430 nm while longer wavelengths were less effective. GTN inhibited mitochondrial respiration though the efficiency of GTN was lower compared to NO concentrations observed in sepsis models. However, GTN inhibition was absolutely insensitive to illumination regardless of wavelength used. Our data show that visible light of short wavelengths efficiently facilitates the recovery of mitochondria inhibited by NO-gas at the levels generated under septic conditions. The inhibition of mitochondrial respiration by GTN is not sensitive to visible light, suggesting an inhibition mechanism other that NO mediation.     

Cellular Damage to Phytophthora infestans in Tomato Leaves Treated with Oxadixyl: an Ultrastructural Investigation

Ultrastructural observations showed massive accumulation of electron dense deposits adjacent to plasmalemma and mitochondria and mitochondrial degeneration, which probably leads to cell death, in some hyphae and haustoria of Phytophthora infestans in infected tomato leaves treated with the systemic fungicide oxadixyl (8 μg/ml). However, no changes in the endoplasmic reticulum and number of ribosomes were observed. The cell walls of some hyphae were markedly thickened after oxadixyl treatment. Degenerated haustoria were partly present in intact host cells. Evidently, fungal cell damage by treatment with oxadixyl is not correlated with host cell damage.     

Mitochondrial alterations related to programmed cell death in tobacco cells under aluminium stress

The present investigation was undertaken to verify whether mitochondria play a significant role in aluminium (Al) toxicity, using the mitochondria isolated from tobacco cells (Nicotiana tabacum, non-chlorophyllic cell line SL) under Al stress. An inhibition of respiration was observed in terms of state-III, state-IV, succinate-dependent, alternative oxidase (AOX)-pathway capacity and cytochrome (CYT)-pathway capacity, respectively, in the mitochondria isolated from tobacco cells subjected to Al stress for 18 h. In accordance with the respiratory inhibition, the mitochondrial ATP content showed a significant decrease under Al treatment. An enhancement of reactive oxygen species (ROS) production under state-III respiration was observed in the mitochondria isolated from Al-treated cells, which would create an oxidative stress situation. The opening of mitochondrial permeability transition pore (MPTP) was seen more extensively in mitochondria isolated from Al-treated cells than in those isolated from control cells. This was Ca(2+) dependent and well modulated by dithioerythritol (DTE) and Pi, but insensitive to cyclosporine A (CsA). The collapse of inner mitochondrial membrane potential (DeltaPsi(m)) was also observed with a release of cytochrome c from mitochondria. A great decrease in the ATP content was also seen under Al stress. Transmission electron microscopy analysis of Al-treated cells also corroborated our biochemical data with distortion in membrane architecture in mitochondria. TUNEL-positive nuclei in Al-treated cells strongly indicated the occurrence of nuclear fragmentation. From the above study, it was concluded that Al toxicity affects severely the mitochondrial respiratory functions and alters the redox status studied in vitro and also the internal structure, which seems to cause finally cell death in tobacco cells.     

The stimulation of hepatic oxidative phosphorylation following dexamethasone treatment of rats

The effect of short-term treatment of rats with the synthetic glucocorticoid, dexamethasone, on mitochondrial oxidative phosphorylation has been examined. Treatment of rats for 3 h increased the oxidative capacity of the subsequently isolated mitochondria such that they displayed increased uncoupled and State 3 rates of respiration with NAD-linked substrates, succinate or durohydroquinone. The oxidation of ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine was unaffected. No change was apparent in the activity of a variety of dehydrogenase enzymes nor was there any increase in the mitochondrial content of cytochromes a, b, c1 or c. The uncoupler-dependent ATPase activity of the mitochondria was slightly enhanced following hormone treatment, but not the basal or the total ATPase activity measured in the presence of Triton X-100 plus Mg2+. The mitochondria prepared from dexamethasone-treated rats also displayed increased intramitochondrial concentrations of Mg2+, K+ and exchangeable adenine nucleotides but not Ca2+. It is suggested that the effect of glucocorticoids on mitochondrial respiration may be both the result of a direct activation of the respiratory chain within Complex III and an elevated intramitochondrial adenine nucleotide concentration. The evidence for the de novo synthesis of mitochondrial proteins which mediate the response remains inconclusive.     

Loss of Sensitivity to Helminthosporium maydis Race T Toxin during Aging of Mitochondria Isolated from Texas Cytoplasm Corn

Helminthosporium maydis Race T toxin caused the expected changes in freshly isolated mitochondria from T cytoplasm corn, namely complete uncoupling of oxidative phosphorylation, pronounced stimulation of succinate and NADH respiration, complete inhibition of malate respiration, and increased mitochondrial swelling. In contrast, identical toxin treatments of the mitochondria after 12 hours aging on ice resulted in partial uncoupling, much lower stimulation of succinate and NADH respiration, no inhibition of malate respiration, and no mitochondrial swelling. Almost all of the toxin sensitivity was lost by 6 hours aging. At this stage, the mitochondria were 208× and 66× less sensitive to toxin-induced changes in coupling of malate respiration and state 4 malate respiration rates, respectively. Loss of toxin sensitivity did not occur when the mitochondria were aged under nitrogen or in the presence of 5 millimolar dithiothreitol. This suggested that the aging effect was due to oxidation, possibly of sulfhydryl groups in one or more mitochondrial membrane proteins.     

Acetaminophen toxicity results in site-specific mitochondrial damage in isolated mouse hepatocytes

Exposure of isolated mouse hepatocytes to a toxic concentration of acetaminophen (5 mM) resulted in damage to the mitochondrial respiratory apparatus. The nature of this damage was investigated by measuring respiration stimulated by site-specific substrates in digitonin-permeabilized hepatocytes after acetaminophen exposure. Respiration stimulated by succinate at energy-coupling site 2 was most sensitive to inhibition and was decreased by 47% after 1 h. Respiration supported by NADH-linked substrates (site 1) was also decreased but to a lesser extent, while there was no decrease in the rate of ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD)-supported respiration (site 3). The loss of mitochondrial respiratory function was accompanied by a decrease in ATP levels and ATP/ADP ratios in the cytosolic compartment and was preceded by a loss of reduced glutathione in both the cytosol and mitochondria. All these effects occurred well before the loss of cell membrane integrity. The putative toxic metabolite of acetaminophen, N-acetyl-p-benzoquinonimine (NAPQI), produced a similar pattern of respiratory dysfunction in isolated hepatic mitochondria. Respiration stimulated by succinate- and NADH-linked substrates was very sensitive to 50 microM NAPQI, while ascorbate + TMPD-supported respiration was unaffected. The interaction between NAPQI and the respiratory chain was further investigated using submitochondrial particles. Succinate dehydrogenase (associated with respiratory complex II) was found to be very sensitive to NAPQI, while NADH dehydrogenase (respiratory complex I) was inhibited to a lesser extent. Our results indicate that a loss of the ability to utilize succinate- and NADH-linked substrates due to attack of the respiratory chain by NAPQI causes a disruption of energy homeostasis in acetaminophen hepatotoxicity.     

Effect of melatonin treatment on oxygen consumption by rat liver mitochondria

Summary. The objective of this study was to examine the in vivo effect of melatonin on rat mitochondrial liver respiration. Two experiments were performed: For experiment 1, adult male rats received melatonin in the drinking water (16 or 50 μg/ml) or vehicle during 45 days. For experiment 2, rats received melatonin in the drinking water (50 μg/ml) for 45 days, or the same amount for 30 days followed by a 15 day-withdrawal period. At sacrifice, a liver mitochondrial fraction was prepared and oxygen consumption was measured polarographically in the presence of excess concentration of DL-3-β-hydroxybutyrate or L-succinate. Melatonin treatment decreased Krebs’ cycle substrate-induced respiration significantly at both examined doses. The stimulation of mitochondrial respiration caused by excess concentration of substrate recovered after melatonin withdrawal. Basal state 4 respiration was not modified by melatonin. Melatonin, by curtailing overstimulation of cellular respiration caused by excess Krebs’ cycle substrates, can protect the mitochondria from oxidative damage.     

Regulation of mitochondrial membrane assembly in Neurospora crassa. Transient expression of a respiratory mutant phenotype.

Cultures of mutant cni-1, a chromosomal mutant of Neurospora crassa, undergo a marked change in respiratory properties as the age of the culture increases. Early log phase cultures have a high level of respiration that is insensitive to inhibition by cyanide or antimycin A. Late log and stationary phase cultures have reduced rates of respiration. A high percentage of this respiration is inhibited by cyanide. Mitochondria from early log phase cni-1 have an excess of cytochrome c and little or no detectable cytochrome aa3. Mitochondria from late log and stationary phase cultures have levels of c-, b-, and a-type cytochromes that are not significantly different in concentration from those found in wild type cells. The cytochrome aa3 content and the cytochrome oxidase activity of cni-1 mitochondria increase 5- to 10-fold as the age of the culture increases. Mitochondria from early log phase cells of cni-1 synthesize only polypeptides of apparent molecular weights 7,000 to 10,000 and donot synthesize any of the mitochondrial components of cytochrome oxidase. Mitochondria from late log and stationary phase cells synthesize the normal complement of mitochondrial translation products including the mitochondrial components of cytochrome oxidase. The assembly of cytochrome oxidase is likely due to the availability of the mitochondrially synthesized components of the enzyme. The regulation of mitochondrial translation in the cni-1 mutant is independent of the nutrient content of the growth medium and is due to the accumulation or depletion of some component within the cell.     

Nitric oxide donors, nitrosothiols and mitochondrial respiration inhibitors induce caspase activation by different mechanisms

We investigated to what extent different types of NO donors induce caspase activation by opening of the mitochondrial permeability transition pore (PTP) or inhibition of mitochondrial respiration. We found that nitrosothiols can directly open the PTP in isolated mitochondria and cause cytochrome c release, whereas NONOate donors can not. In macrophages nitrosothiols cause caspase activation that is blocked by cyclosporin A or calcium chelation, both of which prevent PTP opening, whereas caspase activation caused by NONOates is much less sensitive to these agents. Inhibitors of mitochondrial respiration did not promote PTP opening in isolated mitochondria, and although they cause caspase activation in macrophages, this activation was slower than that caused by NO donors, and was relatively insensitive to cyclosporin and calcium chelators suggesting that PTP opening was not involved.     

Respiration and mitochondrial ATPase in energized mitochondria during isoproterenol-induced cell injury of myocardium.

1. Respiration of mitochondria, membrane potential and mitochondrial ATPase under energized conditions were studied in rat myocardium during cell injury induced by treatment with isoproterenol. 2. Increase in the state 4 rate of respiration and ADP:O ratio, as well as decrease in the state 3 rate and Respiratory Control Ratio (RCR) were found. 3. The optimum pH for RCR and for maximum ATPase activity was shifted to lower values. 4. The state 3 respiration was more sensitive to oligomycin inhibition. 5. The mitochondria showed lower ability to generate membrane potential. 6. An increase in the K0.5 values for catalytic sites II and III of mitochondrial ATPase at pH 7.4 and 5.5 was found. 7. These results are consistent with alterations on the integrity of mitochondrial membrane, and corroborate with the hypothesis of changes on the mitochondrial ATPase during isoproterenol-induced cell injury of myocardium.     

一种用于研究骨骼肌缺血/再灌注损伤的细胞模型

目的:复制L-6TG大鼠肌母细胞缺血/再灌注损伤的细胞模型.方法:将培养的L-6TG大鼠肌母细胞随机分为2组:①正常对照组(C组),②缺血/再灌注组(I/R组),观测了培养上清中乳酸脱氢酶(LDH)、细胞内超氧化物歧化酶(SOD)、黄嘌呤氧化酶(XOD)、Ca2 含量的变化;采用MTT法检测线粒体的功能;在光镜下观察细胞的形态学改变.结果:与对照组相比,L-6TG大鼠肌母细胞IR 4h后培养上清中LDH、细胞内XOD、Ca2 含量明显增加,细胞内SOD及线粒体呼吸功能明显降低,细胞严重受损,明显圆缩,并有脱落现象.结论:应用模拟缺血液和再灌液可成功复制L-6TG大鼠肌母细胞缺血/再灌注损伤的细胞模型.     

In situ respiration and bioenergetic status of mitochondria in primary cerebellar granule neuronal cultures exposed continuously to glutamate

Mitochondria play a central role in neuronal death during pathological exposure to glutamate (excitotoxicity). To investigate the detailed bioenergetics of the in situ mitochondria, a method is described to monitor continuously the respiration of primary cerebellar granule neuron cultures while simultaneously imaging cytoplasmic Ca(2+) and mitochondrial membrane potential. Coverslip-attached cells were perfused in an imaging chamber with upstream and downstream flow-through oxygen electrodes. The bioenergetic consequences of chronic glutamate exposure were investigated, including ATP supply and demand, proton leak, and mitochondrial respiratory capacity during chronic glutamate exposure. In 25 mM K(+) medium supplemented with 10% dialyzed serum, cells utilized 54% of their respiratory capacity in the absence of receptor activation (37% for ATP generation, 12% to drive the mitochondrial proton leak, and the residual 5% was nonmitochondrial). glutamate initially increased mitochondrial respiration from 51 to 68% of capacity, followed by a slow decline. It was estimated that 85% of this increased respiration was because of increased ATP demand, whereas 15% was attributable to a transient mitochondrial proton leak. N-Methyl-D-aspartate receptor activation was only responsible for 62% of the increased respiration. When adjusted for cell death over 3 h of glutamate exposure, respiration of the viable cells remained near basal and protonophore stimulated respiration to the same extent as control cells. Pyruvate-supplemented media protected cells from glutamate excitotoxicity, although this was associated with mitochondrial dysfunction. We conclude that excitotoxicity under these conditions is not because of an ATP deficit or uncoupling. Furthermore, mitochondria maintain the same respiratory capacity as in control cells.