Brain-derived respiring mitochondria exhibit homogeneous, complete and cyclosporin-sensitive permeability transition

The mitochondrial permeability transition (mPT) is increasingly implicated in neuronal cell death. In the present study, isolated respiring brain mitochondria were examined for their ability to undergo calcium-induced mPT and their sensitivity to mPT inhibition by cyclosporin A (CsA). Previous studies have suggested a heterogeneous response to calcium, a limitation of CsA inhibition, and a relative resistance in the ability of respiring brain mitochondria to undergo mPT. Using fluorometric and electron microscopic analyses, we found that virtually the whole population of respiring brain mitochondria readily undergo mPT and swell upon calcium exposure. Further, brain mitochondria were highly sensitive to CsA which potentiated morphological recovery after transient swelling as well as completely blocked mPT induction in the presence of a low concentration of ADP. Using flow cytometry, which allows analysis of individual mitochondria, we demonstrate that both brain and liver mitochondria display homogeneous responses to calcium-induced mPT. We conclude that the mPT is one likely target for the broad in vivo neuroprotective effects displayed by CsA when allowed to penetrate the blood-brain barrier, and that development of compounds inhibiting mPT may prove beneficial for the treatment of severe brain disease.     

Lithium desensitizes brain mitochondria to calcium, antagonizes permeability transition, and diminishes cytochrome C release

Among the numerous effects of lithium on intracellular targets, its possible action on mitochondria remains poorly explored. In the experiments with suspension of isolated brain mitochondria, replacement of KCl by LiCl suppressed mitochondrial swelling, depolarization, and a release of cytochrome c induced by a single Ca2+ bolus. Li+ robustly protected individual brain mitochondria loaded with rhodamine 123 against Ca2+-induced depolarization. In the experiments with slow calcium infusion, replacement of KCl by LiCl in the incubation medium increased resilience of synaptic and nonsynaptic brain mitochondria as well as resilience of liver and heart mitochondria to the deleterious effect of Ca2+. In LiCl medium, mitochondria accumulated larger amounts of Ca2+ before they lost the ability to sequester Ca2+. However, lithium appeared to be ineffective if mitochondria were challenged by Sr2+ instead of Ca2+. Cyclosporin A, sanglifehrin A, and Mg2+, inhibitors of the mitochondrial permeability transition (mPT), increased mitochondrial Ca2+ capacity in KCl medium but failed to do so in LiCl medium. This suggests that the mPT might be a common target for Li+ and mPT inhibitors. In addition, lithium protected mitochondria against high Ca2+ in the presence of ATP, where cyclosporin A was reported to be ineffective. SB216763 and SB415286, inhibitors of glycogen synthase kinase-3beta, which is implicated in regulating reactive oxygen species-induced mPT in cardiac mitochondria, did not increase Ca2+ capacity of brain mitochondria. Altogether, these findings suggest that Li+ desensitizes mitochondria to elevated Ca2+ and diminishes cytochrome c release from brain mitochondria by antagonizing the Ca2+-induced mPT.     

Functional and Pharmacological Characteristics of Permeability Transition in Isolated Human Heart Mitochondria

The objective of the present study was to validate the presence and explore the characteristics of mitochondrial permeability transition (mPT) in isolated mitochondria from human heart tissue in order to investigate if previous findings in animal models of cardiac disorders are translatable to human disease. Mitochondria were rapidly isolated from fresh atrial tissue samples obtained from 14 patients undergoing Maze surgery due to atrial fibrillation. Human heart mitochondria exhibited typical mPT characteristics upon calcium overload such as swelling, evaluated by changes in light scattering, inhibition of respiration and loss of respiratory coupling. Swelling was a morphologically reversible event following transient calcium challenge. Calcium retention capacity (CRC), a quantitative measure of mPT sensitivity assayed by following extramitochondrial [Ca²⁺] and changes in respiration during a continuous calcium infusion, was significantly increased by cyclophilin D (CypD) inhibitors. The thiol-reactive oxidant phenylarsine oxide sensitized mitochondria to calcium-induced mPT. Release of the pro-apoptotic intermembrane protein cytochrome c was increased after, but not before, calcium discharge and respiratory inhibition in the CRC assay. From the present study, we conclude that adult viable heart mitochondria have a CypD- and oxidant-regulated mPT. The findings support that inhibition of mPT may be a relevant pharmacological target in human cardiac disease and may underlie the beneficial effect of cyclosporin A in reperfusion injury.     

Calcium-induced generation of reactive oxygen species in brain mitochondria is mediated by permeability transition

Mitochondrial uptake of calcium in excitotoxicity is associated with subsequent increase in reactive oxygen species (ROS) generation and delayed cellular calcium deregulation in ischemic and neurodegenerative insults. The mechanisms linking mitochondrial calcium uptake and ROS production remain unknown but activation of the mitochondrial permeability transition (mPT) may be one such mechanism. In the present study, calcium increased ROS generation in isolated rodent brain and human liver mitochondria undergoing mPT despite an associated loss of membrane potential, NADH and respiration. Unspecific permeabilization of the inner mitochondrial membrane by alamethicin likewise increased ROS independently of calcium, and the ROS increase was further potentiated if NAD(H) was added to the system. Importantly, calcium per se did not induce a ROS increase unless mPT was triggered. Twenty-one cyclosporin A analogs were evaluated for inhibition of calcium-induced ROS and their efficacy clearly paralleled their potency of inhibiting mPT-mediated mitochondrial swelling. We conclude that while intact respiring mitochondria possess powerful antioxidant capability, mPT induces a dysregulated oxidative state with loss of GSH- and NADPH-dependent ROS detoxification. We propose that mPT is a significant cause of pathological ROS generation in excitotoxic cell death.     

Increased Potassium Conductance of Brain Mitochondria Induces Resistance to Permeability Transition by Enhancing Matrix Volume

Modulation of K⁺ conductance of the inner mitochondrial membrane has been proposed to mediate preconditioning in ischemia-reperfusion injury. The mechanism is not entirely understood, but it has been linked to a decreased activation of mitochondrial permeability transition (mPT). In the present study K⁺ channel activity was mimicked by picomolar concentrations of valinomycin. Isolated brain mitochondria were exposed to continuous infusions of calcium. Monitoring of extramitochondrial Ca²⁺ and mitochondrial respiration provided a quantitative assay for mPT sensitivity by determining calcium retention capacity (CRC). Valinomycin and cyclophilin D inhibition separately and additively increased CRC. Comparable degrees of respiratory uncoupling induced by increased K⁺ or H⁺ conductance had opposite effects on mPT sensitivity. Protonophores dose-dependently decreased CRC, demonstrating that so-called mild uncoupling was not beneficial per se. The putative mitoK_ATP channel opener diazoxide did not mimic the effect of valinomycin. An alkaline matrix pH was required for mitochondria to retain calcium, but increased K⁺ conductance did not result in augmented ΔpH. The beneficial effect of valinomycin on CRC was not mediated by H₂O₂-induced protein kinase Cϵ activation. Rather, increased K⁺ conductance reduced H₂O₂ generation during calcium infusion. Lowering the osmolarity of the buffer induced an increase in mitochondrial volume and improved CRC similar to valinomycin without inducing uncoupling or otherwise affecting respiration. We propose that increased potassium conductance in brain mitochondria may cause a direct physiological effect on matrix volume inducing resistance to pathological calcium challenges.     

Mitochondrial Permeability Transition in the Central Nervous System: Induction by Calcium Cycling-Dependent and -Independent Pathways

Abstract: Isolated rat CNS mitochondria and cultured cortical astrocytes were examined for behavior indicative of a mitochondrial permeability transition (mPT). Exposure of isolated CNS mitochondria to elevated calcium or phosphate or both produced loss of absorbance indicative of mitochondrial swelling. The absorbance decreases were prevented by ADP and Mg²⁺ and reduced by cyclosporin A, dithiothreitol, and N -ethylmaleimide. Ruthenium red prevented calcium cycling-induced, but only attenuated phosphate-induced losses of absorbance. In cultured astrocytes permeabilized with digitonin or treated with the calcium ionophore, 4-bromo-A23187, elevations of external calcium altered mitochondrial morphology visualized with the dye, JC-1, from rod-like to rounded, swollen structures. Similar changes were observed in digitonin-permeabilized astrocytes exposed to phosphate. The incidence of calcium-induced changes in astrocyte mitochondria was prevented by Mg²⁺ and pretreatment with dithiothreitol and N -ethylmaleimide, and was reduced by cyclosporin A, ADP, and butacaine alone or in combinations. Ruthenium red and the Na⁺/Ca²⁺ exchange inhibitor CGP 37157 blocked calcium cycling and prevented mitochondrial shape changes in digitonin-treated, but not ionophore-treated astrocytes. Thus, the demonstrated induction conditions and pharmacological profile indicated the existence of an mPT in brain mitochondria. The mPT occurred consequent to activation of calcium cycling-dependent and -independent pathways. Induction of an mPT could contribute to neuronal injury following ischemia and reperfusion.     

Metabolic and functional differences between brain and spinal cord mitochondria underlie different predisposition to pathology

Mitochondrial dysfunctions contribute to neurodegeneration, the locations of which vary among neurodegenerative diseases. To begin to understand what mechanisms may underlie higher vulnerability of the spinal cord motor neurons in amyotrophic lateral sclerosis, compared with brain mitochondria, we studied three major functions of rat brain mitochondria (BM) and spinal cord mitochondria (SCM) mitochondria: oxidative phosphorylation, Ca(2+) sequestration, and production of reactive oxygen species (ROS), using a new metabolic paradigm (Panov et al., J. Biol. Chem. 284: 14448-14456, 2009). We present data that SCM share some unique metabolic properties of the BM. However, SCM also have several distinctions from the BM: 1) With the exception of succinate, SCM show significantly lower rates of respiration with all substrates studied; 2) immunoblotting analysis showed that this may be due to 30-40% lower contents of respiratory enzymes and porin; 3) compared with BM, SCM sequestered 40-50% less Ca(2+), and the total tissue calcium content was 8 times higher in the spinal cord; 4) normalization for mitochondria from 1 g of tissue showed that BM can sequester several times more Ca(2+) than was available in the brain tissue, whereas SCM had the capacity to sequester only 10-20% of the total tissue Ca(2+); and 5) with succinate and succinate-containing substrate mixtures, SCM showed significantly higher state 4 respiration than BM and generated more ROS associated with the reverse electron transport. We conclude that SCM have an intrinsically higher risk of oxidative damage and overload with calcium than BM, and thus spinal cord may be more vulnerable under some pathologic conditions. (250).     

Upregulation of the transport system for TNFalpha at the blood-brain barrier

The transport system for the cytokine tumor necrosis factor-alpha (TNFalpha) at the blood-brain barrier (BBB) enables an enhanced yet saturable entry of TNFalpha from blood to the CNS. This review focuses on the selective upregulation of the transport system for TNFalpha at the BBB that is specific for type of pathology, region, and time. The upregulation is reflected by increased CNS tissue uptake of radiolabeled TNFalpha after iv injection in mice and by inhibition of this increase with excess non-radiolabeled TNFalpha. (1) Spinal cord injury (SCI): upregulation of TNFalpha uptake after thoracic transection is seen in the delayed phase of BBB disruption at the lumbar spinal cord. Thoracic SCI by compression, however, has a longer lasting impact on TNFalpha transport that involves thoracic and lumbar spinal cord, in contrast to the upregulation confined to the lumbar region in lumbar SCI by compression. Regardless, the uptake of TNFalpha by spinal cord does not parallel BBB disruption as measured by the leakage of radiolabeled albumin. (2) Experimental autoimmune encephalomyelitis (EAE): the increase in the differential permeability to TNFalpha is seen in all CNS regions (brain and cervical, thoracic, and lumbar spinal cord) and has a distinct time course and reversibility. Exogenous TNFalpha has biphasic effects in modulating functional scores. The BBB, a dynamically regulated barrier, is actively involved in disease processes.     

Studying the isolated central nervous system; a report on 35 years: more inquisitive than acquisitive

1. The CNS from invertebrate animals such as slugs, snails, leeches, and cockroaches, can be isolated and kept alive for many hours. 2. The electrical and pharmacological properties of invertebrate CNS neurons have many similarities and it is probable that the basic rules governing the CNS evolved more than 600 million years ago. 3. The nerve cells can show sodium action potentials, calcium action potentials, EPSP, IPSP, biphasic potentials, electrogenic sodium pump potentials, and a variety of potassium, sodium, calcium and chloride currents. 4. Invertebrate CNS ganglia contain identifiable individual nerve cells whose properties and responses to neurotransmitters and drugs are constant and repeatable from preparation to preparation. 5. It was possible to set up an isolated CNS-nerve trunk-muscle preparation and study the transport of radioactive material from the CNS to the muscle and from muscle to CNS. This has provided information about axoplasmic transport in both invertebrate and vertebrate preparations. 6. The methods developed from studies of invertebrate isolated CNS preparations have been applied to vertebrate isolated CNS preparations. 7. In addition to thin slices of the mammalian brain, it is possible to keep 5 cm lengths of the whole mammalian spinal cord and brain stem alive for many hours. 8. The isolated mammalian spinal cord has functional ipsilateral and contralateral reflexes, ascending and descending pathways, extensive sensory integrative local area networks, and inhibitory interneuron circuits. Much of the in vivo circuitry is functional in vitro. 9. The isolated mammalian spinal cord and brain stem can be developed to include functional higher brain circuits that will provide increased understanding of the control and integrative action of the mammalian central nervous system.     

Increased CNS uptake and enhanced antinociception of morphine-6-glucuronide in rats after inhibition of P-glycoprotein

Morphine-6-glucuronide (M6G) is a substrate of P-glycoprotein (P-gp), which forms an outward transporter at the blood-brain barrier. Inhibition of P-gp may therefore be expected to cause increased CNS uptake of M6G. We directly assessed the spinal concentrations of M6G and its antinociceptive effects in rats following pharmacological inhibition of P-gp. Spinal cord tissue concentrations of M6G were assessed by microdialysis with probes transversally implanted through the dorsal horns of the spinal cord at level L4. Ten rats received M6G intravenously (0.018 mg/kg loading dose plus 0.00115 mg/kg/min for an 8-h infusion), five of them together with PSC833 to inhibit P-gp (32-h infusion, starting 24 h before the addition of M6G). Antinociceptive effects were explored by means of formalin tests. After having obtained evidence for enhanced CNS uptake and antinociception of M6G in the presence of PSC833, additional behavioural experiments were performed in another 32 rats to assess the dose dependency of the antinociceptive effects of M6G either with or without PSC833 in comparison with both PSC833 alone and placebo. Inhibition of P-gp increased the M6G concentrations in the spinal cord approximately three-fold whereas the plasma concentrations were increased only by a factor of 1.4, which resulted in a more than doubled spinal cord/plasma concentration ratio (from 0.08 +/- 0.03 for M6G alone to 0.17 +/- 0.08 for M6G plus PSC833). Antinociceptive effects of M6G were significantly enhanced by inhibition of P-gp. Inhibition of P-gp alters the transport of M6G across the blood-brain barrier, resulting in enhanced spinal cord uptake and enhanced antinociception.     

Mechanism of stem cells in the central nervous system

Injury to the central nervous system (CNS) can result in severe functional impairment. The brain and spinal cord, which constitute the CNS, have been viewed for decades as having a very limited capacity for regeneration. However, over the last several years, the body of evidence supporting the concept of regeneration and continuous renewal of neurons in specific regions of the CNS has increased. This evidence has significantly altered our perception of the CNS and has offered new hope for possible cell therapy strategies to repair lost function. Transplantation of stem cells or the recruitment of endogenous stem cells to repair specific regions of the brain or spinal cord is the next exciting research challenge. However, our understanding of the existing stem cell pool in the adult CNS remains limited. This review will discuss the identification and characterization of CNS stem cells in the adult brain and spinal cord.     

STUDIES OF THE MECHANISM OF DEMYELINATION REGIONAL DIFFERENCES IN MYELIN STABILITY IN VITRO1

—Incubation of slices of rat central nervous system in Krebs-Ringer bicarbonate buffer produced a lipoprotein fraction which floated on 10·5% sucrose after homogenization of the slices and centrifugation. This fraction was not found after homogenization and centrifugation of fresh tissue and appeared to depend upon incubation. The amount of the light fraction increased in the following order per 100-mg slice: cerebrum < thalamic area < cerebellum < brain stem < spinal cord. The lipid composition of this fraction was similar to that of myelin, but contained a lower protein content compared to myelin of the corresponding area. This fraction was termed ‘dissociated myelin’. Upon incubation of slices a portion of the basic protein was lost from myelin subsequently isolated, and the dissociated fraction was slightly enriched in basic protein. The distribution of myelin protein among the characteristic three groups (basic, proteolipid and high mol. wt.) was quite different in myelin from spinal cord compared to that from other CNS area. Spinal cord myelin contained about 17% protein compared to about 23% in cerebrum, with brain stem myelin intermediate (19%), and the difference appeared to be due to lesser amounts of proteolipid in the caudal areas. The amount of dissociation after incubation was about 3–5 per cent of the total myelin in the cerebral cortex, 10 per cent in the thalamic area, 20 per cent in cerebellum, 35 per cent in the brain stem, and around 45 per cent in spinal cord. The smaller amount of proteolipid protein in spinal cord myelin may result in a deficiency of cohesive forces holding lipids and proteins together, thus causing greater instability and dissociation. Myelin dissociation increased with time of incubation up to 3 h, was augmented by Ca²⁺, and was substantial at pH 11, reaching a peak at pH 7, then decreased in the acid range. A similar fraction has been isolated previously from fresh CNS tissue made edematous by chronic treatment of rats with triethyl tin. The possible relationship of swelling in the disease process and myelin dissociation are discussed.     

Glial and axonal regeneration following spinal cord injury

Spinal cord injury (SCI) has been regarded clinically as an irreversible damage caused by tissue contusion due to a blunt external force. Past research had focused on the analysis of the pathogenesis of secondary injury that extends from the injury epicenter to the periphery, as well as tissue damage and neural cell death associated with secondary injury. Recent studies, however, have proven that neural stem (progenitor) cells are also present in the brain and spinal cord of adult mammals including humans. Analyses using spinal cord injury models have also demonstrated active dynamics of cells expressing several stem cell markers, and methods aiming at functional reconstruction by promoting the potential self-regeneration capacity of the spinal cord are being explored. Furthermore, reconstruction of the neural circuit requires not only replenishment or regeneration of neural cells but also regeneration of axons. Analysis of the tissue microenvironment after spinal cord injury and research aiming to remove axonal regeneration inhibitors have also made progress. SCI is one of the simplest central nervous injuries, but its pathogenesis is associated with diverse factors, and further studies are required to elucidate these complex interactions in order to achieve spinal cord regeneration and functional reconstruction.     

Acrolein induces oxidative stress in brain mitochondria

Acrolein, a byproduct of lipid peroxidation, has been shown to inflict significant structural and functional damage to isolated guinea pig spinal cord. Reactive oxygen species (ROS) are thought to mediate such detrimental effects. The current study demonstrates that acrolein can directly stimulate mitochondrial oxidative stress. Specifically, exposure of purified brain mitochondria to acrolein resulted in a dose-dependent increase of ROS and decreases in glutathione content and aconitase activity. This effect was not accompanied by significant intramitochondrial calcium influx or mitochondrial permeability transition, but rather by impaired function of the mitochondrial electron transport system. As well, we detected a significant inhibition of mitochondrial adenine nucleotide translocase (ANT) in the presence of acrolein. This inhibition of ANT likely contributes to acrolein-induced ROS elevation since application of atractyloside, a specific ANT inhibitor, induced significant increase of ROS. We hypothesize that inhibition of ANT may mediate, in part, the acrolein-induced ROS increase in mitochondria.     

Purification of brain peroxisomes and localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol biosynthesis. Because proper CNS development depends on de novo cholesterol biosynthesis, peroxisomes must play a critical functional role in this process. Surprisingly, no information is available on the peroxisomal isoprenoid/cholesterol biosynthesis pathway in normal brain tissue or on the compartmentalization of isoprene metabolism in the CNS. This has been due mainly to the lack of a well-defined isolation procedure for brain tissue, and also to the presence of myelin in brain tissue, which results in significant contamination of subcellular fractions. As a first step in characterizing the peroxisomal isoprenoid pathway in the CNS, we have established a purification procedure to isolate peroxisomes and other cellular organelles from the brain stem, cerebellum and spinal cord of the mouse brain. We demonstrate by use of marker enzymes and immunoblotting with antibodies against organelle specific proteins that the isolated peroxisomes are highly purified and well separated from the ER and mitochondria, and are free of myelin contamination. The isolated peroxisomal fraction was purified at least 40-fold over the original homogenate. In addition, we show by analytical subcellular fractionation and immunoelectron microscopy that HMG-CoA reductase protein and activity are localized both in the ER and peroxisomes in the CNS.     

Biocompatible hydrogels in spinal cord injury repair

Spinal cord injury results in a permanent neurological deficit due to tissue damage. Such a lesion is a barrier for "communication" between the brain and peripheral tissues, effectors as well as receptors. One of the primary goals of tissue engineering is to bridge the spinal cord injury and re-establish the damaged connections. Hydrogels are biocompatible implants used in spinal cord injury repair. They can create a permissive environment and bridge the lesion cavities by providing a scaffold for the regeneration of neurons and their axons, glia and other tissue elements. The advantage of using artificial materials is the possibility to modify their physical and chemical properties in order to develop the best implant suitable for spinal cord injury repair. As a result, several types of hydrogels have been tested in experimental studies so far. We review our work that has been done during the last 5 years with various types of hydrogels and their applications in experimental spinal cord injury repair.     

Glial and axonal regeneration following spinal cord injury

Spinal cord injury (SCI) has been regarded clinically as an irreversible damage caused by tissue contusion due to a blunt external force. Past research had focused on the analysis of the pathogenesis of secondary injury that extends from the injury epicenter to the periphery, as well as tissue damage and neural cell death associated with secondary injury. Recent studies, however, have proven that neural stem (progenitor) cells are also present in the brain and spinal cord of adult mammals including humans. Analyses using spinal cord injury models have also demonstrated active dynamics of cells expressing several stem cell markers, and methods aiming at functional reconstruction by promoting the potential self-regeneration capacity of the spinal cord are being explored. Furthermore, reconstruction of the neural circuit requires not only replenishment or regeneration of neural cells but also regeneration of axons. Analysis of the tissue microenvironment after spinal cord injury and research aiming to remove axonal regeneration inhibitors have also made progress. SCI is one of the simplest central nervous injuries, but its pathogenesis is associated with diverse factors, and further studies are required to elucidate these complex interactions in order to achieve spinal cord regeneration and functional reconstruction.Key words: glia, regeneration, spinal cord, injury, axon     

Histochemical localization of FMRFamide-like immunoreactivity in the rat brain

Molluscan cardioexcitatory neuropeptide or FMRFamide is present in the invertebrate central nervous system (CNS) and FMRFamide like peptide has been demonstrated in the mammalian CNS. In this study, the distribution of FMRFamide immunoreactivity was studied in rat brain using the indirect immunofluorescent method. The highest number of FMRFamide staining cell bodies was found in the nucleus (n) arcuatus. N. paraventricularis, n. hypothalamus, n. ventromedialis, n. dorsomedialis and n. tractus solitarii also contained high numbers. FMRFamide positive nerve fibers and terminals were widely distributed. The septal complex contained high densities, especially in n. interstitialis striae terminalis. N. paraventricularis hypothalami, n. paraventricularis, n. hypothalamicus, n. ventromedialis and n. dorsomedialis showed a high to very high degree of immunoreactivity. In myelencephalon, n. tractus solitarii had the densest innervation. Spinal cord had a dense band of FMRFamide positive fibers in lamina I and II of the dorsal horn. The present findings support a neurotransmitter role for a FMRFamide like peptide in the mammalian brain, possibly related to endocrine and autonomic regulation as well as pain modulation.     

Rotenone model of Parkinson disease: multiple brain mitochondria dysfunctions after short term systemic rotenone intoxication

Chronic infusion of rotenone (Rot) to Lewis rats reproduces many features of Parkinson disease. Rot (3 mg/kg/day) was infused subcutaneously to male Lewis rats for 6 days using Alzet minipumps. Control rats received the vehicle only. Presence of 0.1% bovine serum albumin during the isolation procedure completely removed rotenone bound to the mitochondria. Therefore all functional changes observed were aftereffects of rotenone toxicity in vivo. In Rot rat brain mitochondria (Rot-RBM) there was a 30-40% inhibition of respiration in State 3 and State 3U with Complex I (Co-I) substrates and succinate. Rot did not affect the State 4Deltapsi of RBM and rat liver mitochondria (RLM). However, Rot-RBM required two times less Ca2+ to initiate permeability transition (mPT). There was a 2-fold increase in O*2- or H2O2 generation in Rot-RBM oxidizing glutamate. Rot infusion affected RLM little. Our results show that in RBM, the major site of reactive oxygen species generation with glutamate or succinate is Co-I. We also found that Co-II generates substantial amounts of reactive oxygen species that increased 2-fold in the Rot-RBM. Our data suggest that the primary mechanism of the Rot toxic effect on RBM consists in a significant increase of O*2- generation that causes damage to Co-I and Co-II, presumably at the level of 4Fe-4S clusters. Decreased respiratory activity diminishes resistance of RBM to Ca2+ and thus increases probability of mPT and apoptotic cell death. We suggest that the damage to Co-I and Co-II shifts O*2- generation from the CoQ10 sites to more proximal sites, such as flavines, and makes it independent of the RBM functional state.