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We have examined the action of nitric oxide (NO) on the ability of Fenton's reagent (ferrous iron and hydrogen peroxide), to oxidize a number of organic optical probes. We found that NO is able to arrest the oxidation of organic compounds at concentrations of NO found in brain, in vivo. We present evidence that Fenton's reagent proceeds via a ferryl intermediate ([Fe[double bond]O]2+), before the generation of hydroxyl radical *OH. NO reacts rapidly with this ferryl, blocking the production of *OH. We propose that NO has an important role in protecting biological tissues, and the brain in particular, from Fenton chemistry.

Cultured rat and human astrocytes and rat neurones were shown to release reduced glutathione (GSH). In addition, GSH oxidation was retarded by the concomitant release of a factor from the cells. One possibility is that this factor is extracellular superoxide dismutase (SOD). In support of this, the factor was found to bind heparin, have a molecular mass estimated to be between 50 and 100 kDa, and CuZn-type SOD protein and cyanide sensitive enzyme activity were demonstrated in the cell-conditioned medium. In addition, supplementation of native medium with exogenous CuZn-type SOD suppressed GSH oxidation. We propose that preservation of released GSH is essential to allow for maximal up-regulation of GSH metabolism in neurones. Furthermore, cytokine stimulation of astrocytes increased release of the extracellular SOD, and enhanced stability of GSH. This may be a protective strategy occurring in vivo under conditions of oxidative stress, and suggests that SOD mimetics may be of therapeutic use.

The tripeptide glutathione (gamma-glutamylcysteinylglycine; GSH) is the predominant low molecular mass thiol in cells. The function of GSH is of considerable interest, with the molecule being implicated in numerous cellular processes in addition to being a major cellular antioxidant. The enzyme glutamate-cysteine ligase (GCL) is the rate-limiting step in GSH synthesis. The GCL assay described here is based on high-performance liquid chromatography and exploits the electrochemically active nature of gamma-glutamylcysteine (gamma-GC), the product of GCL activity. This method allows for the direct detection of gamma-GC rather than relying on derivatization of the molecule or linked assays. The sensitivity of the assay is sufficient to allow for the measurement of GCL activity in cultured cells. The specific activity of GCL in rat primary culture astrocytes was 9.7 +/- 1.7 nmol gamma-GC synthesized/min/mg protein.

Publisher Summary The inner mitochondrial membrane is the site of the electron-transport chain (ETC), and is where the process of oxidative phosphorylation (OXPHOS) occurs that facilitates ATP synthesis. In view of the key role the ETC plays in energy metabolism, damage to one or more of the respiratory-chain complexes could lead to an impairment of cellular ATP formation. However, each of the complexes of the ETC appears to exert varying degrees of control over respiration. In vitro studies suggest that substantial loss of activity of an individual complex may be required before ATP synthesis is compromised. However, the degree of control a particular complex exerts over respiration may differ between cell types. The chapter presents descriptions of the predominant metabolic pathways located to mitochondria that are responsible for NADH and FADH 2 generation. The optimal mitochondrial function is essential for cell survival. There is an increasing body of evidence to suggest that mitochondrial dysfunction occurs in a number of neurodegenerative disorders.

Cytokine-stimulated astrocytes produce nitric oxide (NO), which, along with its metabolite peroxynitrite (ONOO(-)), can inhibit components of the mitochondrial respiratory chain. We used astrocytes as a source of NO/ONOO(-) and monitored the effects on neurons in coculture. We previously demonstrated that astrocytic NO/ONOO(-) causes significant damage to the activities of complexes II/III and IV of neighbouring neurons after a 24-h coculture. Under these conditions, no neuronal death was observed. Using polytetrafluoroethane filters, which are permeable to gases such as NO but impermeable to NO derivatives, we have now demonstrated that astrocyte-derived NO is responsible for the damage observed in our coculture system. Expanding on these observations, we have now shown that 24 h after removal of NO-producing astrocytes, neurons exhibit complete recovery of complex II/III and IV activities. Furthermore, extending the period of exposure of neurons to NO-producing astrocytes does not cause further damage to the neuronal mitochondrial respiratory chain. However, whereas the activity of complex II/III recovers with time, the damage to complex IV caused by a 48-h coculture with NO-producing astrocytes is irreversible. Therefore, it appears that neurons can recover from short-term damage to mitochondrial complex II/III and IV, whereas exposure to astrocytic-derived NO for longer periods causes permanent damage to neuronal complex IV.

Mutations in GTP-cyclohydrolase I (GTP-CH) have been identified as causing a range of inborn errors of metabolism, including dopa-responsive dystonia. GTP-CH catalyses the first step in the biosynthesis of tetrahydrobiopterin (BH4), a cofactor necessary for the synthesis of catecholamines and serotonin. Current therapy based on monoamine neurotransmitter replacement may be only partially successful in correcting the neurological deficits. The reason might be that BH4 is also a cofactor for nitric oxide synthase. Using a strain of mutant GTP-CH-deficient (hph-1) mice, we demonstrate that in addition to impaired monoamine metabolism, BH4 deficiency is also associated with diminished nitric oxide synthesis in the brain (as evaluated by measuring the levels of cyclic GMP), when compared with wild-type animals. We have found a decline in the levels of BH4 with age in all animals, but no gender-related differences. We found a strong association between the levels of BH4 and cyclic GMP in hph-1 mice but not in wild-type animals. We also demonstrate that acute peripheral administration of BH4 (100 micromol/kg s.c.) in hph-1 mice significantly elevated the brain BH4 concentration and subsequently cyclic GMP levels in cerebellum, with peaks at 2 and 3 h, respectively. We suggest that BH4 administration should be considered in BH4 deficiency states in addition to monoamine replacement therapy.

Previously we reported that the co-culture of non-brain vascular endothelial cells with glioma cells leads to the induction of a more differentiated endothelial cell phenotype which exhibits important properties of the blood-brain barrier (BBB). Recognising the potential for improving the model barrier system with agents known to modify the growth and differentiation of cells in culture we examined the effects of four differentiating agents (butyric acid, dexamethasone, retinoic acid, and dimethyl sulfoxide) on barrier function. Of these agents only butyric acid and dexamethasone resulted in an enhancement (depending on the dose used) of transendothelial electrical resistance (barrier function). The greatest effect was observed with butyric acid in a dose-dependent manner and was slow in onset and only occurred in the endothelial/glial cell co-cultures. These data indicate that butyric acid may be a beneficial agent in optimising conditions necessary for induction of BBB properties in in vitro barrier systems.

Functional mitochondrial respiratory chain and oxidative phosphorylation systems are obligate requirements for the normal function of most eukaryotic cells. When a defect of one of these systems occurs in humans, it results in a variety of clinical symptoms according to the severity of the defect and the tissues involved. Defects of all five multisubunit complcxes comprising the respiratory chain and oxidative phosphorylation systems have been reported [ 1-51, with some patterns emerging [ 61. but generally no clear picture correlating. the clinical symptoms and the biochemical or molecular

Mitochondria are known targets of ischemia-reperfusion injury in adult brain. Although neonates are more resistant to ischemic episodes, the mechanisms accounting for this are not yet fully understood. The aim of this study therefore was to determine whether a difference in post-ischemic mitochondrial function may play a role in the heightened recovery of the neonatal brain following ischemia-reperfusion. We have therefore compared the effects of an in vitro model of ischemia on the enzymes of the mitochondrial respiratory chain in isolated nerve terminals (synaptosomes) from neonatal and adult rats. Ischemia caused a significant, reversible decrease in mitochondrial Complex I activity in both adult and neonatal preparations. In neonatal preparations alone, ischemia also led to a significant decrease in mitochondrial Complexes II-III activity. Following 30 min of reperfusion mitochondrial Complexes II-III and IV exhibited decreased activity in synaptosomes from adult, but not neonatal rats. These data suggest a difference in the susceptibility of adult as compared to neonatal nerve terminal mitochondria to ischemia-reperfusion. These data show for the first time that nerve terminal mitochondria from immature animals remain undamaged following a period of ischemia and reperfusion, in contrast to nerve terminal mitochondria from the adult brain. This adds to the growing body of evidence that mitochondrial function plays a key role in neuronal death following cerebral ischemia reperfusion.

It has been suggested that decreased tetrahydrobiopterin (BH 4 ) availability may be a useful tool for limiting excessive nitric oxide (NO) formation. In order to test this hypothesis we utilised cultured astrocytes derived from the brain of the hph-1 (BH 4 deficient) mouse. In response to treatment with lipopolysaccharide and interferon-γ (LPS/γIFN) levels of BH 4 doubled in both wild type and hph-1 astrocytes. However, levels of BH 4 in hph-1 astrocytes remained only 25% of the wild type astrocytes. Nitric oxide formation, measured with an NO-electrode, was 45% less from LPS/γIFN stimulated hph-1 astrocytes compared with wild type stimulated astrocytes. In contrast, iNOS specific activity and iNOS protein were enhanced in hph-1 stimulated astrocytes by 40 and 60%, respectively when compared with wild type. In conclusion it appears that whilst a decrease in BH 4 may limit NO release per se, the possibility and consequences of long term `over' induction of iNOS protein requires further consideration.

The level of phosphocreatine (PCr) and the intracellular pH (pHi) of superfused cortical brain slices from adult or 10-day-old rats were monitored using 31P NMR. When the glucose in the superfusing medium was replaced by 3-hydroxybutyrate (3HB), there was a significant reduction in PCr of the adult but not the neonatal slices. The level of PCr of the adult slices was reduced by a greater amount by aglycaemic hypoxia compared with the neonatal brain slices and pHi was decreased by the same amount. After aglycaemic hypoxia, the levels of PCr of the neonatal slices recovered to the same extent when perfused with glucose or 3HB alone or a mixture of glucose and 3HB. The recovery of the PCr was significantly more in the neonatal than the adult brain slices with glucose alone after aglycaemic hypoxia, whereas pHi returned to control levels in both tissue types and with all substrates. The relative recovery of the PCr of the adult slices after aglycaemic hypoxia was the same with either 3HB or glucose. However, if glucose and 3HB were applied together, recovery of PCr was significantly improved compared with glucose alone.

After a brief period of global ischemia, the hippocampal CA1 region is more susceptible to irreversible damage than the paramedian neocortex. To test whether primary differences in bioenergetic parameters may be present between these regions, respiration rates and respiratory control activities were measured. In synaptosomal and nonsynaptic mitochondria isolated from the hippocampal CA1 region, state 3 respiration rates and complex IV activities were significantly lower than those present in synaptosomal and nonsynaptic mitochondria from the paramedian neocortex. These results suggest that mitochondria from the CA1 hippocampal area differ in some properties of metabolism compared with the neocortex area, which may render them more susceptible to a toxic insult such as that of ischemia. In addition, when complex I and IV activities were titrated with specific inhibitors, thresholds in ATP synthesis and oxygen respiration became apparent. Complex I and IV activities were decreased by 60% in nonsynaptic mitochondria from the hippocampal CA1 region and paramedian neocortex before oxidative phosphorylation was severely compromised; however, in synaptosomes from these regions, complex I activities had a threshold of 25%, indicating heterogenous behaviour for brain mitochondria. Reduced complex I thresholds in mitochondria, in association with other constitutive defects in energy metabolism, may induce a decreased ATP supply in the synaptic region. The implications of these findings are discussed in relation to delayed neuronal death and processes of neurodegeneration.

Previous results demonstrated that after 2-hour middle cerebral artery occlusion (MCAO) in the rat, 1- to 2-hour recirculation temporarily restored the bioenergetic state and mitochondrial function, but secondary deterioration took place after 4 hours. The authors measured the activity of mitochondrial respiratory chain complexes, citrate synthase, and glutamate dehydrogenase as possible targets of secondary damage. Focal and penumbral tissues were sampled in the control condition, after 2 hours of MCAO, and after 1, 2, or 4 hours of postischemic recirculation; two groups were treated with α-phenyl-N- tert-butyl-nitrone (PBN). Complex IV activity transiently decreased after MCAO, but after recirculation all measured activities returned to control values.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme regulated by many diverse mechanisms. In this study we present evidence that GAPDH activity is stimulated in the presence of oxyhemoglobin (2.3-fold, P

Within the central nervous system, nitric oxide is an important physiological messenger [1] . However, when synthesized excessively in neurones, cell death may occur [2] . An impairment of mitochondrial cytochrome oxidase and subsequent cellular energy depletion seems to be a likely mechanism for this neurotoxicity [3] . Within neurones, nitric oxide is synthesized by the constitutive, Ca2+-dependent form of nitric oxide synthase (nNOS) [4] . Astrocytes, however, possess both the constitutive [5] and the inducible Ca2+-independent NOS (iNOS) 6 , 7 , which is expressed by endotoxin and/or cytokines [8] . In vitro, activation of nNOS rapidly produces neuronal cell death [2] . In contrast to neurones, following induction of iNOS, astrocytes synthesize large quantities of nitric oxide 3 , 9 , but cell death is not apparent despite marked damage to mitochondrial cytochrome oxidase [3] . The resistance of astrocytes to nitric oxide synthase-mediated cell damage may be due to their ability to increase their glycolytic rate when mitochondrial ATP synthesis is compromised [3] . On the basis of this phenomenon, we propose that activated astrocytes represent a suitable system for studying the efficacy of potential therapeutic agents at protecting from nitric oxide synthase-mediated mitochondrial damage [10] .

Within the CNS and under normal conditions, nitric oxide (.NO) appears to be an important physiological signalling molecule. Its ability to increase cyclic GMP concentration suggests that .NO is implicated in the regulation of important metabolic pathways in the brain. Under certain circumstances .NO synthesis may be excessive and .NO may become neurotoxic. Excessive glutamate-receptor stimulation may lead to neuronal death through a mechanism implicating synthesis of both .NO and superoxide (O2.-) and hence peroxynitrite (ONOO-) formation. In response to lipopolysaccharide and cytokines, glial cells may also be induced to synthesize large amounts of .NO, which may be deleterious to the neighbouring neurones and oligodendrocytes. The precise mechanism of .NO neurotoxicity is not fully understood. One possibility is that it may involve neuronal energy deficiency. This may occur by ONOO- interfering with key enzymes of the tricarboxylic acid cycle, the mitochondrial respiratory chain, mitochondrial calcium metabolism, or DNA damage with subsequent activation of the energy-consuming pathway involving poly(ADP-ribose) synthetase. Possible mechanisms whereby ONOO- impairs the mitochondrial respiratory chain and the relevance for neurotoxicity are discussed. The intracellular content of reduced glutathione also appears important in determining the sensitivity of cells to ONOO- production. It is concluded that neurotoxicity elicited by excessive .NO production may be mediated by mitochondrial dysfunction leading to an energy deficiency state.

13C nuclear magnetic resonance spectroscopy was used to study the activity of malic enzyme in isolated brain mitochondria from rat in the presence of unlabelled malate and [U-13C]glutamate. ADP, inorganic phosphate, malate and [U-13C]glutamate were added to a suspension of oxygenated mitochondria. Typical tricarboxylic acid (TCA) cycle constituents (malate, 2-oxoglutarate and succinate) were labelled from [U-13C]glutamate and detected in the superfusion medium. The labelling patterns in the different atom positions of glutamate revealed entry of both unlabelled and labelled acetyl-CoA into the TCA cycle. Unlabelled acetyl-CoA was derived via pyruvate from exogenously applied malate by the action of mitochondrial malic enzyme, while labelled acetyl-CoA was derived from TCA cycle intermediates, most likely by the action of mitochondrial malic enzyme on malate produced from [U-13C]glutamate. The results demonstrate malic enzyme activity and pyruvate recycling in isolated rat brain mitochondria.

Tumor necrosis factor-alpha (TNF-alpha) has been implicated in the breakdown of blood-brain barrier (BBB) function which can occur during various inflammatory conditions. Recent evidence suggests a role for the free radical nitric oxide (NO) in the process of cytokine-induced barrier dysfunction. The mitochondrial enzyme cytochrome oxidase is inhibited by NO, and hence, using a coculture model of the BBB, we have investigated whether TNF-alpha alters barrier function by a NO-mediated mechanism and, if so, whether it is related to a reduction of endothelial cell respiration and ATP synthesis. TNF-alpha mediated a marked reduction in model BBB integrity that was partially prevented by inhibition of NO synthase activity. Additionally, exposure of BBB cultures to authentic gaseous NO also resulted in a progressive decline in barrier integrity. Authentic NO inhibited endothelial cell respiration in a reversible manner. Mitochondrial respiratory chain inhibitors induced significant reductions in endothelial cell respiratory rate and ATP levels, but did not mimic the action of NO on barrier function. We conclude that NO is partially responsible for the detrimental effect of TNF-alpha on BBB function. The mechanism of NO-induced barrier dysfunction does not involve an inhibition of endothelial mitochondrial electron transport chain and reduced energy resources.

Nitric oxide and its highly reactive derivative peroxynitrite have been implicated as non-specific inflammatory mediators of neuronal and oligodendrocyte damage and death in multiple sclerosis. In a cross-sectional study we found levels of the nitric oxide metabolites nitrate and nitrite to be raised in the serum of patients with demyelinating disease (65.6 microM (SD 32.9)), acquired immune deficiency syndrome (57.9 microM (SD 34.9)) and inflammatory neurological disease (57.5 microM (SD 31.3)), compared with normal control subjects (32.8 microM (SD 12.2)) and patients with non-inflammatory neurological disease (41.1 microM (SD 12.3), p < 0.001). Nitric oxide metabolites were raised in all clinical subtypes of multiple sclerosis, as well as in clinically isolated syndromes compatible with demyelination, and were not related to progressive disease or disability. This study provides further evidence for a role of nitric oxide in the immunopathogenesis of inflammatory diseases of the central nervous system, including multiple sclerosis.

In this study, the effect of tetrahydrobiopterin deficiency on the nitric oxide/cGMP pathway has been investigated in cerebellar slices derived from the hph-1 mouse. This animal displays a partial deficiency of tetrahydrobiopterin. Basal levels of cGMP were significantly reduced (-29.5%) in the hph-1 mouse cerebellum compared to controls. Following kainate stimulation (500 microM) cGMP levels increased in both control and hph-1 preparations but were again significantly lower (-29.1%) in the hph-1 mouse. Exposure of slices to the nitric oxide donors, S-nitroso-N-acetylpenicillamine and S-nitroso-glutathione, revealed no difference in cGMP accumulation between the two groups. These findings suggest that the cerebellar nitric oxide/cGMP pathway may be impaired in partial tetrahydrobiopterin deficiency states due to diminished nitric oxide formation.

Nitric oxide may regulate cellular respiration by competition with oxygen at mitochondrial cytochrome oxidase. Using an astrocyte-derived cell line, we have compared the mechanism of action of the nitric oxide-generating compound Roussin's black salt with that of sodium nitroprusside on cellular oxygen consumption. Intense light exposure induced the release of large quantities of nitric oxide from both of the donor compounds. However, in room light only Roussin's black salt generated low levels of the radical. Simultaneous measurement of oxygen consumption and of nitric oxide production demonstrated that sodium nitroprusside only had inhibitory actions when exposed to intense light (nitric oxide release), whereas Roussin's black salt had inhibitory actions in room light. Extracellular haemoglobin did not prevent the inhibition of respiration rate induced by Roussin's black salt even though stimulation of nitric oxide release on light exposure was markedly reduced. Preincubation of cells with Roussin's black salt and subsequent measurement of levels of light-liberated nitric oxide demonstrated that the compound was rapidly internalised. The uptake of sodium nitroprusside was minimal. These data suggest that, in contrast to sodium nitroprusside, the cellular internalisation of Roussin's black salt allows site-directed nitric oxide release and very effective inhibition of cellular respiration.

The role played by external calcium and calcium channels in the recovery from aglycaemic hypoxia in cortical brain slices from 10-day old rats was investigated by 1H and 31P NMR. 30 minutes of aglycaemic hypoxia significantly decreased the levels of phosphocreatine (PCr), ATP, lactate and intracellular pH (pHi). After a 30 minute recovery period there was incomplete recovery of PCr and ATP with lactate increasing by 50% with pHi normal. When the aglycemic hypoxia was carried out in media which had no added calcium (approximately 10 microM) the PCr and ATP recovery was significantly greater. Application of diltiazem or verapamil but not nifedipine significantly improved the recovery from the aglycemic hypoxia. These data suggest that calcium influx through L-type voltage-gated calcium channels is involved in the ischemic damage in neonatal brain which manifests itself as a decrease in the energy state and an increase in lactate.

The effects of hypoxia, aglycaemia, and hypoxia-aglycaemia on intrasynaptosomal free Ca2+ concentration ([Ca2+]i) have been investigated in rat brain synaptosomes prepared from animals aged 5, 10, 15, 20, 25, and 60 days. After 60 min of hypoxia there was no significant difference, when compared with controls, in basal [Ca2+]i or [Ca2+]i following depolarisation in all of the ages studied. Following 60 min of aglycaemia there was no significant difference from controls in [Ca2+]i of synaptosomes prepared from pups ofor = 20 days, although a significant rise in [Ca2+]i was seen in preparations from animals20 days old. Sixty minutes of hypoxia-aglycaemia led to a significant rise in [Ca2+]i only in preparations from animals 15-60 days old. With both aglycaemia and hypoxia-aglycaemia a progressive increase in the magnitude of the rise in [Ca2+]i was seen with development. These data suggest increases in [Ca2+]i in adult nerve terminals following prolonged aglycaemia and hypoxia-aglycaemia but no change following prolonged hypoxia. In contrast, no significant changes in [Ca2+]i values were apparent in neonatal nerve terminals under any of these conditions. In control synaptosomes with glucose and oxygen freely available, a decrease in resting and depolarised [Ca2+]i during development was seen, suggesting a change in calcium homeostasis within the nerve terminal as the brain develops. It is suggested that the mechanism underlying the relative resistance to ischaemic damage of neonatal brain as compared with adult brain may be related to the regulation of calcium at the nerve ending.

The primary function of mitochondria is to generate energy in the form of ATP. This is achieved by the four complexes of the electron transport chain and the ATP synthetase. Damage to one or more of the complexes of the mitochondrial respiratory chain may therefore result in depletion of cellular energy reserves and cell death. Increased cellular formation of oxidizing species may lead to mitochondrial damage, since the components of the mitochondrial respiratory chain are susceptible to oxidative damage in vitro (Zhang et al 1990). Thus it may be conjectured that mitochondrial damage may occur in a number of inborn errors of metabolism where the metabolic block increases oxidative stress. One such group are the inborn errors of glutathione (GSH) biosynthesis, which lead to GSH deficiency. Clinically they are characterized by a neurological syndrome, namely spinocerebellar degeneration, peripheral neuropathy, myopathy and ataxia (Meister and Larsson 1989). Interestingly, depletion (-60%) of brain GSH by the subcutaneous administration of L-buthionine sulphoximine (L-BSO) to pre-weanling rats results in mitochondrial swelling, damage to complex IV (cytochrome oxidase, EC 1.9.3.1) of the mitochondrial electron transport chain and neuronal damage (Jian et al 1991 ; Heales et al 1995). In this study we have expanded on our previous observations (Heales et al 1995) by examining the effect of varying degrees of mitochondrial GSH depletion on complex IV activity.

In this study we have examined the susceptibility of the mitochondrial respiratory chain of astrocytes and astrocytes depleted of glutathione to peroxynitrite exposure. Astrocytes, as reported previously by us, appeared resistant to the actions of peroxynitrite. In contrast, depletion (-94%) of astrocytic glutathione rendered the cells susceptible with mitochondrial complexes I and II/III being decreased in activity by 80 and 64%, respectively, after peroxynitrite exposure. Furthermore, cell death, as judged by lactate dehydrogenase release, was significantly increased (+81%) in the glutathione-depleted astrocytes exposed to peroxynitrite. Glutathione depletion alone had no effect on any of the measured parameters. It is concluded that glutathione is an important intracellular defence against peroxynitrite and that when glutathione levels are compromised the mitochondrial respiratory chain is a vulnerable target and cell death ensues. In view of the relative paucity of neuronal glutathione, it is possible that astrocyte-derived peroxynitrite may, in certain pathological conditions, be released and diffuse into neighboring neurones where mitochondrial damage may occur.

In this study we have investigated the mechanisms leading to mitochondrial damage in cultured neurons following sustained exposure to nitric oxide. Thus, the effects upon neuronal mitochondrial respiratory chain complex activity and reduced glutathione concentration following exposure to either the nitric oxide donor, S-nitroso-N-acetylpenicillamine, or to nitric oxide releasing astrocytes were assessed. Incubation with S-nitroso-N-acetylpenicillamine (1 mM) for 24 h decreased neuronal glutathione concentration by 57%, and this effect was accompanied by a marked decrease of complex I (43%), complex II-III (63%), and complex IV (41%) activities. Incubation of neurons with the glutathione synthesis inhibitor, L-buthionine-[S,R]-sulfoximine caused a major depletion of neuronal glutathione (93%), an effect that was accompanied by a marked loss of complex II-III (60%) and complex IV (41%) activities, although complex I activity was only mildly decreased (34%). In an attempt to approach a more physiological situation, we studied the effects upon glutathione status and mitochondrial respiratory chain activity of neurons incubated in coculture with nitric oxide releasing astrocytes. Astrocytes were activated by incubation with lipopolysaccharide/interferon-gamma for 18 h, thereby inducing nitric oxide synthase and, hence, a continuous release of nitric oxide. Coincubation for 24 h of activated astrocytes with neurons caused a limited loss of complex IV activity and had no effect on the activities of complexes I or II-III. However, neurons exposed to astrocytes had a 1.7-fold fold increase in glutathione concentration compared to neurons cultured alone. Under these coculture conditions, the neuronal ATP concentration was modestly reduced (14%). This loss of ATP was prevented by the nitric oxide synthase inhibitor, NG-monomethyl-L-arginine. These results suggest that the neuronal mitochondrial respiratory chain is damaged by sustained exposure to nitric oxide and that reduced glutathione may be an important defence against such damage.

The effect of glutathione depletion, in vivo, on rat brain nitric oxide synthase activity has been investigated and compared to the effect observed in vitro with cultured neurones. Using L-buthionine sulfoximine rat brain glutathione was depleted by 62%. This loss of glutathione was accompanied by a significant increase in brain nitric oxide synthase activity by up to 55%. Depletion of glutathione in cultured neurones, by approximately 90%, led to a significant 67% increase in nitric oxide synthase activity, as judged by nitrite formation, and cell death. It is concluded that depletion of neuronal glutathione results in increased nitric oxide synthase activity. These findings may have implications for our understanding of the pathogenesis of neurodegenerative disorders in which loss of brain glutathione is considered to be an early event.

In this article we demonstrate the immunocytochemical localization of nitric oxide synthase in non-synaptosomal rat brain mitochondria and rat liver mitochondria, using a monoclonal antibody directed against the endothelial form of nitric oxide synthase. The possibility that nitric oxide synthase located in mitochondria is involved in the regulation of mitochondrial oxidative phosphorylation is discussed.

Cultured astrocytes, activated to express the inducible form of nitric oxide synthase, produced up to 1 microM nitric oxide (NO) measured by a NO-selective electrode, while non-activated cells produced no detectable NO. The production of NO was associated with an inhibition of cellular respiration, measured simultaneously by an oxygen electrode. The inhibition of respiration was rapidly reversed by inhibiting the NO synthase or by binding the NO with haemoglobin. The respiratory inhibition had an NO, oxygen and substrate dependence consistent with NO-inhibition at cytochrome oxidase. This is the first demonstration that cells can reversibly inhibit mitochondrial respiration via NO production. This inhibition is large and potentially important in a range of pathophysiological conditions.

Marked abnormalities of the magnetic resonance intensity of N-acetylaspartate (NAA) have been reported in patients with various neurological disorders, but the neurochemical consequences of these alterations are difficult to assess because the function of NAA remains speculative. The purpose of this study was to examine whether NAA plays a role in protecting neurons against osmotic stress. Intracerebral microdialysis was used to expose a small region of the rat dorsolateral striatum to an increasingly hyposmotic environment and to measure resulting changes in NAA extracellular concentrations. NAA changes in the extracellular fluid (ECF) were compared with those of the amino acids, in particular, taurine, known to be involved in brain osmoregulation. Stepped increases in cellular hydration produced by hyposmotic perfusion media induced a marked increase in ECF NAA, reflecting a redistribution of NAA from intra-to extracellular space. Parallel experiments showed that, of all the extracellular amino acids measured, only taurine markedly increased with hyposmolar perfusion medium, indicating that the ECF NAA increase associated with hyposmotic stress was a specific response and not passive leakage out of the cells. As NAA is predominantly neuronal, it may contribute to the protection of neurons against swelling (i.e., regulatory volume decrease). In conditions with impaired blood-brain barrier and cytotoxic oedema, efflux of intracellular NAA subsequent to sustained cellular swelling might lead to a reduction in total brain NAA detectable by magnetic resonance spectroscopy. Alternatively, redistribution of NAA from intra-to extracellular space implies changes in its chemical environment that may alter its magnetic resonance visibility.

The effect of depletion of reduced glutathione (GSH) on brain mitochondrial function and N-acetyl aspartate concentration has been investigated. Using pre-weanling rats, GSH was depleted by L-buthionine sulfoximine administration for up to 10 days. In both whole brain homogenates and purified mitochondrial preparations complex IV (cytochrome c oxidase) activity was decreased, by up to 27%, as a result of this treatment. In addition, after 10 days of GSH depletion, citrate synthase activity was significantly reduced, by 18%, in the purified mitochondrial preparations, but not in whole brain homogenates, suggesting increased leakiness of the mitochondrial membrane. The whole brain N-acetyl aspartate concentration was also significantly depleted at this time point, by 11%. It is concluded that brain GSH is important for the maintenance of optimum mitochondrial function and that prolonged depletion leads also to loss of neuronal integrity. The relevance of these findings to Parkinson's disease and the inborn errors of glutathione metabolism are also discussed.

cDNA clones encoding the testis-specific form of the rat pyruvate dehydrogenase complex E1 alpha subunit have been isolated. Comparison of the predicted amino acid sequence with those of the somatic and testis-specific E1 alpha forms of man and mouse and the somatic E1 alpha form of rat indicates the change of a serine residue, believed to be phosphorylated in vivo by pyruvate dehydrogenase E1 alpha-specific kinase, to an alanine at position 233. The implications of this change are discussed. Northern blot analysis and RNase protection assays indicate that the expression of mRNA encoding testis-specific E1 alpha subunit is restricted to testis whereas mRNA for the somatic form is found in all tissues analyzed, albeit in very small amounts in testis.

The metabolic capability for the complete oxidation of glucose, i.e. aerobic glycolysis, is highly developed in the brains of neurologically mature (precocial) species at birth, whereas this activity is severely limited in the brains of neurologically immature (non-precocial) species such as the rat and human. The latter utilize a mixture of glucose and ketone bodies for synthetic and energetic activities and the advent of neurological competence associated with the capability for complete dependence on and oxidation of glucose must await the development of key enzymes such as the pyruvate dehydrogenase complex (PDHC). A similar relationship appears to exist with respect to the development of neurological maturity of different brain regions in a single species, the rat. The development of the enzymes of energy metabolism of neonatal rat brain will be discussed with respect to the energy fuels available to the neonatal brain. In particular mechanisms by which the PDHC develops in neonatal brain will be evaluated. Evidence suggests that this is due to a specific increase in enzyme protein in contrast to a general increase in mitochondrial activity.

Twenty-three patients with mitochondrial myopathies and mitochondrial DNA deletions in muscle were studied by means of deletion mapping and sequencing, histochemistry and polarography. Histochemistry showed significantly less focal cytochrome oxidase deficiency relative to number of ragged red fibres when the deletion did not involve reading frames for cytochrome oxidase subunits. Polarography in such patients showed defects exclusively involving complex I, in contrast to the others with larger deletions who generally had more diffuse respiratory chain defects. Analysis of other published histochemical data showed similar findings to our own. It is concluded that translation of a proportion of deleted mitochondrial DNAs occurs in at least some patients with mitochondrial DNA deletions, implying that deleted and normal mitochondrial genomes share transfer RNAs within mitochondria in such cases.

In depolarised anoxic synaptosomes, in which lactate production was significantly raised compared with normoxic conditions, calcium uptake, net acetylcholine release, and the intrasynaptosomal calcium concentration were all significantly lowered. In contrast, lactate production in synaptosomes incubated under aglycaemic- and ischaemic-type conditions was significantly lower and basal calcium uptake, acetylcholine release, and intrasynaptosomal calcium concentration were elevated compared with normoxia. In addition, the increase in intrasynaptosomal calcium concentration under the ischaemic-type condition appeared to be greater than could be accounted for by the rise in calcium uptake alone. Intrasynaptosomal pH reflected the lactate production under each condition investigated. Addition of exogenous lactate to normoxic synaptosomes mimicked the effects observed in anoxia, suggesting that lactate itself may have blocked the calcium uptake, inhibiting the rise in intrasynaptosomal calcium and acetylcholine release occurring in depolarised anoxic synaptosomes. When lactate was added to ischaemic synaptosomes, the large rise in intrasynaptosomal calcium concentration, calcium uptake, and acetylcholine release were decreased, suggesting that lactate may have a protective role in preventing cell death by calcium overload under ischaemic-type conditions. Evidence is presented to suggest that the effect of L-lactate was due to the lactate moiety itself rather than the associated acidosis.

Previously, we have used the isolated perfused rat brain (IPRB) to demonstrate authentic cerebral synthesis of the lipid mediator platelet-activating-factor (Kumar, R., Harvey, S.A.K., Kester, M., Hanahan, D.J. and Olson, M.S. (1988) Biochim. Biophys. Acta 963, 375-383). The present study demonstrates that this fluorocarbon perfusion technique maintains the integrity of the blood-brain barrier (BBB), as evidenced by the small volume (1.77-3.33%) accessible to [carboxyl-14C]inulin. 51-66% of the brain was accessible to 3H2O, except for the spinal cord which is poorly perfused (16% accessible to 3H2O). There is no effective perfusion of muscle tissue associated with the preparation (less than 6% accessible to 3H2O). Fast Fourier Transform analysis of digitized EEG data showed that in low frequency bands (less than 7.5 Hz) the IPRB had reduced electrical activity relative to the whole conscious animal. The GABA antagonist bicuculline, which has convulsant effects in vivo, causes a 3-4-fold increase in overall (root-mean-square) electrical activity, but decreases further the relative amplitude of low frequencies. With appropriate corrections, measurement of the oxygen consumption of the IPRB can be made without the necessity for venous cannulation. Oxygen consumption of the IPRB is flow-dependent. At a perfusion rate of 1.54 ml/min per g, unstimulated oxygen consumption of the IPRB is 2.07-2.23 mumol/min per g, or 67-72% of the consumption of the brain in vivo. Administration of bicuculline to the IPRB causes a 31% increase in lactate efflux, but only a 15% increase in oxygen uptake, suggesting that the preparation becomes functionally ischemic. Measurement of ATP/ADP levels in control and bicuculline-treated IPRBs confirms this. Other workers have used the IPRB as a model for the cerebral effects of pharmacological agents and of metabolic insult. The present study shows that under various experimental conditions oxygen uptake, analytical EEG measurements, and the integrity of the blood-brain barrier all can be monitored.

The energy demands within an actively respiring tissue such as cardiac muscle must be satisfied, for the most part, by mitochondrial ATP synthesis. One of the principal mechanisms for the matching of ATP synthesis with demand is the control of the mitochondrial isocitrate, oxoglutarate and pyruvate dehydrogenases by the mitochondrial matrix Ca2 + concentration which is itself dependent upon cytosolic Ca2+ levels [ 1, 21. Fig. 1 shows the principal elements which form the Ca2+ homoeostasis system in heart mitochondria. The steady-state matrix Ca" concentration is the net sum of both the principal influx (Ca?' uniporter) and efflux pathways (Na+/Ca2+ exchange). From Fig. 1 it is evident that cytosolic Na+ and Ca2+ levels will be important in keeping matrix Ca2+ levels within the physiological norm. In experiments with isolated mitochondria, the response of the organelle to increasing Ca2 + concentration has been clearly established and has arbitrarily been categorized as consisting