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Biomedical subjects

J B Clark

Publications and source records attributed to J B Clark.

At least 73 records · Page 4Linked to original sources
PubMed1997

Nitric oxide-mediated mitochondrial damage in the brain: mechanisms and implications for neurodegenerative diseases.

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.

Animals↗
PubMed1997

Pretreatment of astrocytes with interferon-alpha/beta impairs interferon-gamma induction of nitric oxide synthase.

Excessive nitric oxide/peroxynitrite generation has been implicated in the pathogenesis of multiple sclerosis, and the demonstration of increased astrocytic nitric oxide synthase activity in the postmortem brain of multiple sclerosis patients supports this hypothesis. Exposure of astrocytes, in primary culture, to interferon-gamma results in stimulation of nitric oxide synthase activity and increased nitric oxide release. In contrast to interferon-gamma, interferon-alpha/beta had a minimal effect on astrocytic nitric oxide formation. Furthermore, pretreatment of astrocytes with interferon-alpha/beta inhibited (approximately 65%) stimulation by interferon-gamma of nitric oxide synthase activity and nitric oxide release. Treatment with interferon-alpha/beta at a concentration as low as 10 U/ml caused inhibition of mitochondrial cytochrome c oxidase. Furthermore, the damage to cytochrome c oxidase was prevented by the putative interferon-alpha/beta receptor antagonist oxyphenylbutazone. In view of these observations, our current hypothesis is that the mitochondrial damage caused by exposure to interferon-alpha/beta may impair the ability of astrocytes to induce nitric oxide synthase activity on subsequent interferon-gamma exposure. These results may have implications for our understanding of the mechanisms responsible for the therapeutic effects of interferon-alpha/beta preparations in multiple sclerosis.

Animals↗
PubMed1997

Threshold effects in synaptosomal and nonsynaptic mitochondria from hippocampal CA1 and paramedian neocortex brain regions.

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.

Animals↗
PubMed1997

Activity of mitochondrial respiratory chain enzymes after transient focal ischemia in the rat.

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 alpha-phenyl-N-tert-butyl-nitrone (PBN). Complex IV activity transiently decreased after MCAO, but after recirculation all measured activities returned to control values.

Animals↗
PubMed1996

Impairment of the nitric oxide/cyclic GMP pathway in cerebellar slices prepared from the hph-1 mouse.

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.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1996

Inhibition of N-acetylaspartate production: implications for 1H MRS studies in vivo.

The effect of specific irreversible inhibitors of complexes I, III, IV and V of the mitochondrial respiratory chain, (rotenone, myxothiazol, cyanide and oligomycin, respectively) on mitochondrial N-acetylaspartate production, and its relationship to oxidative phosphorylation (ATP production and oxygen consumption) were investigated in isolated rat brain mitochondria. Mitochondrial N-acetylaspartate production, ATP production and oxygen consumption were all significantly decreased in the presence of each of the inhibitors used compared with control incubations, and correlated positively with each other. It is postulated that decreased N-acetylaspartate levels seen in disease states by 1H NMR spectroscopy in vivo may reflect primarily an impaired mitochondrial energy production rather than neuronal cell loss.

Analysis of Variance↗
PubMed1996

Depletion of brain glutathione results in a decrease of glutathione reductase activity; an enzyme susceptible to oxidative damage.

Loss of the intracellular antioxidant glutathione (GSH) from the substantia nigra is considered to be an early event in the pathogenesis of Parkinson's disease (PD). While the cause of the loss is unclear, an imbalance in the enzymes associated with the synthesis, utilisation, degradation and translocation of GSH has been implicated. The enzyme glutathione reductase is also important in GSH homeostasis: it regenerates GSH from the oxidised from (GSSG). However, to date the activity and regulation of glutathione reductase in conditions such as PD have not been explored. In view of this we have measured the effects of GSH depletion on glutathione reductase activity of the rat brain. Other glutathione related enzymes were also measured. Using pre-weanling rats, brain GSH was depleted by up to 60% by subcutaneous administration of L-buthionine sulfoximine. The only enzyme affected by GSH depletion was glutathione reductase; its activity being reduced by approximately 40%. As GSH inactivates a number of oxidising species including peroxynitrite (ONOO-), we additionally investigated the susceptibility of glutathione reductase to ONOO- in vitro, using purified enzyme. ONOO- decreased glutathione reductase activity in a concentration dependent manner with an apparent 50% inhibition occurring at an initial concentration of 0.09 mM. These data suggest that GSH is important in the maintenance glutathione reductase activity. This may arise in part from its ability to inactivate oxidising agents such as ONOO-.

Animals↗
PubMed1996

Mitochondrial nitric oxide synthase: a ubiquitous regulator of oxidative phosphorylation?

In this article we demonstrate the immunocytochemical localization of nitric oxide synthase in mitochondria isolated from heart, skeletal muscle, and kidney, using a monoclonal antibody directed against the endothelial form of nitric oxide synthase. The possibility that mitochondrially located nitric oxide synthase is a ubiquitous regulator of mitochondrial oxidative phosphorylation in mammalian cells is discussed.

Animals↗
PubMed1996

Glutathione depletion is accompanied by increased neuronal nitric oxide synthase activity.

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.

Animals↗
PubMed1996

Nitric oxide and antioxidant status in glucose and oxygen deprived neonatal and adult rat brain synaptosomes.

Nitric oxide (NO.) has been implicated in the process of cerebral ischemia/reperfusion injury. We have examined the production of NO., as reflected by nitrite (NO2-) + nitrate (NO3-) accumulation, from synaptosomes isolated from neonatal or adult rat brain and subjected to a period of glucose and oxygen deprivation. There was a significant increase in the amount of NO2- + NO3- production from adult synaptosomes under these conditions, whereas there was no difference compared to control in the production of NO2- + NO3- from the neonatal synaptosomes. The total antioxidant status of the synaptosomes at these different stages of brain development was found to be the same. These data suggest that the vulnerability of the adult brain to ischemia/reperfusion injury may be associated with the production of NO. from nerve terminals. The ratios of antioxidant capacity to NO. production under such conditions have been shown here to be different between the neonatal and adult nerve terminals. Thus the well documented resistance of neonatal brain to ischemia/reperfusion injury may involve the neonatal nerve terminal being under less oxidative stress than the adult.

Aging↗
PubMed1996

Assessment of energy metabolism in the developing brain following aglycemic hypoxia by 1H and 31P NMR.

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.

Alanine↗
PubMed1996

Energy metabolism of adult astrocytes in vitro.

In this study we established cultures of astrocytes from the forebrain of the adult rat. The homeostatic regulatory mechanisms of the aerobic and anaerobic pathways of energy metabolism in these cells showed that adult astrocytes express many of the regulatory properties previously demonstrated in neonatal astrocytes. Changes in mitochondrial respiration and ATP production were readily evident upon incubation with the relevant substrates. Inhibition of mitochondrial respiration led to a compensatory increase in anaerobic glycolysis as evidenced by an increased release of lactate. We assessed the role of cytosolic calcium in the regulation of the mitochondrial energy metabolism. Increases in cytosolic calcium concentration in response to ATP or stimulation of mechanical receptors were followed by depolarizations of the mitochondrial membrane potential, whose magnitude reflected the amplitude of the cytosolic calcium response. The changes in mitochondrial membrane potential were largely dependent on the presence of external calcium. These results provide the first evidence of a signalling mechanism in astrocytes by which changes in cytosolic calcium mediate changes in respiration, possibly through mitochondrial calcium uptake and subsequent activation of several mitochondrial dehydrogenases. This signalling pathway would thus ensure that energy demands due to changes in cytosolic calcium concentrations are met by increases in energy production through increases in mitochondrial oxidative phosphorylation.

Animals↗
PubMed1996

Mechanisms of intracellular calcium regulation in adult astrocytes.

Microfluorimetric techniques were used to measure changes in intracellular calcium in astrocytes cultured from the forebrain of the adult rat. Application of ATP consistently raised intracellular calcium. The response persisted in the absence of extracellular calcium, but then quickly declined upon repeated agonist application. Thapsigargin abolished responses to nucleotides following depletion of the endoplasmic reticular calcium stores. Calcium release was inhibited by caffeine, but was dramatically increased through inositol phosphate receptor sensitization by the sulphydryl reagent thimerosal. Responses to repeated nucleotide applications resulted in a gradual decline of peak calcium concentrations, suggesting a (post)receptor-mediated desensitization or gradual depletion of the internal calcium stores. Subsequent application of ionomycin suggested intracellular calcium depletion as the relevant mechanism. Depletion of the internal calcium stores with ATP, ionomycin or thapsigargin failed to reveal a calcium influx pathway. These results suggest that the capacitative mechanism of calcium entry does not operate in response to nucleotide receptor activation in these cells, and that the immediate refilling of the internal calcium stores is primarily determined by re-uptake of cytosolic calcium into the endoplasmic reticulum. A complete refilling of this calcium store by extracellular calcium may be a much slower process. Control of these signal transduction pathways is crucial to the maintenance of the calcium/energy homeostasis of the adult astrocyte in the central nervous system.

Adenosine Triphosphate↗