Pharmacology of Sodium-Dependent High-Affinity l-[3H]Glutamate Transport in Glial Cultures

Abstract: Pharmacological and molecular biological studies provide evidence for subtypes of sodium-dependent high-affinity glutamate (Glu) transport in the mammalian CNS. At least some of these transporters appear to be selectively expressed in different brain regions or by different cell types. In the present study, the properties of l -[³H]Glu transport were characterized using astrocyte-enriched cultures prepared from cerebellum and cortex. In both brain regions, the kinetic data for sodium-dependent transport were consistent with a single site with K_m values of 91 ± 17 µM in cortical glial cells and 66 ± 23 µM in cerebellar glial cells. The capacities were 6.1 ± 1.6 nmol/mg of protein/min in cortical glial cells and 8.4 ± 0.9 nmol/mg of protein/min in cerebellar glial cells. The potencies of ～40 excitatory amino acid analogues for inhibition of sodium-dependent transport into glial cells prepared from cortex and cerebellum were examined, including compounds that are selective inhibitors of transport in synaptosomes prepared from either cerebellum or cortex. Of the analogues tested, 14 inhibited transport activity by >50% at 1 mM concentrations. Unlike l -[³H]Glu transport in synaptosomes prepared from cerebellum or cortex, there were no large differences between the potencies of compounds for inhibition of transport measured in glial cells prepared from these two brain regions. With the exception of (2S,1′R,2′R)-2-(carboxycyclopropyl)glycine and l -α-aminoadipate, all of the compounds examined were ～10–200-fold less potent as inhibitors of l -[³H]Glu transport measured in glial cells than as inhibitors of transport measured in synaptosomes prepared from their respective brain regions. The pharmacology of transport measured in these glial cells differs from the reported pharmacology of the cloned Glu transporters, suggesting the existence of additional uncloned Glu transporters or Glu transporter subunits.     

Autotypical septal contacts of cerebellar glial cells as protective reaction under toxic action of glutamate and nitric oxide

Structural transformations of the frog cerebellar glial cells (astrocytes) in the presence of high concentrations of glutamate and nitric oxide (NO) were studied by electron microscopy method. It was found that under these conditions mimicking a natural stroke, glial processes storing sufficient amounts of glycogen grains could protect the neurons of the molecular layer. Under these conditions glial processes transformed (stretched out) into strands, virtually deprived of cytoplasm. The distance between two membranes of such strands dropped to 25–30 nm, and cross-bridges appeared inside. These structures are called “autotypic septal contact” (ASC). A modified glial process with ASC could wrap injured synapse or synaptic elements (buttons, dendritic spines) and form a tight capsule. Glial processes with ASC can also intrude between two neurons of the cerebellum granular layer, preventing their pathological fusion. The results suggest that ASC can have a protecting role in the toxic action of glutamate and NO-generating agents.     

Nitric oxide synthase induction in glial cells: Effect on neuronal survival

In primary rat cortical glial cell cultures lipopolysaccharide (LPS) induced a dose- and time-dependent increase of intracellular cyclic GMP concentration associated with a release of nitrite. The LPS-induced cyclic GMP and nitrite increase was enhanced by interferon-γ and was prevented by L-N^G- nitroarginine, dexamethasone and cycloheximide. Thus indicates that LPS effect occured via the production of nitric oxide (NO) and involved new protein synthesis suggesting the induction of NO syntahse in these cells. Furthermore this induction was Ca²⁺-independent and was blocked by an inhibitor of the synthesis of tetrahydrobiopterin. The inducible NO synthase was also expressed by C6 glioma cells. In primary mixed cultures containing both neuronal and glial cells, the effects of LPS were less important than in primary glial cell cultures suggesting that glial cells rather than neurons expressed the inducible form of NO synthase. On the other hand no change on neuronal viability was observed after NO synthase induction by LPS in this culture type. This study indicates that glial cells are able to induce NO synthase without affecting neuronal survival.     

Modulation of Mn2+ accumulation in cultured rat neuronal and astroglial cells

The effects of physiological concentrations of K⁺ on Mn²⁺ accumulation were compared in rat glial cells and neurons in culture. Increasing the K⁺ concentration in growth medium increased significantly the Mn²⁺ level of the cultivated cells, with glial cells more affected than neurons. Ethanol markedly increased the Mn²⁺ accumulation within glia but not within neurons while ouabaïn caused inhibition of Mn²⁺ uptake with neurons and glial cells. A modulation of the total protein synthesis by Mn²⁺ and ethanol level in the growth medium was observed with glial cells. These data suggest that the mechanisms involved in Mn²⁺ accumulation in glial cells are different from those present in neurons. Moreover, the results are consistent with the hypothesis that Mn²⁺ plays a regulatory role in glial cell metabolism.     

Responses of crayfish neurons and glial cells to photodynamic impact: Intracellular signaling,ultrastructural changes,and neuroglial interactions

Photodynamic therapy (PDT), an inducer of oxidative stress, is used for treatment of cancer, including brain tumors. To study the mechanisms of photodynamic injury of neurons and glial cells (GC), we used a simple model object — isolated crayfish mechanoreceptor consisting of a single sensory neuron surrounded by a multilayered glial envelope. PDT caused inhibition and elimination of neuronal activity, impairment of intracellular organelles involved in the biosynthetic, bioenergetic, and transport processes and neuroglial interactions, necrosis of neurons and glial cells, and in glial apoptosis. PDT-induced death of a neuron and GC was mediated by intercellular molecular messengers and intracellular signaling cascades. PDT-induced inhibition and elimination of neuronal activity was associated with opening of mitochondrial permeability transition pores, Ca²⁺ release into cytosol, protein kinase C and NO synthase activities. Necrosis of neurons was mediated by protein kinases B/Akt, GSK-3β and mTOR, opening of mitochondrial permeability transition pores and Ca²⁺/calmodulin/CaMKII pathway. NO and GDNF reduced neuronal necrosis. Multiple signal pathways, such as phospholipase C/Ca²⁺, Ca²⁺/calmodulin/CaMKII, Ca²⁺/PKC, Akt/mTOR, MEK/p38, and protein kinase G mediated PDT-induced necrosis both in glial cells and in neurons. NOS/NO and neurotrophic factors NGF and GDNF protected glial cells and demonstrated antinecrotic activity. Glial apoptosis was reduced by neurotrophic factors NGF and GDNF, protein kinase C, and MAP kinase JNK. In contrast, mitochondrial permeability transition pores and phospholipase C, which mobilize intracellular Ca²⁺, NOS/NO/protein kinase G, proteins GSK-3β and mTOR, stimulated apoptosis of glial cells. The schemes of involvement of various inter- and intracellular signaling processes in the responses of neurons and GC to PDT are developed.     

Calcium Transport by Primary Cultured Neuronal and Glial Cells from Chick Embryo Brain

The uptake of calcium was examined in primary cultures of pure neurons and of glial cells from dissociated hemispheres of chick embryo brain. Neuronal cultures took up calcium at a rate of 2.0 nmol per min per mg cell protein at medium concentrations of 1.2 mM-Ca²⁺ and 5.4 mM-K⁺. The rate of calcium entry into neurons was increased 2.7-fold by elevating medium potassium to 60 MM. The effect of high external potassium was to increase the V_max value for calcium transport from 5.5 to 13 nmol per min per mg; the Michaelis constant for calcium, 1.2 mM, was unchanged. The potassium-dependent component of calcium entry into the neuronal cultures was eliminated by addition of 0.1 mM-D-600 (a verapamil derivative) or by 1 mM-CoCl₂, but 0.5 μM-tet-rodotoxin had no significant effect. When choline replaced potassium in uptake medium no change in calcium transport was detected in neurons, nor was the entry of calcium increased when choline replaced sodium. Glial cultures took up calcium at 20% of the basal rate for neuronal cultures on a weight-of-protein basis. Uptake was not increased by potassium; during depolarization by potassium the calcium transport activity of glia was less than 10% that of neurons. It was concluded that cultured neurons contain a depolarization-sensitive, calcium-specific channel. A similar calcium transport activity was not detected in cultured glial cells.     

Myelin-associated glycoprotein activation triggers glutamate uptake by oligodendrocytes in vitro and contributes to ameliorate glutamate-mediated toxicity in vivo

BackgroundMyelin-associated glycoprotein (MAG) is a key molecule involved in the nurturing effect of myelin on ensheathed axons. MAG also inhibits axon outgrowth after injury. In preclinical stroke models, administration of a function-blocking anti-MAG monoclonal antibody (mAb) aimed to improve axon regeneration demonstrated reduced lesion volumes and a rapid clinical improvement, suggesting a mechanism of immediate neuroprotection rather than enhanced axon regeneration. In addition, it has been reported that antibody-mediated crosslinking of MAG can protect oligodendrocytes (OLs) against glutamate (Glu) overload by unknown mechanisms.PurposeTo unravel the molecular mechanisms underlying the protective effect of anti-MAG therapy with a focus on neuroprotection against Glu toxicity.ResultsMAG activation (via antibody crosslinking) triggered the clearance of extracellular Glu by its uptake into OLs via high affinity excitatory amino acid transporters. This resulted not only in protection of OLs but also nearby neurons. MAG activation led to a PKC-dependent activation of factor Nrf2 (nuclear-erythroid related factor-2) leading to antioxidant responses including increased mRNA expression of metabolic enzymes from the glutathione biosynthetic pathway and the regulatory chain of cystine/Glu antiporter system xc^? increasing reduced glutathione (GSH), the main antioxidant in cells. The efficacy of early anti-MAG mAb administration was demonstrated in a preclinical model of excitotoxicity induced by intrastriatal Glu administration and extended to a model of Experimental Autoimmune Encephalitis showing axonal damage secondary to demyelination.ConclusionsMAG activation triggers Glu uptake into OLs under conditions of Glu overload and induces a robust protective antioxidant response.     

Involvement of Na/H exchanger and respiratory burst enzymes NADPH oxidase and NO synthase,in Cd-induced lipid peroxidation and DNA damage in haemocytes of mussels

This study investigated cadmium-induced oxidative and genotoxic effects, such as lipid peroxidation and disturbance of DNA integrity (DNA damage) in haemocytes of mussel Mytilus galloprovincialis and the possible involvement of Na⁺/H⁺ exchanger (NHE), and/or the main enzymes of respiratory burst, NADPH oxidase and nitric oxide (NO) synthase, in the induction of Cd toxic effects. In order to verify the role of either NHE, or NADPH oxidase and NO synthase in Cd-mediated toxicity, inhibitors such as ethyl-N-isopropyl-amiloride (EIPA), diphenyleneiodonium chloride (DPI) and N^G-nitro-l-arginine methyl ester (L-NAME) were used in each case. Moreover, phorbol-myristate acetate (PMA), a well-known protein kinase C (PKC)-mediated NADPH oxidase and NO synthase stimulator, as well as hydrogen peroxide (H₂O₂), a well-known genotoxic agent, was also used for elucidating the modulation of signaling molecules within cells, thus leading to the induction of lipid peroxidation and DNA damage. The results of the present study showed that micromolar concentrations of Cd (0.05–50 μΜ) could enhance both lipid peroxidation and DNA damage, possible via a PKC-mediated signaling pathway with the involvement of NHE, thus leading to the induction of NADPH oxidase and NO synthase activity, since inhibition of either NHE, or NADPH oxidase and NO synthase activity, significantly attenuates Cd-induced toxic effects in each case.     

Cytokine-Induced Accumulation of Very Long-Chain Fatty Acids in Rat C6 Glial Cells: Implication for X-Adrenoleukodystrophy

Abstract: X-Adrenoleukodystrophy (X-ALD) is an inherited metabolic disorder of very long-chain fatty acids (VLCFA) with subsequent manifestation of neuroinflammatory disease. To investigate the possible role of proinflammatory cytokines in the X-ALD disease process, we examined the effect of cytokines on the metabolism of VLCFA in C6 glial cells expressing oligodendrocyte-like properties. C6 glial cells under serum-free conditions were treated with different combinations of cytokines (tumor necrosis factor-α, interleukin-1β, interferon-γ) or cytokine with bacterial lipopolysaccharide (LPS). Cytokine-treated C6 cells had higher concentrations of VLCFA, measured as percent weight and also as C_26:0/C_22:0 ratio, which were 300–400% as compared with the controls. We also found increased levels of C_26:1 in cytokine-treated cells. The accumulation of VLCFA paralleled the decrease (35–55%) in peroxisomal β-oxidation activity and a 12- to 14-fold increase in the production of nitric oxide (NO). Individual cytokines were unable either to produce NO or to increase the levels of VLCFA in C6 cells. Inhibition of cytokine-induced NO production by l -N-methylarginine, an inhibitor of NO synthase (NOS), and N-acetylcysteine, an inhibitor of cytokine-mediated induction of inducible NOS, normalized the peroxisomal β-oxidation activity and the levels of VLCFA, suggesting a role for the proinflammatory cytokines and NO toxicity in the neuropathological changes associated with abnormal VLCFA metabolism (e.g., X-ALD). X-ALD is a peroxisomal disease having deficient oxidation of VLCFA, resulting in the excessive accumulation of VLCFA in all tissues but especially in brain. We observed greater increase in levels of VLCFA in the inflammatory region of ALD brain (in the demyelinating plaque and the area around the plaque) than in the normal-looking area away from the plaque; this also indicates that cytokines in the proinflammatory region may augment the VLCFA defect caused by the inherited abnormality in X-ALD brain. Although C6 glial cultured cells do not reflect the X-ALD model precisely, the observed relationship between the cytokine-induced inhibition of the oxidation of VLCFA, excessive accumulation of VLCFA, and excessive production of NO and their normalization by inhibitors of NOS in C6 glial cells suggests that NO-mediated toxicity may play a role in VLCFA-associated neuroinflammatory diseases (e.g., X-ALD).     

Atrial natriuretic peptide inhibits nitric oxide synthesis in mouse macrophages

The atrial natriuretic peptide (ANP) affects cardiovascular physiology, and, as has been suggested more recently, exerts immunomodulatory activities. In this context, we examined the effect of ANP on nitric oxide (NO) synthesis in murine bone marrow derived macrophages as well as in peritoneal macrophages. Cultured macrophages were stimulated with lipopolysaccharides (LPS 0.1–10 μg/ml) and NO synthesis was monitored by measuring increased concentrations of NO₂ in the medium. In initial experiments employment of N^G-monomethyl-L-arginine (L-NMMA) and dexamethasone, two specific inhibitors of nitric oxide synthase (NOS), confirmed the presence of inducible NOS activity in the cells. Exposure of cells to rat ANP99–126 in the range of 10⁻⁸ to 10⁻⁶ M significantly decreased LPS induced NO synthesis over 24 hours of incubation. Thus, ANP may alter macrophage function by affecting their nitric oxide synthesizing pathway.     

Mechanisms of neuroprotection by hemopexin: modeling the control of heme and iron homeostasis in brain neurons in inflammatory states

Hemopexin provides neuroprotection in mouse models of stroke and intracerebral hemorrhage and protects neurons in vitro against heme or reactive oxygen species (ROS) toxicity via heme oxygenase‐1 (HO1) activity. To model human brain neurons experiencing hemorrhages and inflammation, we used human neuroblastoma cells, heme–hemopexin complexes, and physiologically relevant ROS, for example, H₂O₂ and HOCl, to provide novel insights into the underlying mechanism whereby hemopexin safely maintains heme and iron homeostasis. Human amyloid precursor protein (hAPP), needed for iron export from neurons, is induced ~twofold after heme–hemopexin endocytosis by iron from heme catabolism via the iron‐regulatory element of hAPP mRNA. Heme–hemopexin is relatively resistant to damage by ROS and retains its ability to induce the cytoprotective HO1 after exposure to tert‐butylhydroperoxide, although induction is impaired, but not eliminated, by exposure to high concentrations of H₂O₂ in vitro. Apo‐hemopexin, which predominates in non‐hemolytic states, resists damage by H₂O₂ and HOCl, except for the highest concentrations likely in vivo. Heme–albumin and albumin are preferential targets for ROS; thus, albumin protects hemopexin in biological fluids like CSF and plasma where it is abundant. These observations provide strong evidence that hemopexin will be neuroprotective after traumatic brain injury, with heme release in the CNS, and during the ensuing inflammation. Hemopexin sequesters heme, thus preventing unregulated heme uptake that leads to toxicity; it safely delivers heme to neuronal cells; and it activates the induction of proteins including HO1 and hAPP that keep heme and iron at safe levels in neurons.     

Effects of the peptide toxin from Microcystis aeruginosa on intracellular calcium,pH and membrane integrity in mammalian cells

Extracts of water blooms of the toxic cyanobacterium Microcystis aeruginosa showed a range of toxicities not related to their ability to lyse mammalian red cells. The HPLC-purified heptapeptide toxin (mol. wt. 1035) from Microcystis did not lyse red cells at up to 500-fold higher concentrations than that required to kill mice. This toxin (LD₅₀ 110 μg/kg for male mice) was used to investigate in vitro effects on isolated thymocytes, hepatocytes, mammary alveolar cells, and cultured Swiss 3T3 fibroblasts. Thymocytes were stimulated to progressive Ca²⁺ entry by toxin (0.1–10 μg/ml), reaching a peak after approx. 5 min. No deformation, intracellular pH change, Trypan Blue entry or cell lysis was seen within 60 min at 37°C. Hepatocytes were grossly deformed by the toxin, with a dose/response relationship between 0.1 and 1.0 μg/ml. No progressive Ca²⁺ entry was observed on toxin addition, instead a rapid rise in intracellular Ca²⁺, presumably from intracellular sources. No change in intracellular pH, Trypan Blue exclusion or cell lysis was observed over 60 min. Mammary alveolar cells and 3T3 fibroblasts were unresponsive to toxin at the concentrations tested. No change in protein synthesis or nucleic acid synthesis in thymocytes was observed after culture with 0.5 or 5.0 μg/ml toxin. It was concluded that cytoskeletal changes in deformed hepatocytes (the target cells in vivo) demonstrated the most probable cellular basis for toxicity, rather than changes in membrane permeability or cell metabolism.     

Ischemia-Induced Taurine Release Is Modified by Nitric Oxide-Generating Compounds in Slices from the Developing and Adult Mouse Hippocampus

The novel neurotransmitter/neuromodulator nitric oxide (NO), which is linked to the activation of the N-methyl-D-aspartate class of glutamate receptors, has been shown to modify transmitter release in brain tissue. Release of the inhibitory amino acid taurine is also markedly enhanced by N-methyl-D-aspartate and NO-producing agents under normal conditions in the mouse hippocampus. The release of preloaded [³H]taurine from hippocampal slices from adult (3-month-old) and developing (7-day-old) mice was characterized under ischemic conditions in the presence of different NO-generating compounds, hydroxylamine, sodium nitroprusside, and S-nitroso-N-acetylpenicillamine (SNAP), using a superfusion system. The ischemia-induced taurine release at both ages was markedly enhanced by 1.0 mM nitroprusside and 1.0 mM SNAP, whereas 5.0 mM hydroxylamine was effective only in adults. The nitroprusside- and SNAP-induced releases were reduced by the inhibitors of NO synthase (nitroarginine and 7-nitroindazole) and NO-sensitive soluble guanylyl cyclase [1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one], suggesting involvement of the NO/cGMP pathway. The release in ischemia in the absence of Na⁺ was modified by NO compounds only in adults; the 0.1 mM N-methyl-D-aspartate stimulated taurine release at both ages. The enhanced release of taurine associated with NO production could be beneficial to brain tissue under cell-damaging conditions and corroborates the neuroprotective role of this amino acid, particularly in the immature brain.     

Uptake and metabolism of l-[3H]pyroglutamic acid in neuronal and glial cells in culture

The presence of an efficient uptake system for l-pyroglutamate was demonstrated in cultured glial cells originating from newborn rats. This compound is also transported by a high affinity uptake mechanism in neurons cultured from rat embryos cerebral hemispheres, but the V_max is 6 times lower than for glial cells. It is shown that l-pyroglutamate like l-glutamate is preferentially transported by glial cells, but with a V_max 40 to 60 times lower than for glutamate. The metabolism of l-pyroglutamate was also studied in cultured rat neuronal and glial cells, using l-[³H]pyroglutamate. Pyroglutamate, its metabolites and the various amino acids were separated by thin-layer electrophoresis. [³H]Pyroglutamate is more actively metabolised in glial cells than in neurons and glutamate is the main metabolite. Glutamate maximal specific activity is 4 times higher in glial than in neuronal cultures. It should also be noted that some [³H]pyroglutamate is transformed in [³H]GABA after longer incubation periods, but only in neurons. These results show the importance of glial cells for pyroglutamate uptake and metabolism in nervous tissue. They also suggest that pyroglutamate may interfere with glutamate neurotransmission in vivo.     

ATP Induces NO Production in Hippocampal Neurons by P2X7 Receptor Activation Independent of Glutamate Signaling

To assess the putative role of adenosine triphosphate (ATP) upon nitric oxide (NO) production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2′(3′)-O-(4-Benzoylbenzoyl) ATP (Bz-ATP) elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC₅₀ values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG) or by N^ω-propyl-L-arginine, suggesting the involvement of P2X₇Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA) receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV), but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity.     

Role of astrocyte connexin hemichannels in cortical spreading depression

Cortical spreading depression (CSD) is an intriguing phenomenon consisting of massive slow brain depolarizations that affects neurons and glial cells. It has been recognized since 1944, but its pathogenesis has only been uncovered during the last decade. Acute brain injuries can be further complicated by CSD in > 50% of severe cases. This phenomenon is repetitive and produces a metabolic overload that increments secondary damage. Propagation of CSD is known to be linked to excitotoxicity, but the mechanisms associated with its initiation remain less understood. It has been shown that CSD can be initiated by increases in extracellular [K⁺] ([K⁺]_e), and animal models use high [K⁺]_e to promote CSD. Connexin hemichannel activity increases due to high [K⁺]_e and low extracellular [Ca^2 +], conditions that occur after brain injury. Moreover, glial cell gap junction channels are fundamental in controlling extracellular medium composition, particularly in maintaining normal extracellular glutamate and K⁺ concentrations through “spatial buffering”. However, the role of astrocytic gap junctions under tissue stress can change to damage spread in the acute damage zone whereas the reduced communication in adjacent zone would reduce cell dead propagation. Here, we review the main findings associated with CSD, and discuss the possible involvement of astrocytic connexin-based channels in secondary damage propagation. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

Uptake and metabolism of choline in primary isolated neurons and glial cells in culture

The influx and metabolism of choline have been studied in primary cultures of isolated neurons and glial cells from chick embryo dissociated cerebral hemispheres. The results showed a correlation between both influx and metabolism of choline and the exogenous concentrations of choline. When neurons and glial cells were preincubated (10 min) and incubated in Krebs-Ringer phosphate solution with concentrations of choline lower (0.5 μM) or higher (150 μM) than the one present in the growth medium, the metabolism of choline, as a function of time, approached saturation following unusual kinetics. This suggests a non steady state of the endocellular concentrations of free choline. Moreover, when both neurons and glial cells were preincubated (10 min) with 50 μM choline and then incubated (2 min) with various concentrations of choline, only one uptake mechanism was measured, while the preincubation in the absence of choline followed by the incubation of the cells with various concentrations of choline showed the presence of two apparent K_m's with different affinities.The results also indicate the capacity of glial cells to incorporate choline suggesting a storage function for the cells.     

Reactive oxygen species plasmatic levels in ischemic stroke

Oxidative stress is probably one of the mechanisms involved in neuronal damage induced by ischemia-reperfusion, and the antioxidant activity of plasma may be an important factor providing protection from neurological damage caused by stroke-associated oxidative stress. The aim of this study was to investigate the status of oxidative stress, NO and ONOO⁻ levels in patients with atherothrombotic and lacunar acute ischemic stroke and iNOS, eNOS and nitrotyrosine expression in the same patients. Plasma ONOO⁻ levels were significantly higher in patients than in controls while NO decreases in patients in respect to controls. Densitometric analysis of bands indicated that iNOS and N-Tyr protein levels were significantly higher in patients in respect to controls. This study has highlighted a significant NO decrease in our patients compared with controls and this is most probably due to the increased expression of inducible NO synthase by the effect of thrombotic attack. In fact, the constitutive NO isoforms, which produce small amounts of NO, are beneficial, while activation of the inducible isoform of NO, which produces much more NO, causes injury, being its toxicity greatly enhanced by generation of peroxynitrite. The significant ONOO⁻ increase observed in our patients, compared to controls, is most probably due to reaction of NO with O₂^·−. These findings suggest that free radical production and oxidative stress in ischemic stroke might have a major role in the pathogenesis of ischemic brain injury. Peroxynitrite might be the main marker of brain damage and neurological impairment in acute ischemic stroke.     

Right Ventricular Adaptation Is Associated with the Glu298Asp Variant of the NOS3 Gene in Elite Athletes

Nitric oxide (NO), an important endogenous pulmonary vasodilator is synthetized by the endothelial NO synthase (NOS3). Reduced NO bioavailability and thus the Glu298Asp polymorphism of NOS3 may enhance right ventricular (RV) afterload and hypertrophic remodeling and influence athletic performance. To test this hypothesis world class level athletes (water polo players, kayakers, canoeists, rowers, swimmers, n = 126) with a VO₂ maximum greater than 50ml/kg/min were compared with non-athletic volunteers (n = 155). Cardiopulmonary exercise tests and cardiac magnetic resonance imaging (cMRI) were performed to determine structural or functional changes. Genotype distribution of the NOS3 Glu298Asp polymorphism was not affected by gender or physical performance. Cardiac MRI showed increased stroke volume with eccentric hypertrophy in all athletes regardless of their genotype. However, the Asp allelic variant carriers had increased RV mass index (32±6g versus 27±6g, p<0.01) and larger RV stroke volume index (71±10ml versus 64±10ml, p<0.01) than athletes with a Glu/Glu genotype. Genotype was not significantly associated with athletic performance. In the non-athletic group no genotype related differences were detected. The association between the NOS3 Glu298Asp polymorphism and RV structure and dimension in elite athletes emphasizes the importance of NOS3 gene function and NO bioavailability in sport related cardiac adaptation.     

Inflammatory neurodegeneration mediated by nitric oxide,glutamate, and mitochondria

In inflammatory, infectious, ischemic, and neurodegenerative pathologies of th central nervous system (CNS) glia become “activated” by inflammatory mediators, and express new proteins such as the inducible isoform of nitric oxide synthase (iNOS). Although these activated glia have beneficial roles, in vitro they potently kill cocultured neurons, and there is increasing evidence that they contribute to pathology in vivo. Nitric oxide (NO) from iNOS appears to be a key mediator of such glial-induced neuronal death. The high sensitivity of neurons to NO is partly due to NO causing inhibition of respiration, rapid glutamate release from both astrocytes and neurons, and subsequent excitotoxic death of the neurons. NO is a potent inhibitor of mitochondrial respiration, due to reversible binding of NO to cytochrome oxidase in competition with oxygen, resulting in inhibition of energy production and sensitization to hypoxia. Activated astrocytes or microglia cause a potent inhibition of respiration in cocultured neurons due to glial NO inhibiting cytochrome oxidase within the neurons, resulting in ATP depletion and glutamate release. In some conditions, glutamate-induced neuronal death can itself be mediated by N-methyl-d-aspartate (NMDA)-receptor activation of the neuronal isoform of NO synthase (nNOS) causing mitochondrial damage. In addition NO can be converted to a number of reactive derivatives such as peroxynitrite, NO₂, N₂O₃, and S-nitrosothiols that can kill cells in part by inhibiting mitochondrial respiration or activation of mitochondrial permeability transition, triggering neuronal apoptosis or necrosis.