Excess NO Research Articles

Late-stage systemic immune effectors inPlasmodium bergheiANKA infection: biopterin and oxidative stress

AbstractTo investigate the involvement of systemic oxidative stress in the pathogenesis of murine cerebral malaria, mice were infected with thePlasmodium berghei(P. berghei) ANKA 6653 strain. Serum tryptophan (Trp), kynurenine and urinary biopterin, liver, brain, spleen and serum superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA) and nitrite and nitrate (NOx) levels were measured on day 7 post-inoculation. Our data showed a significant decrease in SOD and an increase in GPx activity and MDA level in all the examined biological materials (p<0.05), except spleen. Conversely, GPx activities in spleen were depleted, while SOD and MDA levels remained unchanged. Increased MDA levels might indicate increased peroxynitrite production, lipid peroxidation and oxidative stress. Also, elevated urinary biopterin, which was accompanied by increased NOx (p<0.05), may support the inhibition of Trp degradation (p>0.05). The excessive NO synthesis inP. bergheiinfection may be related to the up-regulation of inducible NO synthase, which was in accordance with the increased biopterin excretion. Thus, the large quantities of released toxic redox active radicals attack cell membranes and induce lipid peroxidation. AlthoughP. bergheiinfection did not demonstrate systemic Trp degradation and related indoleamine-2,3-dioxygenase activity, it may cause multi-organ failure and death, owing to host-derived severe oxidative stress.

Excess Nitric Oxide (NO) Blunts Presynaptic Adrenergic Transduction in Orthostatic Intolerance (OI)

NO is a fundamental signaling molecule during postural blood redistribution. NO can centrally modulate sympathetic vasoconstriction by reducing sympathetic outflow, and can peripherally reduce sympathetic transduction at pre- and post-junctional sites of the adrenergic synapse. Prior data suggest blunted adrenergic vasoconstriction as a fundamental mechanism in orthostatic intolerance. We hypothesized that the production of NO in OI is enhanced resulting in reduced sympathetic noradrenergic neurotransmission which may be at pre- or post-junctional sites. To investigate this we predilated cutaneous arteries using adenosine as a [mostly] NO-independent vasodilator and local heat as a[mostly] NO-dependent vasodilator. We investigated the dose response to pre-synaptic vasoconstriction with tyramine and post-synaptic vasoconstriction with norepinephrine with or without NOS inhibition using nitro-L-arginine (NLA). The dose-responses of 8 OI (19 ± 3 yrs) patients were compared with 11 healthy controls (21 ±3 yrs). Data from OI and control subjects with and without NLA were compared by repeated measures ANOVA. OI subjects exhibited blunted vasoconstriction responses (P<0.001) to tyramine following adenosine pre-dilation which were similar to control with NLA. Tyramine dose-response following heat pre-dilation were blunted in both OI and controls. Norepinephrine dose-response was similar for OI and controls with and without NLA. The data confirm that NO modulates cutaneous sympathetic nerve transduction both pre- and post-synaptically in OI and controls. However, excessive endogenous NO blunts pre-synaptic adrenergic transduction in OI which may account, in part, for impaired sympathetic vasoconstriction.

De Novo Design of Potent and Selective Neuronal Nitric Oxide Synthase (nNOS) Inhibitors by a Fragment-Based Approach

Nitric oxide, a gaseous free radical molecule (NO) behaves, as a secondary messenger in various tissues. It is responsible for different physiological functions and pathological symptoms. Mammals contain three different nitric oxide synthase (NOS) isoforms: neuronal NOS (nNOS: in the brain, in peripheral nervous system and muscle tissues), inducible NOS (iNOS: in macrophage cells), endothelial NOS (eNOS: in endothelial cells). Under certain pathological conditions and/or after certain ages excessive NO produced in brain causes tissue damage and oxidative stress. It also reacts with other free radicals to create specific molecular modifications. The excessive production of NO, especially by nNOS (in brain) is implicated in various disease states such as neurodegeneration, stroke, migraine and Parkinsons, Alzheimers, and Huntingtons diseases. The active sites of three NOS isoforms show great similarity; therefore, designing of selective nNOS inhibitors is not an easy task. The computational results carried out with all of the docking tools clearly demonstrate that the selected scaffold is a potential candidate for further modifications and optimization for designing selective and potent nNOS inhibitors. Subtle differences in the conformations of amino acid sequences (e.g. ASP597 in nNOS) of the three isoforms in the active site region were the determining factors for the selectivity and the potency of the compounds. In this study several hundred compounds were screened in silico using the ZINCv12 lead library for prioritization of lead candidates. De novo design method was used rationally for the modifications of selected scaffold within a target-binding site in order to enhance its binding affinity and selectivity to nNOS enzyme. The potency and the selectivity of nNOS isoform were achieved by introducing 1-methyl amino group at the forth position of the imidazole moiety of the best inhibitor. The positively charged 1-methyl amino group makes three hydrogen bonds with the two-propionate groups of the heme cofactor, which does not occur in eNOS and iNOS. Removing of 1-methyl amino group from scaffold totally abolished both potency and selectivity for nNOS. Newly designed inhibitor 7 shows nNOS inhibition 23 and 17 fold better than both eNOS and iNOS, respectively.

Glutathione Supplementation Attenuates Oxidative Stress and Improves Vascular Hyporesponsiveness in Experimental Obstructive Jaundice.

We investigated the protective effects and mechanism of glutathione (GSH) on vascular hyporesponsiveness induced by bile duct ligation (BDL) in a rat model. Seventy-two male Sprague-Dawley rats were randomly divided into four groups: a NS group, a GSH group, a BDL + NS group, and a BDL + GSH group. GSH was administrated into rats in the GSH and BDL + GSH groups by gastric gavage. An equal volume of normal saline was, respectively, given in the NS group and BDL + NS group. Blood was gathered for serological determination and thoracic aorta rings were isolated for measurement of isometric tension. Obstructive jaundice led to a significant increase in the serum total bilirubin, AST, and ALT levels. The proinflammatory cytokines levels (TNF-α and IL-1β), concentration of NO, and oxidative stress markers (MDA and 3-NT) were increased as well. All of those were reduced by the treatment of GSH. Meanwhile, contraction of aorta rings to NA and vasorelaxation to ACh or SNP in the BDL group rats were markedly decreased, while GSH administration reversed this change. Our findings suggested that GSH supplementation attenuated overexpressed ONOO(−) from the reaction of excessive NO with O2 ∙- and protected against obstructive jaundice-induced vascular hyporesponsiveness in rats.

Telmisartan attenuates myocardial apoptosis induced by chronic intermittent hypoxia in rats: modulation of nitric oxide metabolism and inflammatory mediators.

NO and NO synthase (NOS) are known to play key roles in the development of myocardial apoptosis induced by ischemia/hypoxia. Current evidence suggests that angiotensin II type 1 receptor blockers, such as telmisartan, lower blood pressure and produce beneficial regulatory effects on NO and NOS. Here, we examined the protective role of telmisartan in myocardial apoptosis induced by chronic intermittent hypoxia (CIH). Adult male Sprague-Dawley rats were subjected to 8h of intermittent hypoxia/day, with/without telmisartan for 8weeks. Myocardial apoptosis, NO and NOS activity, and levels of inflammatory mediators and radical oxygen species were determined. Treatment with telmisartan preserved endothelial NOS expression and inhibited inducible NOS and excessive NO generation, while reducing oxidation/nitration stress and inflammatory responses. Administration of telmisartan before CIH significantly ameliorated the CIH-induced myocardial apoptosis. This study show that pre-CIH telmisartan administration ameliorated myocardial injury following CIH by attenuating CIH-induced myocardial apoptosis via regulation of NOS activity and inhibition of excessive NO generation, oxidation/nitration stress, and inflammatory responses.

Does nitrate fertilization induce nox emission from scots pine (p. sylvestris) shoots?

The possibility of NOx (NO + NO2) emissions from plants and the underlying mechanisms are still under discussion. Excess NO created possibly as a result of nitrite accumulation in plant leaves has been suggested to result in emissions. Such emission has been observed in laboratory conditions due to nitrate fertilization. In this study, we tested whether nitrate fertilization of Scots pine seedlings growing outdoors leads to accumulation of NO3- or NO2- in the needles and subsequent NOx emission. The experiment was done at the SMEAR II station in Southern Finland. The seedlings received nitrate or ammonium fertilizer or neither. Shoot NOx emissions were measured with dynamic chambers. Total dissolved nitrogen, inorganic nitrogen, NO3-, NO2- and NH4+ concentrations in the soil and needles were determined. NOx fluxes from the shoots were, on average, deposition. There was no indication of fertilization-induced NO or NOx emissions. The highest NO fluxes were observed at night and were humidity-related. It seems unlikely that additional nitrate in the soil could cause significant NOx emission from boreal Scots pine forests in field conditions, possibly because of soil chemistry.

Chemical amplification--cavity attenuated phase shift spectroscopy measurements of atmospheric peroxy radicals.

We describe a new instrument for the quantification of atmospheric peroxy radicals (HO2, CH3O2, C2H5O2, etc.) using the chemical amplification method. Peroxy radicals are mixed with high concentrations of NO and CO, causing a chain reaction that produces a measurable increase in NO2 which is quantified by cavity attenuated phase shift (CAPS) spectroscopy, a highly sensitive spectroscopic detection technique. The instrument utilizes two identical reaction chambers, each with a dedicated CAPS NO2 sensor. Similar to all dual-channel chemical amplifiers, one reaction chamber operates in amplification or "ROx" mode and the other in background or "Ox" mode. The peroxy radical mixing ratio is determined by the difference between the two channels' NO2 readings divided by a laboratory-determined chain length. Each reaction chamber alternates between ROx and Ox mode on an anti-synchronized schedule, eliminating the effect of CAPS baseline offsets on the calculated peroxy radical concentrations. The chain length is determined by a new calibration method: peroxyacetyl and methyl peroxy radicals are produced by the photolysis of acetone and quantified as NO2 following reaction with excess NO. We demonstrate the performance of the instrument with results from ambient sampling in Amherst and several diagnostics of its precision. The detection limit while sampling ambient air at a relative humidity (RH) of 40% is 0.6 ppt (1 min average, signal-to-noise ratio =2), with an estimated accuracy of 25% (2σ).

Scavenging properties of neutrophil 4-hydroxyphenylpyruvate dioxygenase are based on a hypothesis that does not stand up to scrutiny

It was previously reported by D’Eufemia et al. [9] that neutrophil preparations from a patient with tyrosinemia type III, i.e. with inherited deficiency of 4-hydroxyphenylpyruvate dioxygenase (HPPD), exhibited a far higher NO release than controls, when NO was estimated in terms of nitrite content in the suspending media. It was hypothesized that HPPD might participate to NO sequestration in neutrophils and that excessive NO release might reflect the lack of the scavenging action in defective cells. In recent control experiments, we found that HPPD activity in neutrophils preparations from healthy subjects is below the detection limit of the enzymatic assay (less than 3nmol product/h per mg protein). This indicates that HPPD concentration in neutrophils is very low, if any, confirming what was already suggested in literature, and rules out the possibility of a prominent role of HPPD as NO scavenger in these cells. Moreover, we found that 500μM l-tyrosine increases nitrite release and accumulation in suspending media of U-937 cells, a human monoblast-like lymphoma cell line which displays many characteristics of macrophages, including the expression of inducible and endothelial nitric oxide synthases. We hypothesize that the increase of nitrite release by patient's neutrophils might be related to the presence of high l-tyrosine concentrations in the blood samples (426μmol/L instead of 52.1±10.9μmol/L as healthy subjects), rather than to HPPD deficiency of in these cells.

Migratory Insertion and Reductive Coupling of Tetraarylruthenium(IV) Complexes

Tetraarylruthenium(IV) complexes have been synthesized and their migratory insertion, reductive coupling, and bromination reactions investigated. Treatment of [Ru(acac)3] (acac– = acetylacetonate) with RMgBr, followed by column chromatography in air afforded the tetraaryl complexes [RuR4] (R = 2,5-dimethylphenyl (R1); 4-methoxy-2-methylphenyl (R2)). Oxidation of [RuR24] with AgBF4 gave the Ru(V) complex [RuVR24](BF4), which has a measured μeff of 1.8 μB. [RuR4] and can catalyze the oxidation of methyl p-tolyl sulfide with PhIO. Reaction of [RuR24] with PhICl2 led to C–C reductive coupling and the formation of the Ru(II) η6-biaryl complex [(η6-R2-R2)RuCl2]2 (1). Treatment of [RuR14] with excess NO gave the tetranuclear Ru(II) aryl-N-nitrosohydroxylaminato complex [RuR1(N){κ2-O,O′-ON(R1)NO}(μ-NO2)]4 (2). Bromination of [RuR14] with N-bromosuccinimide resulted in formation of [RuR′4] (R′ = 4-bromo-2,5-dimethylphenyl) (3). The crystal structures of [RuR24], [RuR24](BF4), and 1–3 have been determined.

Nitric oxide PLIF measurement in a point-to-plane pulsed discharge in vitiated air of a propane/air flame

The effect of a point-to-plane pulsed discharge on the vitiated downstream of a propane/air flame has been investigated by phase-locked NO planar laser-induced-fluorescence (PLIF) measurements. Phase-locked NO PLIF measurements with the variation of pulsed plasma energy, equivalence ratio and applied voltage rise time have been performed. Fast rise time (25 ns) and slower rise time (150 ns) high-voltage pulsers are used to produce NO radical densities greater than the ambient flame-produced NO radicals in lean, balanced and rich premixed flames. The pulsed plasma produced excess NO radical densities were found to decay to 50% level with time constants greater than 250 µs in the burnt gas regions with gas temperatures greater than 1000 K. The super-equilibrium NO populations were dependent on energy deposited and overall equivalence ratio, but independent of voltage pulse rise time for similar energy deposition per pulse. Due to long NO radical density decay lifetimes, super-equilibrium NO populations are convected away from production regions with the ambient flow and observed in downstream exhaust gas regions.

A measurement of total reactive nitrogen, NOy, together with NO₂, NO, and O₃ via cavity ring-down spectroscopy.

We present a sensitive, compact detector that measures total reactive nitrogen (NOy), as well as NO2, NO, and O3. In all channels, NO2 is directly detected by laser diode based cavity ring-down spectroscopy (CRDS) at 405 nm. Ambient O3 is converted to NO2 in excess NO for the O3 measurement channel. Likewise, ambient NO is converted to NO2 in excess O3. Ambient NOy is thermally dissociated at ∼700 °C to form NO2 or NO in a heated quartz inlet. Any NO present in ambient air or formed from thermal dissociation of other reactive nitrogen compounds is converted to NO2 in excess O3 after the thermal converter. We measured thermal dissociation profiles for six of the major NOy components and compared ambient measurements with other instruments during field campaigns in Utah and Alabama. Alabama measurements were made in a rural location with high biogenic emissions, and Utah measurements were made in the wintertime in unusual conditions that form high ozone levels from emissions related to oil and gas production. The NOy comparison in Alabama, to an accepted standard measurement method (a molybdenum catalytic converter/chemiluminescence instrument), agreed to within 12%, which we define as an upper limit to the accuracy of the NOy channel. The 1σ precision is <30 pptv at 1 s and <4 pptv at 1 min time resolution for all measurement channels. The accuracy is 3% for the NO2 and O3 channels and 5% for the NO channel. The precision and accuracy of this instrument make it a versatile alternative to standard chemiluminescence-based NOy instruments.

Calcium-mediated signaling and calmodulin-dependent kinase regulate hepatocyte-inducible nitric oxide synthase expression

BackgroundInduced nitric oxide synthase (iNOS) is induced in hepatocytes by shock and inflammatory stimuli. Excessive NO from iNOS mediates shock-induced hepatic injury and death, so understanding the regulation of iNOS will help elucidate the pathophysiology of septic shock. In vitro, cytokines induce iNOS expression through activation of signaling pathways including mitogen-activated protein kinases and nuclear factor κB. Cytokines also induce calcium (Ca2+) mobilization and activate calcium-mediated intracellular signaling pathways, typically through activation of calmodulin-dependent kinases (CaMK). Calcium regulates NO production in macrophages but the role of calcium and calcium-mediated signaling in hepatocyte iNOS expression has not been defined. Materials and methodsPrimary rat hepatocytes were isolated, cultured, and induced to produce NO with proinflammatory cytokines. Calcium mobilization and Ca2+-mediated signaling were altered with ionophore, Ca2+ channel blockers, and inhibitors of CaMK. ResultsThe Ca2+ ionophore A23187 suppressed cytokine-stimulated NO production, whereas Ethylene glycol tetraacetic acid and nifedipine increased NO production, iNOS messenger RNA, and iNOS protein expression. Inhibition of CaMK with KN93 and CBD increased NO production but the calcineurin inhibitor FK 506 decreased iNOS expression. ConclusionsThese data demonstrate that calcium-mediated signaling regulates hepatocyte iNOS expression and does so through a mechanism independent of calcineurin. Changes in intracellular calcium levels may regulate iNOS expression during hepatic inflammation induced by proinflammatory cytokines.

Exercise-induced cardioprotection: a role for eNOS uncoupling and NO metabolites

Exercise is an efficient strategy for myocardial protection against ischemia-reperfusion (IR) injury. Although endothelial nitric oxide synthase (eNOS) is phosphorylated and activated during exercise, its role in exercise-induced cardioprotection remains unknown. This study investigated whether modulation of eNOS activation during IR could participate in the exercise-induced cardioprotection against IR injury. Hearts isolated from sedentary or exercised rats (5 weeks training) were perfused with a Langendorff apparatus and IR performed in the presence or absence of NOS inhibitors [N-nitro-L-arginine methyl ester, L-NAME or N5-(1-iminoethyl)-L-ornithine, L-NIO] or tetrahydrobiopterin (BH₄). Exercise training protected hearts against IR injury and this effect was abolished by L-NAME or by L-NIO treatment, indicating that exercise-induced cardioprotection is eNOS dependent. However, a strong reduction of eNOS phosphorylation at Ser1177 (eNOS-PSer1177) and of eNOS coupling during early reperfusion was observed in hearts from exercised rats (which showed higher eNOS-PSer1177 and eNOS dimerization at baseline) in comparison to sedentary rats. Despite eNOS uncoupling, exercised hearts had more S-nitrosylated proteins after early reperfusion and also less nitro-oxidative stress, indexed by lower malondialdehyde content and protein nitrotyrosination compared to sedentary hearts. Moreover, in exercised hearts, stabilization of eNOS dimers by BH4 treatment increased nitro-oxidative stress and then abolished the exercise-induced cardioprotection, indicating that eNOS uncoupling during IR is required for exercise-induced myocardial cardioprotection. Based on these results, we hypothesize that in the hearts of exercised animals, eNOS uncoupling associated with the improved myocardial antioxidant capacity prevents excessive NO synthesis and limits the reaction between NO and O₂·- to form peroxynitrite (ONOO⁻), which is cytotoxic.

Denitrification and Potential Nitrous Oxide and Carbon Dioxide Production in Brownfield Wetland Soils

Brownfields, previously developed sites that are derelict, vacant, or underused, are ubiquitous in urban areas. Wetlands on brownfields often retain rain and stormwater longer than the surrounding landscape because they are low-lying; this increases the possibility for these areas to process waterborne contaminants from the urban environment. In the northeastern United States, atmospheric deposition of nitrate (NO) is high. Denitrification, a microbial process common in wetlands, is a means of removing excess NO. Nitrogen gas is the desired end product of denitrification, but incomplete denitrification results in the production of NO, a greenhouse gas. The goal of this study was to investigate the potential of brownfield wetlands to serve as sinks for inorganic nitrogen and sources of greenhouse gases. We examined limitations to denitrification and NO production in brownfield wetland soils in New Jersey. Soil C:N ratios were high (18-40) and intact core denitrification (-0.78 to 11.6 μg NO-N kg dry soil d) and N mineralization (0.11-2.97 mg N kg dry soil d) were low for all sites. However, soil NO increased during dry periods. Nitrate additions to soil slurries increased denitrification rates, whereas labile C additions did not, indicating that soil denitrifiers were nitrogen limited. Incubations indicated that the end product of denitrification was primarily NO and not N. These results indicate that brownfield wetlands can develop significant denitrification capacity, potentially causing NO limitation. They might be significant sinks for atmospheric NO but may also become a significant source of NO if NO deposition were to increase.

O‐GlcNAcylation and p50/p105 binding of c‐Rel are dynamically regulated by LPS and glucosamine in BV2 microglia cells

Previously, we demonstrated that glucosamine (GlcN) exerts a suppressive effect on LPS-induced inducible NOS (iNOS) through the inhibition of NF-κB activation in BV2 mouse microglial cells. The purpose of the present study was to examine the mechanisms by which GlcN inhibits NF-κB activation. BV2 cells were stimulated with LPS with or without GlcN. NF-κB/c-Rel activities were studied by EMSA, nuclear translocation, reporter assay or chromatin immunoprecipitation. Wheat germ agglutinin precipitation or galactosyltransferase assay were used to measure O-linked N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) of c-Rel. Protein-protein interactions were examined by co-immunoprecipitation. LPS stimulated the activation of c-Rel, increased the O-GlcNAcylation of c-Rel and enhanced the binding of c-Rel to the NF-κB site in the iNOS promoter; GlcN attenuated these effects of LPS. O-GlcNAcylation of both nuclear and cytosolic forms of c-Rel was increased by LPS and reduced by GlcN. LPS increased the interaction of c-Rel with O-GlcNAc transferase (OGT) and p50/p105, and GlcN suppressed these interactions. Knockdown of OGT reduced the c-Rel O-GlcNAcylation and c-Rel-p50 interaction in response to LPS, but did not affect either the binding of c-Rel to the iNOS promoter or the transcriptional activity of c-Rel. In BV2 microglial cells, the anti-inflammatory effect of GlcN is mediated by prevention of the prolonged activation of transcription factors, c-Rel and NF-κB. Further clarification of the mechanism by which GlcN exerts this effect will facilitate the development of pharmacological strategies for preventing excessive NO formation when targeting inflammatory diseases of the periphery or CNS.

Decoding the Substrate Supply to Human Neuronal Nitric Oxide Synthase

Nitric oxide, produced by the neuronal nitric oxide synthase (nNOS) from L-arginine is an important second messenger molecule in the central nervous system: It influences the synthesis and release of neurotransmitters and plays an important role in long-term potentiation, long-term depression and neuroendocrine secretion. However, under certain pathological conditions such as Alzheimer’s or Parkinson’s disease, stroke and multiple sclerosis, excessive NO production can lead to tissue damage. It is thus desirable to control NO production in these situations. So far, little is known about the substrate supply to human nNOS as a determinant of its activity. Measuring bioactive NO via cGMP formation in reporter cells, we demonstrate here that nNOS in both, human A673 neuroepithelioma and TGW-nu-I neuroblastoma cells can be fast and efficiently nourished by extracellular arginine that enters the cells via membrane transporters (pool I that is freely exchangeable with the extracellular space). When this pool was depleted, NO synthesis was partially sustained by intracellular arginine sources not freely exchangeable with the extracellular space (pool II). Protein breakdown made up by far the largest part of pool II in both cell types. In contrast, citrulline to arginine conversion maintained NO synthesis only in TGW-nu-I neuroblastoma, but not A673 neuroepithelioma cells. Histidine mimicked the effect of protease inhibitors causing an almost complete nNOS inhibition in cells incubated additionally in lysine that depletes the exchangeable arginine pool. Our results identify new ways to modulate nNOS activity by modifying its substrate supply.

Endothelial NO and O2[rad]− production rates differentially regulate oxidative, nitroxidative, and nitrosative stress in the microcirculation

Endothelial dysfunction causes an imbalance in endothelial NO and O2− production rates and increased peroxynitrite formation. Peroxynitrite and its decomposition products cause multiple deleterious effects including tyrosine nitration of proteins, superoxide dismutase (SOD) inactivation, and tissue damage. Studies have shown that peroxynitrite formation during endothelial dysfunction is strongly dependent on the NO and O2− production rates. Previous experimental and modeling studies examining the role of NO and O2− production imbalance on peroxynitrite formation showed different results in biological and synthetic systems. However, there is a lack of quantitative information about the formation and biological relevance of peroxynitrite under oxidative, nitroxidative, and nitrosative stress conditions in the microcirculation. We developed a computational biotransport model to examine the role of endothelial NO and O2− production on the complex biochemical NO and O2− interactions in the microcirculation. We also modeled the effect of variability in SOD expression and activity during oxidative stress. The results showed that peroxynitrite concentration increased with increase in either O2− to NO or NO to O2− production rate ratio (QO2−/QNO or QNO/QO2−, respectively). The peroxynitrite concentrations were similar for both production rate ratios, indicating that peroxynitrite-related nitroxidative and nitrosative stresses may be similar in endothelial dysfunction or inducible NO synthase (iNOS)-induced NO production. The endothelial peroxynitrite concentration increased with increase in both QO2−/QNO and QNO/QO2− ratios at SOD concentrations of 0.1–100μM. The absence of SOD may not mitigate the extent of peroxynitrite-mediated toxicity, as we predicted an insignificant increase in peroxynitrite levels beyond QO2−/QNO and QNO/QO2− ratios of 1. The results support the experimental observations of biological systems and show that peroxynitrite formation increases with increase in either NO or O2− production, and excess NO production from iNOS or from NO donors during oxidative stress conditions does not reduce the extent of peroxynitrite mediated toxicity.

Calcium-sensing receptor antagonist (calcilytic) NPS 2143 specifically blocks the increased secretion of endogenous Aβ42 prompted by exogenous fibrillary or soluble Aβ25–35 in human cortical astrocytes and neurons—Therapeutic relevance to Alzheimer's disease

The “amyloid-β (Aβ) hypothesis” posits that accumulating Aβ peptides (Aβs) produced by neurons cause Alzheimer's disease (AD). However, the Aβs contribution by the more numerous astrocytes remains undetermined. Previously we showed that fibrillar (f)Aβ25–35, an Aβ42 proxy, evokes a surplus endogenous Aβ42 production/accumulation in cortical adult human astrocytes. Here, by using immunocytochemistry, immunoblotting, enzymatic assays, and highly sensitive sandwich ELISA kits, we investigated the effects of fAβ25–35 and soluble (s)Aβ25–35 on Aβ42 and Aβ40 accumulation/secretion by human cortical astrocytes and HCN-1A neurons and, since the calcium-sensing receptor (CaSR) binds Aβs, their modulation by NPS 2143, a CaSR allosteric antagonist (calcilytic). The fAβ25–35-exposed astrocytes and surviving neurons produced, accumulated, and secreted increased amounts of Aβ42, while Aβ40 also accrued but its secretion was unchanged. Accordingly, secreted Aβ42/Aβ40 ratio values rose for astrocytes and neurons. While slightly enhancing Aβ40 secretion by fAβ25–35-treated astrocytes, NPS 2143 specifically suppressed the fAβ25–35-elicited surges of endogenous Aβ42 secretion by astrocytes and neurons. Therefore, NPS 2143 addition always kept Aβ42/Aβ40 values to baseline or lower levels. Mechanistically, NPS 2143 decreased total CaSR protein complement, transiently raised proteasomal chymotrypsin activity, and blocked excess NO production without affecting the ongoing increases in BACE1/β-secretase and γ-secretase activity in fAβ25–35-treated astrocytes. Compared to fAβ25–35, sAβ25–35 also stimulated Aβ42 secretion by astrocytes and neurons and NPS 2143 specifically and wholly suppressed this effect. Therefore, since NPS 2143 thwarts any Aβ/CaSR-induced surplus secretion of endogenous Aβ42 and hence further vicious cycles of Aβ self-induction/secretion/spreading, calcilytics might effectively prevent/stop the progression to full-blown AD.

English

Introduction In this study, we report on the role of epidermal growth factor receptor (EGFR) transactivation in H. pylori lipopolysaccharide (LPS)-elicited induction in gastric mucosal expression of COX-2 and iNOS. Materials and methods We aimed to demonstrate that the LPS-induced p38 activation along with MMPs are of critical significance to EGFR transactivation that leads to up-regulation in the activation of ERK signalling cascade, amplification in iNOS and COX-2 induction, and consequently to the excessive increase in gastric mucosal PGE2 and NO generation. Results EGFR transactivation results in the amplification of the LPS-induced ERK phosphorylation and up-regulation in ERK-mediated IKK-β activation for the enhanced induction of NFκB-dependent expression of iNOS. The rise in iNOS-dependent NO generation, in turn, leads to an up-regulation in COX-2 activation through Snitrosylation and excessive PGE2 production. Conclusion Taken together, our data provide strong indications of the involvement of EGFR transactivation in the amplification of ERK signalling cascade associated with up-regulation in gastric mucosal induction of iNOS and COX-2, and excessive NO and PGE2 generation in response to H. pylor

The threshold of pentylenetetrazole-induced convulsive seizures, but not that of nonconvulsive seizures, is controlled by the nitric oxide levels in murine brains

Alterations in the NO pathway play an important role in the development of convulsive seizures via the glutamatergic and GABAergic systems in acute pentylenetetrazole (PTZ) seizure animals. We previously reported that the background NO levels under physiological conditions negatively regulate convulsive seizures, while excess NO levels under pathologic conditions positively regulate PTZ-induced convulsive seizures. In this study, the NO content in various brain regions after a single dose injection of PTZ was quantitatively and directly measured using the ex vivo X-band electron paramagnetic resonance method with an NO-trapping agent. Experimental data demonstrated the effects of NO on the convulsive seizure threshold: a 1.5-fold increase in the NO level in all brain regions compared to that observed in the control state showed proconvulsive properties without any involvement with nonconvulsive seizures. The distribution of the background NO content in the normal animals was higher in the temporal region of the cerebral cortex, including the amygdala, than in the hippocampus, cerebellum and other regions of the cerebral cortex. However, the levels of NO after the occurrence of acute PTZ-induced convulsive seizures significantly increased by more than 50% in all brain regions, thus suggesting that the NO levels in all brain regions contribute to PTZ-induced convulsions as a seizure threshold. In a pharmacological study, the inhibitor of neuronal NO synthase and antagonists of ionotropic glutamate receptors prevented PTZ-induced convulsions and excessive NO generation. In addition, therapeutic drugs, such as valproate and ethosuximide used to treat generalized seizures not only inhibited the increase in NO generation induced by PTZ, but also prevented both convulsive and nonconvulsive seizures caused by PTZ. We herein provide novel insight into the involvement of NO in PTZ-seizure susceptibility at the whole-animal level.