Oxide Release Research Articles

γ-Glutamyltransferase catabolism of S-nitrosoglutathione modulates IL-8 expression in cystic fibrosis bronchial epithelial cells

S-nitrosoglutathione (GSNO) is an endogenous nitrosothiol involved in several pathophysiological processes. A role for GSNO has been envisaged in the expression of inflammatory cytokines such as IL-8; however, conflicting results have been reported. γ-Glutamyltransferase (GGT) enzyme activity can hydrolyze the γ-glutamyl bond present in the GSNO molecule thus greatly accelerating the release of bioactive nitric oxide. Expression of GGT is induced by oxidative stress, and activated neutrophils contribute to GGT increase in cystic fibrosis (CF) lung exudates by releasing GGT-containing microvesicles. This study was aimed at evaluating the effect of GSNO catabolism mediated by GGT on production of IL-8 in CF transmembrane regulation protein-mutated IB3-1 bronchial cells. The rapid, GGT-catalyzed catabolism of GSNO caused a decrease in both basal and lipopolysaccharide-stimulated IL-8 production in IB3-1 cells, by modulating both NF-κB and ERK1/2 pathways, along with a decrease in cell proliferation. In contrast, a slow decomposition of GSNO produced a significant increase in both cell proliferation and expression of IL-8, the latter possibly through p38-mediated stabilization of IL-8 mRNA. Our data suggest that the differential GSNO catabolism mediated by GGT enzyme activity can downregulate the production of IL-8 in CF cells. Hence, the role of GGT activity should be considered when evaluating GSNO for both in vitro and in vivo studies, the more so in the case of GSNO-based therapies for cystic fibrosis.

Abstract 208: Altered Cerebrovascular Reactivity Following One Month of Obstructive Sleep Apnea

Obstructive sleep apnea (OSA) has been identified as a significant risk factor for stroke. However, little is known regarding the effects of OSA on the cerebrovascular wall. Using a novel rodent model of OSA we assessed the hypothesis that (1) OSA augments endothelin (ET-1) induced constrictions of cerebral arteries and (2) OSA attenuates dilations of cerebral arteries by agonist-induced nitric oxide (NO) release from the cerebrovascular endothelium. The repetitive airway closures associated with OSA lead to intermittent hypoxia/hypercapnia and reoxygenation, increased negative intrathoracic pressures, and arousals. In order to model the physiological consequences of OSA, we have chronically instrumented rats with inflatable endotracheal obstruction devices. Unanesthetized freely-ranging rats underwent 30 apneas/ hour for 8 hours/ day (sleep phase) for 1 month. During apnea pO 2 decreased from 122±3 to 67±3 mm Hg; pCO 2 increased from 43±1 to 51±1 mm Hg; pH decreased from 7.46±0.00 to 7.38±0.01; and hemoglobin O 2 saturation decreased from 94±1 to 82±1 % (n=5 and p<0.05 for each). Following 1 month of OSA blood pressure, plasma ET-1 and NO levels were similar in sham and OSA rats. Using the pressurized cerebral artery preparation, we observed a 17.5-fold increase in sensitivity to ET-1 (n=5-6, p<0.05) after 1 month of OSA. The increased sensitivity of OSA cerebral arteries to ET-1 was abolished by the ET-B receptor antagonist BQ-788 (n=6, NS). Additionally, constrictions to the ET-B specific agonist IRL-1620 were significantly greater in OSA, versus sham, cerebral arteries (n=6, p<0.05). Dilations to ATP (a P2Y 2 agonist which stimulates NO production in the endothelium) were attenuated in cerebral arteries from OSA rats by 40% (n=5-8, p<0.05). However dilations to the NO-donor MAHMA-NOnoate were similar between groups. In conclusion, 1 month of OSA results in (1) increased sensitivity of cerebral arteries to ET-1, likely through upregulation of ET-B receptors on the vascular smooth muscle and (2) decreased endothelial-derived NO production. These data suggest that OSA results in significant alterations to the cerebrovascular wall in the absence of hypertension.

PROTECTIVE ROLE OF DDAH2 GENE HAPLOTYPES IN PATIENTS WITH MYOCARDIAL INFARCTION

Several polymorphisms of the dimethylarginine dimethylaminohydrolase (DDAH) genes have been studied in cardiovascular diseases. These molecules play an important role in the regulation of nitric oxide synthesis and release. The aim of the present study was to establish the role of DDAH gene

ROLE OF EICOSANOIDS IN REGULATING CAPILLARY PERFUSION: EVALUATION OF CAPILLARY BLOOD VOLUME WITH CONTRAST ULTRASOUND

Contrast-enhanced ultrasound (CEU) can quantify tissue capillary blood volume (CBV). This technique has been used to study functional control of capillaries in response to metabolic stimuli. Increases in muscle CBV in response to insulin are partially regulated by nitric oxide (NO) release. However

1066 – Inflammation as common denominator of ED and MDD

Introduction Comorbidity between ED and depression is high. Causal relation between two is bidirectional. Person with ED can have MDD because of psychological factors. MDD symptoms and associated hypogonadism affect on libido and erectile function. Diseases like ankolysing spondolysis, asthma, rheumatoid arthritis, coronary heart disease, psoriasis, diabetes, and multiple sclerosis have strong co morbid ED and MDD. Inflammation is common overlapping factor in these illnesses. Method An internet search was made on pubmed, and psychiatrist.com, using key words, major depression, endothelial dysfunction, inflammation, erectile dysfunction, and cytokines. This was followed by study of relevant martial in different journal and books. Result Men with ED had high levels of depressive, somatic, and anxious symptoms. Approximately one-third of people with MDD had higher levels of peripheral inflammatory marker. Incidences of ED are two fold higher in depressed people as compared with nondepressed. Discussion Levels of pro inflammatory cytokine like IL-1,I patient L-6,TNF,CRP, interferon-gamma and homosystien are raised in MDD. Cytokines effect on the HPA axis, glucocorticoid resistance, neurotransmission and hippocampal neurogenesis. Patients treated with interferon have higher risk of developing MDD .Antidepressant therapy results in decreased inflammatory response. Erectile function depends on integrity of endothelium which is target of inflammation. Endothelial dysfunction is linked to ED as they share a common pathway through the release of nitric oxide. Proinflammatory cytokines and homosystien play role, both in ED and MDD. Conclusion Inflammation and its markers need to be considered in cases of co morbid ED and MDD.

Novel Role of Neuropeptide Y in the Modulation of Microglia Activity

Microglia are the smallest member of the glia family but greatly responsible for vital physiological responses to brain injury and/or infection. The resulting inflammatory process includes the recruitment of microglial cells to the site of injury, the release of both toxic and trophic molecules and the clearance of invading pathogens or dead cells. The purpose of this review is to detail the role of neuropeptide Y (NPY) over microglia principal activities. In addition, we also summarize the main effects of other neuropeptides, such as galanin, somatostatin, substance P, pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP). Recently, we showed that NPY downregulates the innate immune response, by decreasing the release of interleukin-1 and nitric oxide, migration and phagocytosis by activated microglia cells. A huge body of evidence has also showed that the reduction of microglia inflammatory responses supports regenerative mechanisms aiming at the protection of susceptible neurons in several brain diseases. Therefore, the neuroprotective and immunosuppressive effects of NPY in microglia cells suggest that it can be a key therapeutic target against Central Nervous System injury or disease.

Panax notoginseng saponin attenuates hypoxia/reoxygenation-induced oxidative stress in cortical neurons.

The present study monitored the effect of 2, 10, and 50 mg/L of Panax notoginseng saponin exposure following hypoxia-reoxygenation injury in fetal rat cortical neurons. Results showed that varying doses of Panax notoginseng saponin significantly enhanced the cell viability of neurons, reduced malondialdehyde content, increased superoxide dismutase activity, inhibited mRNA and protein expression of inducible and neuronal nitric oxide synthase, and decreased the release of nitric oxide in hypoxia/reoxygenation injured cells. In particular, 50 mg/L of Panax notoginseng saponin was the most effective dose. These findings suggest that Panax notoginseng saponin can attenuate neuronal oxidative stress injury caused by hypoxia/reoxygenation in a dose-dependent manner.

Bacterial nitrous oxide reductase expressed in transgenic plants: Evidence for sufficient anaerobicity to permit activity

Wan, S., Goto, K., Mottiar, Y., Staebler, J. M., Johnson, A. M., Voronova, A., Blais, D. R., Zaidi, M. A. and Altosaar, I. 2012. Bacterial nitrous oxide reductase expressed in transgenic plants: Evidence for sufficient anaerobicity to permit activity. Can. J. Plant Sci. 92: 1283-1294. Soil nitrogen enrichment practices enhance crop yields but greatly exacerbate the production and release of nitrous oxide (N2O), a potent greenhouse gas. The conversion of N2O to dinitrogen (N2) in soil denitrifiers is normally driven by the anaerobic enzyme nitrous oxide reductase (N2OR). Since this step is often limited in fertilised soils, we have transferred this unique microbial biocatalyst from the soil bacterium Pseudomonas stutzeri to transgenic Nicotiana tabacum plants. Our results confirm that engineered plants expressing the N2OR structural gene nosZ are capable of producing functional reductase enzyme without the involvement of the native accessory gene products. Since bacterial N2OR normally exists in the anaero...

Bacterial nitrous oxide reductase expressed in transgenic plants: Evidence for sufficient anaerobicity to permit activity

Wan, S., Goto, K., Mottiar, Y., Staebler, J. M., Johnson, A. M., Voronova, A., Blais, D. R., Zaidi, M. A. and Altosaar, I. 2012. Bacterial nitrous oxide reductase expressed in transgenic plants: Evidence for sufficient anaerobicity to permit activity. Can. J. Plant Sci. 92: 1283–1294. Soil nitrogen enrichment practices enhance crop yields but greatly exacerbate the production and release of nitrous oxide (N2O), a potent greenhouse gas. The conversion of N2O to dinitrogen (N2) in soil denitrifiers is normally driven by the anaerobic enzyme nitrous oxide reductase (N2OR). Since this step is often limited in fertilised soils, we have transferred this unique microbial biocatalyst from the soil bacterium Pseudomonas stutzeri to transgenic Nicotiana tabacum plants. Our results confirm that engineered plants expressing the N2OR structural gene nosZ are capable of producing functional reductase enzyme without the involvement of the native accessory gene products. Since bacterial N2OR normally exists in the anaerobic environment of the soil bacterium's periplasmic space, this novel in planta activity suggests that plant cells can provide a similar hypoxic refuge for anaerobic enzyme activity. Genetically engineered crops containing N2OR could have considerable potential in the phytoremediation of atmospheric N2O.

The effects of nicotine on nitric oxide induced anxiogenic-like behaviors in the dorsal hippocampus

Nicotine, an active tobacco derived alkaloid, regulates the activity of the neuronal nitric oxide synthase (nNOS) as well as the release of nitric oxide (NO) in the nervous system. Nicotinic acetylcholine receptors and nNOS are abundantly co-expressed in the hippocampal neurons and are found to alter anxiety-like behaviors in rodents. Dorsal hippocampus may be a site for modulation of anxiety. Therefore, in this study, we investigated the possible interactions between nicotine and NO systems of the dorsal hippocampus and the resultant effect on anxiety-like behaviors. The elevated plus-maze (EPM) test has been used to test the anxiety. Intraperitoneal administration of nicotine (0.5mg/kg) decreased the open arm time percentage (%OAT) and open arm entries percentage (%OAE) but not the locomotor activity, indicating an anxiogenic-like response. Intra-CA1 injection of l-arginine (a NO precursor) or l-NAME (a NOS inhibitor) also caused anxiogenic-like effects. On the other hand, injection of the low dose nicotine before different doses of l-arginine or l-NAME blocked the anxiogenic-like response induced by the drugs. Our results suggested that, both NO and nicotinic cholinergic systems not only play a part in the modulation of the anxiety in mice dorsal hippocampus, but also demonstrate a complex interaction in this respect.

P043 Ciliary neurotrophic factor (CNTF) downregulates CAMKII-AMPK pathway protecting MIN6 cells against apoptosis induced by IL-1beta

Introduction Background: Diabetes Mellitus is a metabolic syndrome of multiple etiologies, resulting from a lack of insulin and sometimes associated with an increase in resistance to the hormone by insulin-target tissues. DM1 is characterized by the infiltration of macrophages and T-type CD4+ and T-CD8+ cells in the pancreas, due to the failure of the autoimmune system, causing inflammation and leading to the release of nitric oxide, free radicals, and cytokines such as IL1β and IFNγ. These pro-inflammatory cytokines activate pro-apoptotic mechanisms leading to a loss in functional beta-cell mass. These mechanisms can be reproduced in vitro by the exposure of beta cells to pro-inflammatory cytokines, such as IL-1β. In contrast, CNTF is a cytokine that promotes pancreatic islet cell survival. CaMKII is Calmodulin-Ca2+ activated enzyme that acts in memory formation and release process. This enzyme is important in the insulin secretion and in, together with LKB1, the masters AMPK activators. AMPK is a kinase that acts on pancreatic beta cells as a sensor of the cellular energy state. It is well known that in pancreatic islets AMPK plays an important role in regulating insulin secretion and inhibition of AMPK protects beta cells from apoptosis mediated either by cytokines and/or induced T cell CD8+ and CD4+. Methods MIN6 cells, Wildtype or AMPKkd, were pre-treated with (N Group) ou Without (C Group) 1nM of CNTF before the exposure to IL-1β (10 ng/ml). After the treatment, the proteins and the total mRNA were extract and the protein and mRNA of the AMPK pathway were analyzed. Furthermore, importants apoptosis markers, such as a caspase-3 cleavage, iNOS expression and DNA fragmentation were measured. Results Our results indicate that apoptosis induced by IL-1β in MIN6 cells requires activation of the CaMKII-AMPK pathway; furthermore, CNTF inhibits MIN6 cell apoptosis through downregulation of AMPK decreasing pCaMKII. Moreover, IL1β activate AMPK mainly through CaMKII phosphorylation. Finally, IL1β-induced MIN6 cell apoptosis via an increase in iNOS that was reversed by CNTF. Conclusion IL1β upregulates AMPK-INOS pathway, ultimately leading to beta cell death. In this context, CNTF protects beta cells against apoptosis, induced by either IL1β or Alloxan through the reduction of the activity of the CaMKII-AMPK-INOS pathway.

Large gradient high magnetic field affects FLG29.1 cells differentiation to form osteoclast-like cells

Purpose: We aimed to investigate the effects of different apparent gravities (μ g, 1 g and 2 g) produced by large gradient high magnetic field (LGHMF) on human preosteoclast FLG29.1 cells.Materials and methods: FLG29.1 cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium. Cells were exposed to LGHMF for 72 h. On culture day 1, 2, 3, cell proliferation was detected by 3-(4,5)-dimethylthiahi-azo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method. On day 3, cell apoptosis and necrosis were assayed by Hoechst and propidium iodide (PI) staining. After cells were exposed to LGHMF for 72 h with the induction of 12-o-tetradecanoylphorbol 13-acetate (TPA), Tartrate-Resistant Acid Phosphatase (TRAP) positive cells and nitric oxide (NO) release were detected by TRAP staining and Griess method, respectively. Intracellular TRAP activity was measured using nitrophenylphosphate (pNPP) as the substrate.Results: MTT detection revealed that compared to control, FLG 29.1 cell proliferation in the μ g and 2 g groups were promoted. However, there is no obvious difference between the 1 g and control groups. Hoechst-PI staining showed that LGHMF promoted cell apoptosis and necrosis, especially in the 2 g group. Exposure to LGHMF inhibited the NO concentration of supernatant. Both the TRAP activity and the number of TRAP positive cells were higher in cells of μ g group than those in 2 g group. In the 1 g group, they were decreased significantly compared to control.Conclusions: These findings indicate that LGHMF could directly affect human preosteoclast FLG29.1 cells survival and differentiation. High magnetic flux inhibited osteoclasts formation and differentiation while reduced apparent gravity enhanced osteoclastogenesis.

Identification and characterization of triamcinolone acetonide, a microglial-activation inhibitor

Recent studies show that necrotic neuronal cells (NNC) activate microglia, thereby leading to neuronal cell death. This suggests that chemicals that inhibit microglia activation may be used as neuroprotective drugs. In this context, we screened a chemical library for inhibitors of microglia activation. Using a screening system based on a nitrite assay, we isolated two chemicals that inhibit nitric oxide (NO) release from activated microglia: triamcinolone acetonide (TA) and amcinonide. The half-maximal inhibitory concentrations (IC50) of TA and amcinonide for NO release inhibition were 1.78 nM and 3.38 nM, respectively. These chemicals also inhibited NNC-induced expression of the proinflammatory genes iNOS, TNF-α, and IL-1β in glial cells. A study based on a luciferase assay revealed that TA attenuated NNC-induced microglia activation by blocking the NF-κB signaling pathway. In addition, TA protected cortical neurons in coculture with microglia from LPS/IFN-γ-induced neuronal cell death. In conclusion, TA may inhibit microglia activation and may protect neuronal cells from death induced by microglial activation.

Abstract 377: Soluble CD40 Ligand Reverses the Inhibitory Effect of Early Endothelial Progenitor Cells on Platelet Aggregation

Introduction and hypothesis: Circulating Endothelial progenitor cells (EPCs) interact with other blood cells and platelets and may contribute to limit the thrombo-inflammatory reactions at the sites of vascular injury. Indeed, we have previously shown that EPCs inhibit platelet activation and aggregation. The CD40/CD40L axis is a thrombo-inflammatory mediator that affects platelet and endothelial functions. EPCs express CD40 while platelets represent the major source of soluble CD40 ligand (sCD40L) in the circulation. We aimed to demonstrate the existence of a sCD40L/CD40 signaling pathway in EPCs and to test the hypothesis that sCD40L affects the anti-platelet function of EPCs. Methods and results: Human peripheral blood mononuclear cell-derived early EPCs in culture express CD40 and its adaptor proteins, the tumor necrosis factor associated factors: TRAF1, TRAF2 and TRAF3. sCD40L stimulation of early EPCs had no effect on TRAF2 expression, but increased its binding to CD40. In contrast, sCD40L increased the expression of TRAF1 and affected its binding to CD40; whereas TRAF3 binding was not affected. In functional studies, pretreatment of early EPCs with sCD40L had no effect on their viability, but reduced their inhibitory effect on platelet aggregation. However, the release of the anti-aggregating agents prostacyclin and nitric oxide by early EPCs was not affected by sCD40L treatment. Conclusion: We have revealed the existence of the CD40L/CD40/TRAF axis in early EPCs, which reduced their anti-platelet properties. This was not related to an effect of sCD40L on early EPC survival nor on their release of prostacyclin or nitric oxide.

Octopamine, unlike other trace amines, inhibits responses of astroglia-enriched cultures to lipopolysaccharide via a β-adrenoreceptor-mediated mechanism

Trace amines (TAs), i.e. β-phenylethylamine, tyramine and octopamine, are generally regarded as sympathomimetic compounds with structural and functional analogy with catecholamines. Previous reports have shown particularly high levels of circulating TAs in migraine and cluster headache patients. However, no clues are yet available as to the pathophysiological significance of these alterations. The effect of different TAs on the release of nitric oxide was investigated in rat astroglial cells stimulated with lipopolysaccharide (LPS). Octopamine substantially inhibited the release of NO evoked by LPS. Tyramine and β-PEA were ineffective. The inhibitory effect of octopamine was fully reverted by two selective antagonists of β-adrenergic receptors, while α-adrenergic blockade was ineffective. These data, consistent with a role of octopamine as a modulator of NO release, uncover an interaction between octopamine and β-adrenergic receptors in astroglial cells. These results may have an impact in understanding the mechanisms underlying migraine pathophysiology.

717 Increased Distal Airway Nitric Oxide (NO) Release during Acute Complications in Lung Transplant Recipients (LTxR)

717 Increased Distal Airway Nitric Oxide (NO) Release during Acute Complications in Lung Transplant Recipients (LTxR)

Characterization of the Oxide Films Grown at 260°C in a Simulated Secondary Coolant of a Nuclear Reactor

This paper presents the characteristics of superficial films formed on carbon steel A106 B samples machined from an original tube of a secondary circuit of a CANDU Nuclear Power Station not exposed to the coolant. The oxide layers were grown on a set of coupons over 77 and 560h periods in static stainless steel conditioned autoclaves at 260°C simulating AVT conditions: I) degassed (hydrazine + ethanolamine) solution and II) degassed (hydrazine + morpholine) solution. The oxide films were studied using SEM, DRX and chemical descaling to evaluate metal corrosion, adherent oxide thickness and oxide release. Up to 560h of testing, the samples exposed to medium I) showed a slightly lower metal corrosion and oxide release, although of the same order of magnitude for the two media.

Cytotoxicity of bovine and porcine collagen membranes in mononuclear cells

This study compared the cytotoxicity and the release of nitric oxide induced by collagen membranes in human mononuclear cells. Peripheral blood was collected from each patient and the separation of mononuclear cells was performed by Ficoll. Then, 2x10(5) cells were plated in 48-well culture plates under the membranes in triplicate. The polystyrene surface was used as negative control. Cell viability was assessed by measuring mitochondrial activity (MTT) at 4, 12 and 24 h, with dosage levels of nitrite by the Griess method for the same periods. Data had non-normal distribution and were analyzed by the Kruskal-Wallis test (p<0.05). Statistically significant differences (p<0.05) were observed between the membranes and the control in the experimental period, although there was a significant reduction in viability over time (p<0.01). At 4 and 12 h, the porcine membrane induced a higher release of nitrite compared with the control and bovine membrane, respectively (p<0.01), and this difference was maintained at 24 h (p<0.05). This in vitro study showed that the porcine collagen membrane induces an increased production of proinflammatory mediators by mononuclear cells in the first hours of contact, decreasing with time.

Effects of Chronic Endothelin ETA Receptor Blockade on Blood Pressure and Vascular Formation of Cyclic Guanosine-3’,5’-monophosphate in Spontaneously Hypertensive Rats

Endothelin (ET) mediates vasoconstriction in intact arterial blood vessels with functional endothelium via stimulation of ET(A) receptors, while ET(B) receptor stimulation leads to vasodilation via nitric oxide (NO) release and formation of cyclic guanosine-3',5'-monophosphate (cGMP). In spontaneously hypertensive rats (SHR) the cGMP-forming NO-receptor guanylyl cyclase (sGC) is downregulated. It is unclear whether ET contributes to the hypertensive phenotype of SHR, and whether this involves the disturbed cGMP signaling. The selective ETA receptor antagonist darusentan (CAS 171714-84-4), given orally via drinking water (10 mg kg(-1) d(-1)) for 12 weeks, significantly lowered systolic blood pressure of SHR as determined by radiotelemetry. Neither impaired endothelium-dependent relaxation to acetylcholine was restored nor sGC expression and activity affected when compared to control SHR. While these findings show a role for ETA receptors in blood pressure regulation in genetically elevated blood pressure, downregulation of sGC expression and cGMP-mediated vasorelaxant response in SHR were shown to be independent of ETA receptors. The findings suggest distinct mechanisms of gene expression affecting ET and cGMP mediated vasomotor functions.

Antidiabetes drug metformin is a donor of nitric oxide: EPR measurement of efficiency

It is shown that metformin, which is a drug used for treatment of type 2 diabetes mellitus, metabolizes itself in vivo in the intestine and liver of mice with the release of nitric oxide. Subsequently the released nitric oxide forms paramagnetic mono- and dinitrosyl iron complexes which can be registered by EPR method. It is suggested that nitric oxide is just responsible for multifarious therapeutic action of metformin such as lowering of blood glucose level, reduction of arterial hypertension and other biological effects.