Increased NO Generation Research Articles

Effects of sub-atmospheric pressure on appearance and pollutant formation of inverse diffusion flame within a confined space

Gas-fired boilers operating at high-altitude regions often suffer from inadequate output, decreased thermal efficiency, and excessive NOx emissions. The effect of sub-atmospheric pressure on flame appearance and pollutant formation is the main reason for those problems, and thus needs to be clarified particularly under furnace combustion conditions with a fixed excess air coefficient. Inverse diffusion is a widely employed fuel–air configuration in burners of gas-fired boilers, and therefore the flame appearance, CO generation, and NO generation were experimentally investigated in this paper by adopting a low-pressure quartz tube reactor. Results show that the flame is elongated from reducing pressure under fuel-lean conditions, mainly due to the reduced oxygen mass concentration and the elevated jet velocity. Under fuel-rich combustion conditions, however, the flame is shorted at sub-atmospheric pressure from the suppressed soot formation. The reduced pressure leads to an increase in the global strain rate, making the flame more prone to uplift. With decreasing pressure, the increased air–fuel mixing and flame length facilitate the gas burnout, thus decreasing CO generation. The sub-atmospheric pressure could evidently reduce the NO generation under fuel-rich conditions, but slightly increase it under fuel-lean conditions. Under fuel-lean conditions, the NO major pathways (prompt, thermal, NNH, and N2O) are promoted which leads to an increase in NO generation with decreasing pressure. Under fuel-rich conditions, however, NO formation is suppressed from the decreased rate of reaction N2+CH↔HCN+N.

Potential mechanism of transient receptor potential cation channel subfamilyV member1 combined with an ATP‑sensitive potassium channel in severe preeclampsia.

Severe preeclampsia is one of the most serious obstetric diseases. However, the pathogenesis of the disease is not fully understood. In the present study, placental artery and blood serum was collected from patients with severe preeclampsia, as well as from normal pregnant women. The results of reverse transcription-quantitative (q)PCR, western blotting, and immunohistochemical staining revealed markedly decreased transient receptor potential cation channel subfamily V member 1 (TRPV1), ATP-sensitive potassium channel (KATP) subtype Kir6.1/SUR2B and endothelial nitric oxide synthase (eNOS) expression in severe preeclampsia tissue specimens compared with those in samples from normal pregnant women. The nitrate reduction method indicated lower NO levels in the tissue specimens and serum of patients with severe preeclampsia. Moreover, hematoxylin-eosin staining showed that the endothelial cell layer in the placental artery of patients with severe preeclampsia was notably damaged. To investigate the potential role of TRPV1-KATP channels in severe preeclampsia, HUVECs were used for in vitro experiments. The samples were divided into a control group, a TRPV1 agonist group (capsaicin) and a TRPV1 inhibitor group (capsazepine). qPCR and western blotting revealed that the relative gene and protein expression levels of TRPV1, Kir6.1, SUR2B and eNOS in the control group were significantly lower than those in the capsaicin group and considerably higher than those in the capsazepine group. Based on previous studies and the results of the present study, we hypothesized that impairment of the endothelial TRPV1-KATP channels results in decreased eNOS/NO pathway activity, which may be one of the mechanisms involved in severe preeclampsia. The increase in NO generation mediated by TRPV1-KATP may be a suitable target for the management of severe preeclampsia.

Porous Defective Bi/Bi3NbO7 Nanosheets for Efficient Photocatalytic NO Removal under Visible Light

Since conventional techniques are ineffective for NO removal at low concentrations, photocatalysis has become attractive in this regard, recently. However, in practice, photocatalytic NO removal has drawbacks such as limited light absorption and the proclivity of producing toxic by-products. To address these issues, novel defective Bi/Bi3NbO7 structures with good porosity were fabricated by a solvothermal method and used for enhanced photocatalytic NO removal under visible light irradiation. The morphological and structural properties of the prepared materials were comprehensively analyzed. The optimal photocatalytic activity of pore-defective Bi/Bi3NbO7 for NO removal was 60.3%, when the molar ratios of urea and Bi(NO)3•5H2O to pristine Bi3NbO7 were 1:25 and 1:2, respectively, under the following operational conditions: NO concentration of 700 ppb, catalyst dosage of 50 mg and irradiation time of 14 min. The induced defects and the surface plasmon resonance (SPR) effect of Bi nanodots made remarkable contributions to improving the photocatalytic NO removal as well as inhibiting the toxic byproduct NO2. The photocatalytic NO removal pathway over the prepared photocatalysts was further mechanistically clarified taking advantage of EPR results and scavenging experiments. Considering the increased NO generation in the atmosphere, this work may provide novel insights for designing effective porous photocatalysts to treat gaseous toxic pollutants.

A novel nano-anti-malarial induces redox damage and elicits cytokine response to the parasite

Emergence and spread of resistant parasites to the newest chemotherapeutic anti-malarial agents are the biggest challenges against malaria control programs. Therefore, developing a novel effective treatment to reduce the overgrowing burden of multidrug resistant malaria is a pressing need. Herein, we have developed a biocompatible and biodegradable, non-toxic chitosan-tripolyphosphate-chloroquine (CS-TPP CQ) nanoparticle. CS-TPP CQ nanoparticles effectively kill the parasite through redox generation and induction of the pro- and anti-inflammatory cytokines in both sensitive and resistant parasite in vitro. The in vitro observations showed a strong inhibitory effect (p < 0.01) on pro-inflammatory cytokines more specifically on TNF-α and IFN-γ whereas CS-TPP CQ nanoparticles significantly elevated the anti-inflammatory cytokines- IL-10 and TGF-β. In addition, CS-TPP CQ nanoparticle significantly increased NO generation (p < 0.01) and altered the GSH/GSSG ratio 72 h after parasite co-culture with peripheral blood mononuclear cells culminating in the free radical induced parasite killing. CS-TPP CQ nanoparticle had an effective dose of 100 ng/ml against CQ-sensitive parasite lines (p < 0.001) whereas effective dose against CQ-resistant parasite line was 200 ng/ml CS-TPP CQ with an effective duration of 72 h (p < 0.001). Our studies suggest that CS-TPP CQ nanoparticle has a potential to modulate the pro- and anti-inflammatory responses, and to trigger the redox-mediated parasite killing. It can be a novel nano-based futuristic approach towards malaria control.

Couplet medicines of leech and centipede granules improve erectile dysfunction via inactivation of the CaSR/PLC/PKC signaling in streptozotocin-induced diabetic rats.

Erectile dysfunction (ED) is one of the significant complications of diabetes mellitus (DM), and CASR plays an important role in cellular antiapoptosis and NO production in the vascular endothelium by activating PKC. The present study was aimed to investigate the efficacy of Leech and Centipede Granules (LCG) through the CaSR/PLC/PKC signaling. Fifty male Sprague-Dawley rats were treated with streptozotocin to induce the DM model. After 10 weeks, an apomorphine test was used to confirm DMED. Rats with DMED were administrated with LCG and U73122 for 4 weeks. Fasting blood glucose, body weight, insulin and glucagon levels were measured. Erectile function in rats was assessed by apomorphine. Serums were measured using enzyme-linked immunosorbent assay and flow cytometry, and penile tissues were harvested for histologic and the expression of related targets analyses. After treatment, fasting blood glucose, body weight, insulin, glucagon levels, and erectile function were significantly ameliorated in the LCG groups. The LOX-1, NOX, and EMPs concentrations were significantly decreased with LCG treatment. LCG also continuously increased NO and decreased ET-1 content in penile tissues. LCG and U73122 administration also improved penile fibrosis by significantly decreasing VCAM-1, ICAM-1, and CD62P. The data also showed that LCG reduced the apoptosis level in the penis. Furthermore, the inhibited activation of the CaSR/PLC/PKC pathway was observed in DMED rats with LCG treatment. Collectively, LCG significantly ameliorated erectile function of DMED rats via increased NO generation, inhibiting endothelial cells apoptosis and penile fibrosis, which might benefit from the suppression of CaSR/PLC/PKC pathway in DMED rats.

Augmentation of iNOS expression in myeloid progenitor cells expedites neutrophil differentiation.

Neutrophils play important role in immunity and inflammation through diverse mechanisms. Reports from this lab and others have demonstrated involvement of NO in neutrophil adhesion, chemotaxis, bacterial killing, reactive oxygen species generation, neutrophil extracellular traps' formation, and apoptosis. Constitutive expression of iNOS in human neutrophils has also been documented. The role of NO-iNOS in neutrophil differentiation however remains ill-defined. The present study was undertaken to understand the role of NO generated from iNOS in the neutrophil differentiation by using iNOS-overexpressing K562 cells (K562iNOS ) and iNOS-deficient murine progenitor cells (lineage negative cells; lin-ve ). We observed that iNOS overexpression led to increased neutrophilic differentiation in K562 cells; more specifically an early and accelerated neutrophilic differentiation was spotted in K562iNOS . These observations were further validated using iNOS knockout lin-ve cells or hematopoietic progenitor cells that exhibited delayed neutrophil differentiation in comparison to its wild-type counterpart. In addition, a significant increase in the gene expression of iNOS during neutrophilic differentiation of CD34+ hematopoietic stem and progenitor cells derived from human bone marrow further substantiates importance of iNOS in neutrophil differentiation. Moreover, a significant increase in NO generation during neutrophil differentiation was observed and enhanced neutrophil differentiation with NO donor was also observed, implying the importance of NO in neutrophil differentiation. Collectively, using alternative approaches, we demonstrated that neutrophil differentiation is significantly influenced by iNOS or NO, suggesting the possibility of exploiting this novel link for therapeutic aspects of NO generated from iNOS and neutrophil differentiation in hematopoiesis-related disorders.

Abstract 444: Dusp4 Overexpressed EC Derived Exosomes Promote Angiogenesis: A Novel Therapeutic Strategy For Ischemic Heart Disease

After an ischemia and reperfusion (I/R) injury to the heart, re-establishing blood flow to the infarct region can salvage myocardium, stimulate angiogenesis and recruit progenitor cells for tissue regeneration. Stimulating angiogenesis through the growth of new microcirculation in the peri-infarct area can help preserve and improve cardiac function following myocardial infarction. In this study, we hypothesize that exosomes derived from DUSP4-overexpressed endothelial cells (ECs) stimulate angiogenesis and improve vascular function. DUSP4-derived exosomes (50 μg/ml) promote angiogenesis by increasing tube formation of endothelial cells (2.74 ± 0.14 versus control exosomes 2.17 ± 0.15 in the total length; p &lt; 0.001). Immunoblotting reveals that DUSP4 overexpression in endothelial cells down-regulates p53 (0.59 ± 0.04 versus 1.0 ± 0.01 of control) expression, which modulates the HIF1α pathway and angiogenesis. Moreover, NO is a critical small molecule involved in regulation of vascular tone, vascular growth, platelet aggregation, and modulation of inflammation. The level of NO generation determined using DAF fluorescence microscopy demonstrated that DUSP4 overexpression augments NO generation. The increase in NO generation also correlates to the increase in eNOS protein (1.23 ± 0.20 versus control) and mRNA (5.84 ± 0.37 versus control) expression. The proteomic analysis of control and DUSP4 derived exosomes showed that DUSP4 overexpression up-regulates several key exosomal proteins including HSP70, HSP90, tubulins, Vimentin, and clathrin. More interestingly, several redox proteins were up-regulated in DUSP4 derived exosomes including glutathione S-transferase P, peroxiredoxin-2, peroxiredoxin-4, and protein disulfide-isomerase. Together, these results demonstrate that exosomes derived from DUSP4 reprogrammed ECs are a valuable therapeutic option for the treatment of ischemic heart disease by promoting angiogenesis.

Hazelnut and cocoa spread improves flow-mediated dilatation in smokers.

Hazelnut and cocoa spread is an Italian product containing cocoa and hazelnut. Several epidemiological studies suggest that cocoa and hazelnuts cocoa exert beneficial cardiovascular effects. To investigate whether in smokers, hazelnut and cocoa spread elicits artery dilatation via down-regulation of oxidative stress. Flow-mediated dilatation (FMD), oxidative stress (as assessed by serum isoprostanes excretion, Nox2 activation and NO bioavailability) and antioxidant status [as assessed by vitamin E levels, plasma total polyphenols and H2O2 breaking down activity (HBA)] were studied in 20 smokers in a crossover, single-blind study. Patients were randomly allocated to 60g of Hazelnut and cocoa spread or 60g of milk chocolate (≤ 35% cocoa). FMD, serum isoprostanes, Nox2 activation, NOx, vitamin E, HBA and total polyphenols were assessed at baseline and 2h after chocolate ingestion. After Hazelnut and cocoa spread intake, FMD and NOx significantly increased (from 4.3 ± 2.8 to 8.0 ± 3.2%, p < 0.001 and from 23.1 ± 5.5 to 32.0 ± 12.6µM, p = 0.016, respectively); conversely, serum isoprostanes and Nox2 activation significantly decreased (from 302.8 ± 59.8 to 240.7 ± 90.8pmol/l, p = 0.03 and from 25 ± 4.4 to 22.6 ± 3.2, p = 0.03, respectively). After Hazelnut and cocoa spread intake, serum total polyphenols, vitamin E and HBA significantly increased (from 133.8 ± 49.7 to 202.5 ± 69.5mg/l GAE, p = 0.001; from 3.56 ± 1.4 to 4.5 ± 1.0μmol/mmol cholesterol, p = 0.002 and from 63.3 ± 13.2 to 74.2 ± 12.4%, p = 0.003, respectively). No changes in the above variables were observed after milk chocolate intake. A linear correlation analysis shows that Δ (expressed by difference of values between before and after chocolate intake) of FMD correlates with Δ of total polyphenols and Δ of vitamin E. This study shows that Hazelnut and cocoa spread improves FMD with a mechanism potentially involving downregulation of oxidative stress and eventually increased NO generation in smokers.

Involvement of the NO/sGC/cGMP/K+ channels pathway in vascular relaxation evoked by two non-quinazoline α1-adrenoceptor antagonists

The aim of this study was to explore the α1-adrenoceptor-independent mechanisms involved in the vasorelaxant properties of two non-quinazoline α1-adrenoceptors antagonists (MH-76 and MH-79). Endothelium intact and endothelium denuded rat aorta was contracted with 1 μM phenylephrine to plateau, and the vasodilatory effect of MH-76 and MH-79 was examined in the absence or presence of inhibitors of the different signal transduction pathways. cGMP concetration was measured in rat aorta (enzyme immunoassay kit). In human aortic endothelial cells (HAEC) NO production was examined using a DAF-FM DA fluorescent indicator, whereas in human aortic smooth muscle cells the influence of the title compounds on K+ efflux was evaluated. The vasorelaxant effect of MH-76 and MH-79 was attenuated by endothelium removal, Nω-Nitro-l-arginine methyl ester (L-NAME) and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) pretreatment to the level characteristic for α1-adrenoreceptor blocking activity. In addition, the MH-76 and MH-79 induced relaxation was reduced by K+ channels blockers. In endothelium intact rat aorta, MH-76 and MH-79 caused an increase in cGMP level, whereas in HAEC they increased NO generation. In contrast, the reference, quinazoline based α1-antagonist prazosin, did not influence NO production. Our findings suggest that the mechanisms underlying the vasodilatory properties of non-quinazoline based α1-adrenoceptors antagonists MH-76 and MH-79 involve not only α1-adrenoceptor blocking activity but also the activation of the endothelial NO–cGMP signalling pathway and the subsequent opening of K+ channels. Our studies show that such double mechanism of action is superior to pure α1-adrenoceptor blockade, and may be considered as a promising alternative for the prevention and treatment of cardiovascular diseases.

Effects of exercise training on nitric oxide, blood pressure and antioxidant enzymes.

The relationship between exercise training and nitric oxide-related parameters was examined in a cross-sectional study and an intervention study. A cross-sectional study using 184 employees was conducted to observe the association of exercise habits with serum arginase (ELISA and activity), l-arginine, l-citrulline, l-ornithine, NOx, exhaled nitric oxide, blood pressure, FEV1%, hs-CRP, HDL-cholesterol, IgE, and life style factors. An intervention study was also conducted to evaluate the changes of serum arginase I, nitric oxide-related parameters, and mRNA levels of anti-oxidant enzymes in blood monocytes before and after 1 h of aerobic exercise training per day for a month. Exercise habits were associated with increased arginase activity and a moderate alcohol drinking habit, after adjustment with several covariates. Aerobic exercise training induced a decrease in l-arginine and diastolic blood pressure and induced an increase in NO2− and urea. Moreover, mRNA expression of anti-oxidant enzymes, such as catalase and GPX1, and a life elongation enzyme, SIRT3, were significantly increased after aerobic exercise. The results that aerobic exercise training increased NO generation, reduced blood pressure, and induced anti-oxidant enzymes via SIRT3 suggest that exercise training may be an important factor for the prevention of disease by inducing intrinsic NO and anti-oxidant enzymes.

Cholesterol regulates contractility and inotropic response to β2-adrenoceptor agonist in the mouse atria: Involvement of Gi-protein–Akt–NO-pathway

Majority of cardiac β2-adrenoceptors is located in cholesterol-rich microdomains. Here, we have investigated the underlying mechanisms by which a slight to moderate cholesterol depletion with methyl-β-cyclodextrin (MβCD, 1 and 5mM) interferes with contractility and inotropic effect of β2-adrenergic agonist (fenoterol, 50μM) in the mouse atria. Treatment with MβCD itself increased amplitude of Ca2+ transient but did not change the contraction amplitude due to a clamping action of elevated NO. Cholesterol depletion significantly attenuated the positive inotropic response to fenoterol which is accompanied by increase in NO generation and decrease in Ca2+ transient. Influence of 1mM MβCD on the fenoterol-driven changes in both contractility and NO level was strongly attenuated by inhibition of Gi-protein (pertussis toxin), Akt (Akt 1/2 kinase inhibitor) or NO-synthase (L-NAME). After exposure to 5mM MβCD, pertussis toxin or Akt inhibitor could recover the β2-agonist effects on contractility, NO production and Ca2+ transient, while L-NAME only reduced NO level. An adenylyl cyclase activator (forskolin, 50nM) had no influence on the MβCD-induced changes in the β2-agonist effects. Obtained results suggest that slight cholesterol depletion upregulates Gi-protein/Akt/NO-synthase signaling that attenuates the positive inotropic response to β2-adrenergic stimulation without altering the Ca2+ transient. Whilst moderate cholesterol depletion additionally could suppress the enhancement of the Ca2+ transient amplitude caused by the β2-adrenergic agonist administration in Gi-protein/Akt-dependent but NO-independent manner.

Involvement of endothelium in the vasorelaxant effects of 3,4-DHPEA-EA and 3,4-DHPEA-EDA, two major functional bioactives in olive oil

The olive oil polyphenols 3,4-DHPEA-EA and 3,4-DHPEA-EDA displayed an endothelium-dependent vasorelaxant effect in rat aorta, starting at ~1 µM and abolished by NG-nitro-L-arginine (L-NA) or N-acetylcysteine, and an endothelium-independent vasorelaxant effect, starting at ~10 µM. Hydroxytyrosol only presented an endothelium-independent effect at 100 µM. DHPEA-EA and 3,4-DHPEA-EDA, but not hydroxytyrosol, also increased NO generation within endothelial cells. At higher concentrations, the three compounds reduced arginine-vasopressin-induced increase of cytosolic Ca2+ concentration ([Ca2+]c) in vascular myocytes. By UV-visible spectroscopy, we found that these polyphenols undergo autoxidative processes in organ-bath conditions. Thus, 3,4-DHPEA-EA and 3,4-DHPEA-EDA have an endothelium-dependent vasorelaxant effect caused by an enhanced NO production, probably through a redox mechanism within endothelial cells and an endothelium-independent vasorelaxant effect mediated by a reduction of agonist-induced [Ca2+]c increase in vascular myocytes. Bearing in mind the plasmatic concentrations of these polyphenols following dietary intake of olive oil, these effects could modulate vascular tone in vivo.

The Regulatory Role of Nuclear Factor Kappa B in the Heart of Hereditary Hypertriglyceridemic Rat.

Activation of nuclear factor-κB (NF-κB) by increased production of reactive oxygen species (ROS) might induce transcription and expression of different antioxidant enzymes and also of nitric oxide synthase (NOS) isoforms. Thus, we aimed at studying the effect of NF-κB inhibition, caused by JSH-23 (4-methyl-N 1-(3-phenyl-propyl)-benzene-1,2-diamine) injection, on ROS and NO generation in hereditary hypertriglyceridemic (HTG) rats. 12-week-old, male Wistar and HTG rats were treated with JSH-23 (bolus, 10 μmol, i.v.). After one week, blood pressure (BP), superoxide dismutase (SOD) activity, SOD1, endothelial NOS (eNOS), and NF-κB (p65) protein expressions were higher in the heart of HTG rats compared to control rats. On the other hand, NOS activity was decreased. In HTG rats, JSH-23 treatment increased BP and heart conjugated dienes (CD) concentration (measured as the marker of tissue oxidative damage). Concomitantly, SOD activity together with SOD1 expression was decreased, while NOS activity and eNOS protein expression were increased significantly. In conclusion, NF-κB inhibition in HTG rats led to decreased ROS degradation by SOD followed by increased oxidative damage in the heart and BP elevation. In these conditions, increased NO generation may represent rather a counterregulatory mechanism activated by ROS. Nevertheless, this mechanism was not sufficient enough to compensate BP increase in HTG rats.

Dysregulated flow-mediated vasodilatation in the human placenta in fetal growth restriction.

Increased vascular resistance and reduced fetoplacental blood flow are putative aetiologies in the pathogenesis of fetal growth restriction (FGR); however, the regulating sites and mechanisms remain unclear. We hypothesised that placental vessels dictate fetoplacental resistance and in FGR exhibit endothelial dysfunction and reduced flow-mediated vasodilatation (FMVD). Resistance was measured in normal pregnancies (n = 10) and FGR (n = 10) both in vivo by umbilical artery Doppler velocimetry and ex vivo by dual placental perfusion. Ex vivo FMVD is the reduction in fetal-side inflow hydrostatic pressure (FIHP) following increased flow rate. Results demonstrated a significant correlation between vascular resistance measured in vivo and ex vivo in normal pregnancy, but not in FGR. In perfused FGR placentas, vascular resistance was significantly elevated compared to normal placentas (58 ± 7.7 mmHg and 36.8 ± 4.5 mmHg, respectively; 8 ml min−1; means ± SEM; P < 0.0001) and FMVD was severely reduced (3.9 ± 1.3% and 9.1 ± 1.2%, respectively). In normal pregnancies only, the highest level of ex vivo FMVD was associated with the lowest in vivo resistance. Inhibition of NO synthesis during perfusion (100 μm l-NNA) moderately elevated FIHP in the normal group, but substantially in the FGR group. Human placenta artery endothelial cells from FGR groups exhibited increased shear stress-induced NO generation, iNOS expression and eNOS expression compared with normal groups. In conclusion, fetoplacental resistance is determined by placental vessels, and is increased in FGR. The latter also exhibit reduced FMVD, but with a partial compensatory increased NO generation capacity. The data support our hypothesis, which highlights the importance of FMVD regulation in normal and dysfunctional placentation.Key points A correlation was found between in vivo umbilical artery Doppler velocimetry and resistance to fetal-side flow in the human ex vivo dually perfused placenta, highlighting that the fetoplacental vascular bed is a key site of resistance to umbilico-placental flow in pregnancy. We discovered high resistance and poor flow-mediated vasodilatory responses in placentas from pregnancies associated with fetal growth restriction (FGR). Endothelial cells isolated from the FGR placentas and grown in static and flow culture showed a dysregulated phenotype, with biochemical signalling demonstrating a failed compensatory response to high blood-flow resistance.

Quantitative determination of nitric oxide production in haemocytes: Nitrite reduction activity as a potential pathway of NO formation in haemolymph of Galleria mellonella larvae

The generation of nitric oxide by Galleria mellonella larvae haemocytes has been investigated. For this purpose, a fluorescent method, specific for detection of NO, has been developed. The method is based on the application of fluorescence probe DAF-FM diacetate and nitronyl nitroxyl radical, NNR, which accelerates the NO-dependent formation of fluorescence product, DAF-FM triazole. The key feature of the method is the registration and analysis of differential kinetics, namely, the difference of kinetics obtained in samples with NNR and without NNR. This approach allows us to exclude any other kinetic processes not related to triazole formation. The differential kinetics were calibrated versus NO generation rate and the resulting low limit of method sensitivity was obtained as about 0.4–0.5nM/min. The generation of nitric oxide by the haemocytes of insects treated with LPS in vivo has been detected at a rate of 0.5–0.7nM/min. However, the production of NO in haemocyte suspensions treated with both the substrate, l-arginine, and the inhibitor, l-NAME, of NOS, has not been detected within method sensitivity. These data provide only the upper level of NO generation by haemocytes but cannot be used to draw definite conclusions about NOS as a source of NO. Meanwhile, it is known, that NO can be formed by NOS independent mechanism. Indeed, we have observed a significant increase in NO generation in the samples of haemocytes intracellularly loaded with nitrite. Moreover, adding nitrite to lysed haemocytes results in the higher NO generation rate. After addition of 500μM nitrite, the rates of NO generation in the samples are determined to be 2 and 20–30nM/min, respectively. The nitrite/nitrate content of haemocytes and lymph were found to be 5 and 50μM, respectively. The detected nitrite reduction activity of haemocytes allowed us to estimate the generation rate of nitric oxide as 0.05–0.5nM/min from endogenous nitrite. It is thus assumed that the observed nitrite reduction activity in haemocytes is dominant in the increased NO production during immune response of the G. mellonella larvae.

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.

Mechanism of reversal of high glucose-induced endothelial nitric oxide synthase uncoupling by tanshinone IIA in human endothelial cell line EA.hy926

Endothelial nitric oxide synthase (eNOS) uncoupling plays a causal role in endothelial dysfunction in many cardiovascular and metabolic diseases. Tanshinone IIA (Tan IIA), an active compound from Salvia miltiorrhiza, has been used to treat cardiovascular and metabolic diseases. However, the effects of Tan IIA on eNOS uncoupling have not been reported. We hypothesize that Tan IIA can regulate eNOS uncoupling in endothelium cells under oxidative stress. The results showed that eNOS-mediated NO generation was significantly decreased, accompanied by increased superoxide production and NOX4 expression. The ratio of eNOS dimer to monomer and NOS cofactor tetrahydrobiopterin (BH4) to 7,8-dihydrobiopterin (BH2) as well as expressions of heat-shock protein of 90kDa (HSP90), GTP cyclohydrolase-1 (GTPCH1) and dihydrofolate reductase (DHFR) were significantly decreased. Tan IIA significantly inhibited superoxide production and expression of NOX4, and increased NO generation and eNOS homodimerization, as well as expressions of HSP90, GTPCH1 and DHFR in a concentration-dependent manner. The ratio of BH4 to BH2 was also elevated by Tan IIA. In addition, Tan IIA significantly inhibited the increase in expression of PI3K in high glucose treated cells. Wortmannin, a PI3K inhibitor, significantly inhibited the high glucose induced NOX4 expression. The results demonstrated that Tan IIA restored eNOS uncoupling induced by high glucose by targeting NADPH oxidase, HSP90, GTPCH1 and DHFR, and PI3K pathway, which leads to reduced intracellular oxidative stress and increased NO generation. Tan IIA may be used as a prototype agent to restore eNOS coupling under certain cardiovascular and metabolic diseases.

Binding of XOR to cellular glycosaminoglycans (GAGs) facilitates nitrite reductase activity under oxygenated conditions

Under anoxic conditions, XOR catalyzes the one e − reduction of nitrite ( NO 2 - ) to NO at the Mo-cofactor. However, the conditions that favor e − transfer to NO 2 - at the Mo-cofactor over e − transfer to O 2 at the FAD site have not been defined. Using various detection methods, NO 2 - reduction by purified XOR was analyzed in an O 2 controlled environment. At 0% O 2 , XOR reduced NO 2 - , generating 17.0 ± 3.2 nM/min NO from xanthine while similar rates (22.9 ± 4.8) were observed with NADH. As O 2 tension increased to 2%, O 2 concentration-dependent delays in the onset of NO production occurred. Concurrent measurement of O 2 concentrations revealed that NO 2 - -dependent NO generation did not occur until all O 2 was consumed, suggesting that O 2 decreases NO 2 - reduction by indirect oxidation of the Mo via e − withdrawal at the FAD. Experiments with XDH, which has lower affinity for O 2 , did not demonstrate improved rates of NO formation or reduced lag times. However, immobilization of XO on artificial GAGs conferred the capacity for pronounced and sustained NO formation. In addition, reduction of NO 2 - by GAG-bound XO resulted not only in NO formation but concomitant diminution of XO-derived ROS. GAG-mediated enhancement of nitrite reductase activity and reduction in XO-derived ROS formation was confirmed when XO was associated with endothelial cell GAGs. Combined, these data indicate under inflammatory conditions where vascular XO levels and XO-GAG interactions are enhanced that O 2 -mediated inhibition of XO-catalyzed NO 2 - reduction can be overcome resulting in both increased NO generation and decreased ROS production.

Immune Evasion byHelicobacter pyloriIs Mediated by Induction of Macrophage Arginase II

Helicobacter pylori infection persists for the life of the host due to the failure of the immune response to eradicate the bacterium. Determining how H. pylori escapes the immune response in its gastric niche is clinically important. We have demonstrated in vitro that macrophage NO production can kill H. pylori, but induction of macrophage arginase II (Arg2) inhibits inducible NO synthase (iNOS) translation, causes apoptosis, and restricts bacterial killing. Using a chronic H. pylori infection model, we determined whether Arg2 impairs host defense in vivo. In C57BL/6 mice, expression of Arg2, but not arginase I, was abundant and localized to gastric macrophages. Arg2(-/-) mice had increased histologic gastritis and decreased bacterial colonization compared with wild-type (WT) mice. Increased gastritis scores correlated with decreased colonization in individual Arg2(-/-) mice but not in WT mice. When mice infected with H. pylori were compared, Arg2(-/-) mice had more gastric macrophages, more of these cells were iNOS(+), and these cells expressed higher levels of iNOS protein, as determined by flow cytometry and immunofluorescence microscopy. There was enhanced nitrotyrosine staining in infected Arg2(-/-) versus WT mice, indicating increased NO generation. Infected Arg2(-/-) mice exhibited decreased macrophage apoptosis, as well as enhanced IFN-γ, IL-17a, and IL-12p40 expression, and reduced IL-10 levels consistent with a more vigorous Th1/Th17 response. These studies demonstrate that Arg2 contributes to the immune evasion of H. pylori by limiting macrophage iNOS protein expression and NO production, mediating macrophage apoptosis, and restraining proinflammatory cytokine responses.

The peroxisome proliferator‐activated receptor alpha agonist fenofibrate decreases airway reactivity to methacholine and increases endothelial nitric oxide synthase phosphorylation in mouse lung

In the present study, we have investigated the effect of the peroxisome proliferator-activated receptor α (PPARα) agonist fenofibrate on airway reactivity and the role of the endothelial nitric oxide synthase (eNOS)/NO pathway in this effect. Airway reactivity to methacholine was assessed in C57BL/6 mice treated or not with fenofibrate by whole-body plethysmography. In some experiments, animals were administered with the NOS inhibitor L-NAME, one hour before airway reactivity measurement. Expression and phosphorylation of eNOS were evaluated in lung homogenates from fenofibrate and control animals using Western blotting. Fenofibrate dose and time dependently decreased airway reactivity to methacholine in mice. A statistically significant (P < 0.05) reduction was observed after a treatment of 10 days with a dose of 3 or 15 mg/day fenofibrate. Mice treated with fenofibrate and administered with l-NAME exhibited similar reactivity to methacholine than vehicle-treated mice administered with the NOS inhibitor, suggesting that NO mediates fenofibrate-induced decrease in airway reactivity. eNOS levels remained unchanged in the lung from mice treated with fenofibrate, but phosphorylation of the enzyme at Ser-1177 was increased by 118% (P < 0.05). Taken together, our data demonstrate that fenofibrate downregulates airway reactivity to methacholine in the mouse and suggest that this effect could involve an increase in NO generation through an enhanced eNOS phosphorylation.