Rate Of Nitric Oxide Formation Research Articles

Erythroid anion Exchanger-1 (band 3) transports nitrite for nitric oxide metabolism

Nitrite (NO2−) interacts with hemoglobin (Hb) in various ways to regulate blood flow. During hypoxic vasodilation, nitrite is reduced by deoxyHb to yield nitric oxide (NO). While NO, a hydrophobic gas, could freely diffuse across the cell membrane, how the reactant nitrite anion could permeate through the red blood cell (RBC) membrane remains unclear. We hypothesized that Cl−/HCO3− anion exchanger-1 (AE1; band 3) abundantly embedded in the RBC membrane could transport NO2−, as HCO3− and NO2− exhibit similar hydrated radii. Here, we monitored NO/N2O3 generated from NO2− inside human RBCs by DAF-FM fluorophore. NO2−, not NO3−, increased intraerythrocytic DAF-FM fluorescence. To test the involvement of AE1-mediated transport in intraerythrocytic NO/N2O3 production from nitrite, we lowered Cl− or HCO3− in the RBC-incubating buffer by 20 % and indeed observed slower rise of the DAF-FM fluorescence. Anti-extracellular AE1, but not anti-intracellular AE1 antibodies, reduced the rates of NO formation from nitrite. The AE1 blocker DIDS similarly reduced the rates of NO/N2O3 production from nitrite in a dose-dependent fashion, confirming that nitrite entered RBCs through AE1. Nitrite inside the RBCs reacted with both deoxyHb and oxyHb, as evidenced by 6.1 % decrease in deoxyHb, 14.7 % decrease in oxyHb, and 20.7 % increase in methemoglobin (metHb). Lowering Cl− in the milieu equally delayed metHb production from nitrite-oxyHb and nitrite-deoxyHb reactions. Thus, AE1-mediated NO2− transport facilitates NO2−-Hb reactions inside the red cells, supporting NOx metabolism in circulation.

Zero-dimensional and pseudo-one-dimensional models of atmospheric-pressure plasma jets in binary and ternary mixtures of oxygen and nitrogen with helium background

A zero-dimensional (volume-averaged) and a pseudo-one-dimensional plug-flow (spatially resolved) model are developed to investigate atmospheric-pressure plasma jets operated with He, He/O2, He/N2 and He/N2/O2 mixtures. The models are coupled with the Boltzmann equation under the two-term approximation to self-consistently calculate the electron energy distribution function. An agreement is obtained between the zero-dimensional model calculations and the spatially averaged values of the plug-flow simulation results. The zero-dimensional model calculations are verified against spatially resolved simulation results and validated against a wide variety of measurement data from the literature. The nitric oxide (NO) concentration is thoroughly characterized for a variation of the gas mixture ratio, helium flow rate and absorbed power. An ‘effective’ and a hypothetical larger rate coefficient value for the reactive quenching are used to estimate the role of the species N2(A3Σ, B3Π; v > 0) and multiple higher N2 electronically excited states instead of only N2(A3Σ, B3Π; v = 0) in this quenching. The NO concentration measurements at low power are better and almost identically captured by the simulations using the ‘effective’ and hypothetical values, respectively. Furthermore, the density measurements under the same operation conditions are also better predicted by the simulations adopting these values. It is found that the contribution of the vibrationally excited nitrogen molecules N2(v ⩾ 13) to the net NO formation rate gains more significance at higher power. The vibrational distribution functions (VDFs) of molecular oxygen O2(v < 41) and nitrogen N2(v < 58) are investigated regarding their formation mechanisms and their responses to the variation of operation parameters. It is observed that the N2 VDF shows a stronger response than the O2 VDF. The sensitivity of the simulation results with respect to a variation of the VDF resolutions, wall reaction probabilities and synthetic air impurity levels is presented. The simulated plasma properties are sensitive to the variation, especially for a feed gas mixture containing nitrogen. The plug-flow model is validated against one-dimensional experimental data in the gas flow direction, and it is only used in case an analysis of the spatially resolved plasma properties inside the jet chamber is of interest. The increasing NO spatial concentration in the gas flow direction is saturated at a relatively high power. A stationary O2 VDF is obtained along the direction of the mass flow, while a continuously growing N2 VDF is observed until the jet nozzle.

Shock tube measurement of NO time-histories in nitromethane pyrolysis using a quantum cascade laser at 5.26 µm

The pyrolysis of nitromethane, CH3NO2, was studied at ∼3.5 atm and 1013 K–1418 K in a heated shock tube by measuring the key product nitric oxide (NO) using mid-infrared laser absorption spectroscopy. We used a quantum cascade laser (QCL) at 5.26 µm to exploit the strong NO absorption at 1900.08 cm−1. With the NO absorption cross-section data characterized at 1006 K–1789 K and 2.7 atm–3.5 atm behind reflected shock waves, we measured the NO concentration time-histories during the pyrolysis of nitromethane at two different concentrations (1.05% and 0.6%). The absorption interference from other major products such as CO and H2O was analyzed to be negligible, leading to an interference-free NO diagnostic in nitromethane pyrolysis. A recent kinetic model of Shang et al. (2019) was adopted to interpret the shock tube data. All the NO time-histories measured over the entire temperature range 1013 K–1418 K were well-predicted by this model in terms of the initial NO formation rate and the final plateau level. The rate-of-production, sensitivity, and reaction flux analyses were performed to identify four important reactions (CH3NO2 = CH3 + NO2, CH3NO2 ↔ CH3ONO = CH3O + NO, CH3 + NO2 = CH3O + NO, and NO2 + H = NO + OH) that determine the NO formation during CH3NO2 pyrolysis. The satisfactory agreement between the simulation and shock tube/laser absorption measurement further validated the kinetic mechanism of nitromethane decomposition. The developed mid-infrared NO absorption sensor provides a promising diagnostic tool for studying fuel-nitrogen chemical kinetics in the shock tube experiments.

Combustion and NOx Emission Characteristics of a Bluff Body Hydrogen Burner

Nitric oxide (NOx) emissions from a partial premixed bluff body (PPBB) hydrogen burner under varying operational conditions have been investigated numerically. The PPBB burner employs a conical bluff body to stabilize the flame, and its design allows for the dilution of the fuel–air mixture by internally recirculated flue gas. The degree of premixing can be adjusted via primary and secondary fuel ports that are controlled independently. Steady-state computational fluid dynamic (CFD) simulations were conducted for 12 different combinations of secondary fuel fractions and thermal loads to investigate the complex flow structure in the burner and the source of NOx formation. All simulations were validated against experimental data and underpredicted NOx emissions by 7% on average. A detailed analysis of the combustion characteristics was conducted and showed that primary fuel is burned in a multiregime mode at a wide range of mixture fractions/equivalence ratios, while secondary fuel is burned closer to stoichiometry. Utilizing a secondary fuel fraction of 30% (i.e., the base operational condition) leads to a fuel-rich mixture within the inner recirculation zone compared to a fuel-lean mixture when the burner is operated without secondary fuel. The concave mean curvature of the stoichiometric isosurface between primary and secondary fuel leads furthermore to a local peak of the NOx formation rate when the burner operates at its base conditions. The analysis of the mass flow rates in the inner and outer recirculation zones showed that increasing the thermal load reduces the amount of internally recirculated flue gas. The conducted simulations indicate that these effects contribute to an increase in NOx emissions with increasing thermal load and with increasing secondary fuel fraction.

Intravenous Arginine and Citrulline Supplementation Increases Rate of Nitric Oxide Formation, Near-Infrared Spectroscopy Values and Decreases the Incidence of Necrotizing Enterocolitis in a Premature Piglet Model

Lau, Patricio E. MD; Cruz, Stephanie M. MD; Robinson, Jason L. PhD; Style, Candace C. MD; Stoll, Barbara PhD; Yu, Ling PhD; Burrin, Doug PhD; Olutoye, Oluyinka O. MD, PhD Author Information

Development of a Real-Time Virtual Nitric Oxide Sensor for Light-Duty Diesel Engines

This study describes the development of a semi-physical, real-time nitric oxide (NO) prediction model that is capable of cycle-by-cycle prediction in a light-duty diesel engine. The model utilizes the measured in-cylinder pressure and information obtained from the engine control unit (ECU). From the inputs, the model takes into account the pilot injection burning and mixing, which affects the in-cylinder mixture formation. The representative in-cylinder temperature for NO formation was determined from the mixture composition calculation. The selected temperature and mixture composition was substituted using a simplified form of the NO formation rate equation for the cycle-by-cycle estimation. The reactive area and the duration of NO formation were assumed to be limited by the fuel quantity. The model predictability was verified not only using various steady-state conditions, including the variation of the EGR rate, the boost pressure, the rail pressure, and the injection timing, but also using transient conditions, which represent the worldwide harmonized light vehicles test procedure (WLTC). The WLTC NO prediction results produced less than 3% error with the measured value. In addition, the proposed model maintained its reliability in terms of hardware aging, the changing and artificial perturbations during steady-state and transient engine operations. The model has been shown to require low computational effort because of the cycle-by-cycle, engine-out NO emission prediction and control were performed simultaneously in an embedded system for the automotive application. We expect that the developed NO prediction model can be helpful in emission calibration during the engine design stage or in the real-time controlling of the exhaust NO emission for improving fuel consumption while satisfying NO emission legislation.

Nitric oxide pollutant formation in high hydrogen content (HHC) syngas flames

Three-dimensional direct numerical simulations (DNS) of high hydrogen content (HHC) syngas nonpremixed jet flames with a Reynolds number of Re = 6000 have been carried out to study the nitric oxide (NO) pollutant formation. The detailed chemistry employed is the GRI 3.0 updated with the influence of the NCN radical chemistry using flamelet generated manifolds (FGM). Preferential diffusion effects have been considered via FGM tabulation and the reaction progress variable transport equation.The DNS based quantitative results indicate a strong correlation between the flame temperature and NO concentration for the pure hydrogen flame, in which NO formation is mainly characterised by the Zeldovich mechanism. The results also indicate a rapid decrease of maximum NO values in H2/CO syngas mixtures due to lower temperatures associated with the CO-dilution into H2. Results on NO formation routes in H2/CO syngas flames show that while the Zeldovich mechanism dominates the NO formation at low strain rates, the high NO formation rate at high strain rates is entirely caused by the NNH mechanism. We also found that the Fenimore mechanism has a least contribution on NO formation in H2/CO syngas flames due to absence of CH radicals in the oxidation of CO. It is found that, due to preferential diffusion, NO concentration exhibits higher values near the flame base depending on the hydrogen content in H2/CO syngas fuel mixture.

Interaction between connexin 43 and nitric oxide synthase in mice heart mitochondria.

Connexin 43 (Cx43), which is highly expressed in the heart and especially in cardiomyocytes, interferes with the expression of nitric oxide synthase (NOS) isoforms. Conversely, Cx43 gene expression is down-regulated by nitric oxide derived from the inducible NOS. Thus, a complex interplay between Cx43 and NOS expression appears to exist. As cardiac mitochondria are supposed to contain a NOS, we now investigated the expression of NOS isoforms and the nitric oxide production rate in isolated mitochondria of wild-type and Cx43-deficient (Cx43Cre-ER(T)/fl) mice hearts. Mitochondria were isolated from hearts using differential centrifugation and purified via Percoll gradient ultracentrifugation. Isolated mitochondria were stained with an antibody against the mitochondrial marker protein adenine-nucleotide-translocator (ANT) in combination with either a neuronal NOS (nNOS) or an inducible NOS (iNOS) antibody and analysed using confocal laser scanning microscopy. The nitric oxide formation was quantified in purified mitochondria using the oxyhaemoglobin assay. Co-localization of predominantly nNOS (nNOS: 93 ± 4.1%; iNOS: 24.6 ± 7.5%) with ANT was detected in isolated mitochondria of wild-type mice. In contrast, iNOS expression was increased in Cx43Cre-ER(T)/fl mitochondria (iNOS: 90.7 ± 3.2%; nNOS: 53.8 ± 17.5%). The mitochondrial nitric oxide formation was reduced in Cx43Cre-ER(T)/fl mitochondria (0.14 ± 0.02 nmol/min./mg protein) in comparison to wild-type mitochondria (0.24 ± 0.02 nmol/min./mg). These are the first data demonstrating, that a reduced mitochondrial Cx43 content is associated with a switch of the mitochondrial NOS isoform and the respective mitochondrial rate of nitric oxide formation.

Nitrite Reductase and Nitric-oxide Synthase Activity of the Mitochondrial Molybdopterin Enzymes mARC1 and mARC2

Mitochondrial amidoxime reducing component (mARC) proteins are molybdopterin-containing enzymes of unclear physiological function. Both human isoforms mARC-1 and mARC-2 are able to catalyze the reduction of nitrite when they are in the reduced form. Moreover, our results indicate that mARC can generate nitric oxide (NO) from nitrite when forming an electron transfer chain with NADH, cytochrome b5, and NADH-dependent cytochrome b5 reductase. The rate of NO formation increases almost 3-fold when pH was lowered from 7.5 to 6.5. To determine if nitrite reduction is catalyzed by molybdenum in the active site of mARC-1, we mutated the putative active site cysteine residue (Cys-273), known to coordinate molybdenum binding. NO formation was abolished by the C273A mutation in mARC-1. Supplementation of transformed Escherichia coli with tungsten facilitated the replacement of molybdenum in recombinant mARC-1 and abolished NO formation. Therefore, we conclude that human mARC-1 and mARC-2 are capable of catalyzing reduction of nitrite to NO through reaction with its molybdenum cofactor. Finally, expression of mARC-1 in HEK cells using a lentivirus vector was used to confirm cellular nitrite reduction to NO. A comparison of NO formation profiles between mARC and xanthine oxidase reveals similar Kcat and Vmax values but more sustained NO formation from mARC, possibly because it is not vulnerable to autoinhibition via molybdenum desulfuration. The reduction of nitrite by mARC in the mitochondria may represent a new signaling pathway for NADH-dependent hypoxic NO production.

NO formation in model syngas and biogas blends

A comparative study of syngas and biogas fuel blends, comprising of CH4, H2, CO, and CO2, is conducted to validate current natural gas kinetics models and to assess nitric oxide (NO) production from prompt, thermal, NNH, and N2O formation routes. Experiments are conducted at atmospheric-pressure in jet–wall stagnation flames over fuel–air equivalence ratios of 0.7≲ϕ≲1.4. Measurements of flame burning rate are made using Particle Image Velocimetry (PIV), and NO formation is measured using Planar Laser Induced Fluorescence (PLIF), whereby absolute NO concentrations are calibrated using known seeding levels. The resulting experimental profiles are directly compared to numerical simulations of stagnation flames in order to evaluate the predictive capabilities of several chemical kinetic models: GRI-Mech 3.0, GDF-Kin NCN 3.0, CRECK (Version 1212), and Konnov 0.6. It is found that, within the range of experimental uncertainty, no model is able to provide acceptable predictions over all tested conditions for both NO formation and burning rate. GRI-Mech 3.0, CRECK (Version 1212), and Konnov 0.6 under-predict burning rate in fuels with significant H2 and CO concentration and generally over-predict emissions by more than 50% for the studied conditions, while GDF-Kin 3.0 increasingly over-predicts flame reactivity and burning rate with increasing CH4 composition, but performs the best for NO predictions. The comparative nature of the study also allows for a direct comparison of the NO emission characteristics of the fuels, which shows that the primary NO formation routes are both thermal and prompt in biogas flames and only thermal for syngas; however, NNH and N2O routes have considerable influence at lean equivalence ratios for all fuels considered. Finally, these fuel blends are shown to produce lower levels of NO than methane when compared at equivalent thermal power levels.

Characterization of the East Asian Variant of Aldehyde Dehydrogenase-2: BIOACTIVATION OF NITROGLYCERIN AND EFFECTS OF Alda-1

The East Asian variant of mitochondrial aldehyde dehydrogenase (ALDH2) exhibits significantly reduced dehydrogenase, esterase, and nitroglycerin (GTN) denitrating activities. The small molecule Alda-1 was reported to partly restore low acetaldehyde dehydrogenase activity of this variant. In the present study we compared the wild type enzyme (ALDH2*1) with the Asian variant (ALDH2*2) regarding GTN bioactivation and the effects of Alda-1. Alda-1 increased acetaldehyde oxidation by ALDH2*1 and ALDH2*2 approximately 1.5- and 6-fold, respectively, and stimulated the esterase activities of both enzymes to similar extent as the coenzyme NAD. The effect of NAD was biphasic with pronounced inhibition occurring at ≥5 mm. In the presence of 1 mm NAD, Alda-1 stimulated ALDH2*2-catalyzed ester hydrolysis 73-fold, whereas the NAD-stimulated activity of ALDH2*1 was inhibited because of 20-fold increased inhibitory potency of NAD in the presence of the drug. Although ALDH2*2 exhibited 7-fold lower GTN denitrating activity and GTN affinity than ALDH2*1, the rate of nitric oxide formation was only reduced 2-fold, and soluble guanylate cyclase (sGC) activation was more pronounced than with wild type ALDH2 at saturating GTN. Alda-1 caused slight inhibition of GTN denitration and did not increase GTN-induced sGC activation in the presence of either variant. The present results indicate that Alda-1 stimulates established ALDH2 activities by improving NAD binding but does not improve the GTN binding affinity of the Asian variant. In addition, our data revealed an unexpected discrepancy between GTN reductase activity and sGC activation, suggesting that GTN denitration and bioactivation may reflect independent pathways of ALDH2-catalyzed GTN biotransformation.

Isoform-specific differences in the nitrite reductase activity of nitric oxide synthases under hypoxia

Nitrite (NO(2)(-)) recycling to nitric oxide (NO) is catalysed by a number of enzymes and induces a protective vasodilation effect under hypoxia/ischaemia. In the present work, we tested the in vitro ability of the three NOS (nitric oxide synthase) isoforms to release NO from nitrite under anoxia using electrochemical detection, chemiluminescence and absorption spectroscopy. The release of free NO from anoxic nitrite solutions at 15 muM was specific to the endothelial NOS isoform (eNOS) and did not occur with the neuronal (nNOS) or inducible (iNOS) isoforms. Unlike xanthine oxidase, the eNOS reductase domain did not recycle nitrite to NO, and wild-type eNOS did not reduce nitrate. Our data suggest that structural and, by inference, dynamic differences between nNOS and eNOS in the distal haem side account for eNOS being the only isoform capable of converting nitrite into NO at pH 7.6. In human dermal microvascular endothelial cells under careful control of oxygen tension, the rates of NO formation determined by chemiluminescence were enhanced approximately 3.6- and approximately 8.3-fold under hypoxia (2 p.p.m. O(2)) and anoxia (argon) respectively compared with normoxia ( approximately 22 p.p.m. O(2)) using 10 muM extracellular nitrite. NOS inhibitors inhibited this hypoxic NO release. Our data show that eNOS is unique in that it releases NO under all oxygen levels from normoxia to complete anoxia at physiological micromolar nitrite concentrations. The magnitude of the hypoxic NO release by the endothelial cells suggest that the endothelium could provide an appropriate response to acute episodic ischaemia and may explain the observed eNOS-expression-specific protective effect as a short-term response in animal models of acute hypoxia.

AtNOS/AtNOA1 Is a Functional Arabidopsis thaliana cGTPase and Not a Nitric-oxide Synthase

AtNOS1 was previously identified as a potential nitric-oxide synthase (NOS) in Arabidopsis thaliana, despite lack of sequence similarity to animal NOSs. Although the dwarf and yellowish leaf phenotype of Atnos1 knock-out mutant plants can be rescued by treatment with exogenous NO, doubts have recently been raised as to whether AtNOS1 is a true NOS. Moreover, depending on the type of physiological responses studied, Atnos1 is not always deficient in NO induction and/or detection, as previously reported. Here, we present experimental evidence showing that AtNOS1 is unable to bind and oxidize arginine to NO. These results support the argument that AtNOS1 is not a NOS. We also show that the renamed NO-associated protein 1 (AtNOA1) is a member of the circularly permuted GTPase family (cGTPase). AtNOA1 specifically binds GTP and hydrolyzes it. Complementation experiments of Atnoa1 mutant plants with different constructs of AtNOA1 show that GTP hydrolysis is necessary but not sufficient for the physiological function of AtNOA1. Mutant AtNOA1 lacking the C-terminal domain, although retaining GTPase activity, failed to complement Atnoa1, suggesting that this domain plays a crucial role in planta. cGTPases appear to be RNA-binding proteins, and the closest homolog of AtNOA1, the Bacillus subtilis YqeH, has been shown to participate in ribosome assembly and stability. We propose a similar function for AtNOA1 and discuss it in the light of its potential role in NO accumulation and plant development.

Bioactivation of Nitroglycerin by Purified Mitochondrial and Cytosolic Aldehyde Dehydrogenases

Metabolism of nitroglycerin (GTN) to 1,2-glycerol dinitrate (GDN) and nitrite by mitochondrial aldehyde dehydrogenase (ALDH2) is essentially involved in GTN bioactivation resulting in cyclic GMP-mediated vascular relaxation. The link between nitrite formation and activation of soluble guanylate cyclase (sGC) is still unclear. To test the hypothesis that the ALDH2 reaction is sufficient for GTN bioactivation, we measured GTN-induced formation of cGMP by purified sGC in the presence of purified ALDH2 and used a Clark-type electrode to probe for nitric oxide (NO) formation. In addition, we studied whether GTN bioactivation is a specific feature of ALDH2 or is also catalyzed by the cytosolic isoform (ALDH1). Purified ALDH1 and ALDH2 metabolized GTN to 1,2- and 1,3-GDN with predominant formation of the 1,2-isomer that was inhibited by chloral hydrate (ALDH1 and ALDH2) and daidzin (ALDH2). GTN had no effect on sGC activity in the presence of bovine serum albumin but caused pronounced cGMP accumulation in the presence of ALDH1 or ALDH2. The effects of the ALDH isoforms were dependent on the amount of added protein and, like 1,2-GDN formation, were sensitive to ALDH inhibitors. GTN caused biphasic sGC activation with apparent EC50 values of 42 ± 2.9 and 3.1 ± 0.4 μm in the presence of ALDH1 and ALDH2, respectively. Incubation of ALDH1 or ALDH2 with GTN resulted in sustained, chloral hydrate-sensitive formation of NO. These data may explain the coupling of ALDH2-catalyzed GTN metabolism to sGC activation in vascular smooth muscle.

Dynamics of Nitric Oxide and Peroxynitrite During Global Brain Ischemia/Reperfusion in Rat Hippocampus: NO-sensor Measurement and Modeling Study

The dynamics of nitric oxide (NO) and peroxynitrite concentration changes during brain ischemia/reperfusion are poorly understood. In this paper, a NO-selective sensor was used to measure NO concentration changes in the rat brain hippocampus during global brain ischemia/reperfusion. Four-vessel occlusion model of transient global brain ischemia was used. Global cerebral ischemia was induced by occluding both common carotid arteries with artery nips (for 20 min) and reperfusion was induced by loosening the artery nips. Results showed that the changes of NO concentration during global brain ischemia/reperfusion could be divided into different stages. Together with the effects of O2 tension changes and NO synthase (NOS) on nitric oxide levels, we determined five stages in the NO concentration profile: (1) acute O2-limited decrease stage; (2) O2-limited steady stage; (3) neuronal NOS activation stage; (4) acute O2-recovery elevation stage; and (5) O2-recovery steady stage. In addition, a chemical reaction network model was constructed to simulate the dynamics of peroxynitrite during the reperfusion stage, and the effects of a change in the NO formation rate on the dynamics of peroxynitrite were investigated specifically. Results show the rate of NO formation has a great influence on peroxynitrite dynamics.

Estimation of Nitric Oxide Concentration in Blood for Different Rates of Generation: EVIDENCE THAT INTRAVASCULAR NITRIC OXIDE LEVELS ARE TOO LOW TO EXERT PHYSIOLOGICAL EFFECTS

Endothelium-derived nitric oxide (NO) is a potent vasodilator in the cardiovascular system. Several lines of experimental evidence suggest that NO or NO equivalents may also be generated in the blood. However, blood contains a large amount of hemoglobin (Hb) in red blood cells (RBCs). The RBC-encapsulated Hb can react very quickly with NO, which is only limited by the rate of NO diffusion into the RBCs. It is unclear what the possible NO concentration levels in blood are and how the NO diffusion coefficient (D) and the permeability (Pm) of RBC membrane to NO affect the level of NO concentration. In this study, a steady-state concentration experimental method combined with a spherical diffusion model are presented for determining D and Pm and examining the effect of NO generation rate (V0) and hematocrit (Hct) on NO concentration. It was determined that Pm is 4.5 +/- 1.5 cm/s and D is 3410 +/- 50 microm2/s at 37 degrees C. Simulations based on experimental parameters show that, when the rate of NO formation is as high as 100 nm/s, the maximal NO concentration in blood is below 0.012 nM at Pm = 4.5 cm/s and Hct = 45%. Thus, it is unlikely that NO is directly exported or generated from the RBC as an intravascular signaling molecule, because its concentration would be too low to exert a physiological role. Furthermore, our results suggest that, if RBCs export NO bioactivity, this would be through NO-derived species that can release or form NO rather than NO itself.

Differential effects of mutations in human endothelial nitric oxide synthase at residues Tyr-357 and Arg-365 on l-arginine hydroxylation and [formula omitted]-hydroxy- l-arginine oxidation

Biosynthesis of nitric oxide (NO) is catalyzed by NO synthase (NOS) through a two-step oxidation of l-arginine (Arg) with formation of an intermediate, GN -hydroxy- l-Arg (NHA). In this study we have employed mutagenesis to investigate how residues Y357 and R365 which interact primarily with the substrate Arg and (6R)-5,6,7,8-tetrahydro- l-biopterin (H 4B) modulate these two steps of the NOS reaction. Mutant Y357F preserved most wild-type heme characteristics and NADPH oxidation ability. However, mutation of this residue markedly increased the dissociation constants for both Arg and NHA by 20-fold and decreased the NO synthesis from Arg by 85% compared to that of wild type. Mutation of Y357 had less effect on the rate of NO generated from NHA. Mutant R365L purified in the presence of Arg had a normal heme environment and retained 9 and 55% of the wild-type NO formation rate from Arg and NHA, respectively. When Arg was removed from buffer, R365L instantly became a low-spin state (Soret peak at 418 nm) with the resultant loss of H 4B and instability of the heme–CO complex. The low-spin R365L exhibited an NADPH oxidation rate higher than that of wild type. Its Arg-driven NO formation was decreased to near the limit of detection, whereas the rate of NHA-driven NO synthesis was one third that of wild type. This NHA-driven NO formation completely relied on H 4B and was not sensitive to superoxide dismutase or catalase but was inhibited by imidazole. The wild-type eNOS required 14 μM NHA and 0.39 μM H 4B to reach the half-maximal NHA-driven NO formation rate (EC 50), while R365L needed 59 μM NHA and 0.73 μM H 4B to achieve EC 50. The differential effect of mutation on Arg and NHA oxidation suggests that distinct heme-based active oxidants are responsible for each step of NO synthesis.

Endogenous Methylarginines Regulate Neuronal Nitric-oxide Synthase and Prevent Excitotoxic Injury

Nitric oxide (NO) has a critical role in neuronal function; however, high levels lead to cellular injury. While guanidino-methylated arginines (MA) including asymmetric dimethylarginine (ADMA) and N(G)-methyl-l-arginine (NMA) are potent competitive inhibitors of nitric oxide synthase (NOS) and are released upon protein degradation, it is unknown whether their intracellular concentrations are sufficient to critically regulate neuronal NO production and secondary cellular function or injury. Therefore, we determine the intrinsic neuronal MA concentrations and their effects on neuronal NOS function and excitotoxic injury. Kinetic studies demonstrated that the K(m) for l-arginine is 2.38 microm with a V(max) of 0.229 micromol mg(-1) min(-1), while K(i) values of 0.67 microm and 0.50 microm were determined for ADMA and NMA, respectively. Normal neuronal concentrations of all NOS-inhibiting MA were determined to be approximately 15 microm, while l-arginine concentration is approximately 90 microm. These MA levels result in >50% inhibition of NO generation from neuronal NOS. Down-modulation or up-modulation of these neuronal MA levels, respectively, dramatically enhanced or suppressed NO-mediated excitotoxic injury. Thus, neuronal MA profoundly modulate NOS function and suppress NO mediated injury. Pharmacological modulation of the levels of these intrinsic NOS inhibitors offers a novel approach to modulate neuronal function and injury.

Focusing of nitric oxide mediated nitrosation and oxidative nitrosylation as a consequence of reaction with superoxide.

The impact of nitric oxide (NO) synthesis on different biological cascades can rapidly change dependent on the rate of NO formation and composition of the surrounding milieu. With this perspective, we used diaminonaphthalene (DAN) and diaminofluorescein (DAF) to examine the nitrosative chemistry derived from NO and superoxide (O2-) simultaneously generated at nanomolar to low micromolar per minute rates by spermine/NO decomposition and xanthine oxidase-catalyzed oxidation of hypoxanthine, respectively. Fluorescent triazole product formation from DAN and DAF increased as the ratio of O2- to NO approached equimolar, then decreased precipitously as O2- exceeded NO. This pattern was also evident in DAF-loaded MCF-7 carcinoma cells and when stimulated macrophages were used as the NO source. Cyclic voltammetry analysis and inhibition studies by using the N2O3 scavenger azide indicated that DAN- and DAF-triazole could be derived from both oxidative nitrosylation (e.g., DAF radical + NO) and nitrosation (NO+ addition). The latter mechanism predominated with higher rates of NO formation relative to O2-. The effects of oxymyoglobin, superoxide dismutase, and carbon dioxide were examined as potential modulators of reactant availability for the O2- + NO pathway in vivo. The findings suggest that the outcome of NO biosynthesis in a scavenger milieu can be focused by O2- toward formation of NO adducts on nucleophilic residues (e.g., amines, thiols, hydroxyl) through convergent mechanisms involving the intermediacy of nitrogen dioxide. These modifications may be favored in microenvironments where the rate of O2- production is temporally and spatially contemporaneous with nitric oxide synthase activity, but not in excess of NO generation.

Sources of Intra-Oesophageal Nitric Oxide Production Following Intraluminal Acid Exposure

Background: The aim of the present study was to assess luminal nitric oxide (NO) levels in the oesophagus during baseline and acidic conditions and to clarify the sources of such oesophageal NO formation. Methods: Healthy volunteers received an intra-oesophageal infusion of either HCl (100 mM) or NaCl (150 mM) on two separate study days. After a low nitrate diet, nitrate load or no dietary restrictions/pretreatment, direct intraluminal measurements of NO formation were performed using a tonometric technique. Endoscopy was performed and mucosal biopsies were taken and analysed by means of immunohistochemistry, Western blot and RT-PCR. Results: No intra-oesophageal NO was detected during baseline conditions with pH neutrality. During the infusion of HCl the NO levels rose dramatically to around 12000 ppb. This high rate of NO formation fell by 95% following deviation of saliva. NO formation after an acute nitrate load was almost doubled during acid perfusion compared to control. Immunohistochemistry demonstrated distinct staining for iNOS in the oesophageal squamous epithelial cells, and Western blot and RT-PCR confirmed the presence of iNOS. Conclusion: Two sources exist for intra-oesophageal NO formation, both dependent on the luminal acidity: 1) chemical reduction of salivary nitrite, a mechanism related to dietary intake of nitrate, and 2) NO formation within the oesophageal mucosal epithelium by enzymatic degradation of L-arginine. In the latter case, the NO synthase has antigenic characteristics, indicating the inducible isoform, although a functional behaviour suggests an unconventional subtype.