Oxidation Product Of Nitric Oxide Research Articles

Using Metalloporphyrin Nanosensors for In Situ Monitoring and Measurement of Nitric Oxide and Peroxynitrite in a Single Human Neural Progenitor Cell.

Nitric oxide (NO) is an inorganic signaling molecule that plays a crucial role in the regulation of numerous physiological functions. An oxidation product of the cytoprotective NO is cytotoxic peroxynitrite (ONOO-). In biological systems, the concentrations of NO and ONOO- are typically transient, ranging from nanomolar to micromolar, and these increases are normally followed by a swift return to their basal levels due to their short life spans. To understand the vital physiological role of NO and ONOO- in vitro and in vivo, sensitive and selective methods are necessary for direct and continuous NO and ONOO- measurements in real time. Because electrochemical methods can be adjusted for selectivity, sensitivity, and biocompatibility in demanding biological environments, they are suitable for real-time monitoring of NO and ONOO- release. Metalloporphyrin nanosensors, described here, have been designed to measure the concentration of NO and ONOO- produced by a single human neural progenitor cell (hNPC) in real time. These nanosensors (200-300 nm in diameter) can be positioned accurately in the proximity of 4-5 ± 1 μm from an hNPC membrane. The response time of the sensors is better than a millisecond, while detection limits for NO and ONOO- are 1 × 10-9 and 3 × 10-9 mol/L, respectively, with a linear concentration response of up to about 1 μM. The application of these metalloporphyrin nanosensors for the efficient measurement of the concentrations of NO and ONOO- in hNPCs is demonstrated, providing an opportunity to observe in real time the molecular changes of the two signaling molecules in situ.

Inflammatory Activation and Nitric Oxide Oxidation in Exertional Dyspnea in Southwest Asia Deployed Military Veterans

Post deployment personnel of the Southwest Asia theater of war present with significant exertional dyspnea. While deployed, these service men and women were exposed to a variety of inhaled toxicants including geological dust, burn pit smoke, and heavy metals. Upon examination, a large portion of these veterans display near‐normal pulmonary function and gas exchange in the lung, making diagnosis and treatment difficult. The aim of this study is to define the physiological and biochemical profiles of individuals with unexplained exertional dyspnea. We hypothesized that exposure to airborne toxicants has led to a chronic activated inflammatory state in the systemic vasculature, resulting in increased oxidative stress and reducing the bioavailability of nitric oxide (NO), contributing to the symptoms of dyspnea.Eligible veterans were tested for lung function by spirometry including diffusing capacity for carbon monoxide (DLco). Resting blood samples were taken and immediately separated into plasma and cellular components. Granuloyctes were removed from whole blood by magnetic separation, and subsequent leukocytes were collected and gated on expression of the myeloid marker CD45. Within CD45+ cells, flow cytometry was used to analyze the expression of inflammatory macrophage activation markers CD87, CD11b, CD163, HLA‐DR, CD195, CD192, CD14, CD16. Circulating levels of nitrite and nitrate (oxidation products of NO) were measured in plasma via reductive chemiluminescence, and the ratio of nitrate/nitrite (NOx ratio) was used as a marker for reduced bioavailability of NO. Increased expression of inflammatory marker CD87 in CD11b+ cells was positively correlated with expression of CD163 in HLA‐DR+ and CD14 in CD16+ (rho = .54, p <.01; rho= .36, p= .047; n =30), but not CD195 in CD192+ (rho =‐.3, p =.1). Similar observations were observed in granulocytic cells regarding CD66b expression. This correlation between activation markers within the circulating myeloid‐derived cells is consistent with the presence of monocyte activation in the systemic vasculature. NOx levels were within normally observable bounds, however, the NOx ratio was negatively correlated with FEV1/FVC (a marker of lung obstruction) but not with %predicted DLco(rho =‐.56, p =.016; rho =.01, p = .96, n = 18). The reduction in FEV1/FVC with increased NOx ratio suggests potential loss of endothelial function, which may be contributing to exertional dyspnea and is currently under investigation.

Quantitative Proteome Profiling of a S-Nitrosoglutathione Reductase (GSNOR) Null Mutant Reveals a New Class of Enzymes Involved in Nitric Oxide Homeostasis in Plants.

Nitric oxide (NO) is a short-lived radical gas that acts as a signaling molecule in all higher organisms, and that is involved in multiple plant processes, including germination, root growth, and fertility. Regulation of NO-levels is predominantly achieved by reaction of oxidation products of NO with glutathione to form S-nitrosoglutathione (GSNO), the principal bioactive form of NO. The enzyme S-nitrosoglutathione reductase (GSNOR) is a major route of NADH-dependent GSNO catabolism and is critical to NO homeostasis. Here, we performed a proteomic analysis examining changes in the total leaf proteome of an Arabidopsis thaliana GSNOR null mutant (hot5-2/gsnor1-3). Significant increases or decreases in proteins associated with chlorophyll metabolism and with redox and stress metabolism provide insight into phenotypes observed in hot5-2/gsnor1-3 plants. Importantly, we identified a significant increase in proteins that belong to the aldo-keto reductase (AKR) protein superfamily, AKR4C8 and 9. Because specific AKRs have been linked to NO metabolism in mammals, we expressed and purified A. thaliana AKR4C8 and 9 and close homologs AKR4C10 and 11 and determined that they have NADPH-dependent activity in GSNO and S-nitroso-coenzyme A (SNO-CoA) reduction. Further, we found an increase of NADPH-dependent GSNO reduction activity in hot5-2/gsnor1-3 mutant plants. These data uncover a new, NADPH-dependent component of NO metabolism that may be integrated with NADH-dependent GSNOR activity to control NO homeostasis in plants.

A simple blood microdialysis in freely-moving rats for pharmacokinetic–pharmacodynamic modeling study of Shengmai injection with simultaneous determination of drug concentrations and efficacy levels in dialysate

Microdialysis is a powerful in vivo sampling technique for pharmacokinetic–pharmacodynamic (PK-PD) modeling of drugs in pre-clinical and clinical studies. However, the noticeable limitations of previous studies using microdialysis were that animals anesthesia in the whole experiment and the combination of microdialysis and blood sampling for drug and (or) effect detection, which can obviously influence PK and PD behavior of drugs. In this study, a simple blood microdialysis sampling system in freely-moving rats was established for simultaneous study of PK and PD of Shengmai injection (SMI) effect on inducing real-time nitric oxide (NO) release on isoproterenol (ISO) induced myocardial ischemia rats. The LC–MS/MS and HPLC with fluorescence detection (HPLC-FLD) methods were developed to determine ginsenside Rg1, Rg2, Re, Rf, Rb1, Rd and Rc, the main effective components of SMI, and NOx−, the main oxidation products of NO, in dialysates respectively. Through simultaneous determination of drug concentrations and NO efficacy levels in dialysate, the developed methods were successfully applied to set up concentration-time and effect-time profiles followed by PK-PD modeling of SMI effect on inducing NO release after intravenous administration of 10.8 mL kg−1 SMI in myocardial ischemia rats. The PK-PD modeling characterized the dose-effect relationships of SMI and behaved good prediction ability. The established blood microdialysis in freely-moving rats is an appealing technology for rational PK-PD studies when selecting suitable blood endogenous micromolecule as effect marker.

Evidence of the chemical reaction of 18O-labelled nitrite with CO2 in aqueous buffer of neutral pH and the formation of 18OCO by isotope ratio mass spectrometry

Inorganic nitrite (NO2−, ON-O– ←→ –O-NO) is the autoxidation product of nitric oxide (NO). Nitrite can also be formed from inorganic nitrate (ONO2−), the major oxidation product of NO in erythrocytes, by the catalytic action of bacterial nitrate reductase in gut and oral microflora. Nitrite can be reduced to NO by certain cellular proteins and enzymes, as well as in the gastric juice under acidic conditions. Hemoglobin, xanthine oxidoreductase and carbonic anhydrase (CA) have been reported to convert nitrite to NO. Renal CA isoforms are involved in the reabsorption of nitrite and may, therefore, play an important role in NO homeostasis. Yet, the mechanisms underlying the action of CA on nitrite are incompletely understood. The nitrate/nitrite system is regarded as a reservoir of NO. We have recently shown that nitrite reacts chemically with carbon dioxide (CO2), the regular substrate of CA. The present communication reports a stable isotope ratio mass spectrometry (IRMS) study on the reaction of NO2− and CO2 performed in 50 mM HEPES buffer of pH 7.4 at 37 °C. By using 18O-labelled nitrite (18ON-O–/−18O-NO) and CO2 we observed formation of 18O-labelled CO2. This finding is an unequivocal evidence of the chemical reaction of 18ON-O–/−18O-NO with CO2. The reaction is rapid and involves nucleophilic attack of the negatively charged nitrite via one of its oxygen atoms on the partially positively charged CO2 molecule to form the putative intermediate 18ON-O-CO2–/–O2C-18O-NO. The by far largest fraction of this intermediate decomposes back to 18ON-O–/−18O-NO and CO2. A very small fraction of the intermediate, however, rearranges and finally decomposes to form 18OCO and nitrite. This reaction is slower in the presence of an isolated erythrocytic CA isoform II. In summary, NO2−, CO2 and CA are ubiquitous. The chemical reaction of NO2− with CO2 and its modulation by CA isoforms may play important roles in the transport of nitrite in red blood cells, the kidney and other cells and organs.

Nitrite circumvents canonical cGMP signaling to enhance proliferation of myocyte precursor cells.

Skeletal muscle tissue has a remarkable high regenerative capacity. The underlying cellular events are governed by complex signaling processes, and the proliferation of skeletal myoblasts is a key initial event. The role of nitric oxide (NO) in cell cycle regulation is well-appreciated. Nitrite, an NO oxidation product, is a stable source for NO-like bioactivity particularly in cases when oxygen shortage compromises NO-synthases activity. Although numerous studies suggest that nitrite effects are largely related to NO-dependent signaling, emerging evidence also implicates that nitrite itself can activate protein pathways albeit under physiological, normoxic conditions. This includes a recently demonstrated cyclic guanosine monophosphate-(cGMP)-independent enhancement of endothelial cell proliferation. Whether nitrite itself has the potential to affect myoblast proliferation and metabolism with or without activation of the canonical NO/cGMP pathway to subsequently support muscle cell regeneration is not known. Here we show that nitrite increases proliferation and metabolic activity of murine cultured myoblasts dose-dependently. This effect is not abolished by the NO scavenger 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimida-zoline-1-oxyl-3 oxide and does not affect intracellular cGMP levels, implicating a cGMP-independent mechanism. Nitrite circumvents the rapamycin induced attenuation of myoblast proliferation and enhances mTOR activity. Our results provide evidence for a novel potential physiological and therapeutic approach of nitrite in skeletal muscle regeneration processes under normoxia independent of NO and cGMP.

A pharmacokinetic and pharmacodynamic study of drug–drug interaction between ginsenoside Rg1, ginsenoside Rb1 and schizandrin after intravenous administration to rats

Ethnopharmacological relevanceGinsenoside Rg1, ginsenoside Rb1 and schizandrin are main bioactive components from Panax ginseng and Schisandra chinensis. They have been found in many prescriptions of Traditional Chinese Medicines (TCM) and proven to be effective for prevention and treatment of cardiovascular disease. It is valuable to investigate their pharmacokinetic and pharmacodynamic behavior and potential synergistic effect for better drug development and clinical application. Materials and methodsPharmacokinetic and nitric oxide (NO) release pharmacodynamic drug–drug interactions of ginsenoside Rg1, ginsenoside Rb1 and schisandrin were studied after intravenous administration of each compound with the dose of 10mg/kg and their mixture with the total dose of 10mg/kg to isoproterenol (ISO)-induced myocardial ischemia rats. Drug concentrations in serum were determined using LC–MS method. Nitrite and nitrate (NOx−), the predominant oxidation product of NO in serum was used as an effective marker and quantitated by the method of high-performance liquid chromatography coupled with fluorescence detection (HPLC-FL). The main pharmacokinetic parameters of T1/2β, MRT0−∞, Vd, Cl, and AUC, and the main pharmacodynamic parameters of Cmax, Tmax and AUEC were calculated by non-compartment model. ResultsThe results indicated ginsenoside Rb1 and (or) schisandrin in mixture could significantly postpone the elimination of ginsenoside Rg1 in rat serum. Co-administration of three compounds markedly increased the systemic exposure level of each compound in vivo. Ginsenoside Rg1 and ginsenoside Rb1 had the effect of inducing real-time NO release in rats concentration dependently. Schisandrin had no effect of inducing real-time NO release in this study. The mixture of ginsenoside Rg1, Rb1 and schisandrin administration exhibited synergistic effect of inducing NO release in ISO treated rats. ConclusionsThe result obtained from this study suggested pharmacokinetic and pharmacodynamic drug–drug interactions between ginsenoside Rg1, Rb1 and schisandrin. The study provided valuable information for drug development and clinical application of TCM.

Specific Visualization of Nitric Oxide in the Vasculature with Two-Photon Microscopy Using a Copper Based Fluorescent Probe

To study the role and (sub) cellular nitric oxide (NO) constitution in various disease processes, its direct and specific detection in living cells and tissues is a major requirement. Several methods are available to measure the oxidation products of NO, but the detection of NO itself has proved challenging. We visualized NO production using a NO-sensitive copper-based fluorescent probe (Cu 2FL2E) and two-photon laser scanning microscopy (TPLSM). Cu 2FL2E demonstrated high sensitivity and specificity for NO synthesis, combined with low cytotoxicity. Furthermore, Cu 2FL2E showed superior sensitivity over the conventionally used Griess assay. NO specificity of Cu 2FL2E was confirmed in vitro in human coronary arterial endothelial cells and porcine aortic endothelial cells using various triggers for NO production. Using TPLSM on ex vivo mounted murine carotid artery and aorta, the applicability of the probe to image NO production in both endothelial cells and smooth muscle cells was shown. NO-production and time course was detected for multiple stimuli such as flow, acetylcholine and hydrogen peroxide and its correlation with vasodilation was demonstrated. NO-specific fluorescence and vasodilation was abrogated in the presence of NO-synthesis blocker L-NAME. Finally, the influence of carotid precontraction and vasorelaxation validated the functional properties of vessels. Specific visualization of NO production in vessels with Cu 2FL2E-TPLSM provides a valid method for studying spatial-temporal synthesis of NO in vascular biology at an unprecedented level. This approach enables investigation of the pathways involved in the complex interplay between NO and vascular (dys) function.

P29: Nitrite influences proliferation of myoblasts via its reduction to nitric oxide in skeletal muscle tissue

Skeletal muscle regeneration is governed by complex cellular signaling processes. Proliferation of skeletal myoblasts is a key initial event in muscle regeneration and has been largely related to mammalian target of rapamycin (mTOR) signal transduction mechanisms. Although the precise underlying signaling remains incompletely understood, the role of nitric oxide (NO) in cell cycle regulation is well-appreciated. Nitrite, the oxidation product of NO, is regarded as a stable source for NO-like bioactivity in cases when the oxygen-dependent NO-synthases become dysfunctional. Whether nitrite itself has the potential to affect myoblast proliferation and metabolic activity under normoxic conditions with or without activation of the NO/cyclic guanosine monophosphate (cGMP) pathway is not known. The influence of nitrite and NO on C 2 C 12 myoblast proliferation was observed in cultures grown in DMEM + 10% FCS at 37 °C/5% CO 2 , with or without sodium nitrite, NO-donor DETA-NONOate, NO-scavenger cPTIO, soluble guanylatecyclase (sGC) inhibitor ODQ, or mTOR inhibitor rapamycin. Neutral red staining, propodium iodide staining for cell cycle analysis, WST-8 viability assay, mTOR phosphorylation activity assay and intracellular cGMP measuerements were performed according to modified manufacturer’s protocols. We here show that nitrite increases proliferation and metabolic activity of murine cultured myoblasts dose-dependently in normoxia. This effect is not abolished by the NO scavenger 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) and does not affect intracellular cGMP levels, implicating a cGMP-independent mechanism. Nitrite circumvents the rapamycin induced attenuation of myoblast proliferation and enhances mTOR phosphorylation activity. Our results provide evidence for a novel potential physiological and therapeutic approach of nitrite in skeletal muscle regeneration processes under normoxia independent of NO and cGMP. Nothing to disclose.

P47: AMPK activation by nitrite and metformin increases glut-4 mediated glucose uptake and normalizes pulmonary venous hypertension in a rat model of severe metabolic syndrome

Metabolic syndrome is associated with decreased bioavailability of nitric oxide (NO) and has recently been defined as a risk factor for the development of pulmonary venous hypertension in both humans and animal models. Nitrite, an endogenous circulating oxidation product of NO, has been recognized as an important signaling molecule that regulates blood flow and blood pressure, and exhibits therapeutic efficacy in pre-clinical models of pulmonary hypertension (PH). Nitrite NO 2 - is formed from the reduction of nitrate NO 3 - by oral commensal bacteria and nitrate has recently been reported to improve metabolic syndrome in the eNOS knock out mouse, however, the mechanism for this effect are unknown. In the current study we evaluated the effects of nitrite, the immediate product of nitrate metabolism, on metabolic syndrome in a genetic rat model of severe obesity, diabetes and spontaneously hypertension (ZSF1). Nitrite supplementation (50 and 100 mg/L in drinking water) significantly reduced fasting blood glucose and glycosylated hemoglobin (HbA1c) levels and improved glucose tolerance compared with untreated ZSF1 rats. Nitrite administration had no effect on β-cell mass and plasma insulin levels. We therefore measured the effect of nitrite on insulin sensitivity and glucose metabolism in skeletal muscle, the primary site for insulin-stimulated glucose utilization. Compared with untreated ZSF1 rats, chronic nitrite treatment significantly increased AMPK phosphorylation and membrane translocation of GLUT-4, key regulators of glucose uptake in skeletal muscle. Similar effects were observed in human skeletal muscle cells in culture obtained from normal and obese volunteers. We next developed a novel model of pulmonary hypertension (PH) in the ZSF1 rats using a single treatment with the VEGF inhibitor sugen (100 mg/kg). The ZSF1 rats developed significant pulmonary venous hypertension after 14 weeks. Both nitrite and the canonical AMPK activator metformin normalized pulmonary pressures in this model. Thus, our observations indicate that chronic oral therapy of nitrite improves glucose metabolism, via the AMPK-glut 4 signaling pathway, and improves PH associated severe metabolic syndrome. Disclosure Nothing to disclose.

Pharmacological evidence for the role of nitric oxide in the modulation of stress-induced anxiety by morphine in rats

The present study evaluated the effects of the opioid agonist, morphine on stress induced anxiogenesis and the possible involvement of nitric oxide (NO) in such effects in rats. Acute restraint stress consistently induced an anxiety-like response in the elevated plus maze test, i.e. reduced number of open arm entries and time spent in the open arms as compared to controls. Pretreatment with morphine (1 and 5 mg/kg), attenuated the restraint stress induced anxiogenic response in a dose related manner. Restraint stress induced neurobehavioral suppression was associated with reductions in brain NO oxidation products (NOx) levels, which were also reversed with morphine. Interaction studies showed that sub-effective doses of morphine and l-arginine (a NO precursor) had synergistic effects on stress induced elevated plus maze activity and brain NOx, whereas, l-NAME (a NO synthase inhibitor) neutralized these effects of morphine. Repeated restraint stress (× 5) induced adaptative changes as evidenced by normalization of behavioral suppression and elevations in brain NOx, as compared to acute stress. Pretreatment with morphine in combination with repeated stress (× 5) showed potentiating effects in the induction of behavioral adaptation in the elevatedplus maze and elevations in brain NOx, as compared to repeated stress alone. Further, l-NAME, when administered prior to morphine, blocked this effect of morphine on stress adaptation. These results suggest differential morphine–NO interactions during acute and repeated restraint stress.

Children with screening‐detected coeliac disease show increased levels of nitric oxide products in urine

Increased concentration of nitric oxide (NO) metabolites, nitrite and nitrate, in the urine is a strong indication of ongoing small intestinal inflammation, which is a hallmark of the enteropathy of coeliac disease (CD). It has previously been shown that children with symptomatic, untreated CD have increased levels of NO oxidation products in their urine. The aim of this study was to investigate whether screening-detected, asymptomatic coeliac children display the same urinary nitrite/nitrate pattern. In a multicenter screening study, serum samples were collected from 7208 12-year-old children without previously diagnosed CD. Sera were analysed for anti-human tissue transglutaminase (tTG) of isotype IgA. Small bowel biopsy was performed in antibody-positive children, yielding 153 new cases of CD. In the screening-detected individuals, the sum of nitrite and nitrate concentrations in the urine was analysed and used as an indicator of NO production. For comparison, 73 children with untreated, symptomatic CD were studied. The nitrite/nitrate levels in children with screening-detected CD and those with untreated symptomatic CD did not differ significantly. Both groups had significantly increased urinary nitrite/nitrate concentrations compared to the children with normal small bowel biopsy (p < 0.001). Children with screening-detected CD have increased production of NO just as children with untreated symptomatic CD. High NO metabolite levels in the urine may indicate a pathogenetic feature of CD and be a marker of major clinical importance.

Nitrous oxide-induced NO-dependent neuronal release of β-endorphin from the rat arcuate nucleus and periaqueductal gray

Nitrous oxide (N 2O)-induced antinociception is thought to result from nitric oxide (NO)-dependent neuronal release of endogenous opioid peptides in the central nervous system. The present study employed microdialysis to determine whether exposure to N 2O stimulates proopiomelanocortin (POMC) neurons to release β-endorphin in the arcuate nucleus (ARC) of the hypothalamus and the periaqueductal gray (PAG) of the midbrain. Male Sprague–Dawley rats were stereotaxically implanted with microdialysis probes in the ARC or PAG. Exposure to 70% N 2O significantly increased dialysate levels of oxidation products of NO as well as β-endorphin, compared to levels in fractions collected under room air. These increases in the ARC and PAG were abolished by systemic pretreatment with l-N G-nitro arginine methyl ester ( l-NAME). These findings suggest an association between increased NO activity and the stimulated release of β-endorphin during exposure of rats to N 2O.

Dietary inorganic nitrate reverses features of metabolic syndrome in endothelial nitric oxide synthase-deficient mice

The metabolic syndrome is a clustering of risk factors of metabolic origin that increase the risk for cardiovascular disease and type 2 diabetes. A proposed central event in metabolic syndrome is a decrease in the amount of bioavailable nitric oxide (NO) from endothelial NO synthase (eNOS). Recently, an alternative pathway for NO formation in mammals was described where inorganic nitrate, a supposedly inert NO oxidation product and unwanted dietary constituent, is serially reduced to nitrite and then NO and other bioactive nitrogen oxides. Here we show that several features of metabolic syndrome that develop in eNOS-deficient mice can be reversed by dietary supplementation with sodium nitrate, in amounts similar to those derived from eNOS under normal conditions. In humans, this dose corresponds to a rich intake of vegetables, the dominant dietary nitrate source. Nitrate administration increased tissue and plasma levels of bioactive nitrogen oxides. Moreover, chronic nitrate treatment reduced visceral fat accumulation and circulating levels of triglycerides and reversed the prediabetic phenotype in these animals. In rats, chronic nitrate treatment reduced blood pressure and this effect was also present during NOS inhibition. Our results show that dietary nitrate fuels a nitrate-nitrite-NO pathway that can partly compensate for disturbances in endogenous NO generation from eNOS. These findings may have implications for novel nutrition-based preventive and therapeutic strategies against cardiovascular disease and type 2 diabetes.

Levels of nitric oxide oxidation products are increased in the epithelial lining fluid of children with persistent asthma

Children with severe allergic asthma have persistent airway inflammation and oxidant stress. We hypothesized that children with severe allergic asthma would have increased concentrations of the nitric oxide (NO) oxidation products nitrite, nitrate, and nitrotyrosine in the proximal and distal airway epithelial lining fluid (ELF). We further hypothesized that NO oxidation products would be associated with higher exhaled NO values (fraction of exhaled nitric oxide [F(ENO)]), greater allergic sensitization, and lower pulmonary function. Bronchoalveolar lavage fluid was obtained from 15 children with mild-to-moderate asthma, 30 children with severe allergic asthma, 5 nonasthmatic children, and 20 nonsmoking adults. The bronchoalveolar lavage fluid was divided into proximal and distal portions and nitrite, nitrate, and nitrotyrosine values were quantified. Children with mild-to-moderate and severe allergic asthma had increased concentrations of nitrite (adult control subjects, 15 +/- 3 micromol/L; pediatric control subjects, 23 +/- 4 micromol/L; subjects with mild-to-moderate asthma, 56 +/- 26 micromol/L; subjects with severe asthma, 74 +/- 18 micromol/L), nitrate (37 +/- 13 vs 145 +/- 38 vs 711 +/- 155 vs 870 +/- 168 micromol/L, respectively) and nitrotyrosine (2 +/- 1 vs 3 +/- 1 vs 9 +/- 3 vs 10 +/- 4 micromol/L, respectively) in the proximal ELF. Similar results were seen in the distal ELF, although the concentrations were significantly lower (P < .05 for each). Although univariate analyses revealed no associations between NO oxidation products and clinical features, multivariate analyses revealed F(ENO) values to be a significant predictor of NO oxidation in asthmatic children. NO oxidation products are increased in the ELF of asthmatic children. The relationship between F(ENO) values and airway nitrosative stress is complicated and requires further study.

Status of Lipid Peroxidation, Antioxidants, and Oxidation Products of Nitric Oxide in Equine Babesiosis: Status of Antioxidant and Oxidant in Equine Babesiosis

Equine babesiosis is a tick-borne protozoal disease of horses caused by Theileria equi and Babesia caballi. The disease is endemic in most tropical and subtropical areas. The aim of this paper is to assess the antioxidant status, lipid peroxidation, and oxidation products of nitric oxide (NO) in horses and mules naturally infected with T. equi and B. caballi. East and Southeast Anatolian horses and mules living in rural region of the Eastern border of Turkey were used as the material for this study. These animals are used as pack animal (3–7 years of age). Infected animals were in acute or subacute infection period. In the current study, malondialdehyde (MDA), oxidation products of NO (nitrate and nitrite), serum glutathione (GSH), vitamin E, and retinol levels were analyzed in 58 equids (horse and mule) infected with T. equi and B. caballi as well as in 44 healthy equids. Compared with controls, the level of MDA and nitrate increased significantly ( P < .01, P < .05, respectively), whereas GSH concentration and levels of vitamin E decreased significantly ( P < .01). There was no significant change in the level of nitrite and retinol between two groups. The results of the current study suggest that in equids infected with T. equi and B. caballi, this alteration in the lipid peroxidation, oxidants, and antioxidants may be related to the host's defenses against parasitic infection and may play a central role in the pathologic conditions associated with babesiosis.

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.

Gentamicin Increases Nitric Oxide Production and Induces Hearing Loss in Guinea Pigs

Gentamicin application is an important therapeutic option for Ménière's disease. However, even if given at intervals, a destruction of the cochlea was often observed in various animal models together with an increased content of nitric oxide (NO) and reactive oxygen species. The present study was undertaken to identify the correlation between hearing threshold alteration and the NO production in the lateral wall and organ of Corti of the guinea pig in response to gentamicin application. Prospective animal study in guinea pigs. A single dose of gentamicin (10 mg/kg body weight) was injected intratympanally into male guinea pigs and the auditory brainstem responses were recorded before treatment and 1, 2, and 7 days after application. The organ of Corti and the lateral wall were removed from the bulla, incubated separately for 6 hours in cell culture medium and the amount of NO production was determined by chemiluminescence. Gentamicin application resulted in a hearing threshold shift beginning on the second day after gentamicin application. This hearing impairment correlates simultaneously with an increased NO2(-) content--the stable oxidation product of NO--in the lateral wall. In the organ of Corti, a slight increase in NO2(-) production was seen as early as on day 1 after gentamicin injection. The correlation between hearing threshold shift and NO production in the cochlea leads to the assumption that increased NO contributes to gentamicin-induced hearing impairment.

Evidence for a physiological role of intracellularly occurring photolabile nitrogen oxides in human skin fibroblasts

Nitric oxide (NO) plays a pivotal role in human skin biology. Cutaneous NO can be produced enzymatically by NO synthases (NOS) as well as enzyme independently via photodecomposition of photolabile nitrogen oxides (PNOs) such as nitrite or nitroso compounds, both found in human skin tissue in comparably high concentrations. Although the physiological role of NOS-produced NO in human skin is well defined, nothing is known about the biological relevance or the chemical origin of intracellularly occurring PNOs. We here, for the first time, give evidence that in human skin fibroblasts (FB) PNOs represent the oxidation products of NOS-produced NO and that in human skin fibroblasts intracellularly occurring PNOs effectively protect against the injurious effects of UVA radiation by a NO-dependent mechanism. In contrast, in PNO-depleted FB cultures an increased susceptibility to UVA-induced lipid peroxidation and cell death is observed, whereas supplementation of PNO-depleted FB cultures with physiological nitrite concentrations (10 μM) or with exogenously applied NO completely restores UVA-increased injuries. Thus, intracellular PNOs are biologically relevant and represent an important initial shield functioning in human skin physiology against UVA radiation. Consequently, nonphysiological low PNO concentrations might promote known UVA-related skin injuries such as premature aging and carcinogenesis.

Abstract 251: Endogenous Endocrine Function of Cardiac-derived Nitric Oxide Yields Distant Organ Protection Against Ischemic Injury

Background : eNOS-derived nitric oxide (NO) has long been considered a paracrine signaling molecule only capable of affecting nearby cells due to its short half-life in blood (&lt;2ms). We have recently shown that nitrite, previously thought to be an inert oxidation product of NO, is an important storage form converted to NO under hypoxic conditions via reductase activity. Currently, no studies have revealed endogenously generated NO to possess a clearly defined endocrine function, we therefore evaluated whether generation of NO in the heart could modulate remote physiological actions. Methods: Mice with cardiac-specific (αMHC promoter) eNOS overexpression (CS-eNOS-Tg) and WT littermates were analyzed for NO metabolites in the heart, plasma, and liver (NO 2 − /NO 3 − - ion chromatography; nitroso/NO-heme - gas phase chemiluminescence). WT and CS-eNOS-Tg mice were subjected to 60 min hepatic ischemia and following 5 hr reperfusion (IR) serum ALT and AST were analyzed. Results: Overexpression of eNOS restricted to the heart resulted in a significant increase in nitrite in the heart (2.2 fold), plasma (3.2 fold) and liver (6.5 fold). CS-eNOS-Tg displayed a significant reduction in hepatic IR injury (4.3 fold reduction in ALT and a 3.5 fold reduction in AST). Conclusions: This represents the first report demonstrating that eNOS-derived NO is transported in the blood and stored in remote organs. Importantly, increases in nitrite levels resulted in protection against IR injury. These findings suggest NO possesses endocrine function via transport of nitrite in the blood to distant organs and that transported nitrite exhibits physiological activity.