Reduced Nitric Oxide Bioavailability in Horses with Colic: Evaluation by ESR Spectroscopy

Biophysics - The pathogenesis of diseases of the gastrointestinal tract in horses is accompanied by inflammation and oxidative stress and may be caused by a lack of nitric oxide, which controls...     

Reactions of Nitric Oxide with Nitronyl Nitroxides and Oxygen: Prediction of Nitrite and Nitrate Formation by Kinetic Simulation

Nitric oxide reacts with nitronyl nitroxides (NNO) to form imino nitroxides (INO) and this transformation can be monitored using electron spin resonance spectroscopy. Recently, Akaike et al., reported that NNO such as 2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl (PTIO) and its derivatives (e.g., carboxy-PTIO) react with nitric oxide (·NO) in a 1:1 stoichiometry forming 2-phenyl-4,4,5,5-tetra-methylimidazoline-1-oxyl (PTI) or the respective product (e.g., carboxy-PTI) together with nitrite and nitrate (Akaike et al., Biochemistry 32, 827-832, 1993). In this paper, we reevaluate their results and show that the stoichiometry of the reaction between PTIO and ·NO is 0.63 ± 0.06:1.0. The reason for this discrepancy is due to an erroneous assumption by Akaike et al., that the stoichiometry for the reaction between ·NO and O₂ is 2:1 in aqueous solution. If the data reported by Akaike et al., were recalculated using a 4:1 stoichiometry established for the aqueous oxidation of ·NO, the reaction between ·NO and PTIO would give a stoichiometry of 0.5:1.0 in closer agreement with our data. We propose a mechanism for the reaction between PTIO and ·NO in aqueous solution. This mechanism predicts that the stoichiometry between carboxy-PTIO and ·NO is dependent on the rate of generation of ·NO and is 1:1 only at low rates of ·NO generation (i.e., 10^-13 M/s). However the stoichiometry approaches 0.5:1.0 at higher rates of ·NO production or when it is added as a bolus. The ratio between nitrite and nitrate also varies as a function of the rate of generation of ·NO. The model agrees with previous experimental observations that the aqueous oxidation of ·NO in air saturated solutions will exclusively form nitrite and predicts that ·NO will only generate substantial amounts of nitrate if it is released at a rate less than 10^-17 M/s. This may have important consequences in cellular systems where the concentration of ·NO is typically measured from nitrite production.     

Regional Estimate of Nitric Oxide Emissions Following Woody Encroachment: Linking Imaging Spectroscopy and Field Studies

Woody encroachment, a spatially explicit process of land-cover change, is known to affect the biophysical and biogeochemical properties of ecosystems. However, little information is available on the impacts of woody encroachment on N oxide emissions from savanna regions. We combined hyperspectral remote sensing and field measurements to quantify spatial patterns and estimate regional fluxes of soil N oxide emissions as they covary with vegetation cover and soil type across a semiarid rangeland in north Texas. Soil nitric oxide (NO) emissions were highly correlated with Prosopis canopy cover, allowing the extrapolation of NO fluxes from hyperspectral observations of woody cover. NO emissions were highly variable, ranging from 0 to 550 kg NO-N km^–2 y^–1 across the region, with the lowest emissions from shallow clay soils and highest from deeper upland clay loams. An estimate of annual NO emissions based on remotely derived Prosopis cover, temperature, and precipitation was 160 kg NO-N km^–2 y^–1, almost twice that of the value derived from traditional averaging of field measurements. We conclude that relationships between NO emissions and remotely sensed structure and composition are advantageous for quantifying NO emissions at the regional scale. This study also provides new insight into the role of woody encroachment on biogeochemical processes that are highly variable and otherwise difficult to measure at the regional scale.     

Involvement of Nitric Oxide in the Nociceptive Behavioral Reaction in Mice with Tonic Pain

In experiments on mongrel albino male mice with a nidus of tonic pain created by subcutaneous injection of 5% formalin solution into the hindlimb, we estimated changes in nociceptive behavioral reaction (licking the pain nidus) elicited by i.p. injections of compounds modulating the system of nitric oxide (NO): a blocker of NO synthase, N-nitro-L-arginine (L-NAME), and activator of NO synthesis, L-arginine (L-Arg), as well as NO donors: sodium nitroprusside (SNP) and sodium nitrate (SN). After injections of L-NAME, L-Arg, and SN, the intensity of the nociceptive behavioral reaction dropped by 55-21%, as compared with the control. In contrast, SN significantly increased the intensity of this reaction. Mechanisms responsible for modulation of the nociceptive behavioral reaction with the involvement of NO and specific features of the effects of different NO donors on this reaction (related to a complex nature of these effects) are discussed.     

Studies of Nitric Oxide Production in Rat Tissues in Postnatal Development by Electron Paramagnetic Resonance Spectroscopy

Biophysics - Paramagnetic complexes containing nitric oxide (NO) have been assayed in rat heart and liver tissues by EPR spectroscopy to investigate the time variation of NO production during...     

Nitric Oxide and Cnidarian-Dinoflagellate Symbioses: Pieces of a Puzzle

The presence of nitric oxide synthase (NOS) activity is demonstratedin the tropical marine cnidarian Aiptasia pallida and in itssymbiotic dinoflagellate algae, Symbiodinium bermudense. Enzymeactivity was assayed by measuring the conversion of arginineto citrulline. Biochemical characterization of NOS from Aiptasiawas characterized with respect to cellular localization, substrateand cofactor requirements, inhibitors, and kinetics. In responseto acute temperature shock, anemones retracted their tentacles.Animals subjected to such stress had lower NOS activities thandid controls. Treatment with NOS inhibitors caused tentacularretraction, while treatment with the NOS substrate L-arginineinhibited this response to stress, as did treatment with NOdonors. These results provide a preliminary biochemical characterizationof, and suggest a functional significance for, NOS activityin anthozoan-algal symbiotic assemblages.     

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability^{1, 2}. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation³, and ultimately to oxidative stress leading to cell injury or death⁴. Superoxide radical anion (O₂•-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O₂•- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O₂•- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O₂•- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase⁵. Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO⁶. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O₂•- in a process called uncoupling, which is believed to be caused by oxidation of heme⁷ or the co-factor, tetrahydrobiopterin (BH₄)⁸.There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production⁹.The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO¹⁰, the phosphoryl-substituted DEPMPO¹¹, and the ester-substituted, EMPO¹² and BMPO¹³, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O₂•- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)₂ is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct¹⁴.     

Nitric Oxide Transport in Normal Human Thoracic Aorta: Effects of Hemodynamics and Nitric Oxide Scavengers

Despite the crucial role of nitric oxide (NO) in the homeostasis of the vasculature, little quantitative information exists concerning NO transport and distribution in medium and large-sized arteries where atherosclerosis and aneurysm occur and hemodynamics is complex. We hypothesized that local hemodynamics in arteries may govern NO transport and affect the distribution of NO in the arteries, hence playing an important role in the localization of vascular diseases. To substantiate this hypothesis, we presented a lumen/wall model of the human aorta based on its MRI images to simulate the production, transport and consumption of NO in the arterial lumen and within the aortic wall. The results demonstrated that the distribution of NO in the aorta was quite uneven with remarkably reduced NO bioavailability in regions of disturbed flow, and local hemodynamics could affect NO distribution mainly via flow dependent NO production rate of endothelium. In addition, erythrocytes in the blood could moderately modulate NO concentration in the aorta, especially at the endothelial surface. However, the reaction of NO within the wall could only slightly affect NO concentration on the luminal surface, but strongly reduce NO concentration within the aortic wall. A strong positive correlation was revealed between wall shear stress and NO concentration, which was affected by local hemodynamics and NO reaction rate. In conclusion, the distribution of NO in the aorta may be determined by local hemodynamics and modulated differently by NO scavengers in the lumen and within the wall.     

Inactivation of Tryptophan Hydroxylase by Nitric Oxide: Enhancement by Tetrahydrobiopterin

Abstract: Tryptophan hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is inactivated by the nitric oxide generators sodium nitroprusside, diethylamine/nitric oxide complex, and S -nitroso- N -acetylpenicillamine. Physiological concentrations of tetrahydrobiopterin, the natural and endogenous cofactor for the hydroxylase, significantly enhance the inactivation of the enzyme caused by each of these nitric oxide generators. The substrate tryptophan does not have this effect. The chemically reduced (tetrahydro-) form of the pterin is required for the enhancement, because neither biopterin nor dihydrobiopterin is effective. The 6 S -isomer of tetrahydrobiopterin, which has little cofactor efficacy for tryptophan hydroxylase, does not enhance enzyme inactivation as does the natural 6 R -isomer. A number of synthetic, reduced pterins share with tetrahydrobiopterin the ability to enhance nitric oxide-induced inactivation of tryptophan hydroxylase. The tetrahydrobiopterin effect is not prevented by agents known to scavenge hydrogen peroxide, superoxide radicals, peroxynitrite anions, hydroxyl radicals, or singlet oxygen. On the other hand, cysteine partially protects the enzyme from both the nitric oxide-induced inactivation and the combined pterin/nitric oxide-induced inactivation. These results suggest that the tetrahydrobiopterin cofactor enhances the nitric oxide-induced inactivation of tryptophan hydroxylase via a mechanism that involves attack on free protein sulfhydryls. Potential in vivo correlates of a tetrahydrobiopterin participation in the inactivation of tryptophan hydroxylase can be drawn to the neurotoxic amphetamines.     

Production of Nitric Oxide and Nitrous Oxide During Denitrification by Corynebacterium nephridii

Resting cells of Corynebacterium nephridii reduce nitrate, nitrite, and nitric oxide to nitrous oxide under anaerobic conditions. Nitrous oxide production from nitrite was optimal from pH 7.0 to 7.4. The stoichiometry of nitrous oxide production from nitrite was 99% of the theoretical-two moles of nitrite was used for each mole of nitrous oxide detected. Hydroxylamine increases gas evolution from nitrite but inhibits the reduction of nitric oxide to nitrous oxide. Hydroxylamine is converted to nitrogenous gas(es) by resting cells only in the presence of nitrite. Under certain conditions nitric oxide, as well as nitrous oxide, was detected.     

Role of Nitric Oxide in Methamphetamine Neurotoxicity: Protection by 7-Nitroindazole, an Inhibitor of Neuronal Nitric Oxide Synthase

Abstract: The role of nitric oxide (NO^•) in the neurotoxic effects of methamphetamine (METH) was evaluated using 7-nitroindazole (7-NI), a potent inhibitor of neuronal nitric oxide synthase. Treatment of mice with 7-NI (50 mg/kg) almost completely counteracted the loss of dopamine, 3,4-dihydroxyphenylacetic acid, and tyrosine hydroxylase immunoreactivity observed 5 days after four injections of 10 or 7.5 mg/kg METH. With the higher dose of METH, this protection at 5 days occurred despite the fact that combined administration of METH and 7-NI significantly increased lethality and exacerbated METH-induced dopamine release (as indicated by a greater dopamine depletion at 90 min and 1 day). Combined treatment with 4 × 10 mg/kg METH and 7-NI also slightly increased the body temperature of mice as compared with METH alone. Thus, the neuroprotective effects of 7-NI are independent from lethality, are not likely to be related to a reduction of METH-induced dopamine release, and are not due to a decrease in body temperature. These results indicate that NO^• formation is an important step leading to METH neurotoxicity, and suggest that the cytotoxic properties of NO^• may be directly involved in dopaminergic terminal damage.     

Formation Mechanism of the Free Radical Product and Its Precursor by the Reaction of Dehydro-L-ascorbic Acid with Amino Acid

During the formation of radical A (2) and its precursor (tris(2-deoxy-2-L-ascorbyl)amine, 1) by the reaction of dehydroascorbic acid (DHA) with amino acid, ascorbic acid (AsA) and the reduced red pigment (3) were newly identified, in addition to scorbamic acid (SCA) and the red pigment (4), as intermediate products. The addition of AsA to the DHA-amino acid reaction, as well as to the DHA-SCA reaction, greatly increased the formation of 3 and 1. The reaction of AsA with 4 gave rapidly 3, followed by the gradual production of 1. From these results, a reaction pathway is proposed that 3 formed by the reduction of 4 with AsA is a key intermediate and its condensation with DHA followed by reduction with AsA might produce 2 and 1.     

Regulation of Neuronal Nitric Oxide and Cyclic GMP Formation by Ca2+

Nitric oxide (NO) acts as a messenger molecule in the CNS by activating soluble guanylyl cyclase. Rat brain synaptosomal NO synthase was stimulated by Ca2+ in a concentration-dependent manner with half-maximal effects observed at 0.3 microM and 0.2 microM when its activity was assayed as formation of NO and L-citrulline, respectively. Cyclic GMP formation was apparently inhibited, however, at Ca2+ concentrations required for the activation of NO synthase, indicating a down-regulation of the signal in NO-producing cells. Purified synaptosomal guanylyl cyclase was not inhibited directly by Ca2+, and the effect was not mediated by a protein binding to guanylyl cyclase at low or high Ca2+ concentrations. In cytosolic fractions, the breakdown of cyclic GMP, but not that of cyclic AMP, was highly stimulated by Ca2+, and 3-isobutyl-1-methylxanthine did not block this reaction effectively. The effects of Ca2+ on cyclic GMP hydrolysis and on apparent guanylyl cyclase activities were abolished almost completely in the presence of the calmodulin antagonist calmidazolium, whose effect was attenuated by added calmodulin. Thus, a Ca2+/calmodulin-dependent cyclic GMP phosphodiesterase is highly active in synaptic areas of the brain and may prevent elevations of intracellular cyclic GMP levels in activated, NO-producing neurons.     

Synthesis,Pharmacological Study and Modeling of 7-Methoxyindazole and Related Substituted Indazoles as Neuronal Nitric Oxide Synthase Inhibitors

The synthesis, pharmacological evaluation and modelisation of 7-methoxyindazole (7-MI) and related alkoxy-indazoles as novel inhibitors of neuronal nitric oxide synthase are presented. 7-MI remains the most active compound of this series in an in vitro enzymatic assay of neuronal nitric oxide synthase activity. Modeling studies of the interaction of 7-substituted indazole derivatives complexed with nNOS and the relationship with their respective biological activities suggest that a bulky substitution on position-7 is responsible for a steric hindrance effect which does not allow these compounds to interact with nNOS in the same way as 7-NI and 7-MI.     

Synthesis of Substituted Aminomalonates by the Reaction of Chloramine with Malonate Carbanions

Increased abnormal oocytes due to meiotic chromosome misalignment and spindle defects lead to elevated rates of infertility, miscarriage, and trisomic conceptions. Here, we investigated the effect of biotin deficiency on oocyte quality. Three-week-old female ICR mice were fed a biotin-deficient or control diet (0, 0.004 g biotin/kg diet) for 21 days. On day 22, these mouse oocytes were analyzed by immunofluorescence. Due to biotin, undernutrition increased the frequency of abnormal oocytes (the biotin deficient vs. control: 40 vs. 16%). Next, the remaining mice in the biotin-deficient group were fed a control or biotin-deficient diet from day 22 to 42. Although biotin nutritional status in the recovery group was restored, the frequency of abnormal oocytes in the recovery group was still higher than that in the control group (48 vs. 18%). Our results indicate that steady, sufficient biotin intake is required for the production of high-quality oocytes in mice.     

Production and Consumption of Nitric Oxide by Three Methanotrophic Bacteria

We studied nitrogen oxide production and consumption by methanotrophs Methylobacter luteus (group I), Methylosinus trichosporium OB3b (group II), and an isolate from a hardwood swamp soil, here identified by 16S ribosomal DNA sequencing as Methylobacter sp. strain T20 (group I). All could consume nitric oxide (nitrogen monoxide, NO), and produce small amounts of nitrous oxide (N₂O). Only Methylobacter strain T20 produced large amounts of NO (>250 parts per million by volume [ppmv] in the headspace) at specific activities of up to 2.0 × 10⁻¹⁷ mol of NO cell⁻¹ day⁻¹, mostly after a culture became O₂ limited. Production of NO by strain T20 occurred mostly in nitrate-containing medium under anaerobic or nearly anaerobic conditions, was inhibited by chlorate, tungstate, and O₂, and required CH₄. Denitrification (methanol-supported N₂O production from nitrate in the presence of acetylene) could not be detected and thus did not appear to be involved in the production of NO. Furthermore, cd₁ and Cu nitrite reductases, NO reductase, and N₂O reductase could not be detected by PCR amplification of the nirS, nirK, norB, and nosZ genes, respectively. M. luteus and M. trichosporium produced some NO in ammonium-containing medium under aerobic conditions, likely as a result of methanotrophic nitrification and chemical decomposition of nitrite. For Methylobacter strain T20, arginine did not stimulate NO production under aerobiosis, suggesting that NO synthase was not involved. We conclude that strain T20 causes assimilatory reduction of nitrate to nitrite, which then decomposes chemically to NO. The production of NO by methanotrophs such as Methylobacter strain T20 could be of ecological significance in habitats near aerobic-anaerobic interfaces where fluctuating O₂ and nitrate availability occur.     

Formation of Free Radicals and Nitric Oxide Derivative of Hemoglobin in Rats During Shock Syndrome

Free radicals have been postulated to play an important role as mediators in the pathogenesis of shock syndrome and multiple-organ failure. We attempted to directly detect the increased formation of radicals by Electron Spin Resonance (ESR) in animal models of shock, namely the endotoxin (ETX) shock or the hemorrhagic shock of the rat. In freeze-clamped lung tissue, a small but significant increase of a free radical signal was detected after ETX application. In the blood of rats under ETX shock, a significant ESR signal with a triplet hyperfine structure was observed. The latter ESR signal evolved within several hours after the application of ETX and was localized in the red blood cells. This signal was assigned to a nitric oxide (NO) adduct of hemoglobin with the tentative structur ((a2+ NO)/23+)2. The amount of hemoglobin-NO formed, up to 0.8% of total hemoglobin, indicated that under ETX shock a considerable amount of NO was produced in the vascular system. This NO production was strongly inhibited by the arginine analog N^G-monomethyl-arginine (NMMA). The ESR signal of Hb-NO was also observed after severe hemorrhagic shock. There are three questions, namely (i) the type of vascular cells and the regulation of the process forming such a large amount of NO during ETX shock, (ii) the pathophysiological implications of the formed NO, effects which have been described as cytotoxic mediator, endothelium-derived relaxing factor (EDRF) or inhibitor of platelet aggregation, and (iii) the possible use of Hb-NO for monitoring phases of shock syndrome.     

糖皮质激素对机械通气大鼠肺组织iNOS、NO表达的影响

目的通过观察糖皮质激素对机械通气大鼠肺组织诱导型一氧化氮合酶（iNOS）及一氧化氮（NO）表达的影响,探讨糖皮质激素对呼吸机所致肺损伤（ventilator induced lung injury,VILI）的干预作用。方法 24只雄性Wistar大鼠随机分为对照组、机械通气组、地塞米松（DXM）干预组。用逆转录-聚合酶链反应（RT-PCR）法检测肺组织iNOS mRNA表达,用免疫组织化学染色法检测肺组织iNOS蛋白表达,用硝酸还原酶法测定肺组织和血浆NO含量。结果机械通气组和DXM干预组大鼠肺组织iNOS mRNA及其蛋白表达水平,以及血浆和肺组织NO含量均明显高于对照组（P〈0.01）;DXM干预组上述指标与机械通气组比较均明显降低（P〈0.01）。结论糖皮质激素可通过抑制肺组织iNOS的表达,减少NO的生成,对机械通气大鼠肺组织具有保护作用。