Nitric oxide synthase inhibitors decrease human polymorphonuclear leukocyte luminol-dependent chemiluminescence

Nitric oxide synthase (NOS) inhibitors have been reported to modulate luminol-dependent chemiluminescence (CL) in rat macrophages, whereas the potent oxidant peroxynitrite (ONOO^-) was shown to react with luminol to yield CL in a cellfree system. We evaluated the role of the -arginine/NOS pathway in luminol CL by phorbol ester-activated human polymorpho-nuclear (PMN) leukocytes using the NOS inhibitors N^G-monomethyl- -arginine ( -NMMA) and N-iminoethyl- -omithine ( -NIO). Nitric oxide (·NO) release was determined by oxidation of oxymyoglobin. In addition, the effect of NOS inhibitors on superoxide anion O₂^-) production was measured. Luminol CL was notably diminished by -NMMA in a dose-dependent manner. Superoxide dismutase (SOD) also decreased luminol CL and -NMMA potentiated light emission decrease produced by SOD. Nitric oxide and O₂^·- production was significantly decreased by -NMMA; moreover, luminol-dependent CL but not O₂^·- production was attenuated by -NIO. These data suggest that products of catalytic activity of both ·NO synthase and NADPH oxidase are required to elicit maximal luminol CL in this system. These studies demonstrate that the NOS synthase pathway is involved in luminol CL by human PMN, and they suggest that ONOO would be an unrecognized mediator in this phenomenon.     

Up-regulation of inducible nitric oxide synthase and nitric oxide in Helicobacter pylori-infected human gastric epithelial cells: possible role of interferon-gamma in polarized nitric oxide secretion

Background. Nitric oxide (NO) generated by nitric oxide synthase (NOS) is known to be an important modulator of the mucosal inflammatory response. In this study, we questioned whether Helicobacter pylori infection could up‐regulate the epithelial cell inducible NOS (iNOS) gene expression and whether NO production could show polarity that can be regulated by immune mediators. Materials and Methods. Human gastric epithelial cell lines were infected with H. pylori, and the iNOS mRNA expression was assessed by quantitative RT‐PCR. NO production was assayed by determining nitrite/nitrate levels in culture supernatants. To determine the polarity of NO secretion by the H. pylori‐infected epithelial cells, Caco‐2 cells were cultured as polarized monolayers in transwell chambers, and NO production was measured. Results. iNOS mRNA levels were significantly up‐regulated in the cells infected with H. pylori, and expression of iNOS protein was confirmed by Western blot analysis. Increased NO production in the gastric epithelial cells was seen as early as 18 hours postinfection, and reached maximal levels by 24 hours postinfection. The specific MAP kinase inhibitors decreased H. pylori‐induced iNOS and NO up‐regulation. After H. pylori infection of polarized epithelial cells, NO was released predominantly into the apical compartment, and IL‐8 was released predominantly into basolateral compartment. The addition of IFN‐γ to H. pylori‐infected polarized epithelial cells showed a synergistically higher apical and basolateral NO release. Conclusion. These results suggest that apical NO production mediated by MAP kinase in H. pylori‐infected gastric epithelial cells may influence the bacteria and basolateral production of NO and IL‐8 may play a role in the tissue inflammation.     

Nitric oxide and pH modulation in gynaecological cancer

Nitric oxide plays several roles in cellular physiology, including control of the vascular tone and defence against pathogen infection. Neuronal, inducible and endothelial nitric oxide synthase (NOS) isoforms synthesize nitric oxide. Cells generate acid and base equivalents, whose physiological intracellular concentrations are kept due to membrane transport systems, including Na⁺/H⁺ exchangers and Na⁺/HCO₃^? transporters, thus maintaining a physiological pH at the intracellular (~7.0) and extracellular (~7.4) medium. In several pathologies, including cancer, cells are exposed to an extracellular acidic microenvironment, and the role for these membrane transport mechanisms in this phenomenon is likely. As altered NOS expression and activity is seen in cancer cells and because this gas promotes a glycolytic phenotype leading to extracellular acidosis in gynaecological cancer cells, a pro‐inflammatory microenvironment increasing inducible NOS expression in this cell type is feasible. However, whether abnormal control of intracellular and extracellular pH by cancer cells regards with their ability to synthesize or respond to nitric oxide is unknown. We, here, discuss a potential link between pH alterations, pH controlling membrane transport systems and NOS function. We propose a potential association between inducible NOS induction and Na⁺/H⁺ exchanger expression and activity in human ovary cancer. A potentiation between nitric oxide generation and the maintenance of a low extracellular pH (i.e. acidic) is proposed to establish a sequence of events in ovarian cancer cells, thus preserving a pro‐proliferative acidic tumour extracellular microenvironment. We suggest that pharmacological therapeutic targeting of Na⁺/H⁺ exchangers and inducible NOS may have benefits in human epithelial ovarian cancer.     

Susceptibility of Hyphantria cunea to a granulosis virus isolated from Diacrisia virginica

The dosage-mortality response of Hyphantria cunea larvae to a granulosis virus isolated from Diacrisia virginica was studied. Serial decimal dilutions of the D. virginica granulosis virus were fed to early second-instar H. cunea larvae. the LD₅₀ for this virus (7.9 × 10⁵ capsules/larva) was significantly greater than the LD₅₀ calculated for the H. cunea granulosis virus (7.06 × 10⁴ capsules/larva) against the same instar of H. cunea. Time mortality studies demonstrated that the LT₅₀ values for the D. virginica granulosis were four to six times greater than for comparable dosages of H. cunea granulosis virus. Based on the mortality response of assayed H. cunea, it is possible to distinguish between the D. virginica granulosis virus and the H. cunea granulosis virus.     

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Endogenous nitric oxide (NO) is an important mediator in the processes that control biological clocks and circadian rhythms. The present study was designed to elucidate if NO synthase (NOS) activity in the brain, kidney, testis, aorta, and lungs and plasma NO_x levels in mice are controlled by an endogenous circadian pacemaker. Male BALB/c mice were exposed to two different lighting regimens of either light–dark 14:10 (LD) or continuous lighting (LL). At nine different equidistant time points (commencing at 09:00h) blood samples and tissues were taken from mice. The plasma and tissue homogenates were used to measure the levels of NO₂+ NO₃^? (NO_x) and total protein. The NO_x concentrations were determined by a commercial nitric oxide synthase assay kit, and protein content was assessed in each homogenate tissue sample by the Lowry method. Nitric oxide synthase activity was calculated as pmol/mg protein/h. The resulting patterns were analyzed by the single cosinor method for pre-adjusted periods and by curve-fitting programs to elucidate compound rhythmicity. The NOS activity in kidneys of mice exposed to LD exhibited a circadian rhythm, but no rhythmicity was detected in mice exposed to LL. Aortic NOS activity displayed 24h rhythmicity only in LL. Brain, testis, and lung NOS activity and plasma NO_x levels displayed 24h rhythms both in LD and LL. Acrophase values of NOS activity in brain, kidney, testis, and lungs were at midnight corresponding to their behavioral activities. Compound rhythms were also detected in many of the examined patterns. The findings suggest that NOS activity in mouse brain, aorta, lung, and testis are regulated by an endogenous clock, while in kidney the rhythm in NOS activity is synchronized by the exogenous signals.     

Mitochondrial DNA analysis of the introduced fall webworm, showing its shift in life cycle in Japan

The fall webworm, Hyphantria cunea (Drury) (Lepidoptera: Arctiidae), was introduced from North America into Japan in 1945. For the first three decades after its introduction, its life cycle was bivoltine. Thereafter, its life cycle shifted to trivoltine in south‐western areas of Japan. Two hypotheses have been proposed for the process that led to the shift in voltinism: one based on a single and the other on multiple independent colonizations. To test these hypotheses, mitochondrial (mt)DNA sequences were analyzed in the black‐headed type of 14 Japanese, one Korean and two North American populations of H. cunea. In addition, the same regions of mtDNA were compared with the red‐headed type of two North American populations. In the black‐headed type, mtDNA sequences were the same in all Japanese populations and in the Korean population, but sequences of the North American populations differed from each other and from those of the other populations. These results suggest that the process of the shift in voltinism occurred originally in Japan, and that the Japanese and the Korean population of H. cunea originated from a relatively small area in North America.     

Copper Oxide Nanomaterials Derived from Zanthoxylum armatum DC. and Berberis lycium Royle Plant Species: Characterization,Assessment of Free Radical Scavenging and Antibacterial Activity

Copper oxide nanomaterials were synthesized by a facile sustainable biological method using two plant species (Zanthoxylum armatum DC. and Berberis lycium Royle ). The formation of materials was confirmed by FT‐IR, ATR, UV‐visible, XRD, TEM, SEM, EDX, TGA and PL. The antibacterial activity was evaluated by agar well diffusion method to ascertain the efficacy of plant species extract and extract derived copper oxide nanomaterials against six Gram‐positive bacteria namely Staphylococcus aureus, Streptococcus mutans, Streptococcus pyogenes, Corynebacterium diphtheriae, Corynebacterium xerosis, Bacillus cereus and four Gram‐negative bacteria such as Klebsiella pneumonia, Escherichia coli, Pseudomonas aeruginosa and Proteus vulgaris against the standard drug, Ciprofloxacin for Gram‐positive and Gentamicin for Gram‐negative bacteria, respectively. In both cases, copper oxide nanomaterials were found to be sensitive in all the bacterial species. Sensitivity of copper oxide nanomaterials shows an be higher as compared to plant species extract against different bacteria. Scavenging activity of plant extracts along with nanomaterials have been accessed using previously reported protocols employing ascorbic acid as standard. Scavenging activity of copper oxide nanomaterials shows an increase with increase in concentration. The biological activity (bactericidal and scavenging efficiency) of plant derived copper oxide nanomaterials revealed that these materials can be used as potent antimicrobial agent and DPPH scavengers in industrial as well as pharmacological fields.     

Paramecium caudatum as a source of nitric oxide: Chemiluminescent detection based on Bluestar® Forensic reagent connected with microdialysis

Nitric oxide (NO) chemistry inside the body is the most interesting part of its behavior. NO is involved in controlling blood pressure, and in transmitting nerve signals and a variety of other signaling processes. To explain the behavior of NO, it is necessary to determine its immediate concentration or observe time‐dependent changes in its concentration. In Paramecium caudatum, NO is formed by calcium‐dependent nNOS (NOS1)‐like protein, which is distributed in the cytoplasm. NO synthesis affects the ciliary beat and consequent motility of cells and blocked NO synthesis reduces the ability of cells to move. The possibility of online coupling of microdialysis (of P. caudatum solution) with NO detection is demonstrated. Direct measurement of NO is carried out using dilute Bluestar^® Forensic reagent (luminol–H₂O₂ system; one of the NO detections is based upon the chemiluminescent reaction between NO and the luminol–H₂O₂ system, which is specifically reactive to NO). The effect of a nitric oxide synthase inhibitor, NG‐nitro‐l ‐arginine methyl ester was observed. NO production was inhibited and the movement of P. caudatum was restricted. These effects were time dependent and after a specific time were reversed.     

Expression of the tetrahydrofolate‐dependent nitric oxide synthase from the green alga Ostreococcus tauri increases tolerance to abiotic stresses and influences stomatal development in Arabidopsis

Nitric oxide (NO) is a signaling molecule with diverse biological functions in plants. NO plays a crucial role in growth and development, from germination to senescence, and is also involved in plant responses to biotic and abiotic stresses. In animals, NO is synthesized by well‐described nitric oxide synthase (NOS) enzymes. NOS activity has also been detected in higher plants, but no gene encoding an NOS protein, or the enzymes required for synthesis of tetrahydrobiopterin, an essential cofactor of mammalian NOS activity, have been identified so far. Recently, an NOS gene from the unicellular marine alga Ostreococcus tauri (OtNOS) has been discovered and characterized. Arabidopsis thaliana plants were transformed with OtNOS under the control of the inducible short promoter fragment (SPF) of the sunflower (Helianthus annuus) Hahb‐4 gene, which responds to abiotic stresses and abscisic acid. Transgenic plants expressing OtNOS accumulated higher NO concentrations compared with siblings transformed with the empty vector, and displayed enhanced salt, drought and oxidative stress tolerance. Moreover, transgenic OtNOS lines exhibited increased stomatal development compared with plants transformed with the empty vector. Both in vitro and in vivo experiments indicate that OtNOS, unlike mammalian NOS, efficiently uses tetrahydrofolate as a cofactor in Arabidopsis plants. The modulation of NO production to alleviate abiotic stress disturbances in higher plants highlights the potential of genetic manipulation to influence NO metabolism as a tool to improve plant fitness under adverse growth conditions.     

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 (Cx43^{Cre‐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 Cx43^{Cre‐ER(T)/fl} mitochondria (iNOS: 90.7 ± 3.2%; nNOS: 53.8 ± 17.5%). The mitochondrial nitric oxide formation was reduced in Cx43^{Cre‐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.     

Characterisation of nitric oxide synthase activity in the tropical sea anemone Aiptasia pallida

The presence of nitric oxide synthase (EC 1.14.23 NOS) activity is demonstrated in the tropical marine cnidarian Aiptasia pallida (Verrill). Enzyme activity was assayed by measuring the conversion of [³H]arginine to [³H]citrulline. Optimal NOS activity was found to require NADPH. Activity was inhibited by the competitive NOS inhibitor N^G-methyl- -arginine ( -NMA), but not the arginase inhibitors -valine and -ornithine. NOS activity was predominantly cytosolic, and was characterised by a K_m for arginine of 19.05 μM and a V_max of 2.96 pmol/min per μg protein. Histochemical localisation of NOS activity using NADPH diaphorase staining showed the enzyme to be predominantly present in the epidermal cells and at the extremities of the mesoglea. These results provide a preliminary biochemical characterisation and histochemical localisation of NOS activity in A. pallida, an ecologically important sentinel species in tropical marine ecosystems.     

WD40‐REPEAT 5a functions in drought stress tolerance by regulating nitric oxide accumulation in Arabidopsis

Nitric oxide (NO) generation by NO synthase (NOS) in guard cells plays a vital role in stomatal closure for adaptive plant response to drought stress. However, the mechanism underlying the regulation of NOS activity in plants is unclear. Here, by screening yeast deletion mutants with decreased NO accumulation and NOS‐like activity when subjected to H₂O₂ stress, we identified TUP1 as a novel regulator of NOS‐like activity in yeast. Arabidopsis WD40‐REPEAT 5a (WDR5a), a homolog of yeast TUP1, complemented H₂O₂‐induced NO accumulation of a yeast mutant Δtup1, suggesting the conserved role of WDR5a in regulating NO accumulation and NOS‐like activity. This note was further confirmed by using an Arabidopsis RNAi line wdr5a‐1 and two T‐DNA insertion mutants of WDR5a with reduced WDR5a expression, in which both H₂O₂‐induced NO accumulation and stomatal closure were repressed. This was because H₂O₂‐induced NOS‐like activity was inhibited in the mutants compared with that of the wild type. Furthermore, these wdr5a mutants were more sensitive to drought stress as they had reduced stomatal closure and decreased expression of drought‐related genes. Together, our results revealed that WDR5a functions as a novel factor to modulate NOS‐like activity for changes of NO accumulation and stomatal closure in drought stress tolerance.     

Testosterone induction of male‐typical sexual behavior is associated with increased preoptic NADPH diaphorase and citrulline production in female whiptail lizards

In rodents, male‐typical copulatory behavior is generally dependent on gonadal sex steroids such as testosterone, and it is thought that the mechanism by which the hormone gates the behavior involves the gaseous neurotransmitter nitric oxide. According to one model, testosterone induces an up‐regulation of nitric oxide synthase (NOS) in the preoptic area, increasing nitric oxide synthesis following exposure to a sexual stimulus. Nitric oxide in turn, possibly through its effect on catecholamine turnover, influences the way the stimulus is processed and enables the appropriate copulatory behavioral response. In whiptail lizards (genus Cnemidophorus), administration of male‐typical levels of testosterone to females induces the display of male‐like copulatory responses to receptive females, and we hypothesized that this radical change in behavioral phenotype would be accompanied by a large change in the expression of NOS in the preoptic area. As well as comparing NOS expression using NADPH diaphorase histochemistry between testosterone‐treated females and controls, we examined citrulline immunoreactivity (a marker of recent nitric oxide production) in the two groups, following a sexual stimulus and following a nonsexual stimulus. Substantially more NADPH diaphorase‐stained cells were observed in the testosterone‐treated animals. Citrulline immunoreactivity was greater in testosterone‐implanted animals than in blank‐implanted animals, but only following exposure to a sexual stimulus. This is the first demonstration that not only is NOS up‐regulated by testosterone, but NOS thus up‐regulated is activated during male‐typical copulatory behavior. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Purification, characterisation and distribution of ovine neuronal nitric oxide synthase

Nitric oxide (NO) is an ubiquitous intercellular messenger molecule synthesised from the amino acid arginine by the enzyme nitric oxide synthase (NOS). A number of NOS iso-enzymes have been identified, varying in molecular size, tissue distribution and possible biological role. To further understand the role of NO in the regulation of neuroendocrine function in the sheep, we have purified and characterised ovine neuronal NOS (nNOS) using anion exchange, affinity and size-exclusion chromatography. SDS-PAGE reveals that ovine nNOS has an apparent denatured molecular weight of 150 kDa which correlates well with the other purified nNOS forms such as rat, bovine and porcine. The native molecular weight predicted by size-exclusion chromatography was 200 kD which is in close agreement with that found for porcine and rat nNOS. Internal amino acid sequences generated from tryptic digests of the purified ovine nNOS are highly homologous to rat nNOS. There was no significant difference in the cofactor dependence and kinetic characteristics of ovine nNOS when compared to rat and bovine nNOS, (K_m for arginine 2.8, 2.0 and 2.3 μM respectively). A polyclonal anti-peptide antibody directed toward the C-terminal end of the rat nNOS sequence showed full cross-reactivity with the purified ovine nNOS. Immunohistochemical and Western analysis using this antiserum demonstrate the expression of nNOS in the cortex, cerebellum, hypothalamus and pituitary of the sheep. The lack of staining in the neural and anterior lobes of the pituitary seems to suggest that NOS plays a varied role in the control of endocrine systems between species.     

Elucidating nitric oxide synthase domain interactions by molecular dynamics

Nitric oxide synthase (NOS) is a multidomain enzyme that catalyzes the production of nitric oxide (NO) by oxidizing l ‐Arg to NO and L‐citrulline. NO production requires multiple interdomain electron transfer steps between the flavin mononucleotide (FMN) and heme domain. Specifically, NADPH‐derived electrons are transferred to the heme‐containing oxygenase domain via the flavin adenine dinucleotide (FAD) and FMN containing reductase domains. While crystal structures are available for both the reductase and oxygenase domains of NOS, to date there is no atomic level structural information on domain interactions required for the final FMN‐to‐heme electron transfer step. Here, we evaluate a model of this final electron transfer step for the heme–FMN–calmodulin NOS complex based on the recent biophysical studies using a 105‐ns molecular dynamics trajectory. The resulting equilibrated complex structure is very stable and provides a detailed prediction of interdomain contacts required for stabilizing the NOS output state. The resulting equilibrated complex model agrees well with previous experimental work and provides a detailed working model of the final NOS electron transfer step required for NO biosynthesis.     

Induction of Nitric Oxide Synthase Inhibits Gap Junction Permeability in Cultured Rat Astrocytes

Abstract: Nitric oxide (^?NO) synthase (NOS) was induced in cultured rat astrocytes by incubation with lipopolysaccharide (LPS) for 18 h and gap junction permeability was assessed by the scrape-loading/Lucifer yellow transfer technique. Induction of NOS was confirmed by determining either the N^G-methyl-l -arginine (NMMA)-inhibitable production of nitrites and nitrates or the conversion of l -[³H]arginine to l -[³H]citrulline. Incubation with LPS dose-dependently inhibited gap junction permeability to 63.3% at 0.05 µg/ml LPS and no further inhibition was observed on increasing the LPS concentration up to 0.5 µg/ml. LPS-mediated gap junction inhibition was irreversible but was prevented by incubation with the NOS inhibitor NMMA and with the superoxide anion (O₂^??) scavenger superoxide dismutase. Incubation of the cells with both the ^?NO donor S-nitroso-N-acetylpenicillamine and the O₂^??-generating system xanthine/xanthine oxidase inhibited gap junction permeability. These results suggest that the in situ reaction between ^?NO and O₂^??, to form the peroxynitrite anion (ONOO^?), may be responsible for the inhibition of gap junction permeability. Scavenging the ONOO^? derivative hydroxyl radical (^?OH) with either dimethyl sulfoxide or mannitol prevented the LPS-mediated inhibition of gap junction permeability. Finally, exposure of astrocytes to authentic ONOO^? caused a dose-dependent inhibition of gap junction permeability (65.7% of inhibition at 0.5 mM ONOO^?). The pathophysiological relevance of ONOO^?-mediated inhibition of gap junctional communication in astrocytes after NOS induction by LPS is discussed, stressing the possible role played by this mechanism in some neurodegenerative diseases.