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Sample records for allene oxide synthase

  1. Allene oxide synthase, allene oxide cyclase and jasmonic acid levels in Lotus japonicus nodules.

    Directory of Open Access Journals (Sweden)

    Anna Zdyb

    Full Text Available Jasmonic acid (JA, its derivatives and its precursor cis-12-oxo phytodienoic acid (OPDA form a group of phytohormones, the jasmonates, representing signal molecules involved in plant stress responses, in the defense against pathogens as well as in development. Elevated levels of JA have been shown to play a role in arbuscular mycorrhiza and in the induction of nitrogen-fixing root nodules. In this study, the gene families of two committed enzymes of the JA biosynthetic pathway, allene oxide synthase (AOS and allene oxide cyclase (AOC, were characterized in the determinate nodule-forming model legume Lotus japonicus JA levels were to be analysed in the course of nodulation. Since in all L. japonicus organs examined, JA levels increased upon mechanical disturbance and wounding, an aeroponic culture system was established to allow for a quick harvest, followed by the analysis of JA levels in whole root and shoot systems. Nodulated plants were compared with non-nodulated plants grown on nitrate or ammonium as N source, respectively, over a five week-period. JA levels turned out to be more or less stable independently of the growth conditions. However, L. japonicus nodules formed on aeroponically grown plants often showed patches of cells with reduced bacteroid density, presumably a stress symptom. Immunolocalization using a heterologous antibody showed that the vascular systems of these nodules also seemed to contain less AOC protein than those of nodules of plants grown in perlite/vermiculite. Hence, aeroponically grown L. japonicus plants are likely to be habituated to stress which could have affected JA levels.

  2. Suppression of allene oxide synthase 3 in potato increases degree of arbuscular mycorrhizal fungal colonization.

    Science.gov (United States)

    Morcillo, Rafael Jorge León; Navarrete, María Isabel Tamayo; Bote, Juan Antonio Ocampo; Monguio, Salomé Prat; García-Garrido, José Manuel

    2016-01-15

    Arbuscular mycorrhizal (AM) is a mutually beneficial interaction among higher plants and soil fungi of the phylum Glomeromycota. Numerous studies have pointed that jasmonic acid plays an important role in the development of the intraradical fungus. This compound belongs to a group of biologically active compounds known as oxylipins which are derived from the oxidative metabolism of polyunsaturated fatty acids. Studies of the regulatory role played by oxylipins in AM colonization have generally focused on jasmonates, while few studies exist on the 9-LOX pathway of oxylipins during AM formation. Here, the cDNA of Allene oxide synthase 3 (AOS3), a key enzyme in the 9-LOX pathway, was used in the RNA interference (RNAi) system to transform potato plants in order to suppress its expression. Results show increases in AOS3 gene expression and 9-LOX products in roots of wild type potato mycorrhizal plants. The suppression of AOS3 gene expression increases the percentage of root with mycorrhizal colonization at early stages of AM formation. AOS3 RNA interference lead to an induction of LOXA and 13-LOX genes, a reduction in AOS3 derived 9-LOX oxylipin compounds and an increase in jasmonic acid content, suggesting compensation between 9 and 13-LOX pathways. The results in a whole support the hypothesis of a regulatory role for the 9-LOX oxylipin pathway during mycorrhization. Copyright © 2015 Elsevier GmbH. All rights reserved.

  3. Crystallization and Preliminary X-ray Analysis of Allene Oxide Synthase, Cytochrome P450 CYP74A2, from Parthenium argentatum

    Science.gov (United States)

    Oxylipins are oxygenated derivatives of fatty acids and pivotal signaling molecules in plants and animals. Allene oxide synthase (AOS) is a key cytochrome P450 CYP74 enzyme involved in the biosynthesis of plant oxylipin jasmonates to convert 13(S)-hydroperoxide to allene oxide. Guayule (Parthenium a...

  4. Cellular localization and detergent dependent oligomerization of rice allene oxide synthase-1.

    Science.gov (United States)

    Yoeun, Sereyvath; Kim, Jeong-Il; Han, Oksoo

    2015-01-01

    Allene oxide synthase-1 from Oryza sativa (OsAOS1) localizes to the chloroplast, but lacks a putative chloroplast targeting sequence typically found in dicot AOS. Here, kinetic parameters and the oligomerization state/subunit composition of OsAOS1 were characterized in vitro in the absence or presence of detergent micelles. The catalytic efficiency (k(cat)/K(m)) of OsAOS1 reached a maximum near the critical micelle concentration for polyoxyethylene 10 tridecyl ether. Native gel analysis showed that OsAOS1 exists as a multimer in the absence of detergent micelles. The multimeric form of OsAOS1 was stably cross-linked in the absence of detergents, while only monomeric OsAOS1 was detected in the presence of detergent micelles. Gel filtration analysis indicated that the oligomeric state of OsAOS1 depends strongly on the detergents and that the monomer becomes the predominant form in the presence of detergent micelles. These data suggest that the detergent-dependent oligomeric state of OsAOS1 is an important factor for the regulation of its catalytic efficiency.

  5. Wounding stimulates ALLENE OXIDE SYNTHASE gene and increases the level of jasmonic acid in Ipomoea nil cotyledons

    Directory of Open Access Journals (Sweden)

    Emilia Wilmowicz

    2016-03-01

    Full Text Available Allene oxide synthase (AOS encodes the first enzyme in the lipoxygenase pathway, which is responsible for jasmonic acid (JA formation. In this study we report the molecular cloning and characterization of InAOS from Ipomoea nil. The full-length gene is composed of 1662 bp and encodes for 519 amino acids. The predicted InAOS contains PLN02648 motif, which is evolutionarily conserved and characteristic for functional enzymatic proteins. We have shown that wounding led to a strong stimulation of the examined gene activity in cotyledons and an increase in JA level, which suggest that this compound may be a modulator of stress responses in I. nil.

  6. Modes of Heme-Binding and Substrate Access for Cytochrome P450 CYP74A Revealed by Crystal Structures of Allene Oxide Synthase

    Science.gov (United States)

    Cytochrome P450s exist ubiquitously in all organisms and are involved in many biological processes. Allene oxide synthase (AOS) is a P450 enzyme that plays a key role in the biosynthesis of oxylipin jasmonates which are involved in signal and defense reactions in higher plants. The crystal structure...

  7. Crystallization and preliminary X-ray analysis of allene oxide synthase, cytochrome P450 CYP74A2, from Parthenium argentatum

    International Nuclear Information System (INIS)

    Chang, Zhenzhan; Li, Lenong; Pan, Zhiqiang; Wang, Xiaoqiang

    2008-01-01

    Allene oxide synthase, an atypical cytochrome P450 from Parthenium argentatum, was crystallized and diffraction data were collected to 2.4 Å resolution. Oxylipins are oxygenated derivatives of fatty acids and pivotal signaling molecules in plants and animals. Allene oxide synthase (AOS) is a key cytochrome P450 CYP74 enzyme involved in the biosynthesis of plant oxylipin jasmonates to convert 13(S)-hydroperoxide to allene oxide. Guayule (Parthenium argentatum) AOS, CYP74A2, was expressed in Escherichia coli. Protein was purified using affinity chromatography and size exclusion chromatography, and then crystallized. Two different crystal forms were obtained from 0.2 M (NH 4 )H 2 PO 4 , 50% MPD, 0.1 M Tris, pH 8.5 at 277 K using the hanging-drop vapor-diffusion method. Preliminary X-ray analysis was carried out, and the crystals were found to belong to the tetragonal space group I422 with cell parameters a = b = 126.5, c = 163.9 Å, and the monoclinic space group C2 with cell parameters a = 336.5, b = 184.2, c = 159.0 Å, β = 118.6°. Diffraction data were collected to 2.4 Å resolution from a tetragonal form of crystal using a home X-ray source

  8. Crystallization and preliminary X-ray analysis of allene oxide synthase, cytochrome P450 CYP74A2, from Parthenium argentatum

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Zhenzhan; Li, Lenong [Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401 (United States); Pan, Zhiqiang, E-mail: zpan@ars.usda.gov [USDA, ARS, Natural Products Utilization Research Unit, PO Box 8048, University, MS 38677 (United States); Wang, Xiaoqiang, E-mail: zpan@ars.usda.gov [Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401 (United States)

    2008-07-01

    Allene oxide synthase, an atypical cytochrome P450 from Parthenium argentatum, was crystallized and diffraction data were collected to 2.4 Å resolution. Oxylipins are oxygenated derivatives of fatty acids and pivotal signaling molecules in plants and animals. Allene oxide synthase (AOS) is a key cytochrome P450 CYP74 enzyme involved in the biosynthesis of plant oxylipin jasmonates to convert 13(S)-hydroperoxide to allene oxide. Guayule (Parthenium argentatum) AOS, CYP74A2, was expressed in Escherichia coli. Protein was purified using affinity chromatography and size exclusion chromatography, and then crystallized. Two different crystal forms were obtained from 0.2 M (NH{sub 4})H{sub 2}PO{sub 4}, 50% MPD, 0.1 M Tris, pH 8.5 at 277 K using the hanging-drop vapor-diffusion method. Preliminary X-ray analysis was carried out, and the crystals were found to belong to the tetragonal space group I422 with cell parameters a = b = 126.5, c = 163.9 Å, and the monoclinic space group C2 with cell parameters a = 336.5, b = 184.2, c = 159.0 Å, β = 118.6°. Diffraction data were collected to 2.4 Å resolution from a tetragonal form of crystal using a home X-ray source.

  9. Modes of heme binding and substrate access for cytochrome P450 CYP74A revealed by crystal structures of allene oxide synthase

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lenong; Chang, Zhenzhan; Pan, Zhiqiang; Fu, Zheng-Qing; Wang, Xiaoqiang (US-Agriculture); (SRNF); (Georgia)

    2009-01-12

    Cytochrome P450s exist ubiquitously in all organisms and are involved in many biological processes. Allene oxide synthase (AOS) is a P450 enzyme that plays a key role in the biosynthesis of oxylipin jasmonates, which are involved in signal and defense reactions in higher plants. The crystal structures of guayule (Parthenium argentatum) AOS (CYP74A2) and its complex with the substrate analog 13(S)-hydroxyoctadeca-9Z,11E-dienoic acid have been determined. The structures exhibit a classic P450 fold but possess a heme-binding mode with an unusually long heme binding loop and a unique I-helix. The structures also reveal two channels through which substrate and product may access and leave the active site. The entrances are defined by a loop between {beta}3-2 and {beta}3-3. Asn-276 in the substrate binding site may interact with the substrate's hydroperoxy group and play an important role in catalysis, and Lys-282 at the entrance may control substrate access and binding. These studies provide both structural insights into AOS and related P450s and a structural basis to understand the distinct reaction mechanism.

  10. Biosynthesis of allene oxides in Physcomitrella patens

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    Scholz Julia

    2012-11-01

    Full Text Available Abstract Background The moss Physcomitrella patens contains C18- as well as C20-polyunsaturated fatty acids that can be metabolized by different enzymes to form oxylipins such as the cyclopentenone cis(+-12-oxo phytodienoic acid. Mutants defective in the biosynthesis of cyclopentenones showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis. The initial step in this biosynthetic route is the conversion of a fatty acid hydroperoxide to an allene oxide. This reaction is catalyzed by allene oxide synthase (AOS belonging as hydroperoxide lyase (HPL to the cytochrome P450 family Cyp74. In this study we characterized two AOS from P. patens, PpAOS1 and PpAOS2. Results Our results show that PpAOS1 is highly active with both C18 and C20-hydroperoxy-fatty acid substrates, whereas PpAOS2 is fully active only with C20-substrates, exhibiting trace activity (~1000-fold lower kcat/KM with C18 substrates. Analysis of products of PpAOS1 and PpHPL further demonstrated that both enzymes have an inherent side activity mirroring the close inter-connection of AOS and HPL catalysis. By employing site directed mutagenesis we provide evidence that single amino acid residues in the active site are also determining the catalytic activity of a 9-/13-AOS – a finding that previously has only been reported for substrate specific 13-AOS. However, PpHPL cannot be converted into an AOS by exchanging the same determinant. Localization studies using YFP-labeled AOS showed that PpAOS2 is localized in the plastid while PpAOS1 may be found in the cytosol. Analysis of the wound-induced cis(+-12-oxo phytodienoic acid accumulation in PpAOS1 and PpAOS2 single knock-out mutants showed that disruption of PpAOS1, in contrast to PpAOS2, results in a significantly decreased cis(+-12-oxo phytodienoic acid formation. However, the knock-out mutants of neither PpAOS1 nor PpAOS2 showed reduced fertility, aberrant sporophyte morphology or interrupted sporogenesis

  11. Allenes in Asymmetric Catalysis. Asymmetric Ring-Opening of Meso-Epoxides Catalyzed by Allene-Containing Phosphine Oxides

    Science.gov (United States)

    Pu, Xiaotao; Qi, Xiangbing; Ready, Joseph M.

    2009-01-01

    Unsymmetrically substituted allenes (1,2 dienes) are inherently chiral and can be prepared in optically pure form. Nonetheless, to date the allene framework has not been incorporated into ligands for asymmetric catalysis. Since allenes project functionality differently than either tetrahedral carbon or chiral biaryls, they may create complementary chiral environments. This study demonstrates that optically active C2 symmetric allene-containing bisphosphine oxides can catalyze the addition of SiCl4 to meso epoxides with high enantioselectivity. The epoxide-opening likely involves generation of a Lewis acidic, cationic (bisphosphine oxide)SiCl3 complex. The fact that high asymmetric induction is observed suggests that allenes may represent a new platform for the development of ligands and catalysts for asymmetric synthesis. PMID:19722613

  12. Photolysis of allene-ozone mixtures at 647 nm in cryogenic matrices. Part 1. Formation of allene oxide

    Science.gov (United States)

    Singmaster, Karen A.; Pimentel, George C.

    1989-03-01

    Matrix studies of the photolytic reaction at 647 nm between allene and ozone were carried out at 12 K. Primary photoproducts include carbon monoxide, acrolein ( cis and trans), cyclopropanone, ketene, ethylene, allene oxide and formaldehyde. In Ar and Kr matrices both acrolein and cyclopropanone are produced in high yields, whereas in Xe matrices cyclopropanone is the major product. Infrared spectra for cyclopropanone and its oxygen-18 and deuterium substitutes are reported. The carbonyl stretch for cyclopropanone is observed at 1815 cm -1 in an Ar matrix. Also reported is the first synthesis of allene oxide. The carbon—carbon double bond stretch is observed at 1823.4 cm -1 and it exhibits a small oxygen-18 shift. The change in product distribution is discussed in terms of heavy atom spin—orbit enhancement of singlet—triplet excitation, so that in xenon reaction takes place on a triplet surface, whereas in argon it occurs on a singlet surface.

  13. Photochemical generation and 1H NMR detection of alkyl allene oxides in solution

    International Nuclear Information System (INIS)

    Breen, L.E.; Schepp, N.P.; Tan, C.-H.E.

    2005-01-01

    Irradiation of substituted 5-alkyl-4,5-epoxyvalerophenones leads to the formation of alkyl allene oxides that, in some cases, are sufficiently long-lived to be detected at room temperature by 1 H NMR spectroscopy. Absolute lifetime measurements show that the size of the alkyl group has a significant influence on the reactivity of the allene oxide, with tert-butyl allene oxide having a lifetime of 24 h in CD 3 CN at room temperature that is considerably longer than the 1.5 h lifetime of the ethyl allene oxide. The allene oxides react rapidly with water to give α-hydroxyketones. The mechanism involves nucleophilic attack to the epoxide carbon to give an enol, which can also be detected as an intermediate by 1 H NMR spectroscopy. (author)

  14. Relationship between endothelial nitric oxide synthase gene ...

    African Journals Online (AJOL)

    Introduction: Endothelial nitric oxide synthase (eNOS), the enzyme in charge of nitric oxide production, plays a crucial role in vascular biology. However, the impact of single nucleotide polymorphisms (SNPs) affecting the gene encoding for eNOS (eNOS) on coronary artery diseases remains under debate and no data were ...

  15. Endothelial nitric oxide synthase gene polymorphisms associated ...

    African Journals Online (AJOL)

    Endothelial nitric oxide synthase (NOS3) is involved in key steps of immune response. Genetic factors predispose individuals to periodontal disease. This study's aim was to explore the association between NOS3 gene polymorphisms and clinical parameters in patients with periodontal disease. Genomic DNA was obtained ...

  16. Oxidative allene amination for the synthesis of azetidin-3-ones.

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    Burke, Eileen G; Schomaker, Jennifer M

    2015-10-05

    Regioselectivity in the aziridination of silyl-substituted homoallenic sulfamates is readily diverted to the distal double bond of the allene to yield endocyclic bicyclic methyleneaziridines with excellent stereocontrol. Subsequent reaction with electrophilic oxygen sources initiates facile rearrangement to densely functionalized, fused azetidin-3-ones in excellent d.r., effectively transferring the axial chirality of the allene to central chirality in the products. The steric nature of the silyl group dictates which of the two rings of the fused azetidin-3-one will undergo further functionalization, providing an additional element of diversity for the preparation of enantioenriched azetidine scaffolds with potential biological activity. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Activity Regulation by Heteromerization of Arabidopsis Allene Oxide Cyclase Family Members

    Czech Academy of Sciences Publication Activity Database

    Otto, M.; Naumann, Ch.; Brandt, W.; Wasternack, Claus; Hause, B.

    2016-01-01

    Roč. 5, č. 1 (2016), č. článku 3. ISSN 2223-7747 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Activity regulation * Arabidopsis allene oxide cyclase isoforms * Heteromerization Subject RIV: EB - Genetics ; Molecular Biology

  18. A Comparison of the Effects of Neuronal Nitric Oxide Synthase and Inducible Nitric Oxide Synthase Inhibition on Cartilage Damage

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    Nevzat Selim Gokay

    2016-01-01

    Full Text Available The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg, inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg, or nitric oxide precursor L-arginine (200 mg/kg. After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P=0.044 positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

  19. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Lopez-Figueroa, M.; Hellsten, Ylva

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed ...

  20. Nitric oxide synthase expression and enzymatic activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B; Wanscher, B

    2004-01-01

    and endothelial nitric oxide synthase (NOS)], and enzymatic NO synthase activity. MRI guided biopsies documented more active plaques than macroscopic examination, and histological examination revealed further lesions. Inducible NOS (iNOS) was the dominant IR isoform, while reactive astrocytes were the dominant i...

  1. Localization of nitric oxide synthase in human skeletal muscle

    DEFF Research Database (Denmark)

    Frandsen, Ulrik; Lopez-Figueroa, M.; Hellsten, Ylva

    1996-01-01

    The present study investigated the cellular localization of the neuronal type I and endothelial type III nitric oxide synthase in human skeletal muscle. Type I NO synthase immunoreactivity was found in the sarcolemma and the cytoplasm of all muscle fibres. Stronger immunoreactivity was expressed...... in the sarcolemma as well as the cytoplasm of type I muscle fibres. NADPH diaphorase activity confirmed a higher level of NO synthase activity in the sarcolemma as well as the cytoplasm of type I muscle fibers. Histochemical staining for cytochrome oxidase showed a staining pattern similar to that observed for type...... I NO synthase immunoreactivity and NADPH diaphorase activity. Type III NO synthase immunoreactivity was observed both in the endothelium of larger vessels and of microvessels. The results establish that human skeletal muscle expresses two different constitutive isoforms of NO synthase in different...

  2. Nitric oxide synthase in ferret brain: localization and characterization.

    Science.gov (United States)

    Matsumoto, T.; Mitchell, J. A.; Schmidt, H. H.; Kohlhaas, K. L.; Warner, T. D.; Förstermann, U.; Murad, F.

    1992-01-01

    1. In the present study, we have investigated the distribution of nitric oxide synthase in the ferret brain. Nitric oxide synthase was determined biochemically and immunochemically. 2. In the rat brain, the highest nitric oxide synthase activity has been detected in the cerebellum. However, in the ferret brain, the highest activity was found in the striatum and the lowest in the cerebellum and cerebral cortex. The enzymatic activity was localized predominantly in the cytosolic fractions, it was dependent on NADPH and Ca2+, and inhibited by NG-nitro-L-arginine or NG-methyl-L-arginine. 3. Western blot analysis revealed that all regions of the ferret brain contained a 160 kD protein crossreacting with an antibody to nitric oxide synthase purified from the rat cerebellum, and the levels of relative intensity of staining by the antibody correlated with the distribution of nitric oxide synthase activity. 4. These results indicate that the ferret brain contains a nitric oxide synthase similar to the rat brain, but the distribution of enzymatic activity in the ferret brain differs markedly from the rat brain. Images Figure 1 PMID:1282076

  3. Inhibition of Inducible Nitric Oxide Synthase, Cycleooxygenase-2 ...

    African Journals Online (AJOL)

    HP

    Purpose: To explore the antioxidant properties of the methanol extract of Pericarpium Zanthoxyli and its effect on inducible nitric oxide synthase (iNOS), cycleooxygenase-2 (COX-2) and lipopolysaccharides (LPS)-induced cell damage in macrophage cells. Methods: Anti-oxidant activities were tested by measuring free ...

  4. Inhibition of Inducible Nitric Oxide Synthase, Cycleooxygenase-2 ...

    African Journals Online (AJOL)

    Inhibition of Inducible Nitric Oxide Synthase, Cycleooxygenase-2 and Lipid Peroxidation by Methanol Extract of Pericarpium Zanthoxyli. ... Production of iNOS induced by LPS was significantly (p < 0.05) inhibited by the extract, suggesting that the extract inhibits nitric oxide (NO) production by suppressing iNOS expression.

  5. Role of Endothelial Nitric Oxide Synthase Gene Polymorphisms ...

    African Journals Online (AJOL)

    Background: Previous studies indicated an association between endothelial nitric oxide synthase (eNOS) activity and maintenance of pregnancy, but it is rather controversial whether polymorphisms of the gene encoding for eNOS are associated with recurrent spontaneous abortions (RSAs). Aim: The aim was to investigate ...

  6. Expression of Inducible Nitric Oxide Synthase in the Epithelial ...

    African Journals Online (AJOL)

    Conclusion: iNOS was over expressed in OKCs when compared with DC and RC suggesting that iNOS may contribute to the aggressive behavior of OKC. This is yet another evidence to support that OKC is the neoplasm. Keywords: Dentigerous cyst, Immunohistochemistry, Inducible nitric oxide synthase, Odontogenic ...

  7. Analysis of genetic variation of inducible nitric oxide synthase and ...

    African Journals Online (AJOL)

    The genetic diversity of 100 Malaysian native chickens was investigated using polymerase chain reaction-restriction fragment polymorphism (PCR-RFLP) for two candidate genes: inducible nitric oxide synthase (INOS) and natural resistance-associated macrophage protein 1 (NRAMP1). The two genes were selected ...

  8. Nitric oxide synthase expression and enzymatic activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Broholm, H; Andersen, B; Wanscher, B

    2004-01-01

    We used post-mortem magnetic resonance imaging (MRI) guidance to obtain paired biopsies from the brains of four patients with clinical definite multiple sclerosis (MS). Samples were analyzed for the immunoreactivity (IR) of the three nitric oxide (NO) synthase isoforms [inducible, neuronal...... and sex showed no such changes. Our data support the hypothesis that NO is a pathogenic factor in MS, and that NOS IR is strongly expressed in brain regions appearing normal by MRI...

  9. Nitric oxide synthase isoforms in spontaneous and salt hypertension

    Czech Academy of Sciences Publication Activity Database

    Hojná, Silvie; Kuneš, Jaroslav; Zicha, Josef

    2007-01-01

    Roč. 25, Suppl. 2 (2007), S 338-S 338 ISSN 0263-6352. [European Meeting on Hypertension /17./. 15.06.2007-19.06.2007, Milan] R&D Projects: GA MŠk(CZ) 1M0510 Institutional research plan: CEZ:AV0Z50110509 Keywords : nitric oxide synthase isoforms * spontaneous and salt hypertension Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery

  10. Nitric Oxide Synthase Inhibitors as Antidepressants

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    Vallo Volke

    2010-01-01

    Full Text Available Affective and anxiety disorders are widely distributed disorders with severe social and economic effects. Evidence is emphatic that effective treatment helps to restore function and quality of life. Due to the action of most modern antidepressant drugs, serotonergic mechanisms have traditionally been suggested to play major roles in the pathophysiology of mood and stress-related disorders. However, a few clinical and several pre-clinical studies, strongly suggest involvement of the nitric oxide (NO signaling pathway in these disorders. Moreover, several of the conventional neurotransmitters, including serotonin, glutamate and GABA, are intimately regulated by NO, and distinct classes of antidepressants have been found to modulate the hippocampal NO level in vivo. The NO system is therefore a potential target for antidepressant and anxiolytic drug action in acute therapy as well as in prophylaxis. This paper reviews the effect of drugs modulating NO synthesis in anxiety and depression.

  11. Intestinal nitric oxide synthase activity changes during experimental colon obstruction.

    Science.gov (United States)

    Palásthy, Zsolt; Kaszaki, József; Lázár, György; Nagy, Sándor; Boros, Mihály

    2006-08-01

    The experiments in this study were designed to follow the time course of nitric oxide (NO) synthesis in the large bowel during acute mechanical ileus. Occlusion of the mid-transverse colon was maintained for 420 min in anesthetized dogs. Strain-gauge transducers were used to analyze motility changes on the hepatic and lienal flexures, respectively. Constitutive NO synthase (cNOS) and inducible NOS (iNOS) activities were determined in tissue biopsies, and plasma nitrite/nitrate (NOx) level was measured in the portal blood. Following completion of the baseline studies, the animals were treated with either 7-nitroindazole (7-NI, selective neuronal NOS inhibitor), or N-nitro-L-arginine (NNA, non-selective NOS inhibitor). In the sham-operated group the cNOS activities differed significantly in the oral and aboral tissue samples (oral: 102.9; versus aboral: 62.1 fmol/mg protein/min). The obstruction elicited a significant increase in portal NOx and elevated tissue inducible NO synthase (iNOS) activity. NNA treatment decreased the motility index in both intestinal segments for 60 min, but 120 min later the motility index was significantly elevated (2.5-fold increase in the oral part, and 1.8-fold enhancement in the aboral segment, respectively). Treatment with 7-NI decreased the cNOS activity in the oral and aboral parts by approximately 40% and 70%, respectively, and suppressed the motility increase in the aboral colon segment. The motility of the colon was either significantly increased or decreased, depending on the type and selectivity of the NOS inhibitor compounds applied. NO of neuronal origin is a transmitter that stimulates peristaltic activity; but an increased iNOS/nNOS ratio significantly moderates the obstruction-induced motility increase.

  12. Hyperbaric oxygen upregulates cochlear constitutive nitric oxide synthase

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    Kao Ming-Ching

    2011-02-01

    Full Text Available Abstract Background Hyperbaric oxygen therapy (HBOT is a known adjuvant for treating ischemia-related inner ear diseases. Controversies still exist in the role of HBOT in cochlear diseases. Few studies to date have investigated the cellular changes that occur in inner ears after HBOT. Nitric oxide, which is synthesized by nitric oxide synthase (NOS, is an important signaling molecule in cochlear physiology and pathology. Here we investigated the effects of hyperbaric oxygen on eardrum morphology, cochlear function and expression of NOS isoforms in cochlear substructures after repetitive HBOT in guinea pigs. Results Minor changes in the eardrum were observed after repetitive HBOT, which did not result in a significant hearing threshold shift by tone burst auditory brainstem responses. A differential effect of HBOT on the expression of NOS isoforms was identified. Upregulation of constitutive NOS (nNOS and eNOS was found in the substructures of the cochlea after HBOT, but inducible NOS was not found in normal or HBOT animals, as shown by immunohistochemistry. There was no obvious DNA fragmentation present in this HBOT animal model. Conclusions The present evidence indicates that the customary HBOT protocol may increase constitutive NOS expression but such upregulation did not cause cell death in the treated cochlea. The cochlear morphology and auditory function are consequently not changed through the protocol.

  13. Differential modulation of nitric oxide synthases in aging: therapeutic opportunities

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    Stêfany Bruno De Assis Cau

    2012-06-01

    Full Text Available Vascular aging is the term that describes the structural and functional disturbances of the vasculature with advancing aging. The molecular mechanisms of aging-associated endothelial dysfunction are complex, but reduced nitric oxide (NO bioavailability and altered vascular expression and activity of NO synthase (NOS enzymes have been implicated as major players. Impaired vascular relaxation in aging has been attributed to reduced endothelial NOS (eNOS-derived NO, while increased inducible NOS (iNOS expression seems to account for nitrosative stress and disrupted vascular homeostasis. Although eNOS is considered the main source of NO in the vascular endothelium, neuronal NOS (nNOS also contributes to endothelial cells-derived NO, a mechanism that is reduced in aging. Pharmacological modulation of NO generation and expression/activity of NOS isoforms may represent a therapeutic alternative to prevent the progression of cardiovascular diseases. Accordingly, this review will focus on drugs that modulate NO bioavailability, such as nitrite anions and NO-releasing non-steroidal anti-inflammatory drugs, hormones (dehydroepiandrosterone and estrogen, statins, resveratrol and folic acid, since they may be useful to treat/to prevent aging-associated vascular dysfunction. The impact of these therapies on life quality in elderly and longevity will be discussed.

  14. Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms.

    Science.gov (United States)

    Barouch, Lili A; Harrison, Robert W; Skaf, Michel W; Rosas, Gisele O; Cappola, Thomas P; Kobeissi, Zoulficar A; Hobai, Ion A; Lemmon, Christopher A; Burnett, Arthur L; O'Rourke, Brian; Rodriguez, E Rene; Huang, Paul L; Lima, João A C; Berkowitz, Dan E; Hare, Joshua M

    2002-03-21

    Subcellular localization of nitric oxide (NO) synthases with effector molecules is an important regulatory mechanism for NO signalling. In the heart, NO inhibits L-type Ca2+ channels but stimulates sarcoplasmic reticulum (SR) Ca2+ release, leading to variable effects on myocardial contractility. Here we show that spatial confinement of specific NO synthase isoforms regulates this process. Endothelial NO synthase (NOS3) localizes to caveolae, where compartmentalization with beta-adrenergic receptors and L-type Ca2+ channels allows NO to inhibit beta-adrenergic-induced inotropy. Neuronal NO synthase (NOS1), however, is targeted to cardiac SR. NO stimulation of SR Ca2+ release via the ryanodine receptor (RyR) in vitro, suggests that NOS1 has an opposite, facilitative effect on contractility. We demonstrate that NOS1-deficient mice have suppressed inotropic response, whereas NOS3-deficient mice have enhanced contractility, owing to corresponding changes in SR Ca2+ release. Both NOS1-/- and NOS3-/- mice develop age-related hypertrophy, although only NOS3-/- mice are hypertensive. NOS1/3-/- double knockout mice have suppressed beta-adrenergic responses and an additive phenotype of marked ventricular remodelling. Thus, NOS1 and NOS3 mediate independent, and in some cases opposite, effects on cardiac structure and function.

  15. Decoding the substrate supply to human neuronal nitric oxide synthase.

    Directory of Open Access Journals (Sweden)

    Alexandra Simon

    Full Text Available Nitric oxide, produced by the neuronal nitric oxide synthase (nNOS from L-arginine is an important second messenger molecule in the central nervous system: It influences the synthesis and release of neurotransmitters and plays an important role in long-term potentiation, long-term depression and neuroendocrine secretion. However, under certain pathological conditions such as Alzheimer's or Parkinson's disease, stroke and multiple sclerosis, excessive NO production can lead to tissue damage. It is thus desirable to control NO production in these situations. So far, little is known about the substrate supply to human nNOS as a determinant of its activity. Measuring bioactive NO via cGMP formation in reporter cells, we demonstrate here that nNOS in both, human A673 neuroepithelioma and TGW-nu-I neuroblastoma cells can be fast and efficiently nourished by extracellular arginine that enters the cells via membrane transporters (pool I that is freely exchangeable with the extracellular space. When this pool was depleted, NO synthesis was partially sustained by intracellular arginine sources not freely exchangeable with the extracellular space (pool II. Protein breakdown made up by far the largest part of pool II in both cell types. In contrast, citrulline to arginine conversion maintained NO synthesis only in TGW-nu-I neuroblastoma, but not A673 neuroepithelioma cells. Histidine mimicked the effect of protease inhibitors causing an almost complete nNOS inhibition in cells incubated additionally in lysine that depletes the exchangeable arginine pool. Our results identify new ways to modulate nNOS activity by modifying its substrate supply.

  16. Allene functionalization via bicyclic methylene aziridines.

    Science.gov (United States)

    Boralsky, Luke A; Marston, Dagmara; Grigg, R David; Hershberger, John C; Schomaker, Jennifer M

    2011-04-15

    The oxidative functionalization of olefins is a common method for the formation of vicinal carbon-heteroatom bonds. However, oxidative methods to transform allenes into synthetic motifs containing three contiguous carbon-heteroatom bonds are much less developed. This paper describes the use of bicyclic methylene aziridines (MAs), prepared via intramolecular allene aziridination, as scaffolds for functionalization of all three allene carbons. © 2011 American Chemical Society

  17. Role of Polymorphisms of Inducible Nitric Oxide Synthase and Endothelial Nitric Oxide Synthase in Idiopathic Environmental Intolerances

    Directory of Open Access Journals (Sweden)

    Chiara De Luca

    2015-01-01

    Full Text Available Oxidative stress and inflammation play a pathogenetic role in idiopathic environmental intolerances (IEI, namely, multiple chemical sensitivity (MCS, fibromyalgia (FM, and chronic fatigue syndrome (CFS. Given the reported association of nitric oxide synthase (NOS gene polymorphisms with inflammatory disorders, we aimed to investigate the distribution of NOS2A −2.5 kb (CCTTTn as well as Ser608Leu and NOS3 −786T>C variants and their correlation with nitrite/nitrate levels, in a study cohort including 170 MCS, 108 suspected MCS (SMCS, 89 FM/CFS, and 196 healthy subjects. Patients and controls had similar distributions of NOS2A Ser608Leu and NOS3 −786T>C polymorphisms. Interestingly, the NOS3 −786TT genotype was associated with increased nitrite/nitrate levels only in IEI patients. We also found that the NOS2A −2.5 kb (CCTTT11 allele represents a genetic determinant for FM/CFS, and the (CCTTT16 allele discriminates MCS from SMCS patients. Instead, the (CCTTT8 allele reduces by three-, six-, and tenfold, respectively, the risk for MCS, SMCS, and FM/CFS. Moreover, a short number of (CCTTT repeats is associated with higher concentrations of nitrites/nitrates. Here, we first demonstrate that NOS3 −786T>C variant affects nitrite/nitrate levels in IEI patients and that screening for NOS2A −2.5 kb (CCTTTn polymorphism may be useful for differential diagnosis of various IEI.

  18. Cloning of allene oxide cyclase gene from Leymus mollis and analysis of its expression in wheat?Leymus chromosome addition lines

    OpenAIRE

    Eltayeb Habora, Mohamed Elsadig; Eltayeb, Amin Elsadig; Oka, Mariko; Tsujimoto, Hisashi; Tanaka, Kiyoshi

    2013-01-01

    Leymus mollis (Triticeae; Poaceae) is a useful genetic resource for wheat (Triticum aestivum L.) breeding via wide hybridization to introduce its chromosomes and integrate its useful traits into wheat. Leymus mollis is highly tolerant to abiotic stresses such as drought and salinity and resistant to various diseases, but the genetic mechanisms controlling its physiological tolerance remain largely unexplored. We identified and cloned an allene oxide cyclase (AOC) gene from L. mollis that was ...

  19. Inducible nitric oxide synthase haplotype associated with migraine and aura.

    Science.gov (United States)

    de O S Mansur, Thiago; Gonçalves, Flavia M; Martins-Oliveira, Alisson; Speciali, Jose G; Dach, Fabiola; Lacchini, Riccardo; Tanus-Santos, Jose E

    2012-05-01

    Migraine is a complex neurological disorder with a clear neurogenic inflammatory component apparently including enhanced nitric oxide (NO) formation. Excessive NO amounts possibly contributing to migraine are derived from increased expression and activity of inducible NO synthase (iNOS). We tested the hypothesis that two functional, clinically relevant iNOS genetic polymorphisms (C(-1026)A-rs2779249 and G2087A-rs2297518) are associated with migraine with or without aura. We studied 142 healthy women without migraine (control group) and 200 women with migraine divided into two groups: 148 with migraine without aura (MWA) and 52 with aura (MA). Genotypes were determined by real-time polymerase chain reaction using the Taqman(®) allele discrimination assays. The PHASE 2.1 software was used to estimate the haplotypes. The A allele for the G2087A polymorphism was more commonly found in the MA group than in the MWA group (28 vs. 18%; P 0.05). The haplotype combining both A alleles for the two polymorphisms was more commonly found in the MA group than in the control group or in the MWA group (19 vs. 10 or 8%; P = 0.0245 or 0.0027, respectively). Our findings indicate that the G2087A and the C(-1026)A polymorphism in the iNOS gene affect the susceptibility to migraine with aura when their effects are combined within haplotypes, whereas the G2087A affects the susceptibility to aura in migraine patients. These finding may have therapeutic implications when examining the effects of selective iNOS inhibitors.

  20. Inducible nitric oxide synthase catalyzes ethanol oxidation to alpha-hydroxyethyl radical and acetaldehyde.

    Science.gov (United States)

    Porasuphatana, Supatra; Weaver, John; Rosen, Gerald M

    2006-06-15

    The physiologic function of nitric oxide synthases, independent of the isozyme, is well established, metabolizing L-arginine to L-citrulline and nitric oxide (NO). This enzyme can also transfer electrons to O2, affording superoxide (O2*-) and hydrogen peroxide (H2O2). We have demonstrated that NOS1, in the presence of L-arginine, can biotransform ethanol (EtOH) to alpha-hydroxyethyl radical (CH3*CHOH). We now report that a competent NOS2 with l-arginine can, like NOS1, oxidize EtOH to CH3*CHOH. Once this free radical is formed, it is metabolized to acetaldehyde as shown by LC-ESI-MS/MS and HPLC analysis. These observations suggest that NOS2 can behave similarly to cytochrome P-450 in the catalysis of acetaldehyde formation from ethanol via the generation of alpha-hydroxyethyl radical when L-arginine is present.

  1. Differential effects of nitric oxide synthase inhibitors on endotoxin-induced liver damage in rats

    NARCIS (Netherlands)

    Vos, TA; Gouw, ASH; Klok, PA; Havinga, R; vanGoor, H; Roelofsen, H; Kuipers, F; Jansen, PLM; Moshage, H

    1997-01-01

    Background & Aims: During endotoxemia, expression of inducible nitric oxide synthase (iNOS) and nitric oxide production in the liver is increased, NO has been suggested to have a hepatoprotective function. The aim of this study was to investigate the distribution of iNOS and the effect of different

  2. Expression of inducible nitric oxide synthase in trigeminal ganglion cells during culture

    DEFF Research Database (Denmark)

    Jansen-Olesen, Inger; Zhou, MingFang; Zinck, Tina Jovanovic

    2005-01-01

    Nitric oxide (NO) is an important signalling molecule that has been suggested to be a key molecule for induction and maintenance of migraine attacks based on clinical studies, animal experimental studies and the expression of nitric oxide synthase (NOS) immunoreactivity within the trigeminovascular...

  3. Lack of endothelial nitric oxide synthase aggravates murine accelerated anti-glomerular basement membrane glomerulonephritis

    NARCIS (Netherlands)

    Heeringa, P; van Goor, H; Itoh-Lindstrom, Y; Maeda, N; Falk, RJ; Assmann, KJM; Kallenberg, CGM; Jennette, JC

    Nitric oxide (NO) radicals generated by endothelial nitric oxide synthase (eNOS) are involved in the regulation of vascular tone. In addition, NO radicals derived from eNOS inhibit platelet aggregation and leukocyte adhesion to the endothelium and, thus, may have anti-inflammatory effects. To study

  4. Expression of inducible nitric oxide synthase in trigeminal ganglion cells during culture

    DEFF Research Database (Denmark)

    Jansen-Olesen, Inger; Zhou, MingFang; Zinck, Tina Jovanovic

    2005-01-01

    Nitric oxide (NO) is an important signalling molecule that has been suggested to be a key molecule for induction and maintenance of migraine attacks based on clinical studies, animal experimental studies and the expression of nitric oxide synthase (NOS) immunoreactivity within the trigeminovascul...

  5. Propolis attenuates oxidative injury in brain and lung of nitric oxide synthase inhibited rats

    Directory of Open Access Journals (Sweden)

    Zeliha Selamoglu-Talas

    2015-10-01

    Full Text Available Background: The blocking of nitric oxide synthase (NOS activity may reason vasoconstriction with formation of reactive oxygen species. Propolis has biological and pharmacological properties, such as antioxidant. The aim of this study was to examine the antioxidant effects of propolis which natural product on biochemical parameters in brain and lung tissues of acute nitric oxide synthase inhibited rats by Nω-nitro-L-arginine methyl ester (L-NAME.Methods: Rats have been received L-NAME (40 mg/kg, intraperitoneally, NOS inhibitor for 15 days to produce hypertension and propolis (200mg/kg, by gavage the lastest 5 of 15 days.Results: There  were  the  increase  (P<0.001  in  the  malondialdehyde  levels  in  the  L-NAME treatment groups when compared to control rats, but the decrease (P<0.001 in the catalase activities in both brain and lung tissues. There were statistically changes (P<0.001 in these parameters of L-NAME+propolis treated rats as compared with L-NAME-treated group.Conclusion: The application of L-NAME to the Wistar rats resulted in well developed oxidative stress. Also, propolis may influence endothelial NO production. Identification of such compounds and characterisation of their cellular actions may increase our knowledge of the regulation of endothelial NO production and could provide valuable clues for the prevention or treatment of hypertensive diseases and oxidative stress.

  6. Therapeutic strategies to address neuronal nitric oxide synthase deficiency and the loss of nitric oxide bioavailability in Duchenne Muscular Dystrophy.

    Science.gov (United States)

    Timpani, Cara A; Hayes, Alan; Rybalka, Emma

    2017-05-25

    Duchenne Muscular Dystrophy is a rare and fatal neuromuscular disease in which the absence of dystrophin from the muscle membrane induces a secondary loss of neuronal nitric oxide synthase and the muscles capacity for endogenous nitric oxide synthesis. Since nitric oxide is a potent regulator of skeletal muscle metabolism, mass, function and regeneration, the loss of nitric oxide bioavailability is likely a key contributor to the chronic pathological wasting evident in Duchenne Muscular Dystrophy. As such, various therapeutic interventions to re-establish either the neuronal nitric oxide synthase protein deficit or the consequential loss of nitric oxide synthesis and bioavailability have been investigated in both animal models of Duchenne Muscular Dystrophy and in human clinical trials. Notably, the efficacy of these interventions are varied and not always translatable from animal model to human patients, highlighting a complex interplay of factors which determine the downstream modulatory effects of nitric oxide. We review these studies herein.

  7. Association of Endothelial Nitric Oxide Synthase Gene Polymorphisms With Acute Rejection in Liver Transplant Recipients.

    Science.gov (United States)

    Azarpira, Negar; Namazi, Soha; Malahi, Sayan; Kazemi, Kourosh

    2016-06-01

    Polymorphisms of the endothelial nitric oxide synthase gene have been associated with altered endothelial nitric oxide synthase activity. The purpose of this study was to investigate the relation between endothelial nitric oxide synthase -786T/C and 894G/T polymorphism and their haplotypes on the occurrence of acute rejection episodes in liver transplant recipients. We conducted a case control study in which 100 liver transplant recipients and 100 healthy controls were recruited from Shiraz Transplant Center. The patients used triple therapy including tacrolimus, mycophenolate mofetil, and prednisolone for immunosuppression maintenance. DNA was extracted from peripheral blood and endothelial nitric oxide synthase polymorphisms were determined by polymerase chain reaction and restriction fragment length polymorphism. Patients included 60 men and 40 women (mean age, 32.35 ± 10.2 y). There was a significant association of endothelial nitric oxide synthase 894G/T and acute rejection episode. The GT* gen-otype and acute rejection episodes had a significant association (odds ratio, 2.42; 95% confidence interval, 0.97-6.15; P = .03). The GG and GT* genotype and T* allele frequency were significantly different between patients and control subjects (P = .001). Haplotype TT* was higher in recipients than control subjects (odds ratio, 2.17; 95% confidence interval, 1.12-4.25; P = .01). Haplotype TG was higher in the control group (odds ratio, 0.62; 95% confidence interval, 0.40-0.96; P = .02). Our results suggest a relation between different endothelial nitric oxide synthase geno-types and risk of acute rejection episodes. However, further study is necessary to determine genetic susceptibility for transplant patients.

  8. Anticonvulsion effect of acupuncture might be related to the decrease of neuronal and inducible nitric oxide synthases.

    Science.gov (United States)

    Yang, R; Huang, Z N; Cheng, J S

    1999-01-01

    To measure the levels of hippocampal nitric oxide synthase isoforms in penicillin induced epilepsy and to test the effect of electroacupuncture (EA) on changes of these levels during epilepsy, we injected penicillin into rat hippocampus to make an epilepsy model and performed electroacupuncture treatment on "Feng Fu" (DU 16) and "Jin Suo" (DU 8) points in Wistar rats. Nitric oxide synthase (NOS) mRNA levels of rat hippocampus were determined by reverse transcription-polymerase chain reaction (RT-PCR). The neuronal nitric oxide synthase (nNOS) mRNA markedly increased (pepilepsy, whereas no significant change in epithelial nitric oxide synthase (eNOS) mRNA was observed. EA inhibited the epilepsy and decreased nNOS (pepilepsy caused an increase in nNOS and iNOS, and the EA anticonvulsant effect might be related to the decrease of these nitric oxide synthases.

  9. of endothelial nitric oxide synthase gene and serum level of vascular ...

    African Journals Online (AJOL)

    uwerhiavwe

    Davignon and Ganz, 2004). NO is synthe- sized via a reaction that includes the conversion of L- arginine to L-citruline catalyzed by endothelial nitric oxide synthase (eNOS), which is one of the three isoforms of the enzyme (Mayer and Hemmens, 1997) ...

  10. Nitric oxide synthase expression and apoptotic cell death in brains of AIDS and AIDS dementia patients

    NARCIS (Netherlands)

    Vincent, V. A.; de Groot, C. J.; Lucassen, P. J.; Portegies, P.; Troost, D.; Tilders, F. J.; van Dam, A. M.

    1999-01-01

    To determine the occurrence and cellular localization of inducible nitric oxide synthase (iNOS), NOS activity and its association with cell death in brains of AIDS and AIDS dementia complex (ADC) patients. Post-mortem cerebral cortex tissue of eight AIDS patients, eight ADC patients and eight

  11. Astrocytes and microglia express inducible nitric oxide synthase in mice with experimental allergic encephalomyelitis

    DEFF Research Database (Denmark)

    Tran, E H; Hardin-Pouzet, H; Verge, G

    1997-01-01

    Nitric oxide (NO), produced by inducible NO synthase (iNOS), may play a role in inflammatory demyelinating diseases of the central nervous system (CNS). We show upregulation of iNOS mRNA in CNS of SJL/J mice with experimental allergic encephalomyelitis (EAE). Using antibodies against mouse i...

  12. Chronic nitric oxide synthase inhibition exacerbates renal dysfunction in cirrhotic rats

    DEFF Research Database (Denmark)

    Graebe, Martin; Brond, Lone; Christensen, Sten

    2004-01-01

    The present study investigated sodium balance and renal tubular function in cirrhotic rats with chronic blockade of the nitric oxide (NO) system. Rats were treated with the nonselective NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) starting on the day of common bile duct ligation...

  13. Constitutive expression of inducible nitric oxide synthase in the normal human colonic epithelium

    DEFF Research Database (Denmark)

    Perner, A; Andresen, Lars; Normark, M

    2002-01-01

    Inducible nitric oxide synthase (iNOS) in the human colon is considered expressed only in inflammatory states such as ulcerative or collagenous colitis. As subtle iNOS labelling was previously observed in some colonic mucosal biopsies from a heterogeneous group of controls with non-inflamed bowel...

  14. Investigation on oxidative stress of nitric oxide synthase interacting protein from Clonorchis sinensis.

    Science.gov (United States)

    Bian, Meng; Xu, Qingxia; Xu, Yanquan; Li, Shan; Wang, Xiaoyun; Sheng, Jiahe; Wu, Zhongdao; Huang, Yan; Yu, Xinbing

    2016-01-01

    Numerous evidences indicate that excretory-secretory products (ESPs) from liver flukes trigger the generation of free radicals that are associated with the initial pathophysiological responses in host cells. In this study, we first constructed a Clonorchis sinensis (C. sinensis, Cs)-infected BALB/c mouse model and examined relative results respectively at 3, 5, 7, and 9 weeks postinfection (p.i.). Quantitative reverse transcription (RT)-PCR indicated that the transcriptional level of both endothelial nitric oxide synthase (eNOS) and superoxide dismutase (SOD) gradually decreased with lastingness of infection, while the transcriptional level of inducible NOS (iNOS) significantly increased. The level of malondialdehyde (MDA) in sera of infected mouse significantly increased versus the healthy control group. These results showed that the liver of C. sinensis-infected mouse was in a state with elevated levels of oxidation stress. Previously, C. sinensis NOS interacting protein coding gene (named CsNOSIP) has been isolated and recombinant CsNOSIP (rCsNOSIP) has been expressed in Escherichia coli, which has been confirmed to be a component present in CsESPs and confirmed to play important roles in immune regulation of the host. In the present paper, we investigated the effects of rCsNOSIP on the lipopolysaccharide (LPS)-induced activated RAW264.7, a murine macrophage cell line. We found that endotoxin-free rCsNOSIP significantly promoted the levels of nitric oxide (NO) and reactive oxygen species (ROS) after pretreated with rCsNOSIP, while the level of SOD decreased. Furthermore, rCsNOSIP could also increase the level of lipid peroxidation MDA. Taken together, these results suggested that CsNOSIP was a key molecule which was involved in the production of nitric oxide (NO) and its reactive intermediates, and played an important role in oxidative stress during C. sinensis infection.

  15. Woody Allen kimpus arhitektuuriga

    Index Scriptorium Estoniae

    2000-01-01

    Woody Allen protesteerib oma uue lühifilmiga kavatsuse vastu ehitada 16-korruseline ärihoone tema New Yorgi kodu lähedale. W. Allen hindab New Yorgi ajaloolisi rajoone, mida näitab ka oma filmides

  16. Endothelial nitric oxide synthase gene polymorphisms (G894T) in diabetes mellitus in Egypt

    OpenAIRE

    El-baz1 ; Farouk2; Tag Eldin2; Ezat2

    2010-01-01

    Objective: Diabetic nephropathy (DN) is one of the major microvascular complications of diabetes. Genetic predisposition has been implicated in DN. The eNOS protein synthesizes nitric oxide constitutively via a reaction including the conversion of L-arginine to L-citrulline, which involves the transfer of five electrons provided by nicotinamide adenine dinucleotide phosphate The aim of this study is to evaluate the association of G894T polymorphisms of endothelial nitric oxide synthase(eNOS) ...

  17. Neuronal nitric oxide synthase is involved in the induction of NGF induced neck muscle nociception

    OpenAIRE

    Isaak, Andreas

    2011-01-01

    BACKGROUND: Neck muscle nociception mediated by nitric oxide may play a role in the pathophysiology of tension-type headache.OBJECTIVE: The present study addresses the involvement of neuronal nitric oxide synthase (nNOS) in the facilitation of neck muscle nociception after local application of nerve growth factor (NGF).METHODS: After administration of NGF into semispinal neck muscles, the impact of neck muscle noxious input on brainstem processing was monitored by the jaw-opening reflex in an...

  18. Endothelial nitric oxide synthase: a potential therapeutic target for cerebrovascular diseases.

    Science.gov (United States)

    Zhu, Jinqiang; Song, Wanshan; Li, Lin; Fan, Xiang

    2016-03-22

    Endothelial nitric oxide (NO) is a significant signaling molecule that regulates cerebral blood flow (CBF), playing a pivotal role in the prevention and treatment of cerebrovascular diseases. However, achieving the expected therapeutic efficacy is difficult using direct administration of NO donors. Therefore, endothelial nitric oxide synthase (eNOS) becomes a potential therapeutic target for cerebrovascular diseases. This review summarizes the current evidence supporting the importance of CBF to cerebrovascular function, and the roles of NO and eNOS in CBF regulation.

  19. Endothelial nitric oxide synthase polymorphism G298T in ...

    Indian Academy of Sciences (India)

    resulting in nitrosative stress, oxidative DNA damage and endothelial dysfunction which may contribute to develop- ment of atherosclerosis (Nishevitha et al. 2009). Oxidative. DNA damage includes DNA strand breaks and ... diovascular disease, diabetes, hypertension or renal failure, and 150 patients with angiographically ...

  20. Adhesion Development and the Expression of Endothelial Nitric Oxide Synthase

    Directory of Open Access Journals (Sweden)

    David M. Svinarich

    2001-01-01

    Full Text Available Objective: This study was conducted to determine whether nitric oxide (NO, a potent vasodilator and inhibitor of thrombus formation, is involved in the formation and maintenance of adhesions.

  1. Mitochondrial ATP synthases cluster as discrete domains that reorganize with the cellular demand for oxidative phosphorylation.

    Science.gov (United States)

    Jimenez, Laure; Laporte, Damien; Duvezin-Caubet, Stephane; Courtout, Fabien; Sagot, Isabelle

    2014-02-15

    Mitochondria are double membrane-bounded organelles that form a dynamic tubular network. Mitochondria energetic functions depend on a complex internal architecture. Cristae, inner membrane invaginations that fold into the matrix space, are proposed to be the site of oxidative phosphorylation, reactions by which ATP synthase produces ATP. ATP synthase is also thought to have a role in crista morphogenesis. To date, the exploration of the processes regulating mitochondrial internal compartmentalization have been mostly limited to electron microscopy. Here, we describe ATP synthase localization in living yeast cells and show that it clusters as discrete inner membrane domains. These domains are dynamic within the mitochondrial network. They are impaired in mutants defective in crista morphology and partially overlap with the crista-associated MICOS-MINOS-MITOS complex. Finally, ATP synthase occupancy increases with the cellular demand for OXPHOS. Overall our data suggest that domains in which ATP synthases are clustered correspond to mitochondrial cristae. Being able to follow mitochondrial sub-compartments in living yeast cells opens new avenues to explore the mechanisms involved in inner membrane remodeling, an architectural feature crucial for mitochondrial activities.

  2. Nitric Oxide Synthases Reveal a Role for Calmodulin in Controlling Electron Transfer

    Science.gov (United States)

    Abu-Soud, Husam M.; Stuehr, Dennis J.

    1993-11-01

    Nitric oxide (NO) is synthesized within the immune, vascular, and nervous systems, where it acts as a wide-ranging mediator of mammalian physiology. The NO synthases (EC 1.14.13.39) isolated from neurons or endothelium are calmodulin dependent. Calmodulin binds reversibly to neuronal NO synthase in response to elevated Ca2+, triggering its NO production by an unknown mechanism. Here we show that calmodulin binding allows NADPH-derived electrons to pass onto the heme group of neuronal NO synthase. Calmodulin-triggered electron transfer to heme was independent of substrate binding, caused rapid enzymatic oxidation of NADPH in the presence of O_2, and was required for NO synthesis. An NO synthase isolated from cytokine-induced macrophages that contains tightly bound calmodulin catalyzed spontaneous electron transfer to its heme, consistent with bound calmodulin also enabling electron transfer within this isoform. Together, these results provide a basis for how calmodulin may regulate NO synthesis. The ability of calmodulin to trigger electron transfer within an enzyme is unexpected and represents an additional function for calcium-binding proteins in biology.

  3. Endothelial nitric oxide synthase polymorphism G298T in ...

    Indian Academy of Sciences (India)

    with oxidative DNA damage in coronary atherosclerosis. Rajesh G. Kumar, Mrudula K. Spurthi, Kishore G. Kumar, Sanjib K. Sahu and Surekha H. Rani. J. Genet. 91, 349–352. Table 1. The demographic and clinical data of the CHD patient and control groups. Variable. Patient. Control. Power calculation. Samples. 150. 150.

  4. Endothelial nitric oxide synthase gene Glu298Asp polymorphism ...

    African Journals Online (AJOL)

    Preeclampsia (PE) is the most serious complication of pregnancy that causes maternal and fetal morbidity and mortality. Although the exact pathophysiology of PE is unknown, a large number of studies have shown that abnormalities in nitric oxide (NO) synthesis may contribute to the development of this disorder. There are ...

  5. Antioxidant Functions of Nitric Oxide Synthase in a Methicillin Sensitive Staphylococcus aureus

    Directory of Open Access Journals (Sweden)

    Manisha Vaish

    2013-01-01

    Full Text Available Nitric oxide and its derivative peroxynitrites are generated by host defense system to control bacterial infection. However certain Gram positive bacteria including Staphylococcus aureus possess a gene encoding nitric oxide synthase (SaNOS in their chromosome. In this study it was determined that under normal growth conditions, expression of SaNOS was highest during early exponential phase of the bacterial growth. In oxidative stress studies, deletion of SaNOS led to increased susceptibility of the mutant cells compared to wild-type S. aureus. While inhibition of SaNOS activity by the addition of L-NAME increased sensitivity of the wild-type S. aureus to oxidative stress, the addition of a nitric oxide donor, sodium nitroprusside, restored oxidative stress tolerance of the SaNOS mutant. The SaNOS mutant also showed reduced survival after phagocytosis by PMN cells with respect to wild-type S. aureus.

  6. Middle T antigen-transformed endothelial cells exhibit an increased activity of nitric oxide synthase

    OpenAIRE

    1995-01-01

    Endothelioma cell lines transformed by polyoma virus middle T antigen (mTa) cause cavernous hemangiomas in syngeneic mice by recruitment of host cells. The production of nitric oxide (NO), as measured by nitrite and citrulline production, was significantly higher in mTa-transformed endothelial cells in comparison with nontransformed control cells. The maximal activity of NO synthase (NOS) was about 200-fold higher in cell lysates from the tEnd.1 endothelioma cell line than in lysates from non...

  7. Modular functionalization of allenes to aminated stereotriads.

    Science.gov (United States)

    Adams, Christopher S; Boralsky, Luke A; Guzei, Ilia A; Schomaker, Jennifer M

    2012-07-04

    Nitrogen-containing stereotriads, compounds with three adjacent stereodefined carbons, are commonly found in biologically important molecules. However, the preparation of molecules bearing these motifs can be challenging. Herein, we describe a modular oxidation protocol which converts a substituted allene to a triply functionalized amine of the form C-X/C-N/C-Y. The key step employs a Rh-catalyzed intramolecular conversion of the allene to a strained bicyclic methylene aziridine. This reactive intermediate is further elaborated to the target products, often in one reaction vessel and with effective transfer of the axial chirality of the allene to point chirality in the stereotriad.

  8. Inducible and neuronal nitric oxide synthases exert contrasting effects during rat intestinal recovery following fasting.

    Science.gov (United States)

    Ito, Junta; Uchida, Hiroyuki; Machida, Naomi; Ohtake, Kazuo; Saito, Yuki; Kobayashi, Jun

    2017-04-01

    We investigated the effects of endogenous inducible (iNOS) and neuronal nitric oxide synthase on recovery from intestinal mucosal atrophy caused by fasting-induced apoptosis and decreased cell proliferation during refeeding in rats. Rats were divided into five groups, one of which was fed ad libitum, and four of which underwent 72 h of fasting, followed by refeeding for 0, 6, 24, and 48 h, respectively. iNOS and neuronal nitric oxide synthase mRNA and protein levels in jejunal tissues were measured, and mucosal height was histologically evaluated. Apoptotic indices, interferon-γ (IFN-γ) transcription levels, nitrite levels (as a measure of nitric oxide [NO] production),8-hydroxydeoxyguanosine formation (indicating reactive oxygen species [ROS] levels), crypt cell proliferation, and the motility indices (MI) were also estimated. Associations between mucosal height and NOS protein levels were determined using Spearman's rank correlation test. Notably, we observed significant increases in mucosal height and in neuronal nitric oxide synthase mRNA and protein expression as refeeding time increased. Indeed, there was a significant positive correlation between neuronal nitric oxide synthase protein level and mucosal height during the 48-h refeeding period ( r = 0.725, P fasting. Our finding suggests that refeeding likely repairs fasting-induced jejunal atrophy by suppressing iNOS expression and subsequently inhibiting NO, ROS, and IFN-γ as apoptosis mediators, and by promoting neuronal nitric oxide synthase production and inducing crypt cell proliferation via mechanical stimulation. Impact statement Besides providing new data confirming the involvement of iNOS and nNOS in intestinal mucosal atrophy caused by fasting, this study details their expression and function during recovery from this condition following refeeding. We demonstrate a significant negative correlation between iNOS and nNOS levels during refeeding, and associate this with cell proliferation

  9. Synthesis of N-(Methoxycarbonylthienylmethylthioureas and Evaluation of Their Interaction with Inducible and Neuronal Nitric Oxide Synthase

    Directory of Open Access Journals (Sweden)

    Michael D. Threadgill

    2010-04-01

    Full Text Available Two isomeric N-(methoxycarbonylthienylmethylthioureas were synthesised by a sequence of radical bromination of methylthiophenecarboxylic esters, substitution with trifluoroacetamide anion, deprotection, formation of the corresponding isothiocyanates and addition of ammonia. The interaction of these new thiophene-based thioureas with inducible and neuronal nitric oxide synthase was evaluauted. These novel thienylmethylthioureas stimulated the activity of inducible Nitric Oxide Synthase (iNOS.

  10. Uncoupling of Vascular Nitric Oxide Synthase Caused by Intermittent Hypoxia

    Directory of Open Access Journals (Sweden)

    Mohammad Badran

    2016-01-01

    Full Text Available Objective. Obstructive sleep apnea (OSA, characterized by chronic intermittent hypoxia (CIH, is often present in diabetic (DB patients. Both conditions are associated with endothelial dysfunction and cardiovascular disease. We hypothesized that diabetic endothelial dysfunction is further compromised by CIH. Methods. Adult male diabetic (BKS.Cg-Dock7m +/+ Leprdb/J (db/db mice (10 weeks old and their heterozygote littermates were subjected to CIH or intermittent air (IA for 8 weeks. Mice were separated into 4 groups: IA (intermittent air nondiabetic, IH (intermittent hypoxia nondiabetic, IADB (intermittent air diabetic, and IHDB (intermittent hypoxia diabetic groups. Endothelium-dependent and endothelium-independent relaxation and modulation by basal nitric oxide (NO were analyzed using wire myograph. Plasma 8-isoprostane, interleukin-6 (IL-6, and asymmetric dimethylarginine (ADMA were measured using ELISA. Uncoupling of eNOS was measured using dihydroethidium (DHE staining. Results. Endothelium-dependent vasodilation and basal NO production were significantly impaired in the IH and IADB group compared to IA group but was more pronounced in IHDB group. Levels of 8-isoprostane, IL-6, ADMA, and eNOS uncoupling were ≈2-fold higher in IH and IADB groups and were further increased in the IHDB group. Conclusion. Endothelial dysfunction is more pronounced in diabetic mice subjected to CIH compared to diabetic or CIH mice alone. Oxidative stress, ADMA, and eNOS uncoupling were exacerbated by CIH in diabetic mice.

  11. Antioxidant and nitric oxide synthase activation properties of Ganoderma applanatum.

    Science.gov (United States)

    Acharya, Krishnendu; Yonzone, Parinita; Rai, Manjula; Rupa, Acharya

    2005-10-01

    In vitro evaluation of antioxidant activities of Ganoderma applanatum showed significant inhibition of lipid peroxidation, and potent hydroxyl radical scavenging activity when compared with standard drug catechin. IC50 values of crude, boiled and ethanolic extracts of G. applanatum were 604.8, 624 and 267 microg/ml, respectively in case of hydroxyl radical scavenging activity, and 441, 520.5 and 166.16 microg/ml, respectively in case of lipid peroxidation. Furthermore, crude, boiled and ethanolic extracts also increased significantly nitric oxide production (156.67, 121.88 and 742 pmole/mg dry wt/hr, respectively) over the control. The results of present investigation revealed that G. applanatum have potential therapeutic use.

  12. Increased Salt-Sensitivity in Endothelial Nitric Oxide Synthase-Knockout Mice

    OpenAIRE

    Leonard, Allison M.; Chafe, Linda L.; Montani, Jean-Pierre; Van Vliet, Bruce N.

    2017-01-01

    Background: Although impaired nitric oxide production contributes importantly to salt-sensitivity, the role of the endothelial isoform of nitric oxide synthase (eNOS) has received little attention. In the present study we compared the effects of a high-salt diet on the blood pressure response of eNOS knockout (eNOS−/−) and control (eNOS+/+) mice. Methods: Mean arterial pressure (MAP), heart rate, pulse pressure, and activity levels were recorded by telemetry in mice fed a regular-salt diet (0...

  13. Antenatal insults modify newborn olfactory function by nitric oxide produced from neuronal nitric oxide synthase.

    Science.gov (United States)

    Drobyshevsky, Alexander; Yu, Lei; Yang, Yirong; Khalid, Syed; Luo, Kehuan; Jiang, Rugang; Ji, Haitao; Derrick, Matthew; Kay, Leslie; Silverman, Richard B; Tan, Sidhartha

    2012-10-01

    Newborn feeding, maternal, bonding, growth and wellbeing depend upon intact odor recognition in the early postnatal period. Antenatal stress may affect postnatal odor recognition. We investigated the exact role of a neurotransmitter, nitric oxide (NO), in newborn olfactory function. We hypothesized that olfactory neuron activity depended on NO generated by neuronal NO synthase (NOS). Utilizing in vivo functional manganese enhanced MRI (MEMRI) in a rabbit model of cerebral palsy we had shown previously that in utero hypoxia-ischemia (H-I) at E22 (70% gestation) resulted in impaired postnatal response to odorants and poor feeding. With the same antenatal insult, we manipulated NO levels in the olfactory neuron in postnatal day 1 (P1) kits by administration of intranasal NO donors or a highly selective nNOS inhibitor. Olfactory function was quantitatively measured by the response to amyl acetate stimulation by MEMRI. The relevance of nNOS to normal olfactory development was confirmed by the increase of nNOS gene expression from fetal ages to P1 in olfactory epithelium and bulbs. In control kits, nNOS inhibition decreased NO production in the olfactory system and increased MEMRI slope enhancement. In H-I kits the MEMRI slope did not increase, implicating modification of endogenous NO-mediated olfactory function by the antenatal insult. NO donors as a source of exogenous NO did not significantly change function in either group. In conclusion, olfactory epithelium nNOS in newborn rabbits probably modulates olfactory signal transduction. Antenatal H-I injury remote from delivery may affect early functional development of the olfactory system by decreasing NO-dependent signal transduction. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Smoking and gingivitis: focus on inducible nitric oxide synthase, nitric oxide and basic fibroblast growth factor.

    Science.gov (United States)

    Özdemir, B; Özmeric, N; Elgün, S; Barış, E

    2016-10-01

    Periodontal disease pathogenesis has been associated with smoking. Gingivitis is a mild and reversible form of periodontal disease and it tends to progress to periodontitis only in susceptible individuals. In the present study, we aimed to examine the impact of smoking on host responses in gingivitis and to evaluate and compare the inducible nitric oxide synthase (iNOS) activity in gingival tissue and NO and basic fibroblast growth factor (bFGF) levels in the gingival crevicular fluid of patients with gingivitis and healthy individuals. Forty-one participants were assigned to the gingivitis-smoker (n = 13), gingivitis (n = 13), healthy-smoker (n = 7) and healthy groups (n = 8). Clinical indices were recorded; gingival biopsy and gingival crevicular fluid samples were obtained from papillary regions. iNOS expression was evaluated by immunohistochemical staining. The immunoreactive cells were semiquantitatively assessed. For the quantitative determination of nitrite and nitrate in gingival crevicular fluid, the NO assay kit was used. The amount of bFGF in gingival crevicular fluid was determined by enzyme-linked immunosorbent assay. The gingivitis-smoker group demonstrated a stronger iNOS expression than the non-smoker gingivitis group. iNOS expression intensity was lower in the non-smoker healthy group compared to that in healthy-smokers. No significant gingival crevicular fluid NO and bFGF level changes were observed between groups. Among patients with gingivitis, a positive correlation was detected between gingival crevicular fluid NO and bFGF levels (r = 0.806, p = 0.001). Our data suggest that smoking has significant effects on iNOS expression but not on gingival crevicular fluid NO or bFGF levels in healthy and patients with gingivitis. However, our results suggest that bFGF might be involved in the regulation of NO production via iNOS. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Macula Densa Nitric Oxide Synthase 1β Protects against Salt-Sensitive Hypertension.

    Science.gov (United States)

    Lu, Yan; Wei, Jin; Stec, David E; Roman, Richard J; Ge, Ying; Cheng, Liang; Liu, Eddie Y; Zhang, Jie; Hansen, Pernille B Laerkegaard; Fan, Fan; Juncos, Luis A; Wang, Lei; Pollock, Jennifer; Huang, Paul L; Fu, Yiling; Wang, Shaohui; Liu, Ruisheng

    2016-08-01

    Nitric oxide (NO) is an important negative modulator of tubuloglomerular feedback responsiveness. We recently found that macula densa expresses α-, β-, and γ-splice variants of neuronal nitric oxide synthase 1 (NOS1), and NOS1β expression in the macula densa increases on a high-salt diet. This study tested whether upregulation of NOS1β expression in the macula densa affects sodium excretion and salt-sensitive hypertension by decreasing tubuloglomerular feedback responsiveness. Expression levels of NOS1β mRNA and protein were 30- and five-fold higher, respectively, than those of NOS1α in the renal cortex of C57BL/6 mice. Furthermore, macula densa NO production was similar in the isolated perfused juxtaglomerular apparatus of wild-type (WT) and nitric oxide synthase 1α-knockout (NOS1αKO) mice. Compared with control mice, mice with macula densa-specific knockout of all nitric oxide synthase 1 isoforms (MD-NOS1KO) had a significantly enhanced tubuloglomerular feedback response and after acute volume expansion, significantly reduced GFR, urine flow, and sodium excretion. Mean arterial pressure increased significantly in MD-NOS1KO mice (Psalt diet. After infusion of angiotensin II, mean arterial pressure increased by 61.6 mmHg in MD-NOS1KO mice versus 32.0 mmHg in WT mice (Psalt diet. These results indicate that NOS1β is a primary NOS1 isoform expressed in the macula densa and regulates the tubuloglomerular feedback response, the natriuretic response to acute volume expansion, and the development of salt-sensitive hypertension. These findings show a novel mechanism for salt sensitivity of BP and the significance of tubuloglomerular feedback response in long-term control of sodium excretion and BP. Copyright © 2016 by the American Society of Nephrology.

  16. Nitric oxide donors (nitrates), L-arginine, or nitric oxide synthase inhibitors for acute stroke.

    Science.gov (United States)

    Bath, Philip Mw; Krishnan, Kailash; Appleton, Jason P

    2017-04-21

    Nitric oxide (NO) has multiple effects that may be beneficial in acute stroke, including lowering blood pressure, and promoting reperfusion and cytoprotection. Some forms of nitric oxide synthase inhibition (NOS-I) may also be beneficial. However, high concentrations of NO are likely to be toxic to brain tissue. This is an update of a Cochrane review first published in 1998, and last updated in 2002. To assess the safety and efficacy of NO donors, L-arginine, and NOS-I in people with acute stroke. We searched the Cochrane Stroke Group Trials Register (last searched 6 February 2017), MEDLINE (1966 to June 2016), Embase (1980 to June 2016), ISI Science Citation Indexes (1981 to June 2016), Stroke Trials Registry (searched June 2016), International Standard Randomised Controlled Trial Number (ISRCTN) (searched June 2016), Clinical Trials registry (searched June 2016), and International Clinical Trials Registry Platform (ICTRP) (searched June 2016). Previously, we had contacted drug companies and researchers in the field. Randomised controlled trials comparing nitric oxide donors, L-arginine, or NOS-I versus placebo or open control in people within one week of onset of confirmed stroke. Two review authors independently applied the inclusion criteria, assessed trial quality and risk of bias, and extracted data. The review authors cross-checked data and resolved issues through discussion. We obtained published and unpublished data, as available. Data were reported as mean difference (MD) or odds ratio (OR) with 95% confidence intervals (CI). We included five completed trials, involving 4197 participants; all tested transdermal glyceryl trinitrate (GTN), an NO donor. The assessed risk of bias was low across the included studies; one study was double-blind, one open-label and three were single-blind. All included studies had blinded outcome assessment. Overall, GTN did not improve the primary outcome of death or dependency at the end of trial (modified Rankin Scale (m

  17. Placental Vesicles Carry Active Endothelial Nitric Oxide Synthase and Their Activity is Reduced in Preeclampsia.

    Science.gov (United States)

    Motta-Mejia, Carolina; Kandzija, Neva; Zhang, Wei; Mhlomi, Vuyane; Cerdeira, Ana Sofia; Burdujan, Alexandra; Tannetta, Dionne; Dragovic, Rebecca; Sargent, Ian L; Redman, Christopher W; Kishore, Uday; Vatish, Manu

    2017-08-01

    Preeclampsia, a multisystem hypertensive disorder of pregnancy, is associated with increased systemic vascular resistance. Placentae from patients with preeclampsia have reduced levels of endothelial nitric oxide synthase (eNOS) and, thus, less nitric oxide (NO). Syncytiotrophoblast extracellular vesicles (STBEV), comprising microvesicles (STBMV) and exosomes, carry signals from the syncytiotrophoblast to the mother. We hypothesized that STBEV-bound eNOS (STBEV-eNOS), capable of producing NO, are released into the maternal circulation. Dual-lobe ex vivo placental perfusion and differential centrifugation was used to isolate STBEV from preeclampsia (n=8) and normal pregnancies (NP; n=11). Plasma samples of gestational age-matched preeclampsia and NP (n=6) were used to isolate circulating STBMV. STBEV expressed placental alkaline phosphatase, confirming placental origin. STBEV coexpressed eNOS, but not inducible nitric oxide synthase, confirmed using Western blot, flow cytometry, and immunodepletion. STBEV-eNOS produced NO, which was significantly inhibited by N   G -nitro-l-arginine methyl ester (eNOS inhibitor; P preeclampsia-perfused placentae had lower levels of STBEV-eNOS (STBMV; P preeclampsia women had lower STBEV-eNOS expression compared with that from NP women ( P preeclampsia placentae, as well as in plasma. The lower STBEV-eNOS NO production seen in preeclampsia may contribute to the decreased NO bioavailability in this disease. © 2017 The Authors.

  18. Neuronal Nitric Oxide Synthase Induction in the Antitumorigenic and Neurotoxic Effects of 2-Methoxyestradiol

    Directory of Open Access Journals (Sweden)

    Magdalena Gorska

    2014-08-01

    Full Text Available Objective: 2-Methoxyestradiol, one of the natural 17β-estradiol derivatives, is a novel, potent anticancer agent currently being evaluated in advanced phases of clinical trials. The main goal of the study was to investigate the anticancer activity of 2-methoxy-estradiol towards osteosarcoma cells and its possible neurodegenerative effects. We used an experimental model of neurotoxicity and anticancer activity of the physiological agent, 2-methoxyestradiol. Thus, we used highly metastatic osteosarcoma 143B and mouse immortalized hippocampal HT22 cell lines. The cells were treated with pharmacological (1 μM, 10 μM concentrations of 2-methoxyestradiol. Experimental: Neuronal nitric oxide synthase and 3-nitrotyrosine protein levels were determined by western blotting. Cell viability and induction of cell death were measured by MTT and PI/Annexin V staining and a DNA fragmentation ELISA kit, respectively. Intracellular levels of nitric oxide were determined by flow cytometry. Results: Here we demonstrated that the signaling pathways of neurodegenerative diseases and cancer may overlap. We presented evidence that 2-methoxyestradiol, in contrast to 17β-estradiol, specifically affects neuronal nitric oxide synthase and augments 3-nitrotyrosine level leading to osteosarcoma and immortalized hippocampal cell death. Conclusions: We report the dual facets of 2-methoxyestradiol, that causes cancer cell death, but on the other hand may play a key role as a neurotoxin.

  19. Vasoactive systems in L-NAME hypertension: the role of inducible nitric oxide synthase

    Czech Academy of Sciences Publication Activity Database

    Pecháňová, Olga; Dobešová, Zdenka; Čejka, Jakub; Kuneš, Jaroslav; Zicha, Josef

    2004-01-01

    Roč. 22, č. 1 (2004), s. 167-173 ISSN 0263-6352 R&D Projects: GA ČR GA305/03/0769; GA MŠk LN00A069 Grant - others:VEGA(SK) 2/3185/23; SAV(SK) APVT51-017902 Institutional research plan: CEZ:AV0Z5011922 Keywords : nitric oxide synthase * L-NAME hypertension * aminoguanidine Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery Impact factor: 4.871, year: 2004

  20. Endothelial nitric oxide synthase gene haplotypes and diabetic nephropathy among Asian Indians

    DEFF Research Database (Denmark)

    Ahluwalia, Tarun Veer Singh; Ahuja, Monica; Rai, Taranjit Singh

    2008-01-01

    of the constitutive endothelial nitric oxide synthase gene (eNOS) polymorphisms with type 2 diabetic nephropathy. We genotyped three polymorphisms of eNOS (Two SNPs: -786T > C, 894G > T and one 27-bp repeat polymorphism in Intron 4 (27VNTR)) in type 2 diabetic nephropathy patients (cases: n = 195) and type 2 diabetic...... without nephropathy (controls: n = 255), using validated PCR-RFLP assays. We measured serum NO levels in these subjects and examined its correlation with diabetic nephropathy and eNOS genotypes. The frequency of CC (-786T > C), TT (894G > T) and aa genotypes (27VNTR) were significantly higher in diabetic...

  1. Neuronal Nitric-Oxide Synthase Deficiency Impairs the Long-Term Memory of Olfactory Fear Learning and Increases Odor Generalization

    Science.gov (United States)

    Pavesi, Eloisa; Heldt, Scott A.; Fletcher, Max L.

    2013-01-01

    Experience-induced changes associated with odor learning are mediated by a number of signaling molecules, including nitric oxide (NO), which is predominantly synthesized by neuronal nitric oxide synthase (nNOS) in the brain. In the current study, we investigated the role of nNOS in the acquisition and retention of conditioned olfactory fear. Mice…

  2. High-performance liquid chromatography method with radiochemical detection for measurement of nitric oxide synthase, arginase, and arginine decarboxylase activities

    DEFF Research Database (Denmark)

    Volke, A; Wegener, Gregers; Vasar, E

    2006-01-01

    Nitric oxide has been shown to be involved in numerous biological processes, and many studies have aimed to measure nitric oxide synthase (NOS) activity. Recently, it has been demonstrated that arginase and arginine decarboxylase (ADC), two enzymes that also employ arginine as a substrate, may re...

  3. Nitric oxide synthase and nitric oxide alterations in chronically stressed rats: a model for nitric oxide in major depressive disorder.

    Science.gov (United States)

    Gao, Shang-Feng; Lu, Yun-Rong; Shi, Li-Gen; Wu, Xue-Yan; Sun, Bo; Fu, Xin-Yan; Luo, Jian-Hong; Bao, Ai-Min

    2014-09-01

    Nitric oxide (NO) and NO synthase-1 (NOS1) are involved in the stress response and in depression. We compared NOS-NO alterations in rats exposed to chronic unpredictable stress (CUS) with alterations in major depressive disorder (MDD) in humans. In the hypothalamus of male CUS rats we determined NOS activity, and in the paraventricular nucleus (PVN) we determined NOS1-immunoreactive (ir) cell densities and co-localization of NOS1 with stress-related neuropeptides corticotropin-releasing hormone (CRH), vasopressin (AVP) or oxytocin (OXT). We measured plasma NO levels and cortisol in male medicine-naïve MDD patients and plasma NO and corticosterone (CORT) in CUS rats. In the CUS rat total NOS activity in the hypothalamus (P=0.018) and NOS1-ir cell density in the PVN were both significantly decreased (P=0.018), while NOS1 staining was mainly expressed in OXT-ir neurons in this nucleus. Interestingly, plasma NO levels were significantly increased both in male CUS rats (P=0.001) and in male MDD patients (Pdepression. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Allen Ginsberg kirjutab Ameerikast

    Index Scriptorium Estoniae

    2003-01-01

    Arvustus: Ginsberg, Allen. Ameerika : valik luuletusi 1947-1996 / koostanud ja toimetanud Hasso Krull ; inglise keelest tõlkinud Contra, Lauri Kitsnik, Hasso Krull, Andres Langemets, Margit Langemets, Jürgen Rooste, Tõnu Õnnepalu. Tallinn : Perioodika, 2003

  5. Inducible nitric oxide synthase and guinea-pig ileitis induced by adjuvant

    Directory of Open Access Journals (Sweden)

    N. D. Seago

    1995-01-01

    Full Text Available We sought to establish a model of inflammatory bowel disease by augmenting the activity of the local immune system with Freund's complete adjuvant, and to determine if inducible nitric oxide synthase (iNOS expression and peroxynitrite formation accompanied the inflammatory condition. In anaesthetized guinea-pigs, a loop of distal ileum received intraluminal 50% ethanol followed by Freund's complete adjuvant. Control animals were sham operated. When the animals were killed 7 or 14 days later, loop lavage fluid was examined for nitrite and PGE2 levels; mucosal levels of granulocyte and macrophages were estimated by myeloperoxidase (MPO and N-acetyl-D-glucosaminidase (NAG activity, respectively. Cellular localization if iNOS and peroxynitrite formation were determined by immunohistochemistry with polyclonal antibodies directed against peptide epitopes of mouse iNOS and nitrotyrosine, respectfully. Adjuvant administration resulted in a persistent ileitis, featuring gut thickening, crypt hyperplasia, villus tip swelling and disruption, and cellular infiltration. Lavage levels of PGE2 and nitrite were markedly elevated by adjuvant treatment. Immunoreactive iNOS and nitrotyrosine bordered on detectability in normal animals but were markedly evident with adjuvant treatment at day 7 and particularly day 14. Immunohistochemistry suggested that enteric neurons and epithelia were major sites of iNOS activity and peroxynitrite formation. We conclude that local administration of adjuvant establishes a chronic ileitis. Inducible nitric oxide synthase may contribute to the inflammatory process.

  6. Osteopontin protects against hyperoxia-induced lung injury by inhibiting nitric oxide synthases.

    Science.gov (United States)

    Zhang, Xiang-Feng; Liu, Shuang; Zhou, Yu-Jie; Zhu, Guang-Fa; Foda, Hussein D

    2010-04-05

    Exposure of adult mice to more than 95% O(2) produces a lethal injury by 72 hours. Nitric oxide synthase (NOS) is thought to contribute to the pathophysiology of murine hyperoxia-induced acute lung injury (ALI). Osteopontin (OPN) is a phosphorylated glycoprotein produced principally by macrophages. OPN inhibits inducible nitric oxide synthase (iNOS), which generates large amounts of nitric oxide production. However, the relationship between nitric oxide and endogenous OPN in lung tissue during hyperoxia-induced ALI has not yet been elucidated, thus we examined the role that OPN plays in the hyperoxia-induced lung injury and its relationships with NOS. One hundred and forty-four osteopontin knock-out (KO) mice and their matched wild type background control (WT) were exposed in sealed cages > 95% oxygen or room air for 24- 72 hours, and the severity of lung injury was assessed; expression of OPN, endothelial nitric oxide synthase (eNOS) and iNOS mRNA in lung tissues at 24, 48 and 72 hours of hyperoxia were studied by reverse transcription-polymerase chain reaction (RT-PCR); immunohistochemistry (IHC) was performed for the detection of iNOS, eNOS, and OPN protein in lung tissues. OPN KO mice developed more severe acute lung injury at 72 hours of hyperoxia. The wet/dry weight ratio increased to 6.85 +/- 0.66 in the KO mice at 72 hours of hyperoxia as compared to 5.31 +/- 0.92 in the WT group (P < 0.05). iNOS mRNA (48 hours: 1.04 +/- 0.08 vs. 0.63 +/- 0.09, P < 0.01; 72 hours: 0.89 +/- 0.08 vs. 0.72 +/- 0.09, P < 0.05) and eNOS mRNA (48 hours: 0.62 +/- 0.08 vs. 0.43 +/- 0.09, P < 0.05; 72 hours: 0.67 +/- 0.08 vs. 0.45 +/- 0.09, P < 0.05) expression was more significantly increased in OPN KO mice than their matched WT mice when exposed to hyperoxia. IHC study showed higher expression of iNOS (20.54 +/- 3.18 vs. 12.52 +/- 2.46, P < 0.05) and eNOS (19.83 +/- 5.64 vs. 9.45 +/- 3.82, P < 0.05) in lung tissues of OPN KO mice at 72 hours of hyperoxia. OPN can protect against

  7. Immunocytochemical localization of nitric oxide synthase in the brain of the chicken.

    Science.gov (United States)

    Brüning, G; Funk, U; Mayer, B

    1994-12-20

    The distribution of neuronal nitric oxide synthase (NOS) in the chicken brain was investigated by immunocytochemistry. Strongly stained neurones were concentrated in the paleostriatum augmentatum, lobus parolfactorius, ventral pallidum, olfactory tubercle, parts of the neostriatum, mesencephalic reticular formation and locus coeruleus. Cells in these areas have previously been shown to stain for NADPH-diaphorase, a histochemical activity associated with NOS. However, various structures which NADPH-diaphorase staining has suggested to contain NOS were not immunoreactive: these included the glomeruli of the olfactory bulb, magnocellular preoptic neurones, the median eminence, subcommissural organ and mesencephalic trigeminal neurones. NOS was sparsely present in the hyperstriatum ventrale, providing evidence against the involvement of nitric oxide in certain forms of learning and memory processes known to occur in this region.

  8. Cloning of allene oxide cyclase gene from Leymus mollis and analysis of its expression in wheat-Leymus chromosome addition lines.

    Science.gov (United States)

    Eltayeb Habora, Mohamed Elsadig; Eltayeb, Amin Elsadig; Oka, Mariko; Tsujimoto, Hisashi; Tanaka, Kiyoshi

    2013-03-01

    Leymus mollis (Triticeae; Poaceae) is a useful genetic resource for wheat (Triticum aestivum L.) breeding via wide hybridization to introduce its chromosomes and integrate its useful traits into wheat. Leymus mollis is highly tolerant to abiotic stresses such as drought and salinity and resistant to various diseases, but the genetic mechanisms controlling its physiological tolerance remain largely unexplored. We identified and cloned an allene oxide cyclase (AOC) gene from L. mollis that was strongly expressed under salt stress. AOC is involved in biosynthesis of jasmonic acid, an important signaling compound that mediates a wide range of adaptive responses. LmAOC cDNA consisted of 717 bp, coding for a protein with 238 amino acids that was highly similar to AOCs from barley (Hordeum vulgare) and other monocots. Subcellular localization using Nicotiana benthamiana confirmed it as a chloroplast-localized protein. LmAOC was found to be a multiple-copy gene, and that some copies were conserved and efficiently expressed in wheat-Leymus chromosome addition lines. LmAOC expression was upregulated under drought, heat, cold and wounding stresses, and by jasmonic acid and abscisic acid. Our results suggest that LmAOC plays an important role in L. mollis adaptation to abiotic stresses and it could be useful for wheat improvement.

  9. Cloning of allene oxide cyclase gene from Leymus mollis and analysis of its expression in wheat–Leymus chromosome addition lines

    Science.gov (United States)

    Eltayeb Habora, Mohamed Elsadig; Eltayeb, Amin Elsadig; Oka, Mariko; Tsujimoto, Hisashi; Tanaka, Kiyoshi

    2013-01-01

    Leymus mollis (Triticeae; Poaceae) is a useful genetic resource for wheat (Triticum aestivum L.) breeding via wide hybridization to introduce its chromosomes and integrate its useful traits into wheat. Leymus mollis is highly tolerant to abiotic stresses such as drought and salinity and resistant to various diseases, but the genetic mechanisms controlling its physiological tolerance remain largely unexplored. We identified and cloned an allene oxide cyclase (AOC) gene from L. mollis that was strongly expressed under salt stress. AOC is involved in biosynthesis of jasmonic acid, an important signaling compound that mediates a wide range of adaptive responses. LmAOC cDNA consisted of 717 bp, coding for a protein with 238 amino acids that was highly similar to AOCs from barley (Hordeum vulgare) and other monocots. Subcellular localization using Nicotiana benthamiana confirmed it as a chloroplast-localized protein. LmAOC was found to be a multiple-copy gene, and that some copies were conserved and efficiently expressed in wheat–Leymus chromosome addition lines. LmAOC expression was upregulated under drought, heat, cold and wounding stresses, and by jasmonic acid and abscisic acid. Our results suggest that LmAOC plays an important role in L. mollis adaptation to abiotic stresses and it could be useful for wheat improvement. PMID:23641183

  10. Hyperglycemia adversely modulates endothelial nitric oxide synthase during anesthetic preconditioning through tetrahydrobiopterin- and heat shock protein 90-mediated mechanisms.

    Science.gov (United States)

    Amour, Julien; Brzezinska, Anna K; Jager, Zachary; Sullivan, Corbin; Weihrauch, Dorothee; Du, Jianhai; Vladic, Nikolina; Shi, Yang; Warltier, David C; Pratt, Phillip F; Kersten, Judy R

    2010-03-01

    Endothelial nitric oxide synthase activity is regulated by (6R-)5,6,7,8-tetrahydrobiopterin (BH4) and heat shock protein 90. The authors tested the hypothesis that hyperglycemia abolishes anesthetic preconditioning (APC) through BH4- and heat shock protein 90-dependent pathways. Myocardial infarct size was measured in rabbits in the absence or presence of APC (30 min of isoflurane), with or without hyperglycemia, and in the presence or absence of the BH4 precursor sepiapterin. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells cultured in normal (5.5 mm) or high (20 mm) glucose conditions, with or without sepiapterin (10 or 100 microm). APC decreased myocardial infarct size compared with control experiments (26 +/- 6% vs. 46 +/- 3%, respectively; P < 0.05), and this action was blocked by hyperglycemia (43 +/- 4%). Sepiapterin alone had no effect on infarct size (46 +/- 3%) but restored APC during hyperglycemia (21 +/- 3%). The beneficial actions of sepiapterin to restore APC were blocked by the nitric oxide synthase inhibitor N (G)-nitro-L-arginine methyl ester (47 +/- 2%) and the BH4 synthesis inhibitor N-acetylserotonin (46 +/- 3%). Isoflurane increased nitric oxide production to 177 +/- 13% of baseline, and this action was attenuated by high glucose concentrations (125 +/- 6%). Isoflurane increased, whereas high glucose attenuated intracellular BH4/7,8-dihydrobiopterin (BH2) (high performance liquid chromatography), heat shock protein 90-endothelial nitric oxide synthase colocalization (confocal microscopy) and endothelial nitric oxide synthase activation (immunoblotting). Sepiapterin increased BH4/BH2 and dose-dependently restored nitric oxide production during hyperglycemic conditions (149 +/- 12% and 175 +/- 9%; 10 and 100 microm, respectively). The results indicate that tetrahydrobiopterin and heat shock protein 90-regulated endothelial nitric oxide synthase activity play a central

  11. Inactivation of glycogen synthase kinase-3β (GSK-3β) enhances skeletal muscle oxidative metabolism.

    Science.gov (United States)

    Theeuwes, W F; Gosker, H R; Langen, R C J; Verhees, K J P; Pansters, N A M; Schols, A M W J; Remels, A H V

    2017-12-01

    Aberrant skeletal muscle mitochondrial oxidative metabolism is a debilitating feature of chronic diseases such as chronic obstructive pulmonary disease, type 2 diabetes and chronic heart failure. Evidence in non-muscle cells suggests that glycogen synthase kinase-3β (GSK-3β) represses mitochondrial biogenesis and inhibits PPAR-γ co-activator 1 (PGC-1), a master regulator of cellular oxidative metabolism. The role of GSK-3β in the regulation of skeletal muscle oxidative metabolism is unknown. We hypothesized that inactivation of GSK-3β stimulates muscle oxidative metabolism by activating PGC-1 signaling and explored if GSK-3β inactivation could protect against physical inactivity-induced alterations in skeletal muscle oxidative metabolism. GSK-3β was modulated genetically and pharmacologically in C2C12 myotubes in vitro and in skeletal muscle in vivo. Wild-type and muscle-specific GSK-3β knock-out (KO) mice were subjected to hind limb suspension for 14days. Key constituents of oxidative metabolism and PGC-1 signaling were investigated. In vitro, knock-down of GSK-3β increased mitochondrial DNA copy number, protein and mRNA abundance of oxidative phosphorylation (OXPHOS) complexes and activity of oxidative metabolic enzymes but also enhanced protein and mRNA abundance of key PGC-1 signaling constituents. Similarly, pharmacological inhibition of GSK-3β increased transcript and protein abundance of key constituents and regulators of mitochondrial energy metabolism. Furthermore, GSK-3β KO animals were protected against unloading-induced decrements in expression levels of these constituents. Inactivation of GSK-3β up-regulates skeletal muscle mitochondrial metabolism and increases expression levels of PGC-1 signaling constituents. In vivo, GSK-3β KO protects against inactivity-induced reductions in muscle metabolic gene expression. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Inducible nitric oxide synthase inhibitors abolished histological protection by late ischemic preconditioning in rat retina.

    Science.gov (United States)

    Sakamoto, Kenji; Yonoki, Yuzuru; Kubota, Yuko; Kuwagata, Mayumi; Saito, Maki; Nakahara, Tsutomu; Ishii, Kunio

    2006-03-01

    Brief ischemia was reported to protect retinal cells against injury induced by subsequent ischemia-reperfusion with de novo protein synthesis, and this phenomenon is known as late ischemic preconditioning. The aims of the present study were to determine whether nitric oxide synthase (NOS) was involved in the mechanism of late ischemic preconditioning in rat retina using pharmacological tools. Under anesthesia with pentobarbital sodium, male Sprague-Dawley rats were subjected to 60 min of retinal ischemia by raising intraocular pressure to 130 mm Hg. Ischemic preconditioning was achieved by applying 5 min of ischemia 24 hrs before 60 min of ischemia. Retinal sections sliced into 5 microm thick were examined 7 days after ischemia. Additional groups of rats received NG-nitro-L-arginine and NG-monomethyl-L-arginin, non-selective NO synthase inhibitors, 7-nitroindazole, a neuronal NOS inhibitor, and aminoguanidine and L-N6-(1-iminoethyl) lysine, inducible NO synthase (iNOS) inhibitors before preconditioning, and were subjected to 60 min of ischemia. In the non-preconditioned group, cell loss in the ganglion cell layer and thinning of the inner plexiform and inner nuclear layer were observed 7 days after 60 min of ischemia. Ischemic preconditioning for 5 min completely protected against the histological damage induced by 60 min of ischemia applied 24 hrs thereafter. Treatment of rats with aminoguanidine and L-N6-(1-iminoethyl) lysine, but not NG-nitro-L-arginine, NG-monomethyl-L-arginine or 7-nitroindazole, wiped off the protective effect of ischemic preconditioning. The inhibitory effect of aminoguanidine was abolished by L-arginine, but not D-arginine. The results in the present study suggest that NO synthesized by iNOS is involved in the histological protection by late ischemic preconditioning in rat retina.

  13. Hemodynamic effects of nitric oxide synthase inhibition before and after cardiac arrest in infant piglets.

    Science.gov (United States)

    Schleien, C L; Kuluz, J W; Gelman, B

    1998-04-01

    Using infant piglets, we studied the effects of nonspecific inhibition of nitric oxide (NO) synthase by NG-nitro-L-arginine methyl ester (L-NAME; 3 mg/kg) on vascular pressures, regional blood flow, and cerebral metabolism before 8 min of cardiac arrest, during 6 min of cardiopulmonary resuscitation (CPR), and at 10 and 60 min of reperfusion. We tested the hypotheses that nonspecific NO synthase inhibition 1) will attenuate early postreperfusion hyperemia while still allowing for successful resuscitation after cardiac arrest, 2) will allow for normalization of blood flow to the kidneys and intestines after cardiac arrest, and 3) will maintain cerebral metabolism in the face of altered cerebral blood flow after reperfusion. Before cardiac arrest, L-NAME increased vascular pressures and cardiac output and decreased blood flow to brain (by 18%), heart (by 36%), kidney (by 46%), and intestine (by 52%) compared with placebo. During CPR, myocardial flow was maintained in all groups to successfully resuscitate 24 of 28 animals [P value not significant (NS)]. Significantly, L-NAME attenuated postresuscitation hyperemia in cerebellum, diencephalon, anterior cerebral, and anterior-middle watershed cortical brain regions and to the heart. Likewise, cerebral metabolic rates of glucose (CMRGluc) and of lactate production (CMRLac) were not elevated at 10 min of reperfusion. These cerebral blood flow and metabolic effects were reversed by L-arginine. Flows returned to baseline levels by 60 min of reperfusion. Kidney and intestinal flow, however, remained depressed throughout reperfusion in all three groups. Thus nonspecific inhibition of NO synthase did not adversely affect the rate of resuscitation from cardiac arrest while attenuating cerebral and myocardial hyperemia. Even though CMRGluc and CMRLac early after resuscitation were decreased, they were maintained at baseline levels. This may be clinically advantageous in protecting the brain and heart from the damaging effects of

  14. Oxidative stress inactivates cobalamin-independent methionine synthase (MetE in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Elise R Hondorp

    2004-11-01

    Full Text Available In nature, Escherichia coli are exposed to harsh and non-ideal growth environments-nutrients may be limiting, and cells are often challenged by oxidative stress. For E. coli cells confronting these realities, there appears to be a link between oxidative stress, methionine availability, and the enzyme that catalyzes the final step of methionine biosynthesis, cobalamin-independent methionine synthase (MetE. We found that E. coli cells subjected to transient oxidative stress during growth in minimal medium develop a methionine auxotrophy, which can be traced to an effect on MetE. Further experiments demonstrated that the purified enzyme is inactivated by oxidized glutathione (GSSG at a rate that correlates with protein oxidation. The unique site of oxidation was identified by selectively cleaving N-terminally to each reduced cysteine and analyzing the results by liquid chromatography mass spectrometry. Stoichiometric glutathionylation of MetE by GSSG occurs at cysteine 645, which is strategically located at the entrance to the active site. Direct evidence of MetE oxidation in vivo was obtained from thiol-trapping experiments in two different E. coli strains that contain highly oxidizing cytoplasmic environments. Moreover, MetE is completely oxidized in wild-type E. coli treated with the thiol-oxidizing agent diamide; reduced enzyme reappears just prior to the cells resuming normal growth. We argue that for E. coli experiencing oxidizing conditions in minimal medium, MetE is readily inactivated, resulting in cellular methionine limitation. Glutathionylation of the protein provides a strategy to modulate in vivo activity of the enzyme while protecting the active site from further damage, in an easily reversible manner. While glutathionylation of proteins is a fairly common mode of redox regulation in eukaryotes, very few proteins in E. coli are known to be modified in this manner. Our results are complementary to the independent findings of Leichert

  15. Memory consolidation and inducible nitric oxide synthase expression during different sleep stages in Parkinson disease.

    Science.gov (United States)

    Wu, Dean; Tseng, Ing-Jy; Yuan, Rey-Yue; Hsieh, Chia-Yu; Hu, Chaur-Jong

    2014-01-01

    Parkinson disease (PD) is a neurodegenerative disease characterized by motor and nonmotor dysfunctions, which include sleep disturbances. Rapid eye movement (REM) sleep is associated with numerous physiologic changes such as memory consolidation. Compelling evidence suggests that nitric oxide (NO) is crucial to both sleep regulation and memory consolidation. In our study, we explored changes in biologic molecules during various sleep stages and the effects of sleep on memory consolidation in PD. Ten PD patients and 14 volunteers without PD participated in our study. The gene expression of inducible NO synthase (iNOS) in all sleep stages was measured using realtime polymerase chain reaction (PCR) based on polysomnography (PSG)-guided peripheral blood sampling. In addition, the efficiency of memory consolidation during the sleep of the participants was measured using the Wechsler Memory Scale, third edition (WMS-III). The iNOS expression increased in all sleep stages among the PD patients compared to the control participants, in whom iNOS expression decreased during REM sleep. Regarding memory consolidation, the performance of the controls in logic memory and the patients in visual reproduction tasks improved after sleep. The iNOS synthase expression was different from control participants among PD patients, and the expression was dissimilar in various sleep stages. Sleep might enhance memory consolidation and there are different memory consolidation profiles between PD and control participants demonstrating distinct memory consolidation profiles. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Suppressive Role of PPARγ-Regulated Endothelial Nitric Oxide Synthase in Adipocyte Lipolysis.

    Directory of Open Access Journals (Sweden)

    Yoko Yamada

    Full Text Available Metabolic syndrome causes insulin resistance and is associated with risk factor clustering, thereby increasing the risk of atherosclerosis. Recently, endothelial nitric oxide synthase deficient (eNOS-/- mice have been reported to show metabolic disorders. Interestingly, eNOS has also been reported to be expressed in non-endothelial cells including adipocytes, but the functions of eNOS in adipocytes remain unclear.The eNOS expression was induced with adipocyte differentiation and inhibition of eNOS/NO enhanced lipolysis in vitro and in vivo. Furthermore, the administration of a high fat diet (HFD was able to induce non-alcoholic steatohepatitis (NASH in eNOS-/- mice but not in wild type mice. A PPARγ antagonist increased eNOS expression in adipocytes and suppressed HFD-induced fatty liver changes.eNOS-/- mice induce NASH development, and these findings provide new insights into the therapeutic approach for fatty liver disease and related disorders.

  17. Immunohistochemical localization of endothelial and inducible nitric oxide synthase within neurons of cattle with rabies.

    Science.gov (United States)

    Shin, Taekyun; Weinstock, Daniel; Castro, Marlene D; Hamir, Amir N; Wampler, Thomas; Walter, Mark; Kim, Hyun Young; Acland, Helen

    2004-05-01

    The expression of constitutive endothelial nitric oxide synthase (eNOS) and inducible NOS (iNOS) in the brains of cattle with natural rabies was studied. Increased expression of eNOS was detected in neurons of the brain stem and Purkinje cells of cerebellum. By contrast, iNOS was diffusely localized in the cytoplasm of affected neurons, and some inflammatory cells were positive. eNOS and rabies antigen were co-localized in inclusion bodies (Negri bodies) in neurons. The specific localization of eNOS, but not iNOS, in the Negri bodies suggests that eNOS is involved in the formation of rabies virus inclusion bodies.

  18. Inducible nitric oxide synthase inhibitors from Saposhnikovia divaricata and Panax quinquefolium.

    Science.gov (United States)

    Wang, C N; Shiao, Y J; Kuo, Y H; Chen, C C; Lin, Y L

    2000-10-01

    A series of polyacetylenes, falcarinone, panaxynol, falcarindiol, panaxydol, and panaxytriol, were isolated from Saposhnikovia divaricata (Turcz.) Schischk and Panax quinquefolium L. These polyacetylenes were identified as active principles on the inhibition of nitrite production by inducible nitric oxide synthase (iNOS). Treatment with 10 microM of panaxynol, falcarindiol, panaxydol and panaxytriol decreased the LPS/IFN-gamma-stimulated accumulation of nitrite by 71.92 +/- 3.07, 69.95 +/- 3.68, 45.48 +/- 6.11 and 36.85 +/- 8.80%, respectively. The IC50 value of falcarinone, panaxynol, falcarindiol, panaxydol and panaxytriol was > 20, 2.23, 1.98, 6.58 and 9.85 microM, respectively.

  19. Expression of Inducible Nitric Oxide Synthase in Skin Lesions of Patients with American Cutaneous Leishmaniasis

    Science.gov (United States)

    Qadoumi, Muna; Becker, Inge; Donhauser, Norbert; Röllinghoff, Martin; Bogdan, Christian

    2002-01-01

    Cytokine-inducible (or type 2) nitric oxide synthase (iNOS) is indispensable for the resolution of Leishmania major or Leishmania donovani infections in mice. In contrast, little is known about the expression and function of iNOS in human leishmaniasis. Here, we show by immunohistological analysis of skin biopsies from Mexican patients with local (LCL) or diffuse (DCL) cutaneous leishmaniasis that the expression of iNOS was most prominent in LCL lesions with small numbers of parasites whereas lesions with a high parasite burden (LCL or DCL) contained considerably fewer iNOS-positive cells. This is the first study to suggest an antileishmanial function of iNOS in human Leishmania infections in vivo. PMID:12117977

  20. Pseudoguaianolides isolated from Inula britannica var. chinenis as inhibitory constituents against inducible nitric oxide synthase.

    Science.gov (United States)

    Lee, Hyun-Tai; Yang, Seung-Won; Kim, Kyeong Ho; Seo, Eun-Kyoung; Mar, Woongchon

    2002-04-01

    Three pseudoguaianolide type sesquiterpenes, bigelovin (1), 2,3-dihydroaromaticin (2), and ergolide (3) were isolated as inhibitory constituents against inducible nitric oxide synthase (iNOS) from the flowers of Inula britannica var. chinensis. Bigelovin (1) exhibited a highly potent inhibitory activity on lipopolysaccharide (LPS)-induced iNOS in murine macrophage RAW 264.7 cells with an IC50 value of 0.46 mM, which is about 8 times more potent than the known selective inhibitor of iNOS, L-N6-(1-iminoethyl)lysine (IC50 3.49 microM). 2,3-Dihydroaromaticin (2) and ergolide (3) also exhibited potent inhibitory activities on LPS-induced iNOS with IC50 values of 1.05 and 0.69 microM, respectively.

  1. Pathogenic cycle between the endogenous nitric oxide synthase inhibitor asymmetrical dimethylarginine and the leukocyte-derived hemoprotein myeloperoxidase

    Czech Academy of Sciences Publication Activity Database

    von Leitner, E.C.; Klinke, A.; Atzler, D.; Slocum, J.L.; Lund, N.; Kielstein, J.T.; Maas, R.; Schmidt-Haupt, R.; Pekarová, Michaela; Hellwinkel, O.; Tsikas, D.; D'Alecy, L.G.; Lau, D.; Willems, S.; Kubala, Lukáš; Ehmke, H.; Meinertz, T.; Blankenberg, S.; Schwedhelm, E.; Gadegbeku, C.A.; Boger, R.H.; Baldus, S.; Sydow, K.

    2011-01-01

    Roč. 124, č. 4 (2011), s. 2735-U342 ISSN 0009-7322 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : arteriosclerosis * leukocytes * nitric oxide synthase Subject RIV: BO - Biophysics Impact factor: 14.739, year: 2011

  2. Effects of long-term inhibition of neuronal nitric oxide synthase on blood pressure and renin release

    DEFF Research Database (Denmark)

    Ollerstam, A.; Skøtt, O.; Ek, J.

    2001-01-01

    Nitric oxide (NO) produced by neuronal NO-synthase (nNOS) in macula densa cells may be involved in the control of renin release. 7-Nitro indazole (7-NI) inhibits nNOS, and we investigated the effect of short- (4 days) and long-term (4 weeks) 7-NI treatment on blood pressure (BP), plasma renin...

  3. Expression of inducible nitric oxide synthase in endotoxemic rat hepatocytes is dependent on the cellular glutathione status

    NARCIS (Netherlands)

    Vos, TA; van Goor, H; Tuyt, L; de Jager-Krikken, A; Leuvenink, R; Kuipers, F; Jansen, PLM; Moshage, H

    The inducible nitric oxide synthase (iNOS) promoter contains nuclear factor kappa B (NF-kappa B) binding sites. NF-kappa B activation is determined, in part, by the intracellular redox status, The aim of this study was to determine the importance of the cellular glutathione status in relation to

  4. Neuronal nitric oxide synthase is dislocated in type I fibers of myalgic muscle but can recover with physical exercise training

    DEFF Research Database (Denmark)

    Jensen, L; Andersen, L L; Schrøder, H D

    2015-01-01

    Trapezius myalgia is the most common type of chronic neck pain. While physical exercise reduces pain and improves muscle function, the underlying mechanisms remain unclear. Nitric oxide (NO) signaling is important in modulating cellular function, and a dysfunctional neuronal NO synthase (nNOS) may...

  5. The Regulation of Nitric Oxide Synthase Isoform Expression in Mouse and Human Fallopian Tubes: Potential Insights for Ectopic Pregnancy

    Directory of Open Access Journals (Sweden)

    Junting Hu

    2014-12-01

    Full Text Available Nitric oxide (NO is highly unstable and has a half-life of seconds in buffer solutions. It is synthesized by NO-synthase (NOS, which has been found to exist in the following three isoforms: neuro nitric oxide synthase (nNOS, inducible nitric oxide synthase (iNOS, and endothelial nitric oxide synthase (eNOS. NOS activity is localized in the reproductive tracts of many species, although direct evidence for NOS isoforms in the Fallopian tubes of mice is still lacking. In the present study, we investigated the expression and regulation of NOS isoforms in the mouse and human Fallopian tubes during the estrous and menstrual cycles, respectively. We also measured isoform expression in humans with ectopic pregnancy and in mice treated with lipopolysaccharide (LPS. Our results confirmed the presence of different NOS isoforms in the mouse and human Fallopian tubes during different stages of the estrous and menstrual cycles and showed that iNOS expression increased in the Fallopian tubes of women with ectopic pregnancy and in LPS-treated mice. Elevated iNOS activity might influence ovulation, cilia beats, contractility, and embryo transportation in such a manner as to increase the risk of ectopic pregnancy. This study has provided morphological and molecular evidence that NOS isoforms are present and active in the human and mouse Fallopian tubes and suggests that iNOS might play an important role in both the reproductive cycle and infection-induced ectopic pregnancies.

  6. Macrophages in lung tissue from patients with pulmonary emphysema express both inducible and endothelial nitric oxide synthase

    NARCIS (Netherlands)

    van Straaten, JFM; Postma, DS; Coers, W; Noordhoek, JA; Kauffman, HF; Timens, W

    To provide information concerning a possible biologic role of nitric oxide (NO) in smoking-related emphysema, we performed immunohistochemical studies in lung tissue from control subjects and patients with mild and severe emphysema We studied the presence of inducible and endothelial NO synthases

  7. The expression and activity of nitric oxide synthase in brainstem and hypothalamic nuclei of adult SHR: Endothelial vs. neuronal isoforms

    Czech Academy of Sciences Publication Activity Database

    Hojná, Silvie; Zicha, Josef; Kuneš, Jaroslav

    2006-01-01

    Roč. 24, č. S4 (2006), S345-S346 ISSN 0263-6352. [European Meeting on Hypertension /16./. 12.06.2006-15.06.2006, Madrid] R&D Projects: GA MZd(CZ) NR7786 Keywords : nitric oxide synthase * brain * hypertension Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery

  8. Enhanced growth and improved vascular function in offspring from successive pregnancies in endothelial nitric oxide synthase knockout mice

    NARCIS (Netherlands)

    Longo, M; Jain, [No Value; Langenveld, J; Vedernikov, YP; Garfield, RE; Hankins, GDV; Anderson, GD; Saade, GR

    2004-01-01

    Objective: Transgenic mice that lack endothelial nitric oxide synthase have offspring with growth deficiency and abnormal vascular reactivity in later life. Our objective was to evaluate the role of parity in the modulation of the fetal programming of growth and vascular responses in these

  9. Inhibition of Nitric Oxide Synthase 1 Induces Salt-Sensitive Hypertension in Nitric Oxide Synthase 1α Knockout and Wild-Type Mice.

    Science.gov (United States)

    Wang, Ximing; Chandrashekar, Kiran; Wang, Lei; Lai, En Yin; Wei, Jin; Zhang, Gensheng; Wang, Shaohui; Zhang, Jie; Juncos, Luis A; Liu, Ruisheng

    2016-04-01

    We recently showed that α, β, and γ splice variants of neuronal nitric oxide synthase (NOS1) expressed in the macula densa and NOS1β accounts for most of the NO generation. We have also demonstrated that the mice with deletion of NOS1 specifically from the macula densa developed salt-sensitive hypertension. However, the global NOS1 knockout (NOS1KO) strain is neither hypertensive nor salt sensitive. This global NOS1KO strain is actually an NOS1αKO model. Consequently, we hypothesized that inhibition of NOS1β in NOS1αKO mice induces salt-sensitive hypertension. NOS1αKO and C57BL/6 wild-type (WT) mice were implanted with telemetry transmitters and divided into 7-nitroindazole (10 mg/kg/d)-treated and nontreated groups. All of the mice were fed a normal salt (0.4% NaCl) diet for 5 days, followed by a high-salt diet (4% NaCl). NO generation by the macula densa was inhibited by >90% in WT and NOS1αKO mice treated with 7-nitroindazole. Glomerular filtration rate in conscious mice was increased by ≈ 40% after a high-salt diet in both NOS1αKO and WT mice. In response to acute volume expansion, glomerular filtration rate, diuretic and natriuretic response were significantly blunted in the WT and knockout mice treated with 7-nitroindazole. Mean arterial pressure had no significant changes in mice fed a high-salt diet, but increased ≈ 15 mm Hg similarly in NOS1αKO and WT mice treated with 7-nitroindazole. We conclude that NOS1β, but not NOS1α, plays an important role in control of sodium excretion and hemodynamics in response to either an acute or a chronic salt loading. © 2016 American Heart Association, Inc.

  10. Vasorelaxing Activity of Ulmus davidiana Ethanol Extracts in Rats: Activation of Endothelial Nitric Oxide Synthase

    Science.gov (United States)

    Cho, Eun Jung; Park, Myoung Soo; Kim, Sahng Seop; Kang, Gun; Choi, Sunga; Lee, Yoo Rhan; Chang, Seok Jong; Lee, Kwon Ho; Lee, Sang Do; Park, Jin Bong

    2011-01-01

    Ulmus davidiana var. japonica Rehder (Urticales: Ulmaceae) (UD) is a tree widespread in northeast Asia. It is traditionally used for anticancer and anti-inflammatory therapy. The present study investigated the effect of an ethanol extract of UD on vascular tension and its underlying mechanism in rats. The dried root bark of UD was ground and extracted with 80% ethanol. The prepared UD extract was used in further analysis. The effect of UD on the cell viability, vasoreactivity and hemodynamics were investigated using propidium iodide staining in cultured cells, isometric tension recording and blood pressure analysis, respectively. Low dose of UD (10~100µg/ml) did not affect endothelial cell viability, but high dose of UD reduced cell viability. UD induced vasorelaxation in the range of 0.1~10µg/ml with an ED50 value of 2µg/ml. UD-induced vasorelaxation was completely abolished by removal of the endothelium or by pre-treatment with L-NAME, an inhibitor of nitric oxide synthase. UD inhibited calcium influx induced by phenylephrine and high K+ and also completely abolished the effect of L-NAME. Intravenous injection of UD extracts (10~100 mg/kg) decreased arterial and ventricular pressure in a dose-dependent manner. Moreover, UD extracts reduced the ventricular contractility (+dP/dt) in anesthetized rats. However, UD-induced hypotensive actions were minimized in L-NAME-treated rats. Taken together, out results showed that UD induced vasorelaxation and has antihypertensive properties, which may be due the activation of nitric oxide synthase in endothelium. PMID:22359471

  11. Chlorine gas exposure causes systemic endothelial dysfunction by inhibiting endothelial nitric oxide synthase-dependent signaling.

    Science.gov (United States)

    Honavar, Jaideep; Samal, Andrey A; Bradley, Kelley M; Brandon, Angela; Balanay, Joann; Squadrito, Giuseppe L; MohanKumar, Krishnan; Maheshwari, Akhil; Postlethwait, Edward M; Matalon, Sadis; Patel, Rakesh P

    2011-08-01

    Chlorine gas (Cl(2)) exposure during accidents or in the military setting results primarily in injury to the lungs. However, the potential for Cl(2) exposure to promote injury to the systemic vasculature leading to compromised vascular function has not been studied. We hypothesized that Cl(2) promotes extrapulmonary endothelial dysfunction characterized by a loss of endothelial nitric oxide synthase (eNOS)-derived signaling. Male Sprague Dawley rats were exposed to Cl(2) for 30 minutes, and eNOS-dependent vasodilation of aorta as a function of Cl(2) dose (0-400 ppm) and time after exposure (0-48 h) were determined. Exposure to Cl(2) (250-400 ppm) significantly inhibited eNOS-dependent vasodilation (stimulated by acetycholine) at 24 to 48 hours after exposure without affecting constriction responses to phenylephrine or vasodilation responses to an NO donor, suggesting decreased NO formation. Consistent with this hypothesis, eNOS protein expression was significantly decreased (∼ 60%) in aorta isolated from Cl(2)-exposed versus air-exposed rats. Moreover, inducible nitric oxide synthase (iNOS) mRNA was up-regulated in circulating leukocytes and aorta isolated 24 hours after Cl(2) exposure, suggesting stimulation of inflammation in the systemic vasculature. Despite decreased eNOS expression and activity, no changes in mean arterial blood pressure were observed. However, injection of 1400W, a selective inhibitor of iNOS, increased mean arterial blood pressure only in Cl(2)-exposed animals, suggesting that iNOS-derived NO compensates for decreased eNOS-derived NO. These results highlight the potential for Cl(2) exposure to promote postexposure systemic endothelial dysfunction via disruption of vascular NO homeostasis mechanisms.

  12. The nitrogen oxide synthase effect in endothelium reaction and change in peripheral perfusion under THz radiation of nitrogen oxide occurrence exposure

    OpenAIRE

    Kirichuk, Vyacheslav; Andronov, Evgeny

    2013-01-01

    There is new information about the mechanism of THz radiation of molecular emission and absorption spectrum (MEAS) of 150.176-150.664 GHz nitrogen oxide occurrence exposure. Organized experiments let us establish the fact that a normalizing effect of THz radiation on microvasculature perfusion and functional endothelium condition in male rats under the acute stress condition is not realized after using of nitrogen oxide synthase (NOS) L-NAME. This shows that endogenic nitrogen oxide and endot...

  13. Arginine-based inhibitors of nitric oxide synthase: therapeutic potential and challenges.

    Science.gov (United States)

    Víteček, Jan; Lojek, Antonín; Valacchi, Giuseppe; Kubala, Lukáš

    2012-01-01

    In the past three decades, nitric oxide has been well established as an important bioactive molecule implicated in regulation of cardiovascular, nervous, and immune systems. Therefore, it is not surprising that much effort has been made to find specific inhibitors of nitric oxide synthases (NOS), the enzymes responsible for production of nitric oxide. Among the many NOS inhibitors developed to date, inhibitors based on derivatives and analogues of arginine are of special interest, as this category includes a relatively high number of compounds with good potential for experimental as well as clinical application. Though this group of inhibitors covers early nonspecific compounds, modern drug design strategies such as biochemical screening and computer-aided drug design have provided NOS-isoform-specific inhibitors. With an emphasis on major advances in this field, a comprehensive list of inhibitors based on their structural characteristics is discussed in this paper. We provide a summary of their biochemical properties as well as their observed effects both in vitro and in vivo. Furthermore, we focus in particular on their pharmacology and use in recent clinical studies. The potential of newly designed specific NOS inhibitors developed by means of modern drug development strategies is highlighted.

  14. Arginine-Based Inhibitors of Nitric Oxide Synthase: Therapeutic Potential and Challenges

    Directory of Open Access Journals (Sweden)

    Jan Víteček

    2012-01-01

    Full Text Available In the past three decades, nitric oxide has been well established as an important bioactive molecule implicated in regulation of cardiovascular, nervous, and immune systems. Therefore, it is not surprising that much effort has been made to find specific inhibitors of nitric oxide synthases (NOS, the enzymes responsible for production of nitric oxide. Among the many NOS inhibitors developed to date, inhibitors based on derivatives and analogues of arginine are of special interest, as this category includes a relatively high number of compounds with good potential for experimental as well as clinical application. Though this group of inhibitors covers early nonspecific compounds, modern drug design strategies such as biochemical screening and computer-aided drug design have provided NOS-isoform-specific inhibitors. With an emphasis on major advances in this field, a comprehensive list of inhibitors based on their structural characteristics is discussed in this paper. We provide a summary of their biochemical properties as well as their observed effects both in vitro and in vivo. Furthermore, we focus in particular on their pharmacology and use in recent clinical studies. The potential of newly designed specific NOS inhibitors developed by means of modern drug development strategies is highlighted.

  15. Nitric Oxide Synthase Type III Overexpression By Gene Therapy Exerts Antitumoral Activity In Mouse Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Raúl González

    2015-08-01

    Full Text Available Hepatocellular carcinoma develops in cirrhotic liver. The nitric oxide (NO synthase type III (NOS-3 overexpression induces cell death in hepatoma cells. The study developed gene therapy designed to specifically overexpress NOS-3 in cultured hepatoma cells, and in tumors derived from orthotopically implanted tumor cells in fibrotic livers. Liver fibrosis was induced by CCl4 administration in mice. Hepa 1-6 cells were used for in vitro and in vivo experiments. The first generation adenovirus was designed to overexpress NOS-3 (or GFP and luciferase cDNA under the regulation of murine alpha-fetoprotein (AFP and Rous Sarcoma Virus (RSV promoters, respectively. Both adenoviruses were administered through the tail vein two weeks after orthotopic tumor cell implantation. AFP-NOS-3/RSV-Luciferase increased oxidative-related DNA damage, p53, CD95/CD95L expression and caspase-8 activity in cultured Hepa 1-6 cells. The increased expression of CD95/CD95L and caspase-8 activity was abolished by l-NAME or p53 siRNA. The tail vein infusion of AFP-NOS- 3/RSV-Luciferase adenovirus increased cell death markers, and reduced cell proliferation of established tumors in fibrotic livers. The increase of oxidative/nitrosative stress induced by NOS-3 overexpression induced DNA damage, p53, CD95/CD95L expression and cell death in hepatocellular carcinoma cells. The effectiveness of the gene therapy has been demonstrated in vitro and in vivo.

  16. Role of inducible nitric oxide synthase pathway on methotrexate-induced intestinal mucositis in rodents

    Directory of Open Access Journals (Sweden)

    Siqueira Francisco JWS

    2011-08-01

    Full Text Available Abstract Background Methotrexate treatment has been associated to intestinal epithelial damage. Studies have suggested an important role of nitric oxide in such injury. The aim of this study was to investigate the role of nitric oxide (NO, specifically iNOS on the pathogenesis of methotrexate (MTX-induced intestinal mucositis. Methods Intestinal mucositis was carried out by three subcutaneous MTX injections (2.5 mg/kg in Wistar rats and in inducible nitric oxide synthase knock-out (iNOS-/- and wild-type (iNOS+/+ mice. Rats were treated intraperitoneally with the NOS inhibitors aminoguanidine (AG; 10 mg/Kg or L-NAME (20 mg/Kg, one hour before MTX injection and daily until sacrifice, on the fifth day. The jejunum was harvested to investigate the expression of Ki67, iNOS and nitrotyrosine by immunohistochemistry and cell death by TUNEL. The neutrophil activity by myeloperoxidase (MPO assay was performed in the three small intestine segments. Results AG and L-NAME significantly reduced villus and crypt damages, inflammatory alterations, cell death, MPO activity, and nitrotyrosine immunostaining due to MTX challenge. The treatment with AG, but not L-NAME, prevented the inhibitory effect of MTX on cell proliferation. MTX induced increased expression of iNOS detected by immunohistochemistry. MTX did not cause significant inflammation in the iNOS-/- mice. Conclusion These results suggest an important role of NO, via activation of iNOS, in the pathogenesis of intestinal mucositis.

  17. Molecular cloning and expression pattern of oriental river prawn (Macrobrachium nipponense) nitric oxide synthase.

    Science.gov (United States)

    Rahman, N M A; Fu, H T; Sun, S M; Qiao, H; Jin, S; Bai, H K; Zhang, W Y; Liang, G X; Gong, Y S; Xiong, Y W; Wu, Y

    2016-08-29

    Nitric oxide synthase (NOS) produces nitric oxide (NO) by catalyzing the conversion of l-arginine to l-citrulline, with the concomitant oxidation of nicotinamide adenine dinucleotide phosphate. Recently, various studies have verified the importance of NOS invertebrates and invertebrates. However, the NOS gene family in the oriental river prawn Macrobrachium nipponense is poorly understood. In this study, we cloned the full-length NOS complementary DNA from M. nipponense (MnNOS) and characterized its expression pattern in different tissues and at different developmental stages. Real-time quantitative polymerase chain reaction (RT-qPCR) showed the MnNOS gene to be expressed in all investigated tissues, with the highest levels observed in the androgenic gland (P < 0.05). Our results revealed that the MnNOS gene may play a key role in M. nipponense male sexual differentiation. Moreover, RT-qPCR revealed that MnNOS mRNA expression was significantly increased in post-larvae 10 days after metamorphosis (P < 0.05). The expression of this gene in various tissues indicates that it may perform versatile biological functions in M. nipponense.

  18. Cisplatin upregulates mitochondrial nitric oxide synthase and peroxynitrite formation to promote renal injury

    International Nuclear Information System (INIS)

    Jung, Michaela; Hotter, Georgina; Vinas, Jose Luis; Sola, Anna

    2009-01-01

    The mitochondria are a critical target for cisplatin-associated nephrotoxicity. Though nitric oxide formation has been implicated in the toxicity of cisplatin, this formation has not so far been related to a possible activation of mitochondrial nitric oxide synthase (mNOS). We show here that the upregulation of oxide mNOS and peroxynitrite formation in cisplatin treatment are key events that influence the development of the harmful parameters described in cisplatin-associated kidney failure. We confirm this by isolating the mitochondrial fraction of the kidney and across different access routes such as the use of a specific inhibitor of neuronal NOS, L-NPA, a peroxynitrite scavenger, FeTMPyP, and a peroxynitrite donor, SIN-1. The in vitro studies corroborated the information obtained in the in vivo experiments. The administration of cisplatin reveals a clear upregulation in the transcription of neuronal NOS and an increase in the levels of nitrites in the mitochondrial fractions of the kidneys. The upregulated transcription directly affects the cytoskeleton structure and the apoptosis. The inhibition of neuronal NOS reduces the levels of nitrites, cell death, and cytoskeleton derangement. Peroxynitrite is involved in the mechanism promoting the NOS transcription. In addition, in controls SIN-1 imitates the effects of cisplatin. In summary, we demonstrate that upregulation of mNOS in cisplatin treatment is a key component in both the initiation and the spread of cisplatin-associated damage in the kidney. Furthermore, peroxynitrite formation is directly involved in this process

  19. Differential contributions of nitric oxide synthase isoforms at hippocampal formation to negative feedback regulation of penile erection in the rat

    OpenAIRE

    Chang, Alice Y W; Chan, Julie Y H; Chan, Samuel H H

    2002-01-01

    We established previously that a novel negative feedback mechanism for the regulation of penile erection, which is triggered by ascending sensory inputs initiated by tumescence of the penis, exists in the hippocampal formation (HF). This study further evaluated the participation of nitric oxide (NO) and the contribution of nitric oxide synthase (NOS) isoforms at the HF in this process.Adult, male Sprague-Dawley rats that were anaesthetized and maintained with chloral hydrate were used, and in...

  20. Inhibition of inducible Nitric Oxide Synthase by a mustard gas analog in murine macrophages

    Directory of Open Access Journals (Sweden)

    Smith Milton

    2006-11-01

    Full Text Available Abstract Background 2-Chloroethyl ethyl sulphide (CEES is a sulphur vesicating agent and an analogue of the chemical warfare agent 2,2'-dichlorodiethyl sulphide, or sulphur mustard gas (HD. Both CEES and HD are alkylating agents that influence cellular thiols and are highly toxic. In a previous publication, we reported that lipopolysaccharide (LPS enhances the cytotoxicity of CEES in murine RAW264.7 macrophages. In the present investigation, we studied the influence of CEES on nitric oxide (NO production in LPS stimulated RAW264.7 cells since NO signalling affects inflammation, cell death, and wound healing. Murine macrophages stimulated with LPS produce NO almost exclusively via inducible nitric oxide synthase (iNOS activity. We suggest that the influence of CEES or HD on the cellular production of NO could play an important role in the pathophysiological responses of tissues to these toxicants. In particular, it is known that macrophage generated NO synthesised by iNOS plays a critical role in wound healing. Results We initially confirmed that in LPS stimulated RAW264.7 macrophages NO is exclusively generated by the iNOS form of nitric oxide synthase. CEES treatment inhibited the synthesis of NO (after 24 hours in viable LPS-stimulated RAW264.7 macrophages as measured by either nitrite secretion into the culture medium or the intracellular conversion of 4,5-diaminofluorescein diacetate (DAF-2DA or dichlorofluorescin diacetate (DCFH-DA. Western blots showed that CEES transiently decreased the expression of iNOS protein; however, treatment of active iNOS with CEES in vitro did not inhibit its enzymatic activity Conclusion CEES inhibits NO production in LPS stimulated macrophages by decreasing iNOS protein expression. Decreased iNOS expression is likely the result of CEES induced alteration in the nuclear factor kappa B (NF-κB signalling pathway. Since NO can act as an antioxidant, the CEES induced down-regulation of iNOS in LPS

  1. Phenotype of transgenic mice overexpressed with inducible nitric oxide synthase in the retina.

    Directory of Open Access Journals (Sweden)

    Guey Shuang Wu

    Full Text Available Unlike its constitutive isoforms, including neuronal and endothelial nitric oxide synthase, inducible nitric oxide synthase (iNOS along with a series of cytokines are generated in inflammatory pathologic conditions in retinal photoreceptors. In this study, we constructed transgenic mice overexpressing iNOS in the retina to evaluate the effect of sustained, intense iNOS generation in the photoreceptor damage.For construction of opsin/iNOS transgene in the CMVSport 6 expression vector, the 4.4 kb Acc65I/Xhol mouse rod opsin promoter was ligated upstream to a 4.1 kb fragment encoding the complete mouse cDNA of iNOS. From the four founders identified, two heterozygote lines and one homozygote line were established. The presence of iNOS in the retina was confirmed and the pathologic role of iNOS was assessed by detecting nitrotyrosine products and apoptosis. Commercial TUNEL kit was used to detect DNA strand breaks, a later step in a sequence of morphologic changes of apoptosis process.The insertion and translation of iNOS gene were demonstrated by an intense single 130 kDa band in Western blot and specific immunolocalization at the photoreceptors of the retina. Cellular toxicity in the retinas of transgenic animals was detected by a post-translational modification product, tyrosine-nitrated protein, the most significant one of which was nitrated cytochrome c. Following the accumulation of nitrated mitochondrial proteins and cytochrome c release, marked apoptosis was detected in the photoreceptor cell nuclei of the retina.We have generated a pathologic phenotype with sustained iNOS overexpression and, therefore, high output of nitric oxide. Under basal conditions, such overexpression of iNOS causes marked mitochondrial cytochrome c nitration and release and subsequent photoreceptor apoptosis in the retina. Therefore, the modulation of pathways leading to iNOS generation or its effective neutralization can be of significant therapeutic benefit in the

  2. Askey, Prof. Richard Allen

    Indian Academy of Sciences (India)

    Fellow Profile. Elected: 1988 Honorary. Askey, Prof. Richard Allen. Date of birth: 4 June 1933. Address: 5915, American Parkway, Apt. 228, Madison, WI 43718, U.S.A.. Contact: Office: (+1-608) 262 2880. Residence: (+1-608) 233 7900. Fax: (+1-608) 263 8891. Email: askey@math.wisc.edu. YouTube; Twitter; Facebook ...

  3. Flavone inhibits nitric oxide synthase (NOS) activity, nitric oxide production and protein S-nitrosylation in breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Wenzhen; Yang, Bingwu; Fu, Huiling; Ma, Long; Liu, Tingting; Chai, Rongfei; Zheng, Zhaodi [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China); Zhang, Qunye, E-mail: wz.zhangqy@sdu.edu.cn [Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Ministry of Public Health, Qilu Hospital, Shandong University, Jinan, Shandong (China); Li, Guorong, E-mail: grli@sdnu.edu.cn [Shandong Provincial Key Laboratory of Animal Resistant Biology, School of Life Sciences, Shandong Normal University, Jinan 250014 (China)

    2015-03-13

    As the core structure of flavonoids, flavone has been proved to possess anticancer effects. Flavone's growth inhibitory functions are related to NO. NO is synthesized by nitric oxide synthase (NOS), and generally increased in a variety of cancer cells. NO regulates multiple cellular responses by S-nitrosylation. In this study, we explored flavone-induced regulations on nitric oxide (NO)-related cellular processes in breast cancer cells. Our results showed that, flavone suppresses breast cancer cell proliferation and induces apoptosis. Flavone restrains NO synthesis by does-dependent inhibiting NOS enzymatic activity. The decrease of NO generation was detected by fluorescence microscopy and flow cytometry. Flavone-induced inhibitory effect on NOS activity is dependent on intact cell structure. For the NO-induced protein modification, flavone treatment significantly down-regulated protein S-nitrosylation, which was detected by “Biotin-switch” method. The present study provides a novel, NO-related mechanism for the anticancer function of flavone. - Highlights: • Flavone inhibits proliferation and induces apoptosis in MCF-7 cells. • Flavone decreases nitric oxide production by inhibiting NOS enzymatic activity in breast cancer cells. • Flavone down-regulates protein S-nitrosylation.

  4. Bacterial Nitric Oxide Synthase Is Required for the Staphylococcus aureus Response to Heme Stress.

    Science.gov (United States)

    Surdel, Matthew C; Dutter, Brendan F; Sulikowski, Gary A; Skaar, Eric P

    2016-08-12

    Staphylococcus aureus is a pathogen that causes significant morbidity and mortality worldwide. Within the vertebrate host, S. aureus requires heme as a nutrient iron source and as a cofactor for multiple cellular processes. Although required for pathogenesis, excess heme is toxic. S. aureus employs a two-component system, the heme sensor system (HssRS), to sense and protect against heme toxicity. Upon activation, HssRS induces the expression of the heme-regulated transporter (HrtAB), an efflux pump that alleviates heme toxicity. The ability to sense and respond to heme is critical for the pathogenesis of numerous Gram-positive organisms, yet the mechanism of heme sensing remains unknown. Compound '3981 was identified in a high-throughput screen as an activator of staphylococcal HssRS that triggers HssRS independently of heme accumulation. '3981 is toxic to S. aureus; however, derivatives of '3981 were synthesized that lack toxicity while retaining HssRS activation, enabling the interrogation of the heme stress response without confounding toxic effects of the parent molecule. Using '3981 derivatives as probes of the heme stress response, numerous genes required for '3981-induced activation of HssRS were uncovered. Specifically, multiple genes involved in the production of nitric oxide were identified, including the gene encoding bacterial nitric oxide synthase (bNOS). bNOS protects S. aureus from oxidative stress imposed by heme. Taken together, this work identifies bNOS as crucial for the S. aureus heme stress response, providing evidence that nitric oxide synthesis and heme sensing are intertwined.

  5. The Peptidylarginine Deiminase Inhibitor Cl-Amidine Suppresses Inducible Nitric Oxide Synthase Expression in Dendritic Cells

    Directory of Open Access Journals (Sweden)

    Byungki Jang

    2017-10-01

    Full Text Available The conversion of peptidylarginine into peptidylcitrulline by calcium-dependent peptidylarginine deiminases (PADs has been implicated in the pathogenesis of a number of diseases, identifying PADs as therapeutic targets for various diseases. The PAD inhibitor Cl-amidine ameliorates the disease course, severity, and clinical manifestation in multiple disease models, and it also modulates dendritic cell (DC functions such as cytokine production, antigen presentation, and T cell proliferation. The beneficial effects of Cl-amidine make it an attractive compound for PAD-targeting therapeutic strategies in inflammatory diseases. Here, we found that Cl-amidine inhibited nitric oxide (NO generation in a time- and dose-dependent manner in maturing DCs activated by lipopolysaccharide (LPS. This suppression of NO generation was independent of changes in NO synthase (NOS enzyme activity levels but was instead dependent on changes in inducible NO synthase (iNOS transcription and expression levels. Several upstream signaling pathways for iNOS expression, including the mitogen-activated protein kinase, nuclear factor-κB p65 (NF-κB p65, and hypoxia-inducible factor 1 pathways, were not affected by Cl-amidine. By contrast, the LPS-induced signal transducer and the activator of transcription (STAT phosphorylation and activator protein-1 (AP-1 transcriptional activities (c-Fos, JunD, and phosphorylated c-Jun were decreased in Cl-amidine-treated DCs. Inhibition of Janus kinase/STAT signaling dramatically suppressed iNOS expression and NO production, whereas AP-1 inhibition had no effect. These results indicate that Cl-amidine-inhibited STAT activation may suppress iNOS expression. Additionally, we found mildly reduced cyclooxygenase-2 expression and prostaglandin E2 production in Cl-amidine-treated DCs. Our findings indicate that Cl-amidine acts as a novel suppressor of iNOS expression, suggesting that Cl-amidine has the potential to ameliorate the effects of

  6. Lycopene inhibits LPS-induced proinflammatory mediator inducible nitric oxide synthase in mouse macrophage cells.

    Science.gov (United States)

    Rafi, Mohamed M; Yadav, Prem Narayan; Reyes, Marynell

    2007-01-01

    Lycopene is a fat-soluble red-orange carotenoid found primarily in tomatoes and tomato-derived products, including tomato sauce, tomato paste, and ketchup, and other dietary sources, including dried apricots, guava, watermelon, papaya, and pink grapefruit. In this study, we have demonstrated the molecular mechanism underlying the anti-inflammatory properties of lycopene using a mouse macrophage cell line (RAW 264.7). Treatment with lycopene (10 microM) inhibited lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production (40% compared with the control). Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that lycopene treatment decreased LPS-induced inducible nitric oxide synthase (iNOS) protein and mRNA expression in RAW 264.7 cells, respectively. These results suggest that lycopene has anti-inflammatory activity by inhibiting iNOS proteins and mRNA expressions in mouse macrophage cell lines. Furthermore, cyclooxygenase-2 (COX-2) protein and mRNA expression were not affected by treatment with lycopene.

  7. Activated astrocytes induce nitric oxide synthase-2 in cerebral endothelium via tumor necrosis factor alpha.

    Science.gov (United States)

    Shafer, R A; Murphy, S

    1997-12-01

    Astrocytes under pathological conditions become activated and produce a variety of cytokines and low molecular weight signal molecules. Previously we demonstrated that activated astrocytes release nitric oxide which can downregulate the expression of nitric oxide synthase (NOS)-2 in co-cultured cerebral endothelium, and also release a transcriptionally regulated factor that can induce NOS-2 expression in endothelium (Borgerding and Murphy: J Neurochem 65:1342, 1995). The activity of this NOS-2-inducing factor was impeded by inhibitors of tyrosine kinases and NF-kappaB activation. Tumor necrosis factor (TNF alpha) alone, or in combination with IL-6, induced NOS-2 expression in endothelial cells. A neutralizing antibody against TNF alpha attenuated the NOS-2 expression in endothelial cells exposed to activated astrocytes. These results imply that cytokine-activated astrocytes release TNF alpha which can induce NOS-2 expression in endothelium and suggest that activated astrocytes within the CNS may induce expression of NOS-2 in cells of the adjacent microvasculature. The ensuing alterations in blood-brain barrier properties may be either beneficial or detrimental.

  8. Characterisation of nitric oxide synthase in three cnidarian-dinoflagellate symbioses.

    Directory of Open Access Journals (Sweden)

    Helena Safavi-Hemami

    Full Text Available BACKGROUND: Nitric oxide synthase (NOS is an enzyme catalysing the conversion of L-arginine to L-citrulline and nitric oxide (NO, the latter being an essential messenger molecule for a range of biological processes. Whilst its role in higher vertebrates is well understood little is known about the role of this enzyme in early metazoan groups. For instance, NOS-mediated signalling has been associated with Cnidaria-algal symbioses, however controversy remains about the contribution of enzyme activities by the individual partners of these mutualistic relationships. METHODOLOGY/PRINCIPAL FINDINGS: Using a modified citrulline assay we successfully measured NOS activity in three cnidarian-algal symbioses: the sea anemone Aiptasia pallida, the hard coral Acropora millepora, and the soft coral Lobophytum pauciflorum, so demonstrating a wide distribution of this enzyme in the phylum Cnidaria. Further biochemical (citrulline assay and histochemical (NADPH-diaphorase investigations of NOS in the host tissue of L. pauciflorum revealed the cytosolic and calcium dependent nature of this enzyme and its in situ localisation within the coral's gastrodermal tissue, the innermost layer of the body wall bearing the symbiotic algae. Interestingly, enzyme activity could not be detected in symbionts freshly isolated from the cnidarians, or in cultured algal symbionts. CONCLUSIONS/SIGNIFICANCE: These results suggest that NOS-mediated NO release may be host-derived, a finding that has the potential to further refine our understanding of signalling events in cnidarian-algal symbioses.

  9. Interaction between Mitochondrial Reactive Oxygen Species, Heme Oxygenase, and Nitric Oxide Synthase Stimulates Phagocytosis in Macrophages

    Directory of Open Access Journals (Sweden)

    Andrea Müllebner

    2018-01-01

    Full Text Available BackgroundMacrophages are cells of the innate immune system that populate every organ. They are required not only for defense against invading pathogens and tissue repair but also for maintenance of tissue homeostasis and iron homeostasis.AimThe aim of this study is to understand whether heme oxygenase (HO and nitric oxide synthase (NOS contribute to the regulation of nicotinamide adenine dinucleotide phosphate oxidase (NOX activity and phagocytosis, two key components of macrophage function.MethodsThis study was carried out using resting J774A.1 macrophages treated with hemin or vehicle. Activity of NOS, HO, or NOX was inhibited using specific inhibitors. Reactive oxygen species (ROS formation was determined by Amplex® red assay, and phagocytosis was measured using fluorescein isothiocyanate-labeled bacteria. In addition, we analyzed the fate of the intracellular heme by using electron spin resonance.ResultsWe show that both enzymes NOS and HO are essential for phagocytic activity of macrophages. NOS does not directly affect phagocytosis, but stimulates NOX activity via nitric oxide-triggered ROS production of mitochondria. Treatment of macrophages with hemin results in intracellular accumulation of ferrous heme and an inhibition of phagocytosis. In contrast to NOS, HO products, including carbon monoxide, neither clearly affect NOX activity nor clearly affect phagocytosis, but phagocytosis is accelerated by HO-mediated degradation of heme.ConclusionBoth enzymes contribute to the bactericidal activity of macrophages independently, by controlling different pathways.

  10. Neuronal nitric oxide synthase-deficient mice have impaired Renin release but normal blood pressure

    DEFF Research Database (Denmark)

    Sällström, Johan; Carlström, Mattias; Jensen, Boye L

    2008-01-01

    NOS(-/-)) and wild-type (nNOS(+/+)) mice after 10 days of low (0.01% NaCl) and high (4% NaCl) sodium diets.ResultsThe resting heart rate was reduced in nNOS(-/-) mice, but the two genotypes had similar blood pressure during the low (nNOS(+/+) 104 +/- 2 mm Hg; nNOS(-/-) 103 +/- 2 mm Hg) and high (nNOS(+/+) 107 +/- 3...... for stimulation of renin in response to sodium restriction. Furthermore, nNOS(-/-) mice are normotensive, and their blood pressure responds normally to an increased dietary sodium intake, indicating that nNOS deficiency does not cause salt-sensitive hypertension.American Journal of Hypertension (2008) 21 111......BackgroundNitric oxide deficiency is involved in the development of hypertension, but the mechanisms are currently unclear. This study was conducted to further elucidate the role of neuronal nitric oxide synthase (nNOS) in blood pressure regulation and renin release in relation to different sodium...

  11. Contribution of myeloperoxidase and inducible nitric oxide synthase to pathogenesis of psoriasis

    Directory of Open Access Journals (Sweden)

    Nursel Dilek

    2016-12-01

    Full Text Available Introduction : Histological changes of psoriasis include invasion of neutrophils into the epidermis and formation of Munro abscesses in the epidermis. Neutrophils are the predominant white blood cells in circulation when stimulated; they discharge the abundant myeloperoxidase (MPO enzyme that uses hydrogen peroxide to oxidize chloride for killing ingested bacteria. Aim: To investigate the contribution of neutrophils to the pathogenesis of psoriasis at the blood and tissue levels through inducible nitric oxide synthase (iNOS and MPO. Material and methods: A total of 50 adult patients with a chronic plaque form of psoriasis and 25 healthy controls were enrolled to this study. Serum MPO and iNOS levels were measured using ELISA method. Two biopsy specimens were taken in each patient from the center of the lesion and uninvolved skin. Immunohistochemistry was performed for MPO and iNOS on both normal and psoriasis vulgaris biopsies. Results: While a significant difference between serum myeloperoxidase levels were detected, a similar statistical difference between participants in the serum iNOS levels was not found. In immunohistochemistry, intensely stained leukocytes with MPO and intensely staining with iNOS in psoriatic skin was observed. Conclusions : Neutrophils in psoriasis lesions are actively producing MPO and this indirectly triggers the synthesis of iNOS. Targeting of MPO or synthesis of MPO in the lesion area may contribute to development of a new treatment option.

  12. Extracts of Magnoliae flos inhibit inducible nitric oxide synthase via ERK in human respiratory epithelial cells.

    Science.gov (United States)

    Baek, Jin Ah; Lee, Yang Deok; Lee, Chan Bog; Go, Hyeon Kyu; Kim, Jin Pyo; Seo, Jeong Ju; Rhee, Yang Keun; Kim, A Mi; Na, Dong Jib

    2009-03-01

    Nitric oxide (NO) is a marker of pulmonary inflammation. In asthma, the levels of exhaled NO are elevated and the source of this increased NO is inducible nitric oxide synthase (iNOS) within airway epithelial cells. Epimagnolin and fargesin are compounds isolated from the ethanol extract of Magnoliae flos, the seed of the Magnolia plant and are used to treat nasal congestion, headache and sinusitis in Asian countries. This study investigated whether epimagnolin and fargesin inhibit extracellular signal-regulated kinase (ERK) activation and decrease iNOS expression and NO production in stimulated human respiratory epithelial cells. An immortal Type II alveolar cell line of human origin (A549) was stimulated by cytomix (CM), composed of IL-1beta, TNF-alpha and IFN-gamma, with or without concurrent exposure to M. flos extract (epimagnolin or fargesin). CM-induced levels of NO production, iNOS expression and ERK activation were evaluated. A549 cells stimulated with CM showed increases in iNOS mRNA and protein expression, and NO synthesis. However, treatment with epimagnolin or fargesin decreased levels of iNOS mRNA and protein expression, and NO synthesis. CM stimulated a rapid increase in the activity of ERK, whereas epimagnolin and fargesin inhibited ERK phosphorylation. Epimagnolin and fargesin inhibit iNOS expression and decrease production of NO via ERK pathway in cytokine-stimulated human respiratory epithelial cells.

  13. Effect of 7-nitroindazole, a neuronal nitric oxide synthase inhibitor, on behavioral and physiological parameters.

    Science.gov (United States)

    Brožíčková, C; Mikulecká, A; Otáhal, J

    2014-01-01

    The role of brain derived nitric oxide in the physiology and behavior remains disputable. One of the reasons of the controversies might be systemic side effects of nitric oxide synthase inhibitors. Therefore, under nNOS inhibition by 7-nitroindazole (7-NI) we carried out recordings of blood gasses, blood pressure and spontaneous EEG in conscious adult rats. Locomotion and spontaneous behavior were assessed in an open field. In addition skilled walking and limb coordination were evaluated using a ladder rung walking test. The blood gas analysis revealed a significant increase in pCO(2) 180 min and 240 min after the application of 7-NI. The power and entropy decreased simultaneously with a shift of the mean frequency of the spontaneous EEG toward slow oscillations after 7-NI treatment. The thresholds of evoked potentials underwent a significant drop and a trend towards a slight increase in the I-O curve slope was observed. 7-NI significantly suppressed open field behavior expressed as distance moved, exploratory rearing and grooming. As for the ladder rung walking test the 7-NI treated animals had more errors in foot placement indicating impairment in limb coordination. Therefore our findings suggest that 7-NI increased cortical excitability and altered some physiological and behavioral parameters.

  14. Expression of nitric oxide synthases and effects of L-arginine and L-NMMA on nitric oxide production and fluid transport in collagenous colitis

    DEFF Research Database (Denmark)

    Perner, A; Andresen, Lars; Normark, M

    2001-01-01

    Luminal nitric oxide (NO) is greatly increased in the colon of patients with collagenous and ulcerative colitis. To define the source and consequence of enhanced NO production we have studied expression of NO synthase (NOS) isoforms and nitrotyrosine in mucosal biopsies from these patients...

  15. Opposite effect of oxidative stress on inducible nitric oxide synthase and haem oxygenase-1 expression in intestinal inflammation: anti-inflammatory effect of carbon monoxide

    NARCIS (Netherlands)

    Dijkstra, Gerard; Blokzijl, Hans; Bok, Lisette; Homan, Manon; van Goor, Harry; Faber, Klaas Nico; Jansen, Peter L. M.; Moshage, Han

    2004-01-01

    Inducible nitric oxide synthase (iNOS) is expressed in intestinal epithelial cells (IEC) of patients with active inflammatory bowel disease (IBD) and in IEC of endotoxaemic rats. The induction of iNOS in IEC is an element of the NF-kappaB-mediated survival pathway. Haem oxygenase-1 (HO-1) is an

  16. Expression of inducible nitric oxide synthase and effects of L-arginine on colonic nitric oxide production and fluid transport in patients with "minimal colitis"

    DEFF Research Database (Denmark)

    Perner, Anders; Andresen, Lars; Normark, Michel

    2005-01-01

    Some patients with idiopathic, chronic diarrhoea have minimal, non-specific colonic inflammation. As nitric oxide (NO) acts as a secretagogue in the colon, we studied the expression of inducible NO synthase (iNOS) in mucosal biopsies and the effects of NOS stimulation on colonic transfer of fluid...

  17. Expression of nitric oxide synthases and effects of L-arginine and L-NMMA on nitric oxide production and fluid transport in collagenous colitis

    DEFF Research Database (Denmark)

    Perner, A; Andresen, Lars; Normark, M

    2001-01-01

    Luminal nitric oxide (NO) is greatly increased in the colon of patients with collagenous and ulcerative colitis. To define the source and consequence of enhanced NO production we have studied expression of NO synthase (NOS) isoforms and nitrotyrosine in mucosal biopsies from these patients. In ad...

  18. Premotor nitric oxide synthase immunoreactive pathway connecting lumbar segments with the ventral motor nucleus of the cervical enlargement in the dog.

    Science.gov (United States)

    Marsala, Jozef; Lukácová, Nadezda; Cízková, Dása; Lukác, Imrich; Kuchárová, Karolína; Marsala, Martin

    2004-03-01

    In this study we investigate the occurrence and origin of punctate nitric oxide synthase immunoreactivity in the neuropil of the ventral motor nucleus in C7-Th1 segments of the dog spine, which are supposed to be the terminal field of an ascending premotor propriospinal nitric oxide synthase-immunoreactive pathway. As the first step, nitric oxide synthase immunohistochemistry was used to distinguish nitric oxide synthase-immunoreactive staining of the ventral motor nucleus. Dense, punctate nitric oxide synthase immunoreactivity was found on control sections in the neuropil of the ventral motor nucleus. After hemisection at Th10-11, axotomy-induced retrograde changes consisting in a strong upregulation of nitric oxide synthase-containing neurons were found mostly unilaterally in lamina VIII, the medial part of lamina VII and in the pericentral region in all segments of the lumbosacral enlargement. Concurrently, a strong depletion of the punctate nitric oxide synthase immunopositivity in the neuropil of the ventral motor nucleus ipsilaterally with the hemisection was detected, thus revealing that an uncrossed ascending premotor propriospinal pathway containing a fairly high number of nitric oxide synthase-immunoreactive fibers terminates in the ventral motor nucleus. Application of the retrograde fluorescent tracer Fluorogold injected into the ventral motor nucleus and analysis of alternate sections processed for nitric oxide synthase immunocytochemistry revealed the presence of Fluorogold-labeled and nitric oxide synthase-immunoreactive axons in the ventrolateral funiculus and in the lateral and medial portions of the ventral column throughout the thoracic and upper lumbar segments. A noticeable number of Fluorogold-labeled and nitric oxide synthase-immunoreactive somata detected on consecutive sections were found in the lumbosacral enlargement, mainly in laminae VIII-IX, the medial part of lamina VII and in the pericentral region (lamina X), ipsilaterally with the

  19. Oxidized phospholipids induce ceramide accumulation in RAW 264.7 macrophages: role of ceramide synthases.

    Directory of Open Access Journals (Sweden)

    Lingaraju M Halasiddappa

    Full Text Available Oxidized phospholipids (OxPLs, including 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC and 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphocholine (POVPC are among several biologically active derivatives that are generated during oxidation of low-density lipoproteins (LDLs. These OxPLs are factors contributing to pro-atherogenic effects of oxidized LDLs (OxLDLs, including inflammation, proliferation and death of vascular cells. OxLDL also elicits formation of the lipid messenger ceramide (Cer which plays a pivotal role in apoptotic signaling pathways. Here we report that both PGPC and POVPC are cytotoxic to cultured macrophages and induce apoptosis in these cells which is associated with increased cellular ceramide levels after several hours. In addition, exposure of RAW 264.7 cells to POVPC and PGPC under the same conditions resulted in a significant increase in ceramide synthase activity, whereas, acid or neutral sphingomyelinase activities were not affected. PGPC is not only more toxic than POVPC, but also a more potent inducer of ceramide formation by activating a limited subset of CerS isoforms. The stimulated CerS activities are in line with the C16-, C22-, and C24:0-Cer species that are generated under the influence of the OxPL. Fumonisin B1, a specific inhibitor of CerS, suppressed OxPL-induced ceramide generation, demonstrating that OxPL-induced CerS activity in macrophages is responsible for the accumulation of ceramide. OxLDL elicits the same cellular ceramide and CerS effects. Thus, it is concluded that PGPC and POVPC are active components that contribute to the capacity of this lipoprotein to elevate ceramide levels in macrophages.

  20. Role of nitric oxide synthase inhibition in the acute hypertensive response to intracerebroventricular cadmium

    Science.gov (United States)

    Demontis, Maria Piera; Varoni, Maria Vittoria; Volpe, Anna Rita; Emanueli, Costanza; Madeddu, Paolo

    1998-01-01

    In the rat, intracerebroventricular (i.c.v.) injection of cadmium, a pollutant with long biological half-life, causes a sustained increase in blood pressure at doses that are ineffective by peripheral route. Since cadmium inhibits calcium-calmodulin constitutive nitric oxide (NO) synthase in cytosolic preparations of rat brain, this mechanism may be responsible for the acute pressor action of this heavy metal.To test this possibility, we evaluated the effect of i.c.v. injection of 88 nmol cadmium in normotensive unanaesthetized Wistar rats, which were i.c.v. pre-treated with: (1) saline (control), (2) L-arginine (L-Arg), to increase the availability of substrate for NO biosynthesis, (3) D-arginine (D-Arg), (4) 3-[4-morpholinyl]-sydnonimine-hydrochloride (SIN-1), an NO donor, or (5) CaCl2, a cofactor of brain calcium-calmodulin-dependent cNOSI. In additional experiments, the levels of L-citrulline (the stable equimolar product derived from enzymatic cleavage of L-Arg by NO synthase) were determined in the brain of vehicle- or cadmium-treated rats.The pressor response to cadmium reached its nadir at 5 min (43±4 mmHg) and lasted over 20 min in controls. L-Citrulline/protein content was reduced from 35 up to 50% in the cerebral cortex, pons, hippocampus, striatus, hypothalamus (P<0.01) of cadmium-treated rats compared with controls. Central injection of NG nitro-L-arginine-methylester (L-NAME) also reduced the levels of L-citrulline in the brain.Both the magnitude and duration of the response were attenuated by 1.21 and 2.42 μmol SIN-1 (32±3 and 15±4 mmHg, P<0.05), or 1 μmol CaCl2 (6±4 mmHg, P<0.05). Selectivity of action exerted by SIN-1 was confirmed by the use of another NO donor, S-nitroso-N-acetyl-penicillamine (SNAP). Both L-Arg and D-Arg caused a mild but significant attenuation in the main phase of the pressor response evoked by cadmium. However, only L-Arg reduced the magnitude of the delayed, pressor response. Despite their similarity in

  1. Decreased endothelial nitric oxide synthase expression and function contribute to impaired mitochondrial biogenesis and oxidative stress in fetal lambs with persistent pulmonary hypertension

    OpenAIRE

    Afolayan, Adeleye J.; Eis, Annie; Alexander, Maxwell; Michalkiewicz, Teresa; Teng, Ru-Jeng; Lakshminrusimha, Satyan; Konduri, Girija G.

    2015-01-01

    Impaired vasodilation in persistent pulmonary hypertension of the newborn (PPHN) is characterized by mitochondrial dysfunction. We investigated the hypothesis that a decreased endothelial nitric oxide synthase level leads to impaired mitochondrial biogenesis and function in a lamb model of PPHN induced by prenatal ductus arteriosus constriction. We ventilated PPHN lambs with 100% O2 alone or with inhaled nitric oxide (iNO). We treated pulmonary artery endothelial cells (PAECs) from normal and...

  2. EPICS: Allen-Bradley hardware reference manual

    International Nuclear Information System (INIS)

    Nawrocki, G.

    1993-01-01

    This manual covers the following hardware: Allen-Bradley 6008 -- SV VMEbus I/O scanner; Allen-Bradley universal I/O chassis 1771-A1B, -A2B, -A3B, and -A4B; Allen-Bradley power supply module 1771-P4S; Allen-Bradley 1771-ASB remote I/O adapter module; Allen-Bradley 1771-IFE analog input module; Allen-Bradley 1771-OFE analog output module; Allen-Bradley 1771-IG(D) TTL input module; Allen-Bradley 1771-OG(d) TTL output; Allen-Bradley 1771-IQ DC selectable input module; Allen-Bradley 1771-OW contact output module; Allen-Bradley 1771-IBD DC (10--30V) input module; Allen-Bradley 1771-OBD DC (10--60V) output module; Allen-Bradley 1771-IXE thermocouple/millivolt input module; and the Allen-Bradley 2705 RediPANEL push button module

  3. Neuronal nitric oxide synthase mediates insulin- and oxidative stress-induced glucose uptake in skeletal muscle myotubes.

    Science.gov (United States)

    Kellogg, Dean L; McCammon, Karen M; Hinchee-Rodriguez, Kathryn S; Adamo, Martin L; Roman, Linda J

    2017-09-01

    Previously published studies strongly suggested that insulin- and exercise-induced skeletal muscle glucose uptake require nitric oxide (NO) production. However, the signal transduction mechanisms by which insulin and contraction regulated NO production and subsequent glucose transport are not known. In the present study, we utilized the myotube cell lines treated with insulin or hydrogen peroxide, the latter to mimic contraction-induced oxidative stress, to characterize these mechanisms. We found that insulin stimulation of neuronal nitric oxide synthase (nNOS) phosphorylation, NO production, and GLUT4 translocation were all significantly reduced by inhibition of either nNOS or Akt2. Hydrogen peroxide (H 2 O 2 ) induced phosphorylation of nNOS at the same residue as did insulin, and also stimulated NO production and GLUT4 translocation. nNOS inhibition prevented H 2 O 2 -induced GLUT4 translocation. AMP activated protein kinase (AMPK) inhibition prevented H 2 O 2 activation and phosphorylation of nNOS, leading to reduced NO production and significantly attenuated GLUT4 translocation. We conclude that nNOS phosphorylation and subsequently increased NO production are required for both insulin- and H 2 O 2 -stimulated glucose transport. Although the two stimuli result in phosphorylation of the same residue on nNOS, they do so through distinct protein kinases. Thus, insulin and H 2 O 2 -activated signaling pathways converge on nNOS, which is a common mediator of glucose uptake in both pathways. However, the fact that different kinases are utilized provides a basis for the use of exercise to activate glucose transport in the face of insulin resistance. Copyright © 2017. Published by Elsevier Inc.

  4. Orthostatic responses to nitric oxide synthase inhibition in persons with tetraplegia.

    Science.gov (United States)

    Wecht, Jill M; Radulovic, Miroslav; Lafountaine, Michael F; Rosado-Rivera, Dwindally; Zhang, Run-Lin; Bauman, William A

    2009-08-01

    To determine the effects of 1.0 mg/kg nitro-L-arginine methyl ester (L-NAME) on orthostatic mean arterial pressure (MAP), serum aldosterone, and plasma renin concentrations in persons with chronic tetraplegia compared with nonspinal cord-injured controls. Prospective placebo-controlled intervention study. James J. Peters Veterans Affairs Medical Center. Patients (n=5) with tetraplegia and controls (n=7) participated. The groups were matched for age, height, and weight; the average duration of injury in the tetraplegia group was 22+/-14 years. Subjects with tetraplegia visited the laboratory twice, receiving placebo on day 1 and L-NAME (1.0 mg/kg) on day 2. The agents were infused via an intravenous catheter over 60 minutes with the patient in the supine position. Data were collected during the infusion and then during head-up tilt to 45 degrees for 30 minutes. Control subjects visited the laboratory once for placebo infusion and the head-up tilt maneuver. Orthostatic MAP. Orthostatic MAP was reduced after placebo infusion in subjects with tetraplegia compared with controls (69+/-11 vs 89+/-9 mmHg, respectively; Ptetraplegia group with controls after L-NAME infusion. Orthostatic aldosterone levels were increased after placebo compared with L-NAME infusion in persons with tetraplegia; plasma renin levels did not differ among the groups. These data suggest that nitric oxide synthase inhibition may have clinical potential for treatment of orthostatic hypotension in persons with chronic tetraplegia.

  5. Nitric oxide synthase 2 (NOS2) expression in histologically normal margins of oral squamous cell carcinoma.

    Science.gov (United States)

    Morelatto, Rosana; Itoiz, María-Elina; Guiñazú, Natalia; Piccini, Daniel; Gea, Susana; López-de Blanc, Silvia

    2014-05-01

    The activity of Nitric Oxide Synthase 2 (NOS2) was found in oral squamous cell carcinomas (OSCC) but not in normal mucosa. Molecular changes associated to early carcinogenesis have been found in mucosa near carcinomas, which is considered a model to study field cancerization. The aim of the present study is to analyze NOS2 expression at the histologically normal margins of OSCC. Eleven biopsy specimens of OSCC containing histologically normal margins (HNM) were analyzed. Ten biopsies of normal oral mucosa were used as controls. The activity of NOS2 was determined by immunohistochemistry. Salivary nitrate and nitrite as well as tobacco and alcohol consumption were also analyzed. The Chi-squared test was applied. Six out of the eleven HNM from carcinoma samples showed positive NOS2 activity whereas all the control group samples yielded negative (p=0.005). No statistically significant association between enzyme expression and tobacco and/or alcohol consumption and salivary nitrate and nitrite was found. NOS2 expression would be an additional evidence of alterations that may occur in a state of field cancerization before the appearance of potentially malignant morphological changes.

  6. Intermittent pneumatic compression regulates expression of nitric oxide synthases in skeletal muscles.

    Science.gov (United States)

    Tan, Xiangling; Qi, Wen-Ning; Gu, Xiaosong; Urbaniak, James R; Chen, Long-En

    2006-01-01

    This study investigated the effects of intermittent pneumatic compression (IPC) on expression of nitric oxide synthase (NOS) isoforms in compressed (anterior tibialis, AT) and uncompressed (cremaster muscles, CM) skeletal muscles. Following IPC application of 0.5, 1, and 5h on both legs of rats, the endothelial NOS (eNOS) mRNA expression was significantly up-regulated to 1.2-, 1.8, and 2.7-fold from normal, respectively, in both AT and CM, and protein expression increased more than 1.5-fold of normal at each time point. Similarly, neuronal NOS expression was up-regulated, but to a lesser degree. In contrast, inducible NOS expression was significantly and time-dependently down-regulated in both muscles. After IPC cessation, eNOS levels returned to normal in both AT and CM. The results confirm our hypothesis that IPC-induced vasodilation is mediated by regulating expression of NOS isoforms, in particular eNOS, in both compressed and uncompressed skeletal muscles. The results also suggest the importance of precisely characterizing expression of each NOS isoform in tissue pathophysiology.

  7. Telmisartan activates endothelial nitric oxide synthase via Ser1177 phosphorylation in vascular endothelial cells.

    Directory of Open Access Journals (Sweden)

    Masahiro Myojo

    Full Text Available Because endothelial nitric oxide synthase (eNOS has anti-inflammatory and anti-arteriosclerotic functions, it has been recognized as one of the key molecules essential for the homeostatic control of blood vessels other than relaxation of vascular tone. Here, we examined whether telmisartan modulates eNOS function through its pleiotropic effect. Administration of telmisartan to mice significantly increased the phosphorylation level of eNOS (Ser1177 in the aortic endothelium, but administration of valsartan had no effect. Similarly, telmisartan treatment of human umbilical vein endothelial cells significantly increased the phosphorylation levels of AMP-activated protein kinase (Thr172 and eNOS and the concentration of intracellular guanosine 3',5'-cyclic monophosphate (cGMP. Furthermore, pretreatment with a p38 mitogen-activated protein kinase (p38 MAPK inhibitor suppressed the increased phosphorylation level of eNOS and intracellular cGMP concentration. These data show that telmisartan increases eNOS activity through Ser1177 phosphorylation in vascular endothelial cells mainly via p38 MAPK signaling.

  8. Expression of endothelial nitric oxide synthase in acute radiation-induced cerebral edema

    International Nuclear Information System (INIS)

    Liu Baoguo; Zhang Baomin; Chen Xiaohua; Gao Yabing; Wang Dewen

    2000-01-01

    Objective: To study the relation between changes in expression of endothelial nitric oxide synthase (eNOS) in brain vascular endothelial cells and acute radiation-induced cerebral edema after 60 Co gamma knife irradiation with a high dose. Methods: The right caudate nucleus of rats were irradiated with 200 Gy 60 Co gamma knife. the rats were sacrificed within 14 days after irradiation. By a light microscopy, electron microscopy, immunohistochemistry and in situ hybridization, the authors studied the formation and development of acute radiation-induced cerebral edema as well as the expression of eNOS in brain vascular endothelial cells. Results: Acute radiation-induced cerebral edema was observed morphologically 2 hours after irradiation and peaked on day 3 after irradiation. The expression of eNOS in brain vascular endothelial cells markedly increased 2 hours after irradiation, which could be consistently visualized during the acute developmental phase of radiation-induced cerebral edema. Conclusion: The increased expression of eNOS may be related to the formation and development of acute radiation-induced cerebral edema after 60 Co gamma knife irradiation with a high dose

  9. Expression of monoamine transporters, nitric oxide synthase 3 and neurotrophin genes in antidepressant-stimulated astrocytes

    Directory of Open Access Journals (Sweden)

    Sarah eKittel-Schneider

    2012-04-01

    Full Text Available Background: There is increasing evidence that glial cells play a role in the pathomechanisms of mood disorders and the mode of action of antidepressant drugs. Methods: To examine whether there is a direct effect on the expression of different genes encoding proteins that have been implicated in the pathophysiology of affective disorders, primary astrocyte cell cultures from rats were treated with two different antidepressant drugs, imipramine and escitalopram, and the mRNA expression of brain derived neurotrophic factor (Bdnf, serotonin transporter (5Htt, dopamine transporter (Dat and endothelial nitric oxide synthase (Nos3 was examined. Results: Stimulation of astroglial cell culture with imipramine, a tricyclic antidepressant, lead to a significant increase of the Bdnf mRNA level whereas treatment with escitalopram did not. In contrast, 5Htt was not differentially expressed after antidepressant treatment. Finally, neither Dat nor Nos3 mRNA expression was detected in cultured astrocytes. Conclusions: These data provide further evidence for a role of astroglial cells in the molecular mechanisms of action of antidepressants.

  10. Inducible nitric oxide synthase up-regulates Notch-1 in mouse cholangiocytes: implications for carcinogenesis.

    Science.gov (United States)

    Ishimura, Norihisa; Bronk, Steven F; Gores, Gregory J

    2005-05-01

    Inflammatory mediators and cell fate genes, such as the Notch gene family, both have been implicated in cancer biology. Because cholangiocarcinomas arise in a background of inflammation and express the inflammatory mediator inducible nitric oxide synthase (iNOS), we aimed to determine whether iNOS expression alters Notch expression and signaling. Notch receptor and ligand expression in human liver was evaluated by immunohistochemistry. The effect of iNOS and NO on Notch-1 expression was examined in cell lines. Notch-1, but not other Notch receptors, were up-regulated by cholangiocytes in primary sclerosing cholangitis and cholangiocarcinoma. The colocalization of Notch-1 and iNOS also was observed in large bile ducts from the hilar region of primary sclerosing cholangitis patients. Notch-1 expression in murine cholangiocytes was iNOS dependent. iNOS expression also facilitated Notch signaling by inducing the nuclear translocation of its intracellular domain and the expression of a transcriptional target, hairy and enhancer of split (Hes)-1. The gamma-secretase inhibitor N-[N-(3,5-Difluorophenacetyl-L-alanyl)-S-phenylglycine]-t-butyl ester, which blocks Notch signaling, enhanced tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in cholangiocarcinoma cells. These data implicate a direct link between the inflammatory mediator iNOS and Notch signaling, and have implications for the development and progression of cholangiocarcinoma.

  11. Arginase activity in mitochondria - An interfering factor in nitric oxide synthase activity assays

    Energy Technology Data Exchange (ETDEWEB)

    Venkatakrishnan, Priya; Nakayasu, Ernesto S.; Almeida, Igor C. [Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968 (United States); Miller, R.T., E-mail: tmiller2@utep.edu [Department of Biological Sciences, University of Texas at El Paso, El Paso, TX 79968 (United States)

    2010-04-09

    Previously, in tightly controlled studies, using three independent, yet complementary techniques, we refuted the claim that a mitochondrial nitric oxide synthase (mtNOS) isoform exists within pure, rat liver mitochondria (MT). Of those techniques, the NOS-catalyzed [{sup 14}C]-L-arginine to [{sup 14}C]-L-citrulline conversion assay (NOS assay) with MT samples indicated a weak, radioactive signal that was NOS-independent . Aliquots of samples from the NOS assays were then extracted with acetone, separated by high performance thin-layer chromatography (HPTLC) and exposed to autoradiography. Results obtained from these samples showed no radioactive band for L-citrulline. However, a fast-migrating, diffuse, radioactive band was observed in the TLC lanes loaded with MT samples. In this manuscript, we identify and confirm that this radioactive signal in MT samples is due to the arginase-catalyzed conversion of [{sup 14}C]-L-arginine to [{sup 14}C]-urea. The current results, in addition to reconfirming the absence of NOS activity in rat liver MT, also show the need to include arginase inhibitors in studies using MT samples in order to avoid confounding results when using NOS activity assays.

  12. Occurrence, structure, and evolution of nitric oxide synthase-like proteins in the plant kingdom.

    Science.gov (United States)

    Jeandroz, Sylvain; Wipf, Daniel; Stuehr, Dennis J; Lamattina, Lorenzo; Melkonian, Michael; Tian, Zhijian; Zhu, Ying; Carpenter, Eric J; Wong, Gane Ka-Shu; Wendehenne, David

    2016-03-01

    Nitric oxide (NO) signaling regulates various physiological processes in both animals and plants. In animals, NO synthesis is mainly catalyzed by NO synthase (NOS) enzymes. Although NOS-like activities that are sensitive to mammalian NOS inhibitors have been detected in plant extracts, few bona fide plant NOS enzymes have been identified. We searched the data set produced by the 1000 Plants (1KP) international consortium for the presence of transcripts encoding NOS-like proteins in over 1000 species of land plants and algae. We also searched for genes encoding NOS-like enzymes in 24 publicly available algal genomes. We identified no typical NOS sequences in 1087 sequenced transcriptomes of land plants. In contrast, we identified NOS-like sequences in 15 of the 265 algal species analyzed. Even if the presence of NOS enzymes assembled from multipolypeptides in plants cannot be conclusively discarded, the emerging data suggest that, instead of generating NO with evolutionarily conserved NOS enzymes, land plants have evolved finely regulated nitrate assimilation and reduction processes to synthesize NO through a mechanism different than that in animals. Copyright © 2016, American Association for the Advancement of Science.

  13. Impaired Healing of a Cutaneous Wound in an Inducible Nitric Oxide Synthase-Knockout Mouse

    Directory of Open Access Journals (Sweden)

    Takashi Kitano

    2017-01-01

    Full Text Available Background. We investigated the effects of loss of inducible nitric oxide synthase (iNOS on the healing process of cutaneous excisional injury by using iNOS-null (KO mice. Population of granulation tissue-related cell types, that is, myofibroblasts and macrophages, growth factor expression, and reepithelialization were evaluated. Methods. KO and wild type (WT mice of C57BL/6 background were used. Under general anesthesia two round full-thickness excision wounds of 5.0 mm in diameter were produced in dorsal skin. After specific intervals of healing, macroscopic observation, histology, immunohistochemistry, and real-time reverse transcription-polymerase chain reaction (RT-PCR were employed to evaluate the healing process. Results. The loss of iNOS retards granulation tissue formation and reepithelialization in excision wound model in mice. Detailed analyses showed that myofibroblast appearance, macrophage infiltration, and mRNA expression of transforming growth factor b and of collagen 1α2 were all suppressed by lacking iNOS. Conclusions. iNOS is required in the process of cutaneous wound healing. Lacking iNOS retards macrophage invasion and its expression of fibrogenic components that might further impair fibrogenic behaviors of fibroblasts.

  14. Microangiopathy triggers, and inducible nitric oxide synthase exacerbates dextran sulfate sodium-induced colitis.

    Science.gov (United States)

    Saijo, Hiroki; Tatsumi, Norifumi; Arihiro, Seiji; Kato, Tomohiro; Okabe, Masataka; Tajiri, Hisao; Hashimoto, Hisashi

    2015-07-01

    Ulcerative colitis (UC) is a representative clinical manifestation of inflammatory bowel disease that causes chronic gastrointestinal tract inflammation. Dextran sulfate sodium (DSS)-induced colitis mice have been used to investigate UC pathogenesis, and in this UC model, disturbance and impairment of the mucosal epithelium have been reported to cause colitis. However, how DSS sporadically breaks down the epithelium remains unclear. In this study, we focused on the colonic microcirculation and myenteric neurons of DSS-induced colitis. Moreover, we examined the potential of myenteric neurons as a target to prevent exacerbation of colitis. Fluorescent angiographic and histopathological studies revealed that DSS administration elicited blood vessel disruption before epithelial disorders appeared. Ischemic conditions in the lamina propria induced inducible nitric oxide synthase (iNOS) expression in myenteric neurons as colitis aggravated. When neuronal activity was inhibited with butylscopolamine, neuronal iNOS expression decreased, and the exacerbation of colitis was prevented. These results suggested that DSS-induced colitis was triggered by microcirculatory disturbance in the mucosa, and that excessive neuronal excitation aggravated colitis. During remission periods of human UC, endoscopic inspection of the colonic microcirculation may enable the early detection of disease recurrence, and inhibition of neuronal iNOS expression may prevent the disease from worsening.

  15. Characterisation and vascular expression of nitric oxide synthase 3 in amphibians.

    Science.gov (United States)

    Cameron, Melissa S; Trajanovska, Sofie; Forgan, Leonard G; Donald, John A

    2016-12-01

    In mammals, nitric oxide (NO) produced by nitric oxide synthase 3 (NOS3) localised in vascular endothelial cells is an important vasodilator but the presence of NOS3 in the endothelium of amphibians has been concluded to be absent, based on physiological studies. In this study, a nos3 cDNA was sequenced from the toad, Rhinella marina. The open reading frame of R. marina nos3 encoded an 1170 amino acid protein that showed 81 % sequence identity to the recently cloned Xenopus tropicalis nos3. Rhinella marina nos3 mRNA was expressed in a range of tissues and in the dorsal aorta and pulmonary, mesenteric, iliac and gastrocnemius arteries. Furthermore, nos3 mRNA was expressed in the aorta of Xenopus laevis and X. tropicalis. Quantitative real-time PCR showed that removal of the endothelium of the lateral aorta of R. marina significantly reduced the expression of nos3 mRNA compared to control aorta with the endothelium intact. However, in situ hybridisation was not able to detect any nos3 mRNA in the dorsal aorta of R. marina. Immunohistochemistry using a homologous R. marina NOS3 antibody showed immunoreactivity (IR) within the basal region of many endothelial cells of the dorsal aorta and iliac artery. NOS3-IR was also observed in the proximal tubules and collecting ducts of the kidney but not within the capillaries of the glomeruli. This is the first study to demonstrate that vascular endothelial cells of an amphibian express NOS3.

  16. Citrus nobiletin suppresses inducible nitric oxide synthase gene expression in interleukin-1β-treated hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshigai, Emi [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan); Ritsumeikan Global Innovation Research Organization (R-GIRO), Kusatsu, Shiga (Japan); Machida, Toru [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan); Okuyama, Tetsuya [Ritsumeikan Global Innovation Research Organization (R-GIRO), Kusatsu, Shiga (Japan); Mori, Masatoshi; Murase, Hiromitsu; Yamanishi, Ryota [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan); Okumura, Tadayoshi [Research Organization of Science and Technology, Ritsumeikan University, Kusatsu, Shiga (Japan); Department of Surgery, Kansai Medical University, Hirakata, Osaka (Japan); Ikeya, Yukinobu [Department of Pharmacy, College of Pharmaceutical Sciences, Ritsumeikan University, Kusatsu, Shiga (Japan); Nishino, Hoyoku [Ritsumeikan Global Innovation Research Organization (R-GIRO), Kusatsu, Shiga (Japan); Department of Biochemistry, Kyoto Prefectural University of Medicine, Kyoto (Japan); Nishizawa, Mikio, E-mail: nishizaw@sk.ritsumei.ac.jp [Department of Biomedical Sciences, College of Life Sciences, Kusatsu, Shiga (Japan)

    2013-09-13

    Highlights: •Nobiletin is a polymethoxylated flavone that is abundant in citrus peels. •Nobiletin is a major constituent of the Citrus unshiu peel extract. •Nobiletin suppresses induction of NO and reduces iNOS expression in hepatocytes. •Nobiletin reduces the iNOS promoter activity and the DNA-binding activity of NF-κB. -- Abstract: Background: Nobiletin is a polymethoxylated flavone that is abundant in the peels of citrus fruits, such as Citrus unshiu (Satsuma mandarin) and Citrus sinensis. The dried peels of C. unshiu (chinpi) have been included in several formulae of Japanese Kampo medicines. Nobiletin may suppress the induction of inducible nitric oxide synthase (iNOS), which synthesizes the inflammatory mediator nitric oxide (NO) in hepatocytes. Methods: A C. unshiu peel (CUP) extract was prepared. Primary cultured rat hepatocytes were treated with the CUP extract or nobiletin in the presence of interleukin 1β (IL-1β), which induces iNOS expression. NO production and iNOS gene expression were analyzed. Results: High-performance liquid chromatography analyses revealed that the nobiletin content in the CUP extract was 0.14%. Nobiletin dose-dependently reduced the NO levels and decreased iNOS expression at the protein, mRNA and antisense transcript levels. Flavone, which does not contain any methoxy groups, also suppressed iNOS induction. Nobiletin reduced the transcriptional activity of iNOS promoter-luciferase constructs and the DNA-binding activity of nuclear factor κB (NF-κB) in the nuclei. Conclusions: The suppression of iNOS induction by nobiletin suggests that nobiletin may be responsible for the anti-inflammatory effects of citrus peels and have a therapeutic potential for liver diseases.

  17. Modulating central gain in tinnitus: changes in nitric oxide synthase in the ventral cochlear nucleus.

    Science.gov (United States)

    Coomber, Ben; Kowalkowski, Victoria L; Berger, Joel I; Palmer, Alan Richard; Wallace, Mark Nelson

    2015-01-01

    A significant challenge in tinnitus research lies in explaining how acoustic insult leads to tinnitus in some individuals, but not others. One possibility is genetic variability in the expression and function of neuromodulators - components of neural signaling that alter the balance of excitation and inhibition in neural circuits. An example is nitric oxide (NO) - a free radical and potent neuromodulator in the mammalian brain - that regulates plasticity via both pre-synaptic and postsynaptic mechanisms. Changes in NO have previously been implicated in tinnitus generation, specifically in the ventral cochlear nucleus (VCN). Here, we examined nitric oxide synthase (NOS) - the enzyme responsible for NO production - in the guinea pig VCN following acoustic trauma. NOS was present in most cell types - including spherical and globular bushy cells, small, medium, and large multipolar cells, and octopus cells - spanning the entire extent of the VCN. The staining pattern was symmetrical in control animals. Unilateral acoustic over-exposure (AOE) resulted in marked asymmetries between ipsilateral and contralateral sides of the VCN in terms of the distribution of NOS across the cochlear nuclei in animals with behavioral evidence of tinnitus: fewer NOS-positive cells and a reduced level of NOS staining was present across the whole extent of the contralateral VCN, relative to the ipsilateral VCN. The asymmetric pattern of NOS-containing cells was observed as early as 1 day after AOE and was also present in some animals at 3, 7, and 21 days after AOE. However, it was not until 8 weeks after AOE, when tinnitus had developed, that asymmetries were significant overall, compared with control animals. Asymmetrical NOS expression was not correlated with shifts in the threshold hearing levels. Variability in NOS expression between animals may represent one underlying difference that can be linked to whether or not tinnitus develops after noise exposure.

  18. Modulating central gain in tinnitus: Changes in nitric oxide synthase in the ventral cochlear nucleus

    Directory of Open Access Journals (Sweden)

    Ben eCoomber

    2015-03-01

    Full Text Available A significant challenge in tinnitus research lies in explaining how acoustic insult leads to tinnitus in some individuals, but not others. One possibility is genetic variability in the expression and function of neuromodulators – components of neural signalling that alter the balance of excitation and inhibition in neural circuits. An example is nitric oxide (NO – a free radical and potent neuromodulator in the mammalian brain – that regulates plasticity via both presynaptic and postsynaptic mechanisms. Changes in NO have previously been implicated in tinnitus generation, specifically in the ventral cochlear nucleus (VCN. Here, we examined nitric oxide synthase (NOS – the enzyme responsible for NO production – in the guinea pig VCN following acoustic trauma. NOS was present in most cell types – including spherical and globular bushy cells, small, medium and large multipolar cells, and octopus cells – spanning the entire extent of the VCN. The staining pattern was symmetrical in control animals. Unilateral acoustic over-exposure (AOE resulted in marked asymmetries between ipsilateral and contralateral sides of the VCN in terms of the distribution of NOS across the cochlear nuclei in animals with behavioural evidence of tinnitus: fewer NOS-positive cells and a reduced level of NOS staining was present across the whole extent of the contralateral VCN, relative to the ipsilateral VCN. The asymmetric pattern of NOS-containing cells was observed as early as one day after AOE and was also present in some animals at 3, 7 and 21 days after AOE. However it was not until eight weeks after AOE, when tinnitus had developed, that asymmetries were significant overall, compared with control animals. Asymmetrical NOS expression was not correlated with shifts in the threshold hearing levels. Variability in NOS expression between animals may represent one underlying difference that can be linked to whether or not tinnitus develops after noise

  19. Metabolism via arginase or nitric oxide synthase: two competing arginine pathways in macrophages

    Directory of Open Access Journals (Sweden)

    Meera eRath

    2014-10-01

    Full Text Available Macrophages play a major role in the immune system, both as antimicrobial effector cells and as immunoregulatory cells, which induce, suppress or modulate adaptive immune responses. These key aspects of macrophage biology are fundamentally driven by the phenotype of macrophage arginine metabolism that is prevalent in an evolving or ongoing immune response. M1 macrophages express the enzyme nitric oxide synthase (NOS, which metabolizes arginine to nitric oxide (NO and citrulline. NO can be metabolized to further downstream reactive nitrogen species, while citrulline might be reused for efficient NO synthesis via the citrulline-NO cycle. M2 macrophages are characterized by expression of the enzyme arginase, which hydrolyzes arginine to ornithine and urea. The arginase pathway limits arginine availability for NO synthesis and ornithine itself can further feed into the important downstream pathways of polyamine and proline syntheses, which are important for cellular proliferation and tissue repair. M1 versus M2 polarization leads to opposing outcomes of inflammatory reactions, but depending on the context, M1 and M2 macrophages can be both pro- and antiinflammatory. Notably, M1/M2 macrophage polarization can be driven by microbial infection or innate danger signals without any influence of adaptive immune cells, secondarily driving the T helper (Th1/Th2 polarization of the evolving adaptive immune response. Since both arginine metabolic pathways cross-inhibit each other on the level of the respective arginine break-down products and Th1 and Th2 lymphocytes can drive or amplify macrophage M1/M2 dichotomy via cytokine activation, this forms the basis of a self-sustaining M1/M2 polarization of the whole immune response. Understanding the arginine metabolism of M1/M2 macrophage phenotypes is therefore central to find new possibilities to manipulate immune responses in infection, autoimmune diseases, chronic inflammatory conditions and cancer.

  20. Expression of Inducible Nitric Oxide Synthase (iNOS) in Microglia of the Developing Quail Retina

    Science.gov (United States)

    Sierra, Ana; Navascués, Julio; Cuadros, Miguel A.; Calvente, Ruth; Martín-Oliva, David; Ferrer-Martín, Rosa M.; Martín-Estebané, María; Carrasco, María-Carmen; Marín-Teva, José L.

    2014-01-01

    Inducible nitric oxide synthase (iNOS), which produce large amounts of nitric oxide (NO), is induced in macrophages and microglia in response to inflammatory mediators such as LPS and cytokines. Although iNOS is mainly expressed by microglia that become activated in different pathological and experimental situations, it was recently reported that undifferentiated amoeboid microglia can also express iNOS during normal development. The aim of this study was to investigate the pattern of iNOS expression in microglial cells during normal development and after their activation with LPS by using the quail retina as model. iNOS expression was analyzed by iNOS immunolabeling, western-blot, and RT-PCR. NO production was determined by using DAR-4M AM, a reliable fluorescent indicator of subcellular NO production by iNOS. Embryonic, postnatal, and adult in situ quail retinas were used to analyze the pattern of iNOS expression in microglial cells during normal development. iNOS expression and NO production in LPS-treated microglial cells were investigated by an in vitro approach based on organotypic cultures of E8 retinas, in which microglial cell behavior is similar to that of the in situ retina, as previously demonstrated in our laboratory. We show here that amoeboid microglia in the quail retina express iNOS during normal development. This expression is stronger in microglial cells migrating tangentially in the vitreal part of the retina and is downregulated, albeit maintained, when microglia differentiate and become ramified. LPS treatment of retina explants also induces changes in the morphology of amoeboid microglia compatible with their activation, increasing their lysosomal compartment and upregulating iNOS expression with a concomitant production of NO. Taken together, our findings demonstrate that immature microglial cells express iNOS during normal development, suggesting a certain degree of activation. Furthermore, LPS treatment induces overactivation of amoeboid

  1. Development of radiation-inducible promoters for use in nitric oxide synthase gene therapy of cancer

    International Nuclear Information System (INIS)

    Hirst, D.G.; Worthington, J.; Adams, C.; Robson, T.; Scott, S.D.

    2003-01-01

    Full text: The free radical nitric oxide (NO) at nM concentrations performs multiple signaling roles that are essential for survival. These processes are regulated via the enzymes nNOS and eNOS, but another isoform, inducible nitric oxide synthase (iNOS) is capable of generating much higher concentrations (mM) over longer periods, resulting in the generation of very toxic species such as peroxynitrite. At high concentrations NO has many of the characteristics of an ideal anticancer molecule: it is cytotoxic (pro-apoptotic via peroxynitrite), it is a potent chemical radiosensitizer, it is anti-angiogenic and anti-metastatic. Thus, we see iNOS gene therapy as a strategy for targeting the generation of high concentrations of NO to tumours for therapeutic benefit. iNOS gene therapy should be used in combination with radiotherapy; so it is logical that the use of a radiation-inducible promoter should be part of the targeting strategy. We have tested several candidate promoters in vitro and in vivo. The WAF1 promoter has many of the properties desirable for therapeutic use including: rapid 3-4 fold induction at X-ray doses of 2 and 4Gy and no significant leakiness. WAF1 also has the advantage of being inducible by hypoxia and by the final product, NO. We have also tested the synthetic CArG promoter and demonstrated that, in addition to a high level of radiation inducibility, it is also inducible by NO. We have also been able to demonstrate potent radiosensitization (SER 2.0-2.5) in tumour cells in vitro and in vivo using iNOS gene transfer with constitutive or radiation-inducible promoters. We have also tested the use of iNOS gene therapy in combination with cisplatin and shown significant enhancement

  2. Impact of nutrient excess and endothelial nitric oxide synthase on the plasma metabolite profile in mice

    Directory of Open Access Journals (Sweden)

    Brian E Sansbury

    2014-11-01

    Full Text Available An increase in calorie consumption is associated with the recent rise in obesity prevalence. However, our current understanding of the effects of nutrient excess on major metabolic pathways appears insufficient to develop safe and effective metabolic interventions to prevent obesity. Hence, we sought to identify systemic metabolic changes caused by nutrient excess and to determine how endothelial nitric oxide synthase (eNOS—which has anti-obesogenic properties—affects systemic metabolism by measuring plasma metabolites. Wild-type (WT and eNOS transgenic (eNOS-TG mice were placed on low fat or high fat diets for six weeks, and plasma metabolites were measured using an unbiased metabolomic approach. High fat feeding in WT mice led to significant increases in fat mass, which was associated with significantly lower plasma levels of 1,5-anhydroglucitol, lysophospholipids, 3-dehydrocarnitine, and bile acids, as well as branched chain amino acids (BCAAs and their metabolites. Plasma levels of several lipids including sphingomyelins, stearoylcarnitine, dihomo-linoleate and metabolites associated with oxidative stress were increased by high fat diet. In comparison with low fat-fed WT mice, eNOS-TG mice showed lower levels of several free fatty acids, but in contrast, the levels of bile acids, amino acids, and BCAA catabolites were increased. When placed on a high fat diet, eNOS overexpressing mice showed remarkably higher levels of plasma bile acids and elevated levels of plasma BCAAs and their catabolites compared with WT mice. Treatment with GW4064, an inhibitor of bile acid synthesis, decreased plasma bile acid levels but was not sufficient to reverse the anti-obesogenic effects of eNOS overexpression. These findings reveal unique metabolic changes in response to high fat diet and eNOS overexpression and suggest that the anti-obesity effects of eNOS are likely independent of changes in the bile acid pool.

  3. Nitric oxide synthase modulates CFA-induced thermal hyperalgesia through cytokine regulation in mice.

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    Chen, Yong; Boettger, Michael K; Reif, Andreas; Schmitt, Angelika; Uçeyler, Nurcan; Sommer, Claudia

    2010-03-02

    Although it has been largely demonstrated that nitric oxide synthase (NOS), a key enzyme for nitric oxide (NO) production, modulates inflammatory pain, the molecular mechanisms underlying these effects remain to be clarified. Here we asked whether cytokines, which have well-described roles in inflammatory pain, are downstream targets of NO in inflammatory pain and which of the isoforms of NOS are involved in this process. Intraperitoneal (i.p.) pretreatment with 7-nitroindazole sodium salt (7-NINA, a selective neuronal NOS inhibitor), aminoguanidine hydrochloride (AG, a selective inducible NOS inhibitor), L-N(G)-nitroarginine methyl ester (L-NAME, a non-selective NOS inhibitor), but not L-N(5)-(1-iminoethyl)-ornithine (L-NIO, a selective endothelial NOS inhibitor), significantly attenuated thermal hyperalgesia induced by intraplantar (i.pl.) injection of complete Freund's adjuvant (CFA). Real-time reverse transcription-polymerase chain reaction (RT-PCR) revealed a significant increase of nNOS, iNOS, and eNOS gene expression, as well as tumor necrosis factor-alpha (TNF), interleukin-1 beta (IL-1beta), and interleukin-10 (IL-10) gene expression in plantar skin, following CFA. Pretreatment with the NOS inhibitors prevented the CFA-induced increase of the pro-inflammatory cytokines TNF and IL-1beta. The increase of the anti-inflammatory cytokine IL-10 was augmented in mice pretreated with 7-NINA or L-NAME, but reduced in mice receiving AG or L-NIO. NNOS-, iNOS- or eNOS-knockout (KO) mice had lower gene expression of TNF, IL-1beta, and IL-10 following CFA, overall corroborating the inhibitor data. These findings lead us to propose that inhibition of NOS modulates inflammatory thermal hyperalgesia by regulating cytokine expression.

  4. The endothelial nitric oxide synthase (eNOS) polymorphism in otitis media with effusion (OME).

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    Ates, M; Cevik, C; Dokuyucu, R; Berber, O; Colak, S; Izmirli, M

    2017-02-01

    Otitis media with effusion (OME) is the most common disease after viral infections of upper respiratory tract (URTI) in children. Studies indicate the important role of nitric oxide (NO) in the etiology of hearing loss. However, there is no study that focuses on the role of nitric oxide synthase (eNOS) polymorphisms in the cases with OME. The aim of the present study is to evaluate the eNOS polymorphisms in the pediatric patients with OME. Eighty-nine patients who are diagnosed with otitis media with effusion and 85 healthy subjects who are compatible in terms of age and gender were included in the study. All patients in the study were subjected to complete ear, nose, throat (ENT) and audiological examinations. DNA analysis was performed with polymerase chain reaction (PCR) technique from the blood samples. The PCR product was cut by restriction fragment length polymorphism (RFLP) with BanII enzyme and checked by agarose gel electrophoresis. As a result of genetic analysis, there is no significant difference between patients and the controls in terms of eNOS Glu298Asp polymorphism (G/G, G/T, T/T). When these groups were compared in terms of allele distributions, a significant relationship was found between the patients and the controls (P=0.037). To the best of our knowledge, G allele was identified as predisposing to the development of OME and this is the first report indicates the correlation between the eNOS G894T polymorphism and OME in Turkey. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  5. Nitric oxide synthase modulates CFA-induced thermal hyperalgesia through cytokine regulation in mice

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    Üçeyler Nurcan

    2010-03-01

    Full Text Available Abstract Background Although it has been largely demonstrated that nitric oxide synthase (NOS, a key enzyme for nitric oxide (NO production, modulates inflammatory pain, the molecular mechanisms underlying these effects remain to be clarified. Here we asked whether cytokines, which have well-described roles in inflammatory pain, are downstream targets of NO in inflammatory pain and which of the isoforms of NOS are involved in this process. Results Intraperitoneal (i.p. pretreatment with 7-nitroindazole sodium salt (7-NINA, a selective neuronal NOS inhibitor, aminoguanidine hydrochloride (AG, a selective inducible NOS inhibitor, L-N(G-nitroarginine methyl ester (L-NAME, a non-selective NOS inhibitor, but not L-N(5-(1-iminoethyl-ornithine (L-NIO, a selective endothelial NOS inhibitor, significantly attenuated thermal hyperalgesia induced by intraplantar (i.pl. injection of complete Freund's adjuvant (CFA. Real-time reverse transcription-polymerase chain reaction (RT-PCR revealed a significant increase of nNOS, iNOS, and eNOS gene expression, as well as tumor necrosis factor-alpha (TNF, interleukin-1 beta (IL-1β, and interleukin-10 (IL-10 gene expression in plantar skin, following CFA. Pretreatment with the NOS inhibitors prevented the CFA-induced increase of the pro-inflammatory cytokines TNF and IL-1β. The increase of the anti-inflammatory cytokine IL-10 was augmented in mice pretreated with 7-NINA or L-NAME, but reduced in mice receiving AG or L-NIO. NNOS-, iNOS- or eNOS-knockout (KO mice had lower gene expression of TNF, IL-1β, and IL-10 following CFA, overall corroborating the inhibitor data. Conclusion These findings lead us to propose that inhibition of NOS modulates inflammatory thermal hyperalgesia by regulating cytokine expression.

  6. Endothelial nitric oxide synthase and superoxide mediate hemodynamic initiation of intracranial aneurysms.

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    Nicholas Liaw

    Full Text Available Hemodynamic insults at arterial bifurcations are believed to play a critical role in initiating intracranial aneurysms. Recent studies in a rabbit model indicate that aneurysmal damage initiates under specific wall shear stress conditions when smooth muscle cells (SMCs become pro-inflammatory and produce matrix metalloproteinases (MMPs. The mechanisms leading to SMC activation and MMP production during hemodynamic aneurysm initiation are unknown. The goal is to determine if nitric oxide and/or superoxide induce SMC changes, MMP production and aneurysmal remodeling following hemodynamic insult.Bilateral common carotid artery ligation was performed on rabbits (n = 19, plus 5 sham operations to induce aneurysmal damage at the basilar terminus. Ligated animals were treated with the nitric oxide synthase (NOS inhibitor LNAME (n = 7 or the superoxide scavenger TEMPOL (n = 5 and compared to untreated animals (n = 7. Aneurysm development was assessed histologically 5 days after ligation. Changes in NOS isoforms, peroxynitrite, reactive oxygen species (ROS, MMP-2, MMP-9, and smooth muscle α-actin were analyzed by immunohistochemistry.LNAME attenuated ligation-induced IEL loss, media thinning and bulge formation. In untreated animals, immunofluorescence showed increased endothelial NOS (eNOS after ligation, but no change in inducible or neuronal NOS. Furthermore, during aneurysm initiation ROS increased in the media, but not the intima, and there was no change in peroxynitrite. In LNAME-treated animals, ROS production did not change. Together, this suggests that eNOS is important for aneurysm initiation but not by producing superoxide. TEMPOL treatment reduced aneurysm development, indicating that the increased medial superoxide is also necessary for aneurysm initiation. LNAME and TEMPOL treatment in ligated animals restored α-actin and decreased MMPs, suggesting that eNOS and superoxide both lead to SMC de-differentiation and MMP production

  7. Low Nitric Oxide Synthases (NOS) in Eyes with Age-related Macular Degeneration (AMD)

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    Bhutto, Imran A.; Baba, Takayuki; Merges, Carol; McLeod, D. Scott; Lutty, Gerard A.

    2009-01-01

    Nitric oxide (NO) production by vascular endothelium is important in regulation of blood flow. Reduced production of NO can adversely affect blood flow and other vascular functions. We investigated the expression of three nitric oxide synthase (NOS) isoforms in retina and choroid of aged human eyes and eyes with AMD. Alkaline phosphatase immunohistochemistry was performed using antibodies against inducible (iNOS), neuronal (nNOS), and endothelial (eNOS) NOSs on cryopreserved sections from aged control donor eyes (n= 13) and eyes with AMD (n= 22). CD34 antibody was used as an endothelial cell (EC) marker. Three independent masked observers scored the intensity of the immunohistochemical reaction product. Mean scores from the aged control and AMD eyes were statistically compared. In aged control retinas, nNOS was in ganglion cells (RGCs) and neurons of both nuclear layers. In choroid, perivascular nerve fibers and retinal pigment epithelial (RPE) cells were nNOS+. eNOS and iNOS were confined to the retinal and choroidal vascular ECs. Some cells presumably melanocytes or dendritic cells in choroid were also eNOS+. In AMD eyes, nNOS was significantly lower in RGCs, neurons, retinal vessels and RPE (p≤0.05) compared to the aged control eyes. iNOS and eNOS showed no significant differences between aged control and AMD eyes except that there was significantly less eNOS in choroidal arteries (p=0.006) and choroidal cells (p=0.03) of AMD eyes. Although NO was not measured directly, these findings suggest that there is less NO produced in AMD eyes. The decrease in retinal nNOS in AMD eyes is probably related to neuronal degeneration. The decrease in nNOS and eNOS in AMD choroid could be associated with vasoconstriction and hemodynamic changes. PMID:19836390

  8. Insulin-stimulated phosphorylation of endothelial nitric oxide synthase at serine-615 contributes to nitric oxide synthesis.

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    Ritchie, Stuart A; Kohlhaas, Christine F; Boyd, Alasdair R; Yalla, Krishna C; Walsh, Kenneth; Connell, John M C; Salt, Ian P

    2010-01-27

    Insulin stimulates endothelial NO (nitric oxide) synthesis via PKB (protein kinase B)/Akt-mediated phosphorylation and activation of eNOS (endothelial NO synthase) at Ser-1177. In previous studies, we have demonstrated that stimulation of eNOS phosphorylation at Ser-1177 may be required, yet is not sufficient for insulin-stimulated NO synthesis. We therefore investigated the role of phosphorylation of eNOS at alternative sites to Ser-1177 as candidate parallel mechanisms contributing to insulin-stimulated NO synthesis. Stimulation of human aortic endothelial cells with insulin rapidly stimulated phosphorylation of both Ser-615 and Ser-1177 on eNOS, whereas phosphorylation of Ser-114, Thr-495 and Ser-633 was unaffected. Insulin-stimulated Ser-615 phosphorylation was abrogated by incubation with the PI3K (phosphoinositide 3-kinase) inhibitor wortmannin, infection with adenoviruses expressing a dominant-negative mutant PKB/Akt or pre-incubation with TNFalpha (tumour necrosis factor alpha), but was unaffected by high culture glucose concentrations. Mutation of Ser-615 to alanine reduced insulin-stimulated NO synthesis, whereas mutation of Ser-615 to aspartic acid increased NO production by NOS in which Ser-1177 had been mutated to an aspartic acid residue. We propose that the rapid PKB-mediated stimulation of phosphorylation of Ser-615 contributes to insulin-stimulated NO synthesis.

  9. Chamomile, an anti-inflammatory agent inhibits inducible nitric oxide synthase expression by blocking RelA/p65 activity

    OpenAIRE

    Bhaskaran, Natarajan; Shukla, Sanjeev; Srivastava, Janmejai K; Gupta, Sanjay

    2010-01-01

    Chamomile has long been used in traditional medicine for the treatment of inflammation-related disorders. In this study we aimed to investigate the inhibitory effects of chamomile on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and to explore its potential anti-inflammatory mechanisms using RAW 264.7 macrophages. Chamomile treatment inhibited LPS-induced NO production and significantly blocked IL-1β , IL-6 and TNFα-induced NO levels in RAW 264.7 macropha...

  10. Nitric oxide signalling and neuronal nitric oxide synthase in the heart under stress [version 1; referees: 2 approved

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    Yin Hua Zhang

    2017-05-01

    Full Text Available Nitric oxide (NO is an imperative regulator of the cardiovascular system and is a critical mechanism in preventing the pathogenesis and progression of the diseased heart. The scenario of bioavailable NO in the myocardium is complex: 1 NO is derived from both endogenous NO synthases (endothelial, neuronal, and/or inducible NOSs [eNOS, nNOS, and/or iNOS] and exogenous sources (entero-salivary NO pathway and the amount of NO from exogenous sources varies significantly; 2 NOSs are located at discrete compartments of cardiac myocytes and are regulated by distinctive mechanisms under stress; 3 NO regulates diverse target proteins through different modes of post-transcriptional modification (soluble guanylate cyclase [sGC]/cyclic guanosine monophosphate [cGMP]/protein kinase G [PKG]-dependent phosphorylation, S-nitrosylation, and transnitrosylation; 4 the downstream effectors of NO are multidimensional and vary from ion channels in the plasma membrane to signalling proteins and enzymes in the mitochondria, cytosol, nucleus, and myofilament; 5 NOS produces several radicals in addition to NO (e.g. superoxide, hydrogen peroxide, peroxynitrite, and different NO-related derivatives and triggers redox-dependent responses. However, nNOS inhibits cardiac oxidases to reduce the sources of oxidative stress in diseased hearts. Recent consensus indicates the importance of nNOS protein in cardiac protection under pathological stress. In addition, a dietary regime with high nitrate intake from fruit and vegetables together with unsaturated fatty acids is strongly associated with reduced cardiovascular events. Collectively, NO-dependent mechanisms in healthy and diseased hearts are better understood and shed light on the therapeutic prospects for NO and NOSs in clinical applications for fatal human heart diseases.

  11. Oxidized lipoproteins induce long-lasting inhibition of nitric oxide synthase from a murine endothelioma cell line (bEnd.4).

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    Fogliatto, G; Musanti, R; Pirillo, A; Ghiselli, G

    1995-04-01

    The vascular endothelium produces nitric oxide, which has vasodilatory properties. It has been postulated that some lipoproteins may increase arterial vascular tone by decreasing the availability of endothelium-derived nitric oxide. The mechanism underlying this effect, however, is still poorly understood. We investigated the effect of native and oxidized human low- and high-density lipoproteins on the nitric oxide synthetic activity of an endothelioma cell line (bEnd.4). Oxidized lipoproteins were obtained by incubation with CuSO4. The production of nitric oxide by the cells was monitored by quantifying the nitrite concentration in the medium using Greiss reagent. The synthesis of nitric oxide by the bEnd.4 cell line was calcium-dependent and was abolished by a selective inhibitor of the constitutive nitric oxide synthase. Incubation with oxidized lipoproteins caused a time- and dose-dependent inhibition of nitric oxide synthetic activity. At a concentration of 100 micrograms/ml cholesterol, oxidized low- and high-density lipoproteins inhibited the production of nitric oxide by 27 and 51%, respectively, within 6h. The lipid fraction obtained from the native or the oxidized lipoproteins mimicked the effect of the intact lipoproteins. These results support the involvement of oxidized lipoproteins in the modulation of endothelial functions relevant to the pathogenesis of cardiovascular disease.

  12. Vascular Kinin B1 and B2 Receptors Determine Endothelial Dysfunction through Neuronal Nitric Oxide Synthase

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    Luciano Dos Santos A. Capettini

    2017-04-01

    Full Text Available B1- and B2-kinin receptors are G protein-coupled receptors that play an important role in the vascular function. Therefore, the present study was designed to evaluate the participation of kinin receptors in the acetylcholine (ACh-induced vascular relaxation, focusing on the protein-protein interaction involving kinin receptors with endothelial and neuronal nitric oxide synthases (eNOS and nNOS. Vascular reactivity, nitric oxide (NO· and reactive oxygen species (ROS generation, co-immunoprecipitation were assessed in thoracic aorta from male wild-type (WT, B1- (B1R−/−, B2- (B2R−/− knockout mice. Some vascular reactivity experiments were also performed in a double kinin receptors knockout mice (B1B2R−/−. For pharmacological studies, selective B1- and B2-kinin receptors antagonists, NOS inhibitors and superoxide dismutase (SOD mimetic were used. First, we show that B1- and B2-kinin receptors form heteromers with nNOS and eNOS in thoracic aorta. To investigate the functionality of these protein-protein interactions, we took advantage of pharmacological tools and knockout mice. Importantly, our results show that kinin receptors regulate ACh-induced relaxation via nNOS signaling in thoracic aorta with no changes in NO· donor-induced relaxation. Interestingly, B1B2R−/− presented similar level of vascular dysfunction as found in B1R−/− or B2R−/− mice. In accordance, aortic rings from B1R−/− or B2R−/− mice exhibit decreased NO· bioavailability and increased superoxide generation compared to WT mice, suggesting the involvement of excessive ROS generation in the endothelial dysfunction of B1R−/− and B2R−/− mice. Alongside, we show that impaired endothelial vasorelaxation induced by ACh in B1R−/− or B2R−/− mice was rescued by the SOD mimetic compound. Taken together, our findings show that B1- and B2-kinin receptors regulate the endothelium-dependent vasodilation of ACh through nNOS activity and indicate

  13. Nitric oxide synthase and cyclooxygenase modulate β-adrenergic cutaneous vasodilatation and sweating in young men.

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    Fujii, Naoto; McNeely, Brendan D; Kenny, Glen P

    2017-02-15

    β-Adrenergic receptor agonists such as isoproterenol induce cutaneous vasodilatation and sweating in humans, but the mechanisms underpinning this response remain unresolved. Using intradermal microdialysis, we evaluated the roles of nitric oxide synthase (NOS) and cyclooxygenase (COX) in β-adrenergic cutaneous vasodilatation and sweating elicited by administration of isoproterenol. We show that while NOS contributes to β-adrenergic cutaneous vasodilatation, COX restricts cutaneous vasodilatation. We also show that combined inhibition of NOS and COX augments β-adrenergic sweating These new findings advance our basic knowledge regarding the physiological control of cutaneous blood flow and sweating, and provide important and new information to better understand the physiological significance of β-adrenergic receptors in the skin. β-Adrenergic receptor agonists such as isoproterenol can induce cutaneous vasodilatation and sweating in humans, but the mechanisms underpinning this response remain unresolved. We evaluated the hypotheses that (1) nitric oxide synthase (NOS) contributes to β-adrenergic cutaneous vasodilatation, whereas cyclooxygenase (COX) limits the vasodilatation, and (2) COX contributes to β-adrenergic sweating. In 10 young males (25 ± 5 years), cutaneous vascular conductance (CVC) and sweat rate were evaluated at four intradermal forearm skin sites infused with (1) lactated Ringer solution (control), (2) 10 mm N ω -nitro-l-arginine (l-NNA), a non-specific NOS inhibitor, (3) 10 mm ketorolac, a non-specific COX inhibitor, or (4) a combination of l-NNA and ketorolac. All sites were co-administered with a high dose of isoproterenol (100 μm) for 3 min to maximally induce β-adrenergic sweating (β-adrenergic sweating is significantly blunted by subsequent activations). Approximately 60 min after the washout period, three incremental doses of isoproterenol were co-administered (1, 10 and 100 μm each for 25 min). Increases in CVC induced

  14. Normalization of Supine Blood Pressure After Nitric Oxide Synthase Inhibition in Persons With Tetraplegia

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    Wecht, Jill M; Weir, Joseph P; Krothe, AnnMarie H; Spungen, Ann M; Bauman, William A

    2007-01-01

    Background/Objective: Orthostatic hypotension is a well-defined clinical consequence of spinal cord injury (SCI), particularly in those with tetraplegia. The etiology of orthostatic hypotension is thought to be loss of sympathetic vasomotor control, although other factors may play a role. There is evidence of up-regulation of nitric oxide synthase (NOS) activity after hind-limb suspension in rats, a condition of antigravity that may have similar vascular effects as shown in persons with tetraplegia caused by paralysis. The study objective was to determine the effect of a NOS inhibitor (nitro-L-arginine methyl ester [L-NAME]) on supine mean arterial pressure in persons with chronic tetraplegia compared with non-SCI controls. Methods: Fourteen individuals participated (7 with tetraplegia and 7 controls). Subjects visited the laboratory twice for placebo on day 1 and L-NAME (1 mg/kg) on day 2; both were infused intravenously over 60 minutes. Blood pressure was monitored for 3 hours after infusion at the brachial artery using a standard manual cuff. Results: Mean arterial pressure (MAP) was lower at baseline (P tetraplegia group compared with the control group. L-NAME increased MAP in both groups; however, the relative increase was greater in the tetraplegia group compared with the control group, such that group differences for MAP were eliminated. Supine MAP was normalized with L-NAME, and there was an increased sensitivity to NOS inhibition in the group with tetraplegia. Conclusions: These findings indicate that blood pressure dysregulation in persons with tetraplegia may reflect increased vascular NO and suggest a novel treatment of hypotension using NOS inhibition in this population. PMID:17385265

  15. Adrenalectomy alters regulation of blood pressure and endothelial nitric oxide synthase in sheep: modulation by estradiol.

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    Li, Feng; Wood, Charles E; Keller-Wood, Maureen

    2007-07-01

    Hypoadrenocorticism produces more severe hypotension during the peripartal period in pregnant ewes and women. We hypothesized that estradiol increases the severity of hypotension after withdrawal of corticosteroids and that this results from combined effects of adrenalectomy and estradiol to increase endothelial nitric oxide synthase (eNOS). In study I, blood pressure and eNOS mRNA and protein in aorta, uterine, renal, and mesenteric arteries were measured in intact ewes or adrenalectomized ewes 18-20 h after cessation of infusion of cortisol and aldosterone; half of each group ewes were treated with estradiol. In study II, adrenalectomized ewes were similarly studied 22-28 h after withdrawal of corticosteroids. Estradiol treatment in both studies significantly increased eNOS mRNA and protein in uterine artery, whereas corticosteroid withdrawal decreased expression of eNOS mRNA and protein in uterine artery. In both studies, adrenalectomy and steroid withdrawal decreased mean arterial pressure. In study II, four of six adrenalectomized ewes not treated with estradiol showed dramatic phasic variations in blood pressure and heart rate with a period of approximately 20 s, developing within 22-28 h after corticosteroid withdrawal. Although there was no effect of estradiol on blood pressure in study I, in study II, ewes treated with estradiol did not develop this pattern. Estradiol also slowed both the decline in plasma sodium and the rise in plasma potassium after corticosteroid withdrawal. These results disprove the hypothesis that estradiol increases the severity of hypotension during hypoadrenocorticism. However, the study reveals an important effect of corticosteroid withdrawal on blood pressure, consistent with corticosteroid modulation of baroreflex responsiveness.

  16. Endothelial nitric oxide synthase single nucleotide polymorphism and left ventricular function in early chronic kidney disease.

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    Sourabh Chand

    Full Text Available Chronic kidney disease (CKD is associated with accelerated cardiovascular disease and heart failure. Endothelial nitric oxide synthase (eNOS Glu298Asp single nucleotide polymorphism (SNP genotype has been associated with a worse phenotype amongst patients with established heart failure and in patients with progression of their renal disease. The association of a cardiac functional difference in non-dialysis CKD patients with no known previous heart failure, and eNOS gene variant is investigated.140 non-dialysis CKD patients, who had cardiac magnetic resonance (CMR imaging and tissue doppler echocardiography as part of two clinical trials, were genotyped for eNOS Glu298Asp SNP retrospectively.The median estimated glomerular filtration rate (eGFR was 50 mls/min and left ventricular ejection fraction (LVEF was 74% with no overt diastolic dysfunction in this cohort. There were significant differences in LVEF across eNOS genotypes with GG genotype being associated with a worse LVEF compared to other genotypes (LVEF: GG 71%, TG 76%, TT 73%, p = 0.006. After multivariate analysis, (adjusting for age, eGFR, baseline mean arterial pressure, contemporary CMR heart rate, total cholesterol, high sensitive C-reactive protein, body mass index and gender GG genotype was associated with a worse LVEF, and increased LV end-diastolic and systolic index (p = 0.004, 0.049 and 0.009 respectively.eNOS Glu298Asp rs1799983 polymorphism in CKD patients is associated with relevant sub-clinical cardiac remodelling as detected by CMR. This gene variant may therefore represent an important genetic biomarker, and possibly highlight pathways for intervention, in these patients who are at particular risk of worsening cardiac disease as their renal dysfunction progresses.

  17. Vascular endothelial growth factor and nitric oxide synthase expression in human tooth germ development.

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    Mastrangelo, F; Sberna, M T; Tettamanti, L; Cantatore, G; Tagliabue, A; Gherlone, E

    2016-01-01

    Vascular Endothelia Growth Factor (VEGF) and Nitric Oxide Synthase (NOS) expression, were evaluated in human tooth germs at two different stages of embryogenesis, to clarify the role of angiogenesis during tooth tissue differentiation and growth. Seventy-two third molar germ specimens were selected during oral surgery. Thirty-six were in the early stage and 36 in the later stage of tooth development. The samples were evaluated with Semi-quantitative Reverse Transcription-Polymerase chain Reaction analyses (RT-PcR), Western blot analysis (WB) and immunohistochemical analysis. Western blot and immunohistochemical analysis showed a VEGF and NOS 1-2-3 positive reaction in all samples analysed. VEGF high positive decrease reaction was observed in stellate reticulum cells, ameloblast and odontoblast clusters in early stage compared to later stage of tooth germ development. Comparable VEGF expression was observed in endothelial cells of early and advanced stage growth. NOS1 and NOS3 expressions showed a high increased value in stellate reticulum cells, and ameloblast and odontoblast clusters in advanced stage compared to early stage of development. The absence or only moderate positive reaction of NOS2 was detected in all the different tissues. Positive NOS2 expression showed in advanced stage of tissue development compared to early stage. The action of VEGF and NOS molecules are important mediators of angiogenesis during dental tissue development. VEGF high positive expression in stellate reticulum cells in the early stage of tooth development compared to the later stage and the other cell types, suggests a critical role of the stellate reticulum during dental embryo-morphogenesis.

  18. Prolactin alters blood pressure by modulating the activity of endothelial nitric oxide synthase.

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    Chang, Albert S; Grant, Ruriko; Tomita, Hirofumi; Kim, Hyung-Suk; Smithies, Oliver; Kakoki, Masao

    2016-11-01

    Increased levels of a cleaved form of prolactin (molecular weight 16 kDa) have been associated with preeclampsia. To study the effects of prolactin on blood pressure (BP), we generated male mice with a single-copy transgene (Tg; inserted into the hypoxanthine-guanine phosphoribosyltransferase locus) that enables inducible hepatic production of prolactin and its cleavage product. The Tg is driven by the indole-3-carbinol (I3C)-inducible rat cytochrome P450 1A1 promoter. When the Tg mice were fed normal chow (NC), plasma prolactin concentrations were comparable to those in female WT mice in the last third of pregnancy, and BP was lower than in WT mice (∼95 mm Hg vs. ∼105 mm Hg). When the Tg mice were fed chow containing IC3, plasma prolactin concentrations increased threefold, BP increased to ∼130 mm Hg, and cardiac function became markedly impaired. IC3 chow did not affect the WT mice. Urinary excretion of nitrite/nitrate and the amount of Ser1177-phosphorylated endothelial nitric oxide (NO) synthase (eNOS) were significantly greater in the Tg mice fed NC than in WT mice, as they are during pregnancy. However, when I3C was fed, these indicators of NO production became significantly less in the Tg mice than in WT mice. The effects of increased plasma prolactin were abolished by a genetic absence of eNOS. Thus, a threefold increase in plasma prolactin is sufficient to increase BP significantly and to markedly impair cardiac function, with effects mediated by NO produced by eNOS. We suggest that pregnant women with abnormally high prolactin levels may need special attention.

  19. Nocardia iowensis sp. nov., an organism rich in biocatalytically important enzymes and nitric oxide synthase

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    Lamm, Andrew S.; Khare, Arshdeep; Conville, Patricia; Lau, Peter C. K.; Bergeron, Hélène; Rosazza, John P. N.

    2009-01-01

    Nocardia strain NRRL 5646, isolated from a garden soil sample in Osceola, Iowa, USA, was initially of interest as an antibiotic producer. It contained biocatalytically important enzymes and represented the first described nitric oxide synthase enzyme system in bacteria. The present polyphasic taxonomic study was undertaken to differentiate strain NRRL 5646T from related species of the genus Nocardia. Chemotaxonomic analyses included determinations of the fatty acid methyl ester profile (C16 : 1ω6c/C16 : 1ω7c, C16 : 0, C18 : 1ω9c and C18 : 0 10-methyl as major components), quinone [cyclo MK-8(H4) as the major component], polar lipid (diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside as major components) and mycolic acid. These results supported its placement within the genus Nocardia. Biochemical testing and 16S rRNA, 65-kDa heat-shock protein (hsp65) and preprotein translocase (secA1) gene sequence analyses differentiated strain NRRL 5646T from recognized Nocardia species. Previous studies have demonstrated that other genetic sequences (carboxylic acid reductase, Nocardia phosphopantetheinyl transferase and GTP cyclohydrolase I) from strain NRRL 5646T can also be used to substantiate its uniqueness. The level of 16S rRNA gene sequence similarity between strain NRRL 5646T and the type strains of Nocardia tenerifensis and Nocardia brasiliensis was 98.8 %. However, strain NRRL 5646T could be clearly distinguished from these Nocardia species based on DNA–DNA hybridization data. Consequently, strain NRRL 5646T is considered to represent a novel species of the genus Nocardia, for which the name Nocardia iowensis sp. nov. is proposed. The type strain is NRRL 5646T (=UI 122540T=NRRL B-24671T=DSM 45197T). PMID:19622667

  20. Effect of acute nitric oxide synthase inhibition in the modulation of heart rate in rats

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    A.L. Fellet

    2003-05-01

    Full Text Available Acute nitric oxide synthase inhibition with N G-nitro-L-arginine methyl ester (L-NAME on chronotropic and pressor responses was studied in anesthetized intact rats and rats submitted to partial and complete autonomic blockade. Blood pressure and heart rate were monitored intra-arterially. Intravenous L-NAME injection (7.5 mg/kg elicited the same hypertensive response in intact rats and in rats with partial (ganglionic and parasympathetic blockade and complete autonomic blockade (38 ± 3, 55 ± 6, 54 ± 5, 45 ± 5 mmHg, respectively; N = 9, P = NS. L-NAME-induced bradycardia at the time when blood pressure reached the peak plateau was similar in intact rats and in rats with partial autonomic blockade (43 ± 8, 38 ± 5, 46 ± 6 bpm, respectively; N = 9, P = NS. Rats with combined autonomic blockade showed a tachycardic response to L-NAME (10 ± 3 bpm, P<0.05 vs intact animals, N = 9. Increasing doses of L-NAME (5.0, 7.5 and 10 mg/kg, N = 9 caused a similar increase in blood pressure (45 ± 5, 38 ± 3, 44 ± 9 mmHg, respectively; P = NS and heart rate (31 ± 4, 34 ± 3, 35 ± 4 bpm, respectively; P = NS. Addition of L-NAME (500 µM to isolated atria from rats killed by cervical dislocation and rats previously subjected to complete autonomic blockade did not affect spontaneous beating or contractile strength (N = 9. In vivo results showed that L-NAME promoted a tachycardic response in rats with complete autonomic blockade, whereas the in vitro experiments showed no effect on intrinsic heart rate, suggesting that humoral mechanisms may be involved in the L-NAME-induced cardiac response.

  1. Packed red blood cells are an abundant and proximate potential source of nitric oxide synthase inhibition.

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    Charles F Zwemer

    Full Text Available We determined, for packed red blood cells (PRBC and fresh frozen plasma, the maximum content, and ability to release the endogenous nitric oxide synthase (NOS inhibitors asymmetric dimethylarginine (ADMA and monomethylarginine (LNMMA.ADMA and LNMMA are near equipotent NOS inhibitors forming blood's total NOS inhibitory content. The balance between removal from, and addition to plasma determines their free concentrations. Removal from plasma is by well-characterized specific hydrolases while formation is restricted to posttranslational protein methylation. When released into plasma they can readily enter endothelial cells and inhibit NOS. Fresh rat and human whole blood contain substantial protein incorporated ADMA however; the maximum content of ADMA and LNMMA in PRBC and fresh frozen plasma has not been determined.We measured total (free and protein incorporated ADMA and LNMMA content in PRBCs and fresh frozen plasma, as well as their incubation induced release, using HPLC with fluorescence detection. We tested the hypothesis that PRBC and fresh frozen plasma contain substantial inhibitory methylarginines that can be released chemically by complete in vitro acid hydrolysis or physiologically at 37°C by enzymatic blood proteolysis.In vitro strong-acid-hydrolysis revealed a large PRBC reservoir of ADMA (54.5 ± 9.7 µM and LNMMA (58.9 ± 28.9 μM that persisted over 42-d at 6° or -80°C. In vitro 5h incubation at 37°C nearly doubled free ADMA and LNMMNA concentration from PRBCs while no change was detected in fresh frozen plasma.The compelling physiological ramifications are that regardless of storage age, 1 PRBCs can rapidly release pathologically relevant quantities of ADMA and LNMMA when incubated and 2 PRBCs have a protein-incorporated inhibitory methylarginines reservoir 100 times that of normal free inhibitory methylarginines in blood and thus could represent a clinically relevant and proximate risk for iatrogenic NOS inhibition upon

  2. Involvement of inducible nitric oxide synthase in radiation-induced vascular endothelial damage

    International Nuclear Information System (INIS)

    Hong, Chang-Won; Lee, Joon-Ho; Kim, Suwan; Noh, Jae Myoung; Kim, Young-Mee; Pyo, Hongryull; Lee, Sunyoung

    2013-01-01

    The use of radiation therapy has been linked to an increased risk of cardiovascular disease. To understand the mechanisms underlying radiation-induced vascular dysfunction, we employed two models. First, we examined the effect of X-ray irradiation on vasodilation in rabbit carotid arteries. Carotid arterial rings were irradiated with 8 or 16 Gy using in vivo and ex vivo methods. We measured the effect of acetylcholine-induced relaxation after phenylephrine-induced contraction on the rings. In irradiated carotid arteries, vasodilation was significantly attenuated by both irradiation methods. The relaxation response was completely blocked by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a potent inhibitor of soluble guanylate cyclase. Residual relaxation persisted after treatment with L-N ω -nitroarginine (L-NA), a non-specific inhibitor of nitric oxide synthase (NOS), but disappeared following the addition of aminoguanidine (AG), a selective inhibitor of inducible NOS (iNOS). The relaxation response was also affected by tetraethylammonium, an inhibitor of endothelium-derived hyperpolarizing factor activity. In the second model, we investigated the biochemical events of nitrosative stress in human umbilical-vein endothelial cells (HUVECs). We measured iNOS and nitrotyrosine expression in HUVECs exposed to a dose of 4 Gy. The expression of iNOS and nitrotyrosine was greater in irradiated HUVECs than in untreated controls. Pretreatment with AG, L-N 6 -(1-iminoethyl) lysine hydrochloride (a selective inhibitor of iNOS), and L-NA attenuated nitrosative stress. While a selective target of radiation-induced vascular endothelial damage was not definitely determined, these results suggest that NO generated from iNOS could contribute to vasorelaxation. These studies highlight a potential role of iNOS inhibitors in ameliorating radiation-induced vascular endothelial damage. (author)

  3. Towards the free energy landscape for catalysis in mammalian nitric oxide synthases.

    Science.gov (United States)

    Leferink, Nicole G H; Hay, Sam; Rigby, Stephen E J; Scrutton, Nigel S

    2015-08-01

    The general requirement for conformational sampling in biological electron transfer reactions catalysed by multi-domain redox systems has been emphasized in recent years. Crucially, we lack insight into the extent of the conformational space explored and the nature of the energy landscapes associated with these reactions. The nitric oxide synthases (NOS) produce the signalling molecule NO through a series of complex electron transfer reactions. There is accumulating evidence that protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH, through the FAD and FMN cofactors, to the haem oxygenase domain, where NO is generated. Simple models based on static crystal structures of the isolated reductase domain have suggested a role for large-scale motions of the FMN-binding domain in shuttling electrons from the reductase domain to the oxygenase domain. However, detailed insight into the higher-order domain architecture and dynamic structural transitions in NOS enzymes during enzyme turnover is lacking. In this review, we discuss the recent advances made towards mapping the catalytic free energy landscapes of NOS enzymes through integration of both structural techniques (e.g. cryo-electron microscopy) and biophysical techniques (e.g. pulsed-electron paramagnetic resonance). The general picture that emerges from these experiments is that NOS enzymes exist in an equilibrium of conformations, comprising a 'rugged' or 'frustrated' energy landscape, with a key regulatory role for calmodulin in driving vectorial electron transfer by altering the conformational equilibrium. A detailed understanding of these landscapes may provide new opportunities for discovery of isoform-specific inhibitors that bind at the dynamic interfaces of these multi-dimensional energy landscapes. © 2014 FEBS.

  4. Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection

    Directory of Open Access Journals (Sweden)

    M. Chacur

    2010-04-01

    Full Text Available Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO. In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT. Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test and allodynia (von Frey hair test. Control animals did not present any alteration (sham-animals. The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 µg in 50 µL, blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48% by day 30 in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed’s lamina X and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469%. Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20%, reaching the greatest increase (60% 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

  5. Effects of nitric oxide synthase inhibition on cutaneous vasodilation in response to acupuncture stimulation in humans.

    Science.gov (United States)

    Kimura, Kenichi; Takeuchi, Hayato; Yuri, Kuniko; Wakayama, Ikuro

    2013-03-01

    The aim of the present study was to elucidate the mechanism of cutaneous vasodilation following acupuncture stimulation by investigating the roles of nitric oxide (NO) and axon reflex vasodilation. The subjects were 17 healthy male volunteers. The role of NO was investigated by administering N(G)-nitro-l-arginine methyl ester hydrochloride (L-NAME, 20 mM), an NO synthase inhibitor or Ringer's solution (control site), via intradermal microdialysis (protocol 1; n=7). The role of axon reflex vasodilation by local sensory neurones was investigated by comparing vasodilation at sites treated with 'eutectic mixture of local anaesthetics' (EMLA) cream (2.5% lidocaine and 2.5% prilocaine) with untreated sites (control site) (protocol 2; n=10). After 5 min of baseline recording, acupuncture was applied to PC4 and a control site in proximity to PC4 for 10 min and scanning was performed for 60 min after acupuncture stimulation. Skin blood flow (SkBF) was evaluated by laser Doppler perfusion imaging. Cutaneous vascular conductance (CVC) was calculated from the ratio of SkBF to mean arterial blood pressure. In the first protocol, sites administered L-NAME showed significant reductions in CVC responses following acupuncture stimulation compared to control sites (administered Ringer's solution) (pacupuncture stimulation did not differ significantly between treated sites with EMLA cream and untreated sites (p>0.05). These data suggest that cutaneous vasodilation in response to acupuncture stimulation may not occur through an axon reflex as previously reported. Rather, NO mechanisms appear to contribute to the vasodilator response.

  6. Response of cardiac endothelial nitric oxide synthase to plasma viscosity modulation in acute isovolemic hemodilution

    Directory of Open Access Journals (Sweden)

    Kanyanatt Kanokwiroon

    2014-01-01

    Full Text Available Background: Endothelial nitric oxide synthase (eNOS is generally expressed in endocardial cells, vascular endothelial cells and ventricular myocytes. However, there is no experimental study elucidating the relationship between cardiac eNOS expression and elevated plasma viscosity in low oxygen delivery pathological conditions such as hemorrhagic shock-resuscitation and hemodilution. This study tested the hypothesis that elevated plasma viscosity increases cardiac eNOS expression in a hemodilution model, leading to positive effects on cardiac performance. Materials and Methods: Two groups of golden Syrian hamster underwent an acute isovolemic hemodilution where 40% of blood volume was exchanged with 2% (low-viscogenic plasma expander [LVPE] or 6% (high-viscogenic plasma expander [HVPE] of dextran 2000 kDa. In control group, experiment was performed without hemodilution. All groups were performed in awake condition. Experimental parameters, i.e., mean arterial blood pressure (MAP, heart rate, hematocrit, blood gas content and viscosity, were measured. The eNOS expression was evaluated by eNOS Western blot analysis. Results: After hemodilution, MAP decreased to 72% and 93% of baseline in the LVPE and HVPE, respectively. Furthermore, pO 2 in the LVPE group increased highest among the groups. Plasma viscosity in the HVPE group was significantly higher than that in control and LVPE groups. The expression of eNOS in the HVPE group showed higher intensity compared to other groups, especially compared with the control group. Conclusion: Our results demonstrated that cardiac eNOS has responded to plasma viscosity modulation with HVPE and LVPE. This particularly supports the previous studies that revealed the positive effects on cardiac function in animals hemodiluted with HVPE.

  7. Endothelial Nitric Oxide Synthase Gene Variation Associated With Chronic Kidney Disease After Liver Transplant

    Science.gov (United States)

    Bambha, Kiran; Kim, W. Ray; Rosen, Charles B.; Pedersen, Rachel A.; Rys, Cynthia; Kolbert, Christopher P.; Cunningham, Julie M.; Therneau, Terry M.

    2010-01-01

    OBJECTIVE: To identify single nucleotide polymorphisms (SNPs) associated with risk of developing chronic kidney disease (CKD), a prevalent comorbidity, after liver transplant (LT). PATIENTS AND METHODS: This study consists of a cohort of adult (≥18 years) primary-LT recipients who had normal renal function before LT and who survived 1 year or more after LT at a high-volume US LT program between January 1, 1990, and December 31, 2000. Patients with adequate renal function (estimated glomerular filtration rate, ≥40 mL/min per 1.73 m2 during follow-up; n=308) and patients with incident CKD (estimated glomerular filtration rate, <40 mL/min per 1.73 m2 after LT; n=92) were identified. To investigate the association of 6 candidate genes with post-LT CKD, we selected SNPs that have been associated with renal function in the literature. Hazard ratios were estimated using Cox regression, adjusted for potential confounding variables. RESULTS: The variant allele (298Asp) of the Glu298Asp SNP in the endothelial nitric oxide synthase gene (NOS3) was significantly associated with CKD after LT (P=.05; adjusted for multiple comparisons). The 5-year incidence of CKD was 70% among patients homozygous for the NOS3 variant allele (298Asp) compared with 42% among those not homozygous for the NOS3 variant allele. Specifically, homozygosity for the NOS3 variant allele conferred a 2.5-fold increased risk of developing CKD after LT (P=.005, adjusted for confounding variables). CONCLUSION: Homozygosity for the variant allele of NOS3 (298Asp) is associated with CKD after LT and may be useful for identifying recipients at higher risk of post-LT CKD. PMID:20810793

  8. Deltamethrin-induced testicular apoptosis in rats: the protective effect of nitric oxide synthase inhibitor.

    Science.gov (United States)

    El-Gohary, M; Awara, W M; Nassar, S; Hawas, S

    1999-01-01

    This study is the first to examine and characterize the testicular apoptosis which might be induced due to exposure of male rats to deltamethrin. Furthermore, the role which might be played by nitric oxide (NO), as well as the other reactive oxygen species (ROS) in controlling this testicular apoptosis was assessed. Apoptosis was evaluated by DNA fragmentation detected by agarose gel electrophoresis and cellular morphology on testicular tissue sections. It was found that administration of deltamethrin (1 mg/kg daily for 21 days) to animals resulted in characteristic DNA migration patterns (laddering), thereby providing evidence that apoptosis is the major mechanism of cell death in the testicular tissues. In addition, histopathological examination of testicular tissue sections showed that apoptosis was confined to the basal germ cells, primary and secondary spermatocytes. These changes, in addition to the appearance of Sertoli cell vacuoles in deltamethrin-intoxicated animals, indicates the suppression of spermatogenesis. At the same time, the plasma levels of both NO and lipid peroxides measured as malondialdehyde (MDA) were found to be significantly increased in deltamethrin-treated animals. Administration of NO synthase (NOS) inhibitors such as N(G)-nitro monomethyl L-arginine hydrochloride (L-NMMA, 1 mg/kg) to rats 2 h before exposure to deltamethrin was effective in the reduction of the typically testicular apoptotic DNA fragmentation pattern and the associated histopathological changes. These findings may suggest that deltamethrin-induced testicular apoptosis is mediated by NO. Therefore, the pharmacological manipulation of apoptosis by selective NOS inhibitors such as L-NMMA may offer new possibilities for the control of deltamethrin-induced testicular dysfunction and infertility in the future.

  9. Structural Basis of Neuronal Nitric-oxide Synthase Interaction with Dystrophin Repeats 16 and 17*

    Science.gov (United States)

    Molza, Anne-Elisabeth; Mangat, Khushdeep; Le Rumeur, Elisabeth; Hubert, Jean-François; Menhart, Nick; Delalande, Olivier

    2015-01-01

    Duchenne muscular dystrophy is a lethal genetic defect that is associated with the absence of dystrophin protein. Lack of dystrophin protein completely abolishes muscular nitric-oxide synthase (NOS) function as a regulator of blood flow during muscle contraction. In normal muscles, nNOS function is ensured by its localization at the sarcolemma through an interaction of its PDZ domain with dystrophin spectrin-like repeats R16 and R17. Early studies suggested that repeat R17 is the primary site of interaction but ignored the involved nNOS residues, and the R17 binding site has not been described at an atomic level. In this study, we characterized the specific amino acids involved in the binding site of nNOS-PDZ with dystrophin R16–17 using combined experimental biochemical and structural in silico approaches. First, 32 alanine-scanning mutagenesis variants of dystrophin R16–17 indicated the regions where mutagenesis modified the affinity of the dystrophin interaction with the nNOS-PDZ. Second, using small angle x-ray scattering-based models of dystrophin R16–17 and molecular docking methods, we generated atomic models of the dystrophin R16–17·nNOS-PDZ complex that correlated well with the alanine scanning identified regions of dystrophin. The structural regions constituting the dystrophin interaction surface involve the A/B loop and the N-terminal end of helix B of repeat R16 and the N-terminal end of helix A′ and a small fraction of helix B′ and a large part of the helix C′ of repeat R17. The interaction surface of nNOS-PDZ involves its main β-sheet and its specific C-terminal β-finger. PMID:26378238

  10. Structural Basis of Neuronal Nitric-oxide Synthase Interaction with Dystrophin Repeats 16 and 17.

    Science.gov (United States)

    Molza, Anne-Elisabeth; Mangat, Khushdeep; Le Rumeur, Elisabeth; Hubert, Jean-François; Menhart, Nick; Delalande, Olivier

    2015-12-04

    Duchenne muscular dystrophy is a lethal genetic defect that is associated with the absence of dystrophin protein. Lack of dystrophin protein completely abolishes muscular nitric-oxide synthase (NOS) function as a regulator of blood flow during muscle contraction. In normal muscles, nNOS function is ensured by its localization at the sarcolemma through an interaction of its PDZ domain with dystrophin spectrin-like repeats R16 and R17. Early studies suggested that repeat R17 is the primary site of interaction but ignored the involved nNOS residues, and the R17 binding site has not been described at an atomic level. In this study, we characterized the specific amino acids involved in the binding site of nNOS-PDZ with dystrophin R16-17 using combined experimental biochemical and structural in silico approaches. First, 32 alanine-scanning mutagenesis variants of dystrophin R16-17 indicated the regions where mutagenesis modified the affinity of the dystrophin interaction with the nNOS-PDZ. Second, using small angle x-ray scattering-based models of dystrophin R16-17 and molecular docking methods, we generated atomic models of the dystrophin R16-17·nNOS-PDZ complex that correlated well with the alanine scanning identified regions of dystrophin. The structural regions constituting the dystrophin interaction surface involve the A/B loop and the N-terminal end of helix B of repeat R16 and the N-terminal end of helix A' and a small fraction of helix B' and a large part of the helix C' of repeat R17. The interaction surface of nNOS-PDZ involves its main β-sheet and its specific C-terminal β-finger. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Inducible nitric oxide synthase (iNOS) regulatory region variation in non-human primates.

    Science.gov (United States)

    Roodgar, Morteza; Ross, Cody T; Kenyon, Nicholas J; Marcelino, Gretchen; Smith, David Glenn

    2015-04-01

    Inducible nitric oxide synthase (iNOS) is an enzyme that plays a key role in intracellular immune response against respiratory infections. Since various species of nonhuman primates exhibit different levels of susceptibility to infectious respiratory diseases, and since variation in regulatory regions of genes is thought to play a key role in expression levels of genes, two candidate regulatory regions of iNOS were mapped, sequenced, and compared across five species of nonhuman primates: African green monkeys (Chlorocebus sabaeus), pig-tailed macaques (Macaca nemestrina), cynomolgus macaques (Macaca fascicularis), Indian rhesus macaques (Macaca mulatta), and Chinese rhesus macaques (M. mulatta). In addition, we conducted an in silico analysis of the transcription factor binding sites associated with genetic variation in these two candidate regulatory regions across species. We found that only one of the two candidate regions showed strong evidence of involvement in iNOS regulation. Specifically, we found evidence of 13 conserved binding site candidates linked to iNOS regulation: AP-1, C/EBPB, CREB, GATA-1, GATA-3, NF-AT, NF-AT5, NF-κB, KLF4, Oct-1, PEA3, SMAD3, and TCF11. Additionally, we found evidence of interspecies variation in binding sites for several regulatory elements linked to iNOS (GATA-3, GATA-4, KLF6, SRF, STAT-1, STAT-3, OLF-1 and HIF-1) across species, especially in African green monkeys relative to other species. Given the key role of iNOS in respiratory immune response, the findings of this study might help guide the direction of future studies aimed to uncover the molecular mechanisms underlying the increased susceptibility of African green monkeys to several viral and bacterial respiratory infections. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Gene therapy of transplant arteriopathy by liposome-mediated transfection of endothelial nitric oxide synthase.

    Science.gov (United States)

    Iwata, A; Sai, S; Moore, M; Nyhuis, J; de Fries-Hallstrand, R; Quetingco, G C; Allen, M D

    2000-11-01

    Transplant arteriopathy is the major factor limiting long-term survival after cardiac transplantation. We have previously demonstrated that liposome-mediated gene delivery of endothelial nitric oxide synthase (eNOS) to donor hearts reduces ischemia-reperfusion injury by blocking NFkappaB activation, adhesion molecule expression, and leukocyte infiltration. In this study, we used gene transfer of eNOS in a rabbit carotid transplant model to see whether these same effects would similarly ameliorate transplant arteriopathy. Liposomes complexed to the gene encoding eNOS were injected into donor carotid arterial segments that were transplanted orthotopically into recipient carotid arteries (n = 10). Controls included transplanted carotids transfected with liposomes complexed to empty plasmids (no functional gene) (n = 4) and transplanted carotids treated with saline (n = 6). Transplanted arteries were harvested for processing at 21 days. Intima/media (I/M) area ratios were calculated by computerized image analysis. Infiltrating T-lymphocytes and macrophages, and expression of VCAM-1 and ICAM-1 were quantified on immunocytochemistry. The I/M ratio was significantly reduced in eNOS-transfected arteries compared with arteries transfected with empty plasmids and saline-treated controls. Compared to transplanted control arteries, eNOS-transfected arteries demonstrated significantly reduced T-cell infiltration into the intima and significantly reduced macrophage infiltration into the media. Cell surface expression of VCAM-1 and ICAM-1 were both reduced in eNOS-transfected arteries. ENOS gene delivery can suppress neointimal lesion formation and T-lymphocyte and macrophage infiltration in transplanted arteries, associated with a reduction in relevant adhesion molecule expression. Thus, gene therapy with eNOS may not only reduce ischemia-reperfusion injury but may also ameliorate transplant arteriopathy in transplanted hearts.

  13. Role of heat shock protein 90 and endothelial nitric oxide synthase during early anesthetic and ischemic preconditioning.

    Science.gov (United States)

    Amour, Julien; Brzezinska, Anna K; Weihrauch, Dorothee; Billstrom, Amie R; Zielonka, Jacek; Krolikowski, John G; Bienengraeber, Martin W; Warltier, David C; Pratt, Philip F; Kersten, Judy R

    2009-02-01

    Nitric oxide is known to be essential for early anesthetic preconditioning (APC) and ischemic preconditioning (IPC) of myocardium. Heat shock protein 90 (Hsp90) regulates endothelial nitric oxide synthase (eNOS) activity. In this study, the authors tested the hypothesis that Hsp90-eNOS interactions modulate APC and IPC. Myocardial infarct size was measured in rabbits after coronary occlusion and reperfusion in the absence or presence of preconditioning within 30 min of isoflurane (APC) or 5 min of coronary artery occlusion (IPC), and with or without pretreatment with geldanamycin or radicicol, two chemically distinct Hsp90 inhibitors, or N-nitro-L-arginine methyl ester, a nonspecific nitric oxide synthase NOS inhibitor. Isoflurane-dependent nitric oxide production was measured (ozone chemiluminescence) in human coronary artery endothelial cells or mouse cardiomyocytes, in the absence or presence of Hsp90 inhibitors or N-nitro-L-arginine methyl ester. Interactions between Hsp90 and eNOS, and eNOS activation, were assessed with immunoprecipitation, immunoblotting, and confocal microscopy. APC and IPC decreased infarct size (by 50% and 59%, respectively), and this action was abolished by Hsp90 inhibitors. N-nitro-L-arginine methyl ester blocked APC but not IPC. Isoflurane increased nitric oxide production in human coronary artery endothelial cells concomitantly with an increase in Hsp90-eNOS interaction (immunoprecipitation, immunoblotting, and immunohistochemistry). Pretreatment with Hsp90 inhibitors abolished isoflurane-dependent nitric oxide production and decreased Hsp90-eNOS interactions. Isoflurane did not increase nitric oxide production in mouse cardiomyocytes, and eNOS was below the level of detection. The results indicate that Hsp90 plays a critical role in mediating APC and IPC through protein-protein interactions, and suggest that endothelial cells are important contributors to nitric oxide-mediated signaling during APC.

  14. Spinal motoneuron synaptic plasticity after axotomy in the absence of inducible nitric oxide synthase

    Directory of Open Access Journals (Sweden)

    Zanon Renata G

    2010-05-01

    Full Text Available Abstract Background Astrocytes play a major role in preserving and restoring structural and physiological integrity following injury to the nervous system. After peripheral axotomy, reactive gliosis propagates within adjacent spinal segments, influenced by the local synthesis of nitric oxide (NO. The present work investigated the importance of inducible nitric oxide synthase (iNOS activity in acute and late glial responses after injury and in major histocompatibility complex class I (MHC I expression and synaptic plasticity of inputs to lesioned alpha motoneurons. Methods In vivo analyses were carried out using C57BL/6J-iNOS knockout (iNOS-/- and C57BL/6J mice. Glial response after axotomy, glial MHC I expression, and the effects of axotomy on synaptic contacts were measured using immunohistochemistry and transmission electron microscopy. For this purpose, 2-month-old animals were sacrificed and fixed one or two weeks after unilateral sciatic nerve transection, and spinal cord sections were incubated with antibodies against classical MHC I, GFAP (glial fibrillary acidic protein - an astroglial marker, Iba-1 (an ionized calcium binding adaptor protein and a microglial marker or synaptophysin (a presynaptic terminal marker. Western blotting analysis of MHC I and nNOS expression one week after lesion were also performed. The data were analyzed using a two-tailed Student's t test for parametric data or a two-tailed Mann-Whitney U test for nonparametric data. Results A statistical difference was shown with respect to astrogliosis between strains at the different time points studied. Also, MHC I expression by iNOS-/- microglial cells did not increase at one or two weeks after unilateral axotomy. There was a difference in synaptophysin expression reflecting synaptic elimination, in which iNOS-/- mice displayed a decreased number of the inputs to alpha motoneurons, in comparison to that of C57BL/6J. Conclusion The findings herein indicate that i

  15. A role for endothelial nitric oxide synthase in intestinal stem cell proliferation and mesenchymal colorectal cancer.

    Science.gov (United States)

    Peñarando, Jon; López-Sánchez, Laura M; Mena, Rafael; Guil-Luna, Silvia; Conde, Francisco; Hernández, Vanessa; Toledano, Marta; Gudiño, Victoria; Raponi, Michela; Billard, Caroline; Villar, Carlos; Díaz, César; Gómez-Barbadillo, José; De la Haba-Rodríguez, Juan; Myant, Kevin; Aranda, Enrique; Rodríguez-Ariza, Antonio

    2018-01-10

    Nitric oxide (NO) has been highlighted as an important agent in cancer-related events. Although the inducible nitric oxide synthase (iNOS) isoform has received most attention, recent studies in the literature indicate that the endothelial isoenzyme (eNOS) can also modulate different tumor processes including resistance, angiogenesis, invasion, and metastasis. However, the role of eNOS in cancer stem cell (CSC) biology and mesenchymal tumors is unknown. Here, we show that eNOS was significantly upregulated in VilCre ERT2 Apc fl/+ and VilCre ERT2 Apc fl/fl mouse intestinal tissue, with intense immunostaining in hyperproliferative crypts. Similarly, the more invasive VilCre ERT2 Apc fl/+ Pten fl/+ mouse model showed an overexpression of eNOS in intestinal tumors whereas this isoform was not expressed in normal tissue. However, none of the three models showed iNOS expression. Notably, when 40 human colorectal tumors were classified into different clinically relevant molecular subtypes, high eNOS expression was found in the poor relapse-free and overall survival mesenchymal subtype, whereas iNOS was absent. Furthermore, Apc fl/fl organoids overexpressed eNOS compared with wild-type organoids and NO depletion with the scavenger carboxy-PTIO (c-PTIO) decreased the proliferation and the expression of stem-cell markers, such as Lgr5, Troy, Vav3, and Slc14a1, in these intestinal organoids. Moreover, specific NO depletion also decreased the expression of CSC-related proteins in human colorectal cancer cells such as β-catenin and Bmi1, impairing the CSC phenotype. To rule out the contribution of iNOS in this effect, we established an iNOS-knockdown colorectal cancer cell line. NO-depleted cells showed a decreased capacity to form tumors and c-PTIO treatment in vivo showed an antitumoral effect in a xenograft mouse model. Our data support that eNOS upregulation occurs after Apc loss, emerging as an unexpected potential new target in poor-prognosis mesenchymal colorectal tumors

  16. Allen's astrophysical quantities

    CERN Document Server

    2000-01-01

    This new, fourth, edition of Allen's classic Astrophysical Quantities belongs on every astronomer's bookshelf. It has been thoroughly revised and brought up to date by a team of more than ninety internationally renowned astronomers and astrophysicists. While it follows the basic format of the original, this indispensable reference has grown to more than twice the size of the earlier editions to accommodate the great strides made in astronomy and astrophysics. It includes detailed tables of the most recent data on: - General constants and units - Atoms, molecules, and spectra - Observational astronomy at all wavelengths from radio to gamma-rays, and neutrinos - Planetary astronomy: Earth, planets and satellites, and solar system small bodies - The Sun, normal stars, and stars with special characteristics - Stellar populations - Cataclysmic and symbiotic variables, supernovae - Theoretical stellar evolution - Circumstellar and interstellar material - Star clusters, galaxies, quasars, and active galactic nuclei ...

  17. Inhibition of Nitric Oxide Synthesis and Gene Knockout of Neuronal Nitric Oxide Synthase Impaired Adaptation of Mouse Optokinetic Response Eye Movements

    OpenAIRE

    Katoh, Akira; Kitazawa, Hiromasa; Itohara, Shigeyoshi; Nagao, Soichi

    2000-01-01

    Nitric oxide (NO) plays a key role in synaptic transmission efficiency in the central nervous system. To gain an insight on the role of NO in cerebellar functions, we, here, measured the dynamics of the horizontal optokinetic response (HOKR) and vestibulo-ocular reflex (HVOR), and the adaptation of HOKR in mice locally injected with NG-monomethyl-l-arginine (L-NMMA) that inhibits NO synthesis and in mice devoid of neuronal nitric oxide synthase (nNOS). Local application of L-NMMA into the cer...

  18. Relationship between endothelial nitric oxide synthase (eNOS) and natural history of intracranial aneurysms: meta-analysis.

    Science.gov (United States)

    Paschoal, Eric Homero Albuquerque; Yamaki, Vitor Nagai; Teixeira, Renan Kleber Costa; Paschoal Junior, Fernando Mendes; Jong-A-Liem, Glaucia Suzanna; Teixeira, Manoel Jacobsen; Yamada, Elizabeth Sumi; Ribeiro-Dos-Santos, Ândrea; Bor-Seng-Shu, Edson

    2018-01-01

    The aneurysmal subarachnoid hemorrhage is a major public health problem described as a sudden drastic event with no warning symptoms and high morbidity and mortality rates. The role of the endothelial isoform of nitric oxide synthase gene polymorphism in intracranial aneurysms (IAs) is still a matter of controversy with divergent findings among European, American, and Asian populations. Our study purposed to test the association between intracranial aneurysms formation and nitric oxide gene polymorphisms through a systematic review and meta-analysis. Systematic search on Medline, Lilacs, and EMBASE was performed. The primary search resulted in 139 papers, out of which 9 met our inclusion criteria after a full text analysis. The dominant T786C model found a significant association with IA (OR 1.22, 95 % CI 1.04-1.44, p = 0.01), so did studies of the recessive T786C model (OR 0.37, 95 % CI 0.30-0.45, p < 0.0001) but with opposite effect. Our findings support the presence of the T786C polymorphism as a predictor for the development of intracranial aneurysm in the cerebral vascular system. More studies are necessary in order to elucidate the pathways of the endothelial nitric oxide synthase (eNOS) in cerebrovascular diseases and in defining how different allelic combinations of the eNOS gene single-nucleotide polymorphism (SNP) could favor this pathological process.

  19. Functional muscle ischemia in neuronal nitric oxide synthase-deficient skeletal muscle of children with Duchenne muscular dystrophy

    Science.gov (United States)

    Sander, Mikael; Chavoshan, Bahman; Harris, Shannon A.; Iannaccone, Susan T.; Stull, James T.; Thomas, Gail D.; Victor, Ronald G.

    2000-01-01

    Duchenne muscular dystrophy (DMD) is a fatal disease caused by mutation of the gene encoding the cytoskeletal protein dystrophin. Despite a wealth of recent information about the molecular basis of DMD, effective treatment for this disease does not exist because the mechanism by which dystrophin deficiency produces the clinical phenotype is unknown. In both mouse and human skeletal muscle, dystrophin deficiency results in loss of neuronal nitric oxide synthase, which normally is localized to the sarcolemma as part of the dystrophin–glycoprotein complex. Recent studies in mice suggest that skeletal muscle-derived nitric oxide may play a key role in the regulation of blood flow within exercising skeletal muscle by blunting the vasoconstrictor response to α-adrenergic receptor activation. Here we report that this protective mechanism is defective in children with DMD, because the vasoconstrictor response (measured as a decrease in muscle oxygenation) to reflex sympathetic activation was not blunted during exercise of the dystrophic muscles. In contrast, this protective mechanism is intact in healthy children and those with polymyositis or limb-girdle muscular dystrophy, muscle diseases that do not result in loss of neuronal nitric oxide synthase. This clinical investigation suggests that unopposed sympathetic vasoconstriction in exercising human skeletal muscle may constitute a heretofore unappreciated vascular mechanism contributing to the pathogenesis of DMD. PMID:11087833

  20. Astaxanthin improves cognitive deficits from oxidative stress, nitric oxide synthase and inflammation through upregulation of PI3K/Akt in diabetes rat.

    Science.gov (United States)

    Xu, Lianbao; Zhu, Juan; Yin, Weibing; Ding, Xinsheng

    2015-01-01

    Diabetes-induced cognitive deficit (DICD) is a prevalent disease with substantial morbidity and mortality and as a global health problem with serious economic burdens. Astaxanthin (AST) has a good prospect in production of nutritional, medical, and particularly functional health drug. The present study was aimed to study the effect of AST on DICD in diabetes mellitus (DM) rat through suppression of oxidative stress, nitric oxide synthase (NOS) pathway, inflammatory reaction and upregulation of PI3K/Akt. In the study, Morris water maze teat was used to detect the cognitive function of DM rat. Afterwards, we measured the body weight and blood glucose levels of DM rats. Then, oxidative stress, the activities of eNOS and iNOS, and inflammatory factors were analyzed using a commercial kit in cerebral cortex and hippocampus. Finally, the caspase-3/9 and phosphoinositide 3-kinase (PI3K)/Akt expressions were also checkout with Real Time PCR and immunoblotting, respectively. In this experiment, AST could availably enhance the body weight and reduce blood glucose levels of DM rats. Moreover, AST could observably perfect cognitive function of DM rat. Next, the activities of oxidative stress, nitric oxide synthase and inflammation were distinctly diminution in DM rat, after the treatment of AST. Furthermore, our present results demonstrated that AST had the protective effect on the brain cell of DM rat, decreased the caspase-3/9 expression and promoted the expression of PI3K/Akt in cerebral cortex and hippocampus.

  1. Expression of the inducible isoform of nitric oxide synthase in pigment cell lesions of the skin.

    Science.gov (United States)

    Ahmed, B; Van Den Oord, J J

    2000-03-01

    Nitric oxide (NO) is a small molecule produced during the conversion of L-arginine to L-citrulline by NO synthase (NOS). Several isoforms of NOS exist, of which the Ca2+-independent, inducible NOS (iNOS or NOS2) is most prominently expressed by macrophages. iNOS activity and increased levels of iNOS have been found in various tumours and tumour cell lines but not in normal tissues; however, the precise role of NO in tumour progression has yet to be elucidated. We studied the expression of iNOS in paraffin sections of 41 benign naevi and 52 primary malignant melanomas (MM) of the skin, as well as in 13 metastatic MM. In addition, nitrotyrosine, indicative of NO production and formation of peroxynitrite, was studied in frozen sections of 13 naevi and 30 MM. Virtually all naevi expressed iNOS, but very few expressed nitrotyrosine, indicating either that iNOS in naevi is functionally inactive, or that naevus cells lack reactive oxygen radicals and thus do not form peroxynitrite. Normal melanocytes in adjacent uninvolved skin were unreactive for both markers. In MM, iNOS was most frequently expressed in the 'pure' and 'invasive' radial growth phase (RGP), whereas expression in the vertical growth phase (VGP) and metastatic phase occurred only in 76% of cases; moreover, in these latest phases of tumour progression, iNOS staining was weak and focal. We conclude that iNOS is expressed de novo in most benign pigment cell lesions. In MM (iNOS-generated) NO appears to play an important part in the early steps of invasion (i.e. the 'invasive' RGP), where it may stimulate neo-angiogenesis and may be a prerequisite for further tumour progression; this view is also supported by the finding of iNOS in the associated blood vessels in the papillary dermis. Finally, our data suggest that (iNOS-generated) NO plays a less significant part in the VGP and in metastatic melanoma.

  2. Ultrastructural localization of NADPH diaphorase and nitric oxide synthase in the neuropils of the snail CNS.

    Science.gov (United States)

    Nacsa, Kálmán; Elekes, Károly; Serfőző, Zoltán

    2015-08-01

    Comparative studies on the nervous system revealed that nitric oxide (NO) retains its function through the evolution. In vertebrates NO can act in different ways: it is released solely or as a co-transmitter, released from presynaptic or postsynaptic site, spreads as a volumetric signal or targets synaptic proteins. In invertebrates, however, the possible sites of NO release have not yet been identified. Therefore, in the present study, the subcellular distribution of the NO synthase (NOS) was examined in the central nervous system (CNS) of two gastropod species, the terrestrial snail, Helix pomatia and the pond snail, Lymnaea stagnalis, which are model species in comparative neurobiology. For the visualization of NOS NADPH-diaphorase histochemistry and an immunohistochemical procedure using a universal anti-NOS antibody were applied. At light microscopic level both techniques labeled identical structures in sensory tracts ramifying in the neuropils of central ganglia and cell bodies of the Lymnaea and Helix CNS. At ultrastructural level NADPH-d reactive/NOS-immunoreactive materials were localized on the nuclear envelope and membrane segments of the rough and smooth endoplasmic reticulum, as well as the cell membrane and axolemma of positive perikarya. NADPH-d reactive and NOS-immunoreactive varicosities connected to neighboring neurons with both unspecialized and specialized synaptic contacts. In the varicosities, the majority of the NADPH-d reactive/NOS-immunoreactive membrane segments were detected in round and pleomorph agranular vesicles of small size (50-200 nm). However, only a small portion (16%) of the vesicles displayed the NADPH-d reactivity/NOS-immunoreactivity. No evidence for the postsynaptic location of NOS was found. Our results suggest that the localization of NADPH-diaphorase and NOS is identical in the snail nervous system. In contrast to vertebrates, however, NO seems to act exclusively in an anterograde way possibly released from membrane

  3. Inducible nitric oxide synthase expression is upregulated in oral submucous fibrosis

    Directory of Open Access Journals (Sweden)

    Rajendran R

    2007-01-01

    Full Text Available Objective: We tested the hypothesis that inducible nitric oxide synthase (iNOS modulates angiogenesis in human models and this information could be extrapolated in elucidating the pathophysiology of oral submucous fibrosis (OSF. A hypothesis which looks inadequate, but is deep rooted in literature is the epithelial alteration ("atrophy" seen in OSF and the events that lead to its causation. This aspect was tried to be addressed and an alternative pathogenetic pathway for the disease is proposed. Materials and Methods: This immunohistochemical study sought to investigate the expression of iNOS in OSF samples (n= 30 a using monospecific antibody (SC- 2050, Santa Cruz Biotechnology, Inc to the protein and also to correlate it with different grades of epithelial dysplasia associated with the disease. Twenty (20 healthy adults acted as controls. Results: iNOS staining was not demonstrated in normal oral epithelium. In oral epithelial dysplasia, staining was seen in all cases (100% in the basal layers of the epithelium and in 30% of cases it extended into the parabasal compartments as well. iNOS staining was uniformly positive in moderate dysplasia with an increase in intensity and distribution noted as the severity of dysplasia progressed. There were highly significant differences in overall positivity for iNOS in epithelium between cases and controls (Mann-Whitney U = 11.000, Wilcoxon W = 221.00, P = 0.000. Significant comparisons were made of mild Vs moderate dysplasia (Mann-Whitney U = 48.000, P = 0.014 Conclusions: This study supports our earlier morphological assessment (image analysis of the nature of vascularity in OSF mucosa. The significant vasodilation noticed in these cases argues against the concept of ischemic atrophy of the epithelium. This observation of vascularity and iNOS expression helped to explain the vasodilation noticed (sinusoids in this disease; NO being a net vasodilator. The mechanism of activation of iNOS in dysplasia is

  4. Posttranslational inactivation of endothelial nitric oxide synthase in the transgenic sickle cell mouse penis

    Science.gov (United States)

    Musicki, Biljana; Champion, Hunter C.; Hsu, Lewis L.; Bivalacqua, Trinity J.; Burnett, Arthur L.

    2017-01-01

    INTRODUCTION Sickle cell disease (SCD)-associated priapism is characterized by endothelial nitric oxide synthase (eNOS) dysfunction in the penis. However, the mechanism of decreased eNOS function/activation in the penis in association with SCD is not known. AIMS Our hypothesis in the present study was that eNOS is functionally inactivated in the SCD penis in association with impairments in eNOS posttranslational phosphorylation and the enzyme’s interactions with its regulatory proteins. METHODS Sickle cell transgenic (sickle) mice were used as an animal model of SCD. Wild type (WT) mice served as controls. Penes were excised at baseline for molecular studies. eNOS phosphorylation on Ser-1177 (positive regulatory site) and Thr-495 (negative regulatory site), total eNOS, and phosphorylated AKT (upstream mediator of eNOS phosphorylation on Ser-1177) expressions, and eNOS interactions with heat shock protein 90 (HSP90) and caveolin-1 were measured by Western blot. Constitutive NOS catalytic activity was measured by conversion of L-[14C]arginine-to-L-[14C]citrulline in the presence of calcium. MAIN OUTCOME MEASURES Molecular mechanisms of eNOS dysfunction in the sickle mouse penis. RESULTS eNOS phosphorylated on Ser-1177, an active portion of eNOS, was decreased in the sickle mouse penis compared to WT penis. eNOS interaction with its positive protein regulator HSP90, but not with its negative protein regulator caveolin-1, and phosphorylated AKT expression, as well as constitutive NOS activity, were also decreased in the sickle mouse penis compared to WT penis. eNOS phosphorylated on Thr-495, total eNOS, HSP90, and caveolin-1 protein expressions in the penis were not affected by SCD. CONCLUSION These findings provide a molecular basis for chronically reduced eNOS function in the penis by SCD, which involves decreased eNOS phosphorylation on Ser-1177 and decreased eNOS-HSP90 interaction. PMID:21143412

  5. Nitric oxide synthase and cGMP-mediated stimulation of renin secretion.

    Science.gov (United States)

    Sayago, C M; Beierwaltes, W H

    2001-10-01

    The interaction between nitric oxide (NO) and renin is controversial. cAMP is a stimulating messenger for renin, which is degraded by phosphodiesterase (PDE)-3. PDE-3 is inhibited by cGMP, whereas PDE-5 degrades cGMP. We hypothesized that if endogenous cGMP was increased by inhibiting PDE-5, it could inhibit PDE-3, increasing endogenous cAMP, and thereby stimulate renin. We used the selective PDE-5 inhibitor zaprinast at 20 mg/kg body wt ip, which we determined would not change blood pressure (BP) or renal blood flow (RBF). In thiobutabarbital (Inactin)-anesthetized rats, renin secretion rate (RSR) was determined before and 75 min after administration of zaprinast or vehicle. Zaprinast increased cGMP excretion from 12.75 +/- 1.57 to 18.67 +/- 1.87 pmol/min (P < 0.003), whereas vehicle had no effect. Zaprinast increased RSR sixfold (from 2.95 +/- 1.74 to 17.62 +/- 5.46 ng ANG I. h(-1) x min(-1), P < 0.024), while vehicle had no effect (from 4.08 +/- 2.02 to 3.87 +/- 1.53 ng ANG I x h(-1) x min(-1)). There were no changes in BP or RBF. We then tested whether the increase in cGMP could be partially due to the activity of the neuronal isoform of NO synthase (nNOS). Pretreatment with the nNOS inhibitor 7-nitroindazole (7-NI; 50 mg/kg body wt) did not change BP or RBF but attenuated the renin-stimulating effect of zaprinast by 40% compared with vehicle. In 7-NI-treated animals, zaprinast-stimulated cGMP excretion was attenuated by 48%, from 9.17 +/- 1.85 to 13.60 +/- 2.15 pmol/min, compared with an increase from 10.94 +/- 1.90 to 26.38 +/- 3.61 pmol/min with zaprinast without 7-NI (P < 0.04). This suggests that changes in endogenous cGMP production at levels not associated with renal hemodynamic changes are involved in a renin-stimulatory pathway. One source of this cGMP may be nNOS generation of NO in the kidney.

  6. Cyclooxygenase 2 and neuronal nitric oxide synthase expression in the renal cortex are not interdependent in states of salt deficiency

    DEFF Research Database (Denmark)

    Castrop, H; Kammerl, M; Mann, Birgitte

    2000-01-01

    Neuronal nitric oxide synthase (nNOS) and cyclooxygenase-2 (COX-2) expression in the kidney are localized to the cortical thick ascending limb of the loop of Henle (cTALH), including the macula region, and increase after salt restriction. Because of the similar localization and regulation of n...... excretion. These findings suggest that under these conditions the control of nNOS and COX-2 gene expression in the macula densa regions of the kidney cortex are not dependent on each other....

  7. Cortisol regulates nitric oxide synthase in freshwater and seawater acclimated rainbow trout, Oncorhynchus mykiss

    DEFF Research Database (Denmark)

    Gerber, Lucie; Madsen, Steffen S; Jensen, Frank B

    2017-01-01

    in osmoregulatory tissues by regulating NO synthase (NOS) expression. We evaluated the influence of cortisol treatment on mRNA expression of Nos1 and Nos2 in gill, kidney and middle intestine of both freshwater (FW) and seawater (SW) acclimated rainbow trout and found both tissue- and salinity-dependent effects...

  8. Endothelial nitric oxide synthase mediates the nitric oxide component of reflex cutaneous vasodilatation during dynamic exercise in humans.

    Science.gov (United States)

    McNamara, Tanner C; Keen, Jeremy T; Simmons, Grant H; Alexander, Lacy M; Wong, Brett J

    2014-12-01

    Recent data suggests neuronal nitric oxide synthase (nNOS) mediates the NO component of reflex cutaneous vasodilatation with passive heat stress. We tested the hypothesis that nNOS inhibition would attenuate reflex cutaneous vasodilatation during sustained dynamic exercise in young healthy humans. All subjects first performed an incremental V̇O2, peak test to exhaustion on a custom-built supine cycle ergometer. On a separate day, subjects were instrumented with four intradermal microdialysis fibres on the forearm and each randomly assigned as: (1) lactated Ringer's (control); (2) 20 mm Nω-nitro-l-arginine methyl ester hydrochloride (non-selective NOS inhibitor); (3) 5 mm N-propyl-l-arginine (nNOS inhibitor); and (4) 10 mm N(5)-(1-iminoethyl)-l-ornithine dihydrochloride [endothelial NOS (eNOS) inhibitor]. Following microdialysis placement, subjects performed supine cycling with the experimental arm at heart level at 60% V̇O2, peak for a period sufficient to raise core temperature 0.8°C. At the end of cycling, all microdialysis sites were locally heated to 43°C and sodium nitroprusside was perfused to elicit maximal vasodilatation. Mean arterial pressure, skin blood flow via laser-Doppler flowmetry and core temperature via ingestible telemetric pill were measured continuously; cutaneous vascular conductance (CVC) was calculated as laser-Doppler flowmetry/mean arterial pressure and normalized to maximum. There was no significant difference between control (58 ± 2%CVCmax) and nNOS-inhibited (56 ± 3%CVCmax) sites in response to exercise-induced hyperthermia. The increase in CVC at eNOS-inhibited (41 ± 3%CVCmax) and non-selective NOS-inhibited (40 ± 4%CVCmax) sites were significantly attenuated compared to control and nNOS-inhibited (P exercise. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

  9. Pu-erh Tea Reduces Nitric Oxide Levels in Rats by Inhibiting Inducible Nitric Oxide Synthase Expression through Toll-Like Receptor 4

    Science.gov (United States)

    Xu, Yang; Wang, Guan; Li, Chunjie; Zhang, Min; Zhao, Hang; Sheng, Jun; Shi, Wei

    2012-01-01

    Pu-erh tea undergoes a unique fermentation process and contains theabrownins, polysaccharides and caffeine; although it is unclear about which component is associated with the down regulation of nitric oxide levels or how this process is mediated. To address this question we examined the effects of pu-erh tea on nitric oxide synthase (NOS) genes. Cohorts of rats were separately given four-week treatments of water as control, pu-erh tea, or the tea components: theabrownins, caffeine or polysaccharides. Five experimental groups were injected with lipopolysaccharides (LPS) to induce nitric oxide (NO) production, while the corresponding five control groups were injected with saline as a negative control. The serum and liver NO concentrations were examined and the NOS expression of both mRNA and protein was measured in liver. The results showed that the rats which were fed pu-erh tea or polysaccharides had lower levels of NO which corresponded with the down-regulation of inducible nitric oxide synthase (iNOS) expression. We further demonstrate that this effect is mediated through reduction of Toll-like receptor 4 (TLR4) signaling. Thus we find that the polysaccharide components in pu-erh tea reduce NO levels in an animal model by inhibiting the iNOS expression via signaling through TLR4. PMID:22837686

  10. Sunscreen ingredients inhibit inducible nitric oxide synthase (iNOS): a possible biochemical explanation for the sunscreen melanoma controversy.

    Science.gov (United States)

    Chiang, Thomas M; Sayre, Robert M; Dowdy, John C; Wilkin, Nathaniel K; Rosenberg, E William

    2005-02-01

    Sunscreen products are rated upon their ability to inhibit visible redness of the skin 24 h after measured doses of ultraviolet (UV) exposure (Sun Protection Factor, SPF). Although sunscreens prevent UV-induced redness, their ability to protect against melanoma or the development of moles is less clear. UV-induced redness occurs in part by the action of nitric oxide (NO), synthesized in the skin. NO is also an important immunoregulatory molecule in the induction of the cell-mediated tumour immune response. In this study, various sunscreen ingredients were tested for their ability to inhibit the production of NO. Four of the five sunscreens tested directly inhibited the conversion of arginine to citrulline by inducible nitric oxide synthase (iNOS) in vitro. These findings suggest that sunscreens may prevent redness partly by UV absorption and partly by inhibition of the skin's inflammatory response. As such, sunscreens might promote instead of protect against melanoma.

  11. The Influence of Hyperoxia On Heat Shock Proteins Expression and Nitric Oxide Synthase Activity – the Review

    Directory of Open Access Journals (Sweden)

    Szyller Jakub

    2017-03-01

    Full Text Available Any stay in an environment with an increased oxygen content (a higher oxygen partial pressure, pO2 and an increased pressure (hyperbaric conditions leads to an intensification of oxidative stress. Reactive oxygen species (ROS damage the molecules of proteins, nucleic acids, cause lipid oxidation and are engaged in the development of numerous diseases, including diseases of the circulatory system, neurodegenerative diseases, etc. There are certain mechanisms of protection against unfavourable effects of oxidative stress. Enzymatic and non-enzymatic systems belong to them. The latter include, among others, heat shock proteins (HSP. Their precise role and mechanism of action have been a subject of intensive research conducted in recent years. Hyperoxia and hyperbaria also have an effect on the expression and activity of nitrogen oxide synthase (NOS. Its product - nitrogen oxide (NO can react with reactive oxygen species and contribute to the development of nitrosative stress. NOS occurs as isoforms in various tissues and exhibit different reactions to the discussed factors. The authors have prepared a brief review of research determining the effect of hyperoxia and hyperbaria on HSP expression and NOS activity.

  12. Fluorinated Amine Stereotriads via Allene Amination.

    Science.gov (United States)

    Liu, Lu; Gerstner, Nels C; Oxtoby, Lucas J; Guzei, Ilia A; Schomaker, Jennifer M

    2017-06-16

    The incorporation of fluorine into organic scaffolds often improves the bioactivity of pharmaceutically relevant compounds. C-F/C-N/C-O stereotriad motifs are prevalent in antivirals, neuraminidase inhibitors, and modulators of androgen receptors, but are challenging to install. An oxidative allene amination strategy using Selectfluor rapidly delivers triply functionalized triads of the form C-F/C-N/C-O, exhibiting good scope and diastereoselectivity for all syn products. The resulting stereotriads are readily transformed into fluorinated pyrrolidines and protected α-, β-, and γ-amino acids.

  13. The Effect of Aging and Exogenous Testosterone Replacement on Nitric Oxide Concentration and Activity of Nitric Oxide Synthase in the Rat Corpus Cavernosum

    OpenAIRE

    Shiono, Manabu

    2001-01-01

    The effects of testosterone replacement on the nitric oxide (NO) concentration and activity of NO synthase (NOS) in the penis were investigated. Male Wistar rats (n = 39) were divided into 5 groups: 14-week-old males, 13-month-old males, 15-month-old males, 15-month-old males treated with low-dose testosterone replacement and 15-month-old males treated with high-dose testosterone replacement. The testosterone concentration in serum and the NO concentration in penile tissue were measured, and ...

  14. Neuronal nitric oxide synthase is involved in the induction of nerve growth factor-induced neck muscle nociception.

    Science.gov (United States)

    Isaak, Andreas; Ellrich, Jens

    2011-05-01

    Neck muscle nociception mediated by nitric oxide may play a role in the pathophysiology of tension-type headache. The present study addresses the involvement of neuronal nitric oxide synthase (nNOS) in the facilitation of neck muscle nociception after local application of nerve growth factor (NGF). After administration of NGF into semispinal neck muscles, the impact of neck muscle noxious input on brainstem processing was monitored by the jaw-opening reflex in anesthetized mice. The modulatory effect of preceding and subsequent administration of an inhibitor of neuronal nitric oxide synthase on central facilitation was addressed in a controlled study. With preceding i.p. application of saline or 0.096 mg/kg of the specific nNOS inhibitor Nω-propyl-L-arginine (NPLA), NGF induced a sustained reflex facilitation within 60 minutes. Preceding injection of 0.96 mg/kg or 1.92 mg/kg NPLA completely prevented the potentially facilitatory effect of NGF. Subsequent administration of 0.96 mg/kg NPLA did not affect established NGF-evoked reflex facilitation. Thus, NPLA prevents facilitation of brainstem processing by noxious myofascial input from neck muscles in a dose-dependent manner. These findings suggest that nNOS is involved in the induction but not the maintenance of NGF-evoked facilitation of nociception in the brainstem. These results from an experimental animal model may support the idea of NOS and nNOS as potential targets for pharmacological treatment of tension-type headache. © 2011 American Headache Society.

  15. ONO1714, a new inducible nitric oxide synthase inhibitor, attenuates sepsis-induced diaphragmatic dysfunction in hamsters.

    Science.gov (United States)

    Nishina, K; Mikawa, K; Kodama , S; Obara, H

    2001-04-01

    Sepsis causes impairment of diaphragmatic contractility and endurance capacity. Nitric oxide (NO) produced via inducible NO synthase (iNOS) has been implicated in the pathogenesis. Peroxynitrite, a NO-derived powerful oxidant, may be responsible for infection-induced diaphragmatic muscle failure. Therefore, we examined whether ONO1714, a new selective iNOS inhibitor, prevents sepsis-induced diaphragmatic dysfunction. Fifty male Golden-Syrian hamsters were randomly divided into five groups: hamsters that underwent sham laparotomy alone and received saline injection (Group Sham), those that underwent cecal ligation with puncture (CLP) and received saline injection (Group Sepsis), those that underwent sham laparotomy and received injection of ONO1714 0.3 mg/kg (Group Sham-ONO1714high), those that underwent CLP and received ONO1714 0.1 mg/kg (Group Sepsis-ONO1714low), and those that underwent CLP and received ONO1714 0.3 mg/kg (Group Sepsis-ONO1714high). ONO1714 or saline was intraperitoneally injected 10 min before surgery. Diaphragmatic contractility was assessed in vitro using diaphragm muscle strips excised 24 h after operation. Diaphragm fatigability was assessed by time until tension decreased to 50% of the initial value (T50%) during fatigue trials. Twitch, tetanic tensions, and T50% during fatigue trials were reduced in Group Sepsis. Pretreatment with ONO1714 dose-dependently attenuated sepsis-induced diaphragmatic contractile profiles and endurance capacity. CLP increased plasma nitrite/nitrate (NOx; stable NO metabolites), and diaphragm malondialdehyde (MDA; a product of lipid peroxidation), positive immunostaining for nitrotyrosine (peroxynitrite footprint), and iNOS activity. ONO1714 attenuated the increase. This beneficial effect of ONO1714 may be attributable, in part, to inhibition of peroxynitrite-induced lipid peroxidation in the diaphragm. Sepsis impairs diaphragmatic contractility and endurance capacity, which may be involved in acute respiratory

  16. Dystrophin Threshold Level Necessary for Normalization of Neuronal Nitric Oxide Synthase, Inducible Nitric Oxide Synthase, and Ryanodine Receptor-Calcium Release Channel Type 1 Nitrosylation in Golden Retriever Muscular Dystrophy Dystrophinopathy.

    Science.gov (United States)

    Gentil, Christel; Le Guiner, Caroline; Falcone, Sestina; Hogrel, Jean-Yves; Peccate, Cécile; Lorain, Stéphanie; Benkhelifa-Ziyyat, Sofia; Guigand, Lydie; Montus, Marie; Servais, Laurent; Voit, Thomas; Piétri-Rouxel, France

    2016-09-01

    At present, the clinically most advanced strategy to treat Duchenne muscular dystrophy (DMD) is the exon-skipping strategy. Whereas antisense oligonucleotide-based clinical trials are underway for DMD, it is essential to determine the dystrophin restoration threshold needed to ensure improvement of muscle physiology at the molecular level. A preclinical trial has been conducted in golden retriever muscular dystrophy (GRMD) dogs treated in a forelimb by locoregional delivery of rAAV8-U7snRNA to promote exon skipping on the canine dystrophin messenger. Here, we exploited rAAV8-U7snRNA-transduced GRMD muscle samples, well characterized for their percentage of dystrophin-positive fibers, with the aim of defining the threshold of dystrophin rescue necessary for normalization of the status of neuronal nitric oxide synthase mu (nNOSμ), inducible nitric oxide synthase (iNOS), and ryanodine receptor-calcium release channel type 1 (RyR1), crucial actors for efficient contractile function. Results showed that restoration of dystrophin in 40% of muscle fibers is needed to decrease abnormal cytosolic nNOSμ expression and to reduce overexpression of iNOS, these two parameters leading to a reduction in the NO level in the muscle fibers. Furthermore, the same percentage of dystrophin-positive fibers of 40% was associated with the normalization of RyR1 nitrosylation status and with stabilization of the RyR1-calstabin1 complex that is required to facilitate coupled gating. We concluded that a minimal threshold of 40% of dystrophin-positive fibers is necessary for the reinstatement of central proteins needed for proper muscle contractile function, and thus identified a rate of dystrophin expression significantly improving, at the molecular level, the dystrophic muscle physiology.

  17. Local inhibition of hippocampal nitric oxide synthase does not impair place learning in the Morris water escape task in rats.

    Science.gov (United States)

    Blokland, A; de Vente, J; Prickaerts, J; Honig, W; Markerink-van Ittersum, M; Steinbusch, H

    1999-01-01

    Recent studies have provided evidence that nitric oxide (NO) has a role in certain forms of memory formation. Spatial learning is one of the cognitive abilities that has been found to be impaired after systemic administration of an NO-synthase inhibitor. As the hippocampus has a pivotal role in spatial orientation, the present study examined the role of hippocampal NO in spatial learning and reversal learning in a Morris task in adult rats. It was found that N omega-nitro-L-arginine infusions into the dorsal hippocampus affected the manner in which the rats were searching the submerged platform during training, but did not affect the efficiency to find the spatial location of the escape platform. Hippocampal NO-synthase inhibition did not affect the learning of a new platform position in the same water tank (i.e. reversal learning). Moreover, no treatment effects were observed in the probe trials (i.e. after acquisition and after reversal learning), indicating that the rats treated with N omega-nitro-L-arginine had learned the spatial location of the platform. These findings were obtained under conditions where the NO synthesis in the dorsal hippocampus was completely inhibited. On the basis of the present data it was concluded that hippocampal NO is not critically involved in place learning in rats.

  18. Zinc thiolate reactivity toward nitrogen oxides: insights into the interaction of Zn2+ with S-nitrosothiols and implications for nitric oxide synthase.

    Science.gov (United States)

    Kozhukh, Julia; Lippard, Stephen J

    2012-07-02

    Zinc thiolate complexes containing N(2)S tridentate ligands were prepared to investigate their reactivity toward reactive nitrogen species, chemistry proposed to occur at the zinc tetracysteine thiolate site of nitric oxide synthase (NOS). The complexes are unreactive toward nitric oxide (NO) in the absence of dioxygen, strongly indicating that NO cannot be the species directly responsible for S-nitrosothiol formation and loss of Zn(2+) at the NOS dimer interface in vivo. S-Nitrosothiol formation does occur upon exposure of zinc thiolate solutions to NO in the presence of air, however, or to NO(2) or NOBF(4), indicating that these reactive nitrogen/oxygen species are capable of liberating zinc from the enzyme, possibly through generation of the S-nitrosothiol. Interaction between simple Zn(2+) salts and preformed S-nitrosothiols leads to decomposition of the -SNO moiety, resulting in release of gaseous NO and N(2)O. The potential biological relevance of this chemistry is discussed.

  19. Ferulic acid and its water-soluble derivatives inhibit nitric oxide production and inducible nitric oxide synthase expression in rat primary astrocytes.

    Science.gov (United States)

    Kikugawa, Masaki; Ida, Tomoaki; Ihara, Hideshi; Sakamoto, Tatsuji

    2017-08-01

    We recently reported that two water-soluble derivatives of ferulic acid (1-feruloyl glycerol, 1-feruloyl diglycerol) previously developed by our group exhibited protective effects against amyloid-β-induced neurodegeneration in vitro and in vivo. In the current study, we aimed to further understand this process by examining the derivatives' ability to suppress abnormal activation of astrocytes, the key event of neurodegeneration. We investigated the effects of ferulic acid (FA) derivatives on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in rat primary astrocytes. The results showed that these compounds inhibited NO production and iNOS expression in a concentration-dependent manner and that the mechanism underlying these effects was the suppression of the nuclear factor-κB pathway. This evidence suggests that FA and its derivatives may be effective neuroprotective agents and could be useful in the treatment of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

  20. Targeting RPL39 and MLF2 reduces tumor initiation and metastasis in breast cancer by inhibiting nitric oxide synthase signaling.

    Science.gov (United States)

    Dave, Bhuvanesh; Granados-Principal, Sergio; Zhu, Rui; Benz, Stephen; Rabizadeh, Shahrooz; Soon-Shiong, Patrick; Yu, Ke-Da; Shao, Zhimin; Li, Xiaoxian; Gilcrease, Michael; Lai, Zhao; Chen, Yidong; Huang, Tim H-M; Shen, Haifa; Liu, Xuewu; Ferrari, Mauro; Zhan, Ming; Wong, Stephen T C; Kumaraswami, Muthiah; Mittal, Vivek; Chen, Xi; Gross, Steven S; Chang, Jenny C

    2014-06-17

    We previously described a gene signature for breast cancer stem cells (BCSCs) derived from patient biopsies. Selective shRNA knockdown identified ribosomal protein L39 (RPL39) and myeloid leukemia factor 2 (MLF2) as the top candidates that affect BCSC self-renewal. Knockdown of RPL39 and MLF2 by specific siRNA nanoparticles in patient-derived and human cancer xenografts reduced tumor volume and lung metastases with a concomitant decrease in BCSCs. RNA deep sequencing identified damaging mutations in both genes. These mutations were confirmed in patient lung metastases (n = 53) and were statistically associated with shorter median time to pulmonary metastasis. Both genes affect the nitric oxide synthase pathway and are altered by hypoxia. These findings support that extensive tumor heterogeneity exists within primary cancers; distinct subpopulations associated with stem-like properties have increased metastatic potential.

  1. Constitutive nitric oxide synthase gene polymorphisms and house dust mite respiratory allergy in an Algerian patient group.

    Science.gov (United States)

    Djidjik, R; Ghaffor, M; Brun, M; Gharnaout, M; Salah, S S; Boukouaci, W; Djidjik, H; Benyounes, A; Koumaravelou, K; Krishnamoorthy, R; Abbadi, M C; Charron, D; Tamouza, R

    2008-02-01

    Genetic polymorphisms in neuronal nitric oxide synthase (NOS1) and calmodulin-dependent endothelial NOS (NOS3) genes are known to influence the course of allergic respiratory disorders. We investigated the role of NOS1 -84 G-->A and NOS3 -786 T-->C, 894 G-->T and 27 base pair (bp) repeat polymorphisms in 125 patients suffering from asthma and/or rhinitis and monosensitized against Dermatophagoides pteronyssinus (Dpter) and 111 controls from Algeria. We found a higher frequency of the -786 C NOS3 allele in patients than in controls [corrected P value (Pc) = 0.04], especially in female cases (Pc = 0.02) and that the 'ab' genotype of the 27-bp polymorphism was significantly associated with specific immunoglobulin E production against Dpter (P = 0.006). This study brings further support for the participation of NOS3 gene polymorphism in the pathogenesis of respiratory allergic disorders.

  2. Gene expression profiles of inducible nitric oxide synthase and cytokines in Leishmania major-infected macrophage-like RAW 264.7 cells treated with gallic acid

    NARCIS (Netherlands)

    Radtke, O.A.; Kiderlen, A.F.; Kayser, Oliver; Kolodziej, H

    2004-01-01

    The effects of gallic acid on the gene expressions of inducible nitric oxide synthase (iNOS) and the cytokines interleukin (IL)-1, IL-10, IL-12, IL-18, TNF-alpha, and interferon (IFN)-gamma were investigated by reverse-transcription polymerase chain reaction (RT-PCR). The experiments were performed

  3. New role for L-arginine in regulation of inducible nitric-oxide-synthase-derived superoxide anion production in Raw 264.7 macrophages

    Czech Academy of Sciences Publication Activity Database

    Pekarová, Michaela; Lojek, Antonín; Martíšková, Hana; Vašíček, Ondřej; Binó, Lucia; Klinke, A.; Lau, D.; Kuchta, R.; Kadlec, J.; Vrba, R.; Kubala, Lukáš

    2011-01-01

    Roč. 11, - (2011), s. 2443-2457 ISSN 1537-744X R&D Projects: GA ČR(CZ) GA524/08/1753 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : macrophage s * L-arginine * inducible nitric oxide synthase Subject RIV: BO - Biophysics Impact factor: 1.524, year: 2010

  4. Hypercapnic vasodilatation in isolated rat basilar arteries is exerted via low pH and does not involve nitric oxide synthase stimulation or cyclic GMP production

    DEFF Research Database (Denmark)

    You, J P; Wang, Qian; Zhang, W

    1994-01-01

    The relaxant effect of hypercapnia (15% CO2) was studied in isolated circular segments of rat basilar arteries with intact endothelium. The nitric oxide synthase inhibitor nitro-L-arginine (L-NOARG) and the cytosolic guanylate cyclase inhibitor methylene blue (MB), significantly reduced this rela...

  5. Endothelial Nitric Oxide Synthase Phosphorylation at Threonine 495 and Mitochondrial Reactive Oxygen Species Formation in Response to a High H2O2 Concentration

    DEFF Research Database (Denmark)

    Guterbaum, Thomas Jeremy; Braunstein, Thomas Hartig; Fossum, A

    2013-01-01

    Hydrogen peroxide (H₂O₂) is produced in vessels during ischemia/reperfusion and during inflammation, both leading to vascular dysfunction. We investigated cellular pathways involved in endothelial nitric oxide synthase (eNOS) phosphorylation at Threonine 495 (Thr(495)) in human umbilical vein end...

  6. The expression of neuronal nitric oxide synthase (NNOS) in brainstem and cerebellum of spontaneously hypertensive rats (SHR): The effect of chronic captopril treatment

    Czech Academy of Sciences Publication Activity Database

    Hojná, Silvie; Dobešová, Zdenka; Zicha, Josef; Kuneš, Jaroslav

    2005-01-01

    Roč. 46, č. 4 (2005), s. 895-895 ISSN 0194-911X. [Annual Meeting of the European Council for Cardiovascular Research (ECCR) /10./. 14.10.2005-16.10.2005, La Colle sur Loup] R&D Projects: GA ČR(CZ) GA305/03/0769 Keywords : nitric oxide synthase * brain * captopril * hypertension Subject RIV: ED - Physiology

  7. Distribution of vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, nitric oxide synthase, and their receptors in human and rat sphenopalatine ganglion

    DEFF Research Database (Denmark)

    Csati, A; Tajti, J; Kuris, A

    2012-01-01

    for the demonstration of vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), nitric oxide synthase (NOS), glutamine synthetase (GS), glial fibrillary acidic protein (GFAP), VIP and PACAP common receptors (VPAC1, VPAC2), and PACAP receptor (PAC1). In addition, double labeling...

  8. Folic Acid Promotes Recycling of Tetrahydrobiopterin and Protects Against Hypoxia-Induced Pulmonary Hypertension by Recoupling Endothelial Nitric Oxide Synthase

    Science.gov (United States)

    Chalupsky, Karel; Kračun, Damir; Kanchev, Ivan; Bertram, Katharina

    2015-01-01

    Abstract Aims: Nitric oxide (NO) derived from endothelial NO synthase (eNOS) has been implicated in the adaptive response to hypoxia. An imbalance between 5,6,7,8-tetrahydrobiopterin (BH4) and 7,8-dihydrobiopterin (BH2) can result in eNOS uncoupling and the generation of superoxide instead of NO. Dihydrofolate reductase (DHFR) can recycle BH2 to BH4, leading to eNOS recoupling. However, the role of DHFR and eNOS recoupling in the response to hypoxia is not well understood. We hypothesized that increasing the capacity to recycle BH4 from BH2 would improve NO bioavailability as well as pulmonary vascular remodeling (PVR) and right ventricular hypertrophy (RVH) as indicators of pulmonary hypertension (PH) under hypoxic conditions. Results: In human pulmonary artery endothelial cells and murine pulmonary arteries exposed to hypoxia, eNOS was uncoupled as indicated by reduced superoxide production in the presence of the nitric oxide synthase inhibitor, L-(G)-nitro-L-arginine methyl ester (L-NAME). Concomitantly, NO levels, BH4 availability, and expression of DHFR were diminished under hypoxia. Application of folic acid (FA) restored DHFR levels, NO bioavailability, and BH4 levels under hypoxia. Importantly, FA prevented the development of hypoxia-induced PVR, right ventricular pressure increase, and RVH. Innovation: FA-induced upregulation of DHFR recouples eNOS under hypoxia by improving BH4 recycling, thus preventing hypoxia-induced PH. Conclusion: FA might serve as a novel therapeutic option combating PH. Antioxid. Redox Signal. 23, 1076–1091. PMID:26414244

  9. Effect of quercetin on metallothionein, nitric oxide synthases and cyclooxygenase-2 expression on experimental chronic cadmium nephrotoxicity in rats

    International Nuclear Information System (INIS)

    Morales, Ana I.; Vicente-Sanchez, Cesar; Jerkic, Mirjana; Santiago, Jose M.; Sanchez-Gonzalez, Penelope D.; Perez-Barriocanal, Fernando; Lopez-Novoa, Jose M.

    2006-01-01

    Inflammation can play a key role in Cd-induced dysfunctions. Quercetin is a potent oxygen free radical scavenger and a metal chelator. Our aim was to study the effect of quercetin on Cd-induced kidney damage and metallothionein expression. The study was performed in Wistar rats that were administered during 9 weeks with either cadmium (1.2 mg Cd/kg/day, s.c.), quercetin (50 mg/kg/day, i.p.) or cadmium + quercetin. Renal toxicity was evaluated by measuring blood urea nitrogen concentration and urinary excretion of enzymes marker of tubular damage. Endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) renal expression were assessed by Western blot. Renal expression of metallothionein 1 and 2 (MT-1, MT-2) and eNOS mRNA was assessed by Northern blot. Our data demonstrated that Cd-induced renal toxicity was markedly reduced in rats that also received quercetin. MT-1 and MT-2 mRNA levels in kidney were substantially increased during treatment with Cd, being even higher when the animals received Cd and quercetin. Renal eNOS expression was significantly higher in rats receiving Cd and quercetin than in animals receiving Cd alone or in control rats. In the group that received Cd, COX-2 and iNOS expression was markedly higher than in control rats. In the group Cd + quercetin, no changes in COX-2 and iNOS expression were observed compared with the control group. Our results demonstrate that quercetin treatment prevents Cd-induced overexpression of iNOS and COX-2, and increases MT expression. These effects can explain the protection by quercetin of Cd-induced nephrotoxicity

  10. Inducible nitric oxide synthase inhibition by 1400W limits pain hypersensitivity in a neuropathic pain rat model.

    Science.gov (United States)

    Staunton, C A; Barrett-Jolley, R; Djouhri, L; Thippeswamy, T

    2018-02-13

    What is the central question of this study? Can modulation of inducible NO synthase reduce pain behaviour and pro-inflammatory cytokine signalling in a rat model of neuropathic pain? What is the main finding and its importance? Nitric oxide synthase-based therapies could be effective for the treatment of peripheral neuropathic pain. Peripheral neuropathic pain (PNP), resulting from injury to or dysfunction of a peripheral nerve, is a major health problem that affects 7-8% of the population. It is inadequately controlled by current drugs and is characterized by pain hypersensitivity, which is believed to be attributable to sensitization of peripheral and CNS neurons by various inflammatory mediators. Here we examined, in a rat model of PNP: (i) whether reducing levels of nitric oxide (NO) with 1400W, a highly selective inhibitor of inducible NO synthase (iNOS), would prevent or attenuate pain hypersensitivity; and (ii) the effects of 1400W on plasma concentrations of several cytokines that are secreted after iNOS upregulation during chronic pain states. The L5 spinal nerve axotomy (SNA) model of PNP was used, and 1400W (20 mg kg -1 ) was administered i.p. at 8 h intervals for 3 days starting at 18 h post-SNA. Changes in plasma concentrations of 12 cytokines in SNA rats treated with 1400W were examined using multiplex enzyme-linked immunosorbent assay. The SNA rats developed behavioural signs of mechanical and heat hypersensitivity. Compared with the vehicle/control, 1400W significantly: (i) limited development of mechanical hypersensitivity at 66 h post-SNA and of heat hypersensitivity at 42 h and at several time points tested thereafter; and (ii) increased the plasma concentrations of interleukin (IL)-1α, IL-1β and IL-10 in the SNA rats. The findings suggest that 1400W might exert its analgesic effects by reducing iNOS and altering the balance between the pro-inflammatory (IL-1β and IL-1α) and anti-inflammatory (IL-10) cytokines and that therapies

  11. Oxidative Stress in Homocystinuria Due to Cystathionine ß-Synthase Deficiency: Findings in Patients and in Animal Models.

    Science.gov (United States)

    Faverzani, Jéssica Lamberty; Hammerschmidt, Tatiane Grazieli; Sitta, Angela; Deon, Marion; Wajner, Moacir; Vargas, Carmen Regla

    2017-11-01

    Homocystinuria is an inborn error of amino acid metabolism caused by deficiency of cystathionine ß-synthase (CBS) activity, biochemically characterized by homocysteine (Hcy) and methionine (Met) accumulation in biological fluids and high urinary excretion of homocystine. Clinical manifestations include thinning and lengthening of long bones, osteoporosis, dislocation of the ocular lens, thromboembolism, and mental retardation. Although the pathophysiology of this disease is poorly known, the present review summarizes the available experimental findings obtained from patients and animal models indicating that oxidative stress may contribute to the pathogenesis of homocystinuria. In this scenario, several studies have shown that enzymatic and non-enzymatic antioxidant defenses are decreased in individuals affected by this disease. Furthermore, markers of lipid, protein, and DNA oxidative damage have been reported to be increased in blood, brain, liver, and skeletal muscle in animal models studied and in homocystinuric patients, probably as a result of increased free radical generation. On the other hand, in vitro and in vivo studies have shown that Hcy induces reactive species formation in brain, so that this major accumulating metabolite may underlie the oxidative damage observed in the animal model and human condition. Taken together, it may be presumed that the disruption of redox homeostasis may contribute to the tissue damage found in homocystinuria. Therefore, it is proposed that the use of appropriate antioxidants may represent a novel adjuvant therapy for patients affected by this disease.

  12. Oleic acid increases mitochondrial reactive oxygen species production and decreases endothelial nitric oxide synthase activity in cultured endothelial cells.

    Science.gov (United States)

    Gremmels, Hendrik; Bevers, Lonneke M; Fledderus, Joost O; Braam, Branko; van Zonneveld, Anton Jan; Verhaar, Marianne C; Joles, Jaap A

    2015-03-15

    Elevated plasma levels of free fatty acids (FFA) are associated with increased cardiovascular risk. This may be related to FFA-induced elevation of oxidative stress in endothelial cells. We hypothesized that, in addition to mitochondrial production of reactive oxygen species, endothelial nitric oxide synthase (eNOS)-mediated reactive oxygen species production contributes to oleic acid (OA)-induced oxidative stress in endothelial cells, due to eNOS uncoupling. We measured reactive oxygen species production and eNOS activity in cultured endothelial cells (bEnd.3) in the presence of OA bound to bovine serum albumin, using the CM-H2DCFDA assay and the L-arginine/citrulline conversion assay, respectively. OA induced a concentration-dependent increase in reactive oxygen species production, which was inhibited by the mitochondrial complex II inhibitor thenoyltrifluoroacetone (TTFA). OA had little effect on eNOS activity when stimulated by a calcium-ionophore, but decreased both basal and insulin-induced eNOS activity, which was restored by TTFA. Pretreatment of bEnd.3 cells with tetrahydrobiopterin (BH4) prevented OA-induced reactive oxygen species production and restored inhibition of eNOS activity by OA. Elevation of OA levels leads to both impairment in receptor-mediated stimulation of eNOS and to production of mitochondrial-derived reactive oxygen species and hence endothelial dysfunction. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Inhibition of nitric oxide synthase expression in activated microglia and peroxynitrite scavenging activity by Opuntia ficus indica var. saboten.

    Science.gov (United States)

    Lee, Ming Hong; Kim, Jae Yeon; Yoon, Jeong Hoon; Lim, Hyo Jin; Kim, Tae Hee; Jin, Changbae; Kwak, Wie-Jong; Han, Chang-Kyun; Ryu, Jae-Ha

    2006-09-01

    Activated microglia by neuronal injury or inflammatory stimulation overproduce nitric oxide (NO) by inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS) such as superoxide anion, resulting in neurodegenerative diseases. The toxic peroxynitrite (ONOO-), the reaction product of NO and superoxide anion further contributes to oxidative neurotoxicity. A butanol fraction obtained from 50% ethanol extracts of Opuntia ficus indica var. saboten (Cactaceae) stem (SK OFB901) and its hydrolysis product (SK OFB901H) inhibited the production of NO in LPS-activated microglia in a dose dependent manner (IC50 15.9, 4.2 microg/mL, respectively). They also suppressed the expression of protein and mRNA of iNOS in LPS-activated microglial cells at higher than 30 microg/mL as observed by western blot analysis and RT-PCR experiment. They also inhibited the degradation of I-kappaB-alpha in activated microglia. Moreover, they showed strong activity of peroxynitrite scavenging in a cell free bioassay system. These results imply that Opuntia ficus indica may have neuroprotective activity through the inhibition of NO production by activated microglial cells and peroxynitrite scavenging activity. Copyright (c) 2006 John Wiley & Sons, Ltd.

  14. Increased Inducible Nitric Oxide Synthase Expression in Organs Is Associated with a Higher Severity of H5N1 Influenza Virus Infection

    OpenAIRE

    Burggraaf, Simon; Bingham, John; Payne, Jean; Kimpton, Wayne G.; Lowenthal, John W.; Bean, Andrew G. D.

    2011-01-01

    BACKGROUND: The mechanisms of disease severity caused by H5N1 influenza virus infection remain somewhat unclear. Studies have indicated that a high viral load and an associated hyper inflammatory immune response are influential during the onset of infection. This dysregulated inflammatory response with increased levels of free radicals, such as nitric oxide (NO), appears likely to contribute to disease severity. However, enzymes of the nitric oxide synthase (NOS) family such as the inducible ...

  15. Role of nitric oxide synthase uncoupling at rostral ventrolateral medulla in redox-sensitive hypertension associated with metabolic syndrome.

    Science.gov (United States)

    Wu, Kay L H; Chao, Yung-Mei; Tsay, Shiow-Jen; Chen, Chen Hsiu; Chan, Samuel H H; Dovinova, Ima; Chan, Julie Y H

    2014-10-01

    Metabolic syndrome (MetS), which is rapidly becoming prevalent worldwide, is long known to be associated with hypertension and recently with oxidative stress. Of note is that oxidative stress in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons reside, contributes to sympathoexcitation and hypertension. This study sought to identify the source of tissue oxidative stress in RVLM and their roles in neural mechanism of hypertension associated with MetS. Adult normotensive rats subjected to a high-fructose diet for 8 weeks developed metabolic traits of MetS, alongside increases in sympathetic vasomotor activity and blood pressure. In RVLM of these MetS rats, the tissue level of reactive oxygen species was increased, nitric oxide (NO) was decreased, and mitochondrial electron transport capacity was reduced. Whereas the protein expression of neuronal NO synthase (nNOS) or protein inhibitor of nNOS was increased, the ratio of nNOS dimer/monomer was significantly decreased. Oral intake of pioglitazone or intracisternal infusion of tempol or coenzyme Q10 significantly abrogated all those molecular events in high-fructose diet-fed rats and ameliorated sympathoexcitation and hypertension. Gene silencing of protein inhibitor of nNOS mRNA in RVLM using lentivirus carrying small hairpin RNA inhibited protein inhibitor of nNOS expression, increased the ratio of nNOS dimer/monomer, restored NO content, and alleviated oxidative stress in RVLM of high-fructose diet-fed rats, alongside significantly reduced sympathoexcitation and hypertension. These results suggest that redox-sensitive and protein inhibitor of nNOS-mediated nNOS uncoupling is engaged in a vicious cycle that sustains the production of reactive oxygen species in RVLM, resulting in sympathoexcitation and hypertension associated with MetS. © 2014 American Heart Association, Inc.

  16. Attenuation of acute nitrogen mustard-induced lung injury, inflammation and fibrogenesis by a nitric oxide synthase inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Malaviya, Rama; Venosa, Alessandro [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States); Hall, LeRoy [Drug Safety Sciences, Johnson and Johnson, Raritan, NJ 08869 (United States); Gow, Andrew J. [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States); Sinko, Patrick J. [Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States); Laskin, Jeffrey D. [Department of Environmental and Occupational Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854 (United States); Laskin, Debra L., E-mail: laskin@eohsi.rutgers.edu [Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854 (United States)

    2012-12-15

    Nitrogen mustard (NM) is a toxic vesicant known to cause damage to the respiratory tract. Injury is associated with increased expression of inducible nitric oxide synthase (iNOS). In these studies we analyzed the effects of transient inhibition of iNOS using aminoguanidine (AG) on NM-induced pulmonary toxicity. Rats were treated intratracheally with 0.125 mg/kg NM or control. Bronchoalveolar lavage fluid (BAL) and lung tissue were collected 1 d–28 d later and lung injury, oxidative stress and fibrosis assessed. NM exposure resulted in progressive histopathological changes in the lung including multifocal lesions, perivascular and peribronchial edema, inflammatory cell accumulation, alveolar fibrin deposition, bronchiolization of alveolar septal walls, and fibrosis. This was correlated with trichrome staining and expression of proliferating cell nuclear antigen (PCNA). Expression of heme oxygenase (HO)-1 and manganese superoxide dismutase (Mn-SOD) was also increased in the lung following NM exposure, along with levels of protein and inflammatory cells in BAL, consistent with oxidative stress and alveolar-epithelial injury. Both classically activated proinflammatory (iNOS{sup +} and cyclooxygenase-2{sup +}) and alternatively activated profibrotic (YM-1{sup +} and galectin-3{sup +}) macrophages appeared in the lung following NM administration; this was evident within 1 d, and persisted for 28 d. AG administration (50 mg/kg, 2 ×/day, 1 d–3 d) abrogated NM-induced injury, oxidative stress and inflammation at 1 d and 3 d post exposure, with no effects at 7 d or 28 d. These findings indicate that nitric oxide generated via iNOS contributes to acute NM-induced lung toxicity, however, transient inhibition of iNOS is not sufficient to protect against pulmonary fibrosis. -- Highlights: ► Nitrogen mustard (NM) induces acute lung injury and fibrosis. ► Pulmonary toxicity is associated with increased expression of iNOS. ► Transient inhibition of iNOS attenuates acute

  17. Frances Allen Wins Turing Award

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 12; Issue 8. Frances Allen Wins Turing Award. Priti Shankar. Article-in-a-Box Volume 12 Issue 8 August 2007 pp 5-5. Fulltext. Click here to view fulltext PDF. Permanent link: http://www.ias.ac.in/article/fulltext/reso/012/08/0005-0005. Author Affiliations.

  18. Frances Allen Wins Turing Award

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 12; Issue 8. Frances Allen Wins Turing Award. Priti Shankar. Article-in-a-Box Volume 12 Issue 8 August 2007 pp ... Author Affiliations. Priti Shankar1. Department of Computer Science and Automation, Indian Institute of Science, Bangalore 560 012, India ...

  19. Improvement of Tissue Survival of Skin Flaps by 5α-Reductase Inhibitors: Possible Involvement of Nitric Oxide and Inducible Nitric Oxide Synthase

    Science.gov (United States)

    Karimi, Ali Asghar; Ajami, Marjan; Asadi, Yasin; Aboutaleb, Nahid; Gorjipour, Fazel; Malekloo, Roya; Pazoki-Toroudi, Hamidreza

    2015-01-01

    Background: Skin flap grafting is a popular approach for reconstruction of critical skin and underlying soft tissue injuries. In a previous study, we demonstrated the beneficial effects of two 5α-reductase inhibitors, azelaic acid and finasteride, on tissue survival in a rat model of skin flap grafting. In the current study, we investigated the involvement of nitric oxide and inducible nitric oxide synthase (iNOS) in graft survival mediated by these agents. Methods: A number of 42 male rats were randomly allocated into six groups: 1, normal saline topical application; 2, azelaic acid (100 mg/flap); 3, finasteride (1 mg/flap); 4, injection of L-NG-nitroarginine methyl ester (L-NAME) (i.p., 20 mg/kg); 5, L-NAME (20 mg/kg, i.p.) + azelaic acid (100 mg/flap, topical); 6, L-NAME (20 mg/kg, i.p.) + finasteride (1 mg/flap, topical). Tissue survival, level of nitric oxide, and iNOS expression in groups were measured. Results: Our data revealed that azelaic acid and finasteride significantly increased the expression of iNOS protein and nitric oxide (NO) levels in graft tissue (P azelaic acid- and finasteride-mediated survival of the skin flaps. PMID:25864816

  20. Arsenic toxicity induced endothelial dysfunction and dementia: Pharmacological interdiction by histone deacetylase and inducible nitric oxide synthase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Bhupesh, E-mail: drbhupeshresearch@gmail.com; Sharma, P.M.

    2013-11-15

    Arsenic toxicity has been reported to damage all the major organs including the brain and vasculature. Dementia including Alzheimer's disease (AD) and vascular dementia (VaD) are posing greater risk to the world population as it is now increasing at a faster rate. We have investigated the role of sodium butyrate, a selective histone deacetylase (HDAC) inhibitor and aminoguanidine, a selective inducible nitric oxide synthase (iNOS) inhibitor in pharmacological interdiction of arsenic toxicity induced vascular endothelial dysfunction and dementia in rats. Arsenic toxicity was done by administering arsenic drinking water to rats. Morris water-maze (MWM) test was used for assessment of learning and memory. Endothelial function was assessed using student physiograph. Oxidative stress (aortic superoxide anion, serum and brain thiobarbituric acid reactive species, brain glutathione) and nitric oxide levels (serum nitrite/nitrate) were also measured. Arsenic treated rats have shown impairment of endothelial function, learning and memory, reduction in serum nitrite/nitrate and brain GSH levels along with increase in serum and brain TBARS. Sodium butyrate as well as aminoguanidine significantly convalesce arsenic induced impairment of learning, memory, endothelial function, and alterations in various biochemical parameters. It may be concluded that arsenic induces endothelial dysfunction and dementia, whereas, sodium butyrate, a HDAC inhibitor as well as aminoguanidine, a selective iNOS inhibitor may be considered as potential agents for the management of arsenic induced endothelial dysfunction and dementia. - Highlights: • As has induced endothelial dysfunction (Edf) and vascular dementia (VaD). • As has increased oxidative stress, AChE activity and decreased serum NO. • Inhibitors of HDAC and iNOS have attenuated As induced Edf and VaD. • Both the inhibitors have attenuated As induced biochemical changes. • Inhibitor of HDAC and iNOS has shown good potential

  1. Arsenic toxicity induced endothelial dysfunction and dementia: Pharmacological interdiction by histone deacetylase and inducible nitric oxide synthase inhibitors

    International Nuclear Information System (INIS)

    Sharma, Bhupesh; Sharma, P.M.

    2013-01-01

    Arsenic toxicity has been reported to damage all the major organs including the brain and vasculature. Dementia including Alzheimer's disease (AD) and vascular dementia (VaD) are posing greater risk to the world population as it is now increasing at a faster rate. We have investigated the role of sodium butyrate, a selective histone deacetylase (HDAC) inhibitor and aminoguanidine, a selective inducible nitric oxide synthase (iNOS) inhibitor in pharmacological interdiction of arsenic toxicity induced vascular endothelial dysfunction and dementia in rats. Arsenic toxicity was done by administering arsenic drinking water to rats. Morris water-maze (MWM) test was used for assessment of learning and memory. Endothelial function was assessed using student physiograph. Oxidative stress (aortic superoxide anion, serum and brain thiobarbituric acid reactive species, brain glutathione) and nitric oxide levels (serum nitrite/nitrate) were also measured. Arsenic treated rats have shown impairment of endothelial function, learning and memory, reduction in serum nitrite/nitrate and brain GSH levels along with increase in serum and brain TBARS. Sodium butyrate as well as aminoguanidine significantly convalesce arsenic induced impairment of learning, memory, endothelial function, and alterations in various biochemical parameters. It may be concluded that arsenic induces endothelial dysfunction and dementia, whereas, sodium butyrate, a HDAC inhibitor as well as aminoguanidine, a selective iNOS inhibitor may be considered as potential agents for the management of arsenic induced endothelial dysfunction and dementia. - Highlights: • As has induced endothelial dysfunction (Edf) and vascular dementia (VaD). • As has increased oxidative stress, AChE activity and decreased serum NO. • Inhibitors of HDAC and iNOS have attenuated As induced Edf and VaD. • Both the inhibitors have attenuated As induced biochemical changes. • Inhibitor of HDAC and iNOS has shown good potential in

  2. Effects of Chinese yellow wine on nitric oxide synthase and intercellular adhesion molecule-1 expressions in rat vascular endothelial cells.

    Science.gov (United States)

    Zhao, Fei; Ji, Zheng; Chi, Jufang; Tang, Weiliang; Zhai, Xiaoya; Meng, Liping; Guo, Hangyuan

    2016-02-01

    The objective of this study was to determine similarities in the effect of yellow wine as compared to statin and the possibility that yellow wine inhibits tumour necrosis factor-α (TNF-α)-induced nitric oxide (NO) synthesis, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and intercellular adhesion molecule-1 (ICAM-1) in cultured rat vascular endothelial cells (VECs). We isolated VECs, and cultivated and purified Sprague Dawley (SD) rat thoracic aortas in vitro. We selected the optimal wine concentration using clonogenic and MTT assays to measure cell survival. Next, we divided the cells into 9 groups: (1) control, (2) TNF-α, (3) TNF-α + rosuvastatin (10 μmol/L), (4) TNF-α + ethanol 0.5%, (5) TNF-α + yellow wine 0.5%, (6) TNF-α + ethanol 1.0%, (7) TNF-α + yellow wine 1.0%, (8) TNF-α + ethanol 1.5%, and (9) TNF-α + yellow wine 1.5% and they were given the corresponding treatment for 24 h. We determined NO production with nitrate reductase. We then measured eNOS activity, and detected eNOS, iNOS, and ICAM-1 protein levels by Western blotting. Compared with the TNF-α group, NO production, eNOS activity, and eNOS protein expression in the rosuvastatin, and yellow wine 1.0%, and 1.5% groups were significantly increased. Protein expression of iNOS and ICAM-1 in the rosuvastatin, yellow wine 1.0%, and 1.5% groups were significantly decreased. Compared with the rosuvastatin group, eNOS, iNOS, and ICAM-1 protein expression in the yellow wine (0.5% -1.5%) groups were significantly different. Treatment with yellow wine increased NO production, eNOS activity, and eNOS protein expression, which decreases iNOS and ICAM-1 protein expression. We conclude that yellow wine may have similar beneficial effects as rosuvastatin on the cardiovascular system. These effects may be attributed to their anti-atherosclerotic actions.

  3. Modulation of cerebral RAGE expression following nitric oxide synthase inhibition in rats subjected to focal cerebral ischemia.

    Science.gov (United States)

    Greco, Rosaria; Demartini, Chiara; Zanaboni, Anna Maria; Blandini, Fabio; Amantea, Diana; Tassorelli, Cristina

    2017-04-05

    The receptor for advanced glycation endproducts (RAGE) is a key mediator of neuroinflammation following cerebral ischemia. Nitric oxide (NO) plays a dualistic role in cerebral ischemia, depending on whether it originates from neuronal, inducible or endothelial synthase. Although a dynamic interplay between RAGE and NO pathways exists, its relevance in ischemic stroke has not been investigated. The aim of this study is to evaluate the effect of the NO synthase (NOS) inhibition on RAGE expression in rats subjected to transient middle cerebral artery occlusion (tMCAo). Full-length (fl-RAGE) gene expression was elevated in the striatum and, to a lesser extent, in the cortex of rats undergone tMCAo. The exacerbation of cortical damage caused by systemic administration of L-N-(1-iminoethyl)ornithine (L-NIO), a relatively selective inhibitor of endothelial NOS (eNOS), was associated with elevated mRNA levels of interleukin (IL)-6, tumor necrosis factor (TNF)-α and fl-RAGE in both the cortex and the striatum. Conversely, NG-nitro-l-arginine methyl ester (L-NAME), a non-selective NOS inhibitor, decreased cortical damage, did not affect cerebral cytokine mRNA levels, while it increased fl-RAGE mRNA expression only in the striatum. Fl-RAGE striatal protein levels varied accordingly with observed mRNA changes in the striatum, while in the cortex, RAGE protein levels were reduced by tMCAo and further decreased following L-NIO treatment. Modulation of RAGE expression by different inhibitors of NOS may have opposite effects on transient cortical ischemia: the non selective inhibition of NOS activity is protective, while the selective inhibition of eNOS is harmful, probably via the activation of inflammatory pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Rosiglitazone Affects Nitric Oxide Synthases and Improves Renal Outcome in a Rat Model of Severe Ischemia/Reperfusion Injury

    Directory of Open Access Journals (Sweden)

    Boris Betz

    2012-01-01

    Full Text Available Background. Nitric oxide (NO-signal transduction plays an important role in renal ischemia/reperfusion (I/R injury. NO produced by endothelial NO-synthase (eNOS has protective functions whereas NO from inducible NO-synthase (iNOS induces impairment. Rosiglitazone (RGZ, a peroxisome proliferator-activated receptor (PPAR-γ agonist exerted beneficial effects after renal I/R injury, so we investigated whether this might be causally linked with NOS imbalance. Methods. RGZ (5 mg/kg was administered i.p. to SD-rats (f subjected to bilateral renal ischemia (60 min. Following 24 h of reperfusion, inulin- and PAH-clearance as well as PAH-net secretion were determined. Morphological alterations were graded by histopathological scoring. Plasma NOx-production was measured. eNOS and iNOS expression was analyzed by qPCR. Cleaved caspase 3 (CC3 was determined as an apoptosis indicator and ED1 as a marker of macrophage infiltration in renal tissue. Results. RGZ improves renal function after renal I/R injury (PAH-/inulin-clearance, PAH-net secretion and reduces histomorphological injury. Additionally, RGZ reduces NOx plasma levels, ED-1 positive cell infiltration and CC3 expression. iNOS-mRNA is reduced whereas eNOS-mRNA is increased by RGZ. Conclusion. RGZ has protective properties after severe renal I/R injury. Alterations of the NO pathway regarding eNOS and iNOS could be an explanation of the underlying mechanism of RGZ protection in renal I/R injury.

  5. Altered vasodilator role of nitric oxide synthase in the pancreas, heart and brain of rats with spontaneous type 2 diabetes.

    Science.gov (United States)

    Song, Dongzhe; Yao, Reina; Pang, Catherine C Y

    2008-09-04

    Type 2 diabetes is associated with altered regional blood flow and expression of nitric oxide synthase (NOS). We examined the functional role of constitutive and inducible NOS synthase (cNOS and iNOS, respectively) on regional blood flow in thiobutabarbital-anesthetized Zucker diabetic fatty (ZDF) and control rats via the radioactive microspheres technique. Blood flow was measured at baseline (1 h after surgery), after i.v. administration of 1400W (N-3-aminomethyl-benzyl-acetamidine, selective iNOS inhibitor, 3 mg/kg), and again after i.v. N(G)-nitro-l-arginine methyl ester (L-NAME, non-selective NOS inhibitor, 8 mg/kg). Both groups had similar baseline mean arterial pressure, cardiac output and total peripheral resistance, but the ZDF rats had lower heart rate relative to the control rats (272 versus 305 beats/min). Whereas 1400W did not alter mean arterial pressure or blood flow in either group, L-NAME markedly increased mean arterial pressure and total peripheral resistance, and reduced cardiac output, heart rate, blood flow and arterial conductance in all organs/tissues of both the control and ZDF rats. L-NAME caused greater vasoconstriction in the heart (1.5-times the constriction in control rats) and brain (1.5-times) of the ZDF rats, but less in the pancreas (0.6-times). Thus, cNOS had greater vasodilator control of the heart and brain, but less in the pancreas of the ZDF than control rats. iNOS has negligible influence on blood flow in both groups of rats.

  6. Chamomile: an anti-inflammatory agent inhibits inducible nitric oxide synthase expression by blocking RelA/p65 activity.

    Science.gov (United States)

    Bhaskaran, Natarajan; Shukla, Sanjeev; Srivastava, Janmejai K; Gupta, Sanjay

    2010-12-01

    Chamomile has long been used in traditional medicine for the treatment of inflammation-related disorders. In this study we investigated the inhibitory effects of chamomile on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and explored its potential anti-inflammatory mechanisms using RAW 264.7 macrophages. Chamomile treatment inhibited LPS-induced NO production and significantly blocked IL-1β, IL-6 and TNFα-induced NO levels in RAW 264.7 macrophages. Chamomile caused reduction in LPS-induced iNOS mRNA and protein expression. In RAW 264.7 macrophages, LPS-induced DNA binding activity of RelA/p65 was significantly inhibited by chamomile, an effect that was mediated through the inhibition of IKKβ, the upstream kinase regulating NF-κB/Rel activity, and degradation of inhibitory factor-κB. These results demonstrate that chamomile inhibits NO production and iNOS gene expression by inhibiting RelA/p65 activation and supports the utilization of chamomile as an effective anti-inflammatory agent.

  7. Chamomile, an anti-inflammatory agent inhibits inducible nitric oxide synthase expression by blocking RelA/p65 activity

    Science.gov (United States)

    Bhaskaran, Natarajan; Shukla, Sanjeev; Srivastava, Janmejai K; Gupta, Sanjay

    2010-01-01

    Chamomile has long been used in traditional medicine for the treatment of inflammation-related disorders. In this study we aimed to investigate the inhibitory effects of chamomile on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and to explore its potential anti-inflammatory mechanisms using RAW 264.7 macrophages. Chamomile treatment inhibited LPS-induced NO production and significantly blocked IL-1β , IL-6 and TNFα-induced NO levels in RAW 264.7 macrophages. Chamomile caused reduction in LPS-induced iNOS mRNA and protein expression. In RAW 264.7 macrophages, LPS-induced DNA binding activity of RelA/p65 was significantly inhibited by chamomile, an effect that was mediated through the inhibition of IKKβ , the upstream kinase regulating NF-κ B/Rel activity, and degradation of inhibitory factor-κ B. These results demonstrate that chamomile inhibits NO production and iNOS gene expression by inhibiting RelA/p65 activation and supports the utilization of chamomile as an effective anti-inflammatory agent. PMID:21042790

  8. Effects of Intracerebroventricularly (ICV) Injected Ghrelin on Cardiac Inducible Nitric Oxide Synthase Activity/Expression in Obese Rats.

    Science.gov (United States)

    Sudar Milovanovic, E; Jovanovic, A; Misirkic-Marjanovic, M; Vucicevic, Lj; Janjetovic, K; Isenovic, E R

    2015-11-01

    The aim of this study was to examine the effects of ghrelin on regulation of cardiac inducible nitric oxide synthase (iNOS) activity/expression in high fat (HF), obese rats.For this study, male Wistar rats fed with HF diet (30% fat) for 4 weeks were injected every 24 h for 5 days intracerebroventricularly (ICV) with ghrelin (0.3 nmol/5 µl) or with an equal volume of phosphate buffered saline (PBS). Control rats were ICV injected with an equal volume of PBS. Glucose, insulin and nitric oxide (NO) concentrations were measured in serum, while arginase activity and citrulline concentrations were measured in heart lysate. Protein iNOS and regulatory subunit of nuclear factor-κB (NFκB-p65), phosphorylation of enzymes protein kinase B (Akt) at Ser(473), and extracellular signal-regulated kinases 1/2 (ERK1/2) at Tyr(202)/Tyr(204) were determined in heart lysate by Western blot. For gene expression of iNOS qRT-PCR was used.Results show significantly (parginase activity (pactivity of cardiac iNOS via Akt phosphorylation followed by NFκB activation in HF rats. © Georg Thieme Verlag KG Stuttgart · New York.

  9. Estrous cycle influences the expression of neuronal nitric oxide synthase in the hypothalamus and limbic system of female mice

    Directory of Open Access Journals (Sweden)

    Viglietti-Panzica Carla

    2009-07-01

    Full Text Available Abstract Background Nitric oxide plays an important role in the regulation of male and female sexual behavior in rodents, and the expression of the nitric oxide synthase (NOS is influenced by testosterone in the male rat, and by estrogens in the female. We have here quantitatively investigated the distribution of nNOS immunoreactive (ir neurons in the limbic hypothalamic region of intact female mice sacrificed during different phases of estrous cycle. Results Changes were observed in the medial preoptic area (MPA (significantly higher number in estrus and in the arcuate nucleus (Arc (significantly higher number in proestrus. In the ventrolateral part of the ventromedial nucleus (VMHvl and in the bed nucleus of the stria terminalis (BST no significant changes have been observed. In addition, by comparing males and females, we observed a stable sex dimorphism (males have a higher number of nNOS-ir cells in comparison to almost all the different phases of the estrous cycle in the VMHvl and in the BST (when considering only the less intensely stained elements. In the MPA and in the Arc sex differences were detected only comparing some phases of the cycle. Conclusion These data demonstrate that, in mice, the expression of nNOS in some hypothalamic regions involved in the control of reproduction and characterized by a large number of estrogen receptors is under the control of gonadal hormones and may vary according to the rapid variations of hormonal levels that take place during the estrous cycle.

  10. The Stress Protein/Chaperone Grp94 Counteracts Muscle Disuse Atrophy by Stabilizing Subsarcolemmal Neuronal Nitric Oxide Synthase

    Science.gov (United States)

    Vitadello, Maurizio; Gherardini, Jennifer

    2014-01-01

    Abstract Aims: Redox and growth-factor imbalance fosters muscle disuse atrophy. Since the endoplasmic-reticulum chaperone Grp94 is required for folding insulin-like growth factors (IGFs) and for antioxidant cytoprotection, we investigated its involvement in muscle mass loss due to inactivity. Results: Rat soleus muscles were transfected in vivo and analyzed after 7 days of hindlimb unloading, an experimental model of muscle disuse atrophy, or standard caging. Increased muscle protein carbonylation and decreased Grp94 protein levels (pmuscle atrophy regulators identified 160 kDa neuronal nitric oxide synthase (nNOS) as a new Grp94 partner. Unloading was demonstrated to untether nNOS from myofiber subsarcolemma; here, we show that such nNOS localization, revealed by means of NADPH-diaphorase histochemistry, appeared preserved in unloaded myofibers expressing recombinant Grp94, compared to those transfected with the empty vector or deleted Grp94 cDNA (p<0.02). Innovation: Grp94 interacts with nNOS and prevents its untethering from sarcolemma in unloaded myofibers. Conclusion: Maintenance of Grp94 expression is sufficient to counter unloading atrophy and oxidative stress by mechanistically stabilizing nNOS-multiprotein complex at the myofiber sarcolemma. Antioxid. Redox Signal. 20, 2479–2496. PMID:24093939

  11. Triterpenoic Acids from Apple Pomace Enhance the Activity of the Endothelial Nitric Oxide Synthase (eNOS).

    Science.gov (United States)

    Waldbauer, Katharina; Seiringer, Günter; Nguyen, Dieu Linh; Winkler, Johannes; Blaschke, Michael; McKinnon, Ruxandra; Urban, Ernst; Ladurner, Angela; Dirsch, Verena M; Zehl, Martin; Kopp, Brigitte

    2016-01-13

    Pomace is an easy-accessible raw material for the isolation of fruit-derived compounds. Fruit consumption is associated with health-promoting effects, such as the prevention of cardiovascular disease. Increased vascular nitric oxide (NO) bioavailability, for example, due to an enhanced endothelial nitric oxide synthase (eNOS) activity, could be one molecular mechanism mediating this effect. To identify compounds from apple (Malus domestica Borkh.) pomace that have the potential to amplify NO bioavailability via eNOS activation, a bioassay-guided fractionation of the methanol/water (70:30) extract has been performed using the (14)C-L-arginine to (14)C-L-citrulline conversion assay (ACCA) in the human endothelium-derived cell line EA.hy926. Phytochemical characterization of the active fractions was performed using the spectrophotometric assessment of the total phenolic content, as well as TLC, HPLC-DAD-ELSD, and HPLC-MS analyses. Eleven triterpenoic acids, of which one is a newly discovered compound, were identified as the main constituents in the most active fraction, accompanied by only minor contents of phenolic compounds. When tested individually, none of the tested compounds exhibited significant eNOS activation. Nevertheless, cell stimulation with the reconstituted compound mixture restored eNOS activation, validating the potential of apple pomace as a source of bioactive components.

  12. Diastereoselective Synthesis of the Aminocyclitol Core of Jogyamycin via an Allene Aziridination Strategy.

    Science.gov (United States)

    Gerstner, Nels C; Adams, Christopher S; Grigg, R David; Tretbar, Maik; Rigoli, Jared W; Schomaker, Jennifer M

    2016-01-15

    Oxidative allene amination provides rapid access to densely functionalized amine-containing stereotriads through highly reactive bicyclic methyleneaziridine intermediates. This strategy has been demonstrated as a viable approach for the construction of the densely functionalized aminocyclitol core of jogyamycin, a natural product with potent antiprotozoal activity. Importantly, the flexibility of oxidative allene amination will enable the syntheses of modified aminocyclitol analogues of the jogyamycin core.

  13. Differential contributions of nitric oxide synthase isoforms at hippocampal formation to negative feedback regulation of penile erection in the rat.

    Science.gov (United States)

    Chang, Alice Y W; Chan, Julie Y H; Chan, Samuel H H

    2002-05-01

    We established previously that a novel negative feedback mechanism for the regulation of penile erection, which is triggered by ascending sensory inputs initiated by tumescence of the penis, exists in the hippocampal formation (HF). This study further evaluated the participation of nitric oxide (NO) and the contribution of nitric oxide synthase (NOS) isoforms at the HF in this process. Adult, male Sprague-Dawley rats that were anaesthetized and maintained with chloral hydrate were used, and intracavernous pressure (ICP) recorded from the corpus cavernosum of the penis was employed as our experimental index for penile erection. Microinjection bilaterally of a NO donor, S-nitroso-N-acetylpenicillamine (0.25 or 1 nmoles), or the NO precursor, L-arginine (1 or 5 nmoles), into the hippocampal CA1 or CA3 subfield or dentate gyrus elicited a significant reduction in baseline ICP. Bilateral hippocampal application of a NO trapping agent, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (10 nmoles), significantly potentiated the elevation in ICP induced by intracavernous administration of papaverine (400 microg). Microinjection bilaterally into the HF of equimolar doses (0.5 or 2.5 pmoles) of two selective neuronal NOS inhibitors, 7-nitroindazole or N(omega)-propyl-L-arginine; or equimolar doses (50 or 250 pmoles) of two selective inducible NOS inhibitors, aminoguanidine or S-methylisothiourea, significantly enhanced the magnitude and/or duration of the papaverine-induced elevation in ICP. In contrast, hippocampal application of a potent endothelial NOS inhibitor, N5-(1-iminoethyl)-L-ornithine (18 or 92 nmoles), was ineffective. Neither of these inhibitors, furthermore, affected baseline ICP. These results suggest that NO generated via both neuronal and inducible NOS at the HF may participate in negative feedback regulation of penile erection.

  14. Acanthopanax divaricatus var. chiisanensis reduces blood pressure via the endothelial nitric oxide synthase pathway in the spontaneously hypertensive rat model.

    Science.gov (United States)

    Park, Soo-Yeon; Do, Gyeong-Min; Lee, Sena; Lim, Yeni; Shin, Jae-Ho; Kwon, Oran

    2014-09-01

    In this study, we investigated the antihypertensive effects of Acanthopanax divaricatus var. chiisanensis extract (AE) and its active compound, acanthoside D (AD), on arterial blood pressure (BP) in vivo and endothelial function in vitro. We hypothesized that AE has antihypertensive effects, which is attributed to enhancement of endothelial function via the improvement of nitric oxide synthesis or the angiotensin II (Ang II) response. Spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs) were randomly divided into 7 groups and then fed the following diets for 14 weeks: WKY fed a normal diet (WN); SHR fed a normal diet (SN); SHR fed a high-cholesterol (HC) diet (SH); SHR fed a HC diet with AE of 150, 300, 600 mg/kg body weight (SH-L, SH-M, SH-H); and SHR fed an HC diet with AD of 600 μg/kg body weight (SH-D). Blood pressure was significantly reduced in the SH-H compared with the SH from week 10 until week 14; BP was also significantly decreased in the SHR fed a HC diet with AE of 300 at week 14. Aortic wall thickness showed a tendency to decrease by AE and AD treatment. The SH-H showed increased endothelial nitric oxide synthase (eNOS) expression in the intima and media, compared with the SH. Furthermore, a significant increase in intracellular nitric oxide production was induced by AE and AD treatment in human umbilical vein endothelial cells. A significant increase of phospho-eNOS was found with a high dose of AE in human umbilical vein endothelial cells but not with AD. These results suggest that AE can regulate BP and improve endothelial function via eNOS-dependent vasodilation. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Saturation of Van Allen's belts

    CERN Document Server

    Le Bel, E

    2002-01-01

    The maximum number of electrons that can be trapped in van Allen's belts has been evaluated at CEA-DAM more precisely than that commonly used in the space community. The modelization that we have developed allows to understand the disagreement (factor 50) observed between the measured and predicted electrons flux by US satellites and theory. This saturation level allows sizing-up of the protection on a satellite in case of energetic events. (authors)

  16. CuCl-catalyzed aerobic oxidation of 2,3-allenols to 1,2-allenic ketones with 1:1 combination of phenanthroline and bipyridine as ligands

    Directory of Open Access Journals (Sweden)

    Shengming Ma

    2011-04-01

    Full Text Available A protocol has been developed to prepare 1,2-allenyl ketones using molecular oxygen in air or pure oxygen as the oxidant from 2,3-allenylic alcohols with moderate to good yields under mild conditions. In this reaction CuCl (20 mol % with 1,10-phenanthroline (10 mol % and bipyridine (10 mol % was used as the catalyst. It is interesting to observe that the use of the mixed ligands is important for the higher yields of this transformation: With the monoligand approach developed by Markó et al., the yields are relatively lower.

  17. Hypercapnic vasodilatation in isolated rat basilar arteries is exerted via low pH and does not involve nitric oxide synthase stimulation or cyclic GMP production

    DEFF Research Database (Denmark)

    You, J P; Wang, Qian; Zhang, W

    1994-01-01

    The relaxant effect of hypercapnia (15% CO2) was studied in isolated circular segments of rat basilar arteries with intact endothelium. The nitric oxide synthase inhibitor nitro-L-arginine (L-NOARG) and the cytosolic guanylate cyclase inhibitor methylene blue (MB), significantly reduced this rela......The relaxant effect of hypercapnia (15% CO2) was studied in isolated circular segments of rat basilar arteries with intact endothelium. The nitric oxide synthase inhibitor nitro-L-arginine (L-NOARG) and the cytosolic guanylate cyclase inhibitor methylene blue (MB), significantly reduced...... this relaxation by 54% and 70%, respectively. The effect of L-NOARG was completely reversed by L-arginine. Blockade of nerve excitation with tetrodotoxin (TTX) had no affect on the 15% CO2 elicited vasodilatation. Measurements of cGMP in vessel segments showed no significant increase in cGMP content in response...

  18. Constitutive arginine-dependent nitric oxide synthase activity in different organs of pea seedlings during plant development.

    Science.gov (United States)

    Corpas, Francisco J; Barroso, Juan B; Carreras, Alfonso; Valderrama, Raquel; Palma, José M; León, Ana M; Sandalio, Luisa M; del Río, Luis A

    2006-07-01

    Nitric oxide (NO) is an important signalling molecule in different animal and plant physiological processes. Little is known about its biological function in plants and on the enzymatic source or site of NO production during plant development. The endogenous NO production from L-arginine (NO synthase activity) was analyzed in leaves, stems and roots during plant development, using pea seedlings as a model. NOS activity was analyzed using a novel chemiluminescence-based assay which is more sensitive and specific than previous methods used in plant tissues. In parallel, NO accumulation was analyzed by confocal laser scanning microscopy using as fluorescent probes either DAF-2 DA or DAF-FM DA. A strong increase in NOS activity was detected in stems after 11 days growth, coinciding with the maximum stem elongation. The arginine-dependent NOS activity was constitutive and sensitive to aminoguanidine, a well-known irreversible inhibitor of animal NOS, and this NOS activity was differentially modulated depending on the plant organ and seedling developmental stage. In all tissues studied, NO was localized mainly in the vascular tissue (xylem) and epidermal cells and in root hairs. These loci of NO generation and accumulation suggest novel functions for NO in these cell types.

  19. Cyclooxygenase 2 and neuronal nitric oxide synthase expression in the renal cortex are not interdependent in states of salt deficiency

    DEFF Research Database (Denmark)

    Castrop, H; Kammerl, M; Mann, Birgitte

    2000-01-01

    Neuronal nitric oxide synthase (nNOS) and cyclooxygenase-2 (COX-2) expression in the kidney are localized to the cortical thick ascending limb of the loop of Henle (cTALH), including the macula region, and increase after salt restriction. Because of the similar localization and regulation of n......NOS and COX-2 expression, we have examined whether there is a functional interrelationship between the expression of the two enzymes. Male Sprague Dawley rats were fed for 1 week either a low-salt diet (0.02% w/w) which produced moderate increases of nNOS and COX-2 expression, or low salt combined...... with the angiotensin I converting enzyme inhibitor ramipril (10 mg/kg per day), which produced strong increases of renocortical nNOS and COX-2 expressions. To inhibit nNOS or COX-2 activities, animals received in addition N(G)-nitro-L-arginine methyl ester (L-NAME, 50 mg/kg per day) or rofecoxib (10 mg/kg per day...

  20. A high-lipid diet potentiates left ventricular dysfunction in nitric oxide synthase 3-deficient mice after chronic pressure overload.

    Science.gov (United States)

    Ahmadie, Roien; Santiago, Jon-Jon; Walker, Jonathan; Fang, Tielan; Le, Khuong; Zhao, Zhaohui; Azordegan, Nazila; Bage, Sheri; Lytwyn, Matthew; Rattan, Sunil; Dixon, Ian M C; Kardami, Elissavet; Moghadasian, Mohammed H; Jassal, Davinder S

    2010-08-01

    A high-lipid diet (HLD) may lead to adverse left ventricular (LV) remodeling and endothelial dysfunction in conditions of hemodynamic stress. Although congenital absence of nitric oxide synthase 3 (NOS3) leads to adverse LV remodeling after transverse aortic constriction (TAC), the effects of a HLD in this state remains unknown. Wild-type (WT) and NOS3 knockout mice (NOS3(-/-)) were randomized into the following 4 groups: 1) WT + low-lipid diet (LLD) (10% of energy); 2) WT + HLD (60% of energy); 3) NOS3(-/-) + LLD; and 4) NOS3(-/-) + HLD for a total of 12 wk. After 1 wk of randomization, TAC was performed on all groups. Serial echocardiography revealed a decrease in LV ejection fraction (LVEF) in WT and NOS3(-/-) mice fed the HLD compared with those fed the LLD diet at 12 wk post-TAC. Mice fed the NOS3(-/-) + HLD diet had a lower LVEF compared with mice in the other 3 groups (P < 0.05). There was greater myocyte hypertrophy, interstitial fibrosis, and percentage change in plasma cholesterol concentrations in the NOS3(-/-) + HLD group 12 wk post-TAC compared with the other 3 groups. Although high molecular weight fibroblast growth factor-2, a marker of cardiac hypertrophy, was more upregulated in the NOS3(-/-) + HLD group than in the other groups, markers of the renin-angiotensin system did not differ among them. A HLD potentiates LV dysfunction in NOS3(-/-) mice in a chronic pressure overload state.

  1. Distinctive expression patterns of hypoxia-inducible factor-1α and endothelial nitric oxide synthase following hypergravity exposure.

    Science.gov (United States)

    Yoon, Gun; Oh, Choong Sik; Kim, Hyun-Soo

    2016-06-07

    This study was designed to examine the expression of hypoxia-inducible factor-1α (HIF-1α) and the level and activity of endothelial nitric oxide synthase (eNOS) in the hearts and livers of mice exposed to hypergravity. Hypergravity-induced hypoxia and the subsequent post-exposure reoxygenation significantly increased cardiac HIF-1α levels. Furthermore, the levels and activity of cardiac eNOS also showed significant increase immediately following hypergravity exposure and during the reoxygenation period. In contrast, the expression of phosphorylated Akt (p-Akt) and phosphorylated extracellular signal-regulated kinase (p-ERK) showed significant elevation only during the reoxygenation period. These data raise the possibility that the increase in cardiac HIF-1α expression induced by reoxygenation involves a cascade of signaling events, including activation of the Akt and ERK pathways. In the liver, HIF-1α expression was significantly increased immediately after hypergravity exposure, indicating that hypergravity exposure to causes hepatocellular hypoxia. The hypergravity-exposed livers showed significantly higher eNOS immunoreactivity than did those of control mice. Consistent with these results, significant increases in eNOS activity and nitrate/nitrite levels were also observed. These findings suggest that hypergravity-induced hypoxia plays a significant role in the upregulation of hepatic eNOS.

  2. Immunodetection and localization of nitric oxide synthase in the olfactory center of the terrestrial snail, Helix pomatia.

    Science.gov (United States)

    Nacsa, K; Elekes, K; Serfőző, Z

    2012-01-01

    The procerebrum of stylommatophoran snails produces nitric oxide (NO)-modulated oscillatory local field potentials which are considered the basis of olfactory information processing. Although the function of NO is well characterized in the PC, the identification and distribution of NO synthase (NOS) has not known completely. In the present study, applying a mammalian anti-NOS antibody, a 170 kDa molecular weight NOS-like protein was demonstrated in the procerebrum homogenate of Helix pomatia. NOS-like immunolabeling of the globuli cells, the internal and terminal neuropils displayed an identical distribution compared to that of NADPH-diaphorase reactive material, confirming the specificity of immunohistochemistry. The detailed characteristics of the immunostaining (different intensity of the neural perikarya, a gradual appearance in the terminal neuropil and in the axon bundles of the tentacular nerve, as well as an intense, homogeneous distribution of NOS-like immunoreactivity in the internal neuropil) suggest that NOS is expressed constitutively, maintaining a high level of the enzyme in neuropil areas. NOS accumulation in the internal neuropil suggests that NO plays an important role in delivering olfactory signals extrinsic to the procerebrum, and integrating them with other sensory modalities, respectively. Our results are the first, demonstrating unequivocally the presence of NOS and resolving its differential distribution in the Helix procerebrum.

  3. Negative feedback regulation of lipopolysaccharide-induced inducible nitric oxide synthase gene expression by heme oxygenase-1 induction in macrophages.

    Science.gov (United States)

    Ashino, Takashi; Yamanaka, Rieko; Yamamoto, Masayuki; Shimokawa, Hiroaki; Sekikawa, Kenji; Iwakura, Yoichiro; Shioda, Seiji; Numazawa, Satoshi; Yoshida, Takemi

    2008-04-01

    Heme oxygenase-1 (HO-1) is induced under infectious diseases in macrophages. We performed experiments using various gene deficient mouse-derived macrophages to determine a detailed induction mechanism of HO-1 by lipopolysaccharide (LPS) and the functional role of HO-1 induction in macrophages. LPS (1 microg/mL) maximally induced inducible nitric oxide synthase (iNOS) and HO-1 mRNAs in wild-type (WT) macrophages at 6h and 12h after treatment, respectively, and liberated tumor necrosis factor alpha (TNFalpha) from WT macrophages. LPS also induced iNOS and HO-1 in TNFalpha(-/-) macrophages, but not in iNOS(-/-) macrophages. Interestingly, although LPS strongly induced iNOS, it failed to induce HO-1 almost completely in nuclear-factor erythroid 2-related factor 2 (Nrf2)(-/-) macrophages. The LPS-induced iNOS gene expression was suppressed by pretreatment with HO-1 inducers, hemin and Co-protoporphyrin (CoPP), but not with HO-1 inhibitor, Sn-protoporphyrin in WT macrophages. In the Nrf2(-/-) macrophages, the ability of CoPP to induce HO-1 and its inhibitory effect on the LPS-induced iNOS gene expression were lower than seen in WT macrophages. The present findings suggest that HO-1 is induced via NO-induced nuclear translocation of Nrf2, and the enzymatic function of HO-1 inhibits the overproduction of NO in macrophages.

  4. Epigenetic regulation of nitric oxide synthase 2, inducible (Nos2) by NLRC4 inflammasomes involves PARP1 cleavage.

    Science.gov (United States)

    Buzzo, Carina de Lima; Medina, Tiago; Branco, Laura M; Lage, Silvia L; Ferreira, Luís Carlos de Souza; Amarante-Mendes, Gustavo P; Hottiger, Michael O; De Carvalho, Daniel D; Bortoluci, Karina R

    2017-02-02

    Nitric oxide synthase 2, inducible (Nos2) expression is necessary for the microbicidal activity of macrophages. However, NOS2 over-activation causes multiple inflammatory disorders, suggesting a tight gene regulation is necessary. Using cytosolic flagellin as a model for inflammasome-dependent NOS2 activation, we discovered a surprising new role for NLRC4/caspase-1 axis in regulating chromatin accessibility of the Nos2 promoter. We found that activation of two independent mechanisms is necessary for NOS2 expression by cytosolic flagellin: caspase-1 and NF-κB activation. NF-κB activation was necessary, but not sufficient, for NOS2 expression. Conversely, caspase-1 was necessary for NOS2 expression, but dispensable for NF-κB activation, indicating that this protease acts downstream NF-κB activation. We demonstrated that epigenetic regulation of Nos2 by caspase-1 involves cleavage of the chromatin regulator PARP1 (also known as ARTD1) and chromatin accessibility of the NF-κB binding sites located at the Nos2 promoter. Remarkably, caspase-1-mediated Nos2 transcription and NO production contribute to the resistance of macrophages to Salmonella typhimurium infection. Our results uncover the molecular mechanism behind the constricted regulation of Nos2 expression and open new therapeutic opportunities based on epigenetic activities of caspase-1 against infectious and inflammatory diseases.

  5. Contribution of nitric oxide synthase to luminol-dependent chemiluminescence generated by phorbol-ester-activated Kupffer cells.

    Science.gov (United States)

    Wang, J F; Komarov, P; Sies, H; de Groot, H

    1991-01-01

    Phorbol 12-myristate 13-acetate-induced luminol chemiluminescence in rat Kupffer cells was doubled by the addition of L-arginine and significantly (up to 70%) inhibited by NG-nitro-L-arginine and NG-monomethyl-L-arginine, competitive inhibitors of L-arginine-dependent nitric oxide (NO) formation. The release of superoxide anion (O2-) by NADPH oxidase was neither affected by L-arginine nor by the inhibitors. Only very slight luminol chemiluminescence was detectable in lipopolysaccharide-pretreated Kupffer cells, a condition in which significant amounts of NO were formed but no O2-. In a cell-free system, significant luminol chemiluminescence only occurred when both authentic NO and the O2-/H2O2- generating system xanthine/xanthine oxidase were present. The results indicate that luminol chemiluminescence in phorbol-ester-activated Kupffer cells largely depends on L-arginine metabolism by NO synthase, requiring the concurrent formation of NO and O2-/H2O2. PMID:1718262

  6. Contribution of nitric oxide synthase from coagulase-negative staphylococci to the development of red myoglobin derivatives.

    Science.gov (United States)

    Ras, Geoffrey; Bailly, Xavier; Chacornac, Jean-Paul; Zuliani, Véronique; Derkx, Patrick; Seibert, Tim M; Talon, Régine; Leroy, Sabine

    2018-02-02

    As part of the microbial community of meat or as starter cultures, coagulase-negative staphylococci (CNS) serve several essential technological purposes in meat products, such as color development through the reduction of nitrate to nitrite. As the safety of nitrite as an additive has been questioned, we explored the potential of CNS to develop red myoglobin derivatives such as oxymyoglobin and nitrosomyoglobin. Nitrosoheme was extracted to evaluate NO production. This production could be due to a nitric oxide synthase (NOS) activity. In all CNS strains, a nos gene was identified. The NOS sequences deduced were highly conserved within CNS. A phylogenetic tree based on the NOS sequences revealed that the strains within species were clustered. Ninety-one percent of the strains, whatever the species, were able to form red myoglobin derivatives in aerobic conditions, but a high variability was observed between strains within species. However, NO production was low as nitrosomyoglobin represented 8% to 16% of the red pigments according to the species. Formation of oxymyoglobin, especially under aerobic conditions, was substantial, but varied greatly within species. The mechanism involved in the formation of oxymyoglobin could rely on staphylococcal reductases and remains to be explored. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Fo Shou San, an ancient Chinese herbal decoction, protects endothelial function through increasing endothelial nitric oxide synthase activity.

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    Cathy W C Bi

    Full Text Available Fo Shou San (FSS is an ancient herbal decoction comprised of Chuanxiong Rhizoma (CR; Chuanxiong and Angelicae Sinensis Radix (ASR; Danggui in a ratio of 2:3. Previous studies indicate that FSS promotes blood circulation and dissipates blood stasis, thus which is being used widely to treat vascular diseases. Here, we aim to determine the cellular mechanism for the vascular benefit of FSS. The treatment of FSS reversed homocysteine-induced impairment of acetylcholine (ACh-evoked endothelium-dependent relaxation in aortic rings, isolated from rats. Like radical oxygen species (ROS scavenger tempol, FSS attenuated homocysteine-stimulated ROS generation in cultured human umbilical vein endothelial cells (HUVECs, and it also stimulated the production of nitric oxide (NO as measured by fluorescence dye and biochemical assay. In addition, the phosphorylation levels of both Akt kinase and endothelial NO synthases (eNOS were markedly increased by FSS treatment, which was abolished by an Akt inhibitor triciribine. Likewise, triciribine reversed FSS-induced NO production in HUVECs. Finally, FSS elevated intracellular Ca(2+ levels in HUVECs, and the Ca(2+ chelator BAPTA-AM inhibited the FSS-stimulated eNOS phosphorylation. The present results show that this ancient herbal decoction benefits endothelial function through increased activity of Akt kinase and eNOS; this effect is causally via a rise of intracellular Ca(2+ and a reduction of ROS.

  8. Enhancer-associated long non-coding RNA LEENE regulates endothelial nitric oxide synthase and endothelial function.

    Science.gov (United States)

    Miao, Yifei; Ajami, Nassim E; Huang, Tse-Shun; Lin, Feng-Mao; Lou, Chih-Hong; Wang, Yun-Ting; Li, Shuai; Kang, Jian; Munkacsi, Hannah; Maurya, Mano R; Gupta, Shakti; Chien, Shu; Subramaniam, Shankar; Chen, Zhen

    2018-01-18

    The optimal expression of endothelial nitric oxide synthase (eNOS), the hallmark of endothelial homeostasis, is vital to vascular function. Dynamically regulated by various stimuli, eNOS expression is modulated at transcriptional, post-transcriptional, and post-translational levels. However, epigenetic modulations of eNOS, particularly through long non-coding RNAs (lncRNAs) and chromatin remodeling, remain to be explored. Here we identify an enhancer-associated lncRNA that enhances eNOS expression (LEENE). Combining RNA-sequencing and chromatin conformation capture methods, we demonstrate that LEENE is co-regulated with eNOS and that its enhancer resides in proximity to eNOS promoter in endothelial cells (ECs). Gain- and Loss-of-function of LEENE differentially regulate eNOS expression and EC function. Mechanistically, LEENE facilitates the recruitment of RNA Pol II to the eNOS promoter to enhance eNOS nascent RNA transcription. Our findings unravel a new layer in eNOS regulation and provide novel insights into cardiovascular regulation involving endothelial function.

  9. Inhibition or knock out of Inducible nitric oxide synthase result in resistance to bleomycin-induced lung injury

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    Crimi Nunzio

    2005-06-01

    Full Text Available Abstract Background In the present study, by comparing the responses in wild-type mice (WT and mice lacking (KO the inducible (or type 2 nitric oxide synthase (iNOS, we investigated the role played by iNOS in the development of on the lung injury caused by bleomycin administration. When compared to bleomycin-treated iNOSWT mice, iNOSKO mice, which had received bleomycin, exhibited a reduced degree of the (i lost of body weight, (ii mortality rate, (iii infiltration of the lung with polymorphonuclear neutrophils (MPO activity, (iv edema formation, (v histological evidence of lung injury, (vi lung collagen deposition and (vii lung Transforming Growth Factor beta1 (TGF-β1 expression. Methods Mice subjected to intratracheal administration of bleomycin developed a significant lung injury. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in lungs from bleomycin-treated iNOSWT mice. Results The intensity and degree of nitrotyrosine staining was markedly reduced in tissue section from bleomycin-iNOSKO mice. Treatment of iNOSWT mice with of GW274150, a novel, potent and selective inhibitor of iNOS activity (5 mg/kg i.p. also significantly attenuated all of the above indicators of lung damage and inflammation. Conclusion Taken together, our results clearly demonstrate that iNOS plays an important role in the lung injury induced by bleomycin in the mice.

  10. AVE 3085, a novel endothelial nitric oxide synthase enhancer, attenuates cardiac remodeling in mice through the Smad signaling pathway.

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    Chen, Yili; Chen, Cong; Feng, Cong; Tang, Anli; Ma, Yuedong; He, Xin; Li, Yanhui; He, Jiangui; Dong, Yugang

    2015-03-15

    AVE 3085 is a novel endothelial nitric oxide synthase enhancer. Although AVE 3085 treatment has been shown to be effective in spontaneously restoring endothelial function in hypertensive rats, little is known about the effects and mechanisms of AVE 3085 with respect to cardiac remodeling. The present study was designed to examine the effects of AVE 3085 on cardiac remodeling and the mechanisms underlying the effects of this compound. Mice were subjected to aortic banding to induce cardiac remodeling and were then administered AVE 3085 (10 mg kg day(-1), orally) for 4 weeks. At the end of the treatment, the aortic banding-treated mice exhibited significant elevations in cardiac remodeling, characterized by an increase in left ventricular weight relative to body weight, an increase in the area of collagen deposition, an increase in the mean myocyte diameter, and increases in the gene expressions of the hypertrophic markers atrial natriuretic peptide (ANP) and β-MHC. These indexes were significantly decreased in the AVE 3085-treated mice. Furthermore, AVE 3085 treatment reduced the expression and activation of the Smad signaling pathway in the aortic banding-treated mice. Our data showed that AVE 3085 attenuated cardiac remodeling, and this effect was possibly mediated through the inhibition of Smad signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Role of Endothelial Nitric Oxide Synthase Gene Polymorphisms in Predicting Aneurysmal Subarachnoid Hemorrhage in South Indian Patients

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    Linda Koshy

    2008-01-01

    Full Text Available Endothelial nitric oxide synthase (eNOS gene polymorphisms have been implicated as predisposing genetic factors that can predict aneurysmal subarachnoid hemorrhage (aSAH, but with controversial results from different populations. Using a case-control study design, we tested the hypothesis whether variants in eNOS gene can increase risk of aSAH among South Indian patients, either independently, or by interacting with other risk factors of the disease. We enrolled 122 patients, along with 224 ethnically matched controls. We screened the intron-4 27-bp VNTR, the promoter T-786C and the exon-7 G894T SNPs in the eNOS gene. We found marked interethnic differences in the genotype distribution of eNOS variants when comparing the South Indian population with the reported frequencies from Caucasian and Japanese populations. Genotype distributions in control and patient populations were found to be in Hardy-Weinberg equilibrium. In patients, the allele, genotype and estimated haplotype frequencies did not differ significantly from the controls. Multiple logistic regression indicated hypertension and smoking as risk factors for the disease, however the risk alleles did not have any interaction with these risk factors. Although the eNOS polymorphisms were not found to be a likely risk factor for aSAH, the role of factors such as ethnicity, gender, smoking and hypertension should be evaluated cautiously to understand the genotype to phenotype conversion.

  12. Analysis of Human Bradykinin Receptor Gene and Endothelial Nitric Oxide Synthase Gene Polymorphisms in End-Stage Renal Disease Among Malaysians

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    R. Vasudevan

    2014-06-01

    Full Text Available The aim of this study was to determine the association of the c.894G>T; p.Glu298Asp polymorphism and the variable number tandem repeat (VNTR polymorphism of the endothelial nitric oxide synthase (eNOS gene and c.181C>T polymorphism of the bradykinin type 2 receptor gene (B2R in Malaysian end-stage renal disease (ESRD subjects.

  13. Interleukin 1 beta induces diabetes and fever in normal rats by nitric oxide via induction of different nitric oxide synthases

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    Reimers, J I; Bjerre, U; Mandrup-Poulsen, T

    1994-01-01

    Substantial in vitro evidence suggests that nitric oxide may be a major mediator of interleukin 1 (IL-1) induced pancreatic beta-cell inhibition and destruction in the initial events leading to insulin-dependent diabetes mellitus. Using NG-nitro-L-arginine methyl ester, an inhibitor of both...

  14. Altered contractile response due to increased beta3-adrenoceptor stimulation in diabetic cardiomyopathy: the role of nitric oxide synthase 1-derived nitric oxide.

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    Amour, Julien; Loyer, Xavier; Le Guen, Morgan; Mabrouk, Nejma; David, Jean-Stéphane; Camors, Emmanuel; Carusio, Nunzia; Vivien, Benoît; Andriantsitohaina, Ramaroson; Heymes, Christophe; Riou, Bruno

    2007-09-01

    In the diabetic heart, the positive inotropic response to beta-adrenoceptor stimulation is altered and beta1 and beta2 adrenoceptors are down-regulated, whereas beta3 adrenoceptor is up-regulated. In heart failure, beta3-adrenoceptor stimulation induces a negative inotropic effect that results from endothelial nitric oxide synthase (NOS3)-derived nitric oxide production. The objective of our study was to investigate the role of beta3-adrenoceptor in diabetic cardiomyopathy. beta-Adrenergic responses were investigated in vivo (dobutamine echocardiography) and in vitro (left ventricular papillary muscle) in healthy and streptozotocin-induced diabetic rats. The effect of beta3-adrenoceptor inhibition on the inotropic response was studied in vitro. Immunoblots and NOS activities were performed in heart homogenates (electron paramagnetic resonance) and isolated cardiomyocytes. Data are mean percentage of baseline +/- SD. The impaired positive inotropic effect was confirmed in diabetes both in vivo (121 +/- 15% vs. 160 +/- 16%; P < 0.05) and in vitro (112 +/- 5% vs. 179 +/- 15%; P < 0.05). In healthy rat, the positive inotropic effect was not significantly modified in presence of beta3-adrenoceptor antagonist (174 +/- 20%), nonselective NOS inhibitor (N -nitro-l-arginine methylester [l-NAME]; 183 +/- 19%), or selective NOS1 inhibitor (vinyl-l-N-5-(1-imino-3-butenyl)-l-ornithine [l-VNIO]; 172 +/- 13%). In diabetes, in parallel with the increase in beta3-adrenoceptor protein expression, the positive inotropic effect was partially restored by beta3-adrenoceptor antagonist (137 +/- 8%; P < 0.05), l-NAME (133 +/- 11%; P < 0.05), or l-VNIO (130 +/- 13%; P < 0.05). Nitric oxide was exclusively produced by NOS1 within diabetic cardiomyocytes. NOS2 and NOS3 proteins were undetectable. beta3-Adrenoceptor is involved in altered positive inotropic response to beta-adrenoceptor stimulation in diabetic cardiomyopathy. This effect is mediated by NOS1-derived nitric oxide in diabetic

  15. Electrochemically driven biocatalysis of the oxygenase domain of neuronal nitric oxide synthase in indium tin oxide nanoparticles/polyvinyl alcohol nanocomposite.

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    Xu, Xuan; Wollenberger, Ulla; Qian, Jing; Lettau, Katrin; Jung, Christiane; Liu, Songqin

    2013-12-01

    Nitric oxide synthase (NOS) plays a critical role in a number of key physiological and pathological processes. Investigation of electron-transfer reactions in NOS would contribute to a better understanding of the nitric oxide (NO) synthesis mechanism. Herein, we describe an electrochemically driven catalytic strategy, using a nanocomposite that consisted of the oxygenase domain of neuronal NOS (D290nNOSoxy), indium tin oxide (ITO) nanoparticles and polyvinyl alcohol (PVA). Fast direct electron transfer between electrodes and D290nNOSoxy was observed with the heterogeneous electron transfer rate constant (ket) of 154.8 ± 0.1s(-1) at the scan rate of 5 Vs(-1). Moreover, the substrate N(ω)-hydroxy-L-arginine (NHA) was used to prove the concept of electrochemically driven biocatalysis of D290nNOSoxy. In the presence of the oxygen cosubstrate and tetrahydrobiopterin (BH4) cofactor, the addition of NHA caused the decreases of both oxidation current at +0.1 V and reduction current at potentials ranging from -0.149 V to -0.549 V vs Ag/AgCl. Thereafter, a series of control experiments such as in the absence of BH4 or D290nNOSoxy were performed. All the results demonstrated that D290nNOSoxy biocatalysis was successfully driven by electrodes in the presence of BH4 and oxygen. This novel bioelectronic system showed potential for further investigation of NOS and biosensor applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Modulation of inducible nitric oxide synthase gene expression in RAW 264.7 murine macrophages by Pacific ciguatoxin.

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    Kumar-Roiné, Shilpa; Matsui, Mariko; Chinain, Mireille; Laurent, Dominique; Pauillac, Serge

    2008-08-01

    To investigate the possible involvement of the nitric oxide radical (NO) in ciguatera fish poisoning (CFP), the in vitro effects of the main Pacific ciguatoxin (P-CTX-1B) and bacterial lipopolysaccharide (LPS) were comparatively studied on neuroblastoma Neuro-2a and on macrophage RAW 264.7 cell lines. NO accumulation was quantified by measuring nitrite levels in cellular supernatant using Griess reagent while the up-regulation of inducible nitric oxide synthase (iNOS) at the mRNA level was quantified via Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR). P-CTX-1B caused a concentration- and time-dependent induction of iNOS in RAW 264.7 cells but not in Neuro-2a cells. NO production was evidenced by increased nitrite levels in the 10 microM range after 48 h of RAW 264.7 cells exposure to LPS and P-CTX-1B (0.05 microg/ml and 6 nM, respectively). The expression of iNOS mRNA peaked at 8h for LPS then gradually decreased to low level at 48 h. In contrast, a sustained level was recorded with P-CTX-1B in the 8-48 h time interval. The addition of N(omega)-nitro-L-arginine methyl ester (L-NAME), a stereoselective NOS inhibitor, strongly diminished NO formation but had no effect on iNOS mRNA synthesis. The implication of NO in CFP paves the way for new therapies for both western and traditional medicines.

  17. Promoter polymorphisms in the nitric oxide synthase 3 gene are associated with ischemic stroke susceptibility in young black women.

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    Howard, Timothy D; Giles, Wayne H; Xu, Jianfeng; Wozniak, Marcella A; Malarcher, Ann M; Lange, Leslie A; Macko, Richard F; Basehore, Monica J; Meyers, Deborah A; Cole, John W; Kittner, Steven J

    2005-09-01

    Endothelial nitric oxide exerts a variety of protective effects on endothelial cells and blood vessels, and therefore the nitric oxide synthase 3 gene (NOS3) is a logical candidate gene for stroke susceptibility. We used the population-based Stroke Prevention in Young Women case-control study to assess the association of five NOS3 polymorphisms in 110 cases (46% black) with ischemic stroke and 206 controls (38% black), 15 to 44 years of age. Polymorphisms included 3 single nucleotide polymorphisms (SNPs) in the promoter region (-1468 T>A, -922 G>A, -786 T>C), 1 SNP in exon 7 (G894T), and 1 insertion/deletion polymorphism within intron 4. Significant associations with both the -922 G>A and -786 T>C SNPs with ischemic stroke were observed in the black, but not the white, population. This association was attributable to an increased prevalence of the -922 A allele (OR=3.0, 95% CI=1.3 to 6.8; P=0.005) and the -786 T allele (OR=2.9, 95% CI=1.3 to 6.4; P=0.005) in cases versus controls. These 2 SNPs were in strong linkage disequilibrium (D'=1.0), making it impossible to determine, within the confines of this genetic study, whether 1 or both of these polymorphisms are functionally related to NOS3 expression. Two sets of haplotypes were also identified, 1 of which may confer an increased susceptibility to stroke in blacks, whereas the other appears to be protective. Promoter variants in NOS3 may be associated with ischemic stroke susceptibility among young black women.

  18. Caveolin-1-mediated post-transcriptional regulation of inducible nitric oxide synthase in human colon carcinoma cells

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    EMANUELA FELLEY-BOSCO

    2002-01-01

    Full Text Available Reactive oxygen species are now widely recognized as important players contributing both to cell homeostasis and the development of disease. In this respect nitric oxide (NO is no exception. The discussion here will center on regulation of the inducible form of nitric oxide synthase (iNOS for two reasons. First, only iNOS produces micromolar NO concentrations, amounts that are high by comparison with the picomolar to nanomolar concentrations resulting from Ca2+-controlled NO production by endothelial eNOS or neuronal nNOS. Second, iNOS is not constitutively expressed in cells and regulation of this isoenzyme, in contrast to endothelial eNOS or neuronal nNOS, is widely considered to occur at the transcriptional level only. In particular, we were interested in the possibility that caveolin-1, a protein that functions as a tumor suppressor in colon carcinoma cells (Bender et al., 2002; this issue, might regulate iNOS activity. Our results provide evidence for the existence of a post-transcriptional mechanism controlling iNOS protein levels that involves caveolin-1-dependent sequestration of iNOS within a detergent-insoluble compartment. Interestingly, despite the high degree of conservation of the caveolin-1 scaffolding domain binding motif within all NOS enzymes, the interaction detected between caveolin-1 and iNOS in vitro is crucially dependent on presence of a caveolin-1 sequence element immediately adjacent to the scaffolding domain. A model is presented summarizing the salient aspects of these results. These observations are important in the context of tumor biology, since down-regulation of caveolin-1 is predicted to promote uncontrolled iNOS activity, genotoxic damage and thereby facilitate tumor development in humans

  19. Inhibition of neutral sphingomyelinase decreases elevated levels of inducible nitric oxide synthase and apoptotic cell death in ocular hypertensive rats

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    Aslan, Mutay, E-mail: mutayaslan@akdeniz.edu.tr [Department of Medical Biochemistry, Akdeniz University Faculty of Medicine, Antalya (Turkey); Basaranlar, Goksun [Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya (Turkey); Unal, Mustafa [Department of Ophthalmology, Akdeniz University Faculty of Medicine, Antalya (Turkey); Ciftcioglu, Akif [Department of Pathology, Akdeniz University Faculty of Medicine, Antalya (Turkey); Derin, Narin [Department of Biophysics, Akdeniz University Faculty of Medicine, Antalya (Turkey); Mutus, Bulent [Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario (Canada)

    2014-11-01

    Endoplasmic reticulum (ER) stress and excessive nitric oxide production via induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of neuronal retinal cell death in ocular hypertension. Neutral sphingomyelinase (N-SMase)/ceramide pathway can regulate NOS2 expression, hence this study determined the role of selective neutral sphingomyelinase (N-SMase) inhibition on retinal NOS2 levels, ER stress, apoptosis and visual evoked potentials (VEPs) in a rat model of elevated intraocular pressure (EIOP). NOS2 expression and retinal protein nitration were significantly greater in EIOP and significantly decreased with N-SMase inhibition. A significant increase was observed in retinal ER stress markers pPERK, CHOP and GRP78 in EIOP, which were not significantly altered by N-SMase inhibition. Retinal TUNEL staining showed increased apoptosis in all EIOP groups; however N-SMase inhibition significantly decreased the percent of apoptotic cells in EIOP. Caspase-3, -8 and -9 activities were significantly increased in EIOP and returned to baseline levels following N-SMase inhibition. Latencies of all VEP components were significantly prolonged in EIOP and shortened following N-SMase inhibition. Data confirm the role of nitrative injury in EIOP and highlight the protective effect of N-SMase inhibition in EIOP via down-regulation of NOS2 levels and nitrative stress. - Highlights: • Inhibition of N-SMase decreases NOS2 levels in ocular hypertension. • Inhibition of N-SMase decreases protein nitration in ocular hypertension. • Inhibition of N-SMase decreases caspase activation in ocular hypertension. • Inhibition of N-SMase decreases apoptosis in ocular hypertension.

  20. Role of neuronal nitric oxide synthase in regulating retinal blood flow during flicker-induced hyperemia in cats.

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    Yoshioka, Takafumi; Nagaoka, Taiji; Song, Youngseok; Yokota, Harumasa; Tani, Tomofumi; Yoshida, Akitoshi

    2015-05-01

    To investigate how neuronal nitric oxide synthase (nNOS) contributes to regulation of the retinal circulation during rest and flicker stimulation in cats. Using laser Doppler velocimetry, we measured the vessel diameter and blood velocity simultaneously and calculated the retinal blood flow (RBF) in feline first-order retinal arterioles. After intravitreal injections of Nω-Nitro-L-arginine methyl ester (L-NAME), a nonselective NOS inhibitor, and Nω-propyl-L-arginine (L-NPA), a selective nNOS inhibitor, we continuously monitored the retinal circulation without any perturbations for 2 hours. We then examined the changes in the RBF in response to 16-Hz flicker stimuli for 3 minutes at 2 hours after intravitreal injection of phosphate-buffered saline (PBS) as a control, L-NAME, L-NPA, and thromboxane A2 (TXA2) analogue U46619 as a basal tone-adjusted control. After intravitreal injection of L-NAME and L-NPA, the baseline RBF decreased gradually in a dose-dependent manner. In the PBS group, the RBF increased gradually and reached a maximal level after 2 to 3 minutes of flicker stimuli. After 3 minutes of 16-Hz flicker stimuli, the RBF increased by 53.5% ± 3.4% compared with baseline. In the L-NAME and L-NPA groups, the increases in RBF during flicker stimulation were attenuated significantly compared with the PBS group. In the TXA2 group, the reduction in the flicker-induced increase in RBF was comparable to that in the PBS group. The current results suggested that increased RBF in response to flicker stimulation may be mediated by nitric oxide (NO) production via nNOS activation.

  1. Endothelial nitric oxide synthase gene polymorphisms and cardiovascular damage in hypertensive subjects: an Italian case-control study

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    Pizzo Federica

    2008-05-01

    Full Text Available Abstract Background Nitric oxide (NO synthesized by endothelial nitric oxide synthase (eNOS plays an important role in regulation of endothelial function and in the control of blood pressure. However, the results from some studies on the association between three clinically relevant eNOS gene polymorphisms (G894T, T786C and intron 4b/a and essential hypertension are unclear. We designed a case-control study to evaluate the influence of eNOS polymorphisms on target organ damage in 127 hypertensives and 67 normotensives. Clinical evaluation, biochemical parameters, Urinary Albumin Excretion (UAE and echocardiogram were performed to characterize target organ damage. eNOS polymorphism were recognized by PCR method. Results The distribution of eNOS genotypes was similar in hypertensives and normotensives but 4aa was present in the 2.5% of hypertensives and completely absent in normotensives. Subjects with 4bb, G894T, and T786C genotypes showed an increased prevalence of target organ damage. Moreover prevalence of G894T and introne 4 variants was significantly higher in hypertensives than in normotensives both with cardiovascular damage. Logistic regression analysis didn't show any association between eNOS polymorphisms, Body Mass Index (BMI, hypertension, gender and cardiovascular damage. Only the age (OR 1.11; IC 95% 1.06–1.18 was predictive of cardiovascular damage in our population. Conclusion Our results seem to indicate a lack of association with eNOS variants and cardiovascular damage onset.

  2. Arginase Inhibition Restores Peroxynitrite-Induced Endothelial Dysfunction via L-Arginine-Dependent Endothelial Nitric Oxide Synthase Phosphorylation

    Science.gov (United States)

    Nguyen, Minh Cong; Park, Jong Taek; Jeon, Yeong Gwan; Jeon, Byeong Hwa; Hoe, Kwang Lae; Kim, Young Myeong

    2016-01-01

    Purpose Peroxynitrite plays a critical role in vascular pathophysiology by increasing arginase activity and decreasing endothelial nitric oxide synthase (eNOS) activity. Therefore, the aims of this study were to investigate whether arginase inhibition and L-arginine supplement could restore peroxynitrite-induced endothelial dysfunction and determine the involved mechanism. Materials and Methods Human umbilical vein endothelial cells (HUVECs) were treated with SIN-1, a peroxynitrite generator, and arginase activity, nitrite/nitrate production, and expression levels of proteins were measured. eNOS activation was evaluated via Western blot and dimer blot analysis. We also tested nitric oxide (NO) and reactive oxygen species (ROS) production and performed a vascular tension assay. Results SIN-1 treatment increased arginase activity in a time- and dose-dependent manner and reciprocally decreased nitrite/nitrate production that was prevented by peroxynitrite scavenger in HUVECs. Furthermore, SIN-1 induced an increase in the expression level of arginase I and II, though not in eNOS protein. The decreased eNOS phosphorylation at Ser1177 and the increased at Thr495 by SIN-1 were restored with arginase inhibitor and L-arginine. The changed eNOS phosphorylation was consistent in the stability of eNOS dimers. SIN-1 decreased NO production and increased ROS generation in the aortic endothelium, all of which was reversed by arginase inhibitor or L-arginine. NG-Nitro-L-arginine methyl ester (L-NAME) prevented SIN-1-induced ROS generation. In the vascular tension assay, SIN-1 enhanced vasoconstrictor responses to U46619 and attenuated vasorelaxant responses to acetylcholine that were reversed by arginase inhibition. Conclusion These findings may explain the beneficial effect of arginase inhibition and L-arginine supplement on endothelial dysfunction under redox imbalance-dependent pathophysiological conditions. PMID:27593859

  3. Genetic knockout and pharmacologic inhibition of neuronal nitric oxide synthase attenuate nerve injury-induced mechanical hypersensitivity in mice

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    Tao Yuan-Xiang

    2007-10-01

    Full Text Available Abstract Neuronal nitric oxide synthase (nNOS is a key enzyme for nitric oxide production in neuronal tissues and contributes to the spinal central sensitization in inflammatory pain. However, the role of nNOS in neuropathic pain remains unclear. The present study combined a genetic strategy with a pharmacologic approach to examine the effects of genetic knockout and pharmacologic inhibition of nNOS on neuropathic pain induced by unilateral fifth lumbar spinal nerve injury in mice. In contrast to wildtype mice, nNOS knockout mice failed to display nerve injury-induced mechanical hypersensitivity. Furthermore, either intraperitoneal (100 mg/kg or intrathecal (30 μg/5 μl administration of L-NG-nitro-arginine methyl ester, a nonspecific NOS inhibitor, significantly reversed nerve injury-induced mechanical hypersensitivity on day 7 post-nerve injury in wildtype mice. Intrathecal injection of 7-nitroindazole (8.15 μg/5 μl, a selective nNOS inhibitor, also dramatically attenuated nerve injury-induced mechanical hypersensitivity. Western blot analysis showed that the expression of nNOS protein was significantly increased in ipsilateral L5 dorsal root ganglion but not in ipsilateral L5 lumbar spinal cord on day 7 post-nerve injury. The expression of inducible NOS and endothelial NOS proteins was not markedly altered after nerve injury in either the dorsal root ganglion or spinal cord. Our findings suggest that nNOS, especially in the dorsal root ganglion, may participate in the development and/or maintenance of mechanical hypersensitivity after nerve injury.

  4. Prevalence of endothelial nitric oxide synthase (eNOS) gene exon 7 Glu298Asp variant in North Eastern India

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    Shankarishan, Priyanka; Borah, Prasanta Kumar; Ahmed, Giasuddin; Mahanta, Jagadish

    2011-01-01

    Background & objectives Endothelial nitric oxide is a potent vasodilator and impairment of its generation brought about by gene polymorphism is considered a major predictor for several diseases. A single nucleotide polymorphism G894T within exon 7 of endothelial nitric oxide synthase (eNOS-7) gene, resulting in a replacement of glutamic acid by aspartic acid, has been studied as a putative candidate gene for cardiovascular diseases. The pattern of eNOS-7 Glu298Asp variant in the Indian population is poorly known. The present study was planned to determine the prevalence of the variant of this gene among tea garden community in Assam, North-East India with high prevalence of hypertension. Methods Study participants of both sex aged ≥18 yr were recruited randomly from temporary field clinics established in tea gardens of Dibrugarh, Assam. Genomic DNA was extracted from 409 subjects by the conventional phenol-chloroform method. The prevalence of the eNOS exon 7 Glu298Asp variant was determined by polymerase chain reaction and restriction fragment length polymorphism analysis. Results The study population was in Hardy-Weinberg Equilibrium. The frequency of the eNOS GG, GT and TT genotypes was found to be 75, 22 and 3 per cent respectively and did not show any significant difference in gender wise analysis. Interpretation & conclusions Our results showed that the prevalence of the homozygous GG genotype was high (75%) and the rare mutant genotype (homozygous, TT) was 3 per cent in a population at risk with cardiovascular disease. Such population-based data on various polymorphisms can ultimately be exploited in pharmacogenomics. PMID:21623032

  5. Effect of crocin on nitric oxide synthase expression in post-ischemic isolated rat heart

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    Mahdi Esmaeilizadeh

    2015-08-01

    Full Text Available Objective: Oxidative stress damages cells and brings about the pathogenesis of ischemia/reperfusion injury. This study was carried out to investigate the preconditioning and cardio protective potential effects of crocin and vitamin E by the eNOS and iNOS express gene in ischemia/reperfusion in rats. Material & Methods: Male rats were divided into seven groups, namely: sham, control group and experimental groups treated with crocin(10, 20 and 40 mg/kg, vitamin E (100 mg/kg and combination of crocin (40 mg/kg with vitamin E (100 mg/kg that were gavaged The heart was removed and relocated to a Langendorff apparatus and subjected to global ischemia and then the left ventricular end diastolic pressure (LVEDP were measured as a hemodynamic parameter. Total RNA was extracted from heart frozen tissues. RT-PCR technique was performed by specific primers designed for nitric oxide gene and the results were assessed by agarose gel electrophoresis. Results: Results after ischemia and reperfusion showed that crocin 40 mg/kg produced a significant improvement of LVEDP as a mechanical function (P

  6. Endothelial nitric oxide synthase polymorphisms and adaptation of parasympathetic modulation to exercise training.

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    Silva, Bruno M; Neves, Fabricia J; Negrão, Marcelo V; Alves, Cleber R; Dias, Rodrigo G; Alves, Guilherme B; Pereira, Alexandre C; Rondon, Maria U; Krieger, José E; Negrão, Carlos E; DA Nóbrega, Antonio Claudio Lucas

    2011-09-01

    There is a large interindividual variation in the parasympathetic adaptation induced by aerobic exercise training, which may be partially attributed to genetic polymorphisms. Therefore, we investigated the association among three polymorphisms in the endothelial nitric oxide gene (-786T>C, 4b4a, and 894G>T), analyzed individually and as haplotypes, and the parasympathetic adaptation induced by exercise training. Eighty healthy males, age 20-35 yr, were genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis, and haplotypes were inferred using the software PHASE 2.1. Autonomic modulation (i.e., HR variability and spontaneous baroreflex sensitivity) and peak oxygen consumption (VO(2peak)) were measured before and after training (running, moderate to severe intensity, three times per week, 60 min·day(-1), during 18 wk). Training increased VO(2peak) (P baroreflex sensitivity after training (change: wild type (-786TT) = 2% ± 89% vs polymorphic (-786TC/CC) = -28% ± 60%, median ± quartile range, P = 0.03), and parasympathetic modulation was marginally reduced in subjects with the 894T polymorphic allele (change: wild type (894GG) = 8% ± 67% vs polymorphic (894GT/TT) = -18% ± 59%, median ± quartile range, P = 0.06). Furthermore, parasympathetic modulation percent change was different between the haplotypes containing wild-type alleles (-786T/4b/894G) and polymorphic alleles at positions -786 and 894 (-786C/4b/894T) (-6% ± 56% vs -41% ± 50%, median ± quartile range, P = 0.04). The polymorphic allele at position -786 and the haplotype containing polymorphic alleles at positions -786 and 894 in the endothelial nitric oxide gene were associated with decreased parasympathetic modulation after exercise training.

  7. The activity of inducible nitric oxide synthase in rejected skin xenografts is selectively inhibited by a factor produced by grafted cells

    Czech Academy of Sciences Publication Activity Database

    Holáň, Vladimír; Pindjáková, Jana; Zajícová, Alena; Krulová, Magdalena; Železná, Blanka; Matoušek, Petr; Svoboda, Petr

    2005-01-01

    Roč. 12, č. 3 (2005), s. 227-234 ISSN 0908-665X R&D Projects: GA MZd(CZ) NR7816; GA ČR(CZ) GP310/02/D162; GA ČR(CZ) GD310/03/H147; GA MŠk(CZ) ME 300; GA AV ČR KSK5020115 Institutional research plan: CEZ:AV0Z5052915; CEZ:AV0Z50110509 Keywords : inducible nitric oxide synthase production * nitric oxide * suppressive molecule Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.114, year: 2005

  8. Decreased endothelial nitric oxide synthase expression and function contribute to impaired mitochondrial biogenesis and oxidative stress in fetal lambs with persistent pulmonary hypertension.

    Science.gov (United States)

    Afolayan, Adeleye J; Eis, Annie; Alexander, Maxwell; Michalkiewicz, Teresa; Teng, Ru-Jeng; Lakshminrusimha, Satyan; Konduri, Girija G

    2016-01-01

    Impaired vasodilation in persistent pulmonary hypertension of the newborn (PPHN) is characterized by mitochondrial dysfunction. We investigated the hypothesis that a decreased endothelial nitric oxide synthase level leads to impaired mitochondrial biogenesis and function in a lamb model of PPHN induced by prenatal ductus arteriosus constriction. We ventilated PPHN lambs with 100% O2 alone or with inhaled nitric oxide (iNO). We treated pulmonary artery endothelial cells (PAECs) from normal and PPHN lambs with detaNONOate, an NO donor. We observed decreased mitochondrial (mt) DNA copy number, electron transport chain (ETC) complex subunit levels, and ATP levels in PAECs and lung tissue of PPHN fetal lambs at baseline compared with gestation matched controls. Phosphorylation of AMP-activated kinase (AMPK) and levels of peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α) and sirtuin-1, which facilitate mitochondrial biogenesis, were decreased in PPHN. Ventilation with 100% O2 was associated with larger decreases in ETC subunits in the lungs of PPHN lambs compared with unventilated PPHN lambs. iNO administration, which facilitated weaning of FiO2 , partly restored mtDNA copy number, ETC subunit levels, and ATP levels. DetaNONOate increased eNOS phosphorylation and its interaction with heat shock protein 90 (HSP90); increased levels of superoxide dismutase 2 (SOD2) mRNA, protein, and activity; and decreased the mitochondrial superoxide levels in PPHN-PAECs. Knockdown of eNOS decreased ETC protein levels in control PAECs. We conclude that ventilation with 100% O2 amplifies oxidative stress and mitochondrial dysfunction in PPHN, which are partly improved by iNO and weaning of oxygen. Copyright © 2016 the American Physiological Society.

  9. Antileishmanial Activity and Inducible Nitric Oxide Synthase Activation by RuNO Complex

    Directory of Open Access Journals (Sweden)

    Tatiane Marcusso Orsini

    2016-01-01

    Full Text Available Parasites of the genus Leishmania are capable of inhibiting effector functions of macrophages. These parasites have developed the adaptive ability to escape host defenses; for example, they inactivate the NF-κB complex and suppress iNOS expression in infected macrophages, which are responsible for the production of the major antileishmanial substance nitric oxide (NO, favoring then its replication and successful infection. Metal complexes with NO have been studied as potential compounds for the treatment of certain tropical diseases, such as ruthenium compounds, known to be exogenous NO donors. In the present work, the compound cis-[Ru(bpy2SO3(NO]PF6, or RuNO, showed leishmanicidal activity directly and indirectly on promastigote forms of Leishmania (Leishmania amazonensis. In addition, treatment with RuNO increased NO production by reversing the depletion of NO caused by Leishmania. We also found increased expression of Akt, iNOS, and NF-κB in infected and treated macrophages. These results demonstrated that RuNO was able to kill the parasite by NO release and modulate the transcriptional capacity of the cell.

  10. Attenuation by creatine of myocardial metabolic stress in Brattleboro rats caused by chronic inhibition of nitric oxide synthase.

    Science.gov (United States)

    Constantin-Teodosiu, D; Greenhaff, P L; Gardiner, S M; Randall, M D; March, J E; Bennett, T

    1995-12-01

    1. The present experiment was undertaken to investigate: (a) the effect of nitric oxide synthase (NOS) inhibition, mediated by oral supplementation of the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), on measures of myocardial energy metabolism and function: (b) the effect of oral creatine supplementation on these variables, in the absence and presence of L-NAME. 2. In one series of experiments, 4 weeks oral administration of L-NAME (0.05 mg ml-1 day-1 in the drinking water) to Brattleboro rats caused significant reductions in myocardial ATP, creatine, and total creatine concentrations and an accumulation of tissue lactate when compared with control animals. Administration of creatine (0.63 mg ml-1 day-1 in the drinking water) for 4 weeks elevated myocardial creatine and total creatine concentrations and reduced lactate accumulation, but did not significantly affect ATP or phosphocreatine (PCr). Concurrent treatment with creatine and L-NAME prevented the reduction in creatine and total creatine concentrations, and significantly attenuated the accumulation of lactate and the reduction in ATP seen with L-NAME alone. 3. In a second series of experiments, 4 weeks treatment with L-NAME and creatine plus L-NAME increased mean arterial blood pressure in conscious Brattleboro rats. Hearts isolated from these animals showed decreased coronary flow and left ventricular developed pressure (LVDP), and total mechanical performance. Treatment with creatine alone had no measurable effect on either mean arterial blood pressure or coronary flow in isolated hearts. However, there was an increase in LVDP, but not in total mechanical performance, because there was a bradycardia. 4. These results indicate that creatine supplementation can attenuate the metabolic stress associated with L-NAME administration and that this effect occurs as a consequence of the action of creatine on myocardial energy metabolism.

  11. Mechanisms of suppression of inducible nitric oxide synthase (iNOS) expression in RAW 264.7 cells by andrographolide

    Science.gov (United States)

    Chiou, Wen-Fei; Chen, Chieh-Fu; Lin, Jin-Jung

    2000-01-01

    Andrographolide, an active component found in leaves of Andrographis paniculata, has been reported to exhibit nitric oxide (NO) inhibitory property in endotoxin-stimulated macrophages, however, the detailed mechanisms remain unclear. In the present study we investigated the effect of andrographolide on the expression of inducible NO synthase (iNOS) mRNA, protein, and enzyme activity in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS) plus interferon-γ (IFN-γ).RAW 264.7 cells stimulated with LPS/IFN-γ activated NO production; in this condition andrographolide (1–100 μM) inhibited NO production in a dose-dependent manner with an IC50 value of 17.4±1.1 μM. Andrographolide also reduces the expression of iNOS protein level but without a significant effect on iNOS mRNA. The reduction of iNOS activity is thought to be caused by decreased expression of iNOS protein.In a protein stability assay, andrographolide moderately but significantly reduced the amount of iNOS protein as suggested by accelerating degradation. Furthermore, andrographolide also inhibited total protein de novo synthesis as demonstrated by [35S]-methionine incorporation.As a whole, these data suggest that andrographolide inhibits NO synthesis in RAW 264.7 cells by reducing the expression of iNOS protein and the reduction could occur through two additional mechanisms: prevention of the de novo protein synthesis and decreasing the protein stability via a post-transcriptional mechanism. It is also possible that inhibition of iNOS protein expression and NO production under immune stimulation and/or bacteria infection may explain, in part, the beneficial effects of andrographolide as an anti-inflammatory agent. PMID:10780958

  12. S100A1 deficiency impairs postischemic angiogenesis via compromised proangiogenic endothelial cell function and nitric oxide synthase regulation.

    Science.gov (United States)

    Most, Patrick; Lerchenmüller, Carolin; Rengo, Giuseppe; Mahlmann, Adrian; Ritterhoff, Julia; Rohde, David; Goodman, Chelain; Busch, Cornelius J; Laube, Felix; Heissenberg, Julian; Pleger, Sven T; Weiss, Norbert; Katus, Hugo A; Koch, Walter J; Peppel, Karsten

    2013-01-04

    Mice lacking the EF-hand Ca2+ sensor S100A1 display endothelial dysfunction because of distorted Ca2+ -activated nitric oxide (NO) generation. To determine the pathophysiological role of S100A1 in endothelial cell (EC) function in experimental ischemic revascularization. Patients with chronic critical limb ischemia showed almost complete loss of S100A1 expression in hypoxic tissue. Ensuing studies in S100A1 knockout (SKO) mice subjected to femoral artery resection unveiled insufficient perfusion recovery and high rates of autoamputation. Defective in vivo angiogenesis prompted cellular studies in SKO ECs and human ECs, with small interfering RNA-mediated S100A1 knockdown demonstrating impaired in vitro and in vivo proangiogenic properties (proliferation, migration, tube formation) and attenuated vascular endothelial growth factor (VEGF)-stimulated and hypoxia-stimulated endothelial NO synthase (eNOS) activity. Mechanistically, S100A1 deficiency compromised eNOS activity in ECs by interrupted stimulatory S100A1/eNOS interaction and protein kinase C hyperactivation that resulted in inhibitory eNOS phosphorylation and enhanced VEGF receptor-2 degradation with attenuated VEGF signaling. Ischemic SKO tissue recapitulated the same molecular abnormalities with insufficient in vivo NO generation. Unresolved ischemia entailed excessive VEGF accumulation in SKO mice with aggravated VEGF receptor-2 degradation and blunted in vivo signaling through the proangiogenic phosphoinositide-3-kinase/Akt/eNOS cascade. The NO supplementation strategies rescued defective angiogenesis and salvaged limbs in SKO mice after femoral artery resection. Our study shows for the first time downregulation of S100A1 expression in patients with critical limb ischemia and identifies S100A1 as critical for EC function in postnatal ischemic angiogenesis. These findings link its pathological plasticity in critical limb ischemia to impaired neovascularization, prompting further studies to probe the

  13. Safflor yellow B reduces hypoxia-mediated vasoconstriction by regulating endothelial micro ribonucleic acid/nitric oxide synthase signaling.

    Science.gov (United States)

    Wang, Chaoyun; Yang, Ying; Li, Miao; Liu, Xin; Wang, Qiaoyun; Xin, Wenyu; Sun, Hongliu; Zheng, Qingyin

    2017-11-07

    Hypoxia-induced generation of vasoconstrictors reduces cerebral blood flow (CBF) while nitric oxide (NO) synthase (NOS) and microRNAs (miRNA) in endothelial cells (ECs) suppress vasoconstriction. Safflor yellow B (SYB), a natural plant compound, previously attenuated angiotensin II-mediated injury of ECs and maintained endothelial function. This study investigated the putative involvement of NOS and miRNAs in SYB-mediated resistance to hypoxia-induced vasoconstriction. In vivo , chronic hypoxia was induced in rats, and SYB was administered intravenously. In vitro , rat primary aortic ECs were cultured under oxygen and glucose deprivation. After treatment with anti-microR-199a, as well as the NOS inhibitor, N(G)-nitro-L-arginine methyl ester, SYB, or both, cell viability, NO and peroxynitrite (ONOO-) levels, NOS expression, and miRNA levels were evaluated. SYB significantly alleviated hypoxia-mediated vasoconstriction and increased CBF endothelium-dependently. SYB upregulated miR-199a, increased EC viability, decreased endothelin-1 (ET-1) levels, inhibited protein kinase C (PKC) activity, and suppressed hypoxia inducible factor-1α (HIF-1α) expression. Furthermore, the SYB-mediated reduction of inducible NOS reduced ONOO- levels. In addition, SYB downregulated miR-138 and, thereby, enhanced S100A1 and endothelial NOS activity. Hypoxia-mediated regulation of miR-138 and miR-199a inhibited endothelial NOS expression and activation, which triggered ET-1 release and vasoconstriction. Therefore, SYB treatment reduced hypoxia-induced vasoconstriction through miR-199a/endothelial NOS signaling.

  14. Priming of hypoxia-inducible factor by neuronal nitric oxide synthase is essential for adaptive responses to severe anemia.

    Science.gov (United States)

    Tsui, Albert K Y; Marsden, Philip A; Mazer, C David; Adamson, S Lee; Henkelman, R Mark; Ho, J J David; Wilson, David F; Heximer, Scott P; Connelly, Kim A; Bolz, Steffen-Sebastian; Lidington, Darcy; El-Beheiry, Mostafa H; Dattani, Neil D; Chen, Kevin M; Hare, Gregory M T

    2011-10-18

    Cells sense and respond to changes in oxygen concentration through gene regulatory processes that are fundamental to survival. Surprisingly, little is known about how anemia affects hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular responses to acute hypoxia, we defined the effects of NOS deficiency in acute anemia. In contrast to endothelial NOS or inducible NOS deficiency, neuronal NOS (nNOS)(-/-) mice demonstrated increased mortality during anemia. Unlike wild-type (WT) animals, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS(-/-) mice. At the cellular level, anemia increased expression of HIF-1α protein and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK1) in the brain of WT, but not nNOS(-/-) mice, despite comparable reductions in tissue PO(2). Paradoxically, nNOS(-/-) mice survived longer during hypoxia, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. Real-time imaging of transgenic animals expressing a reporter HIF-α(ODD)-luciferase chimeric protein confirmed that nNOS was essential for anemia-mediated increases in HIF-α protein stability in vivo. S-nitrosylation effects the functional interaction between HIF and pVHL. We found that anemia led to nNOS-dependent S-nitrosylation of pVHL in vivo and, of interest, led to decreased expression of GSNO reductase. These findings identify nNOS effects on the HIF/pVHL signaling pathway as critically important in the physiological responses to anemia in vivo and provide essential mechanistic insight into the differences between anemia and hypoxia.

  15. Morphometric multivariate analysis of GABAergic neurons containing calretinin and neuronal nitric oxide synthase in the mouse hippocampus.

    Science.gov (United States)

    Jinno, S; Kinukawa, N; Kosaka, T

    2001-05-11

    Several studies reported the morphology of calretinin-positive (CR+) neurons and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) labeled or neuronal nitric oxide synthase-positive (nNOS+) neurons in the rodent hippocampus, where these neurons showed similar morphological features. In addition, a previous study reported the frequent colocalization of CR and NADPH-d in the rat hippocampus. In this study, we aimed to examine whether CR+ neurons and nNOS+ neurons belong to a same morphological subpopulation of GABAergic neurons in the mouse hippocampus. Neurons were immunocytochemically classified into three groups, i.e., CR+/nNOS-, CR-/nNOS+ and CR+/nNOS+ groups. The present morphometric analysis was performed in the mouse Ammon's horn, because CR+/nNOS+ neurons were rarely found in the mouse dentate gyrus. We selected three morphometric parameters, i.e., soma area, soma form factor (FF) and number of primary dendrites. Dunnett's post-hoc analysis revealed that soma area, soma FF and number of primary dendrites were significantly larger in CR-/nNOS+ group than in CR+/nNOS- and CR+/nNOS+ groups. The morphometric data of CR+/nNOS+ group were quite similar to those of CR+/nNOS- group. The morphometric multivariate logistic regression analysis revealed that these three morphometric parameters were independent significant variables to discriminate between CR+/nNOS- and CR-/nNOS+ groups, and the majority of CR+/nNOS- and CR-/nNOS+ groups were correctly classified from the morphometric features. The present results clearly indicate that CR+/nNOS- neurons and CR-/nNOS+ neurons belong to different morphological subpopulations, and lead us to speculate that they might play different functional roles in the hippocampal circuit. The further application of morphometric multivariate analysis would be valuable to understand the functional roles of chemically defined neurons in the various brain regions.

  16. Morphometric evaluation of nitric oxide synthase isoforms and their cytokine regulators predict pulmonary dysfunction and survival in systemic sclerosis

    Directory of Open Access Journals (Sweden)

    E.R. Parra

    2013-01-01

    Full Text Available Because histopathological changes in the lungs of patients with systemic sclerosis (SSc are consistent with alveolar and vessel cell damage, we presume that this interaction can be characterized by analyzing the expression of proteins regulating nitric oxide (NO and plasminogen activator inhibitor-1 (PAI-1 synthesis. To validate the importance of alveolar-vascular interactions and to explore the quantitative relationship between these factors and other clinical data, we studied these markers in 23 cases of SSc nonspecific interstitial pneumonia (SSc-NSIP. We used immunohistochemistry and morphometry to evaluate the amount of cells in alveolar septa and vessels staining for NO synthase (NOS and PAI-1, and the outcomes of our study were cellular and fibrotic NSIP, pulmonary function tests, and survival time until death. General linear model analysis demonstrated that staining for septal inducible NOS (iNOS related significantly to staining of septal cells for interleukin (IL-4 and to septal IL-13. In univariate analysis, higher levels of septal and vascular cells staining for iNOS were associated with a smaller percentage of septal and vascular cells expressing fibroblast growth factor and myofibroblast proliferation, respectively. Multivariate Cox model analysis demonstrated that, after controlling for SSc-NSIP histological patterns, just three variables were significantly associated with survival time: septal iNOS (P=0.04, septal IL-13 (P=0.03, and septal basic fibroblast growth factor (bFGF; P=0.02. Augmented NOS, IL-13, and bFGF in SSc-NSIP histological patterns suggest a possible functional role for iNOS in SSc. In addition, the extent of iNOS, PAI-1, and IL-4 staining in alveolar septa and vessels provides a possible independent diagnostic measure for the degree of pulmonary dysfunction and fibrosis with an impact on the survival of patients with SSc.

  17. The effects of tadalafil and pentoxifylline on apoptosis and nitric oxide synthase in liver ischemia/reperfusion injury

    Directory of Open Access Journals (Sweden)

    Sibel Bektas

    2016-07-01

    Full Text Available The aim of this study was to investigate the effects of tadalafil (TDF and pentoxifylline (PTX on hepatic apoptosis and the expressions of endothelial and inducible nitric oxide synthases (eNOS and iNOS after liver ischemia/reperfusion (IR. Forty Wistar albino rats were randomly divided into five groups (n=8 as follows: sham group; IR group with ischemia/reperfusion alone; low-dose and high-dose TDF groups received 2.5 mg/kg and 10 mg/kg TDF, respectively; and PTX group received 40 mg/kg PTX. Blood was collected for the analysis of serum alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, uric acid, malondialdehyde (MDA, and total antioxidant capacity (TAC. MDA and TAC also were measured in liver tissue. Histopathological examination was performed to assess the severity of hepatic injury. Apoptosis was evaluated using the apoptosis protease-activating factor 1 (APAF-1 antibody; the expressions of eNOS and iNOS were also assessed by immunohistochemistry in all groups. Serum alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, uric acid, MDA, and TAC, tissue MDA and TAC levels, hepatic injury, and score for extent and for intensity of eNOS, iNOS, and apoptosis protease-activating factor 1 were significantly different in TDF and PTX groups compared to the IR group. High dose-TDF and PTX have the best protective effect on IR-induced liver tissue damage. This study showed that TDF and PTX supplementation may be helpful in preventing free oxygen radical damage, lipid peroxidation, hepatocyte necrosis, and apoptosis in liver IR injury and minimizing liver damage.

  18. Overexpression myocardial inducible nitric oxide synthase exacerbates cardiac dysfunction and beta-adrenergic desensitization in experimental hypothyroidism.

    Science.gov (United States)

    Shao, Qun; Cheng, Heng-Jie; Callahan, Michael F; Kitzman, Dalane W; Li, Wei-Min; Cheng, Che Ping

    2016-02-01

    Altered nitric oxide synthase (NOS) has been implicated in the pathophysiology of heart failure (HF). Recent evidence links hypothyroidism to the pathology of HF. However, the precise mechanisms are incompletely understood. The alterations and functional effects of cardiac NOS in hypothyroidism are unknown. We tested the hypothesis that hypothyroidism increases cardiomyocyte inducible NOS (iNOS) expression, which plays an important role in hypothyroidism-induced depression of cardiomyocyte contractile properties, [Ca(2+)]i transient ([Ca(2+)]iT), and β-adrenergic hyporesponsiveness. We simultaneously evaluated LV functional performance and compared myocyte three NOS, β-adrenergic receptors (AR) and SERCA2a expressions and assessed cardiomyocyte contractile and [Ca(2+)]iT responses to β-AR stimulation with and without pretreatment of iNOS inhibitor (1400 W, 10(-5)mol/L) in 26 controls and 26 rats with hypothyroidism induced by methimazole (~30 mg/kg/day for 8 weeks in the drinking water). Compared with controls, in hypothyroidism, total serum T3 and T4 were significantly reduced followed by significantly decreased LV contractility (EES) with increased LV time constant of relaxation. These LV abnormalities were accompanied by concomitant significant decreases in myocyte contraction (dL/dtmax), relaxation (dR/dtmax), and [Ca(2+)]iT. In hypothyroidism, isoproterenol (10(-8)M) produced significantly smaller increases in dL/dtmax, dR/dtmax and [Ca(2+)]iT. These changes were associated with decreased β1-AR and SERCA2a, but significantly increased iNOS. Moreover, only in hypothyroidism, pretreatment with iNOS inhibitor significantly improved basal and isoproterenol-stimulated myocyte contraction, relaxation and [Ca(2+)]iT. Hypothyroidism produces intrinsic defects of LV myocyte force-generating capacity and relaxation with β-AR desensitization. Up-regulation of cardiomyocyte iNOS may promote progressive cardiac dysfunction in hypothyroidism. Copyright © 2015 Elsevier

  19. Chronic wheel running reduces maladaptive patterns of methamphetamine intake: regulation by attenuation of methamphetamine-induced neuronal nitric oxide synthase

    Science.gov (United States)

    Engelmann, Alexander J.; Aparicio, Mark B.; Kim, Airee; Sobieraj, Jeffery C.; Yuan, Clara J.; Grant, Yanabel

    2013-01-01

    We investigated whether prior exposure to chronic wheel running (WR) alters maladaptive patterns of excessive and escalating methamphetamine intake under extended access conditions, and intravenous methamphetamine self-administration-induced neurotoxicity. Adult rats were given access to WR or no wheel (sedentary) in their home cage for 6 weeks. A set of WR rats were injected with 5-bromo-2′-deoxyuridine (BrdU) to determine WR-induced changes in proliferation (2-h old) and survival (28-day old) of hippocampal progenitors. Another set of WR rats were withdrawn (WRw) or continued (WRc) to have access to running wheels in their home cages during self-administration days. Following self-administration [6 h/day], rats were tested on the progressive ratio (PR) schedule. Following PR, BrdU was injected to determine levels of proliferating progenitors (2-h old). WRc rats self-administered significantly less methamphetamine than sedentary rats during acquisition and escalation sessions, and demonstrated reduced motivation for methamphetamine seeking. Methamphetamine reduced daily running activity of WRc rats compared with that of pre-methamphetamine days. WRw rats self-administered significantly more methamphetamine than sedentary rats during acquisition, an effect that was not observed during escalation and PR sessions. WR-induced beneficial effects on methamphetamine self-administration were not attributable to neuroplasticity effects in the hippocampus and medial prefrontal cortex, but were attributable to WR-induced inhibition of methamphetamine-induced increases in the number of neuronal nitric oxide synthase expressing neurons and apoptosis in the nucleus accumbens shell. Our results demonstrate that WR prevents methamphetamine-induced damage to forebrain neurons to provide a beneficial effect on drug-taking behavior. Importantly, WR-induced neuroprotective effects are transient and continued WR activity is necessary to prevent compulsive methamphetamine intake

  20. Restricting the conformational freedom of the neuronal nitric-oxide synthase flavoprotein domain reveals impact on electron transfer and catalysis.

    Science.gov (United States)

    Dai, Yue; Haque, Mohammad Mahfuzul; Stuehr, Dennis J

    2017-04-21

    The signaling molecule nitric oxide (NO) is synthesized in animals by structurally related NO synthases (NOSs), which contain NADPH/FAD- and FMN-binding domains. During catalysis, NADPH-derived electrons transfer into FAD and then distribute into the FMN domain for further transfer to internal or external heme groups. Conformational freedom of the FMN domain is thought to be essential for the electron transfer (ET) reactions in NOSs. To directly examine this concept, we utilized a "Cys-lite" neuronal NOS flavoprotein domain and substituted Cys for two residues (Glu-816 and Arg-1229) forming a salt bridge between the NADPH/FAD and FMN domains in the conformationally closed structure to allow cross-domain disulfide bond formation or cross-linking by bismaleimides of various lengths. The disulfide bond cross-link caused a ≥95% loss of cytochrome c reductase activity that was reversible with DTT treatment, whereas graded cross-link lengthening gradually increased activity, thus defining the conformational constraints in the catalytic process. We used spectroscopic and stopped-flow techniques to further investigate how the changes in FMN domain conformational freedom impact the following: (i) the NADPH interaction; (ii) kinetics of electron loading (flavin reduction); (iii) stabilization of open versus closed conformational forms in two different flavin redox states; (iv) reactivity of the reduced FMN domain toward cytochrome c ; (v) response to calmodulin binding; and (vi) the rates of interflavin ET and the FMN domain conformational dynamics. Together, our findings help explain how the spatial and temporal behaviors of the FMN domain impact catalysis by the NOS flavoprotein domain and how these behaviors are governed to enable electron flow through the enzyme. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Overexpression Myocardial Inducible Nitric Oxide Synthase Exacerbates Cardiac Dysfunction and Beta-Adrenergic Desensitization in Experimental Hypothyroidism☆,☆☆

    Science.gov (United States)

    Shao, Qun; Cheng, Heng-Jie; Callahan, Michael F.; Kitzman, Dalane W; Li, Wei-Min; Cheng, Che Ping

    2015-01-01

    Background Altered nitric oxide synthase (NOS) has been implicated in the pathophysiology of heart failure (HF). Recent evidence links hypothyroidism to the pathology of HF. However, the precise mechanisms are incompletely understood. The alterations and functional effects of cardiac NOS in hypothyroidism are unknown. We tested the hypothesis that hypothyroidism increases cadiomyocyte inducible NOS (iNOS) expression, which plays an important role in hypothyroidism-induced depression of cardiomyocyte contractile properties, [Ca2+]i transient ([Ca2+]iT), and β-adrenergic hyporesponsiveness. Methods and Results We simultaneously evaluated LV functional performance and compared myocyte three NOS, β-adrenergic receptors (AR) and SERCA2a expressions and assessed cardiomyocyte contractile and [Ca2+]iT responses to β-AR stimulation with and without pretreatment of iNOS inhibitor (1400W, 10−5 mol/L) in 26 controls and 26 rats with hypothyroidism induced by methimazole (~30 mg/kg/day for 8 weeks in the drinking water). Compared with controls, in hypothyroidism, total serum T3 and T4 were significantly reduced followed by significantly decreased LV contractility (EES) with increased LV time constant of relaxation. These LV abnormalities were accompanied by concomitant significant decreases in myocyte contraction (dL/dtmax), relaxation (dR/dtmax), and [Ca2+]iT. In hypothyroidism, isoproterenol (10−8 M) produced significantly smaller increases in dL/dtmax, dR/dtmax and [Ca2+]iT. These changes were associated with decreased β1-AR and SERCA2a, but significantly increased iNOS. Moreover, only in hypothyroidism, pretreatment with iNOS inhibitor significantly improved basal and isoproterenol-stimulated myocyte contraction, relaxation and [Ca2+]iT. Conclusions Hypothyroidism produces intrinsic defects of LV myocyte force-generating capacity and relaxation with β-AR desensitization. Up-regulation of cadiomyocyte iNOS may promote progressive cardiac dysfunction in

  2. Inducible nitric oxide synthase, Nos2, does not mediate optic neuropathy and retinopathy in the DBA/2J glaucoma model

    Directory of Open Access Journals (Sweden)

    Savinova Olga V

    2007-12-01

    Full Text Available Abstract Background Nitric oxide synthase 2 (NOS2 contributes to neural death in some settings, but its role in glaucoma remains controversial. NOS2 is implicated in retinal ganglion cell degeneration in a rat glaucoma model in which intraocular pressure (IOP is experimentally elevated by blood vessel cauterization, but not in a rat glaucoma model where IOP was elevated by injection of hypertonic saline. To test the importance of NOS2 for an inherited glaucoma, in this study we both genetically and pharmacologically decreased NOS2 activity in the DBA/2J mouse glaucoma model. Methods The expression of Nos2 in the optic nerve head was analyzed at both the RNA and protein levels at different stages of disease pathogenesis. To test the involvement of Nos2 in glaucomatous neurodegeneration, a null allele of Nos2 was backcrossed into DBA/2J mice and the incidence and severity of glaucoma was assessed in mice of each Nos2 genotype. Additionally, DBA/2J mice were treated with the NOS2 inhibitor aminoguanidine and the disease compared to untreated mice. Results Optic nerve head Nos2 RNA levels varied and increased during moderate but decreased at early and severe stages of disease. Despite the presence of a few NOS2 positive cells in the optic nerve head, NOS2 protein was not substantially increased during the glaucoma. Genetic deficiency of Nos2 or aminoguanidine treatment did not alter the IOP profile of DBA/2J mice. Additionally, neither Nos2 deficiency nor aminoguanidine had any detectable affect on the glaucomatous optic nerve damage. Conclusion Glaucomatous neurodegeneration in DBA/2J mice does not require NOS2 activity. Further experiments involving various models are needed to assess the general importance of Nos2 in glaucoma.

  3. Endothelial nitric oxide synthase gene polymorphisms and risk of erectile dysfunction: An updated meta-analysis of genetic association studies.

    Science.gov (United States)

    Yao, Han-Xin; Ma, Fu-Zhe; Tan, Yu-Ying; Liu, Ling-Yun

    2018-04-11

    Endothelial nitric oxide synthase (eNOS) polymorphisms have been implicated as risk factors for erectile dysfunction (ED), but the results of genetic association studies are inconclusive. We performed a meta-analysis of published studies investigating the association between ED and three eNOS polymorphisms, intron 4 VNTR, G894T and T786C in humans. The PubMed, Web of Science, CNKI and Google Scholar databases were searched for relevant studies published up to November 2017. Association studies with case-control design were included. For each study with genotype information we calculated odds ratios (OR) and 95% confidence intervals (CI). The search identified 13 eligible studies. The G894T and T786C polymorphisms showed a significant association with ED risk in Caucasians (GT + TT versus GG for G894T: OR = 2.13, 95% CI = 1.08-4.19; CC versus CT + TT for T786C: OR = 3.29, 95% CI = 2.30-4.72) and Asians (GT + TT versus GG for G894T: OR = 2.08, 95% CI = 1.53-2.84; CC + CT versus TT for T786C: OR = 3.13, 95% CI = 1.35-7.25). In addition, the intron 4 VNTR polymorphism was associated with ED risk only among Caucasian subjects (aa versus bb + ab: OR = 2.38, 95% CI = 1.15-4.93). We found no evidence of publication bias. The robustness of overall analyses was ensured in sensitivity analyses excluding studies deviating from Hardy-Weinberg equilibrium. Our findings suggest that common genetic polymorphisms in the eNOS gene contribute to risk of ED, presumably by effects on eNOS activity and NO availability. Copyright © 2018. Published by Elsevier Ltd.

  4. Nitric Oxide Synthase Enzymes in the Airways of Mice Exposed to Ovalbumin: NOS2 Expression Is NOS3 Dependent

    Directory of Open Access Journals (Sweden)

    Jennifer M. Bratt

    2010-01-01

    Full Text Available Objectives and Design. The function of the airway nitric oxide synthase (NOS isoforms and the lung cell types responsible for its production are not fully understood. We hypothesized that NO homeostasis in the airway is important to control inflammation, which requires upregulation, of NOS2 protein expression by an NOS3-dependent mechanism. Materials or Subjects. Mice from a C57BL/6 wild-type, NOS1−/−, NOS2−/−, and NOS3−/− genotypes were used. All mice strains were systemically sensitized and exposed to filtered air or ovalbumin (OVA aerosol for two weeks to create a subchronic model of allergen-induced airway inflammation. Methods. We measured lung function, lung lavage inflammatory and airway epithelial goblet cell count, exhaled NO, nitrate and nitrite concentration, and airway NOS1, NOS2, and NOS3 protein content. Results. Deletion of NOS1 or NOS3 increases NOS2 protein present in the airway epithelium and smooth muscle of air-exposed animals. Exposure to allergen significantly reduced the expression of NOS2 protein in the airway epithelium and smooth muscle of the NOS3−/− strain only. This reduction in NOS2 expression was not due to the replacement of epithelial cells with goblet cells as remaining epithelial cells did not express NOS2. NOS1−/− animals had significantly reduced goblet cell metaplasia compared to C57Bl/6 wt, NOS2−/−, and NOS3−/− allergen-exposed mice. Conclusion. The airway epithelial and smooth muscle cells maintain a stable airway NO concentration under noninflammatory conditions. This “homeostatic” mechanism is unable to distinguish between NOS derived from the different constitutive NOS isoforms. NOS3 is essential for the expression of NOS2 under inflammatory conditions, while NOS1 expression contributes to allergen-induced goblet cell metaplasia.

  5. Nitric Oxide Synthase Enzymes in the Airways of Mice Exposed to Ovalbumin: NOS2 Expression Is NOS3 Dependent

    Science.gov (United States)

    Bratt, Jennifer M.; Williams, Keisha; Rabowsky, Michelle F.; Last, Michael S.; Franzi, Lisa M.; Last, Jerold A.; Kenyon, Nicholas J.

    2010-01-01

    Objectives and Design. The function of the airway nitric oxide synthase (NOS) isoforms and the lung cell types responsible for its production are not fully understood. We hypothesized that NO homeostasis in the airway is important to control inflammation, which requires upregulation, of NOS2 protein expression by an NOS3-dependent mechanism. Materials or Subjects. Mice from a C57BL/6 wild-type, NOS1−/−, NOS2−/−, and NOS3−/− genotypes were used. All mice strains were systemically sensitized and exposed to filtered air or ovalbumin (OVA) aerosol for two weeks to create a subchronic model of allergen-induced airway inflammation. Methods. We measured lung function, lung lavage inflammatory and airway epithelial goblet cell count, exhaled NO, nitrate and nitrite concentration, and airway NOS1, NOS2, and NOS3 protein content. Results. Deletion of NOS1 or NOS3 increases NOS2 protein present in the airway epithelium and smooth muscle of air-exposed animals. Exposure to allergen significantly reduced the expression of NOS2 protein in the airway epithelium and smooth muscle of the NOS3−/− strain only. This reduction in NOS2 expression was not due to the replacement of epithelial cells with goblet cells as remaining epithelial cells did not express NOS2. NOS1−/− animals had significantly reduced goblet cell metaplasia compared to C57Bl/6 wt, NOS2−/−, and NOS3−/− allergen-exposed mice. Conclusion. The airway epithelial and smooth muscle cells maintain a stable airway NO concentration under noninflammatory conditions. This “homeostatic” mechanism is unable to distinguish between NOS derived from the different constitutive NOS isoforms. NOS3 is essential for the expression of NOS2 under inflammatory conditions, while NOS1 expression contributes to allergen-induced goblet cell metaplasia. PMID:20953358

  6. ARGINASE ENZYMES IN ISOLATED AIRWAYS FROM NORMAL AND NITRIC OXIDE SYNTHASE 2-KNOCKOUT MICE EXPOSED TO OVALBUMIN

    Science.gov (United States)

    Bratt, Jennifer M.; Franzi, Lisa M.; Linderholm, Angela L.; Last, Michael S.; Kenyon, Nicholas J.; Last, Jerold A.

    2009-01-01

    Arginase has been suggested to compete with nitric oxide synthase (NOS) for their common substrate, L-arginine. To study the mechanisms underlying this interaction, we compared arginase expression in isolated airways and the consequences of inhibiting arginase activity in vivo with NO production, lung inflammation, and lung function in both C57BL/6 and NOS2 knockout mice undergoing ovalbumin-induced airway inflammation, a mouse model of asthma. Arginases I and II were measured by western blot in isolated airways from sensitized C57BL/6 mice exposed to ovalbumin aerosol. Physiological and biochemical responses---inflammation, lung compliance, airway hyperreactivity, exhaled NO concentration, arginine concentration--were compared with the responses of NOS2 knockout mice. NOS2 knockout mice had increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity. Both arginase I and arginase II were constitutively expressed in the airways of normal C57BL/6 mice. Arginase I was up-regulated approximately 8-fold in the airways of C57BL/6 mice exposed to ovalbumin. Expression of both arginase isoforms were significantly upregulated in NOS2 knockout mice exposed to ovalbumin, with about 40- and 4-fold increases in arginases I and II, respectively. Arginine concentration in isolated airways was not significantly different in any of the groups studied. Inhibition of arginase by systemic treatment of C57BL/6 mice with a competitive inhibitor, Nω-hydroxy-nor-L-arginine (nor-NOHA), significantly decreased the lung inflammatory response to ovalbumin in these animals. We conclude that NOS2 knockout mice are more sensitive to ovalbumin-induced airway inflammation and its sequelae than are C57BL/6 mice, as determined by increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity, and that these findings are strongly correlated with increased expression of both arginase isoforms in the airways of the NOS2

  7. AVE3085, an enhancer of endothelial nitric oxide synthase, restores endothelial function and reduces blood pressure in spontaneously hypertensive rats

    Science.gov (United States)

    Yang, Qin; Xue, Hong-Mei; Wong, Wing-Tak; Tian, Xiao-Yu; Huang, Yu; Tsui, Stephen KW; Ng, Patrick KS; Wohlfart, Paulus; Li, Huige; Xia, Ning; Tobias, Silke; Underwood, Malcolm John; He, Guo-Wei

    2011-01-01

    BACKGROUND AND PURPOSE Nitric oxide (NO) plays an important role in endothelial function, and impaired NO production is involved in hypertension. Therefore, compounds that regulate endothelial NO synthase (eNOS) may be of therapeutic benefit. A novel, low molecular weight compound AVE3085 is a recently developed compound with the ability to enhance eNOS transcription. The present study investigated the effects of AVE3085 in endothelial dysfunction associated with hypertension. EXPERIMENTAL APPROACH Spontaneously hypertensive rats (SHRs) were treated with AVE 3085 (10 mg·kg·day−1, orally) for 4 weeks. Isometric force measurement was performed on rings of isolated aortae in organ baths. Protein expression of eNOS, phosphorylated-eNOS and nitrotyrosine in the aortae were examined by Western blotting. mRNA for eNOS in rat aortae were examined by reverse-transcriptase polymerase chain reaction (RT-PCR). KEY RESULTS AVE3085 greatly improved endothelium-dependent relaxations in the aortae of SHRs. This functional change was accompanied by up-regulated expression of eNOS protein and mRNA, enhanced eNOS phosphorylation and decreased formation of nitrotyrosine. Furthermore, AVE3085 treatment reduced the blood pressure in SHR without affecting that of hypertensive eNOS−/− mice. CONCLUSIONS AND IMPLICATIONS The eNOS-transcription enhancer AVE3085 restored impaired endothelial function in a hypertensive model. The present study provides a solid basis for the potential development of eNOS-targeting drugs to restore down-regulated eNOS, as a new strategy in hypertension. PMID:21385179

  8. Association of endothelial nitric oxide synthase gene polymorphisms with coronary artery disease: an updated meta-analysis and systematic review.

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    Himanshu Rai

    Full Text Available Several association studies of endothelial nitric oxide synthase (NOS3 gene polymorphisms with respect to coronary artery disease (CAD have been published in the past two decades. However, their association with the disease, especially among different ethnic subgroups, still remains controversial. This prompted us to conduct a systematic review and an updated structured meta-analysis, which is the largest so far (89 articles, 132 separate studies, and a sample size of 69,235, examining association of three polymorphic forms of the NOS3 gene (i.e. Glu298Asp, T786-C and 27 bp VNTR b/a with CAD. In a subgroup analysis, we tested their association separately among published studies originating predominantly from European, Middle Eastern, Asian, Asian-Indian and African ancestries. The pooled analysis confirmed the association of all the three selected SNP with CAD in three different genetic models transcending all ancestries worldwide. The Glu298Asp polymorphism showed strongest association (OR range = 1.28-1.52, and P<0.00001 for all comparisons, followed by T786-C (OR range = 1.34-1.42, and P<0.00001 for all comparisons and 4b/a, (OR range = 1.19-1.41, and P ≤ 0.002 for all comparisons in our pooled analysis. Subgroup analysis revealed that Glu298Asp (OR range = 1.54-1.87, and P<0.004 for all comparisons and 4b/a (OR range = 1.71-3.02, and P<0.00001 for all comparisons have highest degree of association amongst the Middle Easterners. On the other hand, T786-C and its minor allele seem to carry a highest risk for CAD among subjects of Asian ancestry (OR range = 1.61-1.90, and P ≤ 0.01 for all comparisons.

  9. The Van Allen Probes mission

    CERN Document Server

    Burch, James

    2014-01-01

    This collection of articles provides broad and detailed information about NASA’s Van Allen Probes (formerly known as the Radiation Belt Storm Probes) twin-spacecraft Earth-orbiting mission. The mission has the objective of achieving predictive understanding of the dynamic, intense, energetic, dangerous, and presently unpredictable belts of energetic particles that are magnetically trapped in Earth’s space environment above the atmosphere. It documents the science of the radiation belts and the societal benefits of achieving predictive understanding. Detailed information is provided about the Van Allen Probes mission design, the spacecraft, the science investigations, and the onboard instrumentation that must all work together to make unprecedented measurements within a most unforgiving environment, the core of Earth’s most intense radiation regions.
 This volume is aimed at graduate students and researchers active in space science, solar-terrestrial interactions and studies of the up...

  10. The role of nitric oxide in aspirin induced thrombolysis in vitro and the purification of aspirin activated nitric oxide synthase from human blood platelets.

    Science.gov (United States)

    Karmohapatra, Soumendra K; Chakraborty, Kushal; Kahn, Nighat N; Sinha, Asru K

    2007-11-01

    Aspirin, a well-known inhibitor of platelet aggregation, is extensively used for the prevention/treatment of coronary artery disease. The beneficial and antithrombotic effects of the compound are related to the inhibition of platelet cyclooxygenase. It is currently believed that aspirin has no effect on the formed thrombus, which results in coronary artery disease. It was found that the exposure of platelets to 4.0 microM aspirin either in vitro or in vivo resulted in fibrinolysis of the formed "clot" produced by the recalcification of platelet-rich plasma due to the production of NO in these cells by the compound. The lysis of clot in the presence of aspirin was found to be related to the fibrinolysis with simultaneous appearance of fibrin degradation products due to the generation of serine proteinase activity by NO in the assay mixture. The aspirin activated nitric oxide synthase that catalyzed the synthesis of NO in platelets was solubilized by Triton X-100 treatment and purified to homogeneity by chromatography on DEAE cellulose and Sephadex G-50 columns. The enzyme was found to be a single chain polypeptide with M.W. 19 kDa. The treatment of human plasminogen with NO was found to directly activate the zymogen to plasmin with the production of preactivation peptide in the absence of cofactors, or cells without the formation of cyclic GMP in the assay mixture. (c) 2007 Wiley-Liss, Inc.

  11. Lipoplex gene transfer of inducible nitric oxide synthase inhibits the reactive intimal hyperplasia after expanded polytetrafluoroethylene bypass grafting.

    Science.gov (United States)

    Pfeiffer, Tomas; Wallich, Martina; Sandmann, Wilhelm; Schrader, Jürgen; Gödecke, Axel

    2006-05-01

    Intimal hyperplasia (IH) is most commonly the cause of graft occlusion in infrainguinal bypass grafting for arterial occlusive disease. We investigated the influence of nitric oxide on the IH of the arterial vessel wall at the region of prosthetic bypass anastomoses. Experiments were performed in 10 Foxhound dogs. We used a technique of inducible nitric oxide synthase (iNOS) overexpression by a non-virus-mediated, liposome-based iNOS gene transfer. The plasmid pSCMV-iNOS, which drives the expression of iNOS under control of the cytomegalovirus promoter, was complexed with cationic liposomes (lipoplexes). Segments of both carotid arteries were pretreated by intramural injection of a lipoplex solution by using an infiltrator balloon catheter (Infiltrator Drug Delivery Balloon System). In each dog, iNOS was administered at one side, and a control vector (pSCMV2) was administered at the contralateral side. Carotid arteries were ligated, and bypass grafts (expanded polytetrafluoroethylene, 6-mm, ring enforced) were implanted on both sides. The proximal and distal anastomoses (end-to-side fashion; running nonabsorbable sutures) were placed in the pretreated regions. After 6 months, the prostheses were excised, and the intimal thicknesses of 50 cross sections (orcein staining) of each anastomosis were measured planimetrically. The average reduction of the neointima thickness of the iNOS side in proximal anastomoses at the prosthetic wall, suture region, and arterial wall was 43%, 52%, and 81%, respectively. In distal anastomoses, the average reduction was 40%, 47%, and 52%, respectively. All differences of neointima thickness between the iNOS and control sides were statistically significant (Wilcoxon test; P < or = .05). Inducible NOS expression is an efficient approach for inhibition of IH. In contrast to earlier studies, which investigated the efficacy of gene therapeutic NOS expression at 3 to 4 weeks after intervention, the novelty of our findings is that a single

  12. Involvement of neuronal nitric oxide synthase in cross-sensitization between chronic unpredictable stress and ethanol in adolescent and adult mice.

    Science.gov (United States)

    Santos-Rocha, Jaqueline Borges; Rae, Mariana; Teixeira, Ana Maria Aristimunho; Teixeira, Simone Aparecida; Munhoz, Carolina Demarchi; Muscará, Marcelo Nicolas; Marcourakis, Tania; Szumlinski, Karen K; Camarini, Rosana

    2018-05-01

    The peculiar neurochemical profile of the adolescent brain renders it differently susceptible to several stimuli, including stress and/or drug exposure. Among several stress mediators, nitric oxide (NO) has a role in stress responses. We have demonstrated that adolescent mice are less sensitive to ethanol-induced sensitization than adult mice. The present study investigated whether chronic unpredictable stress (CUS) induces behavioral sensitization to ethanol in adolescent and adult Swiss mice, and investigated the influence of Ca 2+ -dependent nitric oxide synthase (NOS) activity in the phenomenon. Adolescent and adult mice were exposed to repeated 1.8 g/kg ethanol or CUS and challenged with saline or ethanol. A neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitroindazole (7NI), was administered along with ethanol and CUS to test its effects on behavioral sensitization. Both adolescent and adult mice displayed cross-sensitization between CUS and ethanol in adult mice, with adolescents showing a lower degree of sensitization than adults. nNOS inhibition by 7NI reduced both ethanol sensitization and cross-sensitization. All age differences in the Ca 2+ -dependent NOS activity in the hippocampus and prefrontal cortex were in the direction of greater activity in adults than in adolescents. Adolescents showed lower sensitivity to cross-sensitization between CUS and ethanol, and the nitric oxide (NO) system seems to have a pivotal role in ethanol-induced behavioral sensitization and cross-sensitization in both adolescent and adult mice. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. The role of nitric-oxide-synthase-derived nitric oxide in multicellular traits of Bacillus subtilis 3610: biofilm formation, swarming, and dispersal

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    Lamprecht-Grandio María

    2011-05-01

    Full Text Available Abstract Background Bacillus subtilis 3610 displays multicellular traits as it forms structurally complex biofilms and swarms on solid surfaces. In addition, B. subtilis encodes and expresses nitric oxide synthase (NOS, an enzyme that is known to enable NO-mediated intercellular signalling in multicellular eukaryotes. In this study, we tested the hypothesis that NOS-derived NO is involved in the coordination of multicellularity in B. subtilis 3610. Results We show that B. subtilis 3610 produces intracellular NO via NOS activity by combining Confocal Laser Scanning Microscopy with the NO sensitive dye copper fluorescein (CuFL. We further investigated the influence of NOS-derived NO and exogenously supplied NO on the formation of biofilms, swarming motility and biofilm dispersal. These experiments showed that neither the suppression of NO formation with specific NOS inhibitors, NO scavengers or deletion of the nos gene, nor the exogenous addition of NO with NO donors affected (i biofilm development, (ii mature biofilm structure, and (iii swarming motility in a qualitative and quantitative manner. In contrast, the nos knock-out and wild-type cells with inhibited NOS displayed strongly enhanced biofilm dispersal. Conclusion The results suggest that biofilm formation and swarming motility in B. subtilis represent complex multicellular processes that do not employ NO signalling and are remarkably robust against interference of NO. Rather, the function of NOS-derived NO in B. subtilis might be specific for cytoprotection against oxidative stress as has been proposed earlier. The influence of NOS-derived NO on dispersal of B. subtilis from biofilms might be associated to its well-known function in coordinating the transition from oxic to anoxic conditions. Here, NOS-derived NO might be involved in fine-tuning the cellular decision-making between adaptation of the metabolism to (anoxic conditions in the biofilm or dispersal from the biofilm.

  14. Involvement of beta 3-adrenoceptor in altered beta-adrenergic response in senescent heart: role of nitric oxide synthase 1-derived nitric oxide.

    Science.gov (United States)

    Birenbaum, Aurélie; Tesse, Angela; Loyer, Xavier; Michelet, Pierre; Andriantsitohaina, Ramaroson; Heymes, Christophe; Riou, Bruno; Amour, Julien

    2008-12-01

    In senescent heart, beta-adrenergic response is altered in parallel with beta1- and beta2-adrenoceptor down-regulation. A negative inotropic effect of beta3-adrenoceptor could be involved. In this study, the authors tested the hypothesis that beta3-adrenoceptor plays a role in beta-adrenergic dysfunction in senescent heart. beta-Adrenergic responses were investigated in vivo (echocardiography-dobutamine, electron paramagnetic resonance) and in vitro (isolated left ventricular papillary muscle, electron paramagnetic resonance) in young adult (3-month-old) and senescent (24-month-old) rats. Nitric oxide synthase (NOS) immunolabeling (confocal microscopy), nitric oxide production (electron paramagnetic resonance) and beta-adrenoceptor Western blots were performed in vitro. Data are mean percentages of baseline +/- SD. An impaired positive inotropic effect (isoproterenol) was confirmed in senescent hearts in vivo (117 +/- 23 vs. 162 +/- 16%; P < 0.05) and in vitro (127 +/- 10 vs. 179 +/- 15%; P < 0.05). In the young adult group, the positive inotropic effect was not significantly modified by the nonselective NOS inhibitor N-nitro-L-arginine methylester (L-NAME; 183 +/- 19%), the selective NOS1 inhibitor vinyl-L-N-5(1-imino-3-butenyl)-L-ornithine (L-VNIO; 172 +/- 13%), or the selective NOS2 inhibitor 1400W (183 +/- 19%). In the senescent group, in parallel with beta3-adrenoceptor up-regulation and increased nitric oxide production, the positive inotropic effect was partially restored by L-NAME (151 +/- 8%; P < 0.05) and L-VNIO (149 +/- 7%; P < 0.05) but not by 1400W (132 +/- 11%; not significant). The positive inotropic effect induced by dibutyryl-cyclic adenosine monophosphate was decreased in the senescent group with the specific beta3-adrenoceptor agonist BRL 37344 (167 +/- 10 vs. 142 +/- 10%; P < 0.05). NOS1 and NOS2 were significantly up-regulated in the senescent rat. In senescent cardiomyopathy, beta3-adrenoceptor overexpression plays an important role in the

  15. Expression of inducible nitric oxide synthase and nitric oxide production in the mud-dwelled air-breathing singhi catfish, Heteropneustes fossilis under condition of water shortage.

    Science.gov (United States)

    Choudhury, Mahua G; Saha, Nirmalendu

    2012-12-01

    Nitric oxide (NO) is known to be an important regulator molecule for regulating the multiple signaling pathways and also to play diverse physiological functions in mammals including that of adaptation to various stresses. The present study reports on the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) enzyme that produces NO from l-arginine in the freshwater air-breathing catfish (Heteropneustes fossilis) while dwelling inside the mud peat under semidry conditions. Desiccation stress, due to mud-dwelling for 2 weeks, led to significant increase of NO concentration in different tissues and in plasma of singhi catfish, and also the increase of NO efflux from the perfused liver with an accompanying increase of toxic ammonia level in different tissues. Mud-dwelling also resulted to induction of iNOS activity, expression of iNOS protein in different tissues after 7 days with further increase after 14 days, which otherwise was not detectable in control fish. Further, mud-dwelling also resulted to a significant expression of iNOS mRNA after 7 days with a more increase of mRNA level after 14 days, suggesting that the desiccation stress caused transcriptional regulation of iNOS gene. Immunocytochemical analysis indicated the zonal specific expression of iNOS protein in different tissues. Desiccation stress also led to activation and nuclear translocation of nuclear factor кB (NFкB) in hepatic cells. These results suggest that the activation of iNOS gene under desiccation-induced stresses such as high ammonia load was probably mediated through the activation of one of the major transcription factors, the NFкB. This is the first report of desiccation-induced induction of iNOS gene, iNOS protein expression leading to more generation of NO while living inside the mud peat under condition of water shortage in any air-breathing teleosts. 2012 Elsevier Inc. All rights reserved

  16. Influence of environmental ammonia on the production of nitric oxide and expression of inducible nitric oxide synthase in the freshwater air-breathing catfish (Heteropneustes fossilis)

    International Nuclear Information System (INIS)

    Choudhury, Mahua G.; Saha, Nirmalendu

    2012-01-01

    Highlights: ► High environmental ammonia caused more production and accumulation of NO in air-breathing catfish (Heteropneustes fossilis). ► Hyper-ammonia stress caused induction and zonal specific expression of iNOS enzyme protein, mRNA expression in different tissues. ► Activation of NFκB that resulted under hyper-ammonia stress was believed to be the cause of induction of iNOS gene. - Abstract: Nitric oxide (NO) is a highly versatile and unique ubiquitous signaling molecule, and is known to play diverse physiological functions in mammals including those of adaptation to various stresses. The present study reports on the influence of exposure to high external ammonia (HEA) on the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS), that produces NO from L-arginine in the freshwater air-breathing catfish (Heteropneustes fossilis), which is reported to tolerate a very HEA. Some levels of NO were found to be present in all the tissues and also in plasma of control fish, which further enhanced significantly in fishes treated with high concentrations of environmental ammonia (25 and 50 mM ammonium chloride) for 7 days, accompanied by more efflux of NO from the perfused liver. This was accomplished by the induction of iNOS activity in different tissues of fish exposed to HEA, which otherwise was not detectable in control fish. Exposure to 25 mM ammonium chloride also led to a significant expression of iNOS protein in different tissues, followed by further increase at 50 mM ammonium chloride. Further, there was an increase in the expression of iNOS mRNA in ammonia-treated fish, thus suggesting that the expression of iNOS gene under hyper-ammonia stress was probably regulated at the transcriptional level. Immunocytochemical analysis indicated that the expression of iNOS in different tissues was zonal specific and not expressed uniformly throughout the organ. Hyper-ammonia stress also led to activation and nuclear

  17. The Effect of Artemisia fragrans Willd: Essential Oil on Inducible Nitric Oxide Synthase Gene Expression and Nitric Oxide Production in Lipopolysaccharide-stimulated Murine Macrophage Cell Line.

    Science.gov (United States)

    Farghadan, Maryam; Ghafoori, Hosein; Vakhshiteh, Faezeh; Shahzadeh Fazeli, Seyed Abolhassan; Farzaneh, Parvaneh; Kokhaei, Parviz

    2016-12-01

    The genus Artemisia is estimated to comprise over 800 species with anti-cancer, anti-fungal, anti-oxidant and anti-inflammatory properties. Artemisia fragrans (A. fragrans), a species that belongs to genus Artemisia, is rich in monoterpenes and sesquiterpenes derivatives. Due to anti-inflammatory properties of monoterpenes and sesquiterpenes, we aimed to investigate the effect of A. fragrans essential oil on mRNA expression of inducible nitric oxide synthase (iNOS) gene and nitric oxide (NO) production in Lipopolysaccharide (LPS) -stimulated RAW264.7 cell line. NO, which is synthesized by iNOS, is the main macrophage-derived inflammatory mediator. The oil obtained from the A. fragrans was prepared from aerial parts of the plant. Chemical composition of essential oil was analyzed by gas chromatography-mass spectrometry (GC/MS).The cytotoxicity of various concentrations of essential oil was evaluated by mitochondrial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test assay. The effect of different doses (1.75-7 mg/mL) of A. fragrans oil on mRNA expression of iNOS gene and NO production in LPS-stimulated RAW 264.7 cells was assessed by real-time PCR method and Griess reagent, respectively. In GC/MS analyses of A. fragrans oil, 32 compounds were identified. The main components of the oil were camphor and 1, 8-cineole. The results demonstrated that the essential oil of A. fragrans (1.75- 7 mg/mL), in a dose-dependent manner, inhibits mRNA expression of iNOS induced by LPS in the RAW264.7 cells without cytotoxic effect even at higher doses. The results of iNOS were consistent with the results of NO production. Our preliminary results suggest the possible anti-inflammatory effect of A. fragrans. Further studies are needed to determine the full pharmacokinetics of A. fragrans activity in vivo.

  18. Influence of environmental ammonia on the production of nitric oxide and expression of inducible nitric oxide synthase in the freshwater air-breathing catfish (Heteropneustes fossilis)

    Energy Technology Data Exchange (ETDEWEB)

    Choudhury, Mahua G. [Biochemical Adaptation Laboratory, Department of Zoology, North-Eastern Hill University, Shillong 793022 (India); Saha, Nirmalendu, E-mail: nsaha@nehu.ac.in [Biochemical Adaptation Laboratory, Department of Zoology, North-Eastern Hill University, Shillong 793022 (India)

    2012-07-15

    Highlights: Black-Right-Pointing-Pointer High environmental ammonia caused more production and accumulation of NO in air-breathing catfish (Heteropneustes fossilis). Black-Right-Pointing-Pointer Hyper-ammonia stress caused induction and zonal specific expression of iNOS enzyme protein, mRNA expression in different tissues. Black-Right-Pointing-Pointer Activation of NF{kappa}B that resulted under hyper-ammonia stress was believed to be the cause of induction of iNOS gene. - Abstract: Nitric oxide (NO) is a highly versatile and unique ubiquitous signaling molecule, and is known to play diverse physiological functions in mammals including those of adaptation to various stresses. The present study reports on the influence of exposure to high external ammonia (HEA) on the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS), that produces NO from L-arginine in the freshwater air-breathing catfish (Heteropneustes fossilis), which is reported to tolerate a very HEA. Some levels of NO were found to be present in all the tissues and also in plasma of control fish, which further enhanced significantly in fishes treated with high concentrations of environmental ammonia (25 and 50 mM ammonium chloride) for 7 days, accompanied by more efflux of NO from the perfused liver. This was accomplished by the induction of iNOS activity in different tissues of fish exposed to HEA, which otherwise was not detectable in control fish. Exposure to 25 mM ammonium chloride also led to a significant expression of iNOS protein in different tissues, followed by further increase at 50 mM ammonium chloride. Further, there was an increase in the expression of iNOS mRNA in ammonia-treated fish, thus suggesting that the expression of iNOS gene under hyper-ammonia stress was probably regulated at the transcriptional level. Immunocytochemical analysis indicated that the expression of iNOS in different tissues was zonal specific and not expressed uniformly

  19. Impairment of nitric oxide synthase but not heme oxygenase accounts for baroreflex dysfunction caused by chronic nicotine in female rats.

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    Mohamed A Fouda

    Full Text Available We recently reported that chronic nicotine impairs reflex chronotropic activity in female rats. Here, we sought evidence to implicate nitric oxide synthase (NOS and/or heme oxygenase (HO in the nicotine-baroreflex interaction. Baroreflex curves relating changes in heart rate to increases (phenylephrine or decreases (sodium nitroprusside in blood pressure were generated in conscious female rats treated with nicotine or saline in absence and presence of pharmacological modulators of NOS or HO activity. Compared with saline-treated rats, nicotine (2 mg/kg/day i.p., for 14 days significantly reduced the slopes of baroreflex curves, a measure of baroreflex sensitivity (BRS. Findings that favor the involvement of NOS inhibition in the nicotine effect were (i NOS inhibition (Nω-Nitro-L-arginine methyl ester, L-NAME reduced BRS in control rats but failed to do so in nicotine-treated rats, (ii L-arginine, NO donor, reversed the BRS inhibitory effect of nicotine. Alternatively, HO inhibition (zinc protoporphyrin IX, ZnPP had no effect on BRS in nicotine- or control rats and failed to reverse the beneficial effect of L-arginine on nicotine-BRS interaction. Similar to female rats, BRS was reduced by L-NAME, but not ZnPP, in male rats and the L-NAME effect was not accentuated after concomitant administration of nicotine. Baroreflex dysfunction caused by nicotine in female rats was blunted after supplementation with hemin (HO inducer but not tricarbonyldichlororuthenium(II dimer (CORM-2, a carbon monoxide (CO releasing molecule, or bilirubin, the breakdown product of heme catabolism. The facilitatory effect of hemin was abolished upon simultaneous treatment with L-NAME or 1H-[1], [2], [4] oxadiazolo[4,3-a] quinoxalin-1-one (inhibitor of soluble guanylate cyclase, sGC. The activities of HO and NOS in brainstem tissues were also significantly increased by hemin. Thus, the inhibition of NOS, but not HO, accounts for the baroreflex depressant of chronic nicotine

  20. Nitric oxide synthase inhibition enhances the antitumor effect of radiation in the treatment of squamous carcinoma xenografts.

    Directory of Open Access Journals (Sweden)

    Robert J G Cardnell

    Full Text Available This study tests whether the nitric oxide synthase (NOS inhibitor, N(G-nitro-L-arginine (L-NNA, combines favorably with ionizing radiation (IR in controlling squamous carcinoma tumor growth. Animals bearing FaDu and A431 xenografts were treated with L-NNA in the drinking water. IR exposure was 10 Gy for tumor growth and survival studies and 4 Gy for ex vivo clonogenic assays. Cryosections were examined immunohistochemically for markers of apoptosis and hypoxia. Blood flow was assayed by fluorescent microscopy of tissue cryosections after i.v. injection of fluorospheres. Orally administered L-NNA for 24 hrs reduces tumor blood flow by 80% (p<0.01. Within 24 hrs L-NNA treatment stopped tumor growth for at least 10 days before tumor growth again ensued. The growth arrest was in part due to increased cell killing since a combination of L-NNA and a single 4 Gy IR caused 82% tumor cell killing measured by an ex vivo clonogenic assay compared to 49% by L-NNA or 29% by IR alone. A Kaplan-Meyer analysis of animal survival revealed a distinct survival advantage for the combined treatment. Combining L-NNA and IR was also found to be at least as effective as a single i.p. dose of cisplatin plus IR. In contrast to the in vivo studies, exposure of cells to L-NNA in vitro was without effect on clonogenicity with or without IR. Western and immunochemical analysis of expression of a number of proteins involved in NO signaling indicated that L-NNA treatment enhanced arginase-2 expression and that this may represent vasculature remodeling and escape from NOS inhibition. For tumors such as head and neck squamous carcinomas that show only modest responses to inhibitors of specific angiogenic pathways, targeting NO-dependent pro-survival and angiogenic mechanisms in both tumor and supporting stromal cells may present a potential new strategy for tumor control.

  1. Delayed testosterone replacement restores nitric oxide synthase-containing nerve fibres and the erectile response in rat penis.

    Science.gov (United States)

    Baba, K; Yajima, M; Carrier, S; Morgan, D M; Nunes, L; Lue, T F; Iwamoto, T

    2000-05-01

    To elucidate the effect of testosterone on penile innervation. Materials and methods Three groups of six rats each were assessed; two groups (1 and 2) were castrated and the third (group 3) underwent a sham operation (control). Eight weeks after castration, group 2 received a subcutaneous injection with testosterone. At 8 weeks, the rats in group 1 and 3 underwent a final functional analysis while those in group 2 did so at 12 weeks. The evaluation included a subcutaneous injection with apomorphine to study centrally mediated erection, and cavernosal nerve electrostimulation and papaverine injection to study peripherally mediated erection. At death a penile mid-shaft specimen was taken for NADPH-diaphorase staining. In the apomorphine study, castration resulted in significantly fewer yawns and erections than in the control, and those in group 2 significantly better central erectile function than in the controls. The mean (SEM) number of nitric oxide synthase (NOS)-containing nerve fibres in the corpora cavernosa and both dorsal nerves of castrated rats, at 46.2 (9.1) and 203 (32.1), respectively, were significantly lower than in rats in group 2, at 84.1 (11.2) and 300.6 (17.1), and than in the controls, at 88.6 (10.9) and 306.3 (22.9), respectively. The intracavernosal pressure decreased significantly in the absence of testosterone, both after electrostimulation and intracavernosal papaverine injection. However, there was no difference between the control and group 2 rats in either the number of NOS-containing nerve fibres or in the peripheral erectile functional study. Testosterone acts on the nervous system to mediate erection; when it is absent there may be down-regulation of both the production and activity of NO, thereby decreasing the response to peripheral stimulation via the NO pathway. The restoration of erectile function seen in rats in group 2 supports this phenomenon. Delayed testosterone replacement has no detrimental effect on the restoration of the

  2. Tissue distribution of migration inhibitory factor and inducible nitric oxide synthase in falciparum malaria and sepsis in African children

    Directory of Open Access Journals (Sweden)

    Liomba N George

    2003-04-01

    Full Text Available Abstract Background The inflammatory nature of falciparum malaria has been acknowledged since increased circulating levels of tumour necrosis factor (TNF were first measured, but precisely where the mediators downstream from this prototype inflammatory mediator are generated has not been investigated. Here we report on the cellular distribution, by immunohistochemistry, of migration inhibitory factor (MIF and inducible nitric oxide synthase (iNOS in this disease, and in sepsis. Methods We stained for MIF and iNOS in tissues collected during 44 paediatric autopsies in Blantyre, Malawi. These comprised 42 acutely ill comatose patients, 32 of whom were diagnosed clinically as cerebral malaria and the other 10 as non-malarial diseases. Another 2 were non-malarial, non-comatose deaths. Other control tissues were from Australian adults. Results Of the 32 clinically diagnosed cerebral malaria cases, 11 had negligible histological change in the brain, and no or scanty intravascular sequestration of parasitised erythrocytes, another 7 had no histological changes in the brain, but sequestered parasitised erythrocytes were present (usually dense, and the remaining 14 brains showed micro-haemorrhages and intravascular mononuclear cell accumulations, plus sequestered parasitised erythrocytes. The vascular walls of the latter group stained most strongly for iNOS. Vascular wall iNOS staining was usually of low intensity in the second group (7 brains and was virtually absent from the cerebral vascular walls of 8 of the 10 comatose patients without malaria, and also from control brains. The chest wall was chosen as a typical non-cerebral site encompassing a range of tissues of interest. Here pronounced iNOS staining in vascular wall and skeletal muscle was present in some 50% of the children in all groups, including septic meningitis, irrespective of the degree of staining in cerebral vascular walls. Parasites or malarial pigment were rare to absent in all chest

  3. Impairment of nitric oxide synthase but not heme oxygenase accounts for baroreflex dysfunction caused by chronic nicotine in female rats.

    Science.gov (United States)

    Fouda, Mohamed A; El-Gowelli, Hanan M; El-Gowilly, Sahar M; Rashed, Laila; El-Mas, Mahmoud M

    2014-01-01

    We recently reported that chronic nicotine impairs reflex chronotropic activity in female rats. Here, we sought evidence to implicate nitric oxide synthase (NOS) and/or heme oxygenase (HO) in the nicotine-baroreflex interaction. Baroreflex curves relating changes in heart rate to increases (phenylephrine) or decreases (sodium nitroprusside) in blood pressure were generated in conscious female rats treated with nicotine or saline in absence and presence of pharmacological modulators of NOS or HO activity. Compared with saline-treated rats, nicotine (2 mg/kg/day i.p., for 14 days) significantly reduced the slopes of baroreflex curves, a measure of baroreflex sensitivity (BRS). Findings that favor the involvement of NOS inhibition in the nicotine effect were (i) NOS inhibition (Nω-Nitro-L-arginine methyl ester, L-NAME) reduced BRS in control rats but failed to do so in nicotine-treated rats, (ii) L-arginine, NO donor, reversed the BRS inhibitory effect of nicotine. Alternatively, HO inhibition (zinc protoporphyrin IX, ZnPP) had no effect on BRS in nicotine- or control rats and failed to reverse the beneficial effect of L-arginine on nicotine-BRS interaction. Similar to female rats, BRS was reduced by L-NAME, but not ZnPP, in male rats and the L-NAME effect was not accentuated after concomitant administration of nicotine. Baroreflex dysfunction caused by nicotine in female rats was blunted after supplementation with hemin (HO inducer) but not tricarbonyldichlororuthenium(II) dimer (CORM-2), a carbon monoxide (CO) releasing molecule, or bilirubin, the breakdown product of heme catabolism. The facilitatory effect of hemin was abolished upon simultaneous treatment with L-NAME or 1H-[1], [2], [4] oxadiazolo[4,3-a] quinoxalin-1-one (inhibitor of soluble guanylate cyclase, sGC). The activities of HO and NOS in brainstem tissues were also significantly increased by hemin. Thus, the inhibition of NOS, but not HO, accounts for the baroreflex depressant of chronic nicotine

  4. Effect of an inhibitor of neuronal nitric oxide synthase 7-nitroindazole on cerebral hemodynamic response and brain excitability in urethane-anesthetized rats

    Czech Academy of Sciences Publication Activity Database

    Brožíčková, Carole; Otáhal, Jakub

    2013-01-01

    Roč. 62, Suppl.1 (2013), S57-S66 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GAP303/10/0999; GA ČR(CZ) GPP304/11/P386; GA ČR(CZ) GBP304/12/G069 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : cerebral hemodynamic response * brain excitability * neuronal nitric oxide synthase * 7-nitroindazole * rat Subject RIV: FH - Neurology Impact factor: 1.487, year: 2013

  5. Falcarindiol impairs the expression of inducible nitric oxide synthase by abrogating the activation of IKK and JAK in rat primary astrocytes

    OpenAIRE

    Shiao, Young-Ji; Lin, Yun-Lian; Sun, Ya-Hui; Chi, Chih-Wen; Chen, Chieh-Fu; Wang, Chuen-Neu

    2004-01-01

    The effects of falcarindiol on the expression of inducible nitric oxide synthase (iNOS) induced by lipopolysaccharide/interferon-γ (LPS/IFN-γ) in rat primary astrocytes were investigated. The molecular mechanisms underlying falcarindiol that confers its effect on iNOS expression were also elucidated.Falcarindiol abrogated the LPS/IFN-γ-mediated induction of iNOS by about 80%. Falcarindiol attenuated the induction of iNOS in a concentration-dependent manner.The inhibitory effect of falcarindio...

  6. Decreased hippocampal homoarginine and increased nitric oxide and nitric oxide synthase levels in rats parallel training in a radial arm maze.

    Science.gov (United States)

    Sase, Ajinkya; Nawaratna, Gayan; Hu, Shengdi; Wu, Guoyao; Lubec, Gert

    2016-09-01

    L-homoarginine (hArg) is derived from enzymatic guanidination of lysine. It was demonstrated that hArg is a substrate for nitric oxide (NO) synthesis, blocks lysine transport and inhibits the uptake of arginine into synaptosomes and modulates GABA responses ex vivo. As there is limited information on its physiological roles in the brain, the aim of the study was to show whether hippocampal or frontal lobe (FL) hArg is paralleling training in the radial arm maze (RAM) or NO formation. Hippocampi and FL of male Sprague-Dawley rats were taken from trained or yoked in a RAM. Then hArg and metabolites, NO and NO synthase (NOS) were determined by standard methods. The animals learned the task in the RAM showing significant reduction of working memory errors. hArg showed decreased levels in both brain regions of trained animals as compared to yoked animals. Nitrate plus nitrite (NOx) concentrations and NOS activity were significantly increased in hippocampi, F(1,36) = 170.5; P ≤ 0.0001 and FL, F(1,36) = 74.67; P ≤ 0.0001 of trained animals as compared to yoked animals. Levels of hArg were negatively correlated with NOx in hippocampus (r = -0.6355; P = 0.0483) but not in FL and with lysine in the FL (r = -0.6650; P = 0.0358). NOx levels were positively correlated with NOS in both the hippocampus (r = 0.7474; P = 0.0129) and FL (r = 0.9563; P ≤  0.0001). These novel findings indicate that hArg is linked to NO formation in hippocampus but not in FL and is paralleling spatial memory in the RAM.

  7. Arginase inhibition reduces interleukin-1β-stimulated vascular smooth muscle cell proliferation by increasing nitric oxide synthase-dependent nitric oxide production

    Energy Technology Data Exchange (ETDEWEB)

    Yoon, Jeongyeon; Ryoo, Sungwoo, E-mail: ryoosw08@kangwon.ac.kr

    2013-06-07

    Highlights: •Arginase inhibition suppressed proliferation of IL-1β-stimulated VSMCs in dose-dependent manner. •NO production from IL-1β-induced iNOS expression was augmented by arginase inhibition, reducing VSMC proliferation. •Incubation with cGMP analogues abolished IL-1β-dependent proliferation of VSMCs. -- Abstract: We investigated whether arginase inhibition suppressed interleukin (IL)-1β-stimulated proliferation in vascular smooth muscle cells (VSMCs) and the possible mechanisms involved. IL-1β stimulation increased VSMC proliferation, while the arginase inhibitor BEC and transfection of the antisense (AS) oligonucleotide against arginase I decreased VSMC proliferation and was associated with increased protein content of the cell cycle regulator p21Waf1/Cip1. IL-1β incubation induced inducible nitric oxide synthase (iNOS) mRNA expression and protein levels in a dose-dependent manner, but did not affect arginase I and II expression. Consistent with this data, IL-1β stimulation resulted in increase in NO production that was significantly augmented by arginase inhibition. The specific iNOS inhibitor 1400W abolished IL-1β-mediated NO production and further accentuated IL-1β-stimulated cell proliferation. Incubation with NO donors GSNO and DETA/NO in the presence of IL-1β abolished VSMCs proliferation and increased p21Waf1/Cip1 protein content. Furthermore, incubation with the cGMP analogue 8-Br-cGMP prevented IL-1β-induced VSMCs proliferation. In conclusion, arginase inhibition augmented iNOS-dependent NO production that resulted in suppression of IL-1β-induced VSMCs proliferation in a cGMP-dependent manner.

  8. Arginase inhibition reduces interleukin-1β-stimulated vascular smooth muscle cell proliferation by increasing nitric oxide synthase-dependent nitric oxide production

    International Nuclear Information System (INIS)

    Yoon, Jeongyeon; Ryoo, Sungwoo

    2013-01-01

    Highlights: •Arginase inhibition suppressed proliferation of IL-1β-stimulated VSMCs in dose-dependent manner. •NO production from IL-1β-induced iNOS expression was augmented by arginase inhibition, reducing VSMC proliferation. •Incubation with cGMP analogues abolished IL-1β-dependent proliferation of VSMCs. -- Abstract: We investigated whether arginase inhibition suppressed interleukin (IL)-1β-stimulated proliferation in vascular smooth muscle cells (VSMCs) and the possible mechanisms involved. IL-1β stimulation increased VSMC proliferation, while the arginase inhibitor BEC and transfection of the antisense (AS) oligonucleotide against arginase I decreased VSMC proliferation and was associated with increased protein content of the cell cycle regulator p21Waf1/Cip1. IL-1β incubation induced inducible nitric oxide synthase (iNOS) mRNA expression and protein levels in a dose-dependent manner, but did not affect arginase I and II expression. Consistent with this data, IL-1β stimulation resulted in increase in NO production that was significantly augmented by arginase inhibition. The specific iNOS inhibitor 1400W abolished IL-1β-mediated NO production and further accentuated IL-1β-stimulated cell proliferation. Incubation with NO donors GSNO and DETA/NO in the presence of IL-1β abolished VSMCs proliferation and increased p21Waf1/Cip1 protein content. Furthermore, incubation with the cGMP analogue 8-Br-cGMP prevented IL-1β-induced VSMCs proliferation. In conclusion, arginase inhibition augmented iNOS-dependent NO production that resulted in suppression of IL-1β-induced VSMCs proliferation in a cGMP-dependent manner

  9. Localization of nitric oxide synthase and haemoxygenase, and functional effects of nitric oxide and carbon monoxide in the pig and human intravesical ureter.

    Science.gov (United States)

    Iselin, C E; Alm, P; Schaad, N C; Larsson, B; Graber, P; Andersson, K E

    1997-01-01

    The distribution of nitric oxide synthase (NOS)-immunoreactive (IR) and haemoxygenase (HO)-IR nerves was investigated in the pig and human intravesical ureter (IVU). NOS activity was measured by monitoring the conversion of [3H]-arginine to [3H]-citrulline. Effects of NO and resulting changes in cyclic nucleotide concentrations were assessed in vitro. The effects of carbon monoxide (CO) on IVU motility was also tested. Immunohistochemistry revealed an abundant overall innervation of the IVU and numerous NOS-IR nerves. Nerve trunks were also found expressing immunoreactivity for HO-1, one of the enzymes synthetising CO. Similar profiles of nerve structures expressing immunoreactivities for NOS and tyrosine-hydroxylase (TH), as well as NOS and vasoactive intestinal peptide (VIP) were demonstrated. In the pig IVU, measurement of NOS activity revealed a moderate calcium-dependent catalytic activity, NO and the NO-donor SIN-1 reduced in a concentration-dependent manner serotonin-induced contractions of pig and human IVU, and the spontaneous contractions of pig IVU. In pig IVU strips precontracted with the thromboxane analogue U-46619, tetrodotoxin-sensitive relaxations were abolished by the NOS inhibitor NG-nitro-L-arginine. CO exerted no significant effect on spontaneous or induced contractions in the pig and human IVU. In precontracted strips of the pig and human IVU exposed to SIN-1 or NO, significant increases of cyclic GMP levels were measured in comparison to control preparations. The results suggest that the L-arginine/NO/cyclic GMP pathway may play a role in the regulation of the valve function in the uretero-vesical junction (UVJ). A role for CO in the UVJ has yet to be established.

  10. High nitric oxide production, secondary to inducible nitric oxide synthase expression, is essential for regulation of the tumour-initiating properties of colon cancer stem cells.

    Science.gov (United States)

    Puglisi, Maria Ausiliatrice; Cenciarelli, Carlo; Tesori, Valentina; Cappellari, Marianna; Martini, Maurizio; Di Francesco, Angela Maria; Giorda, Ezio; Carsetti, Rita; Ricci-Vitiani, Lucia; Gasbarrini, Antonio

    2015-08-01

    Chronic inflammation is a leading cause of neoplastic transformation in many human cancers and especially in colon cancer (CC), in part due to tumour promotion by nitric oxide (NO) generated at inflammatory sites. It has also been suggested that high NO synthesis, secondary to inducible NO synthase (iNOS) expression, is a distinctive feature of cancer stem cells (CSCs), a small subset of tumour cells with self-renewal capacity. In this study we explored the contribution of NO to the development of colon CSC features and evaluated potential strategies to treat CC by modulating NO production. Our data show an integral role for endogenous NO and iNOS activity in the biology of colon CSCs. Indeed, colon CSCs with high endogenous NO production (NO(high)) displayed higher tumourigenic abilities than NO(low) fractions. The blockade of endogenous NO availability, using either a specific iNOS inhibitor or a genetic knock-down of iNOS, resulted in a significant reduction of colon CSC tumourigenic capacities in vitro and in vivo. Interestingly, analysis of genes altered by iNOS-directed shRNA showed that the knockdown of iNOS expression was associated with a significant down-regulation of signalling pathways involved in stemness and tumour progression in colon CSCs. These findings confirm that endogenous NO plays an important role in defining the stemness properties of colon CSCs through cross-regulation of several cellular signalling pathways. This discovery could shed light on the mechanisms by which NO induces the growth and invasiveness of CC, providing new insights into the link between inflammation and colon tumourigenesis. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  11. The effects of nitric oxide synthase--versus lipoxygenase inhibition on coronary flow and nitrite outflow in isolated rat heart.

    Science.gov (United States)

    Jakovljevic, V Lj; Djuric, D M

    2005-06-01

    The aim of this study was to assess the changes of coronary flow (CF) and nitrite outflow under inhibition of nitric oxide synthase (NOS) by Nomega-nitro-L-arginine monomethyl ester (L-NAME) or lipoxygenase (LOX) induced by nordihydroguaiaretic acid (NDGA) in isolated rat heart. The hearts of male Wistar albino rats (n=18, age 8 weeks, body mass 180-200 g) were retrograde perfused according to the Langendorff's technique at gradually increased constant coronary perfusion pressure (CPP) conditions (40-120 cm H2O) which induced flow-dependent nitric oxide (NO) release (nitrite outflow). The experiments were performed during control conditions, in the presence of NO synthesis inhibitor L-NAME (30 micromol/l) or nonspecific LOX inhibitor (NDGA, 0.1 mmol/l) which were administered separately or in combination. CF varied in autoregulatory range from 4.12+/-0.26 ml/min/g wt at 50 cm H2O to 5.22+/-0.26 ml/min/g wt at 90 cm H2O. In autoregulatory range, nitrite outflow varied from 2.05+/-0.17 nmol/min/g wt at 50 cm H2O to 2.52+/-0.21 nmol/min/g wt at 90 cm H2O and was strictly parallel with CPP/CF curve. The autoregulatory range of CF was significantly extended (40-100 cm H2O, 2.22+/-0.12 ml/min/g wt and 2.90+/-0.25 ml/min/g wt, respectively) under the influence of L-NAME. Hemodynamic effects were accompanied by significant decrease in nitrite outflow after L-NAME administration (0.56+/-0.11 nmol/min/g wt at 40 cm H2O to 1.45+/-0.14 nmol/min/g wt at 100 cm H2O). NDGA affected CF in the range of CPP 40-70 cm H2O only (from 42% at 50 cm H2O to 12% at 90 cm H2O, respectively) with no significant changes in nitrite outflow. When L-NAME was applied in combination with NDGA vs. NDGA only, CF was significantly reduced (from 34% at 50 cm H2O to 50% at 90 cm H2O, respectively) with parallel changes in nitrite outflow (from 40% at 50 cm H2O to 51% at 90 cm H2O, respectively). The results showed that CF and nitrite outflow could be decreased under L-NAME administration. Nonselective

  12. Inhibition of muscle glycogen synthase activity and non-oxidative glucose disposal during hypoglycaemia in normal man

    DEFF Research Database (Denmark)

    Ørskov, Lotte; Bak, Jens Friis; Abildgaard, Ulrik

    1996-01-01

    The purpose of the present study was to evaluate the role of muscle glycogen synthase activity in the reduction of glucose uptake during hypoglycaemia. Six healthy young men were examined twice; during 120 min of hyperinsulinaemic (1.5 mU.kg-1. min-1) euglycaemia followed by: 1)240 min of graded ...

  13. Inducible Nitric Oxide Synthase Promoter Haplotypes and Residential Traffic-Related Air Pollution Jointly Influence Exhaled Nitric Oxide Level in Children.

    Directory of Open Access Journals (Sweden)

    Muhammad T Salam

    Full Text Available Exhaled nitric oxide (FeNO, a biomarker of airway inflammation, predicts asthma risk in children. We previously found that the promoter haplotypes in inducible nitric oxide synthase (NOS2 and exposure to residential traffic independently influence FeNO level. Because NOS2 is inducible by environmental exposures such as traffic-related exposure, we tested the hypothesis that common NOS2 promoter haplotypes modulate the relationship between residential traffic-related exposure and FeNO level in children.In a cross-sectional population-based study, subjects (N = 2,457; 7-11 year-old were Hispanic and non-Hispanic white children who participated in the Southern California Children's Health Study and had FeNO measurements. For residential traffic, lengths of local roads within circular buffers (50m, 100m and 200m radii around homes around the subjects' homes were estimated using geographic information system (GIS methods. We interrogated the two most common NOS2 promoter haplotypes that were found to affect FeNO level.The relationship between local road lengths within 100m and 200m circular buffers and FeNO level varied significantly by one of the NOS2 promoter haplotypes (P-values for interaction between road length and NOS2 promoter haplotype = 0.02 and 0.03, respectively. In children who had ≤250m of local road lengths within 100m buffer around their homes, those with two copies of the haplotype had significantly lower FeNO (adjusted geometric mean = 11.74ppb; 95% confidence intervals (CI: 9.99 to 13.80 than those with no copies (adjusted geometric mean = 15.28ppb; 95% CI: 14.04 to 16.63 with statistically significant trend of lower FeNO level with increasing number of haplotype copy (P-value for trend = 0.002. In contrast, among children who had >250m of local road lengths within 100m buffer, FeNO level did not significantly differ by the haplotype copy-number (P-value for trend = 0.34. Similar interactive effects of this haplotype and local

  14. Inducible Nitric Oxide Synthase in Heart Tissue and Nitric Oxide in Serum of Trypanosoma cruzi-Infected Rhesus Monkeys: Association with Heart Injury

    Science.gov (United States)

    Carvalho, Cristiano Marcelo Espinola; Silverio, Jaline Coutinho; da Silva, Andrea Alice; Pereira, Isabela Resende; Coelho, Janice Mery Chicarino; Britto, Constança Carvalho; Moreira, Otacílio Cruz; Marchevsky, Renato Sergio; Xavier, Sergio Salles; Gazzinelli, Ricardo Tostes; da Glória Bonecini-Almeida, Maria; Lannes-Vieira, Joseli

    2012-01-01

    Background The factors contributing to chronic Chagas' heart disease remain unknown. High nitric oxide (NO) levels have been shown to be associated with cardiomyopathy severity in patients. Further, NO produced via inducible nitric oxide synthase (iNOS/NOS2) is proposed to play a role in Trypanosoma cruzi control. However, the participation of iNOS/NOS2 and NO in T. cruzi control and heart injury has been questioned. Here, using chronically infected rhesus monkeys and iNOS/NOS2-deficient (Nos2 −/−) mice we explored the participation of iNOS/NOS2-derived NO in heart injury in T. cruzi infection. Methodology Rhesus monkeys and C57BL/6 and Nos2 −/− mice were infected with the Colombian T. cruzi strain. Parasite DNA was detected by polymerase chain reaction, T. cruzi antigens and iNOS/NOS2+ cells were immunohistochemically detected in heart sections and NO levels in serum were determined by Griess reagent. Heart injury was assessed by electrocardiogram (ECG), echocardiogram (ECHO), creatine kinase heart isoenzyme (CK-MB) activity levels in serum and connexin 43 (Cx43) expression in the cardiac tissue. Results Chronically infected monkeys presented conduction abnormalities, cardiac inflammation and fibrosis, which resembled the spectrum of human chronic chagasic cardiomyopathy (CCC). Importantly, chronic myocarditis was associated with parasite persistence. Moreover, Cx43 loss and increased CK-MB activity levels were primarily correlated with iNOS/NOS2+ cells infiltrating the cardiac tissue and NO levels in serum. Studies in Nos2 −/− mice reinforced that the iNOS/NOS2-NO pathway plays a pivotal role in T. cruzi-elicited cardiomyocyte injury and in conduction abnormalities that were associated with Cx43 loss in the cardiac tissue. Conclusion T. cruzi-infected rhesus monkeys reproduce features of CCC. Moreover, our data support that in T. cruzi infection persistent parasite-triggered iNOS/NOS2 in the cardiac tissue and NO overproduction might contribute to CCC

  15. Inducible nitric oxide synthase in heart tissue and nitric oxide in serum of Trypanosoma cruzi-infected rhesus monkeys: association with heart injury.

    Directory of Open Access Journals (Sweden)

    Cristiano Marcelo Espinola Carvalho

    Full Text Available BACKGROUND: The factors contributing to chronic Chagas' heart disease remain unknown. High nitric oxide (NO levels have been shown to be associated with cardiomyopathy severity in patients. Further, NO produced via inducible nitric oxide synthase (iNOS/NOS2 is proposed to play a role in Trypanosoma cruzi control. However, the participation of iNOS/NOS2 and NO in T. cruzi control and heart injury has been questioned. Here, using chronically infected rhesus monkeys and iNOS/NOS2-deficient (Nos2(-/- mice we explored the participation of iNOS/NOS2-derived NO in heart injury in T. cruzi infection. METHODOLOGY: Rhesus monkeys and C57BL/6 and Nos2(-/- mice were infected with the Colombian T. cruzi strain. Parasite DNA was detected by polymerase chain reaction, T. cruzi antigens and iNOS/NOS2(+ cells were immunohistochemically detected in heart sections and NO levels in serum were determined by Griess reagent. Heart injury was assessed by electrocardiogram (ECG, echocardiogram (ECHO, creatine kinase heart isoenzyme (CK-MB activity levels in serum and connexin 43 (Cx43 expression in the cardiac tissue. RESULTS: Chronically infected monkeys presented conduction abnormalities, cardiac inflammation and fibrosis, which resembled the spectrum of human chronic chagasic cardiomyopathy (CCC. Importantly, chronic myocarditis was associated with parasite persistence. Moreover, Cx43 loss and increased CK-MB activity levels were primarily correlated with iNOS/NOS2(+ cells infiltrating the cardiac tissue and NO levels in serum. Studies in Nos2(-/- mice reinforced that the iNOS/NOS2-NO pathway plays a pivotal role in T. cruzi-elicited cardiomyocyte injury and in conduction abnormalities that were associated with Cx43 loss in the cardiac tissue. CONCLUSION: T. cruzi-infected rhesus monkeys reproduce features of CCC. Moreover, our data support that in T. cruzi infection persistent parasite-triggered iNOS/NOS2 in the cardiac tissue and NO overproduction might contribute

  16. Inhibition of inducible nitric oxide synthase expression and nitric oxide production in plateau pika (Ochotona curzoniae) at high altitude on Qinghai-Tibet Plateau.

    Science.gov (United States)

    Xie, Ling; Zhang, Xuze; Qi, Delin; Guo, Xinyi; Pang, Bo; Du, Yurong; Zou, Xiaoyan; Guo, Songchang; Zhao, Xinquan

    2014-04-30

    Nitric oxide (NO), a potent vasodilator, plays an important role in preventing hypoxia induced pulmonary hypertension. Endogenous NO is synthesized by nitric oxide synthases (NOSs) from l-arginine. In mammals, three different NOSs have been identified, including neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Plateau pika (Ochotona curzoniae) is a typical hypoxia tolerant mammal that lives at 3000-5000 m above sea level on the Qinghai-Tibet Plateau. The aim of this study was to investigate whether NOS expression and NO production are regulated by chronic hypoxia in plateau pika. Quantitative real-time PCR and western blot analyses were conducted to quantify relative abundances of iNOS and eNOS transcripts and proteins in the lung tissues of plateau pikas at different altitudes (4550, 3950 and 3200 m). Plasma NO metabolites, nitrite/nitrate (NO(x)⁻) levels were also examined by Ion chromatography to determine the correlation between NO production and altitude level. The results revealed that iNOS transcript levels were significantly lower in animals at high altitudes (decreased by 53% and 57% at altitude of 3950 and 4550 m compared with that at 3200 m). Similar trends in iNOS protein abundances were observed (26% and 41% at 3950 and 4550 m comparing with at 3200 m). There were no significant differences in eNOS mRNA and protein levels in the pika lungs among different altitudes. The plasma NO(x)⁻ levels of the plateau pikas at high altitudes significantly decreased (1.65±0.19 μg/mL at 3200 m to 0.44±0.03 μg/mL at 3950 m and 0.24±0.01 μg/mL at 4550 m). This is the first evidence describing the effects of chronic hypoxia on NOS expression and NO levels in the plateau pika in high altitude adaptation. We conclude that iNOS expression and NO production are suppressed at high altitudes, and the lower NO concentration at high altitudes may serve crucial roles for helping the plateau pika to survive at hypoxic environment. Copyright © 2014

  17. Grape Consumption Increases Anti-Inflammatory Markers and Upregulates Peripheral Nitric Oxide Synthase in the Absence of Dyslipidemias in Men with Metabolic Syndrome

    Directory of Open Access Journals (Sweden)

    Jiyoung Lee

    2012-12-01

    Full Text Available We evaluated the effects of grape consumption on inflammation and oxidation in the presence or absence of dyslipidemias in metabolic syndrome (MetS. Men with MetS (n = 24, 11 with high triglycerides and low HDL and 13 with no dyslipidemia were recruited and randomly allocated to consume daily either 46 g of lyophilized grape powder (GRAPE, equivalent to 252 g fresh grapes, or placebo with an identical macronutrient composition and caloric value as GRAPE for four weeks. After a three-week washout, participants followed the alternate treatment. We measured changes between placebo and GRAPE periods in inflammatory and oxidative stress markers both in circulation and in gene expression. Changes in plasma adiponectin (p < 0.05, interleukin (IL-10 (p < 0.005 and in mRNA expression of the inducible isoform of nitric oxide synthase (iNOS (p < 0.25 were increased in the GRAPE compared to the placebo period only in those individuals without dyslipidemia. Additionally, plasma IL-10 was negatively correlated with NOX2 expression, a marker of oxidative stress (r = −0.55, p < 0.01, while iNOS expression was positively correlated with the expression of superoxide dismutase 2 (r = 0.642, p < 0.01, a key anti-oxidative enzyme. Grape consumption displayed anti-oxidative and increased anti-inflammatory markers in the absence of the inflammatory milieu associated with dyslipidemias.

  18. New Role for L-Arginine in Regulation of Inducible Nitric-Oxide-Synthase-Derived Superoxide Anion Production in Raw 264.7 Macrophages

    Directory of Open Access Journals (Sweden)

    Michaela Pekarova

    2011-01-01

    Full Text Available Dietary supplementation with L-arginine was shown to improve immune responses in various inflammatory models. However, the molecular mechanisms underlying L-arginine effects on immune cells remain unrecognized. Herein, we tested the hypothesis that a limitation of L-arginine could lead to the uncoupled state of murine macrophage inducible nitric oxide synthase and, therefore, increase inducible nitric-oxide-synthase-derived superoxide anion formation. Importantly, we demonstrated that L-arginine dose- and time dependently potentiated superoxide anion production in bacterial endotoxin-stimulated macrophages, although it did not influence NADPH oxidase expression and activity. Detailed analysis of macrophage activation showed the time dependence between LPS-induced iNOS expression and increased O2∙- formation. Moreover, downregulation of macrophage iNOS expression, as well as the inhibition of iNOS activity by NOS inhibitors, unveiled an important role of this enzyme in controlling O2∙- and peroxynitrite formation during macrophage stimulation. In conclusion, our data demonstrated that simultaneous induction of NADPH oxidase, together with the iNOS enzyme, can result in the uncoupled state of iNOS resulting in the production of functionally important levels of O2∙- soon after macrophage activation with LPS. Moreover, we demonstrated, for the first time that increased concentrations of L-arginine further potentiate iNOS-dependent O2∙- formation in inflammatory macrophages.

  19. New role for L-arginine in regulation of inducible nitric-oxide-synthase-derived superoxide anion production in raw 264.7 macrophages.

    Science.gov (United States)

    Pekarova, Michaela; Lojek, Antonin; Martiskova, Hana; Vasicek, Ondrej; Bino, Lucia; Klinke, A; Lau, D; Kuchta, Radek; Kadlec, Jaroslav; Vrba, Radimir; Kubala, Lukas

    2011-01-01

    Dietary supplementation with L-arginine was shown to improve immune responses in various inflammatory models. However, the molecular mechanisms underlying L-arginine effects on immune cells remain unrecognized. Herein, we tested the hypothesis that a limitation of L-arginine could lead to the uncoupled state of murine macrophage inducible nitric oxide synthase and, therefore, increase inducible nitric-oxide-synthase-derived superoxide anion formation. Importantly, we demonstrated that L-arginine dose- and time dependently potentiated superoxide anion production in bacterial endotoxin-stimulated macrophages, although it did not influence NADPH oxidase expression and activity. Detailed analysis of macrophage activation showed the time dependence between LPS-induced iNOS expression and increased O(2)(∙-) formation. Moreover, downregulation of macrophage iNOS expression, as well as the inhibition of iNOS activity by NOS inhibitors, unveiled an important role of this enzyme in controlling O(2)(∙-) and peroxynitrite formation during macrophage stimulation. In conclusion, our data demonstrated that simultaneous induction of NADPH oxidase, together with the iNOS enzyme, can result in the uncoupled state of iNOS resulting in the production of functionally important levels of O(2)(∙-) soon after macrophage activation with LPS. Moreover, we demonstrated, for the first time that increased concentrations of L-arginine further potentiate iNOS-dependent O(2) (∙-) formation in inflammatory macrophages.

  20. New Role for L-Arginine in Regulation of Inducible Nitric-Oxide-Synthase-Derived Superoxide Anion Production in Raw 264.7 Macrophages

    Science.gov (United States)

    Pekarova, Michaela; Lojek, Antonin; Martiskova, Hana; Vasicek, Ondrej; Bino, Lucia; Klinke, A.; Lau, D.; Kuchta, Radek; Kadlec, Jaroslav; Vrba, Radimir; Kubala, Lukas

    2011-01-01

    Dietary supplementation with L-arginine was shown to improve immune responses in various inflammatory models. However, the molecular mechanisms underlying L-arginine effects on immune cells remain unrecognized. Herein, we tested the hypothesis that a limitation of L-arginine could lead to the uncoupled state of murine macrophage inducible nitric oxide synthase and, therefore, increase inducible nitric-oxide-synthase-derived superoxide anion formation. Importantly, we demonstrated that L-arginine dose- and time dependently potentiated superoxide anion production in bacterial endotoxin-stimulated macrophages, although it did not influence NADPH oxidase expression and activity. Detailed analysis of macrophage activation showed the time dependence between LPS-induced iNOS expression and increased O2∙− formation. Moreover, downregulation of macrophage iNOS expression, as well as the inhibition of iNOS activity by NOS inhibitors, unveiled an important role of this enzyme in controlling O2∙− and peroxynitrite formation during macrophage stimulation. In conclusion, our data demonstrated that simultaneous induction of NADPH oxidase, together with the iNOS enzyme, can result in the uncoupled state of iNOS resulting in the production of functionally important levels of O2∙− soon after macrophage activation with LPS. Moreover, we demonstrated, for the first time that increased concentrations of L-arginine further potentiate iNOS-dependent O2∙− formation in inflammatory macrophages. PMID:22219714

  1. Suppression by Ghrelin of Porphyromonas gingivalis-Induced Constitutive Nitric Oxide Synthase S-Nitrosylation and Apoptosis in Salivary Gland Acinar Cells

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    Bronislaw L. Slomiany

    2010-01-01

    Full Text Available Oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis, and its key virulence factor, LPS, are characterized by a massive rise in epithelial cell apoptosis and the disturbances in NO signaling pathways. Here, we report that the LPS-induced enhancement in rat sublingual salivary gland acinar cell apoptosis and NO generation was associated with the suppression in constitutive nitric oxide synthase (cNOS activity and a marked increase in the activity of inducible nitric oxide synthase (iNOS. We demonstrate that the detrimental effect of the LPS on cNOS was manifested by the enzyme protein S-nitrosylation, that was susceptible to inhibition by iNOS inhibitor, 1400 W. Further, we show that a peptide hormone, ghrelin, countered the LPS-induced changes in apoptosis and cNOS activity. This effect of ghrelin was reflected in the decrease in cNOS S-nitrosylation and the increase in phosphorylation. Our findings imply that P. gingivalis-induced disturbances in the acinar cell NO signaling pathways result from upregulation in iNOS-derived NO that causes cNOS S-nitrosylation that interferes with its activation through phosphorylation. We also show that ghrelin protection against P. gingivalis-induced disturbances involves cNOS activation associated with a decrease in its S-nitrosylation and the increase in phosphorylation.

  2. Inducible nitric-oxide synthase plays a minimal role in lymphocytic choriomeningitis virus-induced, T cell-mediated protective immunity and immunopathology

    DEFF Research Database (Denmark)

    Bartholdy, C; Nansen, A; Christensen, Jeanette Erbo

    1999-01-01

    By using mice with a targetted disruption in the gene encoding inducible nitric-oxide synthase (iNOS), we have studied the role of nitric oxide (NO) in lymphocytic choriomeningitis virus (LCMV)-induced, T cell-mediated protective immunity and immunopathology. The afferent phase of the T cell......-mediated immune response was found to be unaltered in iNOS-deficient mice compared with wild-type C57BL/6 mice, and LCMV- induced general immunosuppression was equally pronounced in both strains. In vivo analysis revealed identical kinetics of virus clearance, as well as unaltered clinical severity of systemic....... This might suggest a role of NO in regulating vascular reactivity in the context of T cell-mediated inflammation. In conclusion, these findings indicate a minimal role for iNOS/NO in the host response to LCMV. Except for a reduced local oedema in the knockout mice, iNOS/NO seems to be redundant...

  3. The effects of folic acid and nitric oxide synthase inhibition on coronary flow and oxidative stress markers in isolated rat heart.

    Science.gov (United States)

    Djurić, Dragan; Vusanović, Ana; Jakovljević, Vladimir

    2007-06-01

    The aim of this study was to assess the effects of folic acid on coronary flow and oxidative stress markers with or without non-specific inhibition of nitric oxide synthase by L-NAME in isolated rat hearts. The hearts of male Wistar albino rats (n = 12, age 8 weeks, body mass 180-200 g) were retrograde perfused according to the Langendorff technique at gradually increased constant perfusion pressure (40-120 cmH2O). Coronary flow and markers of oxidative stress: nitrite outflow, superoxide anion production, and index of lipid peroxidation (by measuring thiobarbituric acid reactive substances) in coronary effluent were calculated. The experiments were performed during control conditions and in presence of folic acid (100 microM) alone or folic acid (100 microM) plus L-NAME (30 microM). Control values of coronary flow varied in range from 4.37 +/- 0.10 ml/min/g wt at 40 cmH2O to 12.05 +/- 0.42 ml/min/g wt at 120 cmH2O. Nitrite outflow varied from 1.68 +/- 0.17 nmol/min/g wt at 40 cmH2O to 3.56 +/- 0.17 nmol/min/g wt at 120 cmH2O and was parallel with coronary perfusion pressure-coronary flow curve. Folic acid significantly increased coronary flow (40-120 cmH2O, 5.63 +/- 0.10 ml/min/g wt and 15.2 +/- 0.42 ml/min/g wt, respectively) and was accompanied by significant increase in nitrite outflow (2.28 +/- 0.29 nmol/min/g wt at 40 cmH2O to 6.66 +/- 0.50 nmol/min/g wt at 120 cmH2O). In addition, folic acid significantly decreased superoxide anion production especially at upper coronary perfusion pressure values (60% at 120 cmH2O) and increased index of lipid peroxidation (37.16% at 120 cmH2O), respectively. Folic acid plus L-NAME did not change control values of coronary flow significantly. However, folic acid plus L-NAME increased nitrite outflow especially at upper coronary perfusion pressure values (43.05% at 120 cmH2O) and did not change significantly superoxide anion production or index of lipid peroxidation versus control values, respectively. The results clearly

  4. The T -786C, G894T, and Intron 4 VNTR (4a/b) Polymorphisms of the Endothelial Nitric Oxide Synthase Gene in Prostate Cancer Cases.

    Science.gov (United States)

    Diler, S B; Öden, A

    2016-02-01

    In previously conducted some studies it has been revealed that nitric oxide (NO) and nitric oxide synthase (NOS) system play a significant role in carcinogenesis. Nitric oxide (NO) is regulated by endothelial nitric oxide synthase (eNOS) enzyme which is one of the isoenzymes of NO synthase (NOS). In this study we have tried to come to a conclusion about whether eNOS gene T -786C, G894T and Intron 4 VNTR (4a/b) polymorphisms might be considered as a risk factor causing prostate cancer (PCa) or not. A total of 200 subjects were included in this research. 84 patients with PCa (mean age 70.0 ± 6.4) and 116 healthy controls (mean age 69.9 ± 7.5) were recruited in this case-control study. Genomic DNA was extracted using the QIAamp DNA Blood Mini Kit (QIAGEN GmbH, Maryland, USA), according to the manufacturer's guidelines. The T-786C, G894T and Intron 4 VNTR (4a/b) polymorphisms were amplified using polymerase chain reation (PCR), detected by restriction fragment length polymorphism (RFLP). For T -786C polymorphism CC genotype [odds ratio (OR): 0.34, 95% confidence interval (CI): 0.15-0.78, P = 0.009)] and allele frequency (OR: 0.631, CI: 0.421-0.946, P = 0.026) is significant for control. In patients with PCa eNOS G894T polymorphism, both GT (OR: 0.069, CI: 0.027-0.174; P = 0.0001) and TT (OR: 0.040, CI: 0.013-0.123; P = = 0.0001) genotype distribution, and also T allele frequency (OR: 0.237, CI: 0.155-0.362, P = 0.0001) were considered significant statistically. While genotype distribution for the other polymorphism eNOS, intron 4 VNTR (4a/b), is insignificant statistically, "a" allele frequency was found out to be significant (OR: 2.223, CI: 1.311-3.769, P = 0.003). In this study we indicated that genotype and allele frequencies of eNOS T -786C and G894T polymorphisms are statistically significant in patients with PCa. eNOS T -786C and G894T polymorphisms may be associated with PCa susceptibility in the Turkish population. In contrast, intron 4 VNTR (4a

  5. Effects of Nitric Oxide Synthase Inhibition on Fiber-Type Composition, Mitochondrial Biogenesis, and SIRT1 Expression in Rat Skeletal Muscle

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    Masataka Suwa, Hiroshi Nakano, Zsolt Radak, Shuzo Kumagai

    2015-09-01

    Full Text Available It was hypothesized that nitric oxide synthases (NOS regulated SIRT1 expression and lead to a corresponding changes of contractile and metabolic properties in skeletal muscle. The purpose of the present study was to investigate the influence of long-term inhibition of nitric oxide synthases (NOS on the fiber-type composition, metabolic regulators such as and silent information regulator of transcription 1 (SIRT1 and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α, and components of mitochondrial biogenesis in the soleus and plantaris muscles of rats. Rats were assigned to two groups: control and NOS inhibitor (Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME, ingested for 8 weeks in drinking water-treated groups. The percentage of Type I fibers in the L-NAME group was significantly lower than that in the control group, and the percentage of Type IIA fibers was concomitantly higher in soleus muscle. In plantaris muscle, muscle fiber composition was not altered by L-NAME treatment. L-NAME treatment decreased the cytochrome C protein expression and activity of mitochondrial oxidative enzymes in the plantaris muscle but not in soleus muscle. NOS inhibition reduced the SIRT1 protein expression level in both the soleus and plantaris muscles, whereas it did not affect the PGC-1α protein expression. L-NAME treatment also reduced the glucose transporter 4 protein expression in both muscles. These results suggest that NOS plays a role in maintaining SIRT1 protein expression, muscle fiber composition and components of mitochondrial biogenesis in skeletal muscle.

  6. Platelet content of nitric oxide synthase 3 phosphorylated at Serine 1177 is associated with the functional response of platelets to aspirin.

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    Javier Modrego

    Full Text Available OBJECTIVE: To analyse if platelet responsiveness to aspirin (ASA may be associated with a different ability of platelets to generate nitric oxide (NO. PATIENTS/METHODS: Platelets were obtained from 50 patients with stable coronary ischemia and were divided into ASA-sensitive (n = 26 and ASA-resistant (n = 24 using a platelet functionality test (PFA-100. RESULTS: ASA-sensitive platelets tended to release more NO (determined as nitrite + nitrate than ASA-resistant platelets but it did not reach statistical significance. Protein expression of nitric oxide synthase 3 (NOS3 was higher in ASA-sensitive than in ASA-resistant platelets but there were no differences in the platelet expression of nitric oxide synthase 2 (NOS2 isoform. The highest NOS3 expression in ASA-sensitive platelets was independent of the presence of T-to-C mutation at nucleotide position -786 (T(-786 → C in the NOS3-coding gene. However, platelet content of phosphorylated NOS3 at Serine (Ser(1177, an active form of NOS3, was higher in ASA-sensitive than in ASA-resistant platelets. The level of platelet NOS3 Ser(1177 phosphorylation was positively associated with the closure time in the PFA-100 test. In vitro, collagen failed to stimulate the aggregation of ASA-sensitive platelets, determined by lumiaggregometry, and it was associated with a significant increase (p = 0.018 of NOS3 phosphorylation at Ser(1177. On the contrary, collagen stimulated the aggregation of ASA-resistant platelets but did not significantly modify the platelet content of phosphorylated NOS3 Ser(1177. During collagen stimulation the release of NO from ASA-sensitive platelets was significantly enhanced but it was not modified in ASA-resistant platelets. CONCLUSIONS: Functional platelet responsiveness to ASA was associated with the platelet content of phosphorylated NOS3 at Ser(1177.

  7. Sesamin Modulates Tyrosine Hydroxylase, Superoxide Dismutase, Catalase, Inducible No Synthase and Interleukin-6 Expression in Dopaminergic Cells Under Mpp+-Induced Oxidative Stress

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    Vicky Lahaie-Collins

    2008-01-01

    Full Text Available Oxidative stress is regarded as a mediator of nerve cell death in several neurodegenerative disorders, such as Parkinson's disease. Sesamin, a lignan mainly found in sesame oil, is currently under study for its anti-oxidative and possible neuroprotective properties. We used 1-methyl-4-phenyl-pyridine (MPP+ ion, the active metabolite of the potent parkinsonism-causing toxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, to produce oxidative stress and neurodegeneration in neuronal PC12 cells, which express dopamine, as well as neurofilaments. Our results show that picomolar doses of sesamin protected neuronal PC12 cells from MPP+-induced cellular death, as revealed by colorimetric measurements and production of reactive oxygen species. We also demonstrated that sesamin acted by rescuing tyrosine hydroxylase levels from MPP+-induced depletion. Sesamin, however, did not modulate dopamine transporter levels, and estrogen receptor-alpha and -beta protein expression. By examining several parameters of cell distress, we found that sesamin also elicited a strong increase in superoxide dismutase activity as well as protein expression and decreased catalase activity and the MPP+ stimulated inducible nitric oxide synthase protein expression, in neuronal PC12 cells. Finally, sesamin possessed significant anti-inflammatory properties, as disclosed by its potential to reduce MPP+-induced interleukin-6 mRNA levels in microglia. From these studies, we determined the importance of the lignan sesamin as a neuroprotective molecule and its possible role in complementary and/or preventive therapies of neurodegenerative diseases.

  8. [Effect of methylene blue on changes in inducible nitric oxide synthase in lung of rats with sepsis].

    Science.gov (United States)

    Dai, Cheng; Wang, Yi; Yu, Xiangyou

    2016-02-01

    To study the time course of effect of methylene blue on inducible nitric oxide synthase (iNOS) mRNA transcription and protein expression in lung tissue of rats with sepsis, and its mechanism. 126 female Wistar rats were randomly divided into sham group, sepsis group and methylene blue group. Each group was subdivided into 0-, 6-, 12-, 18-, 24-, 30-, and 36-hour subgroups according to the time after operation, with 6 rats in each subgroup. A model of sepsis was reproduced by cecal ligation and puncture (CLP), and the rats in sham group were only opened the abdominal cavity and isolated the membrane of the appendix without CLP. Rats in methylene blue group were given injection of 15 mg/kg methylene blue at all time points after CLP, the remaining rats were given 0.9%NaCl solution in same amount. Six hours after the injection, the rats were sacrificed and the lung tissue was harvested immediately. The expression of iNOS mRNA and protein in lung tissues were determined by real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and Western Blot respectively, and the changes in histopathology were observed using hematoxylin and eosin (HE) staining. Compared with sham group, the expression of iNOS mRNA was significantly up-regulated at 6, 12, 18 and 24 hours after CLP in sepsis group (2(-ΔΔCt): 2.42±0.66 vs. 1.00±0.38 at 6 hours, P = 0.002; 2.54±0.76 vs. 1.00±0.27 at 12 hours, P = 0.000; 5.46±2.26 vs. 1.00±0.38 at 18 hours, P = 0.000; 3.03±0.62 vs. 1.00±0.33 at 24 hours, P = 0.001), and iNOS protein expression was significantly up-regulated at 12, 18 and 24 hours (gray value: 2.54±0.45 vs. 1.00±0.35 at 12 hours, P = 0.000; 2.65±0.64 vs. 1.00±0.33 at 18 hours, P = 0.000; 3.03±0.59 vs. 1.00±0.24 at 24 hours, P = 0.000). Compared with sepsis group, the expression of iNOS mRNA was significantly down-regulated at 6, 12, 18 and 24 hours in methylene blue group (2(-ΔΔCt): 1.55±0.82 vs. 2.42±0.66 at 6 hours, P = 0.034; 1.84±0.42 vs. 2

  9. Topical spinal administration of a nitric oxide synthase inhibitor prevents the hyper-reflexia associated with a rat model of persistent visceral pain.

    Science.gov (United States)

    Rice, A S

    1995-03-03

    The effects of a neuronal selective nitric oxide synthase (NOS) inhibitor, L-Ng-nitro arginine p-nitroanilide (L-Napna), upon the hyper-reflexia of a rat model of persistent visceral pain was investigated. A baseline cystometrogram (CMG) was performed by measuring intravesical pressure during vesical inflation. L-Napna (125-1000 micrograms) or vehicle (control) was then administered topically onto the exposed spinal cord, followed by another CMG. The bladder was then inflamed with turpentine and a final CMG performed. Neither L-Napna nor vehicle affected vesical reflexes in the absence of inflammation. However, following inflammation, a vesical hyper-reflexia was demonstrated in the control animals, which was prevented by L-Napna. Therefore, spinal NOS does not have a role in the generation of normal bladder reflexes, however it does modulate them during vesical inflammation.

  10. [Study of the changes of acrosomal enzyme and nitric oxide synthase, superoxide dismutase of infertile patients with positive antisperm antibody in seminal plasma].

    Science.gov (United States)

    Zhang, Hai-feng; Li, Jian-zhong; Zhang, Chun-ying

    2006-04-01

    To study the impacts of positive antisperm antibody (AsAb) in seminal plasma on acrosomal enzyme activity, nitric oxide synthase (NOS) and superoxide dismutase (SOD) levels of spermatozoa. Swatch from 40 infertile patients with positive AsAb in seminal plasma as experimental group, and 40 fertile men as control group. Acrosomal enzyme activity was detected by the BAEE/ADH unitive method, NOS was detected by the redoxreaction assay, and SOD level was measured xanthine oxidase method. Compared with control group, acrosomal enzyme activity of spermatozoa of experimental group was significantly decreased (P <0.01), NOS activity was apparently increased (P < 0.01), and SOD level in seminal plasma was markedly decreased (P<0.01). It may be possible that the positive AsAb in seminal plasma beget infertility through the changes of acrosomal enzyme of spermatozoa, SOD and NOS activities in seminal plasma.

  11. Endothelium-dependent relaxation of rat aorta to a histamine H3 agonist is reduced by inhibitors of nitric oxide synthase, guanylate cyclase and Na+,K+-ATPase

    Directory of Open Access Journals (Sweden)

    D. M. Djuric

    1996-01-01

    Full Text Available The possible involvement of different effector systems (nitric oxide synthase, guanylate cyclase, β-adrenergic and muscarinic cholinergic receptors, cyclooxygenase and lipoxygenase, and Na+,K+-ATPase was evaluated in a histamine H3 receptor agonist-induced ((Rα-methylhistamine, (Rα-MeHA endothelium-dependent rat aorta relaxation assay. (Rα-MeHA (0.1 nM – 0.01 mM relaxed endothelium-dependent rat aorta, with a pD2 value of 8.22 ± 0.06, compared with a pD2 value of 7.98 ± 0.02 caused by histamine (50% and 70% relaxation, respectively. The effect of (Rα-MeHA (0.1 nM – 0.01 mM was competitively antagonized by thioperamide (1, 10 and 30 nM (pA2 = 9.21 ± 0.40; slope = 1.03 ± 0.35 but it was unaffected by pyrilamine (100 nM, cimetidine (1 μM, atropine (10 μM, propranolol (1 μM, indomethacin (10 μM or nordthydroguaiaretic acid (0.1 mM. Inhibitors of nitric oxide synthase, L-NG-monomethylarginine (L-NMMA, 10 μM and NG-nitro-L-arginine methylester (L-NOARG, 10 μM inhibited the relaxation effect of (Rα-MeHA, by approximately 52% and 70%, respectively. This inhibitory effect of L-NMMA was partially reversed by L-arginine (10 μM. Methylene blue (10 μM and ouabain (10 μM inhibited relaxation (Rα-MeHA-induced by approximately 50% and 90%, respectively. The products of cyclooxygenase and lipoxygenase are not involved in (Rα-MeHA-induced endothelium-dependent rat aorta relaxation nor are the muscarinic cholinergic and β-adrenergic receptors. The results also suggest the involvement of NO synthase, guanylate cyclase and Na+,K+-ATPase in (Rα-MeHA-induced endothelium-dependent rat aorta relaxation.

  12. Benzalacetone Synthase

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    Ikuro eAbe

    2012-03-01

    Full Text Available Benzalacetone synthase, from the medicinal plant Rheum palmatum (Polygonaceae (RpBAS, is a plant-specific chalcone synthase (CHS superfamily of type III polyketide synthase (PKS. RpBAS catalyzes the one-step, decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce the C6-C4 benzalacetone scaffold. The X-ray crystal structures of RpBAS confirmed that the diketide-forming activity is attributable to the characteristic substitution of the conserved active-site "gatekeeper" Phe with Leu. Furthermore, the crystal structures suggested that RpBAS employs novel catalytic machinery for the thioester bond cleavage of the enzyme-bound diketide intermediate and the final decarboxylation reaction to produce benzalacetone. Finally, by exploiting the remarkable substrate tolerance and catalytic versatility of RpBAS, precursor-directed biosynthesis efficiently generated chemically and structurally divergent, unnatural novel polyketide scaffolds. These findings provided a structural basis for the functional diversity of the type III PKS enzymes.

  13. Tyrosol Attenuates High Fat Diet-Induced Hepatic Oxidative Stress: Potential Involvement of Cystathionine β-Synthase and Cystathionine γ-Lyase.

    Science.gov (United States)

    Sarna, Lindsei K; Sid, Victoria; Wang, Pengqi; Siow, Yaw L; House, James D; O, Karmin

    2016-05-01

    The Mediterranean diet is known for its cardioprotective effects. Recently, its protective qualities have also been reported in patients with non-alcoholic fatty liver disease (NAFLD). Oxidative stress is one of the important factors responsible for the development and progression of NAFLD. Hydrogen sulfide (H2S), a multifaceted gasotransmitter, has emerged as a potential therapeutic target in NAFLD. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are major enzymes responsible for endogenous H2S synthesis. Since oxidative stress contributes to NAFLD pathogenesis, the objective of this study was to investigate the effect of tyrosol, a major compound in olive oil and white wine, on high fat diet-induced hepatic oxidative stress and the mechanisms involved. Mice (C57BL/6) were fed for 5 weeks with a control diet (10 % kcal fat), a high fat diet (60 % kcal fat, HFD) or a HFD supplemented with tyrosol. High fat diet feeding induced hepatic oxidative stress, as indicated by the significant increase in lipid peroxidation and NADPH oxidase activity. Tyrosol supplementation significantly increased hepatic CBS and CSE expression and H2S synthesis in high fat diet-fed mice. Such effects were associated with the attenuation of high fat diet-induced hepatic lipid peroxidation and the restoration of the redox equilibrium of the antioxidant glutathione. Tyrosol also inhibited palmitic acid-induced oxidative stress in hepatocytes (HepG2 cells). These results suggest that the antioxidant properties of tyrosol may be mediated through functional changes in CBS and CSE activity, which might contribute to the hepatoprotective effect of the Mediterranean diet.

  14. The Arabidopsis male-sterile mutant dde2-2 is defective in the ALLENE OXIDE SYNTHASE gene encoding one of the key enzymes of the jasmonic acid biosynthesis pathway

    DEFF Research Database (Denmark)

    von Malek, Bernadette; van der Graaff, Eric; Schneitz, Kay

    2002-01-01

    The Arabidopsis thaliana (L.) Heynh. mutant delayed-dehiscence2-2 (dde2-2) was identified in an En1/Spm1 transposon-induced mutant population screened for plants showing defects in fertility. The dde2-2 mutant allele is defective in the anther dehiscence process and filament elongation and thus e...

  15. Insulin resistance in mice lacking neuronal nitric oxide synthase is related to an alpha-adrenergic mechanism

    OpenAIRE

    Turini, P.

    2008-01-01

    Rapport de synthèse : Le monoxyde d'azote (NO) joue un rôle important dans la régulation de l'homéostasie du système cardiovasculaire et du glucose. Les souris déficientes pour le gène codant l'isoforme neuronale de la synthase de monoxyde d'azote (nNOS) sont résistantes à l'insuline, mais les mécanismes sous-jacents sont inconnus. Le manque de NO produit par la nNOS pourrait être à l'origine d'une diminution de la perfusion du muscle squelettique et ainsi d'une diminution de l'apport de s...

  16. Rejuvenating Allen's Arc with the Geometric Mean.

    Science.gov (United States)

    Phillips, William A.

    1994-01-01

    Contends that, despite ongoing criticism, Allen's arc elasticity formula remains entrenched in the microeconomics principles curriculum. Reviews the evolution and continuing scrutiny of the formula. Argues that the use of the geometric mean offers pedagogical advantages over the traditional arithmetic mean approach. (CFR)

  17. The Brosimum allene: a structural revision.

    Science.gov (United States)

    Hu, Gaojie; Liu, Kai; Williams, Lawrence J

    2008-12-04

    Insight derived from a synthetic model, calculated (13)C NMR data, and comparison to experimental data indicate that the proposed allenic structure A, originally assigned to an isolate from Brosimum acutifolium Huber, should be revised to B, a natural product and nonallenic substance, mururin C.

  18. 49 ALLEN'S DEFENCE OF THE EXTENDED ORDERING ...

    African Journals Online (AJOL)

    The primary a~m of this paper is to determine the correctness of the ordering claim expressed by the EOR, i.e. the correctness of the claim that no affixes may appear outs ide compounds. Allen (1978 : 232ff.) discusses various morphologically complex words of English in which, apparently, affixes appear outside ...

  19. Increased expressions of ADAMTS-13, neuronal nitric oxide synthase, and neurofilament correlate with severity of neuropathology in Border disease virus-infected small ruminants.

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    Gungor Cagdas Dincel

    Full Text Available Border Disease (BD, caused by Pestivirus from the family Flaviviridae, leads to serious reproductive losses and brain anomalies such as hydranencephaly and cerebellar hypoplasia in aborted fetuses and neonatal lambs. In this report it is aimed to investigate the expression of neuronal nitric oxide synthase (nNOS, A Disintegrin And Metalloprotease with Thrombospondin type I repeats-13 (ADAMTS-13, and neurofilament (NF in the brain tissue in small ruminants infected with Border Disease Virus (BDV and to identify any correlation between hypomyelinogenesis and BD neuropathology. Results of the study revealed that the levels of ADAMTS-13 (p<0.05, nNOS (p<0.05, and NF (p<0.05 were remarkably higher in BDV-infected brain tissue than in the uninfected control. It was suggested that L-arginine-NO synthase pathway is activated after infection by BDV and that the expression of NF and nNOS is associated with the severity of BD. A few studies have focused on ADAMTS-13 expression in the central nervous system, and its function continues to remain unclear. The most prominent finding from our study was that ADAMTS-13, which contain two CUB domains, has two CUB domains and its high expression levels are probably associated with the development of the central nervous system (CNS. The results also clearly indicate that the interaction of ADAMTS-13 and NO may play an important role in the regulation and protection of the CNS microenvironment in neurodegenerative diseases. In addition, NF expression might indicate the progress of the disease. To the best of the authors'knowledge, this is the first report on ADAMTS-13 expression in the CNS of BDV-infected small ruminants.

  20. Noise-Induced “Toughening” Effect in Wistar Rats: Enhanced Auditory Brainstem Responses Are Related to Calretinin and Nitric Oxide Synthase Upregulation

    Science.gov (United States)

    Alvarado, Juan C.; Fuentes-Santamaría, Verónica; Gabaldón-Ull, María C.; Jareño-Flores, Tania; Miller, Josef M.; Juiz, José M.

    2016-01-01

    An appropriate conditioning noise exposure may reduce a subsequent noise-induced threshold shift. Although this “toughening” effect helps to protect the auditory system from a subsequent traumatic noise exposure, the mechanisms that regulate this protective process are not fully understood yet. Accordingly, the goal of the present study was to characterize physiological processes associated with “toughening” and to determine their relationship to metabolic changes in the cochlea and cochlear nucleus (CN). Auditory brainstem responses (ABR) were evaluated in Wistar rats before and after exposures to a sound conditioning protocol consisting of a broad-band white noise of 118 dB SPL for 1 h every 72 h, four times. After the last ABR evaluation, animals were perfused and their cochleae and brains removed and processed for the activity markers calretinin (CR) and neuronal nitric oxide synthase (nNOS). Toughening was demonstrated by a progressively faster recovery of the threshold shift, as well as wave amplitudes and latencies over time. Immunostaining revealed an increase in CR and nNOS levels in the spiral ganglion, spiral ligament, and CN in noise-conditioned rats. Overall, these results suggest that the protective mechanisms of the auditory toughening effect initiate in the cochlea and extend to the central auditory system. Such phenomenon might be in part related to an interplay between CR and nitric oxide signaling pathways, and involve an increased cytosolic calcium buffering capacity induced by the noise conditioning protocol. PMID:27065815

  1. In vitro and in vivo evidences that antioxidant action contributes to the neuroprotective effects of the neuronal nitric oxide synthase and monoamine oxidase-B inhibitor, 7-nitroindazole.

    Science.gov (United States)

    Thomas, Bobby; Saravanan, Karuppagounder S; Mohanakumar, Kochupurackal P

    2008-05-01

    The neuronal nitric oxide synthase (nNOS) inhibitor, 7-nitroindazole (7-NI) is neuroprotective against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism. Monoamine oxidase (MAO)-B inhibitory action partially contributes to this effect. We tested the hypothesis that 7-NI could be a powerful hydroxyl radical (OH) scavenger, and interferes with oxidative stress caused by MPTP. We measured OH, reduced glutathione (GSH), as well as superoxide dismutase (SOD) and catalase activities in the nucleus caudatus putamen and substantia nigra of Balb/c mice following MPTP and/or 7-NI administration. The nNOS inhibitor caused dose-dependent inhibition in the production of OH in (i) Fenton-like reaction employing ferrous citrate in a cell-free system in test tubes, (ii) in isolated mitochondrial preparation in presence of MPP+, and (iii) in the striatum of mice systemically treated with MPTP. An MPTP-induced depletion of GSH in both the nuclei was blocked by 7-NI, which was dose-dependent (10-50mg/kg), but independent of MAO-B inhibition. The nNOS-mediated recovery of GSH paralleled attenuation of MPTP-induced depletion of striatal dopamine. MPTP-induced increase in the activities of striatal or nigral SOD and catalase were significantly attenuated by 7-NI treatment. These results suggest potent antioxidant action of 7-NI in its neuroprotective effects against MPTP-induced neurotoxicity.

  2. Nitric Oxide Synthase Gene Transfer Overcomes the Inhibition of Wound Healing by Sulfur Mustard in a Human Keratinocyte In Vitro Model

    Science.gov (United States)

    Ishida, Hiroshi; Ray, Radharaman; Amnuaysirikul, Jack; Ishida, Keiko; Ray, Prabhati

    2012-01-01

    Sulfur mustard (SM) is a chemical warfare agent that causes extensive skin injury. Previously we reported that SM exposure resulted in suppression of inducible nitric oxide synthase (iNOS) expression to inhibit the healing of scratch wounds in a cultured normal human epidermal keratinocyte (NHEK) model. Based on this finding, the present study was to use adenovirus-mediated gene transfer of iNOS to restore the nitric oxide (NO) supply depleted by exposure to SM and to evaluate the effect of NO on wound healing inhibited by SM in NHEKs. The effect of the iNOS gene transfer on iNOS protein expression and NO generation were monitored by Western blot and flow cytometry, respectively. Wound healing with or without the iNOS gene transfer after SM exposure was assessed by light and confocal microscopy. The iNOS gene transfer via adenovirus resulted in overexpression of the iNOS and an increase in NO production regardless of SM exposure in the NHEK model. The gene transfer was also effective in overcoming the inhibition of wound healing due to SM exposure leading to the promotion of wound closure. The findings in this study suggest that the iNOS gene transfer is a promising therapeutic strategy for SM-induced skin injury. PMID:23762631

  3. Effects of 7-nitroindazole, a selective neural nitric oxide synthase inhibitor, on context-shock associative learning in a two-process contextual fear conditioning paradigm.

    Science.gov (United States)

    Chen, Weihai; Yan, Minmin; Wang, Yan; Wang, Xiaqing; Yuan, Jiajin; Li, Ming

    2016-10-01

    Nitric oxide (NO) is an important retrograde neuronal intracellular messenger which plays an important role in synaptic plasticity and is involved in learning and memory. However, evidence that NO is particularly important for the acquisition of contextual fear conditioning is mixed. Also, little is known about at which stages of the contextual fear conditioning does NO make its contribution. In the present study, we used 7-nitroindazole to temporarily inhibit neural nitric oxide synthase at either the pre-exposure stage or conditioning stage in a two-process paradigm and examined the potential contribution that NO makes to the contextually conditioned fear. Results showed that the expression of contextual fear memory was significantly impaired in rats treated with 7-nitroindazole (30mg/kg, i.p.) prior to the pairing of context-shock (p=0.034, n=8), but not after the conditioning phase (p=0.846, n=8). In addition, the expression of contextual fear memory and reconsolidation was not significantly impaired by 7-nitroindazole administered prior to the context pre-exposure stage or prior to another context-shock learning. These findings suggest that NO is specifically involved in the acquisition but not the consolidation, retrieval or reconsolidation of contextual fear memory. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Transcriptome sequencing of three Pseudo-nitzschia species reveals comparable gene sets and the presence of Nitric Oxide Synthase genes in diatoms.

    Science.gov (United States)

    Di Dato, Valeria; Musacchia, Francesco; Petrosino, Giuseppe; Patil, Shrikant; Montresor, Marina; Sanges, Remo; Ferrante, Maria Immacolata

    2015-07-20

    Diatoms are among the most diverse eukaryotic microorganisms on Earth, they are responsible for a large fraction of primary production in the oceans and can be found in different habitats. Pseudo-nitzschia are marine planktonic diatoms responsible for blooms in coastal and oceanic waters. We analyzed the transcriptome of three species, Pseudo-nitzschia arenysensis, Pseudo-nitzschia delicatissima and Pseudo-nitzschia multistriata, with different levels of genetic relatedness. These species have a worldwide distribution and the last one produces the neurotoxin domoic acid. We were able to annotate about 80% of the sequences in each transcriptome and the analysis of the relative functional annotations allowed comparison of the main metabolic pathways, pathways involved in the biosynthesis of isoprenoids (MAV and MEP pathways), and pathways putatively involved in domoic acid synthesis. The search for homologous transcripts among the target species and other congeneric species resulted in the discovery of a sequence annotated as Nitric Oxide Synthase (NOS), found uniquely in Pseudo-nitzschia multistriata. The predicted protein product contained all the domains of the canonical metazoan sequence. Putative NOS sequences were found in other available diatom datasets, supporting a role for nitric oxide as signaling molecule in this group of microalgae.

  5. Nitric Oxide Synthase in the Central Nervous System and Peripheral Organs of Stramonita haemastoma: Protein Distribution and Gene Expression in Response to Thermal Stress

    Science.gov (United States)

    Toni, Mattia; De Angelis, Federica; Bonaccorsi di Patti, Maria Carmela; Cioni, Carla

    2015-01-01

    Nitric oxide (NO) is generated via the oxidation of l-arginine by the enzyme NO synthase (NOS) both in vertebrates and invertebrates. Three NOS isoforms, nNOS, iNOS and eNOS, are known in vertebrates, whereas a single NOS isoform is usually expressed in invertebrates, sharing structural and functional characteristics with nNOS or iNOS depending on the species. The present paper is focused on the constitutive Ca2+/calmodulin-dependent nNOS recently sequenced by our group in the neogastropod Stramonita haemastoma (ShNOS). In this paper we provide new data on cellular distribution of ShNOS in the CNS (pedal ganglion) and peripheral organs (osphradium, tentacle, eye and foot) obtained by WB, IF, CM and NADPHd. Results demonstrated that NOS-like proteins are widely expressed in sensory receptor elements, neurons and epithelial cells. The detailed study of NOS distribution in peripheral and central neurons suggested that NOS is both intracellular and presynaptically located. Present findings confirm that NO may have a key role in the central neuronal circuits of gastropods and in sensory perception. The physiological relevance of NOS enzymes in the same organs was suggested by thermal stress experiments demonstrating that the constitutive expression of ShNOS is modulated in a time- and organ-dependent manner in response to environmental stressors. PMID:26528988

  6. Nitric Oxide Synthase in the Central Nervous System and Peripheral Organs of Stramonita haemastoma: Protein Distribution and Gene Expression in Response to Thermal Stress

    Directory of Open Access Journals (Sweden)

    Mattia Toni

    2015-10-01

    Full Text Available Nitric oxide (NO is generated via the oxidation of l-arginine by the enzyme NO synthase (NOS both in vertebrates and invertebrates. Three NOS isoforms, nNOS, iNOS and eNOS, are known in vertebrates, whereas a single NOS isoform is usually expressed in invertebrates, sharing structural and functional characteristics with nNOS or iNOS depending on the species. The present paper is focused on the constitutive Ca2+/calmodulin-dependent nNOS recently sequenced by our group in the neogastropod Stramonita haemastoma (ShNOS. In this paper we provide new data on cellular distribution of ShNOS in the CNS (pedal ganglion and peripheral organs (osphradium, tentacle, eye and foot obtained by WB, IF, CM and NADPHd. Results demonstrated that NOS-like proteins are widely expressed in sensory receptor elements, neurons and epithelial cells. The detailed study of NOS distribution in peripheral and central neurons suggested that NOS is both intracellular and presynaptically located. Present findings confirm that NO may have a key role in the central neuronal circuits of gastropods and in sensory perception. The physiological relevance of NOS enzymes in the same organs was suggested by thermal stress experiments demonstrating that the constitutive expression of ShNOS is modulated in a time- and organ-dependent manner in response to environmental stressors.

  7. Impact of Environmental Thermal Stimulation on Activation of Hypothalamic Neuronal Nitric Oxide Synthase during the Prenatal Ontogenesis in Muscovy Ducks

    Directory of Open Access Journals (Sweden)

    Valery Dunai

    2012-01-01

    Full Text Available The aim of the study is to investigate the influence of prenatal temperature stimulation on neuronal NO synthase (nNOS expression in the anterior hypothalamus of Muscovy duck embryos. Experiments were performed on embryonic day (E E20, E23, E28, and E33 using histochemistry for identification of the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d as marker of NOS-containing neurons. Until the experiments, all duck embryos were incubated under standard temperature conditions (37.5∘C. During 3 hours before the start of the experiments, one group was incubated at 37.5∘C (control group, the second was warm-experienced at 39∘C, and the third was cold-experienced at 34∘C. In normal and warm-incubated duck embryos, nNOS activity could be first detected on E23. Particularly, after cold stimulation, a significant increase in nNOS activity was found in all embryos investigated even on day 20. Warm stimulation obviously induces the opposite effect, but at later embryonic age (E33. It can be concluded that probably in late-term bird embryos NO acts as a mediator of the neuronal cold pathway in the anterior hypothalamus, which might be improved by prenatal cold stimulation.

  8. Impact of environmental thermal stimulation on activation of hypothalamic neuronal nitric oxide synthase during the prenatal ontogenesis in Muscovy ducks.

    Science.gov (United States)

    Dunai, Valery; Tzschentke, Barbara

    2012-01-01

    The aim of the study is to investigate the influence of prenatal temperature stimulation on neuronal NO synthase (nNOS) expression in the anterior hypothalamus of Muscovy duck embryos. Experiments were performed on embryonic day (E) E20, E23, E28, and E33 using histochemistry for identification of the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) as marker of NOS-containing neurons. Until the experiments, all duck embryos were incubated under standard temperature conditions (37.5°C). During 3 hours before the start of the experiments, one group was incubated at 37.5°C (control group), the second was warm-experienced at 39°C, and the third was cold-experienced at 34°C. In normal and warm-incubated duck embryos, nNOS activity could be first detected on E23. Particularly, after cold stimulation, a significant increase in nNOS activity was found in all embryos investigated even on day 20. Warm stimulation obviously induces the opposite effect, but at later embryonic age (E33). It can be concluded that probably in late-term bird embryos NO acts as a mediator of the neuronal cold pathway in the anterior hypothalamus, which might be improved by prenatal cold stimulation.

  9. Circulating Microparticles Carry a Functional Endothelial Nitric Oxide Synthase That Is Decreased in Patients With Endothelial Dysfunction

    Science.gov (United States)

    Horn, Patrick; Cortese‐Krott, Miriam Margherita; Amabile, Nicolas; Hundsdörfer, Claas; Kröncke, Klaus‐Dietrich; Kelm, Malte; Heiss, Christian

    2013-01-01

    Background Microparticles (MPs) are circulating membrane particles of less than a micrometer in diameter shed from endothelial and blood cells. Recent literature suggests that MPs are not just functionally inert cell debris but may possess biological functions and mediate the communication between vascular cells. As a significant proportion of MPs originate from platelets and endothelial cells, we hypothesized that MPs may harbor functional enzymes including an endothelial NO synthase (eNOS). Methods and Results Using immunoprecipitation and Western blot analysis, we found that human circulating MPs carry an eNOS. Ca2+ and l‐arginine‐dependent NOS activity of crude enzyme extract from MPs was determined by measuring the conversion of [3H]‐L‐arginine to [3H]‐citrulline and NOS‐dependent nitrite production. NOS‐dependent NO production in intact MPs was assessed by the NO‐specific fluorescent probe MNIP‐Cu. In patients with cardiovascular disease, endothelial dysfunction was associated with an increase in the total number of circulating MPs as well as a significant decrease in the expression and activity of eNOS in MPs. No difference in reactive oxygen species was noted in MPs isolated from either group. Conclusions Our data further support the concept that circulating MPs may not only retain phenotypic markers but also preserve the functionality of enzymes of the cells they originate from, including eNOS. PMID:23525410

  10. Co-ordinate variations in methylmalonyl-CoA mutase and methionine synthase, and the cobalamin cofactors in human glioma cells during nitrous oxide exposure and the subsequent recovery phase.

    Science.gov (United States)

    Riedel, B; Fiskerstrand, T; Refsum, H; Ueland, P M

    1999-07-01

    We investigated the co-ordinate variations of the two cobalamin (Cbl)-dependent enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase (MCM), and measured the levels of their respective cofactors, methylcobalamin (CH3Cbl) and adenosylcobalamin (AdoCbl) in cultured human glioma cells during nitrous oxide exposure and during a subsequent recovery period of culture in a nitrous oxide-free atmosphere (air). In agreement with published data, MS as the primary target of nitrous oxide was inactivated rapidly (initial rate of 0.06 h(-1)), followed by reduction of CH3Cbl (to ordinate distribution of Cbl cofactors during depletion and repletion.

  11. The radiosensitizing effect of immunoadjuvant OM-174 requires cooperation between immune and tumor cells through interferon-gamma and inducible nitric oxide synthase

    International Nuclear Information System (INIS)

    Ridder, Mark de; Verovski, Valeri N.; Chiavaroli, Carlo; Berge, Dirk L. van den; Monsaert, Christinne; Law, Kalun; Storme, Guy A.

    2006-01-01

    Purpose: To explore whether antitumor immunoadjuvant OM-174 can stimulate immune cells to produce interferon-γ (IFN-γ) and thereby radiosensitize tumor cells. Methods and Materials: Splenocytes from BALB/c mice were stimulated by OM-174 at plasma-achievable concentrations (0.03-3 μg/mL), and afterward analyzed for the expression and secretion of IFN-γ by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Stimulated splenocytes were used as a source of IFN-γ to radiosensitize hypoxic EMT-6 tumor cells through the cytokine-inducible isoform of nitric oxide synthase (iNOS). Results: OM-174 activated the production of IFN-γ at high levels that reached 70 ng/mL in normoxia (21% oxygen) and 27 ng/mL in tumor-relevant hypoxia (1% oxygen). This caused up to 2.1-fold radiosensitization of EMT-6 tumor cells, which was associated with the iNOS-mediated production of the radiosensitizing molecule nitric oxide, as confirmed by accumulation of its oxidative metabolite nitrite, Western blot analysis, and reverse transcriptase-polymerase chain reaction. Both iNOS activation and radiosensitization were counteracted by neutralizing antibodies against IFN-γ. The same mechanism of radiosensitization through the IFN-γ secretion pathway was identified for IL-12 + IL-18, which are known to mediate IFN-γ responses. Hypoxia displayed a dual effect on the immune-tumor cell interaction, by downregulating the expression of the IFN-γ gene while upregulating iNOS at transcriptional level. Conclusion: Immunoadjuvant OM-174 is an efficient radiosensitizer of tumor cells through activation of the IFN-γ secretion pathway in immune cells. This finding indicates a rationale for combining immunostimulatory and radiosensitizing strategies and extends the potential therapeutic applications of OM-174

  12. The ferrous-dioxy complex of neuronal nitric oxide synthase. Divergent effects of L-arginine and tetrahydrobiopterin on its stability.

    Science.gov (United States)

    Abu-Soud, H M; Gachhui, R; Raushel, F M; Stuehr, D J

    1997-07-11

    Nitric oxide synthases (NOS) are hemeproteins that catalyze oxidation of L-arginine to nitric oxide (NO) and citrulline. The NOS heme iron is expected to participate in oxygen activation during catalysis, but its interactions with O2 are not characterized. We utilized the heme-containing oxygenase domain of neuronal NOS (nNOSoxy) and stopped-flow methods to study formation and autooxidative decomposition of the nNOSoxy oxygenated complex at 10 degrees C. Mixing ferrous nNOSoxy with air-saturated buffer generated a transient species with absorption maxima at 427 and approximately 560 nm. This species decayed within 1 s to form ferric nNOSoxy. Its formation was first order with respect to O2, monophasic, and gave rate constants for kon = 9 x 10(5) M-1 s-1 and koff = 108 s-1 for an L-arginine- and tetrahydrobiopterin (H4B)-saturated nNOSoxy. Omission of L-arginine and/or H4B did not greatly effect O2 binding and dissociation rates. Decomposition of the oxygenated intermediate was independent of O2 concentration and was either biphasic or monophasic depending on sample conditions. L-Arginine stabilized the oxygenated intermediate (decay rate = 0.14 s-1), while H4B accelerated its decay by a factor of 70 irrespective of L-arginine. The spectral and kinetic properties of the intermediate identify it as the FeIIO2 complex of nNOSoxy. Destabilization of a metallo-oxy species by H4B is unprecedented and may be important regarding the role of this cofactor in NO synthesis.

  13. Deciphering the interplay between cysteine synthase and thiol cascade proteins in modulating Amphotericin B resistance and survival of Leishmania donovani under oxidative stress

    Directory of Open Access Journals (Sweden)

    Kuljit Singh

    2017-08-01

    Full Text Available Leishmania donovani is the causative organism of the neglected human disease known as visceral leishmaniasis which is often fatal, if left untreated. The cysteine biosynthesis pathway of Leishmania may serve as a potential drug target because it is different from human host and regulates downstream components of redox metabolism of the parasites; essential for their survival, pathogenicity and drug resistance. However, despite the apparent dependency of redox metabolism of cysteine biosynthesis pathway, the role of L. donovani cysteine synthase (LdCS in drug resistance and redox homeostasis has been unexplored. Herein, we report that over-expression of LdCS in Amphotericin B (Amp B sensitive strain (S1-OE modulates resistance towards oxidative stress and drug pressure. We observed that antioxidant enzyme activities were up-regulated in S1-OE parasites and these parasites alleviate intracellular reactive oxygen species (ROS efficiently by maintaining the reduced thiol pool. In contrast to S1-OE parasites, Amp B sensitive strain (S1 showed higher levels of ROS which was positively correlated with the protein carbonylation levels and negatively correlated with cell viability. Moreover, further investigations showed that LdCS over-expression also augments the ROS-primed induction of LdCS-GFP as well as endogenous LdCS and thiol pathway proteins (LdTryS, LdTryR and LdcTXN in L. donovani parasites; which probably aids in stress tolerance and drug resistance. In addition, the expression of LdCS was found to be up-regulated in Amp B resistant isolates and during infective stationary stages of growth and consistent with these observations, our ex vivo infectivity studies confirmed that LdCS over-expression enhances the infectivity of L. donovani parasites. Our results reveal a novel crosstalk between LdCS and thiol metabolic pathway proteins and demonstrate the crucial role of LdCS in drug resistance and redox homeostasis of Leishmania. Keywords

  14. Induction by endotoxin of nitric oxide synthase in the rat mesentery: lack of effect on action of vasoconstrictors.

    Science.gov (United States)

    Mitchell, J. A.; Kohlhaas, K. L.; Sorrentino, R.; Warner, T. D.; Murad, F.; Vane, J. R.

    1993-01-01

    1. Male Sprague-Dawley or Wistar rats were injected with bacterial lipopolysaccharide (LPS; 5 mg kg-1, i.p.) and killed after 1, 3, 6, 15, and 24 h. The brains, mesenteries, spleens, lungs, livers, kidneys, hearts, aortae and diaphragms were removed and frozen immediately. Control rats were injected with sterile saline and killed after 6 h. 2. The organs were homogenized in a semi-frozen state and NO synthase (NOS) activity measured in tissues from both LPS-treated and saline-treated groups by the ability of homogenates to convert [3H]-L-arginine to [3H]-L-citrulline in a NADPH-dependent manner. 3. The NOS activity in all organs taken from control animals was found to be calcium-dependent, with the highest activity being in the brain. After LPS-treatment an induced calcium-independent NOS was detected in all tissues tested, with the exception of the brain. The spleen, lung, mesentery and liver had the highest amounts of LPS-induced NOS activity. No induction of calcium-dependent NOS was detected. 4. Induction of NOS was maximum 6 h after administration of LPS and had returned to control levels in 24 h. 5. The constitutive NOS in brain and mesentery and the LPS-induced activities in the spleen, lung, liver and mesentery were inhibited by NG-monomethyl-L-arginine (L-NMMA) or NG-nitro-L-arginine methyl ester (L-NAME) according to concentration. The IC50 for L-NAME was 2.5 microM against the constitutive NOS from brain, and 20-25 microM against the inducible NOS. For L-NMMA the IC50 was 20-25 microM against either NOS isoform.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7684306

  15. Intensification of Doxorubicin-Related Oxidative Stress in the Heart by Hypothyroidism Is Not Related to the Expression of Cytochrome P450 NADPH-Reductase and Inducible Nitric Oxide Synthase, As Well As Activity of Xanthine Oxidase

    Directory of Open Access Journals (Sweden)

    Jaroslaw Dudka

    2012-01-01

    Full Text Available Cytochrome P450 NADPH-reductase (P450R, inducible synthase (iNOS and xanthine oxidase play an important role in the antracycline-related cardiotoxicity. The expression of P450R and iNOS is regulated by triiodothyronine. The aim of this study was to evaluate the effect of methimazole-induced hypothyreosis on oxidative stress secondary to doxorubicin administration. 48 hours after methimazole giving cessation, rats were exposed to doxorubicin (2.0, 5.0 and 15 mg/kg. Blood and heart were collected 4, 48 and 96 h after the drug administration. Animals exposed exclusively to doxorubicin or untreated ones were also assessed. The hypothyreosis (0.025% of methimazole significantly increased the doxorubicin effect on the cardiac carbonyl group and they may increase the glutathione level. An insignificant effect of methimazole was noticed in case of the cardiac lipid peroxidation product, the amount of DNA oxidative damages, iNOS and xanthine oxidase-enzymes responsible for red-ox activation of doxorubicin. However, the concentration of P450R was affected by a lower dose of methimazole in rats administered with doxorubicin. Since in rats receiving doxorubicin changes in oxidative stress caused by methimazole were not accompanied by elevation of bioreductive enzymes, it may be concluded that these changes in the oxidative stress were not related to the tested enzymes.

  16. Role of reactive nitrogen species generated via inducible nitric oxide synthase in vesicant-induced lung injury, inflammation and altered lung functioning

    Energy Technology Data Exchange (ETDEWEB)

    Sunil, Vasanthi R., E-mail: sunilvr@eohsi.rutgers.edu [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy Piscataway, NJ (United States); Shen, Jianliang; Patel-Vayas, Kinal; Gow, Andrew J. [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy Piscataway, NJ (United States); Laskin, Jeffrey D. [Department of Environmental and Occupational Medicine, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ (United States); Laskin, Debra L. [Department of Pharmacology and Toxicology, Rutgers University, Ernest Mario School of Pharmacy Piscataway, NJ (United States)

    2012-05-15

    Pulmonary toxicity induced by sulfur mustard and related vesicants is associated with oxidative stress. In the present studies we analyzed the role of reactive nitrogen species (RNS) generated via inducible nitric oxide synthase (iNOS) in lung injury and inflammation induced by vesicants using 2-chloroethyl ethyl sulfide (CEES) as a model. C57Bl/6 (WT) and iNOS −/− mice were sacrificed 3 days or 14 days following intratracheal administration of CEES (6 mg/kg) or control. CEES intoxication resulted in transient (3 days) increases in bronchoalveolar lavage (BAL) cell and protein content in WT, but not iNOS −/− mice. This correlated with expression of Ym1, a marker of oxidative stress in alveolar macrophages and epithelial cells. In contrast, in iNOS −/− mice, Ym1 was only observed 14 days post-exposure in enlarged alveolar macrophages, suggesting that they are alternatively activated. This is supported by findings that lung tumor necrosis factor and lipocalin Lcn2 expression, mediators involved in tissue repair were also upregulated at this time in iNOS −/− mice. Conversely, CEES-induced increases in the proinflammatory genes, monocyte chemotactic protein-1 and cyclooxygenase-2, were abrogated in iNOS −/− mice. In WT mice, CEES treatment also resulted in increases in total lung resistance and decreases in compliance in response to methacholine, effects blunted by loss of iNOS. These data demonstrate that RNS, generated via iNOS play a role in the pathogenic responses to CEES, augmenting oxidative stress and inflammation and suppressing tissue repair. Elucidating inflammatory mechanisms mediating vesicant-induced lung injury is key to the development of therapeutics to treat mustard poisoning. -- Highlights: ► Lung injury, inflammation and oxidative stress are induced by the model vesicant CEES ► RNS generated via iNOS are important in the CEES-induced pulmonary toxicity ► iNOS −/− mice are protected from CEES-induced lung toxicity and

  17. Role of reactive nitrogen species generated via inducible nitric oxide synthase in vesicant-induced lung injury, inflammation and altered lung functioning

    International Nuclear Information System (INIS)

    Sunil, Vasanthi R.; Shen, Jianliang; Patel-Vayas, Kinal; Gow, Andrew J.; Laskin, Jeffrey D.; Laskin, Debra L.

    2012-01-01

    Pulmonary toxicity induced by sulfur mustard and related vesicants is associated with oxidative stress. In the present studies we analyzed the role of reactive nitrogen species (RNS) generated via inducible nitric oxide synthase (iNOS) in lung injury and inflammation induced by vesicants using 2-chloroethyl ethyl sulfide (CEES) as a model. C57Bl/6 (WT) and iNOS −/− mice were sacrificed 3 days or 14 days following intratracheal administration of CEES (6 mg/kg) or control. CEES intoxication resulted in transient (3 days) increases in bronchoalveolar lavage (BAL) cell and protein content in WT, but not iNOS −/− mice. This correlated with expression of Ym1, a marker of oxidative stress in alveolar macrophages and epithelial cells. In contrast, in iNOS −/− mice, Ym1 was only observed 14 days post-exposure in enlarged alveolar macrophages, suggesting that they are alternatively activated. This is supported by findings that lung tumor necrosis factor and lipocalin Lcn2 expression, mediators involved in tissue repair were also upregulated at this time in iNOS −/− mice. Conversely, CEES-induced increases in the proinflammatory genes, monocyte chemotactic protein-1 and cyclooxygenase-2, were abrogated in iNOS −/− mice. In WT mice, CEES treatment also resulted in increases in total lung resistance and decreases in compliance in response to methacholine, effects blunted by loss of iNOS. These data demonstrate that RNS, generated via iNOS play a role in the pathogenic responses to CEES, augmenting oxidative stress and inflammation and suppressing tissue repair. Elucidating inflammatory mechanisms mediating vesicant-induced lung injury is key to the development of therapeutics to treat mustard poisoning. -- Highlights: ► Lung injury, inflammation and oxidative stress are induced by the model vesicant CEES ► RNS generated via iNOS are important in the CEES-induced pulmonary toxicity ► iNOS −/− mice are protected from CEES-induced lung toxicity and

  18. Immunohistochemical localization of gastrin-releasing peptide, neuronal nitric oxide synthase and neurone-specific enolase in the uterus of the North American opossum, Didelphis virginiana.

    Science.gov (United States)

    Kumano, A; Sasaki, M; Budipitojo, T; Kitamura, N; Krause, W J; Yamada, J

    2005-08-01

    The present study has demonstrated the immunohistochemical localization of gastrin-releasing peptide (GRP), neuronal nitric oxide synthase (nNOS) and neurone-specific enolase (NSE) in the uterus of the North American opossum. Although the presence of GRP, nNOS and NSE has been reported recently in the uterus of eutherian species this is the first description of these peptides in a metatherian species. Metatherian mammals are of interest because in these species it is the prolonged lactation phase of development that is the period of primary reproductive investment rather than intrauterine development as is true of eutherian mammals. The opossum, like other marsupial species, has a very abbreviated gestation period which in Didelphis lasts only 12.5 days. GRP was localized in the cytoplasm of cells forming the surface lining epithelium and the glandular epithelium of the opossum endometrium late in pregnancy, at 11.5 days of gestation. Likewise, immunoreactivities of nNOS and NSE were found primarily within the epithelial cells of the endometrium at 11.5 days of gestation. As these peptides and enzymes appear primarily at the time of establishment of the yolk sac placenta (between day 10 and day 12.5 gestation), the present results strongly suggest that these factors may play a fundamental role in the placentation of the opossum.

  19. Association of endothelial nitric oxide synthase gene polymorphism with the risk of Henoch-Schönlein purpura/Henoch-Schönlein purpura nephritis.

    Science.gov (United States)

    Zhong, Weiqiang; Zhou, Tian-Biao; Jiang, Zongpei

    2015-04-01

    Association between endothelial nitric oxide synthase (eNOS) gene polymorphism and Henoch-Schönlein purpura (HSP)/Henoch-Schönlein purpura nephritis (HSPN) risk is still controversial. A meta-analysis was performed to evaluate the association between eNOS gene polymorphism and HSP/HSPN susceptibility. A predefined literature search and selection of eligible relevant studies were performed to collect data from electronic database. Three articles were identified for the analysis of association between eNOS gene polymorphism and HSPN/HSP risk. eNOS G894T gene polymorphism was not associated with HSPN susceptibility and the risk of patients with HSP developing into HSPN. Interestingly, eNOS G894T T allele and GG genotype were associated with HSP susceptibility, but not the TT genotype. eNOS T786C TT genotype was associated with HSPN susceptibility, but not C allele and CC genotype. Furthermore, eNOS T786C gene polymorphism was not associated with HSP risk and the risk of patients with HSP developing into HSPN. In conclusion, eNOS T786C TT genotype was associated with and eNOS G894T T allele and GG genotype were associated with HSP susceptibility. However, more studies should be performed in the future.

  20. The expression of endothelial and inducible nitric oxide synthase and apoptosis in intestinal ischemia and reperfusion injury under the action of ischemic preconditioning and pentoxifylline.

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    Oliveira, Teresinha Regina Ribeiro de; Oliveira, Geraldo Ferreira de; Simões, Ricardo Santos; Feitosa, Suellen Maurim; Tikazawa, Eduardo Hiroshi; Monteiro, Hugo Pequeno; Fagundes, Djalma José; Taha, Murched Omar

    2017-11-01

    To investigate the expression of nitric oxide synthase (NOS) and apoptosis associated with ischemic preconditioning (IPC) and pentoxifylline (PTX) in intestinal ischemia (I) and reperfusion (R) injury. Thirty male rats were assigned to 5 groups: (CG), no clamping of the superior mesenteric artery (90 minutes); (IR-SS) saline + ischemia (30 minutes) + reperfusion (60 minutes); (IR-PTX) PTX + ischemia (30 minutes) + reperfusion (60 minutes); (IPC-IR-SS) 5 minutes of ischemia + 5 minutes of reperfusion (IPC) + saline + I(30 minutes)+R(60 minutes); and (IPC-IR-PTX) IPC + PTX + I(30 minutes)+ R(60 minutes). The application of IPC and PTX showed a significantly lower immunohistochemistry reaction for active caspase-3 (P0.05). The NOS-2 expression (qRTPCR) in the IR-PTX group (P<0.05) was higher than the values for the IPC+IR-SS and IPC-IR-PTX groups. The NOS-3 expression was significantly upper in the IPC-IR-PTX group than in the CG (P<0.05), the IR-SS (P<0.05) and the IR-PTX (P<0.05) groups. The BCL-2 and active caspase-3 showed beneficial effects on PTX and IPC. The expression of NOS-2 and NOS-3 in the IPC and IPC-PTX groups showed no synergistic effect.

  1. The Coexpression of Reelin and Neuronal Nitric Oxide Synthase in a Subpopulation of Dentate Gyrus Neurons Is Downregulated in Heterozygous Reeler Mice

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    Raquel Romay-Tallón

    2010-01-01

    Full Text Available Reelin is an extracellular matrix protein expressed in several interneuron subtypes in the hippocampus and dentate gyrus. Neuronal nitric oxide synthase (nNOS is also expressed by interneurons in these areas. We investigated whether reelin and nNOS are co-localized in the same population of hippocampal interneurons, and whether this colocalization is altered in the heterozygous reeler mouse. We found colocalization of nNOS in reelin-positive cells in the CA1 stratum radiatum and lacunosum moleculare, the CA3 stratum radiatum, and the dentate gyrus subgranular zone, molecular layer, and hilus. In heterozygous reeler mice, the colocalization of nNOS in reelin-positive cells was significantly decreased only in the subgranular zone and molecular layer. The coexpression of reelin and nNOS in several hippocampal regions suggests that reelin and nNOS may work synergistically to promote glutamatergic function, and the loss of this coexpression in heterozygous reeler mice may underlie some of the behavioral deficits observed in these animals.

  2. Sodium salicylate-induced amelioration of experimental autoimmune encephalomyelitis in Lewis rats is associated with the suppression of inducible nitric oxide synthase and cyclooxygenases.

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    Moon, Changjong; Ahn, Meejung; Jee, Youngheun; Heo, Seungdam; Kim, Seungjoon; Kim, Hyungmin; Sim, Ki-Bum; Koh, Chang-Sung; Shin, Young-Gyun; Shin, Taekyun

    2004-02-12

    We studied the effects of oral administration of sodium salicylate on the expression of the pro-inflammatory mediators, nitric oxide synthase (iNOS) and cyclooxygenase- (COX-) 1 and 2, in rats with experimental autoimmune encephalomyelitis (EAE). Sodium salicylate (200 mg/kg) was administered orally for 13 days after the induction of EAE by immunization with guinea pig myelin basic protein and complete Freund's adjuvant. The onset (P<0.0001) and severity (P<0.05) of EAE paralysis in salicylate-treated animals were delayed and suppressed significantly compared with vehicle-treated controls. Western blot analysis showed that expression of COX-2 and iNOS, but not COX-1, decreased significantly in the spinal cords of salicylate-treated rats compared with vehicle-treated controls (P<0.05) and this finding was paralleled by immunohistochemical observations. These results suggest that the amelioration by salicylate of paralysis in rats with EAE is mediated in part by the suppression of COX and iNOS.

  3. A quantitative structure-activity relationship (QSAR) study on a few series of potent, highly selective inhibitors of nitric oxide synthase.

    Science.gov (United States)

    Bharti, Vishwa Deepak; Gupta, Satya P; Kumar, Harish

    2014-02-01

    QSAR study was performed on a series of 1,2-dihydro-4-quinazolinamines, 4,5-dialkylsubstituted-2-imino-1,3-thiazolidine derivatives and 4,5-disubstituted-1,3-oxazolidin-2-imine derivatives studied by Tinker et al. [J Med Chem (2003), 46, 913-916], Ueda et al. [Bioorg Med Chem (2004) 12, 4101-4116] and Ueda et al. [Bioorg Med Chem Lett (2004) 14, 313-316], respectively, as potent, highly selective inhibitors of inducible nitric oxide synthase (iNOS). The iNOS inhibition activity of the whole series of compounds was analyzed in relation to the physicochemical and molecular properties of the compounds. The QSAR analysis revealed that the inhibition potency of the compounds was controlled by a topological parameter 1chi(v) (Kier's first order valence molecular connectivity index), density (D), surface tension (St) and length (steric parameter) of a substituent. This suggested that the drug-receptor interaction predominantly involved the dispersion interaction, but the bulky molecule would face steric problem because of which the molecule may not completely fit in active sites of the receptor and thus may not have the optimum interaction.

  4. Reduction in libido and fertility of male rats by administration of the nitric oxide (NO) synthase inhibitor N-nitro-L-arginine methyl ester.

    Science.gov (United States)

    Ratnasooriya, W D; Dharmasiri, M G; Wadsworth, R M

    2000-06-01

    The role of nitric oxide (NO) in libido and fertility of male rats was investigated by administration of the NO synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) (25 or 50 mg/kg/day). L-NAME caused marked reduction of precoital sexual behaviour, and a failure of most rats to mount or ejaculate during the test interval. In most matings that were successful, recovered spermatozoa were present in normal numbers. In mating experiments, L-NAME profoundly reduced the fertility of male rats. In those animals that did succeed in mating, the quantal pregnancy and the number of implants were reduced. After cessation of treatment with L-NAME, the fertility parameters returned close to normal. The inactive stereoisomer, D-NAME, caused none of the above effects when administered to rats. The results suggest that NO is essential for the expression of normal libido and fertility in male rats. It is likely that NO is required both in the male reproductive tract and in the brain.

  5. Effects of aminoguanidine, a potent nitric oxide synthase inhibitor, on myocardial and organ structure in a rat model of hemorrhagic shock

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    Mona M Soliman

    2014-01-01

    Full Text Available Background: Nitric oxide (NO has been shown to increase following hemorrhagic shock (HS. Peroxynitrite is produced by the reaction of NO with reactive oxygen species, leads to nitrosative stress mediated organ injury. We examined the protective effects of a potent inhibitor of NO synthase, aminoguanidine (AG, on myocardial and multiple organ structure in a rat model of HS. Materials and Methods: Male Sprague Dawley rats (300-350 g were assigned to 3 experimental groups (n = 6 per group: (1 Normotensive rats (N, (2 HS rats and (3 HS rats treated with AG (HS-AG. Rats were hemorrhaged over 60 min to reach a mean arterial blood pressure of 40 mmHg. Rats were treated with 1 ml of 60 mg/kg AG intra-arterially after 60 min HS. Resuscitation was performed in vivo by the reinfusion of the shed blood for 30 min to restore normo-tension. Biopsy samples were taken for light and electron microscopy. Results: Histological examination of hemorrhagic shocked untreated rats revealed structural damage. Less histological damage was observed in multiple organs in AG-treated rats. AG-treatment decreased the number of inflammatory cells and mitochondrial swollen in myocardial cells. Conclusion: AG treatment reduced microscopic damage and injury in multiple organs in a HS model in rats.

  6. Inhibition of an inducible nitric oxide synthase expression by a hexane extract from perilla frutescens cv. chookyoupjaso mutant induced by mutagenesis with gamma-ray

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    Park, Yong Dae; Kang, Min Ah; Lee, Hyo Jung; Jin, Chang Hyun; Choi, Dae Seong; Kim, Dong Sub; Kang, Si Yong; Byun, Myung Woo; Jeong, Il Yun [Korea Atomic Energy Research Institte, Jeongeup (Korea, Republic of)

    2009-03-15

    In earlier investigations, seeds of Perilla frutescens(L.) Britt. cv. Chookyoupjaso were irradiated with 200 Gy gamma ray to generate mutagenesis. The aim of this study is to investigate the effects of a hexane extract from Perilla frutescens(L.) Britt. cv. Chookyoupjaso mutant 45 on the actions of anti-inflammatory activity on inducible nitric oxide synthase, and an identification of the major active compound. The hexane extract from P. frutescens exhibited activity of inhibition of a NO production (IC{sub 50}, 295.1{mu}g ml{sup -1}). The hexane extract was further divided into sub-fractions by silica-gel chromatogarphy. Inhibition of the NO production by various fractions was assayed in LPS-stimulated RAW 264.7 cells. Among the seven fractions, the 5th fraction was the most effective (IC{sub 50}, 19.5{mu}g ml{sup -1}). The 5th fraction suppressed the expression of protein of iNOS in LPS-induced RAW 264.7 cells, and GC/MS analyses showed that isoegomaketone is a major bio-active compound in the 5th fraction. The result indicated that isoegomaketone has a good potential to be developed as an anti-inflammation agent.

  7. Up-regulation of miR-31 in human atrial fibrillation begets the arrhythmia by depleting dystrophin and neuronal nitric oxide synthase

    Science.gov (United States)

    Carnicer, Ricardo; Recalde, Alice; Muszkiewicz, Anna; Jayaram, Raja; Carena, Maria Cristina; Wijesurendra, Rohan; Stefanini, Matilde; Surdo, Nicoletta C.; Lomas, Oliver; Ratnatunga, Chandana; Sayeed, Rana; Krasopoulos, George; Rajakumar, Timothy; Bueno-Orovio, Alfonso; Verheule, Sander; Fulga, Tudor A.; Rodriguez, Blanca; Schotten, Ulrich

    2016-01-01

    Atrial fibrillation (AF) is a growing public health burden, and its treatment remains a challenge. AF leads to electrical remodeling of the atria, which in turn promotes AF maintenance and resistance to treatment. Although remodeling has long been a therapeutic target in AF, its causes remain poorly understood. We show that atrial-specific up-regulation of microRNA-31 (miR-31) in goat and human AF depletes neuronal nitric oxide synthase (nNOS) by accelerating mRNA decay and alters nNOS subcellular localization by repressing dystrophin translation. By shortening action potential duration and abolishing rate-dependent adaptation of the action potential duration, miR-31 overexpression and/or disruption of nNOS signaling recapitulates features of AF-induced remodeling and significantly increases AF inducibility in mice in vivo. By contrast, silencing miR-31 in atrial myocytes from patients with AF restores dystrophin and nNOS and normalizes action potential duration and its rate dependency. These findings identify atrial-specific up-regulation of miR-31 in human AF as a key mechanism causing atrial dystrophin and nNOS depletion, which in turn contributes to the atrial phenotype begetting this arrhythmia. miR-31 may therefore represent a potential therapeutic target in AF. PMID:27225184

  8. Involvement of extraneural tissues and upregulation of inducible nitric oxide synthase after experimental infection with rabies virus in BALB/c mice and LEW/SsN rats.

    Science.gov (United States)

    Liao, Pi-Hung; Hsu, Yung-Hsiang; Yang, Hui-Hua; Wang, Ming-Hseng; Chen, Li-Kuang

    2012-09-01

    Rabies virus can cause fatal encephalomyelitis, but the involvement of extraneural organs has not been well characterized. In this study, we investigated the histopathological changes and the distribution of viral antigens in extraneural organs after pathogenic rabies virus infection in mouse and rat models. In histopathological examination, classical viral encephalitis and rabies-specific Negri body were observed in the brain. In addition to the central nervous system (CNS), inflammatory responses were found in other organs, such as the heart, kidney, liver, and lung. Similarly, immunohistochemical staining and reverse transcription-polymerase chain reaction revealed the presence of rabies virus in the CNS and extraneural tissues. Moreover, macrophages, especially in the lung and heart, were involved in the infection. Transcriptional analyses of the expression of inducible nitric oxide synthase (iNOS) demonstrated that rabies virus potentiated the gene expression of iNOS in the brain, lung, and heart. The immunoreactive iNOS-positive macrophages were detected adjacent to the infection. These results suggest that macrophages are involved in the extraneural infection and the expression of iNOS in macrophages contributes to the formation of tissue inflammation. Our study indicates the involvement of extraneural organs following rabies virus infection, which may aggravate the progression of this deadly disease. © 2012 The Authors. Pathology International © 2012 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd.

  9. β-Escin sodium inhibits inducible nitric oxide synthase expression via downregulation of the JAK/STAT pathway in A549 cells.

    Science.gov (United States)

    Ji, Deng Bo; Xu, Bo; Liu, Jing Tao; Ran, Fu Xiang; Cui, Jing Rong

    2011-12-01

    β-escin, a triterpene saponin, is one of the major active compounds extracted from horse chestnut (Aesculus hippocastanum) seed. Previous work has found that β-escin sodium has antiinflammatory and antitumor effects. In the present study, we investigated its effect on cell proliferation and inducible nitric-oxide synthase (iNOS) expression in human lung carcinoma A549 cells. β-escin sodium (5-40 µg/mL) inhibited cytokine mixture (CM)-induced nitric oxide (NO) production in A549 cells by reducing the expression of iNOS. β-escin sodium suppressed phosphorylation and nuclear translocation of STAT1 (Tyr701) and STAT3 (Tyr705) induced by CM but did not affect the activation of c-Jun and NF-κB. β-escin sodium inhibited the activation of protein tyrosine kinase JAK2. Pervanadate treatment reversed the β-escin sodium-induced downregulation of STAT3 and STAT1. β-escin sodium treatment enhanced an activating phosphorylation of the phosphatase SHP2. Small interfering RNA-mediated knockdown of SHP2 inhibited β-escin sodium-induced phospho-STAT dephosphorylation. Moreover β-escin sodium reduced the activation of p38 MAPK. Finally, β-escin sodium inhibited the proliferation of A549 cells, did not change the cell membrane's permeability, nuclear morphology and size and the mitochondria's transmembrane potential of A549 cells. Taken together, these results demonstrate that β-escin sodium could downregulate iNOS expression through inhibiting JAK/STAT signaling and p38 MAPK activation in A549 cells. β-escin sodium has a marked antiproliferative effect on A549 cells at least in part by inhibiting the JAK/STAT signaling pathway, but not by a cytotoxic effect. β-escin sodium would be useful as a chemopreventive agent or a therapeutic against inflammatory-associated tumor. © 2011 Wiley Periodicals, Inc. Copyright © 2011 Wiley Periodicals, Inc.

  10. L-arginine stimulates CAT-1-mediated arginine uptake and regulation of inducible nitric oxide synthase for the growth of chick intestinal epithelial cells.

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    Yuan, Chao; Zhang, Xiaoyun; He, Qiang; Li, Junming; Lu, Jianjun; Zou, Xiaoting

    2015-01-01

    L-arginine (L-Arg) uptake is mediated by members of cationic amino acid transporter (CAT) family and may coincide with the induction of nitric oxide synthases (NOS). The present study was conducted to investigate the extracellular concentrations of L-Arg regulating the CAT-1, CAT-4 and inducible NOS (iNOS) in chick intestinal epithelial cells. The cells were cultured for 4 days in Arg-free Dulbecco's modified Eagle's medium containing 10, 100, 200, 400, or 600 μM L-Arg. Cell viability, nitric oxide (NO) concentrations, uptake and metabolism of L-[3H]-Arg as well as expression of CAT-1, CAT-4, and iNOS were determined. Our results showed that L-Arg enhances cell growth with a maximal response at 10-400 μM. Addition of 100, 200, or 400 μM L-Arg increased the L-[3H]-Arg uptake, which was associated with greater conversion of L-[3H]-citrulline and NO production in comparison with 10 μM L-Arg group. Increasing extracellular concentrations of L-Arg from 10 to 400 μM dose dependently increased the levels of CAT-1 mRNA and protein, while no effect on CAT-4 mRNA abundance was found. Furthermore, supplementation of 100, 200, or 400 μM L-Arg upregulated the expression of iNOS mRNA, and the relative protein levels for iNOS in 200 and 400 μM L-Arg groups were higher than those in 10 and 100 μM L-Arg groups. Collectively, we conclude that the CAT-1 isoform plays a role in L-Arg uptake, and L-Arg-mediated elevation of NO via iNOS promotes the growth of chick intestinal epithelial cells.

  11. Features of the inducible nitric oxide synthase expression in paraventricular and supraoptic nuclei of hypothalamus in different models of arterial hypertension

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    Yu. M. Kolesnyk

    2016-08-01

    Full Text Available The regulation of the paraventricular (PVN and supraoptic (SON nuclei’s activity is carried out with a great amount of different neurotransmitters, in particular, with nitric oxide. In order to get clear understanding of the local NO effects in hypothalamus in normal condition and different models of hypertension it is necessary to study all isoforms of NOS in PVN and SON. Our purpose was to find out the features of the inducible nitric oxide synthase (iNOS expression in magnocellular SON and PVN in SHR and endocrine-saline model of hypertension in rats. Materials and methods. For all rats the mean blood pressure (mBP was measured. In Wistar rats mBP was stable during the experiment. In SHR mBP was higher than normal. In animals of the 3rd group with ESM the first measurement (before the modelling demonstrated normal rates of mBP. Since the 7th day of modelling mBP started increase and became steadily increased from the 21st day. We obtained the frontal slices of hypothalamus and performed the assessment of iNOS expression using immunofluorescence assay. The results showed the presence of the constitutive expression of iNOS in the magnocellular neurons of hypothalamus in Wistar rats as well as in both groups of experimental hypertension. The level of iNOS expression in magnocellular nuclei was dependent both on type of hypertension and topography of magnocellular neurons in hypothalamus. In SHR there was high expression of iNOS in PVN and low one in SON, whereas in endocrine-saline model there was high expression in SON and there were no substantial changes of the iNOS expression in PVN. Conclusions. We believe the alteration of iNOS expression in magnocellular nuclei of hypothalamus could participate in development and/or adaptation to hypertension.

  12. Neuronal nitric oxide synthase-rescue of dystrophin/utrophin double knockout mice does not require nNOS localization to the cell membrane.

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    Michelle Wehling-Henricks

    Full Text Available Survival of dystrophin/utrophin double-knockout (dko mice was increased by muscle-specific expression of a neuronal nitric oxide synthase (nNOS transgene. Dko mice expressing the transgene (nNOS TG+/dko experienced delayed onset of mortality and increased life-span. The nNOS TG+/dko mice demonstrated a significant decrease in the concentration of CD163+, M2c macrophages that can express arginase and promote fibrosis. The decrease in M2c macrophages was associated with a significant reduction in fibrosis of heart, diaphragm and hindlimb muscles of nNOS TG+/dko mice. The nNOS transgene had no effect on the concentration of cytolytic, CD68+, M1 macrophages. Accordingly, we did not observe any change in the extent of muscle fiber lysis in the nNOS TG+/dko mice. These findings show that nNOS/NO (nitric oxide-mediated decreases in M2c macrophages lead to a reduction in the muscle fibrosis that is associated with increased mortality in mice lacking dystrophin and utrophin. Interestingly, the dramatic and beneficial effects of the nNOS transgene were not attributable to localization of nNOS protein at the cell membrane. We did not detect any nNOS protein at the sarcolemma in nNOS TG+/dko muscles. This important observation shows that sarcolemmal localization is not necessary for nNOS to have beneficial effects in dystrophic tissue and the presence of nNOS in the cytosol of dystrophic muscle fibers can ameliorate the pathology and most importantly, significantly increase life-span.

  13. NMDA receptor blockade alters the intracellular distribution of neuronal nitric oxide synthase in the superficial layers of the rat superior colliculus

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    R.E. de Bittencourt-Navarrete

    2009-02-01

    Full Text Available Nitric oxide (NO is a molecular messenger involved in several events of synaptic plasticity in the central nervous system. Ca2+ influx through the N-methyl-D-aspartate receptor (NMDAR triggers the synthesis of NO by activating the enzyme neuronal nitric oxide synthase (nNOS in postsynaptic densities. Therefore, NMDAR and nNOS are part of the intricate scenario of postsynaptic densities. In the present study, we hypothesized that the intracellular distribution of nNOS in the neurons of superior colliculus (SC superficial layers is an NMDAR activity-dependent process. We used osmotic minipumps to promote chronic blockade of the receptors with the pharmacological agent MK-801 in the SC of 7 adult rats. The effective blockade of NMDAR was assessed by changes in the protein level of the immediate early gene NGFI-A, which is a well-known NMDAR activity-dependent expressing transcription factor. Upon chronic infusion of MK-801, a decrease of 47% in the number of cells expressing NGFI-A was observed in the SC of treated animals. Additionally, the filled dendritic extent by the histochemical product of nicotinamide adenine di-nucleotide phosphate diaphorase was reduced by 45% when compared to the contralateral SC of the same animals and by 64% when compared to the SC of control animals. We conclude that the proper intracellular localization of nNOS in the retinorecipient layers of SC depends on NMDAR activation. These results are consistent with the view that the participation of NO in the physiological and plastic events of the central nervous system might be closely related to an NMDAR activity-dependent function.

  14. Candidate pathways linking inducible nitric oxide synthase to a basal-like transcription pattern and tumor progression in human breast cancer.

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    Ambs, Stefan; Glynn, Sharon A

    2011-02-15

    Inducible nitric oxide synthase (NOS2) is an inflammation responsive enzyme (EC 1.14.13.39) that is induced during acute and chronic inflammation and tissue injury as part of the host defense and wound healing process. NOS2 up-regulation leads to increased nitric oxide (NO) production, the means by which this enzyme can initiate NO-dependent signal transduction, influence the redox state of cells and induce modifications of proteins, lipids, and DNA. Aberrant expression of NOS2 has been observed in many types of human tumors. In breast cancer, increased NOS2 is associated with markers of poor outcome and decreased survival. Growth factor and cytokine signaling, tissue remodeling, NF-kB activation, and hypoxia are candidate mechanisms that induce NOS2 in tumor epithelial and tumor-infiltrating cells. NOS2 induction will trigger the release of variable amounts of NO into the tumor microenvironment and can activate oncogenic pathways, including the Akt, epidermal growth factor receptor and c-Myc signaling pathways, and stimulate tumor microvascularization. Constitutively increased NO levels may also select for mutant p53 cells to overcome the tumor suppressor function of NO-activated wild-type p53. More recent findings suggest that NO induces stem cell-like tumor characteristics in breast cancer. In this review, we will discuss the effects of NO in tumor biology and disease progression with an emphasis on breast cancer, and will examine the mechanisms that link increased NO to a basal-like transcription pattern in human breast tumors and poor disease outcome.

  15. Altered expression of cyclooxygenase-2, 12-lipoxygenase, inducible nitric oxide synthase-2 and surfactant protein D in lungs of patients with pulmonary injury caused by sulfur mustard.

    Science.gov (United States)

    Tahmasbpour, Eisa; Ghanei, Mostafa; Khor, Abolfazl; Panahi, Yunes

    2018-03-14

    Sulfur mustard (SM) is a strong alkylating toxicant that targets different organs, particularly human lung tissue. Change in genes expression is one of the molecular mechanisms of SM toxicity in damaged tissue. The purpose of this investigation is to characterize the expression of cyclooxygenase-2 (COX-2), 12-lipoxygenase (12-LO), inducible nitric oxide synthase 2 (iNOS2), and surfactant protein D (SFTPD) in lungs of patients who exposed to SM. Lung biopsies were provided from SM-exposed patients (n = 6) and controls (n = 5). Total RNA were extracted from all specimens and then cDNA was synthesized for each sample. Changes in gene expression were measured using RT 2 Profiler ™PCR Array. Pulmonary function tests revealed more obstructive and restrictive spirometric patterns among patients compared to the control group. Expression of COX-2 and 12-LO in the lung of patients was increased by 6.2555 (p = 0.004) and 6.2379-folds (p = 0.002), respectively. In contrast, expression of SF-D and iNOS genes was reduced by 8.5869-fold (p = 0.005) and 2.4466-folds (p = 0.011), respectively. Mustard lungs were associated with overexpression of COX-2 and 12-LO, which are responsible for inflammation, overproduction of free radicals and oxidative stress. Downregulation of iNOS2 and SF-D are probably the reason for lung disease and dysfunction among these patients. Therefore, the expression of these genes could be an important, routine part of the management of such patients.

  16. Inducible nitric oxide synthase (iNOS expression in monocytes during acute Dengue Fever in patients and during in vitro infection

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    Cerqueira Denise IS

    2005-08-01

    Full Text Available Abstract Mononuclear phagocytes are considered to be main targets for Dengue Virus (DENV replication. These cells are activated after infection, producing proinflammatory mediators, including tumour-necrosis factor-α, which has also been detected in vivo. Nitric oxide (NO, usually produced by activated mononuclear phagocytes, has antimicrobial and antiviral activities. Methods The expression of DENV antigens and inducible nitric oxide synthase (iNOS in human blood isolated monocytes were analysed by flow cytometry using cells either from patients with acute Dengue Fever or after DENV-1 in vitro infection. DENV-1 susceptibility to iNOS inhibition and NO production was investigated using NG-methyl L-Arginine (NGMLA as an iNOS inhibitor, which was added to DENV-1 infected human monocytes, and sodium nitroprussiate (SNP, a NO donor, added to infected C6/36 mosquito cell clone. Viral antigens after treatments were detected by flow cytometry analysis. Results INOS expression in activated monocytes was observed in 10 out of 21 patients with Dengue Fever and was absent in cells from ten healthy individuals. DENV antigens detected in 25 out of 35 patients, were observed early during in vitro infection (3 days, significantly diminished with time, indicating that virus replicated, however monocytes controlled the infection. On the other hand, the iNOS expression was detected at increasing frequency in in vitro infected monocytes from three to six days, exhibiting an inverse relationship to DENV antigen expression. We demonstrated that the detection of the DENV-1 antigen was enhanced during monocyte treatment with NGMLA. In the mosquito cell line C6/36, virus detection was significantly reduced in the presence of SNP, when compared to that of untreated cells. Conclusion This study is the first to reveal the activation of DENV infected monocytes based on induction of iNOS both in vivo and in vitro, as well as the susceptibility of DENV-1 to a NO production.

  17. Far red/near infrared light-induced protection against cardiac ischemia and reperfusion injury remains intact under diabetic conditions and is independent of nitric oxide synthase

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    Agnes eKeszler

    2014-08-01

    Full Text Available Far red/near-infrared light (NIR promotes a wide range of biological effects including tissue protection but whether and how NIR is capable of acutely protecting myocardium against ischemia and reperfusion injury in vivo is not fully elucidated. Our previous work indicates that NIR exposure immediately before and during early reperfusion protects the myocardium against infarction through mechanisms that are nitric oxide (NO-dependent. Here we tested the hypothesis that NIR elicits protection in a diabetic mouse model where other cardioprotective interventions such as pre- and postconditioning fail, and that the protection is independent of nitric oxide synthase (NOS. NIR reduced infarct size dose dependently. Importantly, NIR-induced protection was preserved in a diabetic mouse model (db/db and during acute hyperglycemia, as well as in endothelial NOS-/- mice and in wild type mice treated with NOS inhibitor L-NAME. In in vitro experiments NIR light liberates NO from nitrosyl hemoglobin (HbNO and nitrosyl myoglobin (MbNO in a wavelength (660-830 nm and dose-dependent manner. Irradiation at 660 nm yields the highest release of NO, while at longer wavelengths a dramatic decrease of NO release can be observed. Similar wavelength dependence was observed for the protection of mice against cardiac ischemia and reperfusion injury in vivo. NIR-induced NO release from deoxymyoglobin in the presence of nitrite mildly inhibits respiration of isolated mitochondria after hypoxia. In summary, NIR applied during reperfusion protects the myocardium against infarction in an NO dependent, but NOS-independent mechanisms, whereby mitochondria may be a target of NO released by NIR, leading to reduced reactive oxygen species generation during reperfusion. This unique mechanism preserves protection even during diabetes where other protective strategies fail.

  18. Characterization of the reductase domain of rat neuronal nitric oxide synthase generated in the methylotrophic yeast Pichia pastoris. Calmodulin response is complete within the reductase domain itself.

    Science.gov (United States)

    Gachhui, R; Presta, A; Bentley, D F; Abu-Soud, H M; McArthur, R; Brudvig, G; Ghosh, D K; Stuehr, D J

    1996-08-23

    Rat neuronal NO synthase (nNOS) is comprised of a flavin-containing reductase domain and a heme-containing oxygenase domain. Calmodulin binding to nNOS increases the rate of electron transfer from NADPH into its flavins, triggers electron transfer from flavins to the heme, activates NO synthesis, and increases reduction of artificial electron acceptors such as cytochrome c. To investigate what role the reductase domain plays in calmodulin's activation of these functions, we overexpressed a form of the nNOS reductase domain (amino acids 724-1429) in the yeast Pichia pastoris that for the first time exhibits a complete calmodulin response. The reductase domain was purified by 2',5'-ADP affinity chromatography yielding 25 mg of pure protein per liter of culture. It contained 1 FAD and 0.8 FMN per molecule. Most of the protein as isolated contained an air-stable flavin semiquinone radical that was sensitive to FeCN6 oxidation. Anaerobic titration of the FeCN6-oxidized reductase domain with NADPH indicated the flavin semiquinone re-formed after addition of 1-electron equivalent and the flavins could accept up to 3 electrons from NADPH. Calmodulin binding to the recombinant reductase protein increased its rate of NADPH-dependent flavin reduction and its rate of electron transfer to cytochrome c, FeCN6, or dichlorophenolindophenol to fully match the rate increases achieved when calmodulin bound to native full-length nNOS. Calmodulin's activation of the reductase protein was associated with an increase in domain tryptophan and flavin fluorescence. We conclude that many of calmodulin's actions on native nNOS can be fully accounted for through its interaction with the nNOS reductase domain itself.

  19. Long-term dietary L-arginine supplementation increases endothelial nitric oxide synthase and vasoactive intestinal peptide immunoexpression in rat small intestine.

    Science.gov (United States)

    Velickovic, Ksenija; Markelic, Milica; Golic, Igor; Otasevic, Vesna; Stancic, Ana; Jankovic, Aleksandra; Vucetic, Milica; Buzadzic, Biljana; Korac, Bato; Korac, Aleksandra

    2014-04-01

    Nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) are important intestinal neurotransmitters that coexist in the gut enteric nervous system and play an important role in intestinal physiology (e.g., absorption, motility, fluid secretion and smooth muscle relaxation). It is also known that cold exposure alters several aspects of gastrointestinal physiology and induces hyperphagia to meet increased metabolic demands, but there are no data regarding NO and VIP involvement in intestinal response during acclimation to cold. The objective of this study was to determine the influence of long-term L-arginine supplementation on the expression of the three isoforms of nitric oxide synthase (NOS) and VIP in small intestine of rats acclimated to room temperature or cold. Animals (six per group) acclimated to room temperature (22 ± 1 °C) and cold (4 ± 1 °C), respectively, were treated with 2.25% L-arginine, a substrate for NOSs, or with 0.01% N(ω)-nitro-L-arginine methyl ester, an inhibitor of NOSs, for 45 days. The topographical distribution of VIP and NOSs expression in small intestine was studied by immunohistochemistry, and ImageJ software was used for semiquantitative densitometric analysis of their immunoexpression. Long-term dietary L-arginine supplementation increases VIP and NOSs immunoexpression at room temperature while at cold increases the endothelial NOS, inducible NOS and VIP but decrease neuronal NOS in rat small intestine. Our results demonstrate that long-term dietary L-arginine supplementation modulates NOSs and VIP immunoexpression in rat small intestine with respect to ambient temperature, pointing out the eNOS as a predominant NOS isoform with an immunoexpression pattern similar to VIP.

  20. Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Yong Pil; Kim, Hyung Gyun [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Hien, Tran Thi [College of Pharmacy, Chosun University, Gwangju (Korea, Republic of); Jeong, Myung Ho [Heart Research Center, Chonnam National University Hospital, Gwangju (Korea, Republic of); Jeong, Tae Cheon, E-mail: taecheon@ynu.ac.kr [College of Pharmacy, Yeungnam University, Gyungsan (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

    2011-11-15

    The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-{alpha}-stimulated monocytes to endothelial cells and suppressed the TNF-{alpha} induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-{alpha}-induced nuclear factor-{kappa}B activation, which was attenuated by pretreatment with N{sup G}-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: Black-Right-Pointing-Pointer Puerarin induced the phosphorylation of eNOS and the production of NO. Black-Right-Pointing-Pointer Puerarin activated eNOS through ER-dependent PI3-kinase and Ca{sup 2+}-dependent AMPK. Black-Right-Pointing-Pointer Puerarin-induced NO was involved in the inhibition of NF-kB activation. Black-Right-Pointing-Pointer Puerarin may help for prevention of vascular dysfunction and diabetes.

  1. Generation of nitric oxide by the inducible nitric oxide synthase protects gamma delta T cells from Mycobacterium tuberculosis-induced apoptosis.

    Science.gov (United States)

    Sciorati, C; Rovere, P; Ferrarini, M; Paolucci, C; Heltai, S; Vaiani, R; Clementi, E; Manfredi, A A

    1999-08-01

    Gamma delta T cells are early recruited into mycobacterial lesions. Upon microbial Ag recognition, gamma delta cells secrete cytokines and chemokines and undergo apoptosis via CD95/CD95 ligand (CD95L) interaction, possibly influencing the outcome of infection and the characteristics of the disease. In this paper we show that activated phagocytes acquire, upon challenge with Mycobacterium tuberculosis, the ability to inhibit M. tuberculosis-induced gamma delta cell apoptosis. Apoptosis protection was due to NO because it correlated with NO synthase (NOS)-2 induction and activity in scavenger cells and was abrogated by NOS inhibitors. Furthermore, the NO donor S-nitrosoacetylpenicillamine mimicked the effect of enzyme induction. NO left unaffected the expression of CD95 and CD95L, suggesting interference with an event ensuing CD95/CD95L interaction. NO was found to interfere with the intracellular accumulation of ceramide and the activation of caspases, which were involved in gamma delta T cells apoptosis after M. tuberculosis recognition. We propose that NO generated by infected macrophages determines the life span and therefore the function of lymphocytes at the infection site, thus linking innate and adaptive immunity.

  2. Posttranscriptional and transcriptional regulation of endothelial nitric-oxide synthase during hypoxia: the role of microRNAs.

    Science.gov (United States)

    Kalinowski, Leszek; Janaszak-Jasiecka, Anna; Siekierzycka, Anna; Bartoszewska, Sylwia; Woźniak, Marcin; Lejnowski, Dawid; Collawn, James F; Bartoszewski, Rafal

    2016-01-01

    Understanding the cellular pathways that regulate endothelial nitric oxide (eNOS, NOS3 ) expression and consequently nitric oxide (NO) bioavailability during hypoxia is a necessary aspect in the development of novel treatments for cardiovascular disorders. eNOS expression and eNOS-dependent NO cellular signaling during hypoxia promote an equilibrium of transcriptional and posttranscriptional molecular mechanisms that belong to both proapoptotic and survival pathways. Furthermore, NO bioavailability results not only from eNOS levels, but also relies on the presence of eNOS substrate and cofactors, the phosphorylation status of eNOS, and the presence of reactive oxygen species (ROS) that can inactivate eNOS. Since both NOS3 levels and these signaling pathways can also be a subject of posttranscriptional modulation by microRNAs (miRNAs), this class of short noncoding RNAs contribute another level of regulation for NO bioavailability. As miRNA antagomirs or specific target protectors could be used in therapeutic approaches to regulate NO levels, either by changing NOS3 mRNA stability or through factors governing eNOS activity, it is critical to understand their role in governing eNOS activity during hypoxa. In contrast to a large number of miRNAs reported to the change eNOS expression during hypoxia, only a few miRNAs modulate eNOS activity. Furthermore, impaired miRNA biogenesis leads to NOS3 mRNA stabilization under hypoxia. Here we discuss the recent studies that define miRNAs' role in maintaining endothelial NO bioavailability emphasizing those miRNAs that directly modulate NOS3 expression or eNOS activity.

  3. Increased inducible nitric oxide synthase expression in organs is associated with a higher severity of H5N1 influenza virus infection.

    Science.gov (United States)

    Burggraaf, Simon; Bingham, John; Payne, Jean; Kimpton, Wayne G; Lowenthal, John W; Bean, Andrew G D

    2011-01-19

    The mechanisms of disease severity caused by H5N1 influenza virus infection remain somewhat unclear. Studies have indicated that a high viral load and an associated hyper inflammatory immune response are influential during the onset of infection. This dysregulated inflammatory response with increased levels of free radicals, such as nitric oxide (NO), appears likely to contribute to disease severity. However, enzymes of the nitric oxide synthase (NOS) family such as the inducible form of NOS (iNOS) generate NO, which serves as a potent anti-viral molecule to combat infection in combination with acute phase proteins and cytokines. Nevertheless, excessive production of iNOS and subsequent high levels of NO during H5N1 infection may have negative effects, acting with other damaging oxidants to promote excessive inflammation or induce apoptosis. There are dramatic differences in the severity of disease between chickens and ducks following H5N1 influenza infection. Chickens show a high level of mortality and associated pathology, whilst ducks show relatively minor symptoms. It is not clear how this varying pathogenicty comes about, although it has been suggested that an overactive inflammatory immune response to infection in the chicken, compared to the duck response, may be to blame for the disparity in observed pathology. In this study, we identify and investigate iNOS gene expression in ducks and chickens during H5N1 influenza infection. Infected chickens show a marked increase in iNOS expression in a wide range of organs. Contrastingly, infected duck tissues have lower levels of tissue related iNOS expression. The differences in iNOS expression levels observed between chickens and ducks during H5N1 avian influenza infection may be important in the inflammatory response that contributes to the pathology. Understanding the regulation of iNOS expression and its role during H5N1 influenza infection may provide insights for the development of new therapeutic strategies

  4. Increased inducible nitric oxide synthase expression in organs is associated with a higher severity of H5N1 influenza virus infection.

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    Simon Burggraaf

    Full Text Available BACKGROUND: The mechanisms of disease severity caused by H5N1 influenza virus infection remain somewhat unclear. Studies have indicated that a high viral load and an associated hyper inflammatory immune response are influential during the onset of infection. This dysregulated inflammatory response with increased levels of free radicals, such as nitric oxide (NO, appears likely to contribute to disease severity. However, enzymes of the nitric oxide synthase (NOS family such as the inducible form of NOS (iNOS generate NO, which serves as a potent anti-viral molecule to combat infection in combination with acute phase proteins and cytokines. Nevertheless, excessive production of iNOS and subsequent high levels of NO during H5N1 infection may have negative effects, acting with other damaging oxidants to promote excessive inflammation or induce apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: There are dramatic differences in the severity of disease between chickens and ducks following H5N1 influenza infection. Chickens show a high level of mortality and associated pathology, whilst ducks show relatively minor symptoms. It is not clear how this varying pathogenicty comes about, although it has been suggested that an overactive inflammatory immune response to infection in the chicken, compared to the duck response, may be to blame for the disparity in observed pathology. In this study, we identify and investigate iNOS gene expression in ducks and chickens during H5N1 influenza infection. Infected chickens show a marked increase in iNOS expression in a wide range of organs. Contrastingly, infected duck tissues have lower levels of tissue related iNOS expression. CONCLUSIONS/SIGNIFICANCE: The differences in iNOS expression levels observed between chickens and ducks during H5N1 avian influenza infection may be important in the inflammatory response that contributes to the pathology. Understanding the regulation of iNOS expression and its role during H5N1

  5. Interferon-gamma and nitric oxide synthase 2 mediate the aggregation of resident adherent peritoneal exudate cells: implications for the host response to pathogens.

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    Bhagawat S Chandrasekar

    Full Text Available Interferon-gamma (Ifnγ, a key macrophage activating cytokine, plays pleiotropic roles in host immunity. In this study, the ability of Ifnγ to induce the aggregation of resident mouse adherent peritoneal exudate cells (APECs, consisting primarily of macrophages, was investigated. Cell-cell interactions involve adhesion molecules and, upon addition of Ifnγ, CD11b re-localizes preferentially to the sites of interaction on APECs. A functional role of CD11b in enhancing aggregation is demonstrated using Reopro, a blocking reagent, and siRNA to Cd11b. Studies with NG-methyl-L-arginine (LNMA, an inhibitor of Nitric oxide synthase (Nos, NO donors, e.g., S-nitroso-N-acetyl-DL-penicillamine (SNAP or Diethylenetriamine/nitric oxide adduct (DETA/NO, and Nos2-/- mice identified Nitric oxide (NO induced by Ifnγ as a key regulator of aggregation of APECs. Further studies with Nos2-/- APECs revealed that some Ifnγ responses are independent of NO: induction of MHC class II and CD80. On the other hand, Nos2 derived NO is important for other functions: motility, phagocytosis, morphology and aggregation. Studies with cytoskeleton depolymerizing agents revealed that Ifnγ and NO mediate the cortical stabilization of Actin and Tubulin which contribute to aggregation of APECs. The biological relevance of aggregation of APECs was delineated using infection experiments with Salmonella Typhimurium (S. Typhimurium. APECs from orally infected, but not uninfected, mice produce high amounts of NO and aggregate upon ex vivo culture in a Nos2-dependent manner. Importantly, aggregated APECs induced by Ifnγ contain fewer intracellular S. Typhimurium compared to their single counterparts post infection. Further experiments with LNMA or Reopro revealed that both NO and CD11b are important for aggregation; in addition, NO is bactericidal. Overall, this study elucidates novel roles for Ifnγ and Nos2 in regulating Actin, Tubulin, CD11b, motility and morphology during the

  6. Changes in angiotensin AT1 receptor mRNA levels in the rat brain after immobilization stress and inhibition of central nitric oxide synthase.

    Science.gov (United States)

    Kiss, A; Jurkovicova, D; Jezova, D; Krizanova, O

    2001-06-01

    To study functional interactions between angiotensin II AT1 receptors and nitric oxide (NO) activity in different brain areas in rats exposed to immobilization stress. Central inhibition of nitric oxide synthase (NOS) was provided by intracerebroventricular (i.c.v.) administration of (N-omega-nitro-L-arginine-methylester) L-NAME and analysis of AT1 receptor mRNA was performed using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. The immobilization in prone position lasted 2 hrs and the rats were sacrificed 24 hr later. The hypothalamus, hippocampus, thalamus, and cortex were isolated from fresh brains. In the cortex, gene expression of AT1 receptors was unaffected either by L-NAME treatment, or by a single exposure to immobilization stress for 2 hours followed by 24 hours of rest. In the hippocampus, the repeated treatment with L-NAME increased mRNA levels of AT1 receptors approximately 9-times compared to those in the control (untreated) group. Immobilization also increased AT1 receptor mRNA levels in the hippocampus which was similar to that induced by the L-NAME. The increase of AT1 receptor mRNA levels in the hippocampus of immobilized rats was not further altered when the animals were pretreated with L-NAME. In control rats, exposure to immobilization resulted in a significant rise in mRNA levels coding for AT1 receptors in the hypothalamus, but not in the thalamus. L-NAME treatment showed a tendency of increase in AT1 receptor mRNA levels in the hypothalamus. Moreover, when animals treated with L-NAME were subjected to immobilization, a further increase in AT1 receptor mRNA levels was observed in the hypothalamus in comparison with corresponding controls. The present data indicate that a single immobilization stress results in increased gene expression of AT1 receptors in the hypothalamus and hippocampus. The rise in AT1 mRNA levels in the same brain structures after repeated treatment with L-NAME allow to suggest an

  7. Diastereoselective Au-Catalyzed Allene Cycloisomerizations to Highly Substituted Cyclopentenes.

    Science.gov (United States)

    Reeves, Ryan D; Phelps, Alicia M; Raimbach, William A T; Schomaker, Jennifer M

    2017-07-07

    Site- and regiocontrolled Au-catalyzed allene carbocyclizations furnish highly substituted cyclopentenes in >1:1 dr. Significant substitution on the substrate is tolerated, with potential to install five contiguous stereocenters after alkene functionalization. Major challenges include identifying a Au/Cu catalyst that controls both the relative rates of allene epimerization/cyclization and the facial selectivity in addition of a metal enolate to the allene. Experiments to achieve stereodivergent cyclizations and transform key cyclopentenes into useful synthetic building blocks are described.

  8. The interactive contributions of Na(+) /K(+) -ATPase and nitric oxide synthase to sweating and cutaneous vasodilatation during exercise in the heat.

    Science.gov (United States)

    Louie, Jeffrey C; Fujii, Naoto; Meade, Robert D; Kenny, Glen P

    2016-06-15

    Nitric oxide synthase (NOS) contributes to sweating and cutaneous vasodilatation during exercise in the heat. Similarly, reports show that Na(+) /K(+) -ATPase activation can modulate sweating and microvascular circulation. In light of the fact that NO can activate Na(+) /K(+) -ATPase, we evaluated whether there is an interaction between Na(+) /K(+) -ATPase and NOS in the regulation of heat loss responses during an exercise-induced heat stress. We demonstrate that Na(+) /K(+) -ATPase and NOS do not synergistically influence local forearm sweating during moderate intensity (fixed rate of metabolic heat production of 500 W) exercise in the heat (35°C). Conversely, we show an interactive role between NOS and Na(+) /K(+) -ATPase in the modulation of cutaneous vasodilatation. These findings provide novel insight regarding the mechanisms underpinning the control of sweating and cutaneous vasodilatation during exercise in the heat. Given that ouabain may be prescribed as a cardiac glycoside in clinical settings, potential heat loss impairments with ouabain administration should be explored. Nitric oxide (NO) synthase (NOS) contributes to the heat loss responses of sweating and cutaneous vasodilatation. Given that NO can activate Na(+) /K(+) -ATPase, which also contributes to sweating and microvasculature regulation, we evaluated the separate and combined influence of Na(+) /K(+) -ATPase and NOS on sweating and cutaneous vasodilatation. Thirteen young (23±3 years) males performed two 30 min semi-recumbent cycling bouts in the heat (35°C) at a fixed rate of metabolic heat production (500 W) followed by 20 and 40 min recoveries, respectively. Local sweat rate (LSR) and cutaneous vascular conductance (CVC) were measured at four forearm skin sites continuously perfused via intradermal microdialysis with either: (1) lactated Ringer solution (Control); (2) 6 mᴍ ouabain (Ouabain), a Na(+) /K(+) -ATPase inhibitor; (3) 10 mᴍ l-N(G) -nitroarginine methyl ester (l

  9. Endotoxin and interferon-γ inhibit translation in skeletal muscle cells by stimulating nitric oxide synthase activity

    Science.gov (United States)

    Frost, Robert A.; Nystrom, Gerald J.; Lang, Charles H.

    2012-01-01

    The purpose of the present study was to test the hypothesis that endogenous nitric oxide (NO) negatively affects translation in skeletal muscle cells after exposure to a combination of endotoxin (LPS) and interferon (IFN)γ. Individually LPS and IFNγ did not alter protein synthesis but in combination they inhibited protein synthesis by 80% in C2C12 myotubes. The combination of LPS and IFNγ dramatically down regulated the auto-phosphorylation of the mammalian target of rapamycin (mTOR) and its substrates S6K1 and 4EBP-1. The phosphorylation of ribosomal protein S6 was decreased whereas phosphorylation of elongation factor-2 (eEF-2) and raptor was enhanced consistent with defects in both translation initiation and elongation. Reduced S6 phosphorylation occurred 8–18 h after LPS/IFNγ and coincided with a prolonged upregulation of NOS2 mRNA and protein. NOS2 protein expression and the LPS/IFNγ –induced fall in phosphorylated S6 were prevented by the proteasome inhibitor MG132. The general NOS inhibitor L-NAME and the specific NOS2 inhibitor 1400W also prevented the LPS/IFNγ-induced decrease in protein synthesis and restored translational signaling. LPS/IFNγ down regulated the phosphorylation of multiple Akt substrates including the proline rich Akt substrate-40 (PRAS40) while enhancing the phosphorylation of raptor on an AMPK regulated site. The negative effects of LPS/IFNγ were blunted by the AMPK inhibitor compound C. The data suggest that in combination LPS and IFNγ induce a prolonged expression of NOS2 and excessive production of NO that reciprocally alters Akt and AMPK activity and consequently down regulates translation via reduced mTOR signaling. PMID:19295495

  10. Nitric oxide synthase (NOS inhibition during porcine in vitro maturation modifies oocyte protein S-nitrosylation and in vitro fertilization.

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    Jon Romero-Aguirregomezcorta

    Full Text Available Nitric oxide (NO is a molecule involved in many reproductive processes. Its importance during oocyte in vitro maturation (IVM has been demonstrated in various species although sometimes with contradictory results. The objective of this study was to determine the effect of NO during IVM of cumulus oocyte complexes and its subsequent impact on gamete interaction in porcine species. For this purpose, IVM media were supplemented with three NOS inhibitors: NG-nitro-L-arginine methyl ester (L-NAME, NG-monomethyl-L-arginine (L-NMMA and aminoguanidine (AG. A NO donor, S-nitrosoglutathione (GSNO, was also used. The effects on the cumulus cell expansion, meiotic resumption, zona pellucida digestion time (ZPdt and, finally, on in vitro fertilization (IVF parameters were evaluated. The oocyte S-nitrosoproteins were also studied by in situ nitrosylation. The results showed that after 42 h of IVM, AG, L-NAME and L-NMMA had an inhibitory effect on cumulus cell expansion. Meiotic resumption was suppressed only when AG was added, with 78.7% of the oocytes arrested at the germinal vesicle state (P<0.05. Supplementation of the IVM medium with NOS inhibitors or NO donor did not enhance the efficiency of IVF, but revealed the importance of NO in maturation and subsequent fertilization. Furthermore, protein S-nitrosylation is reported for the first time as a pathway through which NO exerts its effect on porcine IVM; therefore, it would be important to determine which proteins are nitrosylated in the oocyte and their functions, in order to throw light on the mechanism of action of NO in oocyte maturation and subsequent fertilization.

  11. Mechanism of endothelial nitric oxide synthase phosphorylation and activation by tentacle extract from the jellyfishCyanea capillata.

    Science.gov (United States)

    Wang, Beilei; Liu, Dan; Wang, Chao; Wang, Qianqian; Zhang, Hui; Liu, Guoyan; Tao, Xia; Zhang, Liming

    2017-01-01

    Our previous study demonstrated that tentacle extract (TE) from the jellyfish Cyanea capillata ( C. capillata ) could cause a weak relaxation response mediated by nitric oxide (NO) using isolated aorta rings. However, the intracellular mechanisms of TE-induced vasodilation remain unclear. Thus, this study was conducted to examine the role of TE on Akt/eNOS/NO and Ca 2+ signaling pathways in human umbilical vein endothelial cells (HUVECs). Our results showed that TE induced dose- and time-dependent increases of eNOS activity and NO production. And TE also induced Akt and eNOS phosphorylation in HUVECs. However, treatment with specific PI3-kinase inhibitor (Wortmannin) significantly inhibited the increases in NO production and Akt/eNOS phosphorylation. In addition, TE also stimulated an increase in the intracellular Ca 2+ concentration ([Ca 2+ ] i ), which was significantly attenuated by either IP 3 receptor blocker (Heparin) or PKC inhibitor (PKC 412). In contrast, extracellular Ca 2+ -free, L-type calcium channel blocker (Nifedipine), or PKA inhibitor (H89) had no influence on the [Ca 2+ ] i elevation. Since calcium ions also play a critical role in stimulating eNOS activity, we next explored the role of Ca 2+ in TE-induced Akt/eNOS activation. In consistent with the attenuation of [Ca 2+ ] i elevation, we found that Akt/eNOS phosphorylation was also dramatically decreased by Heparin or PKC 412, but not affected by Nifedipine or H89. However, the phosphorylation level could also be decreased by the removal of extracellular calcium. Taken together, our findings indicated that TE-induced eNOS phosphorylation and activation were mainly through PI3K/Akt-dependent, PKC/IP 3 R-sensitive and Ca 2+ -dependent pathways.

  12. Mechanism of endothelial nitric oxide synthase phosphorylation and activation by tentacle extract from the jellyfish Cyanea capillata

    Directory of Open Access Journals (Sweden)

    Beilei Wang

    2017-04-01

    Full Text Available Our previous study demonstrated that tentacle extract (TE from the jellyfish Cyanea capillata (C. capillata could cause a weak relaxation response mediated by nitric oxide (NO using isolated aorta rings. However, the intracellular mechanisms of TE-induced vasodilation remain unclear. Thus, this study was conducted to examine the role of TE on Akt/eNOS/NO and Ca2+ signaling pathways in human umbilical vein endothelial cells (HUVECs. Our results showed that TE induced dose- and time-dependent increases of eNOS activity and NO production. And TE also induced Akt and eNOS phosphorylation in HUVECs. However, treatment with specific PI3-kinase inhibitor (Wortmannin significantly inhibited the increases in NO production and Akt/eNOS phosphorylation. In addition, TE also stimulated an increase in the intracellular Ca2+ concentration ([Ca2+]i, which was significantly attenuated by either IP3 receptor blocker (Heparin or PKC inhibitor (PKC 412. In contrast, extracellular Ca2+-free, L-type calcium channel blocker (Nifedipine, or PKA inhibitor (H89 had no influence on the [Ca2+]i elevation. Since calcium ions also play a critical role in stimulating eNOS activity, we next explored the role of Ca2+ in TE-induced Akt/eNOS activation. In consistent with the attenuation of [Ca2+]i elevation, we found that Akt/eNOS phosphorylation was also dramatically decreased by Heparin or PKC 412, but not affected by Nifedipine or H89. However, the phosphorylation level could also be decreased by the removal of extracellular calcium. Taken together, our findings indicated that TE-induced eNOS phosphorylation and activation were mainly through PI3K/Akt-dependent, PKC/IP3R-sensitive and Ca2+-dependent pathways.

  13. Decreased expression of neuronal nitric oxide synthase in the nucleus tractus solitarii inhibits sympathetically mediated baroreflex responses in rat

    Science.gov (United States)

    Lin, Li-Hsien; Nitschke Dragon, Deidre; Jin, Jingwen; Tian, Xin; Chu, Yi; Sigmund, Curt; Talman, William T

    2012-01-01

    Despite numerous studies it remains controversial whether nitric oxide (NO·) synthesized by neuronal NOS (nNOS) plays an excitatory or inhibitory role in transmission of baroreflex signals in the nucleus tractus solitarii (NTS). In the current studies we sought to test the hypothesis that nNOS is involved in excitation of baroreflex pathways in NTS while excluding pharmacological interventions in assessing the influence of nNOS. We therefore developed, validated and utilized a short hairpin RNA (shRNA) to reduce expression of nNOS in the NTS of rats whose baroreflex activity was then studied. We demonstrate downregulation of nNOS through transduction with adeno-associated virus type 2 (AAV2) carrying shRNA for nNOS. When injected bilaterally into NTS AAV2nNOSshRNA significantly reduced reflex tachycardic responses to acute hypotension while not affecting reflex bradycardic responses to acute increases of arterial pressure. Control animals treated with intravenous propranolol to block sympathetically mediated chronotropic responses manifested the same baroreflex responses as animals that had been treated with AAV2nNOSshRNA. Neither AAV2 eGFP nor AAV2nNOScDNA affected baroreflex responses. Blocking cardiac vagal influences with atropine similarly reduced baroreflex-mediated bradycardic responses to increases in arterial pressure both in control animals and in those treated with AAV2nNOSshRNA. We conclude that NO· synthesized by nNOS in the NTS is integral to excitation of baroreflex pathways involved in reflex tachycardia, a largely sympathetically mediated response, but not reflex bradycardia, a largely parasympathetically mediated response. We suggest that, at the basal state, nNOS is maximally engaged. Thus, its upregulation does not augment the baroreflex. PMID:22687614

  14. Woody Allen, serial schlemiel ?

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    Frédérique Brisset

    2011-04-01

    Full Text Available Woody Allen a développé au fil des années une persona cinématographique de schlemiel new-yorkais aisément reconnaissable par le spectateur. Elle marque nombre de ses films, qu’il y apparaisse en tant qu’acteur ou y dirige des substituts comédiens comme déclinaisons de lui-même. Si cette figure prototypique est le fondement de la sérialité dans sa filmographie, il est des traits stylistiques qui en portent trace tout au long de son œuvre : la récurrence annuelle de ses réalisations, la signature formelle symbolisée par ses génériques à la typographie singulière, le rythme de ses dialogues ponctués d’interjections et l’usage de l’autocitation sont autant de procédés qui marquent son cinéma d’un sceau très personnel. Ils fonctionnent comme des clins d’œil au spectateur qui reçoit dès lors LE Woody Allen millésimé comme une invitation à retrouver son microcosme. Ainsi la sérialité se pose comme à la fois initiale et conséquentielle de son système filmique, processus de création unique dans le cinéma américain.Woody Allen has long constructed a cinematographic persona of schlemiel New- Yorker that the audience can easily identify. It impacts most of his films, whether he stars in them or directs “substitute” actors to impersonate his character. If this prototypical figure is the basis of seriality in his cinematography, serial stylistic features can also be found all along his career: the annual recurrence of his productions, the formal signature symbolised by the typography of his singular credit titles, his rhythmical interjection-punctuated dialogues and the use of self-quotation imprint a very personal seal upon his movies. They all work as a recognition signals for the audience who thus receive THE Woody Allen vintage as an invitation to re-enter his microcosm. Seriality is then both initial and consequential to his cinematographic system, a unique creative process in American film history.

  15. Hypoxia-inducible factor-1α, vascular endothelial growth factor, inducible nitric oxide synthase, and endothelin-1 expression correlates with angiogenesis in congenital heart disease

    Directory of Open Access Journals (Sweden)

    Hsin-Ling Yin

    2016-07-01

    Full Text Available In Taiwan, the average prevalence of congenital heart disease (CHD is 13.08/1000 live births. Most children with CHD die before the age of 5 years; therefore, identifying treatment methods to extend the life of CHD patients is an important issue in clinical practice. The objective of this study is to evaluate the roles of hypoxia-inducible factor-1α (HIF-1α, vascular endothelial growth factor (VEGF, inducible nitric oxide synthase (iNOS, endothelin-1 (ET-1, and CD34 in CHD autopsy cases in comparison with autopsy cases without CHD. The study included 19 autopsy cases, which were divided into the following four groups: acyanotic CHD (n = 11, cyanotic CHD (n = 3, CHD associated with chromosomal abnormalities (n = 3, and complex CHD (n = 2. Heart specimens obtained from 10 autopsy cases without CHD were included as controls. Our results indicated that high percentages of HIF-1α (100%, VEGF (89.5%, iNOS (78.9%, and ET-1 (84.2% expressions were observed in CHD autopsy cases and this was found to be significant. HIF-1α induced by hypoxia could play a potential role in relating downstream gene expressions in CHD patients. Upregulation of VEGF by HIF-1α could play an important role in triggering angiogenesis to protect myocardial cell survival in a hypoxic microenvironment. Therefore, HIF-1α could be a significant prognosis marker in CHD and be a prospective candidate in the development of target therapy in cardiovascular diseases.

  16. Tumor Necrosis Factor (TNF -308G>A, Nitric Oxide Synthase 3 (NOS3 +894G>T Polymorphisms and Migraine Risk: A Meta-Analysis.

    Directory of Open Access Journals (Sweden)

    Min Chen

    Full Text Available Conflicting data have been reported on the association between tumor necrosis factor (TNF -308G>A and nitric oxide synthase 3 (NOS3 +894G>T polymorphisms and migraine. We performed a meta-analysis of case-control studies to evaluate whether the TNF -308G>A and NOS3 +894G>T polymorphisms confer genetic susceptibility to migraine.We performed an updated meta-analysis for TNF -308G>A and a meta-analysis for NOS3 +894G>T based on studies published up to July 2014. We calculated study specific odds ratios (OR and 95% confidence intervals (95% CI assuming allele contrast, dominant model, recessive model, and co-dominant model as pooled effect estimates.Eleven studies in 6682 migraineurs and 22591 controls for TNF -308G>A and six studies in 1055 migraineurs and 877 controls for NOS3 +894G>T were included in the analysis. Neither indicated overall associations between gene polymorphisms and migraine risk. Subgroup analyses suggested that the "A" allele of the TNF -308G>A variant increases the risk of migraine among non-Caucasians (dominant model: pooled OR = 1.82; 95% CI 1.15 - 2.87. The risk of migraine with aura (MA was increased among both Caucasians and non-Caucasians. Subgroup analyses suggested that the "T" allele of the NOS3 +894G>T variant increases the risk of migraine among non-Caucasians (co-dominant model: pooled OR = 2.10; 95% CI 1.14 - 3.88.Our findings appear to support the hypothesis that the TNF -308G>A polymorphism may act as a genetic susceptibility factor for migraine among non-Caucasians and that the NOS3 +894G>T polymorphism may modulate the risk of migraine among non-Caucasians.

  17. Nitric Oxide Synthase-Mediated Phytoalexin Accumulation in Soybean Cotyledons in Response to the Diaporthe phaseolorum f. sp. meridionalis Elicitor1

    Science.gov (United States)

    Modolo, Luzia Valentina; Cunha, Fernando Queiroz; Braga, Márcia Regina; Salgado, Ione

    2002-01-01

    Phytoalexin biosynthesis is part of the defense mechanism of soybean (Glycine max) plants against attack by the fungus Diaporthe phaseolorum f. sp. meridionalis (Dpm), the causal agent of stem canker disease. The treatment of soybean cotyledons with Dpm elicitor or with sodium nitroprusside (SNP), a nitric oxide (NO) donor, resulted in a high accumulation of phytoalexins. This response did not occur when SNP was replaced by ferricyanide, a structural analog of SNP devoid of the NO moiety. Phytoalexin accumulation induced by the fungal elicitor, but not by SNP, was prevented when cotyledons were pretreated with NO synthase (NOS) inhibitors. The Dpm elicitor also induced NOS activity in soybean tissues proximal to the site of inoculation. The induced NOS activity was Ca2+- and NADPH-dependent and was sensitive to the NOS inhibitors NG-nitro-l-arginine methyl ester, aminoguanidine, and l-N6-(iminoethyl) lysine. NOS activity was not observed in SNP-elicited tissues. An antibody to brain NOS labeled a 166-kD protein in elicited and nonelicited cotyledons. Isoflavones (daidzein and genistein), pterocarpans (glyceollins), and flavones (apigenin and luteolin) were identified after exposure to the elicitor or SNP, although the accumulation of glyceollins and apigenin was limited in SNP-elicited compared with fungal-elicited cotyledons. NOS activity preceded the accumulation of these flavonoids in tissues treated with the Dpm elicitor. The accumulation of these metabolites was faster in SNP-elicited than in fungal-elicited cotyledons. We conclude that the response of soybean cotyledons to Dpm elicitor involves NO formation via a constitutive NOS-like enzyme that triggers the biosynthesis of antimicrobial flavonoids. PMID:12427995

  18. Effect modification by delta-aminolevulinic acid dehydratase, vitamin D receptor, and nitric oxide synthase gene polymorphisms on associations between patella lead and renal function in lead workers.

    Science.gov (United States)

    Weaver, Virginia M; Lee, Byung-Kook; Todd, Andrew C; Ahn, Kyu-Dong; Shi, Weiping; Jaar, Bernard G; Kelsey, Karl T; Lustberg, Mark E; Silbergeld, Ellen K; Parsons, Patrick J; Wen, Jiayu; Schwartz, Brian S

    2006-09-01

    Genetic polymorphisms that affect lead toxicokinetics or toxicodynamics may be important modifiers of risk for adverse outcomes in lead-exposed populations. We recently reported associations between higher patella lead, which is hypothesized to represent a lead pool that is both bioavailable and cumulative, and adverse renal outcomes in current and former Korean lead workers. In the present study, we assessed effect modification by polymorphisms in the genes encoding for delta-aminolevulinic acid dehydratase (ALAD), the vitamin D receptor (VDR), and endothelial nitric oxide synthase on those associations. Similar analyses were conducted with three other lead biomarkers. Renal function was assessed via blood urea nitrogen, serum creatinine, measured and calculated creatinine clearances, urinary N-acetyl-beta-D-glucosaminidase, and retinol-binding protein. Mean (SD) blood, patella, tibia, and dimercaptosuccinic acid-chelatable lead values were 30.9 (16.7) microg/dl, 75.1 (101.1)and 33.6 (43.4) microg Pb/g bone mineral, and 0.63 (0.75) microg Pb/mg creatinine, respectively, in 647 lead workers. Little evidence of effect modification by genotype on associations between patella lead and renal outcomes was observed. The VDR polymorphism did modify associations between the other lead biomarkers and the serum creatinine and calculated creatinine clearance. Higher lead dose was associated with worse renal function in participants with the variant B allele. Models in two groups, dichotomized by median age, showed that this effect was present in the younger half of the population. Limited evidence of effect modification by ALAD genotype was observed; higher blood lead levels were associated with higher calculated creatinine clearance among participants with the ALAD(1-2) genotype. In conclusion, VDR and/or ALAD genotypes modified associations between all the lead biomarkers, except patella lead, and the renal outcomes.

  19. Impact of prenatal and postnatal exposure to the pesticide chlorpyrifos on the contraction of rat ileal muscle strips: involvement of an inducible nitric oxide synthase-dependent pathway.

    Science.gov (United States)

    Darwiche, W; Delanaud, S; Dupont, S; Ghamlouch, H; Ramadan, W; Joumaa, W; Bach, V; Gay-Quéheillard, J

    2017-02-01

    Prenatal/postnatal exposure to insecticides has been linked to developmental disorders in adulthood. Chlorpyrifos (CPF) is a widely used organophosphorus acetylcholinesterase (AChE)-inhibiting insecticide. The present study established whether prenatal and postnatal exposure to CPF is associated with intestinal motor dysfunction in adult rats. Three groups of pregnant rats were exposed to either CPF (1 or 5 mg/kg/day; the CPF1 and CPF5 groups) or vehicle (the control group) by gavage from gestational day 1 until weaning. At weaning, the pups were separated from their dams and individually gavaged (with the same dose) until postnatal day 60. We then measured in vivo intestinal transit and the in vitro contractile responses of ileal smooth muscle strips to electrical field stimulation. Expression of inducible nitric oxide synthase (iNOS) in the ileum was determined using qRT-PCR and immunoblots. Morphometry and AChE assays were also performed. At adulthood, the mean body mass was lower in the CPF1 and CPF5 groups than in controls. CPF5 exposure was associated with weaker in vitro contraction of ileal muscle strips, which was reversed by adding the NOS inhibitor (L-NAME). There was no significant intergroup difference in the mean in vivo transit time. Exposure to CPF was associated with greater iNOS expression, lower AChE activity and reduced circular and longitudinal smooth muscle thickness. Prenatal and postnatal exposure to CPF in the rat is associated with weaker contraction of ileal longitudinal smooth muscle via a nitrergic mechanism with increased iNOS expression. © 2016 John Wiley & Sons Ltd.

  20. Elevated CO2-induced production of nitric oxide (NO) by NO synthase differentially affects nitrate reductase activity in Arabidopsis plants under different nitrate supplies.

    Science.gov (United States)

    Du, Shaoting; Zhang, Ranran; Zhang, Peng; Liu, Huijun; Yan, Minggang; Chen, Ni; Xie, Huaqiang; Ke, Shouwei

    2016-02-01

    CO2 elevation often alters the plant's nitrate reductase (NR) activity, the first enzyme acting in the nitrate assimilation pathway. However, the mechanism underlying this process remains unknown. The association between elevated CO2-induced alterations of NR activity and nitric oxide (NO) was examined in Col-0 Arabidopsis fed with 0.2-10 mM nitrate, using NO donors, NO scavenger, and NO synthase (NOS) inhibitor. The noa1 mutant, in which most NOS activity was lost, and the NR activity-null mutant nia1 nia2 were also used to examine the above association. In response to CO2 elevation, NR activity increased in low-nitrate Col-0 plants but was inhibited in high-nitrate Col-0 plants. NO scavenger and NOS inhibitor could eliminate these two responses, whereas the application of NO donors mimicked these distinct responses in ambient CO2-grown Col-0 plants. Furthermore, in both low- and high-nitrate conditions, elevated CO2 increased NOS activity and NO levels in Col-0 and nia1 nia2 plants but had little effect on NO level and NR activity in noa1 plants. Considering all of these findings, this study concluded that, in response to CO2 elevation, either the NR activity induction in low-nitrate plants or the NR activity inhibition in high-nitrate plants is regulated by NOS-generated NO. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  1. Effects of extremely low frequency electromagnetic field (ELF-EMF) on catalase, cytochrome P450 and nitric oxide synthase in erythro-leukemic cells.

    Science.gov (United States)

    Patruno, Antonia; Tabrez, Shams; Pesce, Mirko; Shakil, Shazi; Kamal, Mohammad A; Reale, Marcella

    2015-01-15

    Extremely low frequency electromagnetic fields (ELF-EMFs) are widely employed in electrical appliances and different equipment such as television sets, mobile phones, computers and microwaves. The molecular mechanism through which ELF-EMFs can influence cellular behavior is still unclear. A hypothesis is that ELF-EMFs could interfere with chemical reactions involving free radical production. Under physiologic conditions, cells maintain redox balance through production of ROS/RNS and antioxidant molecules. The altered balance between ROS generation and elimination plays a critical role in a variety of pathologic conditions including neurodegenerative diseases, aging and cancer. Actually, there is a disagreement as to whether there is a causal or coincidental relationship between ELF-EMF exposure and leukemia development. Increased ROS levels have been observed in several hematopoietic malignancies including acute and chronic myeloid leukemias. In our study, the effect of ELF-EMF exposure on catalase, cytochrome P450 and inducible nitric oxide synthase activity and their expression by Western blot analysis in myelogenous leukemia cell line K562 was evaluated. A significant modulation of iNOS, CAT and Cyt P450 protein expression was recorded as a result of ELF-EMF exposure in both phorbol 12-myristate 13-acetate (PMA)-stimulated and non-stimulated cell lines. Modulation in kinetic parameters of CAT, CYP-450 and iNOS enzymes in response to ELF-EMF indicates an interaction between the ELF-EMF and the enzymological system. These new insights might be important in establishing a mechanistic framework at the molecular level within which the possible effects of ELF-EMF on health can be understood. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Expression of the neuronal isoform of nitric oxide synthase (nNOS) and its inhibitor, protein inhibitor of nNOS, in pigment cell lesions of the skin.

    Science.gov (United States)

    Ahmed, B; Van Den Oord, J J

    1999-07-01

    Nitric oxide (NO) is involved in many physiological processes. In cancer, low levels of NO are thought to enhance tumour progression and metastasis. NO is generated from arginine by NO synthase (NOS); the Ca2+-dependent neuronal isoform or nNOS (expressed by neurones and inhibited by the protein inhibitor of nNOS, PIN), is also expressed by cultured normal melanocytes and by all malignant melanoma (MM) cell lines. We studied the expression of nNOS and PIN in paraffin sections of 177 and 58 pigment cell lesions, respectively, using immunohistochemistry; the activity of the necessary cofactor NADPH was studied in 26 frozen cases. Normal melanocytes in situ lacked nNOS and PIN expression, but were NADPH +. Almost half of common acquired benign naevi expressed nNOS; however, halo naevi and congenital naevi expressed nNOS very frequently. Dysplastic naevi and MM showed variable nNOS immunoreactivity in 72% and 83% of cases, respectively. Early (Clark I and Clark II) MM displayed nNOS staining most frequently, and all MM with an invasive radial growth phase expressed nNOS in the papillary dermis. In contrast, only 67% of metastatic MM were nNOS +. PIN was coexpressed with nNOS in 40 of 58 lesions. NADPH activity was present in all nNOS + naevi, but in two malignant cases, NADPH activity was not accompanied by nNOS expression. We conclude that nNOS expression is induced de novo in benign and malignant pigment cell lesions which have all the requirements (NADPH, PIN) necessary for the production and modulation of NO. We postulate that the frequent expression of nNOS in the junctional part of dysplastic naevi may be responsible for their particular histological features. NO generated by the neoplastic dermal cells in the invasive radial growth phase may contribute to the increased number of blood vessels in the papillary dermis.

  3. 4G/5G variant of plasminogen activator inhibitor-1 gene and severe pregnancy-induced hypertension: subgroup analyses of variants of angiotensinogen and endothelial nitric oxide synthase.

    Science.gov (United States)

    Kobashi, Gen; Ohta, Kaori; Yamada, Hideto; Hata, Akira; Minakami, Hisanori; Sakuragi, Noriaki; Tamashiro, Hiko; Fujimoto, Seiichiro

    2009-01-01

    Pregnancy-induced hypertension (PIH) is a common cause of perinatal mortality. It is believed to result from the interaction of several factors, including those related to the blood coagulation system. We performed genotyping and subgroup analyses to determine if the 4G/5G genotypes of the plasminogen activator inhibitor-1 gene (PAI-1) play a role in the pathogenesis of PIH, and to evaluate possible interactions of the PAI-1 polymorphisms with those of the angiotensinogen gene (AGT) and the endothelial nitric oxide synthase gene (NOS3). An association study of PAI-1 polymorphism, and subgroup analyses of common variants of AGT and NOS3, among 128 patients with PIH and 376 healthy pregnant controls. No significant differences were found between the cases and controls in the frequencies of allele 4G or the 4G/4G genotype. In subgroup analyses, after adjustment for multiple comparison, a significant association with the AGT TT genotype was found among women with the PAI-1 4G/4G genotype, and an association with the NOS3 GA+AA genotype was found among women with the 5G/5G or 4G/5G genotypes. Our findings suggest that there are at least 2 pathways in the pathogenesis of severe PIH. However, with respect to early prediction and prevention of severe PIH, although the PAI-1 4G/4G genotype alone was not a risk factor for severe PIH, the fact that PAI-1 genotypes are associated with varying risks for severe PIH suggests that PAI-1 genotyping of pregnant women, in combination with other tests, may be useful in the development of individualized measures that may prevent severe PIH.

  4. Role of Nitric Oxide Synthase on Blood Pressure Regulation and Vascular Function in Pregnant Rats on a High-Fat Diet.

    Science.gov (United States)

    Palei, Ana C; Spradley, Frank T; Granger, Joey P

    2017-03-01

    While obesity is a leading risk factor for preeclampsia, the mechanisms whereby obese women are more susceptible to pregnancy-induced hypertension are unclear. As high-fat diet (HFD) is an important contributor to the development of obesity, we tested the hypothesis that pregnant rats on HFD have hypertension and endothelial dysfunction due to reduced nitric oxide synthase (NOS). Twelve-week-old Sprague-Dawley female rats were fed normal diet (ND, 13% fat kcal) or HFD (40% fat kcal) for 9 weeks. Timed-pregnant rats were then generated and the effect of HFD on mean arterial blood pressure (MAP) and vascular function was assessed on gestational day (GD) 19. MAP was not different between HFD and ND pregnant rats. Intriguingly, sensitivity to acetylcholine-induced endothelium-dependent vasorelaxation was enhanced in small mesenteric arteries of HFD dams compared to ND controls (logEC50 -7.9 ± 0.3 vs. -6.7 ± 0.3 M; P hydrochloride (100 mg/l, drinking water) from GD 14 to 19. It was found that NOS inhibition increased MAP equally in HFD and ND groups. Contrary to our initial hypothesis, HFD dams were normotensive and presented increased endothelial function and NO/NOS3 levels. This enhanced NOS-mediated vascular function does not appear to have a major impact on blood pressure regulation of HFD-fed pregnant rats. © American Journal of Hypertension, Ltd 2017. All rights reserved. For Permissions, please email: journals.permissions@oup.com

  5. Glycated human serum albumin induces NF-κB activation and endothelial nitric oxide synthase uncoupling in human umbilical vein endothelial cells.

    Science.gov (United States)

    Rodiño-Janeiro, Bruno K; Paradela-Dobarro, Beatriz; Raposeiras-Roubín, Sergio; González-Peteiro, Mercedes; González-Juanatey, José R; Álvarez, Ezequiel

    2015-01-01

    Non-enzymatic glycated proteins could mediate diabetes vascular complications, but the molecular mechanisms are unknown. Our objective was to find new targets involved in the glycated human serum albumin (gHSA)-enhanced extracellular reactive oxygen species (ROS) production in human endothelial cells. Some nuclear factors and phosphorylation cascades were analysed. gHSA activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which up-regulated NOX4 and P22PHOX and enhanced ROS production. Pharmacological inhibition of NF-κB reversed gHSA-enhanced NOX4 expression and decreased gHSA-induced ROS production in extra- and intracellular spaces. The inhibition of activator protein-1 (AP-1) induced a rise in NOX4 and P22PHOX subunit expression and a down-regulation of endothelial nitric oxide synthase (eNOS). AP-1 inhibition also enhanced extracellular ROS production in the presence of serum albumin, but not with gHSA. These results were explained by the eNOS uncoupling induced by gHSA, also demonstrated in this study. Phosphatidylinositol 3-kinase or mitogen-activated protein kinase kinase 1/2 did not show to be involved in gHSA-induced ROS production. All together, the results suggested that gHSA-enhanced ROS production in endothelium is mediated by: 1) NF-κB activation and subsequence up-regulation of NADPH oxidase, 2) eNOS uncoupling. AP-1, although is not directly affected by gHSA, is another target for regulating NADPH oxidase and eNOS expression in endothelial cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Falcarindiol impairs the expression of inducible nitric oxide synthase by abrogating the activation of IKK and JAK in rat primary astrocytes.

    Science.gov (United States)

    Shiao, Young-Ji; Lin, Yun-Lian; Sun, Ya-Hui; Chi, Chih-Wen; Chen, Chieh-Fu; Wang, Chuen-Neu

    2005-01-01

    The effects of falcarindiol on the expression of inducible nitric oxide synthase (iNOS) induced by lipopolysaccharide/interferon-gamma (LPS/IFN-gamma) in rat primary astrocytes were investigated. The molecular mechanisms underlying falcarindiol that confers its effect on iNOS expression were also elucidated. Falcarindiol abrogated the LPS/IFN-gamma-mediated induction of iNOS by about 80%. Falcarindiol attenuated the induction of iNOS in a concentration-dependent manner. The inhibitory effect of falcarindiol on iNOS induction was attributable to decrease in the protein content and the mRNA level of iNOS. Treatment with 50 microM of falcarindiol for 30 min decreased LPS/IFN-gamma-induced nuclear factor-kappaB (NF-kappaB) activation by 32%. Treatment with 50 microM of falcarindiol for 60 min diminished the LPS/IFN-gamma-mediated activation of IkappaB kinase-alpha (IKK-alpha) and IKK-beta by 28.2 and 29.7%, respectively. Falcarindiol modulated the nuclear translocation of signal transducer and activator of transcription 1 (Stat1) in a time-dependent manner. Falcarindiol (50 microM) decreased the tyrosine phosphorylation of janus kinase 1 (JAK1) by 84.8% at 5 min. Falcarindiol also abrogated the tyrosine phoshorylation of JAK2 by 82.3% at 10 min.The present study demonstrates that falcarindiol attenuated the activation of IKK and JAK contributing to the blockade of activation of NF-kappaB and Stat1, thereby leading to the suppression of iNOS expression.

  7. Selective versus non-selective suppression of nitric oxide synthase on regional hemodynamics in rats with or without LPS-induced endotoxemia.

    Science.gov (United States)

    Cheng, Xing; Leung, Susan W S; Lo, Lawrence S; Pang, Catherine C Y

    2003-04-01

    The late phase of severe septic shock is associated with reduced cardiac output (CO) and activation of the inducible isoform of nitric oxide synthase (NOS). This study examined the effects of 1400 W (N-3-aminomethyl-benzyl-acetamidine), a new selective inhibitor of inducible NOS (iNOS), relative to those of N(G)-nitro-L-arginine (L-NNA, non-selective inhibitor of NOS) and the vehicle, on mean arterial pressure (MAP), CO, total peripheral resistance (TPR) and tissue blood flow (BF) in thiobutabarbital-anesthetized rats with lipopolysaccharide (LPS, 10 mg/kg, i.v.) induced endotoxemia. At 2.5 as well as 4 h after injection of LPS, MAP, CO, and BF of the stomach, skeletal muscle and skin were decreased, but TPR was increased, BF to the heart and kidneys were also decreased at 4 h after injection of LPS. Treatment of endotoxemic rats with 1400 W (3 mg/kg followed by 3 mg/kg/h, i.v.) at 2.5 h after endotoxin challenge prevented the late phase fall in MAP without exacerbating the decreases in CO and tissue BF. In contrast, treatment with L-NNA (8 mg/kg followed by 3 mg/kg/h, i.v.) at 2.5 h did not prevent the decline in MAP in the LPS-treated rats. Furthermore, CO drastically decreased, TPR markedly increased, and BF to the heart, brain, intestine and skeletal muscle were decreased at 4 h relative to the readings in saline- or 1400 W-treated endotoxemic rats. Therefore, selective inhibition of iNOS by 1400 W restores MAP without compromising CO, but non-selective inhibition of NOS is detrimental at the late stage of septic shock.

  8. Attempting to Compensate for Reduced Neuronal Nitric Oxide Synthase Protein with Nitrate Supplementation Cannot Overcome Metabolic Dysfunction but Rather Has Detrimental Effects in Dystrophin-Deficient mdx Muscle.

    Science.gov (United States)

    Timpani, Cara A; Trewin, Adam J; Stojanovska, Vanesa; Robinson, Ainsley; Goodman, Craig A; Nurgali, Kulmira; Betik, Andrew C; Stepto, Nigel; Hayes, Alan; McConell, Glenn K; Rybalka, Emma

    2017-04-01

    Duchenne muscular dystrophy arises from the loss of dystrophin and is characterized by calcium dysregulation, muscular atrophy, and metabolic dysfunction. The secondary reduction of neuronal nitric oxide synthase (nNOS) from the sarcolemma reduces NO production and bioavailability. As NO modulates glucose uptake, metabolism, and mitochondrial bioenergetics, we investigated whether an 8-week nitrate supplementation regimen could overcome metabolic dysfunction in the mdx mouse. Dystrophin-positive control (C57BL/10) and dystrophin-deficient mdx mice were supplemented with sodium nitrate (85 mg/l) in drinking water. Following the supplementation period, extensor digitorum longus and soleus were excised and radioactive glucose uptake was measured at rest (basal) and during contraction. Gastrocnemius was excised and mitochondrial respiration was measured using the Oroboros Oxygraph. Tibialis anterior was analyzed immunohistochemically for the presence of dystrophin, nNOS, nitrotyrosine, IgG and CD45+ cells, and histologically to assess areas of damage and regeneration. Glucose uptake in the basal and contracting states was normal in unsupplemented mdx muscles but was reduced following nitrate supplementation in mdx muscles only. The mitochondrial utilization of substrates was also impaired in mdx gastrocnemius during phosphorylating and maximal uncoupled respiration, and nitrate could not improve respiration in mdx muscle. Although nitrate supplementation reduced mitochondrial hydrogen peroxide emission, it induced mitochondrial uncoupling in red gastrocnemius, increased muscle fiber peroxynitrite (nitrotyrosine), and promoted skeletal muscle damage. Our novel data suggest that despite lower nNOS protein expression and likely lower NO production in mdx muscle, enhancing NO production with nitrate supplementation in these mice has detrimental effects on skeletal muscle. This may have important relevance for those with DMD.

  9. Type I Interferon Supports Inducible Nitric Oxide Synthase in Murine Hepatoma Cells and Hepatocytes and during Experimental Acetaminophen-Induced Liver Damage

    Directory of Open Access Journals (Sweden)

    Malte Bachmann

    2017-07-01

    Full Text Available Cytokine regulation of high-output nitric oxide (NO derived from inducible NO synthase (iNOS is critically involved in inflammation biology and host defense. Herein, we set out to characterize the role of type I interferon (IFN as potential regulator of hepatic iNOS in vitro and in vivo. In this regard, we identified in murine Hepa1-6 hepatoma cells a potent synergism between pro-inflammatory interleukin-β/tumor necrosis factor-α and immunoregulatory IFNβ as detected by analysis of iNOS expression and nitrite release. Upregulation of iNOS by IFNβ coincided with enhanced binding of signal transducer and activator of transcription-1 to a regulatory region at the murine iNOS promoter known to support target gene expression in response to this signaling pathway. Synergistic iNOS induction under the influence of IFNβ was confirmed in alternate murine Hepa56.1D hepatoma cells and primary hepatocytes. To assess iNOS regulation by type I IFN in vivo, murine acetaminophen (APAP-induced sterile liver inflammation was investigated. In this model of acute liver injury, excessive necroinflammation drives iNOS expression in diverse liver cell types, among others hepatocytes. Herein, we demonstrate impaired iNOS expression in type I IFN receptor-deficient mice which associated with diminished APAP-induced liver damage. Data presented indicate a vital role of type I IFN within the inflamed liver for fine-tuning pathological processes such as overt iNOS expression.

  10. No evidence of a role for neuronal nitric oxide synthase in the nucleus tractus solitarius in ventilatory responses to acute or chronic hypoxia in awake rats

    Science.gov (United States)

    Pamenter, Matthew E.; Go, Ariel; Fu, Zhenxing

    2015-01-01

    When exposed to a hypoxic environment, the body's first response is a reflex increase in ventilation, termed the hypoxic ventilatory response (HVR). With chronic sustained hypoxia (CSH), such as during acclimatization to high altitude, an additional time-dependent increase in ventilation occurs, which increases the HVR and is termed ventilatory acclimatization to hypoxia (VAH). This secondary increase persists after exposure to CSH and involves plasticity within the circuits in the central nervous system that control breathing. The mechanisms of HVR plasticity are currently poorly understood. We hypothesized that changes in neuronal nitric oxide synthase (nNOS) activity or expression in the nucleus tractus solitarius contribute to this plasticity and underlie VAH in rats. To test this, we treated rats held in normoxia or 10% O2 (CSH, PiO2 = 70 Torr) for 7–9 days and measured ventilation in conscious, unrestrained animals before and after microinjecting the general NOS antagonist L-NG-Nitroarginine methyl ester into the nucleus tractus solitarius (NTS) or systemically injecting the nNOS-specific antagonist S-methyl-l-thiocitrulline. Localization of injection sites in the NTS was confirmed by histology following the experiment. We found that 1) neither NTS-specific nor systemic nNOS antagonism had any effect on hypoxia-mediated changes in breathing or metabolism (P > 0.05), but 2) nNOS protein expression was increased in the middle and caudal NTS by CSH. A persistent HVR after nNOS blockade in the NTS contrasts with results in awake mice, and our findings do not support the hypotheses that nNOS in the NTS contribute to the HVR or VAH in awake rats. PMID:25571988

  11. A common variant of endothelial nitric oxide synthase (Glu298Asp) is associated with collateral development in patients with chronic coronary occlusions

    International Nuclear Information System (INIS)

    Lamblin, Nicolas; Cuilleret, François J; Helbecque, Nicole; Dallongeville, Jean; Lablanche, Jean-Marc; Amouyel, Philippe; Bauters, Christophe; Van Belle, Eric

    2005-01-01

    Experimental studies support an important role for endothelial nitric oxide synthase (eNOS) in the regulation of angiogenesis. In humans, a common polymorphism exists in the eNOS gene that results in the conversion of glutamate to aspartate for codon 298. In vitro and in vivo studies have suggested a decreased NOS activity in patients with the Asp 298 variant. We hypothesized that a genetic-mediated decreased eNOS activity may limit collateral development in patients with chronic coronary occlusions. We selected 291 consecutive patients who underwent coronary angiography and who had at least one chronic (>15 days) total coronary occlusion. Collateral development was graded angiographically using two different methods: the collateral flow grade and the recipient filling grade. Genomic DNA was extracted from white blood cells and genotyping was performed using previously published techniques. Collateral development was lower in patients carrying the Asp 298 variant than in Glu-Glu homozygotes (collateral flow grade: 2.64 ± 0.08 and 2.89 ± 0.08, respectively, p = 0.04; recipient filling grade: 3.00 ± 0.08 and 3.24 ± 0.07, respectively, p = 0.04). By multivariable analysis, three variables were independently associated with the collateral flow grade: female gender, smoking, and the Asp 298 variant (p = 0.03) while the Asp 298 variant was the sole variable independently associated with the recipient filling grade (p = 0.03). Collateral development is lower in patients with the Asp 298 variant. This may be explained by the decreased NOS activity in patients with the Asp 298 variant. Further studies will have to determine whether increasing eNOS activity in humans is associated with coronary collateral development

  12. Inducible nitric oxide synthase is key to peroxynitrite-mediated, LPS-induced protein radical formation in murine microglial BV2 cells.

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    Kumar, Ashutosh; Chen, Shih-Heng; Kadiiska, Maria B; Hong, Jau-Shyong; Zielonka, Jacek; Kalyanaraman, Balaraman; Mason, Ronald P

    2014-08-01

    Microglia are the resident immune cells in the brain. Microglial activation is characteristic of several inflammatory and neurodegenerative diseases including Alzheimer's disease, multiple sclerosis, and Parkinson's disease. Though lipopolysaccharide (LPS)-induced microglial activation in models of Parkinson's disease is well documented, the free radical-mediated protein radical formation and its underlying mechanism during LPS-induced microglial activation are not known. Here we have used immuno-spin trapping and RNA interference to investigate the role of inducible nitric oxide synthase (iNOS) in peroxynitrite-mediated protein radical formation in murine microglial BV2 cells treated with LPS. Treatment of BV2 cells with LPS resulted in morphological changes, induction of iNOS, and increased protein radical formation. Pretreatments with FeTPPS (a peroxynitrite decomposition catalyst), L-NAME (total NOS inhibitor), 1400W (iNOS inhibitor), and apocynin significantly attenuated LPS-induced protein radical formation and tyrosine nitration. Results obtained with coumarin-7-boronic acid, a highly specific probe for peroxynitrite detection, correlated with LPS-induced tyrosine nitration, which demonstrated involvement of peroxynitrite in protein radical formation. A similar degree of protection conferred by 1400W and L-NAME led us to conclude that only iNOS, and no other forms of NOS, is involved in LPS-induced peroxynitrite formation. Subsequently, siRNA for iNOS, the iNOS-specific inhibitor 1400W, the NF-κB inhibitor PDTC, and the p38 MAPK inhibitor SB202190 was used to inhibit iNOS directly or indirectly. Inhibition of iNOS precisely correlated with decreased protein radical formation in LPS-treated BV2 cells. The time course of protein radical formation also matched the time course of iNOS expression. Taken together, these results prove the role of iNOS in peroxynitrite-mediated protein radical formation in LPS-treated microglial BV2 cells. Copyright © 2014

  13. Metformin Ameliorates Dysfunctional Traits of Glibenclamide- and Glucose-Induced Insulin Secretion by Suppression of Imposed Overactivity of the Islet Nitric Oxide Synthase-NO System.

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    Ingmar Lundquist

    Full Text Available Metformin lowers diabetic blood glucose primarily by reducing hepatic gluconeogenesis and increasing peripheral glucose uptake. However, possible effects by metformin on beta-cell function are incompletely understood. We speculated that metformin might positively influence insulin secretion through impacting the beta-cell nitric oxide synthase (NOS-NO system, a negative modulator of glucose-stimulated insulin release. In short-time incubations with isolated murine islets either glibenclamide or high glucose augmented insulin release associated with increased NO production from both neural and inducible NOS. Metformin addition suppressed the augmented NO generation coinciding with amplified insulin release. Islet culturing with glibenclamide or high glucose revealed pronounced fluorescence of inducible NOS in the beta-cells being abolished by metformin co-culturing. These findings were reflected in medium nitrite-nitrate levels. A glucose challenge following islet culturing with glibenclamide or high glucose revealed markedly impaired insulin response. Metformin co-culturing restored this response. Culturing murine islets and human islets from controls and type 2 diabetics with high glucose or high glucose + glibenclamide induced a pronounced decrease of cell viability being remarkably restored by metformin co-culturing. We show here, that imposed overactivity of the beta-cell NOS-NO system by glibenclamide or high glucose leads to insulin secretory dysfunction and reduced cell viability and also, importantly, that these effects are relieved by metformin inhibiting beta-cell NO overproduction from both neural and inducible NOS thus ameliorating a concealed negative influence by NO induced by sulfonylurea treatment and/or high glucose levels. This double-edged effect of glibenclamide on the beta-cellsuggests sulfonylurea monotherapy in type 2 diabetes being avoided.

  14. Single-dose rosuvastatin ameliorates lung ischemia-reperfusion injury via upregulation of endothelial nitric oxide synthase and inhibition of macrophage infiltration in rats with pulmonary hypertension.

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    Matsuo, Satoshi; Saiki, Yuriko; Adachi, Osamu; Kawamoto, Shunsuke; Fukushige, Shinichi; Horii, Akira; Saiki, Yoshikatsu

    2015-03-01

    Lung ischemia-reperfusion (IR) injury during cardiopulmonary surgery is associated with postoperative morbidity and mortality, particularly in patients with pulmonary hypertension (PH). Using a rat model for monocrotaline-induced PH, we investigated the protective effect of rosuvastatin against IR injury in lungs affected by PH and attempted to elucidate its mechanism of action. Male Sprague-Dawley monocrotaline-treated rats were divided into 4 groups (n = 8-9): sham, control + IR, statin + IR, and statin + mevalonolactone + IR. Lung ischemia was induced by left pulmonary artery occlusion (1 hour), followed by reperfusion (4 hours). Rosuvastatin (2 mg/kg) was injected 18 hours before reperfusion and mevalonolactone (1 mg/kg) was injected immediately before reperfusion. The arterial oxygen tension/inspired oxygen fraction ratio was used as a measure of lung oxygenation. Left lung tissue was analyzed for the wet-to-dry lung weight ratio and protein expression of endothelial nitric oxide synthase (eNOS) and phospho-eNOS. Macrophage recruitment was assessed by CD68 immunostaining. Our results showed that rosuvastatin decreased IR lung injury (control + IR vs statin + IR) in terms of the arterial oxygen tension/inspired oxygen fraction ratio (272 ± 43 vs 442 ± 13), wet-to-dry ratio (5.7 ± 0.7 vs 4.8 ± 0.6), and macrophage infiltration (8.0 ± 0.6/field vs 4.0 ± 0.5/field) (P < .05 for all). eNOS and phospho-eNOS were downregulated by IR, which was blocked by rosuvastatin. Effects of rosuvastatin were blunted by mevalonolactone. Single-dose rosuvastatin decreased IR injury in lungs affected by PH via 2 anti-inflammatory mechanisms: preserving eNOS function and inhibiting macrophage infiltration. Copyright © 2015 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

  15. Exercise training provides cardioprotection by activating and coupling endothelial nitric oxide synthase via a β3-adrenergic receptor-AMP-activated protein kinase signaling pathway

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    Larry A Barr

    2017-01-01

    Full Text Available Exercise training confers sustainable protection against ischemia/reperfusion injury. However, the mechanism by which this process occurs is not fully understood. Previously, it was shown that β3-adrenergic receptors (β3-ARs play a critical role in regulating the activation of endothelial nitric oxide synthase (eNOS in response to exercise and play a critical role in exercise-mediated cardioprotection. Intriguingly, a deficiency in β3-ARs led to increased myocardial injury following exercise training. The purpose of the current study was to determine mechanisms by which β3-ARs are linked to eNOS activation and to determine the mechanism responsible for the exacerbated ischemia/reperfusion injury displayed by β3-AR deficient (β3-AR KO mice after exercise training. Wild-type (n = 37 and β3-AR KO ( n = 40 mice were subjected to voluntary wheel running for 4 weeks. Western blot analysis revealed that neither protein kinase B nor protein kinase A linked β3-ARs to eNOS following exercise training. However, analysis revealed a role for AMP-activated protein kinase (AMPK. Specifically, exercise training increased the phosphorylation of AMPK in the hearts of wild-type mice, but failed to do so in the hearts of β3-AR KO mice. Additional studies revealed that exercise training rendered eNOS less coupled and increased NOS-dependent superoxide levels in β3-AR KO mice. Finally, supplementing β3-AR KO mice with the eNOS coupler, tetrahydrobiopterin, during the final week of exercise training reduced myocardial infarction. These findings provide important information that exercise training protects the heart in the setting of myocardial ischemia/reperfusion injury by activating and coupling eNOS via the stimulation of a β3-AR-AMPK signaling pathway.

  16. Inhibitor of neuronal nitric oxide synthase improves gas exchange in ventilator-induced lung injury after pneumonectomy.

    Science.gov (United States)

    Suborov, Evgeny V; Smetkin, Alexey A; Kondratiev, Timofey V; Valkov, Andrey Y; Kuzkov, Vsevolod V; Kirov, Mikhail Y; Bjertnaes, Lars J

    2012-06-21

    Mechanical ventilation with high tidal volumes may cause ventilator-induced lung injury (VILI) and enhanced generation of nitric oxide (NO). We demonstrated in sheep that pneumonectomy followed by injurious ventilation promotes pulmonary edema. We wished both to test the hypothesis that neuronal NOS (nNOS), which is distributed in airway epithelial and neuronal tissues, could be involved in the pathogenesis of VILI and we also aimed at investigating the influence of an inhibitor of nNOS on the course of VILI after pneumonectomy. Anesthetized sheep underwent right pneumonectomy, mechanical ventilation with tidal volumes (VT) of 6 mL/kg and FiO2 0.5, and were subsequently randomized to a protectively ventilated group (PROTV; n = 8) keeping VT and FiO2 unchanged, respiratory rate (RR) 25 inflations/min and PEEP 4 cm H2O for the following 8 hrs; an injuriously ventilated group with VT of 12 mL/kg, zero end-expiratory pressure, and FiO2 and RR unchanged (INJV; n = 8) and a group, which additionally received the inhibitor of nNOS, 7-nitroindazole (NI) 1.0 mg/kg/h intravenously from 2 hours after the commencement of injurious ventilation (INJV + NI; n = 8). We assessed respiratory, hemodynamic and volumetric variables, including both the extravascular lung water index (EVLWI) and the pulmonary vascular permeability index (PVPI). We measured plasma nitrite/nitrate (NOx) levels and examined lung biopsies for lung injury score (LIS). Both the injuriously ventilated groups demonstrated a 2-3-fold rise in EVLWI and PVPI, with no significant effects of NI. In the INJV group, gas exchange deteriorated in parallel with emerging respiratory acidosis, but administration of NI antagonized the derangement of oxygenation and the respiratory acidosis significantly. NOx displayed no significant changes and NI exerted no significant effect on LIS in the INJV group. Inhibition of nNOS improved gas exchange, but did not reduce lung water extravasation following

  17. Inhibitor of neuronal nitric oxide synthase improves gas exchange in ventilator-induced lung injury after pneumonectomy

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    Suborov Evgeny V

    2012-06-01

    Full Text Available Abstract Background Mechanical ventilation with high tidal volumes may cause ventilator-induced lung injury (VILI and enhanced generation of nitric oxide (NO. We demonstrated in sheep that pneumonectomy followed by injurious ventilation promotes pulmonary edema. We wished both to test the hypothesis that neuronal NOS (nNOS, which is distributed in airway epithelial and neuronal tissues, could be involved in the pathogenesis of VILI and we also aimed at investigating the influence of an inhibitor of nNOS on the course of VILI after pneumonectomy. Methods Anesthetized sheep underwent right pneumonectomy, mechanical ventilation with tidal volumes (VT of 6 mL/kg and FiO2 0.5, and were subsequently randomized to a protectively ventilated group (PROTV; n = 8 keeping VT and FiO2 unchanged, respiratory rate (RR 25 inflations/min and PEEP 4 cm H2O for the following 8 hrs; an injuriously ventilated group with VT of 12 mL/kg, zero end-expiratory pressure, and FiO2 and RR unchanged (INJV; n = 8 and a group, which additionally received the inhibitor of nNOS, 7-nitroindazole (NI 1.0 mg/kg/h intravenously from 2 hours after the commencement of injurious ventilation (INJV + NI; n = 8. We assessed respiratory, hemodynamic and volumetric variables, including both the extravascular lung water index (EVLWI and the pulmonary vascular permeability index (PVPI. We measured plasma nitrite/nitrate (NOx levels and examined lung biopsies for lung injury score (LIS. Results Both the injuriously ventilated groups demonstrated a 2–3-fold rise in EVLWI and PVPI, with no significant effects of NI. In the INJV group, gas exchange deteriorated in parallel with emerging respiratory acidosis, but administration of NI antagonized the derangement of oxygenation and the respiratory acidosis significantly. NOx displayed no significant changes and NI exerted no significant effect on LIS in the INJV group. Conclusion Inhibition of nNOS improved gas exchange

  18. Regional age-related changes in neuronal nitric oxide synthase (nNOS, messenger RNA levels and activity in SAMP8 brain

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    Guidon Gérard

    2006-12-01

    Full Text Available Abstract Background Nitric oxide (NO is a multifunctional molecule synthesized by three isozymes of the NO synthase (NOSs acting as a messenger/modulator and/or a potential neurotoxin. In rodents, the role of NOSs in sleep processes and throughout aging is now well established. For example, sleep parameters are highly deteriorated in senescence accelerated-prone 8 (SAMP8 mice, a useful animal model to study aging or age-associated disorders, while the inducible form of NOS (iNOS is down-regulated within the cortex and the sleep-structures of the brainstem. Evidence is now increasing for a role of iNOS and resulting oxidative stress but not for the constitutive expressed isozyme (nNOS. To better understand the role of nNOS in the behavioural impairments observed in SAMP8 versus SAMR1 (control animals, we evaluated age-related variations occurring in the nNOS expression and activity and nitrites/nitrates (NOx- levels, in three brain areas (n = 7 animals in each group. Calibrated reverse transcriptase (RT and real-time polymerase chain reaction (PCR and biochemical procedures were used. Results We found that the levels of nNOS mRNA decreased in the cortex and the hippocampus of 8- vs 2-month-old animals followed by an increase in 12-vs 8-month-old animals in both strains. In the brainstem, levels of nNOS mRNA decreased in an age-dependent manner in SAMP8, but not in SAMR1. Regional age-related changes were also observed in nNOS activity. Moreover, nNOS activity in hippocampus was found lower in 8-month-old SAMP8 than in SAMR1, while in the cortex and the brainstem, nNOS activities increased at 8 months and afterward decreased with age in SAMP8 and SAMR1. NOx- levels showed profiles similar to nNOS activities in the cortex and the brainstem but were undetectable in the hippocampus of SAMP8 and SAMR1. Finally, NOx- levels were higher in the cortex of 8 month-old SAMP8 than in age-matched SAMR1. Conclusion Concomitant variations occurring in NO levels

  19. Gender-based reciprocal expression of transforming growth factor-β1 and the inducible nitric oxide synthase in a rat model of cyclophosphamide-induced cystitis

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    Loughran Patricia A

    2009-08-01

    Full Text Available Abstract Background The pluripotent cytokine transforming growth factor-β1 (TGF-β1 is the central regulator of inducible Nitric Oxide Synthase (iNOS that is responsible for nitric oxide (NO production in inflammatory settings. Previous studies have implicated a role for NO, presumably derived from iNOS, in cyclophosphamide (CYP-induced cystitis in the bladder. TGF-β1 is produced in latent form and requires dissociation from the latency-associated peptide (LAP to act as primary anti-inflammatory and pro-healing modulator following tissue injury in the upper urinary tract. Since the role of TGF-β1 in lower urinary tract inflammation is currently unknown, and since gender-based differences exist in the setting of interstitial cystitis (IC, the present study examined the relationship between TGF-β1 and iNOS/NO in the pathogenesis of CYP-induced cystitis in both male and female rats. Methods Sprague-Dawley rats, 4 months of age, of either gender were given 150 mg/kg CYP intraperitoneally. Urinary and bladder tissue TGF-β1 and NO reaction products (NO2-/NO3- were quantified as a function of time following CYP. Expression of active and latent TGF-β1 as well as iNOS in harvested bladder tissue was assessed by immunohistochemistry. Results Female rats had significantly higher levels of NO2-/NO3- in urine even at baseline as compared to male rats (p 2-/NO3- and TGF-β1. Male rats responded to CYP with significantly lower levels of NO2-/NO3- and significantly higher levels of TGF-β1 in urine (p 2-/NO3- after CYP were inversely correlated to latent and active TGF-β1 (Pearson coefficient of -0.72 and -0.69 in females and -0.89 and -0.76 in males, respectively; p Conclusion The results of this study suggest that there exists an inverse relationship between the expression of TGF-β1 and iNOS/NO2-/NO3- in CYP-inflamed bladder. The gender of the animal appears to magnify the differences in urine levels of TGF-β1 and NO2-/NO3- in this inflammatory

  20. Process-driven bacterial community dynamics are key to cured meat colour formation by coagulase-negative staphylococci via nitrate reductase or nitric oxide synthase activities.

    Science.gov (United States)

    Sánchez Mainar, María; Leroy, Frédéric

    2015-11-06

    The cured colour of European raw fermented meats is usually achieved by nitrate-into-nitrite reduction by coagulase-negative staphylococci (CNS), subsequently generating nitric oxide to form the relatively stable nitrosomyoglobin pigment. The present study aimed at comparing this classical curing procedure, based on nitrate reductase activity, with a potential alternative colour formation mechanism, based on nitric oxide synthase (NOS) activity, under different acidification profiles. To this end, meat models with and without added nitrate were fermented with cultures of an acidifying strain (Lactobacillus sakei CTC 494) and either a nitrate-reducing Staphylococcus carnosus strain or a rare NOS-positive CNS strain (Staphylococcus haemolyticus G110), or by relying on the background microbiota. Satisfactory colour was obtained in the models prepared with added nitrate and S. carnosus. In the presence of nitrate but absence of added CNS, however, cured colour was only obtained when L. sakei CTC 494 was also omitted. This was ascribed to the pH dependency of the emerging CNS background microbiota, selecting for nitrate-reducing Staphylococcus equorum strains at mild acidification conditions but for Staphylococcus saprophyticus strains with poor colour formation capability when the pH decrease was more rapid. This reliance of colour formation on the composition of the background microbiota was further explored by a side experiment, demonstrating the heterogeneity in nitrate reduction of a set of 88 CNS strains from different species. Finally, in all batches prepared with S. haemolyticus G110, colour generation failed as the strain was systematically outcompeted by the background microbiota, even when imposing milder acidification profiles. Thus, when aiming at colour formation through CNS metabolism, technological processing can severely interfere with the composition and functionality of the meat-associated CNS communities, for both nitrate reductase and NOS activities

  1. 33 CFR 80.1440 - Port Allen, Kauai, HI.

    Science.gov (United States)

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Port Allen, Kauai, HI. 80.1440 Section 80.1440 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY INTERNATIONAL NAVIGATION RULES COLREGS DEMARCATION LINES Pacific Islands § 80.1440 Port Allen, Kauai, HI. A line drawn from...

  2. Gold-catalyzed intermolecular hydroamination of allenes with sulfonamides

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    Chen Zhang

    2013-05-01

    Full Text Available A co-catalyst of (PPh3AuCl/AgOTf for the intermolecular hydroamination of allenes with sulfonamides is shown. The reaction proceeded smoothly under mild conditions for differently substituted allenes giving N-allylic sulfonamides in good yields with high regioselectivity and E-selectivity.

  3. Oxidant stress evoked damage in rat hepatocyte leading to triggered nitric oxide synthase (NOS levels on long term consumption of aspartame

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    Iyaswamy Ashok

    2015-12-01

    Full Text Available This study investigates how long-term (40 mg/kg b.wt consumption of aspartame can alter the antioxidant status, stress pathway genes, and apoptotic changes in the liver of Wistar albino rats. Numerous controversial reports are available on the use of aspartame as it releases methanol as one of its metabolites during metabolism. To mimic the human methanol metabolism the methotrexate treated rats were included to study the aspartame effects. The aspartame treated methotrexate (MTX animals showed a marked significant increase in the superoxide dismutase (SOD, catalase (CAT, lipid peroxidation (LPO, and Glutathione peroxidase (GPx activity in the liver from control and MTX control animals, and showed a significant decrease in reduced glutathione (GSH and protein thiol in aspartame treated animals. The aspartame treated MTX animals showed a marked significant decrease in the body weight, brain, and liver weight. The aspartame treated MTX animals showed a marked increase in the inducible nitric oxide (iNOS, neuronal nitric oxide (nNOS, c-fos, Heat shock protein (Hsp 70 Tumour necrosis Factor (TNFα, caspase 8, c-jun N terminal kinases (JNK 3 and Nuclear factor kappa B (NFkB gene expression in the liver from control and MTX control animals. The aspartame treated MTX animals showed a marked increase in the c-fos, Hsp 70, iNOS Caspase 8, and JNK 3 protein expression in the liver from control and MTX control animals indicating the enhancement of stress and apoptosis. The aspartame treated MTX animals showed a streak of marked DNA fragmentation in the liver. On immunohistochemical analysis aspartame treated animals showed brown colored positive hepatocytes indicating the stress specific and apoptotic protein expression. Since aspartame consumption is on the rise among people, it is essential to create awareness regarding the usage of this artificial sweetener.

  4. Allen diesel standby sets at Sellafield

    International Nuclear Information System (INIS)

    Ruff, G.

    1982-01-01

    As part of the extension and improvement plans for the British Nuclear Fuels Ltd reprocessing facilities at Sellafield, three Allen diesel engines have been installed for essential standby duty. They are 16-cylinder V-form engines with a continuous rating of 2,832 bhp and drive a 2000kW 11,000 volt 3 phase 50Hz salient pole synchronous alternator at 750r/min. They are arranged for running in parallel and with the public electricity mains supply and are also controlled to operate as standby in the event of mains failure. To prevent radioactive contamination from the atmosphere, primary and secondary air filtration has been supplied for each engine. Other operational facilities and safeguards are mentioned and the cooling system described. (U.K.)

  5. The cardioprotective effects of thoracal epidural anestesia are induced by the expression of vascular endothelial growth factor and inducible nitric oxide synthase in cardiopulmonary bypass surgery.

    Science.gov (United States)

    Gonca, S; Kiliçkan, L; Dalçik, C; Dalçik, H; Bayindir, O

    2007-02-01

    The cardioprotective effects of thoracal epidural anesthesia (TEA) are induced by the expression of vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (i-NOS) in cardiopulmonary bypass (CPB) surgery. When general anaesthesia (GA) is combined with TEA during coronary artery bypass graft, we investigated whether TEA together with GA play a role on VEGF and i-NOS expression in human heart tissue in cardiac ischemia. Right atrial biopsy samples were taken before CPB, before aortic cross clamp (ACC) and at 15 min after ACC release (after ischemia and reperfusion). Human heart tissues were obtained from the TEA+GA and GA groups. Immunocytochemistry was performed using antibodies for VEGF and i-NOS. Both VEGF and i-NOS immunoreactivity was observed in cardiomyocytes and arteriol walls. Although VEGF and i-NOS immunoreactivity was apparent in both groups,, immunostaining intensity was greater in the TEA+GA group than the GA group. Between groups, at 4 h and at 24 h after the end of CPB, the cardiac index (CI) was significantly higher in the TEA+GA group than GA group (3.4+/-0.8 L/min/m(2) vs 2.5+/-0.8 L/min/m(2); P0.05), (2.6+/-0.8 L/min/m(2) vs 3.1+/-1.1 L/min/m(2); P>0.05) respectively. After ACC release, 11/40 (27.5%) patients in the TEA+GA group showed ventricular fibrillation (VF), atrial fibrillation or heart block versus 25/40 (62.5%) of those in the GA group. VF after ACC release in the TEA+GA group (9/20 patients, 22.5%) was significantly lower than in the GA group (21/40 patients, 52.5%); (P<0.006). Sinus rhythm after ACC release in the TEA+GA group (29/40 patients, 72.5%) was significantly higher than in the GA group (15/40 patients, 37.5%); (P<0.002). The results of the present study indicate that TEA plus GA in coronary surgery preserve cardiac function via increased expression of VEGF and i-NOS, improved hemodynamic function and reduced arrhythmias after ACC release.

  6. Heme oxygenase-1 plays a pro-life role in experimental brain stem death via nitric oxide synthase I/protein kinase G signaling at rostral ventrolateral medulla

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    Dai Kuang-Yu

    2010-09-01

    Full Text Available Abstract Background Despite its clinical importance, a dearth of information exists on the cellular and molecular mechanisms that underpin brain stem death. A suitable neural substrate for mechanistic delineation on brain stem death resides in the rostral ventrolateral medulla (RVLM because it is the origin of a life-and-death signal that sequentially increases (pro-life and decreases (pro-death to reflect the advancing central cardiovascular regulatory dysfunction during the progression towards brain stem death in critically ill patients. The present study evaluated the hypothesis that heme oxygnase-1 (HO-1 may play a pro-life role as an interposing signal between hypoxia-inducible factor-1 (HIF-1 and nitric oxide synthase I (NOS I/protein kinase G (PKG cascade in RVLM, which sustains central cardiovascular regulatory functions during brain stem death. Methods We performed cardiovascular, pharmacological, biochemical and confocal microscopy experiments in conjunction with an experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol bilaterally into RVLM of adult male Sprague-Dawley rats. Results Western blot analysis coupled with laser scanning confocal microscopy revealed that augmented HO-1 expression that was confined to the cytoplasm of RVLM neurons occurred preferentially during the pro-life phase of experimental brain stem death and was antagonized by immunoneutralization of HIF-1α or HIF-1β in RVLM. On the other hand, the cytoplasmic presence of HO-2 in RVLM neurons manifested insignificant changes during both phases. Furthermore, immunoneutralization of HO-1 or knockdown of ho-1 gene in RVLM blunted the augmented life-and-death signals exhibited during the pro-life phase. Those pretreatments also blocked the upregulated pro-life NOS I/PKG signaling without affecting the pro-death NOS II/peroxynitrite cascade in RVLM. Conclusions We conclude that transcriptional

  7. Expression of p53, inducible nitric oxide synthase and vascular endothelial growth factor in gastric precancerous and cancerous lesions: correlation with clinical features

    International Nuclear Information System (INIS)

    Feng, Chang Wei; Wang, Li Dong; Jiao, Lian Hua; Liu, Bin; Zheng, Shu; Xie, Xin Ji

    2002-01-01

    The growth and metastasis of tumors depend on the development of an adequate blood supply via angiogenesis. Recent studies indicate that the inducible nitric oxide synthase (iNOS), vascular endothelial growth factor (VEGF) and the tumor suppressor p53 are fundamental play-markers of the angiogenic process. Overexpression of iNOS and VEGF has been shown to induce angiogenesis in tumors. P53 suppresses angiogenesis by down-regulating VEGF and iNOS. The correlation of expression of p53, VEGF and iNOS and clinical features in gastric carcinogenesis, however, has not been well characterized. The expression of p53, iNOS and VEGF in gastric precancerous and cancerous lesions and its relation with the clinical features was determined with immunohistochemistry (avidin-biotin-peroxidase complex method) on 55 randomly selected GC patients and 60 symptom-free subjects from the mass survey in the high-incidence area for GC in Henan, northern China. The positive immunostainig rates for p53, iNOS and VEGF in gastric carcinomas were 51%, 44% and 51%, respectively, and correlated well with TNM stages, but did not show significant difference among the groups with different degrees of gastric wall invasion depth by GC. A positive immunostaining reaction for the iNOS protein was significantly correlated with lymph node metastasis (p = 0.019; Spearman correlation coefficient). P53 protein accumulation was higher in the poorly-differentiated gastric carcinoma than in well-differentiated one. In gastric biopsies, no positive immunosatining was observed for p53, iNOS and VEGF in the histologically normal tissue and chronic superficial gastritis (CSG). However, p53, iNOS and VEGF positive immunostaining was observed in the tissues with different severities of lesions of chronic atrophic gastritis (CAG), intestinal metaplasia (IM) and dysplasia (DYS), and the positive rates increased with the lesion progression from CAG to IM to DYS. A high coincidental positive and negative immunostaining

  8. Protein kinase A-dependent Neuronal Nitric Oxide Synthase Activation Mediates the Enhancement of Baroreflex Response by Adrenomedullin in the Nucleus Tractus Solitarii of Rats

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    Ho I-Chun

    2011-05-01

    Full Text Available Abstract Background Adrenomedullin (ADM exerts its biological functions through the receptor-mediated enzymatic mechanisms that involve protein kinase A (PKA, or neuronal nitric oxide synthase (nNOS. We previously demonstrated that the receptor-mediated cAMP/PKA pathway involves in ADM-enhanced baroreceptor reflex (BRR response. It remains unclear whether ADM may enhance BRR response via activation of nNOS-dependent mechanism in the nucleus tractus solitarii (NTS. Methods Intravenous injection of phenylephrine was administered to evoke the BRR before and at 10, 30, and 60 min after microinjection of the test agents into NTS of Sprague-Dawley rats. Western blotting analysis was used to measure the level and phosphorylation of proteins that involved in BRR-enhancing effects of ADM (0.2 pmol in NTS. The colocalization of PKA and nNOS was examined by immunohistochemical staining and observed with a laser confocal microscope. Results We found that ADM-induced enhancement of BRR response was blunted by microinjection of NPLA or Rp-8-Br-cGMP, a selective inhibitor of nNOS or protein kinase G (PKG respectively, into NTS. Western blot analysis further revealed that ADM induced an increase in the protein level of PKG-I which could be attenuated by co-microinjection with the ADM receptor antagonist ADM22-52 or NPLA. Moreover, we observed an increase in phosphorylation at Ser1416 of nNOS at 10, 30, and 60 min after intra-NTS administration of ADM. As such, nNOS/PKG signaling may also account for the enhancing effect of ADM on BRR response. Interestingly, biochemical evidence further showed that ADM-induced increase of nNOS phosphorylation was prevented by co-microinjection with Rp-8-Br-cAMP, a PKA inhibitor. The possibility of PKA-dependent nNOS activation was substantiated by immunohistochemical demonstration of co-localization of PKA and nNOS in putative NTS neurons. Conclusions The novel finding of this study is that the signal transduction cascade that

  9. Induced nitric oxide synthase (iNOS and indoleamine 2,3-dioxygenase (IDO detection in circulating monocyte subsets from Brazilian patients with Dengue-4 virus

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    Luciana Gomes Fialho

    2017-06-01

    Full Text Available Among viral diseases transmitted by mosquitoes, dengue is characterized by its rapid dispersion around the world. Dengue severity is associated to a cytokine “storm” leading to vascular hemorrhagic manifestations, plasma leakage and shock, but also producing viral clearance. Macrophage/monocyte activation occurs during infection. Monocyte lineage cells are among those that allow virus replication. We investigated circulating human monocyte subsets - classical CD14+CD16− and non-classical CD14+CD16+ - during DENV-4 infection in patients. Intracellular inducible nitric oxide synthase (iNOS and indoleamine 2,3–dioxygenase (IDO were detected in both monocyte subsets. Circulating CD14+CD16+ monocyte frequency is mildly increased during DENV-4 infection. INOS is more intensely detected in CD14+CD16− than in CD16+ monocytes and IDO in CD14+CD16+. DENV-4 patients show increase in NO, TNF-α, IFN-y, IP-10/CXL10, IL-10 and MCP-1/CCL2 plasma levels when compared to healthy individuals. The classical monocyte subset, CD14+CD16− was shown to be inversely correlated with IL-10 and IP-10/CXCL10 levels, while the non-classical CD14+CD16+ is positively correlated with IL-10 cytokine. TNF-α, IL-10 cytokines and IP-10/CXL10 chemokine are positively correlated with the CD14+iNOS+ monocyte population. Both CD14+ cells - CD16−iNOS+ and CD16+iNOS+ subsets - presented positive correlation with IL-10, IP-10/CXL10 and MCP-1/CCL2, besides TNF-α associated with CD16−iNOS+ cells. CD14+CD16−IDO+and CD16+IDO+ populations correlated positively with IL-10. Furthermore, CD16−IDO+ monocyte subset also presented a positive correlation with TNF-α and IP-10/CXCL10. According to these data, we considered that iNOS and IDO are activated in monocyte CD16− and CD16+ subsets, likely exerting both antiviral effects and modulating exacerbated immunological responses during dengue fever.

  10. Feasibility and dosimetry studies for 18F-NOS as a potential PET radiopharmaceutical for inducible nitric oxide synthase in humans.

    Science.gov (United States)

    Herrero, Pilar; Laforest, Richard; Shoghi, Kooresh; Zhou, Dong; Ewald, Gregory; Pfeifer, John; Duncavage, Eric; Krupp, Kitty; Mach, Robert; Gropler, Robert

    2012-06-01

    Nitric oxide (NO), the end product of the inducible form of NO synthase (iNOS), is an important mediator of a variety of inflammatory diseases. Therefore, a radiolabeled iNOS radiopharmaceutical for assessing iNOS protein concentration as a marker for its activity would be of value to the study and treatment of NO-related diseases. We recently synthesized an (18)F-radiolabeled analog of the reversible NOS inhibitor, 2-amino-4-methylpyridine ((18)F-NOS), and confirmed its utility in a murine model of lung inflammation. To determine its potential for use in humans, we measured (18)F-NOS myocardial activity in patients after orthotopic heart transplantation (OHT) and correlated it with pathologic allograft rejection, tissue iNOS levels, and calculated human radiation dosimetry. Two groups were studied-a kinetic analysis group and a dosimetry group. In the kinetic analysis group, 10 OHT patients underwent dynamic myocardial (18)F-NOS PET/CT, followed by endomyocardial biopsy. Myocardial (18)F-NOS PET was assessed using volume of distribution; standardized uptake values at 10 min; area under the myocardial moment curve (AUMC); and mean resident time at 5, 10, and 30 min after tracer injection. Tissue iNOS levels were measured by immunohistochemistry. In the dosimetry group, the biodistribution and radiation dosimetry were calculated using whole-body PET/CT in 4 healthy volunteers and 12 OHT patients. The combined time-activity curves were used for residence time calculation, and organ doses were calculated with OLINDA. Both AUMC at 10 min (P < 0.05) and tissue iNOS (P < 0.0001) were higher in patients exhibiting rejection than in those without rejection. Moreover, the (18)F-NOS AUMC at 10 min correlated positively with tissue iNOS at 10 min (R(2) = 0.42, P < 0.05). (18)F-NOS activity was cleared by the hepatobiliary system. The critical organ was the bladder wall, with a dose of 95.3 μGy/MBq, and an effective dose of 15.9 μSv/MBq was calculated. Myocardial (18)F

  11. Nitric Oxide Synthase 1 Modulates Basal and β-Adrenergic-Stimulated Contractility by Rapid and Reversible Redox-Dependent S-Nitrosylation of the Heart.

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    Alejandra Z Vielma

    Full Text Available S-nitrosylation of several Ca2+ regulating proteins in response to β-adrenergic stimulation was recently described in the heart; however the specific nitric oxide synthase (NOS isoform and signaling pathways responsible for this modification have not been elucidated. NOS-1 activity increases inotropism, therefore, we tested whether β-adrenergic stimulation induces NOS-1-dependent S-nitrosylation of total proteins, the ryanodine receptor (RyR2, SERCA2 and the L-Type Ca2+ channel (LTCC. In the isolated rat heart, isoproterenol (10 nM, 3-min increased S-nitrosylation of total cardiac proteins (+46±14% and RyR2 (+146±77%, without affecting S-nitrosylation of SERCA2 and LTCC. Selective NOS-1 blockade with S-methyl-L-thiocitrulline (SMTC and Nω-propyl-l-arginine decreased basal contractility and relaxation (-25-30% and basal S-nitrosylation of total proteins (-25-60%, RyR2, SERCA2 and LTCC (-60-75%. NOS-1 inhibition reduced (-25-40% the inotropic response and protein S-nitrosylation induced by isoproterenol, particularly that of RyR2 (-85±7%. Tempol, a superoxide scavenger, mimicked the effects of NOS-1 inhibition on inotropism and protein S-nitrosylation; whereas selective NOS-3 inhibitor L-N5-(1-Iminoethylornithine had no effect. Inhibition of NOS-1 did not affect phospholamban phosphorylation, but reduced its oligomerization. Attenuation of contractility was abolished by PKA blockade and unaffected by guanylate cyclase inhibition. Additionally, in isolated mouse cardiomyocytes, NOS-1 inhibition or removal reduced the Ca2+-transient amplitude and sarcomere shortening induced by isoproterenol or by direct PKA activation. We conclude that 1 normal cardiac performance requires basal NOS-1 activity and S-nitrosylation of the calcium-cycling machinery; 2 β-adrenergic stimulation induces rapid and reversible NOS-1 dependent, PKA and ROS-dependent, S-nitrosylation of RyR2 and other proteins, accounting for about one third of its inotropic effect.

  12. Effects of Supervised Structured Aerobic Exercise Training Program on Interleukin-6, Nitric Oxide Synthase-1, and Cyclooxygenase-2 in Type 2 Diabetes Mellitus.

    Science.gov (United States)

    Karimi, Hossein; Rehman, Syed Shakil Ur; Gillani, Syed Amir

    2017-06-01

    To determine the effects of supervised structured aerobic exercise training (SSAET) program on interleukin-6 (IL-6), nitric oxide synthase 1 (NOS-1), and cyclooxygenase-2 (COX-2) in type 2 diabetes mellitus (T2DM). Randomized controlled trial. Riphah Rehabilitation and Research Centre, Railways General Hospital, Rawalpindi, from January 2015 to June 2016. Patients of either gender of minimum one year history of T2DM ranging from 40-70 years of age were included. Those with chronic systemic diseases, history of regular exercise, smoking, and those on dietary plan were excluded. Atotal of 195 patients were screened; 120 were selected and 102 agreed to participate in the study. They were randomly placed into experimental and control groups. SSAETprogram, routine medication, and dietary plan were applied in experimental group; whereas, control group was managed with routine medication and dietary plan for 25 weeks. IL-6, NOS-1, and COX-2 were assessed at baseline and 25 weeks. SSAET program, routine medication and dietary plan showed significantly improved IL-6 (pre-mean=0.25 ±0.11ng/ml, post-mean=0.19 ±0.04 ng/ml), NOS-1 (pre-median=4.65 ng/ml, IQ range=1.04 ng/ml), (post-median=2.72 ng/ml, IQ range=1.60 ng/ml), and COX-2 (pre-mean=18.72 ±4.42 ng/ml, post-mean=15.18 ±2.63 ng/ml) in experimental group, as compared with control group managed by routine medication and dietary plan, where deterioration was noted in IL-6 (pre-mean=0.23 ±0.08 ng/ml, post-mean=0.27 ±0.08 ng/ml) and COX-2 (pre-mean=18.49 ±4.56 ng/ml, postmean=19.10 ±4.76 ng/ml), while NOS-1 slight improvement (pre-mean=4.99 ng/ml, IQ range=2.67 ng/ml), (postmean=4.56 ng/ml, IQ range=3.85 ng/ml). Statistically at the baseline the p-values were not significant (p>0.05) in both experimental and control groups for IL-6, COX-2 and NOS-1; while after 25 weeks of intervention, the experimental group showed significant improvement (p<0.05) in comparison with the control group. SSAET program, routine

  13. Changes in the interstitial cells of Cajal and neuronal nitric oxide synthase positive neuronal cells with aging in the esophagus of F344 rats.

    Science.gov (United States)

    Kim, Hee Jin; Kim, Nayoung; Kim, Yong Sung; Nam, Ryoung Hee; Lee, Sun Min; Park, Ji Hyun; Choi, Daeun; Hwang, Young-Jae; Lee, Jongchan; Lee, Hye Seung; Kim, Min-Seob; Lee, Moon Young; Lee, Dong Ho

    2017-01-01

    The aging-associated cellular and molecular changes in esophagus have not been established, yet. Thus we evaluated histological structure, interstitial cells of Cajal (ICCs), neuronal nitric oxide synthase (nNOS)-positive cells, and contractility in the esophagus of Fischer 344 rat at different ages (6-, 31-, 74-weeks, and 2-years). The lamina propria thickness and endomysial area were calculated. The immunoreactivity of c-Kit, nNOS and protein gene product (PGP) 9.5 was counted after immunohistochemistry. Expression of c-Kit, stem cell factor (SCF), nNOS and PGP 9.5 mRNA was measured by real-time PCR, and expression of c-Kit and nNOS protein was detected by Western blot. Isovolumetric contractile force measurement and electrical field stimulation (EFS) were conducted. The lamina propria thickness increased (6 week vs 2 year, P = 0.005) and the endomysial area of longitudinal muscle decreased with aging (6 week vs 2 year, Paging (6 week vs 2 year; Pchange of PGP 9.5-immunopositiviy. The expressions of nNOS, c-Kit and SCF mRNA also reduced with aging (6 week vs 2 year; P = 0.006, P = 0.001 and P = 0.006, respectively), while the change of PGP 9.5 mRNA expression was not significant. Western blot showed the significant decreases of nNOS and c-Kit protein expression with aging (6 week vs 2 year; P = 0.008 and P = 0.012, respectively). The EFS-induced esophageal contractions significantly decreased in 2-yr-old rat compared with 6-wk-old rats, however, L-NG-Nitroarginine methylester did not significantly increase the spontaneous and EFS-induced contractions in the 6-wk- and 2-yr-old rat esophagus. In conclusion, an increase of lamina propria thickness, a decrease of endomysial area, c-Kit, SCF and NOS expression with preserved total enteric neurons, and contractility in aged rat esophagus may explain the aging-associated esophageal dysmotility.

  14. Effect of Multicomponent Training on Blood Pressure, Nitric Oxide, Redox Status, and Physical Fitness in Older Adult Women: Influence of Endothelial Nitric Oxide Synthase (NOS3 Haplotypes

    Directory of Open Access Journals (Sweden)

    Atila Alexandre Trapé

    2017-01-01

    Full Text Available The purpose of this study was to verify the influence of the genotype or haplotype (interaction of the NOS3 polymorphisms [-786T>C, 894G>T (Glu298Asp, and intron 4b/a] on the response to multicomponent training (various capacities and motor skills on blood pressure (BP, nitrite concentration, redox status, and physical fitness in older adult women. The sample consisted of 52 participants, who underwent body mass index and BP assessments. Physical fitness was evaluated by six-minute walk, elbow flexion, and sit and stand up tests. Plasma/blood samples were used to evaluate redox status, nitrite concentration, and genotyping. Associations were observed between isolated polymorphisms and the response of decreased systolic and diastolic BP and increased nitrite concentration and antioxidant activity. In the haplotype analysis, the group composed of ancestral alleles (H1 was the only one to present improvement in all variables studied (decrease in systolic and diastolic BP, improvement in nitrite concentration, redox status, and physical fitness, while the group composed of variant alleles (H8 only demonstrated improvement in some variables of redox status and physical fitness. These findings suggest that NOS3 polymorphisms and physical training are important interacting variables to consider in evaluating redox status, nitric oxide availability and production, and BP control.

  15. James Van Allen The First Eight Billion Miles

    CERN Document Server

    Foerstner, Abigail

    2009-01-01

    Astrophysicist and space pioneer James Van Allen (1914-2006), for whom the Van Allen radiation belts were named, was among the principal scientific investigators for twenty-four space missions, including Explorer I in 1958, the first successful U.S. satellite; Mariner 2's 1962 flyby of Venus, the first successful mission to another planet; and the 1970's Pioneer 10 and Pioneer 11, missions that surveyed Jupiter and Saturn. Abigail Foerstner blends space science, drama, military agenda's, cold war politics, and the events of Van Allen's lengthy career to create the first biography of this highl

  16. Increased expression and local accumulation of the Prion Protein, Alzheimer Aβ peptides, superoxide dismutase 1, and Nitric oxide synthases 1 & 2 in muscle in a rabbit model of diabetes

    Directory of Open Access Journals (Sweden)

    Bitel Claudine L

    2010-09-01

    Full Text Available Abstract Background Muscle disease associated with different etiologies has been shown to produce localized accumulations of amyloid and oxidative stress-related proteins that are more commonly associated with neurodegeneration in the brain. In this study we examined changes in muscle tissue in a classic model of diabetes and hyperglycemia in rabbits to determine if similar dysregulation of Alzheimer Aβ peptides, the prion protein (PrP, and superoxide dismutase 1 (SOD1, as well as nitric oxide synthases is produced in muscle in diabetic animals. This wild-type rabbit model includes systemic physiological expression of human-like Alzheimer precursor proteins and Aβ peptides that are considered key in Alzheimer protein studies. Results Diabetes was produced in rabbits by injection of the toxic glucose analogue alloxan, which selectively enters pancreatic beta cells and irreversibly decreases insulin production, similar to streptozotocin. Quadriceps muscle from rabbits 16 wks after onset of diabetes and hyperglycemia were analyzed with biochemical and in situ methods. Immunoblots of whole muscle protein samples demonstrated increased PrP, SOD1, as well as neuronal and inducible Nitric oxide synthases (NOS1 and NOS2 in diabetic muscle. In contrast, we detected little change in Alzheimer Aβ precursor protein expression, or BACE1 and Presenilin 1 levels. However, Aβ peptides measured by ELISA increased several fold in diabetic muscle, suggesting a key role for Aβ cleavage in muscle similar to Alzheimer neurodegeneration in this diabetes model. Histological changes in diabetic muscle included localized accumulations of PrP, Aβ, NOS1 and 2, and SOD1, and evidence of increased central nuclei and cell infiltration. Conclusions The present study provides evidence that several classic amyloid and oxidative stress-related disease proteins coordinately increase in overall expression and form localized accumulations in diabetic muscle. The present study

  17. A Summing Up. Allen Memorial Art Museum Addition, Oberlin, Ohio

    Science.gov (United States)

    Progressive Architecture, 1977

    1977-01-01

    Venturi and Rauch's addition to the Allen Art Museum at Oberlin College is in two separate parts: a loft that houses new facilities for the art department and a gallery for contemporary art. (Author/MLF)

  18. Effect of supervised structured aerobic exercise training program of interleukin-6, nitric oxide synthase-1, and cyclooxygenase-2 in type 2 diabetes mellitus

    International Nuclear Information System (INIS)

    Karimi, H.; Gillani, S.A.; Rehman, S.S.U.

    2017-01-01

    To determine the effects of supervised structured aerobic exercise training (SSAET) program on interleukin-6 (IL-6), nitric oxide synthase 1 (NOS-1), and cyclooxygenase-2 (COX-2) in type 2 diabetes mellitus (T2DM). Study Design: Randomized controlled trial. Place and Duration of Study: Riphah Rehabilitation and Research Centre, Railways General Hospital, Rawalpindi, from January 2015 to June 2016. Methodology: Patients of either gender of minimum one year history of T2DM ranging from 40-70 years of age were included. Those with chronic systemic diseases, history of regular exercise, smoking, and those on dietary plan were excluded. A total of 195 patients were screened; 120 were selected and 102 agreed to participate in the study. They were randomly placed into experimental and control groups. SSAET program, routine medication, and dietary plan were applied in experimental group; whereas, control group was managed with routine medication and dietary plan for 25 weeks. IL-6, NOS-1, and COX-2 were assessed at baseline and 25 weeks. Results: SSAET program, routine medication and dietary plan showed significantly improved IL-6 (pre-mean=0.25 +-0.11ng/ml, post-mean=0.19 +-0.04 ng/ml), NOS-1 (pre-median=4.65 ng/ml, IQ range=1.04 ng/ml), (post-median=2.72 ng/ml, IQ range=1.60 ng/ml), and COX-2 (pre-mean=18.72 +-4.42 ng/ml, post-mean=15.18 +-2.63 ng/ml) in experimental group, as compared with control group managed by routine medication and dietary plan, where deterioration was noted in IL-6 (pre-mean=0.23 +-0.08 ng/ml, post-mean=0.27 +-0.08 ng/ml) and COX-2 (pre-mean=18.49 +-4.56 ng/ml, post-mean=19.10 +-4.76 ng/ml), while NOS-1 slight improvement (pre-mean=4.99 ng/ml, IQ range=2.67 ng/ml), (post-mean=4.56 ng/ml, IQ range=3.85 ng/ml). Statistically at the baseline the p-values were not significant (p>0.05) in both experimental and control groups for IL-6, COX-2 and NOS-1; while after 25 weeks of intervention, the experimental group showed significant improvement (p<0

  19. [Effect of hypoxia-inducible factor-1α, endothelin-1 and inducible nitric oxide synthase in the pathogenesis of hypoxia-induced pulmonary hypertension of the neonatal rats].

    Science.gov (United States)

    Sang, Kui; Zhou, Ying; Li, Ming-xia

    2012-12-01

    To study the effect of hypoxia-inducible factor-1α (HIF-1α) in the pathogenesis of hypoxia-induced pulmonary hypertension (HPH) of the neonatal rats through the study on the expression level of HIF-1α and its regulation factors: endothelin-1 (ET-1) and inducible nitric oxide synthase (iNOS) in blood serum and lung tissue. To make an HPH model of neonatal rats, 120 newborn Wistar rats were divided at random into two groups: HPH group and the regular oxygen controlled group with the same birthday. The rats of the two groups were put in the condition of hypoxia for 3, 5, 7, 10, 14, 21 days and then 10 rats of HPH group and control group were picked up, their mean pulmonary arterial pressure (mPAP), serum HIF-1α, and iNOS, and ET-1 content were tested, and finally their lung tissue was taken after they were sacrificed and the expression level of the gene mRNA of HIF-1α, iNOS and ET-1. (1) The rats experienced hypoxia for 3, 5, 7, 10, 14 or 21 days had an increasing mPAP: [8.47 ± 1.45, 10.04 ± 1.69, 10.89 ± 2.97, 16.96 ± 1.97, 13.01 ± 1.93, 21.04 ± 2.13 (mm Hg)], which had a significant differences compared with control groups [5.11 ± 1.06, 8.12 ± 1.11, 8.77 ± 0.92, 12.23 ± 1.78, 8.89 ± 0.89, 11.09 ± 1.64 (mm Hg)] (P rats in hypoxia group had a higher serum HIF-1α [0.83 ± 0.07, 0.84 ± 0.17, 0.97 ± 0.13, 1.10 ± 0.30, 0.92 ± 0.19 (pg/nmol)] than the control group [0.26 ± 0.20, 0.37 ± 0.16, 0.44 ± 0.18, 0.41 ± 0.23, 0.66 ± 0.18 (pg/nmol)] as they experienced hypoxia for 3, 5, 7, 10, and 14 days (P 0.05), and the content of serum iNOS after hypoxia for 14 or 21 days (4.56 ± 0.96, 5.86 ± 1.76) µmol/L was lower than that of the control group (10.35 ± 1.99, 8.44 ± 2.76) µmol/L (P rats and causedn a imbalance of ET-1 and NO. HIF-1α, ET-1 and iNOS altogether contributed to the occurrence and development of HPH in neonatal rats.

  20. Substituent effects on dynamics at conical intersections: Allene and methyl allenes

    Energy Technology Data Exchange (ETDEWEB)

    Neville, Simon P. [Department of Chemistry, University of Ottawa, 10 Marie Curie, Ottawa, Ontario K1N 6N5 (Canada); Wang, Yanmei [State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071 (China); Boguslavskiy, Andrey E.; Stolow, Albert, E-mail: albert.stolow@nrc-cnrc.gc.ca [Department of Chemistry, University of Ottawa, 10 Marie Curie, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, 100 Sussex Drive, Ottawa, Ontario K1A 0R6 (Canada); Department of Physics, University of Ottawa, 150 Louis Pasteur, Ottawa ON K1N 6N5 (Canada); Schuurman, Michael S., E-mail: michael.schuurman@nrc-cnrc.gc.ca [Department of Chemistry, University of Ottawa, 10 Marie Curie, Ottawa, Ontario K1N 6N5 (Canada); National Research Council of Canada, 100 Sussex Drive, Ottawa, Ontario K1A 0R6 (Canada)

    2016-01-07

    We report a joint experimental and theoretical study on the ultrafast excited state dynamics of allene and a series of its methylated analogues (1,2-butadiene, 1,1-dimethylallene, and tetramethylallene) in order to elucidate the conical intersection mediated dynamics that give rise to ultrafast relaxation to the ground electronic state. We use femtosecond time-resolved photoelectron spectroscopy (TRPES) to probe the coupled electronic-vibrational dynamics following UV excitation at 200 nm (6.2 eV). Ab initio multiple spawning (AIMS) simulations are employed to determine the mechanistic details of two competing dynamical pathways to the ground electronic state. In all molecules