scholarly journals l-Arginine Availability Regulates Inducible Nitric Oxide Synthase-Dependent Host Defense against Helicobacter pylori

2007 ◽  
Vol 75 (9) ◽  
pp. 4305-4315 ◽  
Author(s):  
Rupesh Chaturvedi ◽  
Mohammad Asim ◽  
Nuruddeen D. Lewis ◽  
Holly M. Scott Algood ◽  
Timothy L. Cover ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Helicobacter Pylori ◽  
Protein Expression ◽  
Translation Initiation Factor ◽  
No Production ◽  
Dependent Manner ◽  
Inos Protein ◽  
Inos Protein Expression ◽  
H Pylori ◽  
Inducible Nitric Oxide

ABSTRACT Helicobacter pylori infection of the stomach causes an active immune response that includes stimulation of inducible nitric oxide (NO) synthase (iNOS) expression. Although NO can kill H. pylori, the bacterium persists indefinitely, suggesting that NO production is inadequate. We determined if the NO derived from iNOS in macrophages was dependent on the availability of its substrate, l-arginine (l-Arg). Production of NO by H. pylori-stimulated RAW 264.7 cells was dependent on the l-Arg concentration in the culture medium, and the 50% effective dose for l-Arg was 220 μM, which is above reported plasma l-Arg levels. While iNOS mRNA induction was l-Arg independent, iNOS protein increased in an l-Arg-dependent manner that did not involve changes in iNOS protein degradation. l-Lysine, an inhibitor of l-Arg uptake, attenuated H. pylori-stimulated iNOS protein expression, translation, NO levels, and killing of H. pylori. While l-Arg starvation suppressed global protein translation, at concentrations of l-Arg at which iNOS protein was only minimally expressed in response to H. pylori, global translation was fully restored and eukaryotic translation initiation factor α was dephosphorylated. H. pylori lacking the gene rocF, which codes for a bacterial arginase, induced higher levels of NO production by increasing iNOS protein levels. When murine gastric macrophages were activated with H. pylori, supraphysiologic levels of l-Arg were required to permit iNOS protein expression and NO production. These findings indicate that l-Arg is rate limiting for iNOS translation and suggest that the levels of l-Arg that occur in vivo do not permit sufficient NO generation by the host to kill H. pylori.

scholarly journals Elevated gaseous luminal nitric oxide and circulating IL-8 as features of Helicobacter pylori-induced gastric inflammation

2021 ◽  
Vol 126 (1) ◽  
Author(s):  
Hiwa K. Saaed ◽  
Lisa Chiggiato ◽  
Dominic-Luc Webb ◽  
Ann-Sofie Rehnberg ◽  
Carlos A. Rubio ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Helicobacter Pylori ◽  
No Production ◽  
Histopathological Changes ◽  
Immunoblot Assay ◽  
Blood Biomarker ◽  
H Pylori ◽  
Oxide Synthase ◽  
Inflammatory Changes ◽  
Inducible Nitric Oxide

Background: Gastric nitric oxide (NO) production in response to Helicobacter pylori via inducible nitric oxide synthase (iNOS) is suggested as a biomarker of inflammation and cytotoxicity. The aim of this study was to investigate relationships between gastric [NO], immunological biomarkers and histopathology. Materials and methods: Esophagogastroduodenoscopy was done in 96 dyspepsia patients. Luminal [NO] was measured by chemiluminescence. Biopsies were taken from gastric antrum and corpus for culture and histopathology. H. pylori IgG was detected by immunoblot assay. Biobanked plasma from 76 dyspepsia patients (11 H. pylori positives) was analyzed for 39 cytokines by multiplexed ELISA. Results: H. pylori-positive patients had higher [NO] (336 ± 26 ppb, mean ± 95% CI, n = 77) than H. pylori-negative patients (128 ± 47 ppb, n = 19) (P < 0.0001). Histopathological changes were found in 99% of H. pylori-positive and 37% of H. pylori-negative patients. Histopathological concordance was 78–100% between corpus and antrum. Correlations were found between gastric [NO] and severity of acute, but not chronic, inflammation. Plasma IL-8 (increased in H. pylori positives) had greatest difference between positive and negative groups, with eotaxin, MIP-1β, MCP-4, VEGF-A, and VEGF-C also higher (P < 0.004 to P < 0.032). Diagnostic odds ratios using 75% cut-off concentration were 7.53 for IL-8, 1.15 for CRP, and 2.88 for gastric NO. Conclusions: Of the parameters tested, increased gastric [NO] and circulating IL-8 align most consistently and selectively in H. pylori-infected patients. Severity of mucosal inflammatory changes is proportional to luminal [NO], which might be tied to IL-8 production. It is proposed that IL-8 be further investigated as a blood biomarker of treatment outcomes.

scholarly journals Indomethacin augments lipopolysaccharide-induced expression of inflammatory molecules in the mouse brain

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10391
Author(s):  
Mona Yasin Mohamed ◽  
Willias Masocha
Keyword(s):  
Nitric Oxide ◽  
Protein Expression ◽  
Mrna Expression ◽  
Calcium Binding ◽  
Necrosis Factor Alpha ◽  
Inos Protein ◽  
Inos Protein Expression ◽  
Anti Inflammatory ◽  
The Brain ◽  
Inducible Nitric Oxide

Indomethacin and other non-steroidal anti-inflammatory drugs (NSAIDs) are used to relieve pain and fever including during infections. However, some studies suggest that NSAIDs protect against neuroinflammation, while some find no effects or worsening of neuroinflammation. We evaluated the effect of indomethacin alone on in combination with minocycline, a drug that inhibits neuroinflammation, on the expression of transcripts of neuroinflammatory molecules-induced by lipopolysaccharide (LPS) in the brain of mice. Inoculation of male BALB/c mice with LPS induced the expression of the microglia marker ionized calcium binding adaptor molecule protein, mRNA expression of the genes for cytokines interleukin-1beta (Il1b) and tumor necrosis factor-alpha (Tnf) and inducible nitric oxide synthase gene (Nos2), but not Il10, in the brain. Treatment with indomethacin had no significant effect on the cytokines or Nos2 mRNA expression in naïve animals. However, pretreatment with indomethacin increased LPS-induced Nos2 mRNA and inducible nitric oxide (iNOS) protein expression, but had no significant effect on LPS-induced mRNA expression of the cytokines. Minocycline reduced LPS-induced Il1b and Tnf, but not Nos2, mRNA expression. Treatment with indomethacin plus minocycline had no effect on LPS-induced Il1b, Tnf and Nos2 mRNA expression. In conclusion these results show that indomethacin significantly augments LPS-induced Nos2 mRNA and iNOS protein expression in the brain. In the presence of indomethacin, minocycline could not inhibit LPS-induced pro-inflammatory cytokine expression. Thus, indomethacin could exacerbate neuroinflammation by increasing the expression of iNOS and also block the anti-inflammatory effects of minocycline.

scholarly journals iNOS Associates With Poor Survival in Melanoma: A Role for Nitric Oxide in the PI3K-AKT Pathway Stimulation and PTEN S-Nitrosylation

2021 ◽  
Vol 11 ◽  
Author(s):  
Zhen Ding ◽  
Dai Ogata ◽  
Jason Roszik ◽  
Yong Qin ◽  
Sun-Hee Kim ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Protein Expression ◽  
Nitrosative Stress ◽  
Akt Pathway ◽  
Melanoma Tumor ◽  
Inos Protein ◽  
Akt Activation ◽  
Inos Protein Expression ◽  
Patient Prognosis

We previously showed that inducible nitric oxide synthase (iNOS) protein expression in melanoma tumor cells is associated with poor patient prognosis. Here, we analyzed the association between iNOS and the oncogenic PI3K-AKT pathway. TCGA data show that iNOS and phospho-Akt Ser473 expression were associated significantly only in the subset of tumors with genetically intact PTEN. Employing a stage III melanoma TMA, we showed that iNOS protein presence is significantly associated with shorter survival only in tumors with PTEN protein expression. These findings led to our hypothesis that the iNOS product, nitric oxide (NO), suppresses the function of PTEN and stimulates PI3K-Akt activation. Melanoma cells in response to NO exposure in vitro exhibited enhanced AKT kinase activity and substrate phosphorylation, as well as attenuated PTEN phosphatase activity. Biochemical analysis showed that NO exposure resulted in a post-translationally modified S-Nitrosylation (SNO) PTEN, which was also found in cells expressing iNOS. Our findings provide evidence that NO-rich cancers may exhibit AKT activation due to post-translational inactivation of PTEN. This unique activation of oncogenic pathway under nitrosative stress may contribute to the pathogenesis of iNOS in melanoma. Significance: Our study shows that iNOS expression is associated with increased PI3K-AKT signaling and worse clinical outcomes in melanoma patients with wt (intact) PTEN. Mutated PTEN is already inactivated. We also demonstrate that NO activates the PI3K-AKT pathway by suppressing PTEN suppressor function concurrent with the formation of PTEN-SNO. This discovery provides insight into the consequences of inflammatory NO produced in human melanoma and microenvironmental cells. It suggests that NO–driven modification provides a marker of PTEN inactivation, and represents a plausible mechanism of tumor suppressor inactivation in iNOS expressing subset of cancers.

scholarly journals Vaccine-Induced Reduction of Helicobacter pylori Colonization in Mice Is Interleukin-12 Dependent but Gamma Interferon and Inducible Nitric Oxide Synthase Independent

2003 ◽  
Vol 71 (2) ◽  
pp. 910-921 ◽  
Author(s):  
Christine A. Garhart ◽  
Frederick P. Heinzel ◽  
Steven J. Czinn ◽  
John G. Nedrud
Keyword(s):  
Nitric Oxide ◽  
Helicobacter Pylori ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Bacterial Load ◽  
Gamma Interferon ◽  
Ifn Γ ◽  
H Pylori ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

ABSTRACT Previous studies with mice have shown that major histocompatibility complex class II (MHC-II) is required for protection from Helicobacter pylori, while MHC-I and antibodies are not. Thus, CD4+ T cells are presumed to play an essential role in protective immunity via secretion of cytokines. To determine which cytokines are associated with a reduction of bacterial load in immunized mice, gastric cytokine expression was examined by semiquantitative reverse transcription-PCR in protected (defined as ≥2-log-unit decrease in bacterial load) and unprotected mice 4 weeks after challenge. Elevated levels of mRNA for interleukin-12p40 (IL-12p40), gamma interferon (IFN-γ), tumor necrosis factor alpha, and inducible nitric oxide synthase (iNOS) were associated with protection in immunized-challenged (I/C) mice, but Th2 cytokine (IL-4, IL-5, IL-10, and IL-13) and chemokine (KC, MIP-2, and MCP-1) expression was not associated with protection. Despite the association of IFN-γ and iNOS message with protection, I/C mice genetically lacking either of these products were able to reduce the bacterial load as well as the wild-type I/C controls. The I/C mice lacking IL-12p40 were not protected compared to unimmunized-challenged mice. All I/C groups developed gastritis. We conclude that neither IFN-γ nor iNOS is essential for vaccine-induced protection from H. pylori infection. The p40 subunit of IL-12, which is a component of both IL-12 and IL-23, is necessary for protection in immunized mice. These findings suggest a novel IFN-γ-independent function of IL-12p40 in effective mucosal immunization against H. pylori.

Tetrahydrobiopterin synthesis and inducible nitric oxide production in pulmonary artery smooth muscle

1994 ◽  
Vol 266 (4) ◽  
pp. L455-L460 ◽  
Author(s):  
D. K. Nakayama ◽  
D. A. Geller ◽  
M. Di Silvio ◽  
G. Bloomgarden ◽  
P. Davies ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Pulmonary Artery ◽  
De Novo ◽  
Interleukin 1 ◽  
No Production ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Pulmonary Artery Smooth Muscle ◽  
Inducible Nitric Oxide

We recently reported (Am. J. Respir. Cell Mol. Biol. 7: 471-476, 1992) that a mixture of lipopolysaccharide (LPS) and cytokines produced a time-dependent increase in mRNA and protein expression of inducible nitric oxide synthase (iNOS) in cultured rat pulmonary artery smooth muscle cells (RPASM). In the current study we extend observations on regulation of iNOS in RPASM by showing that de novo synthesis of tetrahydrobiopterin (BH4) is critical for LPS and cytokine-induced NO production. A mixture of LPS and the cytokines gamma-interferon, interleukin-1 beta, and tumor necrosis factor-alpha increased steady-state levels of mRNA of GTP-cyclohydrolase-I (GTP-CH), the rate-limiting enzyme in BH4 biosynthesis. Levels of mRNA to GTP-CH became detectable by 4 h, with further increases at 24 h by Northern blot analysis and reverse-transcriptase polymerase chain reaction. Total intracellular biopterin levels, undetectable under basal conditions, increased after 24 h exposure to LPS and cytokines (to 32.3 +/- 0.8 pmol/mg protein). LPS and cytokine-induced NO production, determined by nitrite concentrations in the medium, was decreased in a concentration-dependent manner by the GTP-CH inhibitor, 2,4-diamino-6-hydroxypyrimidine (DAHP) at 24 h. DAHP also inhibited completely the LPS- and cytokine-induced accumulation of intracellular biopterins. Sepiapterin, which supplies BH4 through a salvage pathway independent of GTP-CH, reversed the effect of DAHP on LPS and cytokine-induced NO production.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Withaninsams A and B: Phenylpropanoid Esters from the Roots of Indian Ginseng (Withania somnifera)

Plants ◽  
2019 ◽  
Vol 8 (12) ◽  
pp. 527 ◽  
Author(s):  
Su Cheol Baek ◽  
Seoyoung Lee ◽  
Sil Kim ◽  
Mun Seok Jo ◽  
Jae Sik Yu ◽  
...  
Keyword(s):  
Protein Expression ◽  
Withania Somnifera ◽  
No Production ◽  
Ionization Mass ◽  
Inos Protein ◽  
Bioactive Natural Products ◽  
Indian Ginseng ◽  
Ionization Mass Spectroscopy ◽  
Inos Protein Expression ◽  
Anti Inflammatory

Withania somnifera (L.) Dunal (Solanaceae), known as Indian ginseng or ashwagandha, has been used in Indian Ayurveda for the treatment of a variety of disorders, such as diabetes and reproductive and nervous system disorders. It is particularly used as a general health tonic, analgesic, and sedative. As part of continuing projects to discover unique bioactive natural products from medicinal plants, phytochemical investigation of the roots of W. somnifera combined with a liquid chromatography–mass spectrometry (LC/MS)-based analysis has led to the isolation of two novel phenylpropanoid esters, Withaninsams A (1) and B (2), as an inseparable mixture, along with three known phenolic compounds (3, 4, and 6) and a pyrazole alkaloid (5). The structures of the new compounds were elucidated using a combination of spectroscopic methods, including one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy (HR-ESIMS). Withaninsams A (1) and B (2) are phenylpropanoid esters that contain a side chain, 4-methyl-1,4-pentanediol unit. To the best of our knowledge, the present study is the first to report on phenylpropanoid esters with 4-methyl-1,4-pentanediol unit. The anti-inflammatory activity of the isolated compounds (1–6) was evaluated by determining their inhibitory effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, where compound 3 inhibited LPS-induced NO production (IC50 = 33.3 μM) and TNF-α production, a pro-inflammatory cytokine (IC50 = 40.9 μM). The anti-inflammatory mechanism through the inhibition of transcriptional iNOS protein expression was confirmed by western blotting experiments for the active compound 3, which showed decreased iNOS protein expression.

Anti-Nociceptive and Anti-Inflammatory Effects of Angelicae Dahuricae Radix Through Inhibition of the Expression of Inducible Nitric Oxide Synthase and NO Production

2008 ◽  
Vol 36 (05) ◽  
pp. 913-928 ◽  
Author(s):  
Ok-Hwa Kang ◽  
Hee-Sung Chae ◽  
You-Chang Oh ◽  
Jang-Gi Choi ◽  
Young-Seob Lee ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Formalin Test ◽  
No Production ◽  
Dependent Manner ◽  
Paw Edema ◽  
Sleeping Time ◽  
Anti Inflammatory ◽  
Angelicae Dahuricae Radix ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

The extract of Angelicae Dahuricae Radix has traditionally been used as an anti-noceptive remedy in China. In this study, the methanol extract of Angelicae Dahuricae Radix (MEAD) was evaluated to determine if it has anti-noceptive and anti-inflammatory action. The anti-nociceptive activities of MEAD were evaluated by determining the writhing response and sleeping time, as well as by a formalin test. In addition, the anti-inflammatory activities of MEAD were evaluated by a vascular permeability test as well as by measuring the carrageenan-induced paw edema and conducting a myeloperoxidase (MPO) assay. MEAD (600 and 1200 mg/kg) exhibited anti-inflammatory effects on acetic acid-induced vascular permeability, carrageenan-induced paw edema, and MPO activity. Moreover, the results of the formalin test, the acetic acid-induced writhing response and the pentobarbital-induced sleeping time indicated that MEAD had anti-nociceptive effects that occurred in a concentration-dependent manner. To determine the mechanism by which MEAD exerted its effects on the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide (NO) by treated murine macrophage RAW 264.7 cells was evaluated. Similar to the in vivo activities, both the iNOS expression and NO production were significantly suppressed by MEAD in a dose-dependent manner. Furthermore, MEAD inhibited the activating phosphorylation of ERK1/2. These results provide a scientific basis that explains the mechanism by which Angelicae Dahuricae Radix relieves inflammatory pain.

Regulation of nitric oxide production in limb and ventilatory muscles during chronic exercise training

2003 ◽  
Vol 284 (3) ◽  
pp. L452-L457 ◽  
Author(s):  
T. Vassilakopoulos ◽  
G. Deckman ◽  
M. Kebbewar ◽  
G. Rallis ◽  
R. Harfouche ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Exercise Training ◽  
Protein Expression ◽  
Experimental Period ◽  
Whole Body ◽  
Control Group ◽  
No Production ◽  
Inos Protein Expression ◽  
Nnos Expression ◽  
Ventilatory Muscles

In this study, we evaluated the differential influence of chronic treadmill training (30 m/min, 15% incline, 1 h/day, 5 days/wk) on nitric oxide (NO) production and NO synthase (NOS) isoform expression as well as 3-nitrotyrosine formation (footprint of peroxynitrite) both in limb (gastrocnemius) and ventilatory (diaphragm) muscles. A group of exercise-trained rats and a control group (no training) were examined after a 4-wk experimental period. Exercise training elicited an approximate fourfold rise in gastrocnemius NOS activity and augmented protein expression of the endothelial (eNOS) and neuronal (nNOS) isoforms of NOS to ∼480% and 240%, respectively. Qualitatively similar but quantitatively smaller elevations in NOS activity and eNOS and nNOS expression were observed in the diaphragm. No detectable inducible NOS (iNOS) protein expression was found in any of the muscle samples. Training increased the intensity of 3-nitrotyrosine only in the gastrocnemius muscle. We conclude that whole body exercise training enhances both limb and ventilatory muscle NO production and that constitutive and not iNOS isoforms are responsible for increased protein tyrosine nitration in trained limb muscles.

Bi-directional effects of the elevation of intracellular calcium on the expression of inducible nitric oxide synthase in J774 macrophages exposed to low and to high concentrations of endotoxin

2001 ◽  
Vol 354 (2) ◽  
pp. 351-358 ◽  
Author(s):  
Riku KORHONEN ◽  
Hannu KANKAANRANTA ◽  
Aleksi LAHTI ◽  
Mari LÄHDE ◽  
Richard G. KNOWLES ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Cell Activation ◽  
Inos Mrna ◽  
No Production ◽  
Ionophore A23187 ◽  
Inos Protein ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Nitric oxide produced through the action of inducible nitric oxide synthase (iNOS) is an important mediator in immune responses of the host. Various extracellular factors, including inflammatory stimuli, affect intracellular free Ca2+ levels ([Ca2+]i), modulating cellular signalling and gene expression. In the present study we investigated the effects of increased [Ca2+]i on NO production through the iNOS pathway in J774 macrophages. Thapsigargin (TG), a Ca2+-ATPase inhibitor, and the Ca2+ ionophore A23187 were used as tools to induce an increase in [Ca2+]i in the cytosol. This increase was confirmed by the fura 2 method. The production of NO was measured as accumulated nitrite in the cell culture medium; iNOS protein and iNOS mRNA were detected by Western blotting and reverse-transcriptase-mediated PCR respectively. The activation of nuclear factor κB (NF-κB) was investigated by electrophoretic mobility-shift assay. TG (100nM) induced a marked synthesis of iNOS mRNA, iNOS protein and NO in cells primed with a low concentration of endotoxin [lipopolysaccharide (LPS) 1ng/ml], which on its own induced barely detectable NO synthesis. Stimulation by a high concentration of LPS (100ng/ml) induced a marked expression of iNOS and NO production. Under these conditions, treatment with TG hindered the synthesis of iNOS protein and NO production by accelerating the degradation of iNOS mRNA. Treatment with TG (100nM) did not affect the NF-κB activity induced by low (1ng/ml) or high (100ng/ml) concentrations of LPS. Viability of the cells was confirmed by the 2,3-bis[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxyaniline (‘XTT’) method; apoptosis was ruled out by propidium iodide staining and flow cytometry. A23187 (1µM) also transiently increased [Ca2+]i and had opposite effects on NO production depending on the LPS concentration. Our results show that increased [Ca2+]i induced the stimulation or suppression of NO production through iNOS in macrophages depending on the state of cell activation. These findings suggest that the receptor-mediated increase in [Ca2+]i might be an important factor in the control of the balance between the up-regulation and down-regulation of inflammatory genes, including that encoding iNOS, depending on the phase of the inflammatory response.

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