UNIPROT:P43026 - FACTA Search

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Query: UNIPROT:P43026 (lipopolysaccharide)
62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

NO synthase (NOS) is a unique P-450-type enzyme containing both a reductase and a heme domain on a single polypeptide. We show that ebselen [Ebs, 2-phenyl-1,2-benzisoselenazol-3-(2H) one], a nontoxic selenoorganic compound known to break a cysteine thiolate/Fe bond of some of P-450 enzymes, is a relatively selective inhibitor of endothelial isoform of NOS. In rings of rabbit aorta, Ebs irreversibly blocked both the basal as well as acetylcholine- or calcium ionophore A23187-stimulated release of nitric oxide with an IC50 of 6 microM. In homogenates of bovine aortic endothelial cells, Ebs inhibited the activity of NOS, assayed by monitoring conversion of L-[2,3-3H]arginine to L-[2,3-3H]citrulline, with an IC50 of 8.5 microM. The inhibitory action of Ebs was prevented by glutathione, N-acetyl-L-cysteine or dithiothreitol (30-500 microM). The prevention by thiols of Ebs-induced inhibition of NOS suggests that these are competing with a thiol group of NOS that is essential for the catalytic activity of the enzyme. The consequence of the presence of thiols is the "trapping" of Ebs in the form of inactive selenyl sulfides. Consistent with the proposed mechanism of action of Ebs is lack of activity of diselenide of Ebs, which also demonstrates that the action of Ebs is independent of its glutathione peroxidase-like activity. In comparison to endothelial preparations, IC50 values of Ebs for inhibition of soluble isoforms of NOS present in homogenates of porcine cerebellum and of spleens obtained from lipopolysaccharide-treated rats were more than 30-fold higher.
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PMID:Inhibition of endothelial nitric oxide synthase by ebselen. Prevention by thiols suggests the inactivation by ebselen of a critical thiol essential for the catalytic activity of nitric oxide synthase. 750 26

Recent demonstration of cytokine-inducible production of nitric oxide (NO) in vascular smooth muscle cells (VSMC) from rat aorta has implicated VSMC-derived NO as a key mediator of hypotension in septic shock. Our studies to determine whether an inducible NO pathway exists in human VSMC have revealed a novel cytokine-inducible, NO-independent pathway of guanylate cyclase activation in VSMC from human saphenous vein (HSVSMC). Interleukin 1 (IL-1), tumor necrosis factor (TNF), interferon gamma (IFN-gamma) and Escherichia coli lipopolysaccharide (LPS) increased cGMP at 24 h, whereas IL-2 and IL-6 were ineffective. The effect of IL-1 on cyclic guanosine 3',5'-monophosphate (cGMP) was delayed, occurring after 6 h of exposure, and was maximal after 10 h. Methylene blue and LY83583 reversed the IL-1-induced increase in cGMP, suggesting that it was mediated by activation of soluble guanylate cyclase. However, IL-1-induced cGMP in HSVSMC was not inhibited by extracellular hemoglobin. Also, the effect of IL-1 on cGMP was not reversed by nitro- or methyl-substituted L-arginine analogs, aminoguanidine, or diphenyleneiodonium, all of which inhibit IL-1-induced NO synthase in rat aortic VSMC (RAVSMC). IL-1-induced cGMP in HSVSMC was also independent of tetrahydrobiopterin and extracellular L-arginine, as it was not affected by 2,4-diamino-6-hydroxyprytimidine, an inhibitor of tetrahydrobiopterin biosynthesis, and was similar in L-arginine-free and L-arginine-containing media. Analysis of NO synthase mRNA with the use of polymerase chain reaction indicates that levels of mRNA for inducible NO synthase are several orders of magnitude lower in IL-1-treated human HSVSMC than in IL-1-treated RAVSMC. IL-1-induced cGMP was also NO independent in human umbilical artery VSMC, and NO dependent in rat vena cava VSMC. Together these results indicate that IL-1 activates a novel NO-independent pathway of soluble guanylate cyclase activation in human VSMC.
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PMID:Interleukin 1 activates soluble guanylate cyclase in human vascular smooth muscle cells through a novel nitric oxide-independent pathway. 750 3

The effect of cloricromene, a coumarin derivative, was investigated on the lipopolysaccharide-stimulated nitric oxide (NO) synthase induction in intact aortas from endotoxin shocked rats and in the murine macrophage cell line J774. Rings of thoracic aortas from lipopolysaccharide (4 mg/kg, i.v.)-shocked rats, contracted with phenylephrine, showed a progressive decrease in tone, that was of a greater magnitude than that of aortas from naive rats. Moreover, a decreased response to the constrictor effect of phenylephrine was observed in aortas from shocked rats. In vivo treatment with cloricromene (2 mg/kg, i.v.) 30 min before lipopolysaccharide administration partially prevented the loss in tone of aortic rings and improved their reactivity to phenylephrine. Murine J774 macrophages activated with lipopolysaccharide (100 ng/ml) produced significant amounts of nitrites (NO2-; 28.2 +/- 3.5 nmol/10(6) cells per 24 h). Cloricromene (2, 20 or 200 microM) added to the cells concomitantly with lipopolysaccharide inhibited NO2- production in a concentration-dependent manner. Maximum inhibition (84.0 +/- 8.0%) was observed when cloricromene (200 microM) was added to the cells 6 h before lipopolysaccharide, whereas it was ineffective when given 6 h after endotoxin. These results demonstrate that cloricromene inhibits the expression but not the activity of the inducible NO synthase.
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PMID:Cloricromene inhibits the induction of nitric oxide synthase. 750 14

An enhanced formation of nitric oxide (NO) due to induction of a calcium-independent (inducible) NO synthase (iNOS) contributes importantly to the cardiovascular failure caused by bacterial endotoxin. Repeated challenges with small doses of endotoxin result in tolerance to both peripheral vascular failure and death caused by subsequent injection of a higher dose of endotoxin. Here we investigate whether tolerance to endotoxin is associated with a lack of induction of iNOS in vivo and whether endogenous glucocorticoids play a role in the development of endotoxin tolerance. In anesthetized rats, i.v. administration of Escherichia coli endotoxin [lipopolysaccharide (LPS); 2 mg.kg-1] resulted in a prolonged decrease in mean arterial blood pressure (MAP) and hyporeactivity to the contractile responses elicited by norepinephrine (NE; 10 nM) in aortic rings ex vivo. Hyporeactivity to NE was partially reversed by NG-nitro-L-arginine methyl ester (0.3 mM) in vitro, suggesting that an enhanced formation of NO contributes to this hyporeactivity. There was a substantial increase in the activity of iNOS in the lung 3 h after i.v. injection of LPS (0.2 +/- 0.1 to 6.6 +/- 0.6 pmol.mg-1.min-1; n = 5; P < 0.01). Rats injected i.p. with LPS (0.5 mg.kg-1) for 4 consecutive days became tolerant to an i.v. injection of LPS (2 mg.kg-1) in that both hypotension and vascular hyporeactivity to NE were significantly attenuated. Moreover, in these endotoxin-tolerant rats, the induction of iNOS by LPS in the lung was attenuated by 63% +/- 6%. Injection of LPS caused a 9-fold increase in plasma corticosterone (CCS) levels within 2 h and CCS levels remained significantly elevated 6 and 24 h after LPS. Animals rendered tolerant to endotoxin by administration of a low dose of LPS (0.5 mg.kg-1, i.p.) for 4 days still had a 6-fold increase in plasma CCS levels 24 h after the last injection of LPS. When endotoxin-tolerant rats were treated with the glucocorticoid receptor antagonist RU 486 (50 mg.kg-1, p.o. 3 h prior to LPS), there was a restoration of the effects of LPS (2 mg.kg-1, i.v.) in causing hypotension, vascular hyporeactivity to NE, and iNOS induction in the lung. However, in control rats RU 486 enhanced neither the decrease in MAP nor the induction of iNOS in response to LPS (2 mg.kg-1, i.v.). Thus, cardiovascular tolerance to endotoxin is accompanied and explained by reduced induction of iNOS in vivo due to the elevation of endogenous glucocorticoid levels.
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PMID:Attenuation of the induction of nitric oxide synthase by endogenous glucocorticoids accounts for endotoxin tolerance in vivo. 750 16

Nitric oxide (NO) is a potent mediator involved in many biological functions including inflammation and non-specific immunity. Murine macrophages possess the prototype of high-output NO synthase which is not constitutively expressed but induced within a few hours by immunological stimuli. In this study, we explored the possibility of controlling the activity of the inducible NO synthase by interfering with the transduction signal which triggers its induction, in the RAW 264.7 macrophage cell line. We found that nicotinamide, an inhibitor of ADP-ribosylation, prevented NO synthase induction in RAW 264.7 cells after stimulation with interferon gamma (IFN-gamma) and lipopolysaccharide (LPS). Furthermore, the level of NO synthase mRNA was measured by Northern-blot analysis and we found that nicotinamide prevents expression of NO synthase mRNA in IFN-gamma- and LPS-stimulated cells. Nicotinamide was also found to inhibit other macrophage functions expressed in response to IFN-gamma, i.e. tumour necrosis factor secretion and the expression of the Ia antigen of the major histocompatibility complex. Analysis of the pattern of ADP-ribosylated proteins revealed that nicotinamide as well as cholera toxin prevented the ADP-ribosylation of a 107-117 kDa protein found constitutively ADP-ribosylated in stimulated and non-stimulated macrophage extracts. Together, our results indicate ADP-ribosylation as a crucial point of the signalling pathway which leads to NO synthase mRNA induction.
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PMID:Nicotinamide inhibits nitric oxide synthase mRNA induction in activated macrophages. 750 33

Uptake of oxidized low-density lipoprotein (LDL) by monocyte/macrophages to form "foam" cells has been implicated in atherogenesis. Activated monocyte/macrophages synthesize nitric oxide (NO) from L-arginine. NO is cytotoxic, antiproliferative, and a vasodilator that inhibits platelet and monocyte adhesion. NO synthase mRNA, protein, and enzyme activity were induced in J774.A1 macrophages activated with lipopolysaccharide and gamma interferon. When macrophages were incubated with oxidized LDL for 24 hours and activated, there was a dose- and time-dependent inhibition of NO synthesis, assessed as nitrite accumulation in the media. When activated cells were incubated with nontoxic doses of lipoprotein (25 micrograms/mL), neither native LDL nor acetyl LDL inhibited NO production, whereas oxidized LDL produced 50% inhibition. Levels of enzyme protein were unchanged by Western blot. Inhibition was a function of the degree of oxidation of LDL but was independent of cholesteryl esterification by the cells. Incubations of oxidized LDL with cells that had been pretreated with dextran sulfate or cytochalasin B yielded no evidence that inhibition was dependent on the scavenger receptor or directed endocytosis. Kinetic studies of inducible NO synthase from J774.A1 cells that were incubated with increasing doses of oxidized LDL indicated a pattern of noncompetitive inhibition. Inhibition of the enzyme was produced by lipids extracted from oxidized LDL but not by lipids extracted from native LDL. Because phosphatidylcholine (PC) is converted to lysophosphatidylcholine (LPC) during the oxidation of LDL, the effects of LPC were investigated. PC vesicles containing LPC did not inhibit enzyme activity but produced modest reductions in nitrite accumulation from cells. In contrast, PC vesicles had no significant effect. The data indicate that oxidized LDL lipid inhibits the activity of inducible NO synthase in activated macrophages. NO production by this enzyme and its inhibition by oxidized LDL lipid may influence cell-to-cell interactions and vasomotor tone in atherosclerotic blood vessels.
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PMID:Inhibition of inducible nitric oxide synthase in macrophages by oxidized low-density lipoproteins. 750 15

1. The effect of endotoxin (E. coli lipopolysaccharide) on the induction of nitric oxide synthase (NOS) and the changes in vascular permeability in the colon and jejunum over a 5 h period have been investigated in the rat. 2. Under resting conditions, a calcium-dependent constitutive NOS, determined by the conversion of radiolabelled L-arginine to citrulline, was detected in homogenates of both colonic and jejunal tissue. 3. Administration of endotoxin (3 mg kg-1, i.v.) led, after a 2 h lag period, to the appearance of calcium-independent NOS activity in the colon and jejunum ex vivo, characteristic of the inducible NOS enzyme. 4. Administration of endotoxin led to an increase in colonic and jejunal vascular permeability after a lag period of 3 h, determined by the leakage of radiolabelled albumin. 5. Pretreatment with dexamethasone (1 mg kg-1 s.c., 2 h prior to challenge) inhibited both the induction of NOS and the vascular leakage induced by endotoxin. 6. Administration of the NO synthase inhibitor NG-monomethyl-L-arginine (12.5-50 mg kg-1, s.c.) 3 h after endotoxin injection, dose-dependently reduced the subsequent increase in vascular permeability in jejunum and colon, an effect reversed by L-arginine (300 mg kg-1, s.c.). 7. These findings suggest that induction of NOS is associated with the vascular injury induced by endotoxin in the rat colon and jejunum.
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PMID:The induction of nitric oxide synthase and intestinal vascular permeability by endotoxin in the rat. 750 78

Nitric oxide (NO) contributes to the antitumor, antimicrobial, and immunosuppressive activity of macrophages. An inducible form of NO synthase (iNOS) is responsible for high output generation of nitric oxide by macrophages after stimulation with cytokines and/or lipopolysaccharide (LPS). In the present study, we demonstrate that interleukin 4 (IL-4) suppressed production of NO by primary mouse peritoneal macrophages exposed to IFN-gamma with or without LPS, even while synergizing with IFN-gamma to increase the secretion of TNF-alpha. Suppression of NO production was paralleled by decreases in iNOS enzyme activity and iNOS antigen. IL-4 did not inhibit induction of iNOS mRNA 4-6 h after exposure to IFN-gamma, but strongly reduced iNOS mRNA at later times of stimulation (24-72 h), without increasing its turnover. The conditions for maximal suppression of iNOS expression by IL-4 and the mechanisms of suppression differed from those determined in parallel for transforming growth-factor-beta as described elsewhere. These results illustrate the diversity of phenotypes of macrophages deactivated by different cytokines, and demonstrate that IL-4 has the potential to reduce one component of the anti-tumor, antimicrobial, and immunosuppressive activities of macrophages.
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PMID:Mechanism of suppression of nitric oxide synthase expression by interleukin-4 in primary mouse macrophages. 750 68

1. Effects of dexamethasone on induction of nitric oxide (NO) synthase and L-arginine transport by lipopolysaccharide (LPS) were examined in a murine cultured macrophage cell line J774. Metabolism of L-arginine to L-citrulline and subsequent changes in intracellular amino acids pools were correlated with changes in nitrite production. 2. Despite a high intracellular concentration of arginine in activated J774 cells, LPS (1 microgram ml-1, 8 h) induced a 2.4 fold increase in arginine transport. Treatment of cells with cycloheximide (1 microgram ml-1) inhibited the time-dependent (1-8 h) induction of NO synthase and arginine transport mediated by LPS. 3. Induction of NO synthase by LPS (1 microgram ml-1, 24 h) alone was accompanied by a marked increase in arginine utilisation leading to decreased intracellular arginine levels and elevated intracellular and extracellular L-citrulline levels. These changes were further enhanced in the presence of interferon-gamma (IFN-gamma, 100 units ml-1, 24 h). 4. Dexamethasone (1 microM) abolished the increases in both nitrite and citrulline production induced by LPS alone but only partially reversed the combined effects of LPS and IFN-gamma. In contrast, treatment of cells with dexamethasone (10 microM) had no effect on the LPS-mediated induction of arginine transport or the decrease in intracellular arginine concentration. 5. We conclude that induction of arginine transporter activity in LPS-stimulated J774 cells involves de novo synthesis of carrier proteins, which increases transport of exogenous arginine during enhanced NO production. Moreover, the intracellular signalling pathways mediating induction of arginine transport and of NO synthase by LPS in activated macrophages diverge, since only the latter is sensitive to dexamethasone.
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PMID:Selective inhibition by dexamethasone of induction of NO synthase, but not of induction of L-arginine transport, in activated murine macrophage J774 cells. 750 26

The purpose of these studies was to determine whether the induction of NO synthase activity in murine K-1735 melanoma cells correlated with their metastatic potential. Nonmetastatic, metastatic, and somatic cell hybrids (produced by fusion of nonmetastatic and metastatic cells) were injected i.v. into syngeneic C3H/HeN mice. Metastatic cells survived to produce experimental lung metastases, whereas nonmetastatic cells did not. The various clones and somatic cell hybrids were incubated in vitro with combinations of tumor necrosis factor, interleukin 1, gamma-interferon, and lipopolysaccharide. Nonmetastatic cells exhibited high levels of inducible NO synthase activity and NO, whereas metastatic cells did not. Both the cytotoxic effects of the cytokines and NO production were inhibited by the addition of NG-monomethyl-L-arginine, a specific inhibitor of NO synthase. These data demonstrate an inverse correlation between production of endogenous NO and the ability of K-1735 cells to survive in syngeneic mice to produce lung metastases.
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PMID:Inverse correlation between expression of inducible nitric oxide synthase activity and production of metastasis in K-1735 murine melanoma cells. 750 36

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