Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The enhanced nitric oxide (NO) and prostaglandin (PG) generation of activated macrophages is controlled by glucocorticoid-sensitive inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively. Negative feedback regulation of iNOS expression by the products of both pathways has been suggested, but their effects on COX-2 expression have not been examined. We hae investigated the effect of E- and l-series prostaglandins that activate adenylate cyclase (AC), forskolin (a direct activator of AC), and other agents that influence the cyclicAMP/cyclicGMP systems on the ability of E. coli endotoxin (lipopolysaccharide, LPS) to induce iNOS and COX-2 in the murine macrophage cell line J774. After a 2-hr pretreatment before adding endotoxin, PGE2, PGI2, forskolin, IBMX (isobutylmethylxanthine, a cyclicAMP/cyclicGMP phosphodiesterase inhibitor), 8-bromo cyclicAMP, and arachidonic acid itself all inhibited the expression of both iNOS and COX-2 (as shown by Western blotting) and reduced NO release and COX activity, whereas PGF2 alpha and 8-bromo cyclic GMP were only weakly effective. The effects of PGE2, PGI2, and forskolin were enhanced by cotreatment with IBMX. The suppression of LPS-induced iNOS induction by PGE2 was functionally significant, in that it protected against the mild cytotoxicity of the NO generated in response to endotoxin. These results provide the first direct evidence for the feedback regulatory suppression of COX-2 induction by a PG-driven cAMP-mediated process, and show that the modulation of iNOS and COX-2 induction shares common features. They also suggest that such modulation is normally held in check by high phosphodiesterase activity within these cells.
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PMID:Repression of inducible nitric oxide synthase and cyclooxygenase-2 by prostaglandin E2 and other cyclic AMP stimulants in J774 macrophages. 910

1. DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furan one) was identified as a novel orally active and highly selective cyclo-oxygenase-2 (COX-2) inhibitor. 2. In CHO cells stably transfected with human COX isozymes, DFU inhibited the arachidonic acid-dependent production of prostaglandin E2 (PGE2) with at least a 1,000 fold selectivity for COX-2 (IC50 = 41 +/- 14 nM) over COX-1 (IC50 > 50 microM). Indomethacin was a potent inhibitor of both COX-1 (IC50 = 18 +/- 3 nM) and COX-2 (IC50 = 26 +/- 6 nM) under the same assay conditions. The large increase in selectivity of DFU over indomethacin was also observed in COX-1 mediated production of thromboxane B2 (TXB2) by Ca2+ ionophore-challenged human platelets (IC50 > 50 microM and 4.1 +/- 1.7 nM, respectively). 3. DFU caused a time-dependent inhibition of purified recombinant human COX-2 with a Ki, value of 140 +/- 68 microM for the initial reversible binding to enzyme and a kappa 2 value of 0.11 +/- 0.06 s-1 for the first order rate constant for formation of a tightly bound enzyme-inhibitor complex. Comparable values of 62 +/- 26 microM and 0.06 +/- 0.01 s-1, respectively, were obtained for indomethacin. The enzyme-inhibitor complex was found to have a 1:1 stoichiometry and to dissociate only very slowly (t1/2 = 1-3 h) with recovery of intact inhibitor and active enzyme. The time-dependent inhibition by DFU was decreased by co-incubation with arachidonic acid under non-turnover conditions, consistent with reversible competitive inhibition at the COX active site. 4. Inhibition of purified recombinant human COX-1 by DFU was very weak and observed only at low concentrations of substrate (IC50 = 63 +/- 5 microM at 0.1 microM arachidonic acid). In contrast to COX-2, inhibition was time-independent and rapidly reversible. These data are consistent with a reversible competitive inhibition of COX-1. 5. DFU inhibited lipopolysaccharide (LPS)-induced PGE2 production (COX-2) in a human whole blood assay with a potency (IC50 = 0.28 +/- 0.04 microM) similar to indomethacin (IC50 = 0.68 +/- 0.17 microM). In contrast, DFU was at least 500 times less potent (IC50 > 97 microM) than indomethacin at inhibiting coagulation-induced TXB2 production (COX-1) (IC50 = 0.19 +/- 0.02 microM). 6. In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 microM), DFU inhibited COX-1 with an IC50 value of 13 +/- 2 microM as compared to 20 +/- 1 nM for indomethacin. CGP 28238, etodolac and SC-58125 were about 10 times more potent inhibitors of COX-1 than DFU. The order of potency of various inhibitors was diclofenac > indomethacin approximately naproxen > nimesulide approximately meloxicam approximately piroxicam > NS-398 approximately SC-57666 > SC-58125 > CGP 28238 approximately etodolac > L-745,337 > DFU. 7. DFU inhibited dose-dependently both the carrageenan-induced rat paw oedema (ED50 of 1.1 mg kg-1 vs 2.0 mg kg-1 for indomethacin) and hyperalgesia (ED50 of 0.95 mg kg-1 vs 1.5 mg kg-1 for indomethacin). The compound was also effective at reversing LPS-induced pyrexia in rats (ED50 = 0.76 mg kg-1 vs 1.1 mg kg-1 for indomethacin). 8. In a sensitive model in which 51Cr faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no significant effect was detected after oral administration of DFU (100 mg kg-1, b.i.d.) for 5 days, whereas chromium leakage was observed with lower doses of diclofenac (3 mg kg-1), meloxicam (3 mg kg-1) or etodolac (10-30 mg kg-1). A 5 day administration of DFU in squirrel monkeys (100 mg kg-1) did not affect chromium leakage in contrast to diclofenac (1 mg kg-1) or naproxen (5 mg kg-1). 9. The results indicate that COX-1 inhibitory effects can be detected for all selective COX-2 inhibitors tested by use of a sensitive assay at low substrate concentration. The novel inhibitor DFU shows the lowest inhibitory potency against COX-1, a consistent high selectivity of inhibition of COX-2 over COX-1 (>300 fold) with enzyme, whole cell and whole blood assays, with no detectable loss of integrity of the gastrointestinal tract at doses >200 fold higher than efficacious doses in models of inflammation, pyresis and hyperalgesia. These results provide further evidence that prostanoids derived from COX-1 activity are not important in acute inflammatory responses and that a high therapeutic index of anti-inflammatory effect to gastropathy can be achieved with a selective COX-2 inhibitor.
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PMID:Biochemical and pharmacological profile of a tetrasubstituted furanone as a highly selective COX-2 inhibitor. 914 94

1. Within vessels, the formation of nitric oxide (NO) or prostaglandins is normally catalysed in the endothelium by constitutive isoforms of NO synthase (eNOS) and cyclo-oxygenase (COX-1), respectively. However, during inflammatory conditions, the underlying smooth muscle acquires the ability to release NO and prostaglandins after the expression of inducible isoforms of NOS (iNOS) and COX (COX-2). The co-induction of iNOS and COX-2 has been studied over 24 h in isolated vascular smooth muscle cells in vitro. However, due to the limitation of using cultured cells, the relationship between the activities of iNOS and COX over longer periods has not been addressed. Moreover, the relative contribution of the endothelium to the production of NO and prostaglandins under inflammatory conditions is not completely understood. 2. Here using an organ culture system, we have determined the profile of COX (6-keto prostaglandin F1 alpha (6-keto PGF1 alpha), PGE2, thromboxane B2 (TXB2) and NOS (nitrite and nitrate) metabolites released over a period of 10 days from segments of rat aorta. In each case, segments from the same animal were left untreated or treated with bacterial lipopolysaccharide (LPS; 10 micrograms ml-1) in order to induce iNOS and COX-2. Prostaglandins were measured by radioimmunoassay whilst nitrite and nitrate were measured, respectively, by Greiss reaction alone, or following a nitrate reductase step. The isoforms of NOS and COX responsible for metabolite release were characterized pharmacologically by use of inhibitors and at the molecular level by reverse transcription polymerase chain reaction with specific primers for iNOS, eNOS, COX-1 and COX-2. In separate experiments the role of the endothelium in the release of nitrite, nitrate and prostaglandins and in the expression of iNOS, eNOS, COX-1 and COX-2 was determined by comparing responses in endothelium denuded and endothelium-intact segments of rat aorta. 3. Under control culture conditions vessels released prostaglandins in the following rank order 6-keto PGF1 alpha = PGE2 > > TXB2. LPS increased the release of 6-keto PGF1 alpha and PGE2 but not of TXB2, an effect that was inhibited by the protein synthesis inhibitor cycloheximide (1 microM), the anti-inflammatory steroid dexamethason (1 microM), the nonsteroidal anti-inflammatory drug indomethacin (30 microM) and, where tested, the selective COX-2 inhibitor NS-398 (30 microM). Similarly, segments of rat aorta released detectable levels of nitrite and nitrate, which were reduced by NG-nitro-L-arginine methyl ester (L-NAME, 1 mM), which inhibits all isoforms of NOS, and by dexamethasone (1 microM), which inhibits the induction of iNOS. The proportion of nitrate to nitrite released over the 10 day period varied greatly from approximately 1:1 on days 5 to 8 to 5:1 on day 9. However, the sum of nitrite and nitrate (NOx) as well as PGE2 remained elevated over the whole 10 day period. The formation of 6-keto PGF1 alpha peaked on days 1 and 2. 4. In freshly prepared tissue, mRNAs for eNOS, COX-1, iNOS and COX-2 were detected. After 24 h in culture, there was an apparent increase in the level of mRNAs for iNOS and COX-2 but not for eNOS or COX-1, an effect that was further enhanced when LPS was included in the culture medium. The expressions of mRNA for eNOS, COX-1, iNOS or COX-2 were not greatly different in vessels with intact or disrupted endothelium. Similarly the release of NOx or PGE2 by vessels after the 1st or 9th day in culture were not significantly different from vessels prepared with or without endothelium. 5. Thus, COX-2 and iNOS are co-induced in intact vessels in culture, with the vascular smooth muscle being the main site of mediator generation. In contrast to data from isolated cells in culture (observed usually over 1 day), both COX and NOS activities in cultured blood vessels were elevated for at least 10 days. Also, unlike isolated cells in culture, the COX and NOS pathways were active independently; L-NAME had little effect on the activity of COX and indomethacin had little effect on the activity of NOS.
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PMID:Characterization of the induction of nitric oxide synthase and cyclo-oxygenase in rat aorta in organ culture. 914 96

Prostaglandin (PG) release is characteristic of most inflammatory diseases. The committed step in the formation of free arachidonic acid into PG products is catalyzed by cyclooxygenase (COX, prostaglandin H2 synthase, PGHS), which exists as two genetically distinct isoforms. COX-1 is constitutively expressed and produces PGs and thromboxane A2 during normal physiologic activities, while COX-2 is an inducible enzyme stimulated by growth factors, lipopolysaccharide, and cytokines during inflammation or cell injury. Proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) released into the amniotic fluid in the setting of infection have been proposed to signal amnion and decidual cells to produce PGs that may culminate in preterm labor. However, since the molecular control of this phenomenon has not been established, this study used amnion-derived WISH cells to determine if TNF-alpha promoted the formation of PGs through COX-2 activity. Treatment of WISH cells with TNF-alpha (0.1 ng/mL-100 ng/mL) caused a dose-dependent increase in COX-2 expression and the subsequent biosynthesis of PGE2 that persisted for at least 48 hrs. In contrast, COX-1 mRNA and protein levels were unaltered by TNF-alpha treatment as determined by RT-PCR and immunoblot analysis, respectively. TNF-alpha-stimulated COX-2 expression and the subsequent formation of PGE2 were inhibited by dexamethasone (0.1 microM). In addition, indomethacin (1 microM) and the novel COX-2-selective inhibitor, NS-398 (IC50 approximately 1.1 x 10(-9) M), attenuated TNF-alpha-elicited PGE2 production. Results presented here demonstrate that TNF-alpha elicits prolonged and regulatable induction of COX-2 in WISH cells, while COX-1 is constitutively expressed and unchanged in response to TNF-alpha stimulation.
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PMID:Tumor necrosis factor-alpha promotes sustained cyclooxygenase-2 expression: attenuation by dexamethasone and NSAIDs. 944 Jan 35

Inducible cyclooxygenase 2 (COX 2) converts arachidonic acid to prostaglandins, which are thought to mediate various peripheral lipopolysaccharide (LPS)-induced central effects, including generation of fever and activation of the hypothalamic-pituitary-adrenal axis. To localize prostaglandin production in the brain following peripheral LPS administration, COX 2 mRNA expression was examined by in situ hybridization histochemistry in rats injected intraperitoneally (i.p.) or intravenously (i.v.) with various doses of LPS or saline. Constitutive expression of COX 2 mRNA was found in neurons of cortex, hippocampus, and amygdala, but not in cells of the blood vessels. COX 2 mRNA levels were not altered in saline-injected animals as compared to non-injected controls. In LPS-injected animals, no consistent changes of neuronal COX 2 mRNA expression were observed. COX 2 mRNA expression appeared ex novo at 0.5-h post-injection in cells closely associated with blood vessels, however, ex novo labeling of the number of labeled cells increased to a peak at 2 h and subsided gradually to basal levels by 24 h. Initially, labeling was observed in cells comprising major surface-lying blood vessels and meninges. Later, vascular and perivascular cells associated with smaller penetrating blood vessels were labeled. This pattern of COX 2 mRNA induction is independent of the route and dose of the LPS injection. The induced COX 2 mRNA producing cells are identified as endothelial and leptomeningeal cells. Changes in COX 2 mRNA expression were not observed in circumventricular organs. These results suggest that peripheral LPS induces a rapid increase in COX 2 production throughout the vasculatures of the brain, which could affect the neuronal activity of widespread brain regions by elevating the levels of prostaglandins.
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PMID:Cyclooxygenase 2 mRNA expression in rat brain after peripheral injection of lipopolysaccharide. 974 70

Fever is a coordinated endocrine, autonomic, and behavioral response organized by the brain in response to inflammatory stimuli. We examined the mechanism of the febrile response to intravenous lipopolysaccharide (LPS) in the rat. LPS caused activation of microglia and tissue macrophages in the meninges and along penetrating blood vessels. The microglia produce cytokines, such as tumor necrosis factor-alpha, and cyclooxygenase type 2. The latter produces prostaglandins, which may cross the blood-brain barrier. We found that inhibition of COX at the preoptic area prevented fever, whereas injection of PGE2 at this site produced fever. Either i.v. LPS or PGE2 into the preoptic area activated a specific set of pathways, including the ventromedial preoptic area, which we believe to be a key regulatory site, and the paraventricular nucleus, which we believe produces autonomic and endocrine responses that cause elevation of body temperature. We hypothesize that the pathway connecting these two sites involves a double inhibitory relay through temperature-sensitive GABAergic neurons in the hypothalamus. This pathway would essentially "turn up the thermostat" during a fever, causing an increase in body temperature via normal thermoregulatory pathways.
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PMID:Neurobiological basis of fever. 991 69

Various lines of evidence have implicated inducible cyclooxygenase-2 (COX-2) in fever production. Thus, its expression is selectively enhanced in brain after peripheral exogenous (e.g., lipopolysaccharide [LPS]) or endogenous (e.g., interleukin-1) pyrogen administration, while selective COX-2 inhibitors suppress the fever induced by these pyrogens. In this study, we assessed the febrile response to LPS of congenitally constitutive COX-1 (COX-1-/-) and COX-2 (COX-2-/-)-deficient C57BL/6J-derived mice. COX-1+/- and COX-2+/- mice were also evaluated; controls were wild-type C57BL/6J mice (Jackson Labs.). All the animals were pretrained daily for two weeks to the experimental procedures. LPS was injected intraperitoneally at 1 microgram/mouse; pyrogen-free saline (PFS) was the vehicle and control solution. Core temperatures (Tcs) were recorded using thermocouples inserted 2 cm into the colon. The presence of the COX isoforms was determined in cerebral blood vessels immunocytochemically after the experiments, without knowledge of the functional results. The data showed that the wild-type, COX-1+/-, and COX-1-/- mice all responded to LPS with a 1 degrees C rise in Tc within 1 h; the fever gradually abated over the next 4 h. By contrast, COX-2+/- and COX-2-/- mice displayed no Tc rise after LPS. PFS did not affect the Tc of any animal. It would appear therefore that COX-2 is necessary for LPS-induced fever production.
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PMID:The febrile response to lipopolysaccharide is blocked in cyclooxygenase-2(-/-), but not in cyclooxygenase-1(-/-) mice. 1021 76

Elevated levels of nitric oxide (NO*) produced by expression of inducible nitric oxide synthase (iNOS/NOS type 2) and high levels of prostaglandins (PGs) generated by expression of inducible cyclooxygenase (COX-2/PGH2 synthase-2) are important mediators of immune and inflammatory responses. Previous studies have shown that endogenous levels of NO* can influence the formation of PGs. We examined the mechanism by which NO* regulates PG biosynthesis in macrophages. Treatment of a murine macrophage cell line (ANA-1) with lipopolysaccharide (LPS, 10 ng/mL) and interferon-gamma (IFN-gamma, 10 U/mL) for 20 h elicited high levels of nitrite (NO2-) and prostaglandin E2 (PGE2) that were inhibited in a dose-dependent fashion by the NOS inhibitor, aminoguanidine (AG), with IC50 values of 15.06 and 0.38 microM for NO2- and PGE2, respectively. Stimulation of cultures with LPS and IFN-gamma for 20 h induced de novo iNOS protein expression that was not altered by the addition of AG (0.1, 10, or 1000 microM). In contrast, treatment of cultures with LPS and IFN-gamma for 20 h promoted COX-2 mRNA and protein expression that were decreased in a dose-dependent fashion by AG (P < 0.05 with 10 and 1000 microM). LPS and IFN-gamma-induced COX-2 protein expression was not decreased in cultures treated with AG for 2 h, illustrating that AG does not inhibit the formation of COX-2 protein. Analysis of partially purified enzyme extracts demonstrated that AG did not directly inhibit the enzymatic activity of COX. Additional experiments revealed that NO* donors (S-nitroso-N-aceytl-D-L-pencillamine, SNAP, at 0.1, 10, and 1000 microM) did not induce de novo COX-2 protein expression or potentiate COX-2 expression in cells treated with LPS and/or IFN-gamma. Our results suggest that, while endogenous NO* is not required for de novo COX-2 mRNA and protein expression, NO* is necessary for maintaining prolonged COX-2 gene expression.
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PMID:Blockade of nitric oxide formation down-regulates cyclooxygenase-2 and decreases PGE2 biosynthesis in macrophages. 1038 Sep 1

Effects of auranofin, an orally active chrysotherapeutic agent, were examined on the production of prostaglandin E2 (PGE2) and nitric oxide (NO) in rat peritoneal macrophages and in RAW 264.7 cells, a murine macrophage-like cell line. Auranofin (1-10 microM) inhibited PGE2 production in rat peritoneal macrophages stimulated with 12-O-tetra-decanoylphorbol 13-acetate (TPA, 16.2 nM) at 8-20 h, but did not affect PGE2 production at 4 h. However, in non-stimulated rat peritoneal macrophages, auranofin increased PGE2 production at 4 h and had no effect on PGE2 production at 8-20 h. It was proved that auranofin (1-10 microM) increased COX (cyclooxygenase)-1-dependent PGE2 production and inhibited COX-2-dependent PGE2 production in rat peritoneal macrophages. Auranofin showed no effect on the enzyme activities of the purified COX-1 and COX-2 proteins. Furthermore, auranofin did not affect the COX-1 protein level, but inhibited the TPA-induced expression of COX-2 protein. Therefore, it was suggested that auranofin inhibited PGE2 production by inhibiting the COX-2 protein induction in TPA-stimulated macrophages. In RAW 264.7 cells, auranofin (0.3-3 microM) inhibited lipopolysaccharide-induced NO synthesis by inhibiting the induction of NO synthase (NOS) protein expression. Auranofin did not affect the enzyme activity of iNOS (inducible NOS). Finally, using rat peritoneal macrophages, the effects of auranofin on PGE2 production and NO production were determined. Auranofin (10 microM) strongly inhibited the production of PGE2 and NO, and the induction of COX-2 protein and NOS protein by TPA. Indomethacin, a COX inhibitor, partially inhibited NO production at the concentration at which PGE2 production was completely inhibited. On the other hand, L-NG-monomethyl-L-arginine acetate (L-NMMA), a NOS inhibitor, partially inhibited PGE2 production. NO production was completely inhibited at the same concentration as shown above. These findings suggest that PGE2 production and NO production partially affect each other. Therefore, the inhibition of PGE2 production by auranofin might be partly due to the inhibition of NO production, and the inhibition of NO production by auranofin be partly due to the inhibition of PGE2 production. In conclusion, auranofin inhibits both PGE2 production and NO production by inhibiting the upregulation of mRNA levels of COX-2 and NOS.
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PMID:[Analysis of the mechanism for the anti-inflammatory effect of the anti-rheumatic drug auranofin]. 1072 68

Prostaglandin (PG) formation by the inducible (type 2) cyclooxygenase (COX-2) and reactive oxygen species (ROS) have been proposed to play important roles in cerebrovascular pathological processes. To explore the relationship between ROS and COX-2 expression, adenovirus (Ad) vectors containing cDNA for human antioxidant enzymes including catalase (AdCAT:), copper/zinc superoxide dismutase (AdCu/ZnSOD), and manganese superoxide dismutase (AdMnSOD) were transferred into murine cerebral microvascular endothelial cells. AdCAT: (100 multiplicity of infection) infection increased the content and enzymatic activity of cellular Cat threefold and decreased the intracellular peroxide level. The expression of COX-2 mRNA and protein in cell lysates was up-regulated, and the amount of PGE(2) formed from exogenous arachidonic acid increased following AdCAT: infection in a dose-dependent manner, paralleling the expression of COX-2 protein. The AdCAT:-induced increase in PGE(2) formation was inhibited by NS-398, a selective inhibitor of COX-2 enzymatic activity. AdCAT: infection did not change the expression of the constitutive (type 1) COX protein. Although AdCu/ZnSOD and AdMnSOD infection increased the expression of superoxide dismutase proteins, COX-2 expression was not induced. An in vitro nuclear transcription assay indicated that overexpression of the Cat gene increases the transcription of the COX-2 gene. Furthermore, the stability of COX-2 mRNA induced by lipopolysaccharide was increased after AdCAT: gene transfer. These results indicate that AdCAT: gene transfer induces the transcriptional activation of the COX-2 gene and increases COX-2 mRNA stability. Therefore, peroxide may have regulatory effect on COX-2 function in the cerebral microcirculation.
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PMID:Induction of cyclooxygenase-2 by overexpression of the human catalase gene in cerebral microvascular endothelial cells. 1089 36


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