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)

Cyclooxygenase (Cox), also known as prostaglandin (PG) H synthase (EC 1.14.99.1), catalyzes the rate-limiting step in the formation of inflammatory PGs. A major regulatory step in PG biosynthesis is at the level of Cox: growth factors, cytokines, and tumor promoters induce Cox activity. We have cloned the second form of the Cox gene (Cox-2) from human umbilical vein endothelial cells (HUVEC). The cDNA encodes a polypeptide of 604 amino acids that is 61% identical to the previously isolated human Cox-1 polypeptide. In vitro translation of the human (h)Cox-2 transcript in rabbit reticulocyte lysates resulted in the synthesis of a 70-kDa protein that is immunoprecipitated by antiserum to ovine Cox. Expression of the hCox-2 open reading frame in Cos-7 monkey kidney cells results in the elaboration of cyclooxygenase activity. hCox-2 cDNA hybridizes to a 4.5-kilobase mRNA species in HUVEC, whereas the hCox-1 cDNA hybridizes to 3- and 5.3-kilobase species. Both Cox-1 and Cox-2 mRNAs are expressed in HUVEC, vascular smooth muscle cells, monocytes, and fibroblasts. Cox-2 mRNA was preferentially induced by phorbol 12-myristate 13-acetate and lipopolysaccharide in human endothelial cells and monocytes. Together, these data demonstrate that the Cox enzyme is encoded by at least two genes that are expressed and differentially regulated in a variety of cell types. High-level induction of the hCox-2 transcript in mesenchymal-derived inflammatory cells suggests a role in inflammatory conditions.
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PMID:Human cyclooxygenase-2 cDNA. 138 Jan 56

Several tumor-derived murine macrophage cell lines were evaluated in vitro as cloned prototypes of tissue macrophages for their ability to metabolize arachidonic acid. Unexpectedly, two cell lines, J774A.1 and WR19M.1, rapidly converted exogenous 14C-arachidonic acid (AA) to a single major prostaglandin metabolite. The compound, PGD2, was positively identified by TLC, HPLC, and GC-MS. The enzymatic formation of the PGD2 was shown by inhibition of its formation by indomethacin and reduced formation of 14C-PGD2 from 14C-PGH2 in boiled cells. When J774A.1 cells were prelabeled with 3H-AA, cultured for 24 hours, and stimulated with lipopolysaccharide (LPS), PGD2 was again the predominant product. No other tumor derived cell lines, including several other murine macrophage lines, produced significant amounts of PGD2. Elicited and activated murine peritoneal macrophages produced only small amounts of PGD2, but resident peritoneal macrophages produced modest amounts of PGD2. Exaggerated formation of PGD2 by J774A.1 and WR19M.1 cells may be a consequence of neoplastic transformation or the clonal expansion of a minor subpopulation of normal tissue macrophages.
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PMID:Production of prostaglandin D2 by murine macrophage cell lines. 393 82

The effect of aniso-osmotic exposure on the level of inducible cyclooxygenase (Cox-2) and on prostanoid synthesis was studied in cultured rat liver macrophages (Kupffer cells). In lipopolysaccharide (LPS)- or phorbol 12-myristate 13-acetate-stimulated Kupffer cells, hyperosmotic (355 mosmol/l) exposure, due to addition of NaCl or impermeant sugars, markedly increased prostaglandin (PG) E2, D2 and thromboxane B2 synthesis in a time- and osmolarity-dependent manner. Increased prostanoid production was observed about 8 h after exposure to LPS in hyperosmotic medium compared to Kupffer cells treated with LPS under normotonic (305 mosmol/l) conditions. A similar stimulatory effect of hyperosmolarity on PGE2 production was also seen when arachidonate was added exogenously. Hyperosmotic stimulation of PGE2 production was accompanied by a strong induction of Cox-2 mRNA levels and an increase in immunoreactive Cox-2, whereas the levels of immunoreactive phospholipase A2 and cyclooxygenase-1 did not change significantly. Dexamethasone, indomethacin and the selective Cox-2 inhibitor, NS-398, abolished the hypertonicity-induced stimulation of PGE2 formation; dexamethasone also prevented the increase in Cox-2 mRNA and protein. The increase of immunoreactive Cox-2 lasted for about 24 h and was also blocked by actinomycin D or cycloheximide, but not by brefeldin A. Tunicamycin or treatment with endoglucosidase H reduced the molecular mass of hypertonicity-induced Cox-2 by 5 kDa. Tunicamycin treatment also suppressed the hypertonicity-induced stimulation of PGE2 production. The hyperosmolarity/LPS-induced stimulation of prostaglandin formation was partly sensitive to protein kinase C inhibition but was not accompanied by an increase in the cytosolic free Ca2+ concentration. The data suggest that osmolarity may be a critical factor in the regulation of Cox-2 expression and prostanoid production in activated rat liver macrophages.
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PMID:Hyperosmolarity stimulates prostaglandin synthesis and cyclooxygenase-2 expression in activated rat liver macrophages. 749 3

Administration of Escherichia coli lipopolysaccharide (LPS; 10 mg/kg i.v.) to male Wistar rats caused within 240 min (i) a sustained fall (approximately 30 mmHg) in mean arterial blood pressure, (ii) a reduction (> 75%) in the pressor responses to norepinephrine (1 microgram/kg i.v.), and (iii) an induction of nitric oxide synthase (iNOS) as measured in the lung. Dexamethasone (1 mg/kg i.p. at 2 h prior to LPS) attenuated the hypotension and the vascular hyporeactivity to norepinephrine and reduced (by approximately 77%) the expression of iNOS in the lung. These effects of dexamethasone were prevented by pretreatment of LPS-treated rats with a neutralizing antiserum to lipocortin 1 (anti-LC1; 60 mg/kg s.c. at 24 h prior to LPS) but not by a control nonimmune sheep serum. Stimulation of J774.2 macrophages with LPS (1 microgram/ml for 24 h) caused the expression of iNOS and cyclooxygenase 2 (COX-2) protein and significantly increased nitrite generation; this was prevented by dexamethasone (0.1 microM at 1 h prior to LPS), which also increased cell surface lipocortin 1. Pretreatment of J774.2 cells with anti-LC1 (1:60 dilution at 4 h prior to LPS) also abolished the inhibitory effect of dexamethasone on iNOS expression and nitrite accumulation but not that on COX-2 expression. A lipocortin 1 fragment (residues 1-188 of human lipocortin 1; 20 micrograms/ml at 1 h prior to LPS) also blocked iNOS in J774.2 macrophages activated by LPS (approximately 78% inhibition), and this too was prevented by anti-LC1. We conclude that the extracellular release of endogenous lipocortin 1 (i) mediates the inhibition by dexamethasone of the expression of iNOS, but not of COX-2, and (ii) contributes substantially to the beneficial actions of dexamethasone in rats with endotoxic shock.
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PMID:Lipocortin 1 mediates the inhibition by dexamethasone of the induction by endotoxin of nitric oxide synthase in the rat. 753 34

Nitric oxide synthesized by inducible nitric oxide synthase (iNOS) has been implicated as a mediator of inflammation in rheumatic and autoimmune diseases. We report that exposure of lipopolysaccharide-stimulated murine macrophages to therapeutic concentrations of aspirin (IC50 = 3 mM) and hydrocortisone (IC50 = 5 microM) inhibited the expression of iNOS and production of nitrite. In contrast, sodium salicylate (1-3 mM), indomethacin (5-20 microM), and acetaminophen (60-120 microM) had no significant effect on the production of nitrite at pharmacological concentrations. At suprapharmacological concentrations, sodium salicylate (IC50 = 20 mM) significantly inhibited nitrite production. Immunoblot analysis of iNOS expression in the presence of aspirin showed inhibition of iNOS expression (IC50 = 3 mM). Sodium salicylate variably inhibited iNOS expression (0-35%), whereas indomethacin had no effect. Furthermore, there was no significant effect of these nonsteroidal anti-inflammatory drugs on iNOS mRNA expression at pharmacological concentrations. The effect of aspirin was not due to inhibition of cyclooxygenase 2 because both aspirin and indomethacin inhibited prostaglandin E2 synthesis by > 75%. Aspirin and N-acetylimidazole (an effective acetylating agent), but not sodium salicylate or indomethacin, also directly interfered with the catalytic activity of iNOS in cell-free extracts. These studies indicate that the inhibition of iNOS expression and function represents another mechanism of action for aspirin, if not for all aspirin-like drugs. The effects are exerted at the level of translational/posttranslational modification and directly on the catalytic activity of iNOS.
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PMID:The mode of action of aspirin-like drugs: effect on inducible nitric oxide synthase. 754 10

The aim of our study was to characterize a model of human prostaglandin endoperoxide synthase-2 (PGHS-2) expression allowing the assessment of pharmacological inhibition in vitro and ex vivo. Heparinized human whole blood samples were incubated with lipopolysaccharide (LPS, 0.1-50 micrograms/ml) for 0 to 24 hr at 37 degrees C. The contribution of platelet PGHS-1 was suppressed by either pretreating the subjects with aspirin (300 mg 48 hr before sampling) or adding aspirin (10 micrograms/ml) in vitro at time 0. PGE2 was measured by radioimmunoassay. LPS induced expression of cyclooxygenase activity in a time- and concentration-dependent fashion. After 24 hr at 10 micrograms/ml LPS, PGE2 production averaged 12.1 +/- 6.2 ng/ml (mean +/- S.D., n = 7). Cyclooxygenase activity increased in parallel with the mass of a monocyte protein doublet analyzed by Western blot using antibodies directed against the carboxyl-terminal portion of human PGHS-2. Dexamethasone (2 microM) inhibited LPS-induced PGE2 production by 96 +/- 4% (mean +/- S.D., n = 3). Four different inhibitors were tested in vitro on the cyclooxygenase activity of LPS-induced monocyte PGH-2 and thrombin-stimulated platelet PGHS-1. IC50 values (microM) for inhibition of PGHS-1 and PGHS-2 were: indomethacin, 0.70 +/- 0.20 vs 0.36 +/- 0.10 (P < .05); S-indobufen, 0.64 +/- 0.22 vs. 14.9 +/- 8 (P < .05), R-indobufen, 38 +/- 18 vs. 230 +/- 68 (P < .01), 6-methoxy-2-naphthyl acetic acid (the active metabolite of nabumetone), 278 +/- 96 vs. 187 +/- 96.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Biochemical and pharmacological characterization of the cyclooxygenase activity of human blood prostaglandin endoperoxide synthases. 799 88

Macrophage production of nitric oxide (.N = O) leads to considerable alterations of vital metabolic pathways in various target cells. The present study tested whether .N = O synthesis by Kupffer cells (KCs), the resident macrophages of the liver, interferes with the secretory function of these cells. As in other macrophage-type cells, the combination of lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) was a potent stimulus of .N = O synthesis by KC. Treatment with LPS and IFN-gamma also induced significant production of prostaglandin E2 (PGE2), thromboxane B2 (TBX2), tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6. Inhibition of .N = O synthesis by KC. Treatment with LPS and IFN-gamma also induced significant production of prostaglandin E2 (PGE2), thromboxane B2 (TBX2), tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6. Inhibition of .N = O synthesis by the L-arginine analogue of NG-monomethyl-L-arginine (NMA) resulted in a further increase of PGE2, TXB2, and IL-6 but not IL-1 and TNF-alpha production, indicating specific inhibitory effects of endogenous .N = O synthesis on the secretory activity of KCs. PGE2 production was most sensitive to the suppressive effect of .N = O and increased 24 h after stimulation with LPS and IFN-gamma from 16.3 +/- 4.9 ng/10(6) KCs without NMA to 94.3 +/- 17.9 ng/10(6) KCs with NMA. This effect of NMA was reversed by a 10-fold increase of the L-arginine concentration. No recovery of PGE2 production was seen when .N = O synthesis was blocked after 24 h. NMA treatment increased cyclooxygenase activity more than threefold, suggesting that .N = O inhibits PGE2 and TXB2 production through diminished PGH2 availability. .N = O synthesis did not significantly affect total protein synthesis or viability of the KCs. These results show that .N = O influences the production of specific inflammatory mediators by KCs.
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PMID:Endogenous nitric oxide inhibits the synthesis of cyclooxygenase products and interleukin-6 by rat Kupffer cells. 844 28

1. We have evaluated the selectivity of ketoprofen and two novel nonsteroidal anti-inflammatory drugs, N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulphonamide (NS-398) and 5-methanesulphonamido-6-(2,4-difluorothiophenyl)-1-indano ne (L-745,337), in inhibiting the cyclo-oxygenase activity of prostaglandin endoperoxide synthase-2 (PGHS-2) vs PGHS-1 in human blood monocytes and platelets, respectively. 2. Heparinized whole blood samples were drawn from healthy volunteers pretreated with aspirin, 300 mg 48 h before sampling, to suppress the activity of platelet PGHS-1 and incubated at 37 degrees C for 24 h with increasing concentrations of the test compounds in the presence of lipopolysaccharide (LPS, 10 micrograms ml-1). Immunoreactive PGE2 levels were measured in plasma by a specific radioimmunoassay as an index of the cyclo-oxygenase activity of LPS-induced monocyte PGHS-2. 3. The effects of the same inhibitors on platelet PGHS-1 activity were assessed by allowing whole blood samples, drawn from the same subjects in aspirin-free periods, to clot at 37 degrees C for 1 h in the presence of the compounds and measuring immunoreactive thromboxane B2 (TXB2) levels in serum by a specific radioimmunoassay. 4. Under these experimental conditions, ketoprofen enantioselectively inhibited the cyclo-oxygenase activity of both PGHS-1 and PGHS-2 with equal potency (IC50 ratio: approx. 0.5 for both enantiomers), while L-745,337 and NS-398 achieved selective inhibition of monocyte PGHS-2 (IC50 ratio: > 150). L-745,337 and NS-398 did not affect LPS-induced monocyte PGHS-2 biosynthesis to any detectable extent. 5. We conclude that L-745,337 and NS-398 are selective inhibitors of the cyclo-oxygenase activity of human monocyte PGHS-2. These compounds may provide adequate tools to test the contribution of this novel pathway of arachidonate metabolism to human inflammatory disease.
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PMID:Effects of the novel anti-inflammatory compounds, N-[2-(cyclohexyloxy)-4-nitrophenyl] methanesulphonamide (NS-398) and 5-methanesulphonamido-6-(2,4-difluorothio-phenyl)-1-inda none (L-745,337), on the cyclo-oxygenase activity of human blood prostaglandin endoperoxide synthases. 858 Dec 80

In this study, PGE2 levels in lipopolysaccharide (LPS)-challenged human whole blood and TxB2 levels following blood coagulation were measured as biochemical index for cyclooxygenase (Cox)-2 and Cox-1 activity respectively. Incubation of human mononuclear cells isolated from whole blood with LPS (100 mu g/mL) induced a time-dependent increase in the expression of Cox-2 protein (>100 fold at 24 hr). This is associated with increases in PGE2 production and free arachidonate release in the plasma. Cox-1 protein was detected in the human mononuclear cells at time zero but was not induced by either LPS or PBS. Most non-steroidal anti-inflammatory drugs (NSAIDs) are more potent at inhibiting Cox-1 than Cox-2. Five experimental compounds CGP-28238, Dup-697, NS-398, SC-58125 and L-745,337, have a greater selectivity for Cox-2. Indomethacin at a single oral dose (25 mg) inhibited approximately 90% the whole blood Cox-2 and Cox-1 activities ex vivo in healthy subjects. These results support the use of this assay to assess the biochemical efficacy of selective Cox-2 inhibitors in clinical trials.
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PMID:A human whole blood assay for clinical evaluation of biochemical efficacy of cyclooxygenase inhibitors. 890 87

Tetracyclines have recently been shown to have "chondroprotective" effects in inflammatory arthritides in animal models. Since nitric oxide (NO) is spontaneously released from human cartilage affected by osteoarthritis (OA) or rheumatoid arthritis in quantities sufficient to cause cartilage damage, we evaluated the effect of tetracyclines on the expression and function of human OA-affected nitric oxide synthase (OA-NOS) and rodent inducible NOS (iNOS). Among the tetracycline group of compounds, doxycycline > minocycline blocked and reversed both spontaneous and interleukin 1 beta-induced OA-NOS activity in ex vivo conditions. Similarly, minocycline > or = doxycycline inhibited both lipopolysaccharide- and interferon-gamma-stimulated iNOS in RAW 264.7 cells in vitro, as assessed by nitrite accumulation. Although both these enzyme isoforms could be inhibited by doxycycline and minocycline, their susceptibility to each of these drugs was distinct. Unlike acetylating agents or competitive inhibitors of L-arginine that directly inhibit the specific activity of NOS, doxycycline or minocycline has no significant effect on the specific activity of iNOS in cell-free extracts. The mechanism of action of these drugs on murine iNOS expression was found to be, at least in part, at the level of RNA expression and translation of the enzyme, which would account for the decreased iNOS protein and activity of the enzyme. Tetracyclines had no significant effect on the levels of mRNA for beta-actin and glyceraldehyde-3-phosphate dehydrogenase nor on levels of protein of beta-actin and cyclooxygenase 2 expression. These studies indicate that a novel mechanism of action of tetracyclines is to inhibit the expression of NOS. Since the overproduction of NO has been implicated in the pathogenesis of arthritis, as well as other inflammatory diseases, these observations suggest that tetracyclines should be evaluated as potential therapeutic modulators of NO for various pathological conditions.
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PMID:A novel mechanism of action of tetracyclines: effects on nitric oxide synthases. 894 52


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