title: Modulation of PGE(2) and TNFalpha by nitric oxide and LPS-activated RAW 264.7 cells.
abstract: Prostaglandins (PGs), the arachidonic acid (AA) metabolites of the cyclooxygenase (COX) pathway, and the cytokine TNFalpha play major roles in inflammation and they are synthesised mainly by macrophages. Their syntheses have been shown to be regulated by several factors, including nitric oxide, a further important macrophage product. Since both positive and negative regulations of PGs and TNFalpha synthesis by NO have been reported, we sought to understand the mechanisms underlying these opposite NO effects by using a recent class of NO releasing compounds, the NONOates, which have been shown to release NO in a controlled fashion. To this aim, we analysed the effect of NO released from PAPA/NO (t1/2 15 min) and DETA/NO (t1/2 20 h) in RAW 264.7 cells. Both NONOates were used at the same concentrations allowing the cell cultures to be exposed either at high levels of NO for brief time (PAPA/NO) or at low levels of NO for long time (DETA/NO). We found that the two NONOates had opposite effect on basal TNFalpha release, being increased by PAPA/NO and decreased by DETA/NO, while they did not affect the release stimulated by LPS. At variance, both NONOates increased the basal PGE(2) production, while the LPS-stimulated production was slightly increased only by PAPA/NO. The modulation of PGE(2) synthesis was the result of the distinct effects of the two NO-donors on either arachidonic acid (AA) release or cyclooxygense-2 (COX-2) expression, the precursor and synthetic enzyme of PGs, respectively. Indeed, in resting cultures AA release was enhanced only by PAPA/NO whereas COX-2 expression was moderately upregulated by both donors. In LPS activated cells, both NONOates induced AA release, although with different kinetics and potencies, but only DETA/NO significantly increased COX-2 expression. In conclusion, by comparing the activities of these two NONOates, our observations indicate that level and time of exposure to NO are both crucial in determining the molecular target and the final result of the interactions between NO and inflammatory molecules.
authors.affiliation: Laboratory of Environmental Hygiene, Institito Superiore di Sanità, Rome, Italy. Cecilia@iss.it
authors.forename: Cecilia
authors.initials: C
authors.lastname: Guastadisegni
authors.forename: Alessia
authors.initials: A
authors.lastname: Nicolini
authors.forename: Maria
authors.initials: M
authors.lastname: Balduzzi
authors.forename: Maria Antonietta
authors.initials: MA
authors.lastname: Ajmone-Cat
authors.forename: Luisa
authors.initials: L
authors.lastname: Minghetti
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005609:Free Radicals; D007527:Isoenzymes / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism*; D051379:Mice; D009499:Neutral Red / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D005609:Free Radicals; D007527:Isoenzymes; D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D009499:Neutral Red; D016718:Arachidonic Acid; D009569:Nitric Oxide; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone