title: Blockade of nitric oxide formation down-regulates cyclooxygenase-2 and decreases PGE2 biosynthesis in macrophages.
abstract: 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.
authors.affiliation: Department of Medicine, Duke University, Durham, North Carolina, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Perkins
authors.forename: D A
authors.initials: DA
authors.lastname: Kniss
mesh_terms: D000818:Animals; D002460:Cell Line; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000096:biosynthesis*; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006146:Guanidines / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D007527:Isoenzymes / Q000096:biosynthesis / Q000502:physiology*; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000502:physiology; D052247:Nitric Oxide Synthase Type II; D010544:Peroxidases / Q000502:physiology; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000502:physiology*
chemical_list: D004791:Enzyme Inhibitors; D006146:Guanidines; D007527:Isoenzymes; D009569:Nitric Oxide; D007371:Interferon-gamma; D010544:Peroxidases; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone; C004479:pimagedine