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-γ (IFN-γ, 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 μM for NO2- and PGE2, respectively. Stimulation of cultures with LPS and IFN-γ for 20 h induced de novo iNOS protein expression that was not altered by the addition of AG (0.1, 10, or 1000 μM). In contrast, treatment of cultures with LPS and IFN-γ 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 μM). LPS and IFN-γ-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-acetyl-D-L-pencillamine, SNAP, at 0.1, 10, and 1000 μM) did not induce de novo COX-2 protein expression or potentiate COX-2 expression in cells treated with LPS and/or IFN-γ. 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.