Nitric oxide activates cyclooxygenase enzymes

We have evaluated the role of nitric oxide (NO) on the activity of the constitutive and induced forms of cyclooxygenase (COX; COX-1 and COX-2, respectively). Induction of NO synthase (NOS) and COX (COX-2) in the mouse macrophage cell line RAW264.7 by Escherichia coli lipopolysaccharide (1 μg/ml, 18 h) caused an increase in the release of nitrite (NO^-₂) and prostaglandin E₂ (PGE₂), products of NOS and COX, respectively. Production of both NO^-₂ and PGE₂ was blocked by the NOS inhibitors N^G-monomethyl-L-arginine or aminoguanidine. The effects of N^G-monomethyl-L-argmine or aminoguanidine were reversed by coincubation with L-Arg, the precursor for NO synthesis, but not by D-Arg. RAW264.7 cells stimulated for 18 h with lipopolysaccharide in L-Arg-free medium (to reduce NO generation by the endogenous NOS pathway) failed to release NO^-₂ and accumulated at least 4-fold less PGE₂ when compared to cells in the presence of L-Arg. PGE₂ production elicited by a 15-min arachidonic acid treatment of lipopolysaccharide-induced RAW264.7 cells in L-Arg-deficient medium was decreased 3-fold when compared to the release obtained with cells induced in medium containing L-Arg. To examine the NO activation of the induced form of COX in the absence of an endogenous L-Arg, human fetal fibroblasts were first stimulated for 18h with interleukin 1β. These cells released PGE2 but not NO^-₂, consistent with the induction of COX but not NOS in the fibroblast. Exogenous NO either as a gaseous solution or released by a NO donor, sodium nitroprusside or glyceryl trinitrate, increased COX activity in the interleukin 1β-stimuiated fibroblasts by 5-fold; these effects were abolished by coincubation with hemoglobin (10 μM), which binds and inactivates NO, but not by methylene blue, an inhibitor of the soluble guanylate cyclase. Furthermore, sodium nitroprusside (0.25-1 mM) increased arachidonic acid-stimulated PGE₂ production by murine recombinant COX-1 and COX-2. These results demonstrate that NO enhances COX activity through a mechanism independent of cGMP and suggest that, in conditions in which both the NOS and COX systems are present, there is an NO-mediated increase in the production of proinflammatory prostaglandins that may result in an exacerbated inflammatory response. The data suggest that NO directly interacts with COX to cause an increase in the enzymatic activity.