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 th e mouse macrophage cell line RAW264.7 by Escherichia coli lipopolysacc haride (1 mug/ml, 18 h) caused an increase in the release of nitrite ( NO2-) and prostaglandin E2 (PGE2), products of NOS and COX, respective ly. Production of both NO2- and PGE2 was blocked by the NOS inhibitors N(G)-monomethyl-L-arginine or aminoguanidine. The effects of N(G)-mon omethyl-L-arginine or aminoguanidine were reversed by coincubation Wit h L-Arg, the precursor for NO synthesis, but not by D-Arg. RAW264.7 ce lls stimulated for 18 h with lipopolysaccharide in L-Arg-free medium ( to reduce NO generation by the endogenous NOS pathway) failed to relea se NO2- and accumulated at least 4-fold less PGE2 when compared to cel ls in the presence of L-Arg. PGE2 production elicited by a 15-min arac hidonic acid treatment of lipopolysaccharide-induced RAW264.7 cells in L-Arg-deficient medium was decreased 3-fold when compared to the rele ase 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 end ogenous L-Arg, human fetal fibroblasts were first stimulated for 18 h with interleukin 1beta. These cells released PGE2 but not NO2-, consis tent with the induction of COX but not NOS in the fibroblast. Exogenou s NO either as a gaseous solution or released by a NO donor, sodium ni troprusside or glyceryl trinitrate, increased COX activity in the inte rleukin 1beta-stimulated fibroblasts by 5-fold; these effects were abo lished by coincubation with hemoglobin (10 muM), which binds and inact ivates NO, but not by methylene blue, an inhibitor of the soluble guan ylate 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 condition s in which both the NOS and COX systems are present, there is an NO-me diated increase in the production of proinflammatory prostaglandins th at may result in an exacerbated inflammatory response. The data sugges t that NO directly interacts with COX to cause an increase in the enzy matic activity.