REGULATION OF THE EXPRESSION OF CYCLOOXYGENASE-2 BY NITRIC-OXIDE IN RAT PERITONEAL-MACROPHAGES

Activation of rat peritoneal macrophages by LPS resulted in time-depen dent production of nitric oxide and enhancement of cyclooxygenase (Cox ) activity. This stimulation was accompanied by increased expression o f inducible enzymes, NO synthase, and Cox-2, contrasting with no varia tion in constitutive Cox-1. Inhibition of NO production in LPS-treated macrophages by either the L-arginine analogue N-G-monomethyl-L-argini ne (L-NMMA) or aminoguanidine was accompanied by an additional enhance ment of Cox activity parallel to the expression of Cox-2 protein analy zed by Western blot. Addition of NO donors (3-morpholinosydnonimine (S in-1), sodium nitroprusside, or S-nitrosoglutathione) reversed the eff ects of L-NMMA, confirming the role of NO on Cox-2 expression. Specifi c immunoprecipitation of Cox-2 showed a pattern of protein expression similar to that observed in intact cells. Enzyme activity tested on th e immunoprecipitates was correlated with the enzyme mass. In contrast, there was no variation in immunoprecipitated Cox-1 protein or in acti vity. Levels of mRNA for Cox-2 were increased in macrophages stimulate d by LPS in the presence of L-NMMA compared with LPS alone. Metabolic labeling using [S-35]methionine showed that inhibition of NO formation resulted in enhanced de novo synthesis of the S-35-labeled Cox-2. The amount of Cox-2 protein induced in the presence or absence of L-NMMA did not change for at least 6 h, suggesting that NO does not modify th e t1/2 of the enzyme. These results provide evidence that NO participa tes in PG production by negative regulation of Cox-2 expression.