INDUCIBLE NITRIC-OXIDE SYNTHASE FROM A RAT ALVEOLAR MACROPHAGE CELL-LINE IS INHIBITED BY NITRIC-OXIDE

The objective of this study was to determine whether inducible nitric oxide (NO) synthase from a rat alveolar macrophage cell line (NR8383) activated by LPS plus IFN-gamma could be regulated by NO, one of the t wo products of the enzymatic reaction. This study was based on previou s observations in this laboratory that NO is a negative feedback modul ator of constitutive NO synthase from rat cerebellum. NO synthase acti vity was determined by monitoring the formation of H-3-L-citrulline fr om H-3-L-arginine in the presence of added cofactors. NO synthase cata lyzed the conversion Of L-arginine to equimolar quantities of NO and L -Citrulline. NO and S-nitrosothiols inhibited NO synthase activity and this effect was enhanced by superoxide dismutase and attenuated by ox yhemoglobin. Nitrite and nitrate, the oxidation products of NO, as wel l as L-citrulline, the amino acid end-product, produced no significant effects on NO synthase activity. The inhibitory effect of NO on NO sy nthase appeared to be partially reversible upon addition of oxyhemoglo bin. The inhibitory effect of NO was mimicked by other heme ligands in cluding carbon monoxide, cyanide, and manganese-protoporphyrin IX. The se observations indicate that (1) enzyme-bound heme plays a mechanisti c role in the catalytic conversion of L-arginine to NO plus L-citrulli ne; (2) NO may function as a negative feedback modulator of inducible NO synthase by interacting with enzyme-bound heme; and (3) negative fe edback modulation by NO may represent a mechanism by which the potenti ally toxic L-arginine-NO pathway in activated alveolar macrophages is turned off.