IDENTIFICATION OF N-IMINOETHYL-L-ORNITHINE AS AN IRREVERSIBLE INHIBITOR OF NITRIC-OXIDE SYNTHASE IN PHAGOCYTIC-CELLS

1 The synthesis of nitric oxide (NO) from L-arginine by rat peritoneal neutrophils (PMN) and the murine macrophage cell-line J774 and the inhibition of this synthesis by N-iminoethyl-L-ornithine (L-NIO), N(G)-monomethyl-L-arginine (L-NMMA), N(G)-nitro-L-arginine (L-NNA) and its methyl ester (L-NAME) were investigated. 2 L-NIO was the most potent inhibitor in both types of cells while L-NMMA was less active. L-NNA and L-NAME had no significant effect in PMN and L-NNA produced only approximately 40% inhibition of the generation of NO in the J774 cells at the highest concentration tested (300-mu-M). 3 The inhibitory effect of L-NIO was rapid in onset, requiring 10 min pre-incubation to achieve its full inhibitory activity, while the other compounds required 20-60 min pre-incubation to achieve their full effect. 4 The inhibitory effect of L-NIO (10-mu-M) on intact cells could not be reversed by L-arginine (300-mu-M) but could be prevented by concomitant incubation with this compound (300-mu-M), while the effect of the other inhibitors could be reversed by a 3-5 fold molar excess of L-arginine. 5 The NO synthase from both PMN and J774 cells was cytosolic and NADPH- but not Ca2+-dependent, with K(m) values for L-arginine of 3.3 +/- 0.8 and 4.2 +/- 1.1-mu-M respectively. 6 L-NIO was the most potent inhibitor of the neutrophil and J774 enzymes with IC50 values of 0.8 +/- 0.1 and 3 +/- 0.5-mu-M respectively. Furthermore, the effect of L-NIO was irreversible. The other three compounds were less potent, reversible inhibitors. 7 The inhibitory effects of all these compounds were enantiomerically specific. 8 These data indicate that L-NIO is a novel, potent, rapid in onse and irreversible inhibitor of NO synthase in phagocytic cells. The rapid uptake of L-NIO compared with the other compounds indicates that phagocytic cells have different uptake mechanisms for L-arginine analogues.