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), NG-monomethyl-L-arginine (L-NMMA), NG-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 microM). 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 microM) on intact cells could not be reversed by L-arginine (300 microM) but could be prevented by concomitant incubation with this compound (300 microM), 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 Km values for L-arginine of 3.3 +/- 0.8 and 4.2 +/- 1.1 microM 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 microM 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 onset 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.
McCall
T B
TB
Wellcome Research Laboratories, Langley Court, Beckenham, Kent.
Feelisch
M
M
Palmer
R M
RM
Moncada
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S
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D002460:Cell Line / Q000187:drug effects; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase; D009952:Ornithine / Q000031:analogs & derivatives* / Q000494:pharmacology; D010586:Phagocytes / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D010974:Platelet Aggregation / Q000187:drug effects; D051381:Rats; D011919:Rats, Inbred Strains; D019323:omega-N-Methylarginine
D008070:Lipopolysaccharides; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; C065027:N(G)-iminoethylornithine; D007371:Interferon-gamma; D001120:Arginine; D009952:Ornithine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases