Effects of the new arginase inhibitor N(omega)-hydroxy-nor-L-arginine on NO synthase activity in murine macrophages.
In stimulated murine macrophage, arginase and nitric oxide synthase (NOS) compete for their common substrate, l-arginine. The objectives of this study were (i) to test the new alpha-amino acid N(omega)-hydroxy-nor-l-arginine (nor-NOHA) as a new selective arginase inhibitor and (ii) to elucidate the effects of arginase inhibition on l-arginine utilization by an inducible NOS. Nor-NOHA is about 40-fold more potent than N(omega)-hydroxy-l-arginine (NOHA), an intermediate in the l-arginine/NO pathway, to inhibit the hydrolysis of l-arginine to l-ornithine catalyzed by unstimulated murine macrophages (IC(50) values 12 +/- 5 and 400 +/- 50 microM, respectively). Stimulation of murine macrophages with interferon-gamma and lipopolysaccharide (IFN-gamma + LPS) results in clear expression of an inducible NOS (iNOS) and to an increase in arginase activity. Nor-NOHA is also a potent inhibitor of arginase in IFN-gamma + LPS-stimulated macrophage (IC(50) value 10 +/- 3 microM). In contrast to NOHA, nor-NOHA is neither a substrate nor an inhibitor for iNOS and it appears as a useful tool to study the interplays between arginase and NOS. Inhibition of arginase by nor-NOHA increases nitrite and l-citrulline accumulation for incubation times higher than 12 h, under our conditions. Our results allow the determination of the kinetic parameters of the two competitive pathways and the proposal of a simple model which readily explains the differences observed between experiments. This model readily accounts for the observed effects and should be useful to predict the consequences of arginase inhibition in the presence of an active NOS on l-arginine availability.
Tenu
J P
JP
UMR 8619 CNRS, Batiment 430, Universite Paris-Sud XI, Orsay Cedex, F-91405, France.
Lepoivre
M
M
Moali
C
C
Brollo
M
M
Mansuy
D
D
Boucher
J L
JL
D000818:Animals; D001119:Arginase / Q000037:antagonists & inhibitors*; D001120:Arginine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007371:Interferon-gamma / Q000494:pharmacology; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000201:enzymology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II
D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; C404735:N(omega)-hydroxynorarginine; C068309:N(omega)-hydroxyarginine; D007371:Interferon-gamma; D001120:Arginine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D001119:Arginase