Ž . Biochimica et Biophysica Acta 1334 1997 51–56 Nitric oxide generation from extracellularly applied N G -hydroxy-L-arginine in LPS-activated RAW 264 macrophages Petr Vetrovsky a , Andrei L. Kleschyov a , Gustav Entlicher b , Philippe Poindron c , Jean-Claude Stoclet a, ) a Laboratoire de Pharmacologie et de Physiopathologie Cellulaires-CNRS URA600, Faculte de Pharmacie, B.P. 24, UniÕersite Louis ´ ´ Pasteur de Strasbourg, 74 route du Rhin, 67401 Illkirch, France b Department of Biochemistry, Charles UniÕersity, Prague, Czech Republic c Departement d’Immunologie, Immunopharmacologie et Pathologie, UniÕersite Louis Pasteur de Strasbourg, 67401 Illkirch, France ´ ´ Received 22 March 1996; revised 1 July 1996; accepted 17 July 1996 Abstract Ž . Ž . Lipopolysaccharide LPS -activated but not control RAW 264 macrophages produced nitric oxide NO from extracellu- G Ž . larly-applied N -hydroxy-L-arginine L-NOHA in a concentration-dependent manner, as measured by EPR spin trapping and assays for NO y and NO y . This production was inhibited by N G -nitro-L-arginine methyl ester and N G -monomethyl-L- 2 3 arginine, NO-synthase inhibitors, as well as by L-lysine, a competitor for the y q amino acid carrier system. No significant differences were found between L-NOHA and L-arginine with respect to the rate of NO production and the effects of inhibitors. These results provide evidence that extracellular L-NOHA can enter LPS-activated RAW 264 macrophages via a cationic amino acid carrier system and be metabolized to NO by NO-synthase. The data also suggest that no alternative pathway exists for NO production from L-NOHA in non-activated RAW 264 macrophages. Keywords: Nitric oxide production; N G -Hydroxy-L-arginine; LPS; Macrophage 1. Introduction G Ž . N -hydroxy-L-arginine L-NOHA is a stable in- termediate in the enzymatic production of nitric oxide Ž . Ž . wx NO from L-arginine L-Arg 1 . In some conditions, Abbreviations: L-NOHA, N G -hydroxy-L-arginine; L-Arg, L- arginine; L-Lys, L-lysine; LPS, lipopolysaccharide; NOS, NO- synthase; iNOS, inducible NO-synthase; NOx, nitrite plus nitrate; L-NMMA, N G -monomethyl-L-arginine; L-NAME, N G -nitro-L- arginine methyl ester; EPR, electron paramagnetic resonance; DETC, diethyldithiocarbamate; NADPH, b-nicotinamide adenine dinucleotide phosphate reduced form ) Corresponding author. Fax: q33 88 664633. L-NOHA can be liberated from the active site of Ž . wx NO-synthase NOS 2 . It is released into culture Ž . medium by cells expressing inducible NOS iNOS wx wx 3 , and into the circulation of LPS-treated rats 4. While the physiological relevance of such liberated L-NOHA is unclear, it is known that L-NOHA is able to inhibit the generation of activated oxygen species wx by NOS 2 . Furthermore it is a potent inhibitor of w x arginase 5,6 and can be metabolized into NO by w x Ž cytochrome P-450 7,8 or into NOx nitrite plus . wx w x nitrate by peroxidase 9 and lipoxygenase 10 , ap- parently via formation of NO as an intermediate. Thus, it may be that L-NOHA can enter other cells and exert paracrine effects. 0304-4165r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. Ž . PII S0304-4165 96 00074-8