Regulation and subcellular location of nitrogen oxide synthases in RAW264.7 macrophages.
In nitrinergic signal transduction, nitrogen oxide (NO) synthases (NOS) (EC 1.14.23) catalyze the conversion of L-arginine to L-citrulline and NO, which in turn activates soluble guanylyl cyclase. Macrophages were reported to contain a single isoform of NOS (type II, soluble, Ca(2+)-independent, 130-kDa) and only upon activation of the cells by interferon-gamma (INF) and lipopolysaccharides (LPS). By a mechanism involving L-type Ca2+ channels, calmodulin, and serine proteases, INF/LPS also induce a cytotoxic activation of macrophages. In RAW264.7 macrophages, NO release was detected upon activation of the cells by INF/LPS but also, although at a 20-fold lower level, in control cells. The latter constitutive NOS activity and NO release were Ca2+ dependent and were decreased in INF/LPS-activated RAW264.7 cells or with increasing passage number. RAW264.7 cells did not express soluble guanylyl cyclase, suggesting other target molecules for NO. In INF/LPS-activated cells, NOS activities and NO release were Ca2+ independent (type II) and coinduced with NADPH-diaphorase activities both in the soluble and in the particulate fractions. The NOS-II activities corresponded to a 130-kDa protein, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was not recognized in a protein immunoblot with anti-NOS-I antibody. The serine protease inhibitor tosyl-lysyl chloromethyl ketone abolished the induction of NOS-II by INF/LPS, by depleting intracellular thiol pools and interfering with protein synthesis. Induction of NOS-II by INF/LPS was transcriptionally based and, for maximal enzyme activity, required increased intracellular tetrahydrobiopterin levels, intracellular Ca2+ mobilization, and activation of non-L-type Ca2+ channels but, unlike the induction of macrophage-mediated cytotoxicity, neither L-type-Ca2+ channels nor calmodulin.
Schmidt
H H
HH
Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611.
Warner
T D
TD
Nakane
M
M
Förstermann
U
U
Murad
F
F
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D006152:Cyclic GMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004791:Enzyme Inhibitors; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009249:NADP / Q000378:metabolism; D019001:Nitric Oxide Synthase; D009589:Nitrogen Oxides / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational
D004791:Enzyme Inhibitors; D009589:Nitrogen Oxides; D000001:Calcimycin; D009249:NADP; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D006152:Cyclic GMP