Induction of NADPH-dependent diaphorase and nitric oxide synthase activity in aortic smooth muscle and cultured macrophages.
Lipopolysaccharide (LPS), either alone or in combination with cytokines, induces nitric oxide (NO) synthase activity in cells that normally release little or no NO. In arterial smooth muscle cells and various macrophage cell lines, NO synthase activity is induced after several hours of incubation with LPS. In brain, NADPH-dependent diaphorase activity has been associated with constitutive NO synthase. Here we show that incubation of rat aorta or cultured macrophages with LPS causes a time-dependent induction of NO synthase. The NO synthase activity in both rat aorta and macrophages was calcium independent and inhibited by NG-monomethyl-L-arginine and NG-nitro-L-arginine. We also found that LPS caused a time-dependent induction in NADPH-dependent diaphorase activity in both rat aorta and cultured macrophages. The diaphorase activity was mainly NADPH dependent and NADH independent. NO synthase activity and NADPH-diaphorase activity in crude cytosol from LPS-treated macrophages were found to co-purify, using 2',5'-ADP-Sepharose followed by Superose-6 gel permeation chromatography.
Mitchell
J A
JA
Department 47S, Abbott Laboratories, Abbott Park, Illinois 60064.
Kohlhaas
K L
KL
Matsumoto
T
T
Pollock
J S
JS
Förstermann
U
U
Warner
T D
TD
Schmidt
H H
HH
Murad
F
F
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000201:enzymology; D002478:Cells, Cultured; D004790:Enzyme Induction; D066298:In Vitro Techniques; D007700:Kinetics; D008070:Lipopolysaccharides; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D009252:NADPH Dehydrogenase / Q000096:biosynthesis* / Q000187:drug effects / Q000378:metabolism; D019001:Nitric Oxide Synthase; D009638:Norepinephrine / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors
D008070:Lipopolysaccharides; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D009252:NADPH Dehydrogenase; D009638:Norepinephrine