Inhibition of nitric oxide formation and superoxide generation during reduction of LY83583 by neuronal nitric oxide synthase.
6-Anilino-5,8-quinolinedione (LY83583) has been widely used as an agent to reduce levels of nitric oxide (NO)-dependent cGMP in tissues. We report here that suppression of NO formation and production of superoxide during enzymatic reduction of LY83583 by neuronal NO synthase appeared to be potentially involved in the pharmacological action caused by LY83583. LY83583 suppressed neuronal NO synthase activity of 20,000 x g rat cerebellar supernatant preparation in a concentration-dependent manner (IC50 value = 12.9 microM). A kinetic study revealed that LY83583 is a competitive inhibitor with respect to NADPH, with a Ki value of 2.57 microM. With purified neuronal NO synthase it was found that LY83583 was a potent inhibitor of NO formation by the enzyme and served as efficient substrate for reduction with a specific activity of 173 nmol of NADPH oxidized per mg of protein per minute. The reductase activity was stimulated about 19.8-fold by addition of CaCl2/calmodulin, indicating that the presence of CaCl2/calmodulin is essential to express maximal activity of LY83583 reduction. Although LY83583 was a good substrate for both NADPH-cytochrome P450 reductase (P450 reductase) and DT-diaphorase, these flavin enzymes-catalyzed reductions of LY83583 were less than the neuronal NO synthase-mediated reduction in the presence of CaCl2/calmodulin. Enzymatic generation of superoxide during reduction of LY83583 by neuronal NO synthase, P450 reductase or DT-diaphorase was confirmed by electron spin resonance (ESR) experiments. Thus the present results indicate that a benzoquinone derivative LY83583 appears to interact with the P450 reductase domain on neuronal NO synthase, resulting in inhibition of NO formation and superoxide generation, which is involved in suppression of intracellular cGMP content.
Kumagai
Y
Y
Department of Environmental Medicine, Institute of Community Medicine, University of Tsukuba, Ibaraki, Japan. yk-em-tu@md.tsukuba.ac.jp
Midorikawa
K
K
Nakai
Y
Y
Yoshikawa
T
T
Kushida
K
K
Homma-Takeda
S
S
Shimojo
N
N
D000634:Aminoquinolines / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D005420:Flavoproteins / Q000378:metabolism; D008297:Male; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000037:antagonists & inhibitors / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D009474:Neurons / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010084:Oxidation-Reduction; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*
D000634:Aminoquinolines; D004791:Enzyme Inhibitors; D005420:Flavoproteins; D017382:Reactive Oxygen Species; D013481:Superoxides; D009569:Nitric Oxide; D009249:NADP; C041715:6-anilino-5,8-quinolinedione; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase; D016660:NAD(P)H Dehydrogenase (Quinone)