Inhibition of nitric oxide formation by neuronal nitric oxide synthase by quinones: nitric oxide synthase as a quinone reductase.
Inhibitory action of a variety of quinoid compounds on neuronal nitric oxide synthase (nNOS) activity was examined with a 20000g rat cerebellar supernatant preparation and purified nNOS. The inhibition of citrulline formation from l-arginine by quinones, which exhibit one-electron reduction potentials (E17) ranging between -240 and -100 mV, increased at a more positive one-electron reduction potential, suggesting that quinone appears to act as an electron acceptor for nNOS. Among the quinones tested, 9,10-phenanthraquinone (PQ), corresponding to an E17 value of -124 mV, exhibited the most potent inhibiton of citrulline formation (IC50 value = 10 microM). A kinetic study revealed that PQ is a competitive inhibitor with respect to NADPH, with a Ki value of 0.38 +/- 0.12 microM, and a noncompetitive inhibitor with respect to l-arginine, with a Ki value of 9.63 +/- 0.20 microM. Partial purification of the enzymes which are responsible for reducing PQ in 20000g supernatant of rat cerebellum by anion-exchange column chromatography indicated that one catalyst for PQ reduction was nNOS. Reductase activity of PQ by purified nNOS required CaCl2/calmodulin and was markedly suppressed by the flavoprotein inhibitor diphenyleneiodonium but not by l-nitroarginine which is a specific inhibitor for NO formation. nNOS effectively reduced the quinones as well as PQ causing a marked decrease in the production of NO from l-arginine, while 1, 4-benzoquinone, 9,10-anthraquinone, mitomycin C, and lapachol, which show negligible inhibitory action on nNOS activity, were poor substrates for the enzyme on reduction. These results indicate that PQ and other quinones used in the present study interact with the NADPH-cytochrome P450 reductase domain on nNOS and thus probably inhibit NO formation by shunting electrons away from the normal catalytic pathway. Therefore, our study suggests that quinones could possibly affect NO-dependent physiological and/or pathophysiological actions in vivo.
Kumagai
Y
Y
Department of Environmental Medicine, Institute of Community Medicine, and Master's Program in Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan. yk-em-tu@md.tsukuba.ac.jp
Nakajima
H
H
Midorikawa
K
K
Homma-Takeda
S
S
Shimojo
N
N
D000818:Animals; D008297:Male; D009243:NAD / Q000378:metabolism; D009474:Neurons / Q000201:enzymology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D010616:Phenanthrenes / Q000494:pharmacology*; D011808:Quinone Reductases / Q000037:antagonists & inhibitors*; D011809:Quinones / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar
D010616:Phenanthrenes; D011809:Quinones; D009243:NAD; D009569:Nitric Oxide; C005399:9,10-phenanthrenequinone; D019001:Nitric Oxide Synthase; D011808:Quinone Reductases