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 cerebel lar supernatant preparation and purified nNOS. The inhibition of citru lline formation from L-arginine by quinones, which exhibit one-electro n reduction potentials (E-7(1)) ranging between -240 and -100 mV, incr eased at a more positive one-electron reduction potential, suggesting that quinone appears to act as an electron acceptor for nNOS. Among th e quinones tested, 9,10-phenanthraquinone (PQ), corresponding to an E- 7(1) value of -124 mV, exhibited the most potent inhibiton of citrulli ne formation (IC50 value = 10 mu M). A kinetic study revealed that PQ is a competitive inhibitor with respect to NADPH, with a K-i value of 0.38 +/- 0.12 mu M, and a noncompetitive inhibitor with respect to L-a rginine, with a K-i value of 9.63 +/- 0.20 mu M. Partial purification of the enzymes which are responsible for reducing PQ in 20000g superna tant of rat cerebellum by anion-exchange column chromatography indicat ed that one catalyst for PQ reduction was nNOS. Reductase activity of PQ by purified nNOS required CaCl2/calmodulin and was markedly suppres sed by the flavoprotein inhibitor diphenyleneiodonium but not by L-nit roarginine which is a specific inhibitor for NO formation. nNOS effect ively 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-ant hraquinone, mitomycin C, and lapachol, which show negligible inhibitor y action on nNOS activity, were poor substrates for the enzyme on redu ction. These results indicate that PQ and other quinones used in the p resent study interact with the NADPH-cytochrome P450 reductase domain on nNOS and thus probably inhibit NO formation by shunting electrons a way from the normal catalytic pathway. Therefore, our study suggests t hat quinones could possibly affect NO-dependent physiological and/or p athophysiological actions in vivo.