Neuronal nitric oxide synthase (NNOS) catalyzes one-electron reduction of 2,4,6-trinitrotoluene, resulting in decreased nitric oxide production and increased nNOS gene expression: implication for oxidative stress.
To determine the mechanism of 2,4,6-trinitrotoluene (TNT)-induced oxidative stress involving neuronal nitric oxide synthase (nNOS), we examined alterations in enzyme activity and gene expression of nNOS by TNT, with an enzyme preparation and rat cerebellum primary neuronal cells. TNT inhibited nitric oxide formation (IC(50) = 12.4 microM) as evaluated by citrulline formation in a 20,000 g cerebellar supernatant preparation. A kinetic study revealed that TNT was a competitive inhibitor with respect to NADPH and a noncompetitive inhibitor with respect to L-arginine. It was found that purified nNOS was capable of reducing TNT, with a specific activity of 3900 nmol of NADPH oxidized/mg/min, but this reaction required CaCl(2)/calmodulin (CaM). An electron spin resonance (ESR) study indicated that superoxide (O(2)(.-)) was generated during reduction of TNT by nNOS. Exposure of rat cerebellum primary neuronal cells to TNT (25 microM) caused an intracellular generation of H(2)O(2), accompanied by a significant increase in nNOS mRNA levels. These results indicate that CaM-dependent one-electron reduction of TNT is catalyzed by nNOS, leading to a reduction in NO formation and generation of H(2)O(2) derived from O(2)(.-). Thus, it is suggested that upregulation of nNOS may represent an acute adaptation to an increase in oxidative stress during exposure to TNT.
Kumagai
Yoshito
Y
Social and Environmental Medicine, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. yk-em-tu@md.tsukuba.ac.jp
Kikushima
Makoto
M
Nakai
Yumi
Y
Shimojo
Nobuhiro
N
Kunimoto
Manabu
M
D000818:Animals; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism; D004583:Electrons; D015971:Gene Expression Regulation, Enzymologic; D006861:Hydrogen Peroxide / Q000378:metabolism; D008297:Male; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D014303:Trinitrotoluene / Q000737:chemistry* / Q000378:metabolism*
D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D014303:Trinitrotoluene; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat