title: High concentration of L-arginine suppresses nitric oxide synthase activity and produces reactive oxygen species in NB9 human neuroblastoma cells.
abstract: Hereditary argininemia manifests as neurological disturbance and mental retardation, features not observed in other amino acidemias. The cytotoxic effect of a high concentration of L-arginine (L-Arg) was investigated using NB9 human neuroblastoma cells (NB9), which express neuronal nitric oxide synthase (nNOS). When the concentration of L-Arg in the medium increased from 50 microM to 2 mM after incubation for 48 hr, the intracellular concentration of L-Arg increased from 68.0 +/- 1 pmol/10(6) cells to 1310.0 +/- 5 pmol/10(6) cells and that of L-citrulline (L-Cit) from undetectable levels to 47.1 +/- 0.2 pmol/10(6) cells (mean +/- SD of three independent analyses). This increase in intracellular L-Arg levels caused a decrease in NOS activity by approximately 71%. Flow cytometric analysis showed that reactive oxygen species (ROS) are produced in NB9 exposed to 2 mM L-Arg. The production of ROS was abolished by a NOS inhibitor, NG-nitro-L arginine-methylester. Production of ROS was also observed when NB9 were treated with L-Cit for 48 hr. To investigate the effect of L-Cit on the activity of NOS, a kinetic study on nNOS was conducted using cellular extracts from NB9. The apparent Km value of nNOS for L-Arg was 8.4 microM, with a Vmax value of 8.2 pmol/min/mg protein. L-Cit competitively inhibited NOS activity, as indicated by an apparent Ki value of 65 nM. These results suggest that L-Cit formed by nNOS in L-Arg-loaded neuronal cells inhibits NOS activity and nNOS in these L-Arg-loaded cells functions as a NADPH oxidase to produce ROS, which may cause neurotoxicity in argininemia.
authors.affiliation: Department of Biochemistry and Molecular Biology in Disease, Nagasaki University School of Medicine, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Todoroki
authors.forename: S
authors.initials: S
authors.lastname: Goto
authors.forename: Y
authors.initials: Y
authors.lastname: Urata
authors.forename: K
authors.initials: K
authors.lastname: Komatsu
authors.forename: K
authors.initials: K
authors.lastname: Sumikawa
authors.forename: T
authors.initials: T
authors.lastname: Ogura
authors.forename: I
authors.initials: I
authors.lastname: Matsuda
authors.forename: T
authors.initials: T
authors.lastname: Kondo
mesh_terms: D001120:Arginine / Q000494:pharmacology*; D002956:Citrulline / Q000494:pharmacology; D004249:DNA Damage; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009447:Neuroblastoma / Q000201:enzymology* / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D012333:RNA, Messenger / Q000032:analysis; D017382:Reactive Oxygen Species / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
chemical_list: D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D018808:Transcription Factor AP-1; D002956:Citrulline; D001120:Arginine; C496320:NOS1 protein, human; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D019331:NG-Nitroarginine Methyl Ester