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High Concentration of L-Arginine Suppresses Nitric Oxide Synthase Activity and Produces Reactive Oxygen Species in NB9 Human Neuroblastoma Cells

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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 µM to 2 mM after incubation for 48 hr, the intracellular concentration of L-Arg increased from 68.0 ± 1 pmol/106 cells to 1310.0 ± 5 pmol/106 cells and that of L-citrulline (L-Cit) from undetectable levels to 47.1 ± 0.2 pmol/106 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 µM, 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.

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Acknowledgments

This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan and The Chiyoda Life Social Welfare Foundation.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Nagasaki, 852-8523, Japan

    Sachiko Todoroki, Shinji Goto, Yoshishige Urata, Kazuki Komatsu &  Takahito Kondo

  2. Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki, Japan

    Sachiko Todoroki & Koji Sumikawa

  3. Division of Cancer Therapy Development, National Cancer Institute, Chiba, Japan

    Tsutomu Ogura

  4. Department of Pediatrics, Kumamoto University School of Medicine, Kumamoto, Japan

    Ichiro Matsuda

Authors
  1. Sachiko Todoroki
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  2. Shinji Goto
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  3. Yoshishige Urata
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  4. Kazuki Komatsu
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  5. Koji Sumikawa
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  6. Tsutomu Ogura
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  7. Ichiro Matsuda
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  8. Takahito Kondo
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Correspondence to Takahito Kondo.

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Communicated by E. Buetler.

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Todoroki, S., Goto, S., Urata, Y. et al. High Concentration of L-Arginine Suppresses Nitric Oxide Synthase Activity and Produces Reactive Oxygen Species in NB9 Human Neuroblastoma Cells. Mol Med 4, 515–524 (1998). https://doi.org/10.1007/BF03401756

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