Overexpression of V-1 prevents nitric oxide-induced cell death: Involvement of enhanced tetrahydrobiopterin biosynthesis

Kohei Yuyama, Hideko Yamamoto, Kazuhiro Nakamura, Itone Nishizaki, Tohru Yamakuni, Si Young Song, Ichiro Sora, Toshiharu Nagatsu, Toshifumi Yamamoto

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Abstract

Previously we reported that the synthesis of catecholamines, dopamine, and noradrenaline was enhanced by overexpression of V-1 protein, a neuronal protein active in the initial stage of development of the rat cerebellum, in the neuronal cell line PC12D, a model of dopamine cells (Yamakuni et al. [1998] J. Biol. Chem. 273:27051-27054). To investigate the physiological role of this protein, we examined the effect of V-1 overexpression on cell toxicity induced by nitric oxide (NO) used at low concentrations. Two clones of PC12D cells overexpressing V-1, transfectants termed V1-46 and V1-69, were significantly more resistant to NOR3 (an NO donor) but not to etoposide (an inhibitor of topoisomerase II)induced apoptotic cell death than the control cells (termed C-7 and C-9) that had been transfected with the vector alone. The addition of L-DOPA, dopamine, or noradrenaline to the medium did not abolish NOR3-induced cell death in PC12D cells. Moreover, pretreatment of V1-46 and V1-69 cells with L-α-methyl-p-tyrosine (α-MPT), an inhibitor of tyrosine hydroxylase, to inhibit catecholamine biosynthesis did not affect the resistance to NO toxicity. These results indicate that the catecholamine levels increased by V-1 overexpression did not produce the protection against NOR3-induced toxicity. We further showed that overexpression of V-1 enhanced the synthesis of (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4). In addition, pretreatment with BH4 or with sepiapterin, which is converted to BH4 intracellularly, significantly protected PC12D cells in a dosedependent manner. The increased BH4 synthesis by V-1 overexpression was dose dependently inhibited by pretreatment with diaminohydroxypyrimidine (DAHP), an inhibitor of GTP-cyclohydrolase I, which is the rate-limiting enzyme for the biosynthesis of BH4, concomitantly with the loss of protective effect afforded by V-1 overexpression. Furthermore, the addition of BH4 or sepiapterin to DAHP-pretreated V146 and V1-69 cells restored cell viability. Taken together, these results indicate that V1 protein plays an important role in protection against cell death induced by NO at low levels by promoting the synthesis of BH4. Moreover, these findings suggest the up-regulation of V1 expression as a possible therapeutic target for protection against the insult of NO-induced oxidative stress.

Original languageEnglish
Pages (from-to)716-725
Number of pages10
JournalJournal of Neuroscience Research
Volume72
Issue number6
DOIs
Publication statusPublished - 2003 Jun 15

Keywords

  • Biopterin
  • Cell death
  • DAHP
  • NOR3
  • V-1

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience

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    Yuyama, K., Yamamoto, H., Nakamura, K., Nishizaki, I., Yamakuni, T., Song, S. Y., Sora, I., Nagatsu, T., & Yamamoto, T. (2003). Overexpression of V-1 prevents nitric oxide-induced cell death: Involvement of enhanced tetrahydrobiopterin biosynthesis. Journal of Neuroscience Research, 72(6), 716-725. https://doi.org/10.1002/jnr.10625