Overexpression of V-1 prevents nitric oxide-induced cell death: involvement of enhanced tetrahydrobiopterin biosynthesis.
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-alpha-methyl-p-tyrosine (alpha-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 (BH(4)). In addition, pretreatment with BH(4) or with sepiapterin, which is converted to BH(4) intracellularly, significantly protected PC12D cells in a dose-dependent manner. The increased BH(4) 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 BH(4), concomitantly with the loss of protective effect afforded by V-1 overexpression. Furthermore, the addition of BH(4) 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 BH(4). 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.
Yuyama
Kohei
K
Laboratory of Molecular Recognition, Graduate School of Integrated Science, Yokohama City University, Yokohama, Japan.
Yamamoto
Hideko
H
Nakamura
Kazuhiro
K
Nishizaki
Itone
I
Yamakuni
Tohru
T
Song
Si-Young
SY
Sora
Ichiro
I
Nagatsu
Toshiharu
T
Yamamoto
Toshifumi
T
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000096:biosynthesis* / Q000235:genetics; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics; D009569:Nitric Oxide / Q000633:toxicity*; D020030:Nitric Oxide Donors / Q000633:toxicity; D016716:PC12 Cells; D051381:Rats
D036341:Intercellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D020030:Nitric Oxide Donors; C065700:myotrophin; D001708:Biopterins; D009569:Nitric Oxide; C003402:sapropterin