Tetrahydrobiopterin deficiency increases neuronal vulnerability to hypoxia

Tetrahydrobiopterin (BH 4 ) is an essential co‐factor for nitric oxide synthases (NOS). The aim of the present work was to study whether BH 4 deficiency affects the vulnerability of neurones in primary culture to hypoxia. Intracellular BH 4 levels were depleted by pre‐incubating neurones with 5 m m 2,4‐diamino‐6‐hydroxypyrimidine (DAHP) for 18 h, after which cells were exposed for 1 h to normoxic or hypoxic conditions. Our results showed that whereas neurones were resistant to hypoxia‐induced cellular damage, BH 4 deficiency in neurones led to oxidative stress, mitochondrial depolarization, ATP depletion and necrosis after 1 h of hypoxia. Indeed, hypoxia specifically inhibited mitochondrial complex IV activity in BH 4 ‐deficient neurones. All these effects were counteracted whenneuronal BH 4 levels were restored by incubating cells with exogenous BH 4 during the hypoxic period. Moreover, hypoxia‐induced damage in BH 4 ‐deficient neurones was prevented when N ω ‐nitro‐ l ‐arginine monomethyl ester (NAME), haemoglobin or superoxide dismutase plus catalase were present during the hypoxic period, suggesting that peroxynitrite might be involved in the process. In fact, BH 4 deficiency elicited neuronal NO dysfunction, resulting in an increase in peroxynitrite generation by cells, as shown by the enhancement in tyrosine nitration; this was prevented by supplements of BH 4 , NAME, haemoglobin or superoxide dismutase plus catalase during hypoxia. Our results suggest that BH 4 deficiency converts neuronal NOS into an efficient peroxynitrite synthase, which is responsible for the increase in neuronal vulnerability tohypoxia‐induced mitochondrial damage and necrosis.