Hydrogen peroxide stimulates tetrahydrobiopterin synthesis through the induction of GTP-cyclohydrolase I and increases nitric oxide synthase activity in vascular endothelial cells

Tetrahydrobiopterin (BH4), which is an essential cofactor for nitric oxide synthase (NOS), is generally accepted as an important molecular target for oxidative stress. This study examined whether hydrogen peroxide (H₂O₂), one of the reactive oxygen species (ROS), affects the BH4 level in vascular endothelial cells (ECs). Interestingly, the addition of H₂O₂ to ECs markedly increased the BH4 level, but not its oxidized forms. The H₂O₂-induced increase in the BH4 level was blocked by the inhibitor of GTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme of BH4 synthesis. Moreover, H₂O₂ induced the expression of GTPCH mRNA, and the inhibitors of protein synthesis blocked the H₂O₂-induced increase in the BH4 level. The expression of the inducible isoform of NOS (iNOS) was slightly induced by the treatment with H₂O₂. Additionally, the L-citrulline formation from L-arginine, which is the marker for NO synthesis, was stimulated by the treatment with H₂O₂, and the H₂O₂-induced L-citrulline formation was strongly attenuated by NOS or GTPCH inhibitor. These results suggest that H₂O₂ induces BH4 synthesis via the induction of GTPCH, and the increased BH4 is coupled with NO production by coinduced iNOS. H₂O₂ appears to be one of the important signaling molecules to regulate the BH4-NOS system.