Reactive oxygen species mediate endothelium-dependent relaxations in Tetrahydrobiopterin-deficient mice

(6R)-5,6,7,8-Tetrahydro-biopterin (H 4B) is essential for the catalytic activity of all NO synthases. The hyperphenylalaninemic mouse mutant (hph-1) displays 90% deficiency of the GTP cyclohydrolase I, the rate-limiting enzyme in H 4B synthesis. A relative shortage of H 4B may shift the balance between endothelial NO synthase (eNOS)-catalyzed generation of NO and reactive oxygen species. Therefore, the hph- 1 mouse represents a unique model to assess the effect of chronic H 4B deficiency on endothelial function. Aortas from 8-week-old hph-1 and wild-type mice (C57BL×CBA) were compared. H 4B levels were determined by high-performance liquid chromatography and NO synthase activity by [ 3H]citrulline assay in homogenized tissue. Superoxide production by the chemiluminescence method was measured. Isometric tension was continuously recorded. The intracellular levels of H 4B as well as constitutive NO synthase activity were significantly lower in hph-1 compared with wild-type mice. Systolic blood pressure was increased in hph-1 mice. However, endothelium-dependent relaxations to acetylcholine were present in both groups and abolished by inhibition of NO synthase with N Gnitro-L-arginine methyl ester as well. Only in hph-1 mice were the relaxations inhibited by catalase and enhanced by superoxide dismutase. After incubation with exogenous H 4B, the differences between the 2 groups disappeared. Our findings demonstrate that H 4B deficiency leads to eNOS dysfunction with the formation of reactive oxygen species, which become mediators of endothelium-dependent relaxations. A decreased availability of H 4B may favor an impaired activity of eNOS and thus contribute to the development of vascular diseases.