Reactive Oxygen Species Mediate Endothelium-Dependent Relaxations in Tetrahydrobiopterin-Deficient Mice

Abstract

Abstract—(6 R)-5,6,7,8-Tetrahydro-biopterin (H 4 B) 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 4 B synthesis. A relative shortage of H 4 B 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 4 B deficiency on endothelial function. Aortas from 8-week-old hph-1 and wild-type mice (C57BL×CBA) were compared. H 4 B levels were determined by high-performance liquid chromatography and NO synthase activity by [3 H]citrulline assay in homogenized tissue. Superoxide production by the chemiluminescence method was measured. Isometric tension was continuously recorded. The intracellular levels of H 4 B 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 NG-nitro-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 4 B, the differences between the 2 groups disappeared. Our findings demonstrate that H 4 B deficiency leads to eNOS dysfunction with the formation of reactive oxygen species, which become mediators of endothelium-dependent relaxations. A decreased availability of H 4 B may favor an impaired activity of eNOS and thus contribute to the development of vascular diseases.