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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:496.)
© 2001 American Heart Association, Inc.

Vascular Biology

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

Francesco Cosentino; Jane E. Barker; Michael P. Brand; Simon J. Heales; Ernst R. Werner; John R. Tippins; Nick West; Keith M. Channon; Massimo Volpe; Thomas F. Lüscher

From Cardiology, Cardiovascular Research (F.C., T.F.L.), University Hospital, Zürich, Switzerland; IRCCS Neuromed (F.C., M.V.), Pozzilli, Italy; Medical Chemistry and Biochemistry (E.R.W.), University of Innsbruck, Innsbruck, Austria; Neurochemistry (J.E.B., M.P.B., S.J.H.), Institute of Neurology, Queen Square, London, UK; Biochemistry (J.R.T.), Imperial College, London, UK; and Cardiovascular Medicine (N.W., K.M.C.), University of Oxford, Oxford, UK.

Correspondence to Thomas F. Lüscher, MD, FHRCP, Professor and Head of Cardiology, University Hospital, CH-8091 Zürich, Switzerland. E-mail cardiotfl{at}gmx.ch

Abstract—(6R)-5,6,7,8-Tetrahydro-biopterin (H₄B) 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 H₄B synthesis. A relative shortage of H₄B 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 H₄B deficiency on endothelial function. Aortas from 8-week-old hph-1 and wild-type mice (C57BLxCBA) were compared. H₄B 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 H₄B 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^G-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 H₄B, the differences between the 2 groups disappeared. Our findings demonstrate that H₄B deficiency leads to eNOS dysfunction with the formation of reactive oxygen species, which become mediators of endothelium-dependent relaxations. A decreased availability of H₄B may favor an impaired activity of eNOS and thus contribute to the development of vascular diseases.

Key Words: NO synthase • superoxide anion • hydrogen peroxide • catalase GTP cyclohydrolase I

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