Tetrahydrobiopterin (BH₄) and superoxide dismutase (SOD) synergistically augment endothelium-dependent relaxations in canine middle cerebral artery

BH₄ is an essential cofactor required for activation of nitric oxide synthase. The present study was designed to determine the effect of an increase in endogenous BH₄ on endothelium-dependent relaxations in canine middle cerebral arteries. Segments of the isolated arteries were incubated (24 h at 37°C) in minimum essential medium in the presence and in the absence of a tetrahydrobiopterin precursor, sepiapterin (10^-4M), and/or SOD (150 U/ml). The arteries were suspended for isometric tension recording. BH₄ levels were assayed by high performance liquid chromatography. Levels of cGMP were measured by radioimmunoassay. All experiments were performed in the presence of indomethacin (10^-5 M). Incubation with sepiapterin markedly increased intracellular biopterin levels (Control, 363±273 vs. Sepiapterin, 118960±27151 pmol/g wet weight, n=5, P<0.01). In the sepiapterin-treated arteries, endothelium-dependent relaxations to calcium ionophore A23187 and intracellular cGMP levels were significantly decreased. SOD alone did not affect those responses. However, incubation with SOD plus sepiapterin significantly augmented the endothelium-dependent relaxations as well as the cGMP production. This augmentation was observed only in rings with (but not without) endothelium. The removal of Ca²⁺ from the medium abolished the synergistic effect of BH4 and SOD. These results demonstrate that in the presence of SOD, increased availability of endogenous BH4 may activate endothelial nitric oxide synthase.