Sepiapterin reductase regulation of endothelial tetrahydrobiopterin and nitric oxide bioavailability

Sepiapterin reductase (SPR) catalyzes the final step of tetrahydrobiopterin (H 4 B) biosynthesis and the first step of H 4 B regeneration from an exogenous precursor sepiapterin. Despite the potential significance of SPR in regulating H 4 B-dependent nitric oxide (NO • ) production, the endothelium-specific sequence and functions of SPR remain elusive. We first cloned endothelial SPR cDNA from bovine aortic endothelial cells (Genebank: DQ978331). In cells transiently transfected with SPR gene, SPR activity (HPLC) was dramatically increased by 19-fold, corresponding to a significant increase in endothelial H 4 B content (HPLC) and NO • production (electron spin resonance). In vivo delivery of SPR gene significantly increased vascular SPR protein expression (mouse vs. bovine antibodies to differentiate endogenous vs. exogenous), activity, H 4 B content, and NO • production, as well as NO • -dependent vasorelaxation. In endothelial cells transfected with small interfering RNA specific for SPR, ∼87% of mRNA were attenuated (real-time quantitative RT-PCR), corresponding to a significant reduction in SPR protein expression and activity, which was associated with decreases in both intracellular H 4 B content and NO • level. Exogenous administration of sepiapterin to endothelial cells significantly upregulated H 4 B and NO • levels, which were attenuated by SPR RNA interference (RNAi). H 4 B-stimulated increase in NO • production, however, was SPR RNAi independent. GTP cyclohydrolase 1 expression and activity, as well as dihydrofolate reductase expression, were not affected by SPR RNAi, whereas dihydrofolate reductase activity was significantly downregulated. These data represent the first to study endothelial SPR functionally and clearly demonstrate an important role of endothelial SPR in modulating H 4 B and NO • bioavailability.