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This Article Full Text Full Text (PDF) All Versions of this Article: 297/1/H331    most recent 00007.2009v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Gao, L. Articles by Cai, H. PubMed PubMed Citation Articles by Gao, L. Articles by Cai, H.

Sepiapterin reductase regulation of endothelial tetrahydrobiopterin and nitric oxide bioavailability

Division of Molecular Medicine, Departments of Anesthesiology and Medicine, Cardiovascular Research Laboratories, University of California Los Angeles, Los Angeles, California

Submitted 6 January 2009 ; accepted in final form 28 April 2009

Sepiapterin reductase (SPR) catalyzes the final step of tetrahydrobiopterin (H₄B) biosynthesis and the first step of H₄B regeneration from an exogenous precursor sepiapterin. Despite the potential significance of SPR in regulating H₄B-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 H₄B 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, H₄B 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 H₄B content and NO level. Exogenous administration of sepiapterin to endothelial cells significantly upregulated H₄B and NO levels, which were attenuated by SPR RNA interference (RNAi). H₄B-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 H₄B and NO bioavailability.

endothelial nitric oxide synthase; aortic endothelial cells; guanosine 5'-triphosphate cyclohydrolase 1; dihydrofolate reductase