Abstract 1655: Regulation of Tetrahydrobiopterin Synthesis by Shear Stress
Unidirectional laminar shear stress acutely activates the endothelial nitric oxide synthase (eNOS) to produce NO and over the long term increases eNOS expression. Tetrahydrobiopterin (H4B) is an essential co-factor for eNOS and increased H4B would be necessary to allow for increased NO production in response to prolonged endothelial shear. We therefore examined the effect of shear on H4B biosynthesis in human aortic endothelial cells. Unidirectional laminar shear (15 dynes/cm2) dramatically increased H4B (5.84 ± 0.91 vs. 0.1 ± 0.07 pmoles/mg, shear vs. static n=7). Activity of the first enzyme in H4B biosynthesis, GTP cyclohydrolase 1 (GTPCH-1), was increased 30-fold by shear (0.57 ± 0.15 vs. 0.02 ± 0.01 uU/mg, sheared vs. static cells, n=5–6). In contrast to this dramatic increase in GTPCH-1 activity, shear only increased GTPCH-1 mRNA and protein levels by 3 to 5 fold. Shear did not alter expression or activity of 6-pyruvol-tetrahydropterin synthase and sepiapterin reductase, downstream enzymes in the de-novo pathway of H4B synthesis and paradoxically decreased dihydrofolate reductase (DHFR), the endothelial salvage enzyme for conversion of oxidized H2B to H4B. Moreover, inhibition of DHFR with methotrexate did not prevent the shear-induced increase in H4B. GPTCH-1 contains putative sites for protein kinase C and casein kinase II phosphorylation. The shear induced increase in H4B and GTPCH-1 levels was dose-dependently diminished by the casein kinase II inhibitor 4,5,6,7-tetrabromobenzotriazole but not by protein kinase C inhibition with chelerythrine. The increase in H4B caused by shear is essential in allowing proper function of eNOS, because GPTCH-1 blockade with 2,4-diamino-6-hydroxypyrimidine during shear inhibited dimer formation of eNOS, significantly decreased NO-production as detected by Fe2+ (DETC)2 ESR (1126 ± 49 vs. 2440 ± 227 signal/mg in non-treated cells n=5) and increased superoxide formation. Exercise increases cardiac output and aortic shear, and we found that exercise training in mice also augmented aortic H4B levels. Thus, not only does shear stress and exercise increase eNOS but also its critical co-factor H4B. This is dependent on a marked stimulation of GPTCH-1 enzyme activity and is essential for eNOS function.