Abstract 1497: Differential Modulation of Endothelial Nitric Oxide Synthase (eNOS) Signaling Pathways by Tetrahydrobiopterin Synthesis vs. Salvage

Abstract

Tetrahydrobiopterin (BH4) is an essential redox-active cofactor for eNOS that can be both synthesized de novo or salvaged by enzymatic reduction of the oxidized compound. The endothelial dysfunction associated with diabetes is accompanied a decrease in the abundance of bioactive BH4. De novo biosynthesis of BH4 is catalyzed by GTP cyclohydrolase-1 (GTPCH1); recycling of BH4 is catalyzed by dihydrofolate reductase (DHFR). The relative roles of de novo BH4 synthesis and BH4 redox recycling in regulation of eNOS bioactivity remain incompletely defined. In the present study, we have used siRNA methods to investigate the effects of BH4 “knockdown” on eNOS regulation and endothelial signal transduction pathways in bovine aortic endothelial cells. We transfected duplex siRNA constructs designed to specifically target DHFR and GTPCH1, and suppressed levels of these proteins by ~90% (n = 37) relative to control siRNA-transfected cells. Transfection of siRNA constructs targeting DHFR or GTPCH1 suppressed VEGF-induced eNOS activity (using [ 3 H]-citrulline assay) or NO production (using an electrochemical NO sensor) by 90 ± 9% (n = 8, p < 0.01). siRNA-mediated knockdown of either DHFR or GTPCH1 had no effect on the abundance of stability of eNOS dimers, assessed using low-temperature SDS-PAGE (n = 4). DHFR knockdown completely blocked VEGF-induced eNOS dephosphorylation at the inhibitory phosphoserine residue 116 (n = 4, p < 0.01), but had no effect on agonist-modulated eNOS phosphorylation at the activating phosphoserine residue 1179. GTPCH1 knockdown had no effect either on phosphorylation or dephosphorylation of eNOS at these residues. Phosphorylation of Akt was decreased by 85 ± 4% by DHFR knockdown (p < 0.001, n = 4) but Akt phosphorylation was unaffected by GTPCH1 knockdown. These studies demonstrate for the first time a striking contrast in the consequences for eNOS signaling pathways from the suppression of BH4 salvage/reduction vs. de novo BH4 synthetic pathways. The abrogation of VEGF-mediated Akt activation by siRNA-mediated DHFR knockdown indicates that alterations in BH4 recycling may have broad effects on cell signaling pathways, with important consequences for the development of endothelial dysfunction in vascular disease states.