Abstract 1917: Tetrahydrobiopterin Recycling is a Key Determinant of eNOS-dependent Pathways for Oxidative and Nitrosative Stress in Vascular Endothelium
Tetrahydrobiopterin (BH4) is a key redox-active cofactor for eNOS, and is an important determinant of NO-dependent signaling pathways in the vascular wall. BH4 can be synthesized de novo by the enzyme GTP cyclohydrolase-1 (GTPCH1), or recycled by enzymatic reduction of the oxidized product, dihydrobiopterin (BH2), by dihydrofolate reductase (DHFR). BH4 oxidation is seen in vascular cells in the setting of oxidative stress associated with diabetes and hypertension. However, the relative roles of de novo BH4 synthesis and BH4 redox recycling in regulation of eNOS bioactivity remain incompletely defined. We studied the consequences of small interfering RNA (siRNA)-mediated “knockdown” of enzymes involved in BH4 synthesis and BH4 recycling on eNOS regulation in cultured aortic endothelial cells (EC). Transfection of siRNA constructs targeting DHFR or GTPCH1 decreased levels of these proteins by > 90% (n = 40). Knockdown of either DHFR or GTPCH1 attenuated VEGF-induced eNOS activity (quantitated in the [3H]-arginine/[3H]-citrulline assay) and NO production (determined with an electrochemical NO sensor) by 90 ± 9% (n = 8, p < 0.01); these effects were recovered by supplementation of EC with 10 μM BH4. In contrast, supplementation with BH2 (10 μM) abolished VEGF-induced NO production. DHFR but not GTPCH1 knockdown increased hydrogen peroxide (H2O2) production (quantitated using the Amplex Red fluorescence assay) by 77 ± 12% (n = 8, p < 0.01). The increase in H2O2 production seen with siRNA-mediated DHFR knockdown was abolished either by simultaneous siRNA-mediated knockdown of eNOS, or by supplementing the cells with BH4. In contrast, addition of BH2 increased H2O2 production by 119 ± 6% (n = 10, p < 0.01); this effect of BH2 was inhibited by BH4 supplementation. These studies demonstrate a striking contrast in the consequences for eNOS regulation from the selective modulation of BH4 salvage/reduction vs. de novo BH4 synthetic pathways. Our findings suggest that the simple depletion of BH4 is not sufficient to promote endothelial dysfunction, but rather that it is the concentration of intracellular BH2, as well as the relative concentrations of BH4 and BH2, that together play a determining role in the redox regulation of eNOS-modulated endothelial responses.
This research has received full or partial funding support from the American Heart Association, AHA Founders Affiliate (Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Rhode Island, Vermont).