Abstract 1491: Tetrahydrobiopterin Availability Directly Regulates eNOS Coupling In Tet-regulatable Cells
Tetrahydrobiopterin (BH4) is a critical determinant of eNOS activity. Loss of NO bioavailability, due to reduced synthesis and increased scavenging by reactive oxygen species, is a cardinal feature of endothelial dysfunction in vascular disease. When intracellular BH4 levels are limiting, eNOS becomes ‘uncoupled’ and generates superoxide rather than NO. However, it is unclear whether the absolute intracellular concentration of BH4 or the ratio of BH4 to oxidized biopterins are the key determinant of eNOS coupling, and the stoichiometry of BH4/eNOS interactions is not established. To address these questions we have developed a novel murine fibroblast cell line with tetracycline regulated expression of human GCH cDNA (GCH-tet), the rate limiting enzyme in BH4 synthesis, to generate a range of intracellular BH4 levels. These cells were then stably co-transfected with a human eNOS-GFP construct (GCH eNOS-tet). Addition of doxycycline completely abolished GTPCH mRNA expression within 3 hours and GTPCH protein was undetectable after 10 days. Doxycycline markedly diminished total biopterin levels (quantified by HPLC with electrochemical and fluorescence detection) and the ratio of BH4 to oxidised species was significantly reduced (Figure, A⇓). Superoxide generation from eNOS, as assessed by L-NAME inhibitable 2-hydroxyethidium generation (HPLC), was significantly elevated following 10 days of doxycycline treatment indicating eNOS uncoupling (Figure, B⇓). This was reversed by withdrawal of doxycycline. Using this specific, reductionist approach these data indicate that eNOS coupling is directly regulated by BH4 availability, even in the absence of oxidative stress.