Abstract 4879: Overexpression of GTP-Cyclohydrolase I in Vascular Endothelium Prevents Oxidative Stress-Induced eNOS Translocation From Caveolae to Cytosol

Abstract

GTP-Cyclohydrolase I (GTPCH I) is the rate-limiting enzyme for tetrahydrobiopterin biosynthesis, an essential co-factor required for enzymatic activity of endothelial nitric oxide synthase (eNOS). Recent studies have suggested that endothelium-targeted overexpression of GTPCH I has vasoprotective effects. Therefore, we hypothesized that in endothelium GTPCH I localization in caveolae protects eNOS against oxidative stress. Human umbilical vein endothelial cells (HUVECs) were incubated with recombinant adenovirus encoding the human GTPCH I gene (Ad-GTPCH I, 100 MOI) or control recombinant adenoviral vector (Ad-ΔE1) for 12 hours in serum-free media. Cells were then fed with growth medium for 48 hours prior to analysis. Using sucrose gradient ultracentrifugation we detected both protein expression and enzymatic activity of GTPCH I in caveolar microdomains of HUVECs. Treatment of Ad-ΔE1 transduced endothelial cells for 24 hours with LY83583 (1 μ M), a superoxide anion generator, significantly decreased enzymatic activity of GTPCH I in caveolae-enriched membranes (0.49±0.12 pmol neopterin/mg protein; P<0.05 vs. control: 0.92±0.07 pmol neopterin/mg). Furthermore, LY83583 translocated eNOS from caveolae to cytosol in Ad-ΔE1 treated endothelial cells (P<0.05; n=3). This effect was specific because there was no change in caveolin-1 expression and no decrease in cell viability. Treatment of HUVECs with Ad-GTPCH I significantly increased GTPCH I activity in caveolae-enriched membranes (1.37±0.22 pmol neopterin/mg; P<0.05; n=4). Interestingly, LY83583-induced dislocation of eNOS from caveolar membrane fraction was significantly prevented by overexpression of GTPCH I (P<0.05; n=3). Our results suggest that overexpression of GTPCH I increases its enzymatic activity in caveolae. This effect preserves strategically key localization of eNOS in cell membrane during oxidative stress. This research has received full or partial funding support from the American Heart Association, National Center.