Abstract 1499: Analysis of Subcellular Localization of GTP Cyclohydrolase I in Vascular Endothelium
GTP Cyclohydrolase I (GTPCH I) is the rate-limiting enzyme for biosynthesis of tetrahydrobiopterin (BH4), 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, however the exact subcellular localization of GTPCH I is unknown. Using sucrose gradient ultracentrifugation we detected both protein expression and enzymatic activity of GTPCH I in caveolar microdomains of human umbilical vein endothelial cells (HUVECs) suggesting that GTPCH I co-localizes with eNOS and caveolin-1 (n=3; P<0.05). Indeed, further analysis by confocal microscopy of HUVECs transduced with an adenovirus encoding a haemagglutinin-tagged GTPCH I also demonstrated co-localization of GTPCH I with caveolin-1. Electron microscopy and immunogold labeling revealed that GTPCH I is localized in the membrane region and within the cytoplasm of the aortic endothelium of wild-type mice. Most importantly, caveolin-1 protein was located in close proximity to GTPCH I. Consistent with in vitro findings, expression of GTPCH I in caveolae was significantly elevated in GTPCH I transgenic mice (n=3; P<0.05). To determine functional relevance of GTPCH I co-localization with caveolin-1 we examined biosynthesis of BH4 (as determined by HPLC analysis) in caveolin-1-deficient mice. Interestingly, BH4 levels were significantly increased in aortas from caveolin-1-deficient mice (9.0±0.5 pmol/mg protein; P<0.05 vs wild-type: 6.3±0.2 pmol/mg; n=4). Although expression of GTPCH I protein was not affected by genetic inactivation of caveolin-1, enzymatic activity of GTPCH I was significantly augmented in aortas of caveolin-1-deficient mice (1.4±0.1 pmol neopterin/mg protein; P<0.05 vs wild-type: 0.73±0.1 pmol neopterin/mg; n=4). This observation indicates that the selective increase in BH4 levels was caused by the increased de-novo biosynthesis of BH4 via GTPCH I. Our studies demonstrate caveolar localization of GTPCH I and biosynthesis of BH4. In addition, it appears that in vascular endothelium caveolin-1 exerts an inhibitory effect on enzymatic activity of GTPCH I. 1896 characters (max. 1950 characters allowed).