Abstract 327: Differential Enrichment of NOS3 Protein in Caveolar Membrane Regulates Responses Post-shear: Effects of NOS3 Glu298Asp Single Nucleotide Polymorphism
Human endothelial nitric oxide synthase (NOS3) gene polymorphism at Exon 7 (Glu298Asp) has been linked to vascular endothelial cell (EC) dysfunction but the cellular mechanisms involved are not defined. We have previously demonstrated that the presence of this SNP in human endothelial cells (ECs) alters their responses to increased shear and causes altered NOS3-Caveolin-1 interactions. Here we tested the hypothesis that altered enrichment of NOS3 in the caveolar membrane defines Glu298Asp genotype-specific EC responses and NOS3 activity. ECs were exposed to high shear for 4h and NOx production was estimated by Griess reaction. Basal caveolar membrane enrichment was carried out to quantitate the NOS3 protein level in the caveolar membrane. Phosphorylation status of NOS3 (pNOS3) was quantified by standard western blotting methods. We observed no genotype dependent pNOS3 levels pre and post-shear. In contrast, caveolar membrane fractions from the variant genotypes had less NOS3 protein as compared to the wild-type ECs (NOS3/Cav-1 ratios in caveolar membrane fractions: 2.40±0.05, 1.65±0.05 & 1.41±0.12 in Glu/Glu, Glu/Asp & Asp/Asp (p<0.05)). There was a significant correlation between basal NOS3 activity and basal caveolar NOS3 levels (p<0.05), but not with basal pNOS3 levels. Moreover the NOx production rate post-shear also showed a significant correlation with the basal caveolar membrane enrichment. There was no apparent correlation between NOx production post-shear and activation of NOS3 by phosphor-ylation post-shear (pNOS3). These data demonstrate EC functional consequences of the Glu298Asp NOS3 variation and further define disruption of NOS3 caveolar localization and shear-induced mobilization as the primary mechanism responsible for these differences. These impaired interactions may provide the basis for increased risk for endothelial dysfunction due to the presence of this polymorphism in humans.