Abstract 15521: FXYD1 Protects Against Redox-Dependent Uncoupling of Endothelial Nitric Oxide Synthase
Background: Vascular health is dependent on the vasoprotective molecule, nitric oxide (NO), produced by endothelial NO synthase (eNOS). Under conditions of oxidative stress such as diabetes and hypertension, eNOS becomes “uncoupled” via glutathionylation, preferentially producing the damaging free radical, superoxide (O2.-). Discovery of protective mechanisms against this will have critical implications for vascular function in health and disease. We hypothesised that caveolar co-localisation of FXYD1 and eNOS may result in functional interaction and redox signalling in the vasculature.
Methods and results: FXYD1-eNOS co-localisation was demonstrated by both proximity ligation assay and co-immunoprecipitation in human umbilical vein endothelial cells (HUVECs). eNOS glutathionylation, assessed by co-immunoprecipitation of eNOS and glutathione, was significantly increased (~1.5 fold, p=0.04, n=3) after silencing of FXYD1 using siRNA and this was associated with reduced NO bioavailability, as assessed by diaminofluorescein stain after acetylcholine stimulation (~60% reduction, p<0.001, n=3) and ~50% increase in NADPH-stimulated O2.- production, determined by lucigenin enhanced chemiluminescence (n = 3, P<0.001). Likewise, aortic AngII-induced eNOS glutathionylation was elevated and this was associated with ~doubling in O2.- production in FXYD1 knockout (KO) mice (n = 5, P<0.05). In vivo, blood pressure decreases in anaesthetized mice, measured by carotid artery cannulation, in response to intravenous bolus doses of bradykinin (endothelium-dependent vasodilation) were reduced in KO compared to wild type (WT) mice (max decrease at 10 μg/kg dose: KO 9.6 ± 1.4 mmHg vs. WT 16.6 ± 1.7 mmHg, P<0.001, n=7). However responses to NO donor, sodium nitroprusside, were similar suggesting that reduced NO bioavailability is the cause of endothelial dysfunction in KO mice.
Conclusions: Our findings demonstrated a novel functional partnership of FXYD1 with eNOS, protecting this vital enzyme from glutathionylation-mediated uncoupling. This has important implications for our understanding of ROS-signalling in the vasculature and could lead to novel treatment options with recombinant FXYD1 for redox-dependent vascular disease.
Author Disclosures: K.J. Bubb: None. O. Tang: None. T. Hansen: None. K. Karimi Galougahi: None. G.A. Figtree: None.
- © 2016 by American Heart Association, Inc.