Introduction/Aims:
FXYD1 has been shown to be a master regulator of the sodium potassium pump activity in the caveolae of heart tissue, protecting the pump from glutathionylation and oxidative inhibition. However, very little is known about its expression and function in the endothelium, particularly in regard to its potential interaction with other caveolae proteins. Given the molecular mechanism of eNOS uncoupling is via glutathionylation, we examined whether FXYD1 was expressed in endothelial cells, and whether it had a functional partnership with eNOS.
Methods:
Co-immunoprecipitation was used to study the interaction of FXYD1 and eNOS. GSH antibody was used for immunodetection of eNOS glutathionylation. Silencing of FXYD1 using siRNA followed by DAF staining or spin trap were used to study the effects of FXYD1 on eNOS function. Enzyme-linked immunosorbent assay was also employed to quantify eNOS glutathionylation level in human erythrocyte samples post exposure to angiotensin II (0 to 500 nM) ex vivo.
Results:
FXYD1 co-immunoprecipitated with eNOS in human umbilical vein endothelial cells (HUVECs). Silencing of FXYD1 resulted in significantly increased eNOS glutathionylation, associated with significantly reduced NO bioavailability under baseline and acetylcholine stimulated conditions (1μM, 20mins). Furthermore, a dose-dependent increase in eNOS glutathionylation level was also observed in human erythrocyte exposed to angiotensin II.
Discussion:
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. Furthermore, FXYD1 expression/eNOS glutathionylation may serve as important biomarkers for oxidative stress.
Angiotensin II degrades myeloid cell leukemia 1 in human umbilical vein endothelial cells
