Endothelin‐1 Inhibits Nox1 in Human Abdominal Aortic Endothelial cells: a Novel Function of ETB1 receptors

Abstract

Hypothesis.Endothelin‐1 (ET‐1) inhibits Nox1 activity and decreases superoxide production and cell proliferation in human abdominal aortic endothelial cells (HAAECs).Methods and Results.HAAECs were exposed ET‐1 (10–30 nM) at different time intervals and superoxide production was measured by flow cytometry. Intracellular reactive oxygen species (ROS) was examined by confocal microscopy using 2′,7′‐dichlorodihydrofluorescein diacetate. NADPH oxidase activity was determined using lucigenin enhanced chemiluminescence in the presence and absence of siRNA for ETB1 receptors or Nox1. ET1 significantly attenuated Nox1 activity, ROS generation, and cell proliferation which could be abolished by silence of Nox1 gene. Furthermore, RNAi silence of ETB1 receptors significantly increased Nox1 activity and blocked the inhibitory effect of ET1 on Nox1 activity. ET1 enhanced bioavailability of intracellular NO measured using DAF‐2. ET1 did not affect Nox1 protein expression. Nox2 was not dectable in HAAECs. Activation of ETB1 receptors by ET1 suppressed protein phosphorylation of pyk2 (Y402) and Rac1, suggesting that ET1 inhibits Nox1 activity via ETB1‐Pyk2‐Rac1 pathway.Conclusions.This study demonstrated, for the first time, that that ET1 inhibited Nox1 activity, ROS production, and ell proliferation in HAAECs via ETB1‐Pyk2‐Rac1 pathway.