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Endothelin (ET)-1 Inhibits Nicotinamide Adenine Dinucleotide Phosphate Oxidase Activity in Human Abdominal Aortic Endothelial Cells: A Novel Function of ET_B1 Receptors

Department of Physiology, College of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104

Address all correspondence and requests for reprints to: Zhongjie Sun, M.D., Ph.D., F.A.H.A., Department of Physiology, BMSB 662A, Box 26901, College of Medicine, University of Oklahoma Health Sciences Center, 940 S. L. Young Boulevard, Oklahoma City, Oklahoma 73126-0901. E-mail: Zhongjie-sun{at}ouhsc.edu.

Endothelin (ET)-1 stimulates nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and increases superoxide production in some cells such as vascular smooth muscle cells. Here, we reported that ET1 inhibited NADPH oxidase activity, superoxide generation, and cell proliferation in human abdominal aortic endothelial cells (HAAECs) via the ET_B1-Pyk2-Rac1-Nox1 pathway. Superoxide production was determined by assessing ethidium fluorescence using flow cytometry in HAAECs exposed to ET1 (10–30 nM) at different time intervals. ET1 significantly decreased superoxide production in HAAECs in the presence of N^G-nitro-L-arginine methyl ester, indicating that ET1 suppressed superoxide generation independent of nitric oxide synthase. ET1 significantly attenuated NADPH oxidase activity and cell proliferation, which could be abolished by silence of Nox1 gene, suggesting that ET1-induced inhibition of NADPH oxidase activity was mediated by Nox1. Furthermore, RNA interference silence of ET_B1 receptors significantly increased NADPH oxidase activity, and blocked the inhibitory effect of ET1 on NADPH oxidase activity. Activation of ET_B1 receptors by ET1 suppressed protein phosphorylation of pyk2 (Y402) and Rac1, suggesting that ET1 inhibited NADPH oxidase activity via ET_B1-Pyk2-Rac1 pathway. Indeed, inhibition of Pyk2 by AG-17 abolished ET1-induced suppression of NADPH oxidase activity. ET1 also attenuated angiotensin II-induced activation of NADPH oxidase and cell proliferation. This study demonstrated, for the first time, that ET1, via ET_B1, inhibited NADPH oxidase activity in HAAECs by suppressing the Pyk2-Rac1-Nox1 pathway. This finding reveals a novel function of ET_B1 receptors in regulating endothelial NADPH oxidase activity, superoxide production, and cell proliferation, opening a new avenue for understanding the role of ET_B1 receptors in protecting endothelial cells.