Abstract 1495: Loss of Endothelial NO Synthase Serine 1179 Phosphorylation and Function in ER Stress

Abstract

Endothelial NO synthase (eNOS) function is critically modulated by protein phosphorylation. In particular, phosphorylation of serine 1179 (S1179, bovine)/1177 (S1177, human and rat) by Akt has emerged as a central mechanism of eNOS regulation under both physiological and pathological conditions. Endoplasmic reticulum (ER) stress is a fundamental unfolded protein response occurred in various diseases. Whether and how ER stress affects eNOS phosphorylation is unknown. To address this issue, we induced ER stress in bovine aortic endothelial cells (BAECs) with Brefeldin A (BFA, 5 μg/ml), a compound blocking protein transport from ER to Golgi apparatus. BFA time-dependently induced ER stress in BAECs as evidenced by the markedly increased expressions of ER chaperon Grp78. Parallel to the time course of ER stress, a progressive loss of eNOS S1179 phosphorylation was seen. ER stress-induced eNOS dephosphorylation was specific to S1179 because the phosphorylation status of eNOS T497 or S635 was unchanged. In cells exposed to BFA for 4 hr, eNOS S1179 phosphorylation was decreased more than 5 fold (17.9±0.1% of control, P <0.01, n=5). As a result, eNOS activity was diminished (from 3.32±0.28 to 0.85±0.08 pmol/mg/min, P <0.01, n=3). Further studies revealed that ER stress caused Akt T308 and S473 dephosphorylation leading to Akt deactivation. Besides BFA, the loss of eNOS and Akt phosphorylation was also measured in ER stress induced by depleting ER Ca 2+ content with A23187 (2 μM) or perturbing ER oxidative environment with DTT (5 mM). To determine if these findings from cell culture occur in vivo, we monitored ER stress and eNOS S1177 phosphorylation in postischemic rat hearts. Indeed, severe ER stress and corresponding loss of eNOS S1177 phosphorylation and activity were detected in the infarcted areas of hearts after 1-hr coronary artery (LAD) occlusion followed by 24-hr reperfusion. Collectively, these results demonstrate that ER stress decreases eNOS S1179 phosphorylation and function via Akt deactivation. Ischemia/reperfusion cause ER stress, which, at least in part, accounts for the loss of eNOS S1179 phosphorylation and function in hearts. Thus, reducing ER stress may be an important approach to prevent eNOS dysfunction in postischemic hearts.