High-density lipoprotein and apolipoprotein AI increase endothelial NO synthase activity by protein association and multisite phosphorylation

Abstract

NO propagates a number of antiatherogenic effects in the endothelium, and diminished availability has been associated with vascular disease. Recently it has been reported that phosphorylation of endothelial NO synthase (eNOS) at Ser-1179 is required to increase activity in response to stimuli, including high-density lipoprotein (HDL). The current study was undertaken to further examine the mechanism by which HDL activates eNOS and to specifically determine the role of the major apolipoprotein of HDL, apolipoprotein AI (ApoAI). Phosphorylation of eNOS residues Ser-116, Ser-617, Ser-635, Ser-1179, and Thr-497 after incubation with ApoAI and HDL was examined. There were significant increases in phosphorylation at Ser-116 in response to both HDL and ApoAI and similar magnitudes of dephosphorylation at Thr-497. Ser-1179 phosphorylation increased transiently but returned to basal level after 2.5 min. Data demonstrating activation of AMP activated protein kinase (AMPK) during HDL and ApoAI incubation suggests that AMPK may play a role in activation of eNOS. NO release in response to HDL and ApoAI stimulation in endothelial cells paralleled the time frames of phosphorylation, suggesting a causal relationship. Furthermore, ApoAI was found to associate with eNOS in endothelial cells and bind transfected eNOS in Chinese hamster ovary cells, whereas confocal data demonstrates colocalization of ApoAI and eNOS in the perinuclear region, suggesting a protein-protein interaction. Collectively, the results indicate that HDL and ApoAI increase eNOS activity by multisite phosphorylation changes, involving AMPK activation after protein association between ApoAI and eNOS. D ecreased bioavailability of endothelium-derived NO is an important antecedent to atherosclerosis (1). NO inhibits events that promote atherosclerotic progression, including vasoconstriction, monocyte adhesion, and smooth muscle cell proliferation (2). The bulk of endothelium-derived NO is generated from L-arginine conversion by endothelial NO synthase (eNOS, NOS III) (3). Activity of eNOS is modulated by complex mechanisms including phosphorylation, protein-protein interactions, substrate availability, and intracellular Ca 2ϩ flux. Numerous biological agents have been associated with changes in eNOS activity, including caveolin (4), Ca 2ϩ calmodulin (5), HSP90 (6), Dynamin-2 (7), bradykinin (8), and more recently high-density lipoprotein (HDL) (9). HDL plays a major role in reversing and preventing progression of vascular disease through its role in reverse cholesterol transport and its involvement in signaling͞receptor pathways of cholesterol metabolism (10). Possibly, some cardiovascular protective effects of HDL are mediated via activation of eNOS, although the precise nature of this interaction remains unclear. The current study was undertaken to examine the mechanism by which HDL activates eNOS and to determine whether the major apolipoprotein of HDL, apolipoprotein AI (ApoAI), mediates the response.Endothelial cells incubated with HDL exhibit an increase in...

Adiponectin-Induced Endothelial Nitric Oxide Synthase Activation and Nitric Oxide Production Are Mediated by APPL1 in Endothelial Cells

“…AMPK stimulates eNOS activity by phosphorylating eNOS at Ser 1177 and promoting its association with HSP90. Several vasoprotective agents, such as the antidiabetic drug metformin (40), 17␤-estradiol (34), high-density lipoprotein, and apolipoprotein AI (41), have recently been shown to activate AMPK in endothelial cells. Consistent with the previously reported findings (12,16), our results also demonstrated that AMPK is the principal kinase responsible for adiponectin-evoked eNOS phosphorylation at Ser 1177 , eNOS association with HSP90, and NO production in HUVECs (Fig.…”

Inability of HDL from type 2 diabetic patients to counteract the inhibitory effect of oxidised LDL on endothelium-dependent vasorelaxation

“…On the other hand, HDL can stimulate NO production. eNOS activity can be upregulated by HDL through regulation of phosphorylation sites [23]. Different signalling pathways are likely to be involved in this process.…”

Apolipoprotein A-I stimulates AMP-activated protein kinase and improves glucose metabolism

“…APOA-I is also involved in the delivery of cholesteryl esters to the liver via scavenger receptor class B type I (SR-BI) [4]. A decreased bioavailability of endothelium-derived NO is an important antecedent to atherosclerosis, and it was recently reported that APOA-I could upregulate the activity of endothelial nitric oxide synthase (eNOS), likely through specific phosphorylation of AMP-activated protein kinase (AMPK) in endothelial cells [5]. However, the intracellular function of APOA-I is largely uncharacterised.…”