Abstract 5644: Adenosine Diphosphate Signaling in Vascular Endothelial Cells: A Novel Role for AMP-activated Protein Kinase in eNOS Regulation
Adenosine diphosphate (ADP) responses underlie therapeutic approaches to many cardiovascular diseases, yet the pathways of ADP signaling in endothelial cells remain incompletely understood. In cultured aortic endothelial cells, we found that ADP rapidly promotes phosphorylation of eNOS at Ser1179 (4.7±0.7-fold increase; p<0.05; n=4) and Ser635 (4.4±0.5-fold increase; p<0.01; n=3), as well as dephosphorylation of eNOS at Ser116 (51±2% decrease; p<0.01; n=4), with an EC50 of ~15μM. eNOS enzyme activity is stimulated by both ADP (3.2±0.1-fold increase; p<0.05; n=6) and ATP (3.0±0.1-fold increase; p<0.05; n=4). However, only ADP but not ATP signaling is inhibited by the P2Y1 receptor antagonist MRS 2179 and by siRNA-mediated knockdown of P2Y1. These data distinguish ADP from ATP responses. We analyzed signaling pathways involving PI3K/Akt, ERK1/2, Src, phospholipase C, and calcium/calmodulin-dependent kinase kinase β (CaMKKβ), using the inhibitors wortmannin, PD90859, PP2, U-73122, and STO-609, respectively. None of these inhibitors alters ADP-modulated eNOS phosphorylation. siRNA-mediated knockdown of AMP-activated protein kinase (AMPK) does not affect ADP-dependent eNOS Ser1179 phosphorylation but significantly inhibits eNOS Ser635 phosphorylation and eNOS activity (52±2% decrease; p<0.01; n=5). Importantly, the AMPK enzyme inhibitor compound C has no effect on ADP-induced eNOS activity, despite completely blocking AMPK activity. Similarly, CaMKKβ knockdown suppresses ADP-stimulated eNOS activity, while complete inhibition of CaMKKβ kinase activity using STO-609 has no effect on ADP signaling to eNOS. ADP activates the small GTPase Rac1 and promotes endothelial cell migration (2.5±0.9-fold increase; p<0.001; n=4). ADP-promoted cell migration is abrogated by siRNA-mediated knockdown of Rac1. Notably, siRNA knockdown of Rac1 blocks ADP-mediated eNOS Ser1179 and Ser635 phosphorylation (43±8% decrease; p<0.05; n=4) as well as eNOS activation (55±14% decrease; p<0.05; n=4). These data suggest that the expression- but not the kinase activity- of AMPK and CaMKKβ is necessary for ADP signaling to eNOS. These findings also establish a novel role for Rac1 in ADP regulation of eNOS activity and cell migration in endothelial cells.