ADP signaling in vascular endothelial cells: ADP-dependent activation of the endothelial isoform of nitric-oxide synthase requires the expression but not the kinase activity of AMP-activated protein kinase.
ADP responses underlie therapeutic approaches to many cardiovascular diseases, and ADP receptor antagonists are in widespread clinical use. The role of ADP in platelet biology has been extensively studied, yet ADP signaling pathways in endothelial cells remain incompletely understood. We found that ADP promoted phosphorylation of the endothelial isoform of nitric-oxide synthase (eNOS) at Ser(1179) and Ser(635) and dephosphorylation at Ser(116) in cultured endothelial cells. Although eNOS activity was stimulated by both ADP and ATP, only ADP signaling was significantly inhibited by the P2Y(1) receptor antagonist MRS 2179 or by knockdown of P2Y(1) using small interfering RNA (siRNA). ADP activated the small GTPase Rac1 and promoted endothelial cell migration. siRNA-mediated knockdown of Rac1 blocked ADP-dependent eNOS Ser(1179) and Ser(635) phosphorylation, as well as eNOS activation. We analyzed pathways known to regulate eNOS, including phosphoinositide 3-kinase/Akt, ERK1/2, Src, and calcium/calmodulin-dependent kinase kinase-beta (CaMKKbeta) using the inhibitors wortmannin, PD98059, PP2, and STO-609, respectively. None of these inhibitors altered ADP-modulated eNOS phosphorylation. In contrast, siRNA-mediated knockdown of AMP-activated protein kinase (AMPK) inhibited ADP-dependent eNOS Ser(635) phosphorylation and eNOS activity but did not affect eNOS Ser(1179) phosphorylation. Importantly, the AMPK enzyme inhibitor compound C had no effect on ADP-stimulated eNOS activity, despite completely blocking AMPK activity. CaMKKbeta knockdown suppressed ADP-stimulated eNOS activity, yet inhibition of CaMKKbeta kinase activity using STO-609 failed to affect eNOS activation by ADP. These data suggest that the expression, but not the kinase activity, of AMPK and CaMKKbeta is necessary for ADP signaling to eNOS.
Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Connie Ng
CN
Hess
Ruqin
R
Kou
Rosalyn P
RP
Johnson
Gordon K
GK
Li
Thomas
T
Michel
D055372:AMP-Activated Protein Kinases / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase / Q000378:metabolism; D002417:Cattle; D002465:Cell Movement; D042783:Endothelial Cells / Q000166:cytology*; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D053644:Naphthalimides / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms; D015398:Signal Transduction
D001562:Benzimidazoles; D053644:Naphthalimides; D020033:Protein Isoforms; C458525:STO 609; D000244:Adenosine Diphosphate; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D055372:AMP-Activated Protein Kinases