+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

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 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

Journal of Biological Chemistry 284(47): 32209-32224

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.

Please choose payment method:

(PDF emailed within 0-6 h: $19.90)

Accession: 051293184

Download citation: RISBibTeXText

PMID: 19783664

DOI: 10.1074/jbc.m109.032656

Related references

Hepatocyte Growth Factor Stimulates Nitric Oxide Production through Endothelial Nitric Oxide Synthase Activation by the Phosphoinositide 3-Kinase/Akt Pathway and Possibly by Mitogen-Activated Protein Kinase Kinase in Vascular Endothelial Cells. Hypertension Research 27(11): 887-895, 2004

Hepatocyte growth factor stimulates nitric oxide production through endothelial nitric oxide synthase activation by the phosphoinositide 3-kinase/Akt pathway and possibly by mitogen-activated protein kinase kinase in vascular endothelial cells. Hypertension Research 27(11): 887-895, 2004

Endothelin-1 impairs nitric oxide signaling in endothelial cells through a protein kinase Cdelta-dependent activation of STAT3 and decreased endothelial nitric oxide synthase expression. Dna and Cell Biology 28(11): 543-553, 2009

Glycosylphosphatidylinositol toxin of Plasmodium induces nitric oxide synthase expression in macrophages and vascular endothelial cells by a protein tyrosine kinase-dependent and protein kinase C-dependent signaling pathway. Journal of Immunology 156(5): 1897-1907, 1996

Activation of the mitogen activated protein kinase extracellular signal-regulated kinase 1 and 2 by the nitric oxide-cGMP-cGMP-dependent protein kinase axis regulates the expression of matrix metalloproteinase 13 in vascular endothelial cells. Molecular Pharmacology 62(4): 927-935, 2002

Activation of AMP-activated protein kinase by vascular endothelial growth factor mediates endothelial angiogenesis independently of nitric-oxide synthase. Journal of Biological Chemistry 285(14): 10638-10652, 2010

Akt-dependent phosphorylation of serine 1179 and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 cooperatively mediate activation of the endothelial nitric-oxide synthase by hydrogen peroxide. Molecular Pharmacology 63(2): 325-331, 2003

Vascular endothelial growth factor governs endothelial nitric-oxide synthase expression via a KDR/Flk-1 receptor and a protein kinase C signaling pathway. Journal of Biological Chemistry 274(46): 33057-33063, 1999

Proinsulin C-peptide increases nitric oxide production by enhancing mitogen-activated protein-kinase-dependent transcription of endothelial nitric oxide synthase in aortic endothelial cells of Wistar rats. Diabetologia 46(12): 1698-1705, 2003

Androgen receptor-dependent activation of endothelial nitric oxide synthase in vascular endothelial cells: role of phosphatidylinositol 3-kinase/akt pathway. Endocrinology 151(4): 1822-1828, 2010

Protein kinase Cα phosphorylates a novel argininosuccinate synthase site at serine 328 during calcium-dependent stimulation of endothelial nitric-oxide synthase in vascular endothelial cells. Journal of Biological Chemistry 287(31): 26168-26176, 2012

Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase. Toxicology and Applied Pharmacology 246(3): 171-183, 2010

Nectandrin B activates endothelial nitric-oxide synthase phosphorylation in endothelial cells: role of the AMP-activated protein kinase/estrogen receptor α/phosphatidylinositol 3-kinase/Akt pathway. Molecular Pharmacology 80(6): 1166-1178, 2011

Fluid shear stress attenuates the activity and expression of the hydroxy-methylglutaryl coenzyme a reductase in endothelial cells via the nitric oxide-dependent activation of the AMP-activated protein kinase. Circulation 108(17 Suppl.): IV-191, 2003

Nectandrin B Activates Endothelial Nitric-Oxide Synthase Phosphorylation in Endothelial Cells Role of the AMP-Activated Protein Kinase/Estrogen Receptor alpha/Phosphatidylinositol 3-kinase/Akt Pathway. 2011