Mechanism of purinergic activation of endothelial nitric oxide synthase in endothelial cells.
BACKGROUND
Decreased endothelial nitric oxide (NO) synthase (eNOS) activity and NO production are critical contributors to the endothelial dysfunction and vascular complications observed in many diseases, including diabetes mellitus. Extracellular nucleotides activate eNOS and increase NO generation; however, the mechanism of this observation is not fully clarified.


RESULTS
To elucidate the signaling pathway(s) leading to nucleotide-mediated eNOS phosphorylation at Ser-1177, human umbilical vein endothelial cells were treated with several nucleotides, including ATP, UTP, and ADP, in the presence or absence of selective inhibitors. These experiments identified P2Y1, P2Y2, and possibly P2Y4 as the purinergic receptors involved in eNOS phosphorylation and demonstrated that this process was adenosine independent. Nucleotide-induced eNOS phosphorylation and activity were inhibited by BAPTA-AM (an intracellular free calcium chelator), rottlerin (a protein kinase Cdelta inhibitor), and protein kinase Cdelta siRNA. In contrast, blockade of AMP-activated protein kinase, calcium/calmodulin-dependent kinase II, calcium/calmodulin-dependent kinase kinase, serine/threonine protein kinase B, protein kinase A, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase did not affect nucleotide-mediated eNOS phosphorylation.


CONCLUSIONS
The present study indicates that extracellular nucleotide-mediated eNOS phosphorylation is calcium and protein kinase Cdelta dependent. This newly identified signaling pathway opens new therapeutic avenues for the treatment of endothelial dysfunction.
da Silva
Cleide Gonçalves
CG
Center for Vascular Biology Research and the Division of Vascular Surgery, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA.
Specht
Anke
A
Wegiel
Barbara
B
Ferran
Christiane
C
Kaczmarek
Elzbieta
E
D002118:Calcium / Q000502:physiology*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000201:enzymology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology*; D006801:Humans; D009569:Nitric Oxide / Q000096:biosynthesis; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D009711:Nucleotides / Q000494:pharmacology; D010766:Phosphorylation; D051745:Protein Kinase C-delta / Q000502:physiology*; D011983:Receptors, Purinergic; D015398:Signal Transduction
D009711:Nucleotides; D011983:Receptors, Purinergic; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D051745:Protein Kinase C-delta; D002118:Calcium