Thrombin and histamine stimulate endothelial nitric-oxide synthase phosphorylation at Ser1177 via an AMPK mediated pathway independent of PI3K-Akt.
Histamine and thrombin cause phosphorylation and activation of endothelial NO-synthase (eNOS) on Ser1177. We tested the role of various protein kinases in mediating this effect in human umbilical vein endothelial cells. Inhibition of the Ca2+/calmodulin-dependent protein kinase II or phosphoinositide 3-kinase (PI3K) had no effect. H89, an inhibitor of both protein kinase A (PKA) and 5'-AMP-activated protein kinase (AMPK), strongly inhibited phosphorylation and activity of eNOS. Conversely, the PKA inhibitor Rp-adenosine 3 '5'-cyclic monophosphate (cAMPS) had no effect and eNOS was not phosphorylated by treatments that affect cAMP levels. Thrombin and histamine caused phosphorylation of AMPK on Thr172 as well as on its downstream target acetyl-CoA carboxylase. Activation of AMPK using AICAR or CCCP also resulted in eNOS phosphorylation. We conclude that histamine and thrombin cause eNOS phosphorylation in an AMPK mediated manner, independent of P13K-Akt.
Institute of Pharmacy, Pharmacology and Toxicology, University of Iceland, Hagi Hofsvallagotu 53, Reykjavik, Iceland. brynhit@hi.is
Brynhildur
B
Thors
Haraldur
H
Halldórsson
Gudmundur
G
Thorgeirsson
D055372:AMP-Activated Protein Kinases; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology*; D006801:Humans; D009097:Multienzyme Complexes / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D015398:Signal Transduction / Q000187:drug effects*; D013917:Thrombin / Q000494:pharmacology*
D055372:AMP-Activated Protein Kinases; D002118:Calcium; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D004815:Epidermal Growth Factor; D006632:Histamine; D009097:Multienzyme Complexes; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D010768:Phosphoserine; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D013917:Thrombin; C496314:NOS3 protein, human; C494918:AKT1 protein, human