Phosphorylation of eNOS initiates excessive NO production in early phases of portal hypertension.
Akt, also known as protein kinase B, is a serine/threonine kinase. Akt becomes active when phosphorylated by the activation of receptor tyrosine kinases, G protein-coupled receptors, and mechanical forces such as shear stress. Studies in vitro have shown that Akt can directly phosphorylate endothelial nitric oxide (NO) synthase (eNOS) and activate the enzyme, leading to NO production. The aim of this study was to test the hypothesis that the phosphorylation of eNOS plays a role in the enhanced NO production observed in early portal hypertension. Male Sprague-Dawley rats were subjected to either sham or portal vein ligation (PVL), and mesenteric arterial beds were used for ex vivo perfusion studies. Mesenteric arterial beds from PVL rats had an approximately 60-70% decrease in response to methoxamine (an alpha(1)-agonist and vasoconstrictor) compared with the sham group (P < 0.01). When N(G)-monomethyl-L-arginine (a NOS inhibitor) was added to the perfusion, the difference in perfusion pressure between the two groups was abolished, suggesting that enhanced NO production in the PVL group blunted the response to the vasoconstrictor. The reduced responsiveness in PVL was not due to changes in eNOS expression but was due to an increase in enzyme-specific activity, suggesting posttranslational modification of eNOS. The phosphorylation of eNOS at Ser(1176) was significantly increased by twofold (P < 0.05) in the PVL group. Furthermore, PVL significantly increased Akt phosphorylation (an active form of Akt) by threefold (P < 0.05). When vessels were treated with wortmannin (10 nM) to block the phosphatidylinositol-3-OH-kinase/Akt pathway, NO-induced vasodilatation was significantly reduced. These results suggest that the phosphorylation of eNOS by Akt activates the enzyme and may be the first step leading to an initial increase in NO production in portal hypertension.
Iwakiri
Yasuko
Y
Department of Pharmacology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06536, USA.
Tsai
Ming-Hung
MH
McCabe
Timothy J
TJ
Gratton
Jean-Philippe
JP
Fulton
David
D
Groszmann
Roberto J
RJ
Sessa
William C
WC
D058646:Adrenergic alpha-1 Receptor Agonists; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006975:Hypertension, Portal / Q000201:enzymology*; D008026:Ligation; D008297:Male; D017538:Mesenteric Artery, Superior / Q000201:enzymology; D008729:Methoxamine / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D011169:Portal Vein / Q000601:surgery; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017207:Rats, Sprague-Dawley; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D000077191:Wortmannin; D019323:omega-N-Methylarginine / Q000494:pharmacology
D058646:Adrenergic alpha-1 Receptor Agonists; D000730:Androstadienes; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D014662:Vasoconstrictor Agents; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D008729:Methoxamine; D000077191:Wortmannin