title: cAMP signal transduction induces eNOS activation by promoting PKB phosphorylation.
abstract: The objective of this study was to determine whether activation of protein kinase B (PKB) is involved in the production of nitric oxide (NO) induced by cAMP signal transduction. Mongrel dogs were used for this study. Coronary microvessels were isolated from the left ventricular free wall of these dog hearts. Forskolin (an activator of adenylyl cyclase that increases intracellular cAMP level) and 8-bromo-cAMP (a membrane-permeable cAMP analog) were used to stimulate NO release and activation of PKB and endothelial NO synthase (eNOS) in these blood vessels. We found that forskolin and 8-bromo-cAMP increased NO release (quantified by using the Griess reaction) from coronary microvessels by 80 +/- 6 and 78 +/- 11 pmol/mg (mean +/- SE), respectively (P < 0.05 vs. control). Western blot analysis showed that forskolin elicited a significant increase in eNOS phosphorylation (59 +/- 11%) at serine-1177 (a positively regulatory phosphorylation site for eNOS) and a significant increase in dephosphorylation (28 +/- 6%) at threonine-495 (a negatively regulatory phosphorylation site of eNOS) (P < 0.05 vs. control). Interestingly, forskolin also increased the phosphorylation of PKB at serine-473 (by 49 +/- 17%) and threonine-308 (by 53 +/- 17%), respectively (P < 0.05 vs. control; phosphorylation of both sites is required for a full activation of PKB). N(omega)-nitro-l-arginine methyl ester (an NOS inhibitor) blocked NO formation, Rp diastereomer of cAMP (a PKA inhibitor), and LY-294002 [a PI3-kinase (an activator of PKB) inhibitor] prevented the production of NO, phosphorylation of PKB, and eNOS induced by forskolin. Our data clearly show an involvement of PKB activation in cAMP signal-induced NO production. We are reporting for the first time that cAMP signal transduction stimulates eNOS activation through a PKB-mediated mechanism.
authors.affiliation: Department of Physiology, New York Medical College, Valhalla, NY 10595, USA. xpz789@hotmail.com
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Zhang
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Hintze
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002867:Chromones / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D003326:Coronary Circulation / Q000502:physiology; D003331:Coronary Vessels / Q000201:enzymology / Q000502:physiology; D000242:Cyclic AMP / Q000502:physiology*; D004285:Dogs; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D009025:Morpholines / Q000494:pharmacology; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D005576:Colforsin; D019319:Okadaic Acid; D015124:8-Bromo Cyclic Adenosine Monophosphate; D009569:Nitric Oxide; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D000242:Cyclic AMP; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt; D010749:Phosphoprotein Phosphatases; D019331:NG-Nitroarginine Methyl Ester