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This Article Full Text Full Text (PDF) All Versions of this Article: 290/6/H2376    most recent 00614.2005v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (3) Citing Articles via Google Scholar Google Scholar Articles by Zhang, X.-P. Articles by Hintze, T. H. Search for Related Content PubMed PubMed Citation Articles by Zhang, X.-P. Articles by Hintze, T. H.

cAMP signal transduction induces eNOS activation by promoting PKB phosphorylation

Department of Physiology, New York Medical College, Valhalla, New York

Submitted 8 June 2005 ; accepted in final form 9 January 2006

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-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.

nitric oxide; microvessels; protein kinases; adenylyl cyclase; protein phosphatases; adenosine 3',5'-cyclic monophosphate; protein kinase B; endothelial nitric oxide synthase

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