cAMP signal transduction induces eNOS activation by promoting PKB phosphorylation

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.