Our group has previously demonstrated that production of nitric oxide (NO) by the endothelial nitric oxide synthase (eNOS) in response to shear stress depends on the protein kinase A (PKA)-mediated phosphorylation of eNOS on Ser1177. A highly heterogeneous group of scaffolding proteins, called A kinase anchoring proteins (AKAPs), regulate PKA activity by tethering the regulatory subunit of type 2 PKA (PKA-RII). The AKAPs also bind different effectors of the kinase, localising them close to one another to facilitate signalling. It is estimated that most cell types express six to ten AKAPs, but surprisingly little is known on the expression and function of AKAPs in the endothelium. We therefore determined whether or not the PKA-dependent phosphorylation of eNOS in response to fluid shear stress involves the binding of PKA to AKAPs.In human umbilical vein endothelial cells, fluid shear stress (12 dyne/cm2, 10 minutes) enhanced the phosphorylation of eNOS on Ser1177 leading to an increased NO production as measured by cyclic GMP production. The phosphorylation and activation of eNOS was inhibited by the PKA inhibitors H89 and Rp-cAMP but global endothelial cAMP levels failed to increase, indicating a more subtle regulation of PKA activity in response to fluid shear stress. Indeed, immuno-staining showed that PKA was localised in the peri-nuclear region as well as within punctate membrane domains in cultured endothelial cells. To address the role of AKAPs more directly, we generated adenoviruses encoding a small peptide (Ht31) derived from AKAP13 that effectively disrupts the interaction between AKAPs and PKA-RII. Endothelial cell transduction with control adenoviruses did not prevent the shear stress induced phosphorylation of eNOS on Ser1177 while the expression of Ht31 abrogated the response. Interestingly, Ht31 had no effect on the shear stress-induced phosphorylation of Akt, confirming our previous results using a dominant negative Akt mutant; the PKA inhibitor H89 elicited similar effects. In a cDNA microarray we have been able to detect the expression of AKAPs 9, 10, 12, 13 and ezrin in endothelial cells. In addition, in a proteomics approach using Strep-Tactin labeled PKA-RII we were able to pull down two further, as yet unidentified, AKAPs.Taken together, our findings indicate that interfering with the assembly of the eNOS signalosome by targeting AKAPs can affect endothelial cell function. We are currently assessing which of the endothelial AKAPs can specifically bind to eNOS in cultured and native endothelial cells.