Role of cAMP-dependent protein kinase A activity in endothelial cell cytoskeleton rearrangement

To examine signaling mechanisms relevant to cAMP/protein kinase A (PKA)-dep endent endothelial cell barrier regulation, we investigated the impact of t he cAMP/PKA inhibitors Rp diastereomer of adenosine 3', 5'-cyclic monophosp horothioate (Rp-cAMPS) and PKA inhibitor (PKI) on bovine pulmonary artery a nd bovine lung microvascular endothelial cell cytoskeleton reorganization. Rp-cAMPS as well as PKI significantly increased the formation of actin stre ss fibers and intercellular gaps but did not alter myosin light chain (MLC) phosphorylation, suggesting that the Rp-cAMPS-induced contractile phenotyp e evolves in an MLC-independent fashion. We next examined the role of extra cellular signal-regulated kinases (ERKs) in Rp-cAMPS- and PKI-induced actin rearrangement. The activities of both ERK1/2 and its upstream activator Ra f-1 were transiently enhanced by Rp-cAMPS and linked to the phosphorylation of the well-known ERK cytoskeletal target caldesmon. Inhibition of the Raf -1 target ERK kinase (MEK) either attenuated or abolished Rp-cAMPS- and PKI -induced ERK activation, caldesmon phosphorylation, and stress fiber format ion. In summary, our data elucidate the involvement of the p42/44 ERK pathw ay in cytoskeletal rearrangement evoked by reductions in PKA activity and s uggest the involvement of significant cross talk between cAMP- and ERK-depe ndent signaling pathways in endothelial cell cytoskeletal organization and barrier regulation.