PKA and Epac activation mediates cAMP-induced vasorelaxation by increasing endothelial NO production.
Vascular relaxation induced by 3',5'-cyclic adenosine monophosphate (cAMP) is both endothelium-dependent and endothelium-independent, although the underlying signaling pathways are not fully understood. Aiming to uncover potential mechanisms, we performed contraction-relaxation experiments on endothelium-denuded and intact rat aorta rings and measured NO levels in isolated human endothelial cells using single cell fluorescence imaging. The vasorelaxant effect of forskolin, an adenylyl cyclase activator, was decreased after selective inhibitor of protein kinase A (PKA), a cAMP-activated kinase, or L-NAME, an endothelial nitric oxide synthase (eNOS) inhibitor, only in intact aortic rings. Both selective activation of PKA with 6-Bnz-cAMP and exchange protein directly activated by cAMP (Epac) with 8-pCPT-2'-O-Me-cAMP significantly relaxed phenylephrine-induced contractions. The vasorelaxant effect of the Epac activator, but not that of the PKA activator, was reduced by endothelium removal. Forskolin, dibutyryl cAMP (a cAMP analogue), 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP increased NO levels in endothelial cells and the forskolin effect was significantly inhibited by inactivation of both Epac and PKA, and eNOS inhibition. Our results indicate that the endothelium-dependent component of forskolin/cAMP-induced vasorelaxation is partially mediated by an increase in endothelial NO release due to an enhanced eNOS activity through PKA and Epac activation in endothelial cells.
Farmacología Cardiovascular y Plaquetaria, Centro de Investigación en Medicina Molecular y Enfermedades Crónicas, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
Verónica
V
García-Morales
Farmacología Cardiovascular y Plaquetaria, Centro de Investigación en Medicina Molecular y Enfermedades Crónicas, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
Andrea
A
Cuíñas
Farmacología Cardiovascular y Plaquetaria, Centro de Investigación en Medicina Molecular y Enfermedades Crónicas, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain.
Jacobo
J
Elíes
Farmacología Cardiovascular y Plaquetaria, Centro de Investigación en Medicina Molecular y Enfermedades Crónicas, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain. Electronic address: manuel.campos@usc.es.
Manuel
M
Campos-Toimil
D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008297:Male; D009569:Nitric Oxide / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D009924:Organ Culture Techniques; D051381:Rats; D011921:Rats, Inbred WKY; D014664:Vasodilation / Q000187:drug effects / Q000502:physiology*
D020662:Guanine Nucleotide Exchange Factors; C526973:Rapgef3 protein, rat; D009569:Nitric Oxide; D000242:Cyclic AMP; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D017868:Cyclic AMP-Dependent Protein Kinases
Cyclic AMP; Endothelium; Epac proteins; Protein kinase A; eNOS