Signalling mechanisms of Epac1-mediated vascular responses

Abstract

Cyclic adenosine monophosphate (cAMP) is an important intracellular secondary messenger. The major target of cAMP was traditionally considered as protein kinase (PK) A. This belief has been challenged by the discovery of exchange protein activated by cAMP 1 (Epac1), a cAMP-dependent guanine-nucleotide-exchange factor (GEF).

Epac1 is ubiquitously expressed in all tissues and plays important roles particularly in the cardiovascular system. As cAMP activates both PKA and Epac1, the development of 8-pCPT-2'-O-Me-cAMP (8-pCPT), which has 107-fold higher affinity to bind and activate Epac1 than PKA, aids the researches on Epac1-mediated responses.

In the present study, the protein expressions of Epac1 in the porcine coronary arteries and rat aortas were confirmed by Western blot analysis. In organ chambers, 8-pCPT induced acute relaxations in isolated porcine coronary arteries contracted to thromboxane receptor (TP-receptor) antagonists, and the relaxation was endothelium-independent. The 8-pCPT-induced Epac1 activation selectively altered the vasoactive responses to the TP-receptor agonists. The Epac1-mediated relaxation was found not related to PKA, PKG and the opening of ATP-sensitive potassium channels.

Although Epac1 was first cloned as a Rap-linked GEF, in the porcine coronary artery, small GTPase Rac1 is the downstream target of Epac1 instead of Rap1 for relaxation. Activation of TP-receptors stimulates Rho-kinase to cause contraction, and the 8-pCPT-induced relaxation was Rho-kinase dependent, probably through pathway that is distinct from Rac1. Activation of Epac1 also inhibited the contraction to PKC, which is also downstream of TP-receptor but independent to Rho-kinase activity.

On the contrary, in the aorta from male Sprague-Dawley rats aged 10-12 weeks, 8-pCPT induced relaxation in rings contracted to phenylephrine (PE) and the relaxation was endothelium-dependent. The relaxation depended mainly on endothelial nitric oxide synthase (eNOS) and partly on cyclooxygenase (COX). Western blot analysis found that 8-pCPT did not enhance eNOS phosphorylation, which is one of the mechanisms for eNOS activation. Activation of Epac1 also did not alter the phosphorylation of Akt and ERK1/2 which play important roles in cAMP-dependent eNOS. More experiments are needed to examine whether or not Epac1 alters nitric oxide (NO) and prostanoids synthesis, which are the major endothelium-derived mediators responsible for vascular tone regulation.

In summary, the selective Epac activator 8-pCPT induced significant relaxations by distinct mechanisms in porcine coronary arteries and rat aortas. It is most likely that the relaxing effects of Epac1 activator are tissue and/or species specific. Owing to the effects of 8-pCPT on vascular relaxation, Epac1 might be an alternative therapeutic target for the treatment of vasospasm and hypertension. Further studies are necessary to explore the detailed mechanisms of Epac1 and its in vivo effects and in diseased models.