This raises the question as to under what conditions a cAMP-mediated Epac-dependent inhibition of vascular KATP channel activity would occur. protein kinase (PKA) (Beavo & Brunton, 2002). Two isoforms of Epac are known to exist: the ubiquitously expressed Epac1 (also known as cAMP-GEFI) and the closely related Epac2 (cAMP-GEFII) (de Rooij 1998; Kawasaki 1998). These proteins contain an N-terminal cAMP-binding site (one on Epac1 and two on Epac2) and a C-terminal guanine-nucleotide exchange factor (GEF) domain that promotes GDP/GTP exchange on Rap1/2. Appreciation of the potential importance of Epac has come with the recent development of cell-permeant, Epac-specific cAMP analogues that allow discrimination between the different cAMP-dependent pathways (Enserink 2002; Christensen 2003). These synthetic analogues exploit small but significant structural differences between the conserved cAMP binding pockets found in PKA and CNG ion channels and those found in Epacs (Yagura & PARP14 inhibitor H10 Miller, 1981; Enserink 2002; Dao 2006). The use of such compounds in the selective activation of Epac has revealed previously unrecognized roles in a range of cellular processes including exocytosis, Ca2+ mobilization and the regulation of ion channel function (reviewed in Holz (2006)). This latter Rabbit polyclonal to MAPT point is of particular interest PARP14 inhibitor H10 given that until recently, cAMP was known to influence channel behaviour by only two mechanisms; direct binding, as in the case of CNG channels, or through PKA-mediated phosphorylation of channel subunits. Here we investigate whether vascular ATP-sensitive potassium (KATP) channels, which have a particularly high dependence on cAMP for their normal physiological function, are regulated by the activation of this novel cAMP effector. KATP channels are sensitive to intracellular levels of adenosine nucleotides and thus link changes in cellular metabolism to membrane excitability (Nichols, 2006). They are expressed in pancreatic -cells, certain types of neurones, cardiac, skeletal and smooth muscle and their physiological roles include regulation of insulin secretion, glucose-sensing in the hypothalamus, ischaemic cardioprotection and the control of blood flow (Quayle 1997; Yokoshiki 1998; Miki & Seino, 2005). Vascular KATP channels provide a background K+ conductance important in the regulation of membrane potential and so smooth muscle contractility and blood flow (Quayle 1997; Clapp & Tinker, 1998; Yokoshiki 1998). Pharmacological inhibition of KATP channels has been shown to increase vascular resistance in the systemic and coronary circulations (Samaha 1992; Duncker 2001) and drugs that open vascular KATP channels are used to treat angina and hypertension. Genetically engineered mice that lack vascular KATP channel subunits develop hypertension and die prematurely from coronary vasospasm, a phenotype resembling vasospastic (Prinzmetal or variant) angina in humans (Chutkow 2002; Miki 2002). A substantial part of the physiological regulation of vascular KATP channels occurs via vasoactive transmitters. Endogenous vasodilators, including calcitonin gene-related peptide (CGRP), -adrenoceptor agonists and adenosine, increase KATP channel activity by acting at Gs-coupled receptors to stimulate adenylyl cyclase and elevate intracellular levels of cAMP (Miyoshi & Nakaya, 1993; Quayle 1994; Kleppisch & Nelson, 1995; Wellman 1998). These cAMP-initiated effects are attributed to the activation of PKA, with experiments on cloned KATP channels suggesting that channel activity is increased by PKA-dependent phosphorylation at sites on both its pore-forming and regulatory subunits (Quinn 2004). Even in the absence of vasodilators arterial KATP channels are subject to a tonic PKA-dependent activation, which arises from sustained cAMP production originating from basal adenylyl cyclase turnover (Hayabuchi 20012004). To date no comparable data exist on the role of Epac in the regulation of vascular KATP channel activity. Here, using the well-characterized, Epac-specific cAMP analogue 8-(4-chloro-phenylthio)-2-2002; Christensen 2003), we show that cAMP also modulates vascular KATP channel activity by a mechanism independent of PKA. We show that cAMP-mediated activation of Epac inhibits rat aortic KATP channels via a Ca2+-dependent mechanism involving the activation of Ca2+-sensitive phosphatase 2B (PP-2B, calcineurin). Since vasodilator-induced elevation of intracellular cAMP levels and activation of PKA is associated with KATP channel activation (Miyoshi & Nakaya, 1993; Quayle 1994; Kleppisch & Nelson, 1995; Wellman 1998), these data suggest that under certain conditions cAMP conveys opposite, inhibitory information to the channel. While cAMP affinity between Epac and cAMP is similar (Dao 2006), the concentration of cAMP required for half-maximal activation of Epac1 is reported to be considerably higher than that required to activate PKA (de Rooij 2000; Enserink 2002; Rehmann 2003). We PARP14 inhibitor H10 discuss the possibility that Epac and PKA are differentially activated by different concentrations of cAMP and that Epac acts physiologically as a feedback regulator of KATP channel function. We also discuss an alternative pathophysiological role for Epac in the development of vascular hypertrophy. Methods Antibodies, polyacrylamide gel electrophoresis and immunoblotting The following antibodies were used: anti-Epac (sc-28366), anti-Epac2 (sc-28326) and anti-SUR2B (sc-5793) (Santa Cruz Biotechnology), horseradish peroxidase (HRP)conjugated anti-goat.
This raises the question as to under what conditions a cAMP-mediated Epac-dependent inhibition of vascular KATP channel activity would occur