Epac-Mediated Mobilization of Intracellular Calcium in Vascular Myocytes and the Downstream Effects on Arterial KATP Channels

Exchange proteins directly activated by cyclic AMP (Epac or cAMP-GEFs) are a family of novel cAMP-binding effector proteins [1]. Using the Epac-specific cAMP analogue 8-pCPT-2¯-O-Me-cAMP we show cAMP-mediated but PKA-independent mobilization of Ca2+i within vascular myocytes and downstream effects that culminate in the inhibition of ATP-sensitive potassium (KATP) channels.Application of 8-pCPT-2¯-O-Me-cAMP (5μM) caused a 41.6 ± 4.7% inhibition of pinacidil-evoked whole-cell KATP currents recorded in isolated rat aortic smooth muscle cells. Inclusion of the Ca2+ chelator BAPTA (20μM) in the pipette-filling solution reduced the inhibition to 8.7 ± 4.4%, consistent with the idea that Epac mediates its effects by elevating [Ca2+]i. In support of this, 8-pCPT-2¯-O-Me-cAMP (5μM) caused a transient 171.0 ± 18.0nM increase in [Ca2+]i in Fura-2-loaded myocytes, which persisted in the absence of extracellular Ca2+. Caffeine-induced Ca2+ transients triggered in the presence of 8-pCPT-2¯-O-Me-cAMP typically showed a secondary Ca2+ increase, reminiscent of ectopic Ca2+ transients observed in Epac-activated cardiac myocytes [2]. While Ca2+ transients returned to baseline after 15-20s, the inhibition of KATP current was sustained, suggesting that Ca2+per se does not affect channel activity and implicating the involvement of Ca2+-activated enzymes. Preincubation with calcineurin inhibitors cyclosporin A (10μM) and ascomycin (5μM), significantly reduced the ability of 8-pCPT-2¯-O-Me-cAMP to inhibit KATP currents (inhibition 10.8 ± 2.8% and 7.3 ± 1.6%).These findings suggest cAMP-mediated Epac activation in vascular smooth muscle mobilizes Ca2+ from internal stores and inhibits KATP channels through the activation of the Ca2+-sensitive enzyme, calcineurin.1. Bos JL (2006). Trends in Biological Sciences31:680-6862. Hothi et al (2008). Pflugers Archiv.457:253-270.Supported by the BHF