The relaxation of vascular smooth muscle, which increases blood vessel diameter, is often mediated through vasodilator-induced elevations of intracellular cyclic AMP (cAMP) [1]. The vasculature expresses three distinct cAMP effectors: cAMP-dependent protein kinase (PKA), cyclic nucleotide-gated (CNG) ion channels and the more recently discovered exchange proteins directly activated by cAMP (Epacs) [2]. The mechanisms by which cAMP induces vasorelaxation are thus complex and diverse. Here we investigate the hypothesis that Epac activation increases the frequency of subsurface Ca2+ sparks within rat mesenteric smooth muscle cells, activating large-conductance Ca2+-activated potassium (BKCa) channels and inducing membrane hyperpolarization and vasorelaxation.In Fluo-4-AM-loaded mesenteric myocytes, application of the Epac-specific cAMP analogue 8-pCPT-2'-O-Me-cAMP-AM (10μM) increased spark frequency from 0.045 ± 0.008 sparks/s/μm under basal conditions to 0.103 ± 0.022 sparks/s/μm (p<0.05). Importantly this increase also occurred in the presence of myristoylated PKI amide (14-22), a potent and selective inhibitor of PKA.Application of 8-pCPT-2'-O-Me-cAMP-AM (5μM) reversibly increased both the frequency (0.94 ± 0.25 to 2.30 ± 0.72 s−1) and amplitude (23.9 ± 3.3 to 35.8 ± 7.7 pA) of spontaneous transient outward currents (STOCs) recorded in isolated mesenteric myocytes (n=7; p<0.05). These currents were sensitive to the selective BKCa channel blocker, iberiotoxin (100nM), and to ryanodine (30μM). In addition, current clamp recordings of isolated myocytes showed a 7.43 ± 0.96 mV (n=4) hyperpolarization in response to exposure to 8-pCPT-2'-O-Me-cAMP-AM (5μM).Our data suggest a novel cAMP-dependent mechanism in mesenteric smooth muscle cells whereby activation of Epac facilitates localized Ca2+ release which activates surface BKCa channels to modulate membrane potential and vascular tone.1. Morgado, M et al (2012). Cell. Mol. Life Sci. 69:2472. Bos, JL (2006). Trends in Biol. Sci. 31:680-686Supported by the British Heart Foundation