Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCB313

Poster Communications

Activation of calcium/calmodulin-dependent kinase 2 (CaMKII) mediates Epac-induced increases in spontaneous transient outward current in rat mesenteric artery smooth muscle

E. S. Humphries1, T. Kamishima2, J. M. Quayle2, C. Dart1

1. Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom. 2. Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom.

Exchange protein directly activated by cAMP (Epac), a major cAMP effector, induces smooth muscle relaxation by increasing the frequency of localised Ca2+ release from ryanodine-sensitive Ca2+ release channels (RyRs) located on the peripheral sarcoplasmic reticulum [1]. These subsurface Ca2+ sparks activate large conductance Ca2+-activated K+ (BKCa) channels in the plasma membrane, evoking spontaneous transient outward currents (STOCs) that hyperpolarize the cell and reduce voltage-dependent Ca2+ entry. Here we investigate how Epac activation increases spark/STOC activity. In immunoblots of rat mesenteric artery using phospho-specific antibodies, the selective Epac activator 8-pCPT-2`-O-Me-cAMP (8-pCPT; 10μM) induced phosphorylation of calcium/calmodulin-dependent kinase 2 (CaMKII) at Thr286/7, an autophosphorylation site that indicates CaMKII activation (n=3). Of the four immunoreactive bands detected by pan-specific CaMKII antibodies, only one showed increased phosphorylation levels, suggesting Epac activates specific CaMKII isoforms. In whole-cell recordings from single, freshly isolated rat mesenteric artery smooth muscle cells (RMASMCs), application of the CaMKII inhibitor KN-93 (500nM) reversed the increase in STOC frequency (1.63 ± 0.51 to 0.71 ± 0.52 s-1; mean ± SD) and amplitude (32.6 ± 6.7 to 25.9 ± 5.4pA) induced by 8-pCPT-AM (5μM; p<0.05, paired t-test, n=5). This was not mimicked by application of KN-92 (500nM), an inactive KN-93 analogue (n=3). Additionally, inclusion in the pipette-filling solution of autocamtide-2-inhibitory peptide (1μM), a highly selective CaMKII inhibitor, blocked the ability of 8-pCPT-AM to increase STOC frequency/amplitude (n=4). Inhibition of protein kinase C (PKC), a known CaMKII activator, with bisindolylmaleimide IX (250nM) had no effect on 8-pCPT-AM-induced changes in STOC activity (n=3). 8-pCPT-AM, however, was unable to increase STOC activity in RMASMCs pre-incubated in 2-aminoethoxydiphenyl borate (2-APB; 100μM), an IP3 receptor (IP3R) inhibitor. 2-APB application following STOC activation with 8-pCPT-AM caused an initial rapid increase in STOC frequency followed by a decline to levels significantly below those measured in 8-pCPT-AM alone (p<0.05, n=6). These data suggest that Epac-induced CaMKII activation is independent of PKC and may be triggered by Ca2+ release from intracellular stores via IP3Rs. Application of 2-APB alone caused a transient increase in basal STOC frequency (p<0.01, n=4), which may indicate a constant Ca2+ store leak via IP3Rs which, when blocked, alters store content and RyR activity. In conclusion, our data suggest that Epac increases spark/STOC frequency in RMASMCs via CaMKII activation. CaMKII may phosphorylate downstream targets involved in regulating store load and/or RyR activity.

Where applicable, experiments conform with Society ethical requirements