Abstract
Activation of the major cAMP effector, exchange protein directly activated by cAMP (Epac), induces vascular smooth muscle relaxation by increasing the activity of ryanodine (RyR)-sensitive release channels on the peripheral sarcoplasmic reticulum. Resultant Ca2+ sparks activate plasma membrane Ca2+-activated K+ (BKCa) channels, evoking spontaneous transient outward currents (STOCs) that hyperpolarize the cell and reduce voltage-dependent Ca2+ entry. In this study we investigate the mechanism by which Epac increases STOC activity. We show that the selective Epac activator 8-pCPT-AM induces autophosphorylation (activation) of calcium/calmodulin-dependent kinase 2 (CaMKII), and that inhibition of CaMKII abolishes 8-pCPT-AM-induced increases in STOC activity. Epac-induced CaMKII activation is likely initiated by IP3-mobilized Ca2+:8-pCPT-AM fails to induce CaMKII activation following intracellular Ca2+ store depletion, and inhibition of IP3 receptors blocks both 8-pCPT-AM-mediated CaMKII phosphorylation and STOC activity. 8-pCPT-AM does not directly activate BKCa channels, but STOCs cannot be generated by 8-pCPT-AM in the presence of ryanodine. Further, exposure to 8-pCPT-AM significantly slows the initial rate of [Ca2+]i rise induced by the RyR activator caffeine without significantly affecting the caffeine-induced Ca2+ transient amplitude, a measure of Ca2+ store content. We conclude that Epac-mediated STOC activity (i) occurs via activation of CaMKII, and (ii) is driven by changes in the underlying behaviour of RyR channels. To our knowledge this is the first report of CaMKII initiating cellular activity linked to vasorelaxation and suggests novel roles for this Ca2+ and redox-sensing enzyme in the regulation of vascular tone and blood flow.
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