Epac2-dependent mobilization of intracellular Ca2+by glucagon-like peptide-1 receptor agonist exendin-4 is disrupted in β-cells of phospholipase C-ε knockout mice

Calcium can be mobilized in pancreatic beta-cells via a mechanism of Ca2+-induced Ca2+ release (CICR), and cAMP-elevating agents such as exendin-4 facilitate CICR in beta-cells by activating both protein kinase A and Epac2. Here we provide the first report that a novel phosphoinositide-specific phospholipase C-epsilon (PLC-epsilon) is expressed in the islets of Langerhans, and that the knockout (KO) of PLC-epsilon gene expression in mice disrupts the action of exendin-4 to facilitate CICR in the beta-cells of these mice. Thus, in the present study, in which wild-type (WT) C57BL/6 mouse beta-cells were loaded with the photolabile Ca2+ chelator NP-EGTA, the UV flash photolysis-catalysed uncaging of Ca2+ generated CICR in only 9% of the beta-cells tested, whereas CICR was generated in 82% of the beta-cells pretreated with exendin-4. This action of exendin-4 to facilitate CICR was reproduced by cAMP analogues that activate protein kinase A (6-Bnz-cAMP-AM) or Epac2 (8-pCPT-2'-O-Me-cAMP-AM) selectively. However, in beta-cells of PLC-epsilon KO mice, and also Epac2 KO mice, these test substances exhibited differential efficacies in the CICR assay such that exendin-4 was partly effective, 6-Bnz-cAMP-AM was fully effective, and 8-pCPT-2'-O-Me-cAMP-AM was without significant effect. Importantly, transduction of PLC-epsilon KO beta-cells with recombinant PLC-epsilon rescued the action of 8-pCPT-2'-O-Me-cAMP-AM to facilitate CICR, whereas a K2150E PLC-epsilon with a mutated Ras association (RA) domain, or a H1640L PLC-epsilon that is catalytically dead, were both ineffective. Since 8-pCPT-2'-O-Me-cAMP-AM failed to facilitate CICR in WT beta-cells transduced with a GTPase activating protein (RapGAP) that downregulates Rap activity, the available evidence indicates that a signal transduction 'module' comprised of Epac2, Rap and PLC-epsilon exists in beta-cells, and that the activities of Epac2 and PLC-epsilon are key determinants of CICR in this cell type.