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 β-cells via a mechanism of Ca(2+)-induced Ca(2+) release (CICR), and cAMP-elevating agents such as exendin-4 facilitate CICR in β-cells by activating both protein kinase A and Epac2. Here we provide the first report that a novel phosphoinositide-specific phospholipase C- (PLC-) is expressed in the islets of Langerhans, and that the knockout (KO) of PLC- gene expression in mice disrupts the action of exendin-4 to facilitate CICR in the β-cells of these mice. Thus, in the present study, in which wild-type (WT) C57BL/6 mouse β-cells were loaded with the photolabile Ca(2+) chelator NP-EGTA, the UV flash photolysis-catalysed uncaging of Ca(2+) generated CICR in only 9% of the β-cells tested, whereas CICR was generated in 82% of the β-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 β-cells of PLC- 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- KO β-cells with recombinant PLC- rescued the action of 8-pCPT-2'-O-Me-cAMP-AM to facilitate CICR, whereas a K2150E PLC- with a mutated Ras association (RA) domain, or a H1640L PLC- that is catalytically dead, were both ineffective. Since 8-pCPT-2'-O-Me-cAMP-AM failed to facilitate CICR in WT β-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- exists in β-cells, and that the activities of Epac2 and PLC- are key determinants of CICR in this cell type.
Department of Medicine, State University of New York Upstate Medical University, Syracuse, NY, USA.
Igor
I
Dzhura
Oleg G
OG
Chepurny
Grant G
GG
Kelley
Colin A
CA
Leech
Michael W
MW
Roe
Elvira
E
Dzhura
Parisa
P
Afshari
Sundeep
S
Malik
Michael J
MJ
Rindler
Xin
X
Xu
Youming
Y
Lu
Alan V
AV
Smrcka
George G
GG
Holz
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D000077270:Exenatide; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D000067757:Glucagon-Like Peptide-1 Receptor; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D010455:Peptides / Q000494:pharmacology*; D054801:Phosphoinositide Phospholipase C / Q000235:genetics / Q000378:metabolism*; D018027:Receptors, Glucagon / Q000819:agonists*; D019284:Thapsigargin / Q000494:pharmacology; D014688:Venoms / Q000494:pharmacology*
C473217:8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate; C000593834:Glp1r protein, mouse; D000067757:Glucagon-Like Peptide-1 Receptor; D020662:Guanine Nucleotide Exchange Factors; D010455:Peptides; C486707:Rapgef4 protein, mouse; D018027:Receptors, Glucagon; D014688:Venoms; D019284:Thapsigargin; D000077270:Exenatide; D000242:Cyclic AMP; D054801:Phosphoinositide Phospholipase C; C506061:Plch1 protein, mouse; D020558:GTP Phosphohydrolases; D002118:Calcium