Abstract
The second messenger cAMP exerts powerful stimulatory effects on Ca(2+) signaling and insulin secretion in pancreatic beta-cells. Previous studies of beta-cells focused on protein kinase A (PKA) as a downstream effector of cAMP action. However, it is now apparent that cAMP also exerts its effects by binding to cAMP-regulated guanine nucleotide exchange factors (Epac). Although one effector of Epac is the Ras-related G protein Rap1, it is not fully understood what the functional consequences of Epac-mediated signal transduction are at the cellular level. 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate (8-pCPT-2'-O-Me-cAMP) is a newly described cAMP analog, and it activates Epac but not PKA. Here we demonstrate that 8-pCPT-2'-O-Me-cAMP acts in human pancreatic beta-cells and INS-1 insulin-secreting cells to mobilize Ca(2+) from intracellular Ca(2+) stores via Epac-mediated Ca(2+)-induced Ca(2+) release (CICR). The cAMP-dependent increase of [Ca(2+)](i) that accompanies CICR is shown to be coupled to exocytosis. We propose that the interaction of cAMP and Epac to trigger CICR explains, at least in part, the blood glucose-lowering properties of an insulinotropic hormone (glucagon-like peptide-1, also known as GLP-1) now under investigation for use in the treatment of type-2 diabetes mellitus.
Highlights
The second messenger cAMP exerts powerful stimulatory effects on Ca2؉ signaling and insulin secretion in pancreatic -cells
RIP2-enhanced yellow fluorescent protein (EYFP) was incorporated into an adenoviral vector, and expression of EYFP was conferred to human -cells by adenovirus-mediated gene transfer using AdRIP2EYFP (Fig. 1, C and D)
Confocal microscopy in combination with fluorescence immunocytochemistry demonstrated that expression of EYFP was restricted to the insulin-immunoreactive -cells and not the glucagon-reactive ␣-cells in whole islets of Langerhans derived from rat (Fig. 2)
Summary
Exchange protein activated by cAMP; PKA, protein kinase A; CICR, Ca2ϩ-induced Ca2ϩ release; FBS, fetal bovine serum; EYFP, enhanced yellow fluorescent protein; CRE, cAMP-response elements; TRITC, tetramethylrhodamine isothiocyanate; SES, standard extracellular saline; CREB, cAMP-response element-binding protein; IP3, inositol trisphosphate; RYR, ryanodine receptors; IP3-R, IP3 receptors; IP, inositol phosphate; ER, endoplasmic reticulum. Two isoforms of Epac have been described (Epac, Epac2) [1, 2], and each is proposed to mediate the PKA-independent signal transduction properties of cAMP. Recent structure-function analyses of cAMP action demonstrate that introduction of a 2Ј-methoxyl group in place of the 2Ј-hydroxyl group of cAMP confers Epac specificity to the cyclic nucleotide [4]. The properties of 8-pCPT-2Ј-O-Me-cAMP in living cells have been evaluated only with respect to its ability to promote Epac-mediated activation of Rap1 [4]. 8-pCPT-2Ј-O-Me-cAMP is likely to serve as a specific pharmacological tool for analyses of PKAindependent signaling properties of cAMP in the regulation of intracellular Ca2ϩ signaling and exocytosis
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
