Enhanced Rap1 Activation and Insulin Secretagogue Properties of an Acetoxymethyl Ester of an Epac-selective Cyclic AMP Analog in Rat INS-1 Cells

Abstract

Epac1 and Epac2 are guanine nucleotide exchange factors activated by adenosine-3Ј,5Ј-cyclic monophosphate (cAMP), and which are known to be expressed in numerous mammalian cell types (1, 2). An accumulating body of evidence indicates that the existence of Epac may explain novel protein kinase A (PKA) 2 independent actions of cAMP that underlie cellular responsiveness to hormones, neurotransmitters, and pharmacological agents of therapeutic importance (3). Selective activation of Epac may be achieved through the use of 8-(4-chlorophenylthio)-2Ј-O-methyladenosine-3Ј,5Ј-cyclic monophosphate, also known as 8-pCPT-2Ј-O-Me-cAMP (4, 5). This cAMP analog, which incorporates a 2Ј-O-methyl group on the ribose ring of the nucleotide, as well as a 4-chlorophenylthio group on position 8 of the adenine moiety, acts as a "superactivator" of Epac while having a greatly diminished ability to activate PKA (4). Thus, 8-pCPT-2Ј-O-Me-cAMP is an Epac-selective cAMP analog (ESCA) (6).8-pCPT-2Ј-O-Me-cAMP can cross the plasma membrane and is able to alter diverse cellular functions that include Rap1 GTPase activity, PKB, and ERK1/2 protein kinase activity, phospholipase C⑀ activity, Ca 2ϩ signaling, ion channel activity, exocytosis, cell adhesion, and gene expression (7-9). Although no selective antagonist of Epac activation exists, these effects of 8-pCPT-2Ј-O-Me-cAMP are believed to be Epac-mediated because they are observed under conditions in which PKA activity is blocked, whereas they are reduced or eliminated when Epac gene expression is down-regulated. Furthermore, such actions of 8-pCPT-2Ј-O-Me-cAMP are measurable in cells that do not express the cyclic nucleotide-regulated ion channels that constitute an alternative target of cAMP action.Interestingly, published findings exist in which cAMP-elevating agents were found to exert actions not attributable to * This work was supported, in whole or in part, by National Institutes of Health Grants DK045817 and DK069575 (to G. G. H.). This work was also supported by a American Diabetes Association Research Award (to C. A. L. -89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; Luc, luciferase; PDE, cyclic nucleotide phosphodiesterase; YFP, yellow fluorescent protein; CFP, cyan fluorescent protein; SES, standard extracellular saline; RIP1, rat insulin 1 gene promoter; Epac, exchange protein directly activated by cAMP.

Isoform-specific antagonists of exchange proteins directly activated by cAMP

“…The excitation light was delivered at 435/9 nm (455 nm cutoff), and the emitted light was detected at 485/15 nm (CFP) or 535/15 nm (YFP). For live-cell imaging of FRET in single cells of the HEK cell clones reported here, the methods of analyses were identical to those reported by Chepurny et al in prior reports (40,41).…”

cAMP Analogs and Their Metabolites Enhance TREK-1 mRNA and K+Current Expression in Adrenocortical Cells

“…In this regard, it has become apparent that, in some cells, significantly higher concentrations are required for effective activation of Epac2 (Holz et al, 2008;Chepurny et al, 2009). We have shown previously that in AZF cells, Sp-8CPT-2Ј-OMe-cAMP (100 M) activated Rap1, a downstream effector of Epac2 ).…”

The Epac-Rap1 Signaling Pathway Controls cAMP-mediated Exocytosis of Weibel-Palade Bodies in Endothelial Cells

“…and thereby abolishes VWF secretion (12). Epac, the exchange protein activated by cAMP for the small GTPases Rap1 and Rap2, is involved in regulation of endothelial barrier function (27)(28)(29) and endothelial cell adhesion (30) but also regulated secretion of insulin in pancreatic beta-cells (31). Endothelial cells selectively express Epac1 but not Epac2 (28,29).…”