title: Enhanced Rap1 activation and insulin secretagogue properties of an acetoxymethyl ester of an Epac-selective cyclic AMP analog in rat INS-1 cells: studies with 8-pCPT-2'-O-Me-cAMP-AM.
abstract: To ascertain the identities of cyclic nucleotide-binding proteins that mediate the insulin secretagogue action of cAMP, the possible contributions of the exchange protein directly activated by cAMP (Epac) and protein kinase A (PKA) were evaluated in a pancreatic beta cell line (rat INS-1 cells). Assays of Rap1 activation, CREB phosphorylation, and PKA-dependent gene expression were performed in combination with live cell imaging and high throughput screening of a fluorescence resonance energy transfer-based cAMP sensor (Epac1-camps) to validate the selectivity with which acetoxymethyl esters (AM-esters) of cAMP analogs preferentially activate Epac or PKA. Selective activation of Epac or PKA was achieved following exposure of INS-1 cells to 8-pCPT-2'-O-Me-cAMP-AM or Bt(2)cAMP-AM, respectively. Both cAMP analogs exerted dose-dependent and glucose metabolism-dependent actions to stimulate insulin secretion, and when each was co-administered with the other, a supra-additive effect was observed. Because 2.4-fold more insulin was secreted in response to a saturating concentration (10 microm) of Bt(2)cAMP-AM as compared with 8-pCPT-2'-O-Me-cAMP-AM, and because the action of Bt(2)cAMP-AM but not 8-pCPT-2'-O-Me-cAMP-AM was nearly abrogated by treatment with 3 microm of the PKA inhibitor H-89, it is concluded that for INS-1 cells, it is PKA that acts as the dominant cAMP-binding protein in support of insulin secretion. Unexpectedly, 10-100 microm of the non-AM-ester of 8-pCPT-2'-O-Me-cAMP failed to stimulate insulin secretion and was a weak activator of Rap1 in INS-1 cells. Moreover, 10 microm of the AM-ester of 8-pCPT-2'-O-Me-cAMP stimulated insulin secretion from mouse islets, whereas the non-AM-ester did not. Thus, the membrane permeability of 8-pCPT-2'-O-Me-cAMP in insulin-secreting cells is so low as to limit its biological activity. It is concluded that prior reports documenting the failure of 8-pCPT-2'-O-Me-cAMP to act in beta cells, or other cell types, need to be re-evaluated through the use of the AM-ester of this cAMP analog.
authors.affiliation: Departments of Medicine and Pharmacology, State University of New York, Upstate Medical University, Syracuse, New York 13210, USA.
authors.forename: Oleg G
authors.initials: OG
authors.lastname: Chepurny
authors.forename: Colin A
authors.initials: CA
authors.lastname: Leech
authors.forename: Grant G
authors.initials: GG
authors.lastname: Kelley
authors.forename: Igor
authors.initials: I
authors.lastname: Dzhura
authors.forename: Elvira
authors.initials: E
authors.lastname: Dzhura
authors.forename: Xiangquan
authors.initials: X
authors.lastname: Li
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Rindler
authors.forename: Frank
authors.initials: F
authors.lastname: Schwede
authors.forename: Hans G
authors.initials: HG
authors.lastname: Genieser
authors.forename: George G
authors.initials: GG
authors.lastname: Holz
mesh_terms: D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D004789:Enzyme Activation; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D007546:Isoquinolines / Q000378:metabolism; D051379:Mice; D047428:Protein Kinase Inhibitors / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013449:Sulfonamides / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C473217:8-(4-chloro-phenylthio)-2'-O-methyladenosine-3'-5'-cyclic monophosphate; D020662:Guanine Nucleotide Exchange Factors; D007328:Insulin; D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; C526973:Rapgef3 protein, rat; D011993:Recombinant Fusion Proteins; D013449:Sulfonamides; D000242:Cyclic AMP; D020827:rap1 GTP-Binding Proteins; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide