Abstract
Rap1 is a member of the Ras superfamily of small GTP-binding proteins and is localized on pancreatic zymogen granules. The current study was designed to determine whether GTP-Rap1 is involved in the regulation of amylase secretion. Rap1A/B and the two Rap1 guanine nucleotide exchange factors, Epac1 and CalDAG-GEF III, were identified in mouse pancreatic acini. A fraction of both Rap1 and Epac1 colocalized with amylase in zymogen granules, but only Rap1 was integral to the zymogen granule membranes. Stimulation with cholecystokinin (CCK), carbachol, and vasoactive intestinal peptide all induced Rap1 activation, as did calcium ionophore A23187, phorbol ester, forskolin, 8-bromo-cyclic AMP, and the Epac-specific cAMP analog 8-pCPT-2'-O-Me-cAMP. The phospholipase C inhibitor U-73122 abolished carbachol- but not forskolin-induced Rap1 activation. Co-stimulation with carbachol and 8-pCPT-2'-O-Me-cAMP led to an additive effect on Rap1 activation, whereas a synergistic effect was seen on amylase release. Although the protein kinase A inhibitor H-89 abolished forskolin-stimulated CREB phosphorylation, it did not modify forskolin-induced GTP-Rap1 levels, excluding PKA participation. Overexpression of Rap1 GTPase-activating protein, which blocked Rap1 activation, reduced the effect of 8-bromo-cyclic AMP, 8-pCPT-2'-O-Me-cAMP, and vasoactive intestinal peptide on amylase release by 60% and reduced CCK- as well as carbachol-stimulated pancreatic amylase release by 40%. These findings indicate that GTP-Rap1 is required for pancreatic amylase release. Rap1 activation not only mediates the cAMP-evoked response via Epac1 but is also involved in CCK- and carbachol-induced amylase release, with their action most likely mediated by CalDAG-GEF III.
Highlights
Members of this superfamily of proteins, Rap1 cycles between an inactive GDP-bound and an active GTP-bound form
Rap1 Activation—We studied whether CCK, carbachol, and Rap1 Is Activated by the Epac-specific cAMP Analog 8-pCPT
These findings indicate that forskolin effect on Rap1 activation is most likely Epac-mediated, because it was not modified by the inhibitor of Protein kinase A (PKA)
Summary
Materials—Collagenase was purchased from Crescent Chemical Company (Islandia, NY), bovine serum albumin (BSA) and soybean trypsin inhibitor (STI) were from Sigma, and Dulbecco’s modified Eagle’s medium was from Invitrogen. Either 8-Br-cAMP or the Epac-selective cAMP analog 8-pCPT-2Ј-O-Me-cAMP (CPT-Me-cAMP) was added to isolated pancreatic acini, and both Rap activation (A) and CREB phosphorylation (B) were determined. Expressing the Rap1-specific GTPase-activating protein, Rap1GAP, in isolated pancreatic acini. Samples were cen- overexpression of Rap1GAP-green fluorescence protein in isotrifuged for 30 s in a microcentrifuge, and the supernatant was lated pancreatic acini was analyzed by immunofluorescence assayed for amylase activity with Phadebas reagents (Magle Life and by immunoblotting using polyclonal Rap1GAP antibody as Sciences, Lund, Sweden). Ca2ϩ, DAG, and cAMP Induce an BRAIN BRAIN KIDNEY PPAAKNNICCDRRNEEEAAYSS ACINI ACINI
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