Rap1 activation plays a regulatory role in pancreatic amylase secretion.
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.
Sabbatini
Maria E
ME
Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109-0622, USA. mesabba@umich.edu
Chen
Xuequn
X
Ernst
Stephen A
SA
Williams
John A
JA
D000681:Amylases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D020536:Enzyme Activators / Q000494:pharmacology; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism; D007546:Isoquinolines / Q000494:pharmacology; D051379:Mice; D008813:Mice, Inbred ICR; D046790:Pancreas, Exocrine / Q000201:enzymology*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D022142:Secretory Vesicles / Q000201:enzymology / Q000235:genetics; D013449:Sulfonamides / Q000494:pharmacology; D020668:rap GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020827:rap1 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
C492004:Creb1 protein, mouse; D017362:Cyclic AMP Response Element-Binding Protein; D020536:Enzyme Activators; C482601:Epac protein, mouse; D020662:Guanine Nucleotide Exchange Factors; D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D017868:Cyclic AMP-Dependent Protein Kinases; D000681:Amylases; C492136:Rap1b protein, mouse; D020668:rap GTP-Binding Proteins; D020827:rap1 GTP-Binding Proteins; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide