Rap1 Activation Plays a Regulatory Role in Pancreatic Amylase Secretion

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 GTPRap1 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 cAMPevoked response via Epac1 but is also involved in CCK-and carbachol-induced amylase release, with their action most likely mediated by CalDAG-GEF III.Rap1 is a member of the Ras superfamily of small GTP-binding proteins and is known to be involved in cell adhesion, proliferation, and differentiation (1). Two isoforms of Rap1 exist, Rap1A and Rap1B, which are 95% identical at the amino acid sequence and appear to mediate similar actions (1). Like other members of this superfamily of proteins, Rap1 cycles between an inactive GDP-bound and an active GTP-bound form. A variety of intracellular signals regulate the cycle through unique guanine-nucleotide exchange factors (GEFs), 2 which promote dissociation of GDP from Rap1 followed by binding of GTP, and GTPase-activating proteins (GAPs), which induce hydrolysis of GTP on Rap1 (1). Increases in intracellular levels of Ca 2ϩ and diacylglycerol (DAG) activate Ca 2ϩ -and DAG-binding GEFs (CalDAG-GEF I and CalDAG-GEF III) (2), whereas increases in intracellular levels of cAMP activate the exchange proteins activated by cAMP (Epac1 and Epac2) (3, 4). Protein kinase A (PKA), another mediator of cAMP action, can phosphorylate Rap1, which is necessary for Rap1 activation in certain cell types, including neutrophils, fibroblasts, thyroid and enteroendocrine cells (5-8).Evidence indicates that different endogenous Rap1GEFs exert physiological functions in various cells. For example, in neuronal PC12D cells, Ca...

Over-Expression of Pancreatic Pituitary Adenylate Cyclase–Activating Polypeptide (PACAP) Aggravates Cerulein-Induced Acute Pancreatitis in Mice

“…PACAP or CCK alone has little effect on amylase secretion from pancreatic acinar cells, while in combination they markedly increase the amylase secretion by sensitizing the acinar cells to CCK-induced zymogen activation (12). Epac/Rap1 signals play an essential role as a common signal for both CCK-and cAMP-evoked amylase secretions in pancreatic acinar cells (24). In our previous study, we demonstrated that cAMP /Epac /Rap1 may be involved in PACAP-induced gene expression and neurite extension in PC12 cells (25).…”

Generation of Cyclic ADP-ribose and Nicotinic Acid Adenine Dinucleotide Phosphate by CD38 for Ca2+ Signaling in Interleukin-8-treated Lymphokine-activated Killer Cells

“…Rap activation is important for lymphocyte migration in vivo (53,54). The Rap-dependent signaling pathway activated by Epac is known to be cell-specific (55,56), and there is evidence for Rap-mediated action of Epac to stimulate mitogen-activated protein kinases (p38, extracellular signal-regulated kinase 1/2) in neurons, endocrine cells, and T-cells (57)(58)(59). Rap1 was reported to be activated by PKA through the phosphorylation of Rap1GAP (60), consistent with our finding that treatment of LAK cells with IL-8 or PKA activator did not show Rap1 phosphorylation.…”

CCK activates RhoA and Rac1 differentially through Gα13 and Gαq in mouse pancreatic acini

“…Peroxidase activity was visualized using the SuperSignal West Femto sensitivity substrate kit (Pierce). Rap1 activation was determined as previously described (35).…”

“…After overnight incubation, isolated mouse pancreatic acini were allowed to settle in test tubes and then fixed for 1 h at room temperature with 4% paraformaldehyde in PBS. Acinar preparations were rinsed with PBS, cryoprotected, and frozen as previously described (35). Cryostat sections were incubated with Alexa 594-conjugated phalloidin (1:50 dilution), which stains filamentous actin (F-actin).…”