Rap1 Activation Plays a Regulatory Role in Pancreatic Amylase Secretion

Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109-0622, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 07/2008; 283(35):23884-94. DOI: 10.1074/jbc.M800754200
Source: PubMed


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.

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    • "The question remains whether the differential involvement of Epac1 or Epac2 might depend on the species involved. Furthermore, since activation of either Epac1 or Epac2 has been implicated in regulating secretion in various tissues, [56]–[58] it seems likely that both isoforms could mediate sensitization of sensory neurons depending the endpoints measured or the type of injury involved. "
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    ABSTRACT: We examined whether nerve growth factor (NGF), an inflammatory mediator that contributes to chronic hypersensitivity, alters the intracellular signaling that mediates the sensitizing actions of PGE2 from activation of protein kinase A (PKA) to exchange proteins directly activated by cAMP (Epacs). When isolated sensory neurons are grown in the absence of added NGF, but not in cultures grown with 30 ng/ml NGF, inhibiting protein kinase A (PKA) activity blocks the ability of PGE2 to augment capsaicin-evoked release of the neuropeptide CGRP and to increase the number of action potentials (APs) evoked by a ramp of current. Growing sensory neurons in culture in the presence of increasing concentrations of NGF increases the expression of Epac2, but not Epac1. An intradermal injection of complete Freund's adjuvant into the rat hindpaw also increases the expression of Epac2, but not Epac1 in the dorsal root ganglia and spinal cord: an effect blocked by intraplantar administration of NGF antibodies. Treating cultures grown in the presence of 30 ng/ml NGF with Epac1siRNA significantly reduced the expression of Epac1, but not Epac2, and did not block the ability of PGE2 to augment capsaicin-evoked release of CGRP from sensory neurons. Exposing neuronal cultures grown in NGF to Epac2siRNAreduced the expression of Epac2, but not Epac1 and prevented the PGE2-induced augmentation of capsaicin and potassium-evoked CGRP release in sensory neurons and the PGE2-induced increase in the number of APs generated by a ramp of current. In neurons grown with no added NGF, Epac siRNAs did not attenuate PGE2-induced sensitization. These results demonstrate that NGF, through increasing Epac2 expression, alters the signaling cascade that mediates PGE2-induced sensitization of sensory neurons, thus providing a novel mechanism for maintaining PGE2-induced hypersensitivity during inflammation.
    Full-text · Article · Aug 2014 · PLoS ONE
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    • "Stimulation of acinar cells with the physiologic ligand cholecystokinin (CCK) at supraphysiologic levels (10–100x physiologic) or its orthologue cerulein (CER), increases cAMP levels and cellular PKA activity [4]. Further, Rap1, a small GTP binding protein activated by cAMP stimulation of EPAC, modulates acinar cell amylase secretion [5] and is associated with zymogen granule membranes [6], [7]. Although these studies demonstrated effects of cAMP on acinar cell responses, they did not examine the potential contribution by specific cAMP pools on pancreatitis responses. "
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    ABSTRACT: An early feature of acute pancreatitis is activation of zymogens, such as trypsinogen, within the pancreatic acinar cell. Supraphysiologic concentrations of the hormone cholecystokinin (CCK; 100 nM), or its orthologue cerulein (CER), induce zymogen activation and elevate levels of cAMP in pancreatic acinar cells. The two classes of adenylyl cyclase, trans-membrane (tmAC) and soluble (sAC), are activated by distinct mechanisms, localize to specific subcellular domains, and can produce locally high concentrations of cAMP. We hypothesized that sAC activity might selectively modulate acinar cell zymogen activation. sAC was identified in acinar cells by PCR and immunoblot. It localized to the apical region of the cell under resting conditions and redistributed intracellularly after treatment with supraphysiologic concentrations of cerulein. In cerulein-treated cells, pre-incubation with a trans-membrane adenylyl cyclase inhibitor did not affect zymogen activation or amylase secretion. However, treatment with a sAC inhibitor (KH7), or inhibition of a downstream target of cAMP, protein kinase A (PKA), significantly enhanced secretagogue-stimulated zymogen activation and amylase secretion. Activation of sAC with bicarbonate significantly inhibited secretagogue-stimulated zymogen activation; this response was decreased by inhibition of sAC or PKA. Bicarbonate also enhanced secretagogue-stimulated cAMP accumulation; this effect was inhibited by KH7. Bicarbonate treatment reduced secretagogue-stimulated acinar cell vacuolization, an early marker of pancreatitis. These data suggest that activation of sAC in the pancreatic acinar cell has a protective effect and reduces the pathologic activation of proteases during pancreatitis.
    Full-text · Article · Jul 2012 · PLoS ONE
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    ABSTRACT: Isolated pancreatic acini and isolated acinar cells have become the preparation of choice for many studies of acinar cell function including digestive enzyme synthesis and secretion, signal transduction events including measurement of intracellular Ca 2+ , and ligand binding studies. Isolated or dispersed exocrine pancreatic cells were first described by Amsterdam and Jamieson in 1972 and have been used by multiple investigators since then (1,2,11,33) followed by the description of isolated acini in 1978 which are now the standard preparation (6,24,30,34). Acini have been prepared using similar technique from pancreas of a variety of species including humans (15,21), pig (20), and duck (32,36) but the focus in this entry is on rodent (rat, mouse and guinea pig) pancreas. Dissociation involves the use of collagenase and proteases to digest the extracellular matrix after which acinar units can be prepared by mild shearing forces. Acini are purified away from ducts, islets and blood vessels based on size and density although some contamination from other cell types remains. Isolated acini which are usually composed of 8-20 cells retain their junctional complexes (tight and adherens junctions) and three dimensional architecture for short term studies of 2-6 hours. They can be cultured in suspension in tissue culture media for 24-48 hours but show marked loss of polarity and some functions over time (8,16,23).
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