Cholecystokinin (CCK) has been shown to activate RhoA and Rac1, as well as reorganize the actin cytoskeleton and, thereby, modify acinar morphology and amylase secretion in mouse pancreatic acini. The aim of the present study was to determine which heterotrimeric G proteins activate RhoA and Rac1 upon CCK stimulation. Galpha(13), but not Galpha(12), was identified in mouse pancreatic acini by RT-PCR and Western blotting. Using specific assays for RhoA and Rac1 activation, we showed that only active Galpha(13) activated RhoA. By contrast, active Galpha(13) and Galpha(q), but not Galpha(s), slightly increased GTP-bound Rac1 levels. A greater increase in Rac1 activation was observed when active Galpha(13) and active Galpha(q) were coexpressed. Galpha(i) was not required for CCK-induced RhoA or Rac1 activation. The regulator of G protein signaling (RGS) domain of p115-Rho guanine nucleotide exchange factor (p115-RGS), a specific inhibitor of Galpha(12/13)-mediated signaling, abolished CCK-stimulated RhoA activation. By contrast, both RGS-2, an inhibitor of Galpha(q), and p115-RGS abolished CCK-induced Rac1 activation, which was PLC pathway-independent. Active Galpha(q) and Galpha(13), but not Galpha(s), induced morphological changes and actin redistribution similar to 1 nM CCK. CCK-induced actin cytoskeletal reorganization was inhibited by RGS-2, but not by p115-RGS, whereas CCK-induced amylase secretion was blocked by both inhibitors. Together, these findings indicate that, in mouse pancreatic acini, Galpha(13) links CCK stimulation to the activation of RhoA, whereas both Galpha(13) and Galpha(q) link CCK stimulation to the activation of Rac1. CCK-induced actin cytoskeletal reorganization is mainly mediated by Galpha(q). By contrast, Galpha(13) and Galpha(q) signaling are required for CCK-induced amylase secretion.
"We have recently shown that Gα13 mediates CCK-induced RhoA activation in mouse pancreatic acini . To study whether Gα13 is also involved in CCK-induced RhoA translocation, acini were infected with adenoviruses encoding the RGS like domain of p115-RhoGEF, p115-RGS, which selectively binds and inhibits Gα13 , . "
[Show abstract][Hide abstract] ABSTRACT: RhoA and Rac1 have been implicated in the mechanism of CCK-induced amylase secretion from pancreatic acini. In all cell types studied to date, inactive Rho GTPases are present in the cytosol bound to the guanine nucleotide dissociation inhibitor RhoGDI. Here, we identified the switch mechanism regulating RhoGDI1-Rho GTPase dissociation and RhoA translocation upon CCK stimulation in pancreatic acini. We found that both Gα13 and PKC, independently, regulate CCK-induced RhoA translocation and that the PKC isoform involved is PKCα. Both RhoGDI1 and RhoGDI3, but not RhoGDI2, are expressed in pancreatic acini. Cytosolic RhoA and Rac1 are associated with RhoGDI1, and CCK-stimulated PKCα activation releases the complex. Overexpression of RhoGDI1, by binding RhoA, inhibits its activation, and thereby, CCK-induced apical amylase secretion. RhoA translocation is also inhibited by RhoGDI1. Inactive Rac1 influences CCK-induced RhoA activation by preventing RhoGDI1 from binding RhoA. By mutational analysis we found that CCK-induced PKCα phosphorylation on RhoGDI1 at Ser96 releases RhoA and Rac1 from RhoGDI1 to facilitate Rho GTPases signaling.
PLoS ONE 06/2013; 8(6):e66029. DOI:10.1371/journal.pone.0066029 · 3.23 Impact Factor
[Show abstract][Hide abstract] 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).
[Show abstract][Hide abstract] ABSTRACT: A variety of neurotransmitters, gastrointestinal hormones, and metabolic signals are known to potentiate insulin secretion through GPCRs. We show here that beta cell-specific inactivation of the genes encoding the G protein alpha-subunits Galphaq and Galpha11 resulted in impaired glucose tolerance and insulin secretion in mice. Interestingly, the defects observed in Galphaq/Galpha11-deficient beta cells were not restricted to loss of muscarinic or metabolic potentiation of insulin release; the response to glucose per se was also diminished. Electrophysiological recordings revealed that glucose-induced depolarization of isolated beta cells was impaired in the absence of Galphaq/Galpha11, and closure of KATP channels was inhibited. We provide evidence that this reduced excitability was due to a loss of beta cell-autonomous potentiation of insulin secretion through factors cosecreted with insulin. We identified as autocrine mediators involved in this process extracellular nucleotides such as uridine diphosphate acting through the Gq/G11-coupled P2Y6 receptor and extracellular calcium acting through the calcium-sensing receptor. Thus, the Gq/G11-mediated signaling pathway potentiates insulin secretion in response to glucose by integrating systemic as well as autocrine/paracrine mediators.
The Journal of clinical investigation 06/2010; 120(6):2184-93. DOI:10.1172/JCI41541 · 13.22 Impact Factor
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