Rho and Rac promote acinar morphological changes, actin reorganization, and amylase secretion

Supramaximal stimulation of isolated pancreatic acini with specific agonists such as CCK induces the formation of large basolateral blebs, redistributes filamentous actin, and inhibits secretion. Rho family small G proteins are well documented for their function in actin reorganization that determines cell shape and have been suggested to play a role in secretion. Here, we determined whether Rho and Rac are involved in the morphological changes, actin redistribution, and inhibition of amylase secretion induced by high concentrations of CCK. Introduction of constitutively active RhoV14 and RacV12 but not Cdc42V12 in mouse pancreatic acini by adenoviral vectors stimulated acinar morphological changes including basolateral protrusions, increased the total amount of F-actin, and reorganized the actin cytoskeleton. Dominant-negative RhoN19, Clostridium botulinum C3 exotoxin, which inhibits Rho, and dominant-negative RacN17 all partially blocked CCK-induced acinar morphological changes and actin redistribution. To study the correlation between actin polymerization and acinar shape changes, two marine toxins were employed. Jasplakinolide, a reagent that facilitates actin polymerization and stabilizes F-actin, stimulated acinar basolateral protrusions, whereas latrunculin, which sequesters actin monomers, blocked CCK-induced acinar blebbing. Unexpectedly, RhoV14, RacV12, and jasplakinolide all increased amylase secretion by CCK from 30 pM to 10 nM. The data suggest that Rho and Rac are involved in CCK-evoked changes in acinar morphology, actin redistribution, and secretion and that inhibition of secretion by high concentrations of CCK is not directly coupled to the changes in acinar morphology.     

Cholecystokinin-Mediated RhoGDI Phosphorylation via PKCα Promotes both RhoA and Rac1 Signaling

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.     

Regulation of CCK-induced amylase release by PKC-delta in rat pancreatic acinar cells

PKC is known to be activated by pancreatic secretagogues such as CCK and carbachol and to participate along with calcium in amylase release. Four PKC isoforms, alpha, delta, epsilon, and zeta, have been identified in acinar cells, but which isoforms participate in amylase release are unknown. To identify the responsible isoforms, we used translocation assays, chemical inhibitors, and overexpression of individual isoforms and their dominant-negative variants by means of adenoviral vectors. CCK stimulation caused translocation of PKC-alpha, -delta, and -epsilon, but not -zeta from soluble to membrane fraction. CCK-induced amylase release was inhibited approximately 30% by GF109203X, a broad spectrum PKC inhibitor, and by rottlerin, a PKC-delta inhibitor, but not by G?6976, a PKC-alpha inhibitor, at concentrations from 1 to 5 microM. Neither overexpression of wild-type or dominant-negative PKC-alpha affected CCK-induced amylase release. Overexpression of PKC-delta and -epsilon enhanced amylase release, whereas only dominant-negative PKC-delta inhibited amylase release by 25%. PKC-delta overexpression increased amylase release at all concentrations of CCK, but dominant-negative PKC-delta only inhibited the maximal concentration; both similarly affected carbachol and JMV-180-induced amylase release. Overexpression of both PKC-delta and its dominant-negative variant affected the late but not the early phase of amylase release. GF109203X totally blocked the enhancement of amylase release by PKC-delta but had no further effect in the presence of dominant-negative PKC-delta. These results indicate that PKC-delta is the PKC isoform involved with amylase secretion.     

Laminin-1 activates Cdc42 in the mechanism of laminin-1-mediated neurite outgrowth

Here, we investigated the role of the small Rho GTPases Rac, Cdc42, and Rho in the mechanism of laminin-1-mediated neurite outgrowth in PC12 cells. PC12 cells were transfected with plasmids expressing wild-type and dominant-negative mutants of Rac (RacN17), Cdc42 (Cdc42N17), or Rho (RhoN19). Over 90% of the dominant-negative Rho- and Rac-transfected cells extended neurites when plated on laminin-1; however, none of the PC12 cells transfected with the dominant-negative Cdc42 mutant extended neurites. In cells cotransfected with plasmids expressing c-Jun N-terminal kinase and wild-type Cdc42, laminin-1 treatment stimulated detectable levels of c-Jun phosphorylation. Further, cotransfection with c-Jun N-terminal kinase and the dominant-negative Cdc42 mutant blocked laminin-1-mediated c-Jun phosphorylation. Transfection with either wild-type Rac or the dominant-negative Rac did not effect c-Jun phosphorylation. These data demonstrate that Cdc42 is activated by laminin-1 and that Cdc42 activation is required in the mechanism of laminin-1-mediated neurite outgrowth.     

Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca2+ entry in mouse pancreatic acinar cells

We recently reported that store-operated Ca²⁺ entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca²⁺ channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as "secretion-like coupling." As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stimulated by the physiological agonist CCK-8. BoNT A significantly reduced Ca²⁺ entry induced by store depletion using thapsigargin or CCK-8. In addition, treatment with BoNT A once SOCE had been activated reduced Ca²⁺ influx, indicating that SNAP-25 is needed for both the activation and maintenance of SOCE in pancreatic acinar cells. VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. Both proteins associate with the cytoskeleton upon Ca²⁺ store depletion, although only VAMP-2 seems to be sensitive to TeTx. Treatment of pancreatic acinar cells with TeTx reduced the activation of SOCE without affecting its maintenance. These findings support a role for SNAP-25 and VAMP-2 in the activation of SOCE in pancreatic acinar cells and show parallels between this process and secretion in a specialized secretory cell type. synaptosome-associated protein; vesicle-associated membrane protein; pancreatic acinar cells; cytoskeleton; calcium entry     

CCK-A receptor activates RhoA through G alpha 12/13 in NIH3T3 cells

Cholecystokinin (CCK) is a major regulator of pancreatic acinar cells and was shown previously to be capable of inducing cytoskeletal changes in these cells. In the present study, using NIH3T3 cells stably transfected with CCK-A receptors as a model cell, we demonstrate that CCK can induce actin stress fibers through a G₁₃- and RhoA-dependent mechanism. CCK induced stress fibers within minutes similar to those induced by lysophosphatidic acid (LPA), the active component of serum. The effects of CCK were mimicked by active RhoV14 and blocked by dominant-negative RhoN19, Clostridium botulinum C3 transferase, and the Rho-kinase inhibitor Y-27632. CCK rapidly induced active Rho in cells as shown with a pull-down assay using the Rho binding domain of rhotekin and by a serum response element (SRE)-luciferase reporter assay. To evaluate the G protein mediating the action of CCK, cells were transfected with active -subunits; G₁₃ and G₁₂ but not G_q induced stress fibers and in some cases cell rounding. A p115 Rho guanine nucleotide exchange factor (GEF) regulator of G protein signaling (RGS) domain known to interact with G_12/13 inhibited active _12/13-and CCK-induced stress fibers, whereas RGS2 and RGS4, which are known to inhibit G_q, had no effect. Cotransfection with plasmids coding for the G protein -subunit carboxy-terminal peptide from ₁₃ and, to a lesser extent ₁₂, also inhibited the effect of CCK, whereas the peptide from _q did not. These results show that in NIH3T3 cells bearing CCK-A receptors, CCK activates Rho primarily through G₁₃, leading to rearrangement of the actin cytoskeleton. actin; cholecystokinin; Rho; Rho-kinase; stress fibers     

Effect of AA861, a 5-lipoxygenase inhibitor,on amylase secretion from rat pancreatic acini

We have examined the effects of 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone (AA861), a selective inhibitor of 5-lipoxygenase, on the action of cholecystokinin (CCK) and other secretagogues in the stimulation of amylase secretion from dispersed rat pancreatic acini. AA861 inhibited amylase secretion caused by CCK, carbamylcholine (carbachol), bombesin or calcium ionophore A23187 but failed to affect amylase secretion by vasoactive intestinal peptide or 12-O-tetradecanoyl-phorbol 13-acetate. Inhibition by AA861 of CCK or carbachol-induced amylase secretion was confined to the relatively lower concentrations of these secretagogues. AA861 did not inhibit receptor binding of CCK or alter the cellular calcium mobilization induced by CCK. In kinetic studies, AA861 was effective only on amylase secretion from pancreatic acini incubated with CCK for more than 5 min. Indomethacin, a known inhibitor of cyclooxygenase, did not affect the amylase secretion caused by all secretagogues used. These results indicate that the 5-lipoxygenase pathway of arachidonate metabolism may be involved in the actions of calcium-dependent secretagogues of amylase secretion in rat dispersed pancreatic acini, especially for sustaining stimulation of amylase secretion by CCK.     

Ethanol impairs the assembly and disassembly of actin cytoskeleton and cell adhesion via the RhoA signaling pathway, catenin p120 and E-cadherin in CCK-stimulated pancreatic acini

The purpose of the present study was to evaluate the effects of EtOH on RhoA, actin cytoskeleton, catenin p120 and E-cadherin and their interactions in CCK-stimulated rat pancreatic acini. In isolated rat pancreatic acinar cells, CCK stimulation enhanced protein expression and association of RhoA, G_α13, Vav-2, catenin p120 and E-cadherin. CCK induced translocation and activation of RhoA and actin-filamentous assembly and disassembly. RhoA was diffusely localized throughout the acinar cell in the resting state and redistributed to the apical site in response to submaximal CCK stimulation and to a lesser extent in response to supramaximal CCK stimulation. Ethanol and subsequent submaximal CCK stimulation mimicked the effect of supramaximal CCK stimulation in terms of amylase secretion and morphologic effects. However, inhibition of RhoA translocation and activation were observed only with ethanol pretreatment. Ethanol followed by supramaximal CCK stimulation disrupted the well-defined localization of catenin p120 and E-cadherin around the lateral plasma membrane. These data suggest that ethanol impaired the assembly and disassembly of actin cytoskeleton and impaired cell–cell adhesion via the RhoA signaling pathways, catenin p120 and E-cadherin in CCK-stimulated pancreatic acini.     

Aqueous extract of black tea (Camellia sinensis) prevents ethanol+cholecystokinin-induced pancreatitis in a rat model

Black Tea Extract (BTE), a phytocompound has been attributed with a plethora of health-promoting actions. We have previously demonstrated that BTE inhibits chronic hepatitis in a rat model induced with high-fat and ethanol (EtOH). This study reports that BTE prevents altered pancreatic acinar cell functions, oxidative stress, inflammatory changes and DNA damage in the EtOH+cholecystokinin (CCK)-induced model of pancreatitis. The EtOH+CCK model rats were administered with BTE, and were examined the activity of pancreatic digestive enzymes (amylase and lipase), proinflammatory cytokines (IL-6 and TNF-alpha), oxidative and antioxidative enzymes (nitric oxide, NO; malondialdehyde, MDA; superoxide dismutase, SOD; catalase, CAT), antioxidant level (glutathione, GSH), histopathological changes and the integrity of genomic DNA. Results show that because of chronic EtOH treatment, serum level of amylase and lipase (two biomarkers for pancreatitis) and pancreatic levels of MDA and NO (two biomarkers of oxidative stress) increased significantly, which could be effectively blunted by BTE. BTE could normalize EtOH+CCK-induced suppressed activities of SOD and CAT, and GSH content of pancreatic tissue. Also, histopathological and inflammatory changes during EtOH+CCK-induced pancreatitis could be blunted by BTE. Furthermore, BTE could effectively reduce EtOH+CCK-induced increase in DNA fragmentation and damage. These findings suggest that BTE prevents pancreatitis caused by chronic EtOH+CCK toxicity presumably by enhancing antioxidant, anti-inflammatory and antiapoptotic activity in rats.     

Hypotonicity induces membrane protrusions and actin remodeling via activation of small GTPases Rac and Cdc42 in Rat-1 fibroblasts

An important consequence of cell swelling is the reorganization of the F-actin cytoskeleton in different cell types. We demonstrate in this study by means of rhodamine-phalloidin labeling and fluorescence microscopy that a drastic reorganization of F-actin occurs in swollen Rat-1 fibroblasts: stress fibers disappear and F-actin patches are formed in peripheral extensions at the cell border. Moreover, we demonstrate that activation of both Rac and Cdc42, members of the family of small Rho GTPases, forms the link between the hypotonic stimulation and F-actin reorganization. Indeed, inhibition of the small GTPases RhoA, Rac, and Cdc42 (by Clostridium difficile toxin B) prevents the hypotonicity-induced reorganization of the actin cytoskeleton, whereas inhibition of RhoA alone (by C. limosum C3 exoenzyme) does not preclude this rearrangement. Second, a direct activation and translocation toward the actin patches underneath the plasma membrane is observed for endogenous Rac and Cdc42 (but not for RhoA) during cell swelling. Finally, transfection of Rat-1 fibroblasts with constitutively active RhoA, dominant negative Rac, or dominant negative Cdc42 abolishes the swelling-induced actin reorganization. Interestingly, application of cRGD, a competitor peptide for fibronectin-integrin association, induces identical membrane protrusions and changes in the F-actin cytoskeleton that are also inhibited by C. difficile toxin B and dominant negative Rac or Cdc42. Moreover, cRGD also induces a redistribution of endogenous Rac and Cdc42 to the newly formed submembranous F-actin patches. We therefore conclude that hypotonicity and cRGD remodel the F-actin cytoskeleton in Rat-1 fibroblasts in a Rac/Cdc42-dependent way. Rho; actin; swelling     

Effects of osmotic shrinkage on voltage-gated Ca2+ channel currents in rat anterior pituitary cells

Increased extracellular osmolarity ([Os]_e) suppresses stimulated hormone secretion from anterior pituitary cells. Ca²⁺ influx may mediate this effect. We show that increase in [Os]_e (by 18–125%) differentially suppresses L-type and T-type Ca²⁺ channel currents (I_L and I_T, respectively); I_L was more sensitive than I_T. Hyperosmotic suppression of I_L depended on the magnitude of increase in [Os]_e and was correlated with the percent decrease in pituitary cell volume, suggesting that pituitary cell shrinkage can modulate L-type currents. The hyperosmotic suppression of I_L and I_T persisted after incubation of pituitary cells either with the actin-disrupter cytochalasin D or with the actin stabilizer phalloidin, suggesting that the actin cytoskeleton is not involved in this modulation. The hyperosmotic suppression of Ca²⁺ influx was not correlated with changes in reversal potential, membrane capacitance, and access resistance. Together, these results suggest that the hyperosmotic suppression of Ca²⁺ influx involves Ca²⁺ channel proteins. We therefore recorded the activity of L-type Ca²⁺ channels from cell-attached patches while exposing the cell outside the patch pipette to hyperosmotic media. Increased [Os]_e reduced the activity of Ca²⁺ channels but did not change single-channel conductance. This hyperosmotic suppression of Ca²⁺ currents may therefore contribute to the previously reported hyperosmotic suppression of hormone secretion. L-type Ca²⁺ channels; osmosensitivity; mechanosensitivity; osmolarity; hyperosmolarity     

Dominant negative Rab3D inhibits amylase release from mouse pancreatic acini

Rab3 proteins are believed to play an important role in regulated exocytosis and previous work has demonstrated the presence of Rab3D on pancreatic zymogen granules. To further understand the function of Rab3D in acinar cell exocytosis, adenoviral constructs were prepared encoding hemagglutinin-tagged wild type Rab3D and three mutant forms, N135I and T36N (both deficient in guanine nucleotide binding) and Q81L (deficient in GTP hydrolysis), which also expressed enhanced green fluorescent protein driven by a separate promoter. When isolated mouse pancreatic acini were cultured with 5 x 10(6) pfu/ml adenovirus, nearly 100% of acini were infected as visualized by expression of green fluorescent protein. Cultured acini showed a biphasic dose-response to cholecystokinin (CCK); basal amylase secretion was 1.8 +/- 0.3%/30 min, peak release was 7.3 +/- 0.2%/30 min at 30 pm CCK and reduced secretion was observed at higher CCK concentrations. Control beta-galactosidase virus infection had no effect on either basal or CCK-induced secretion in the titer range from 0.5 to 10 x 10(6) pfu/ml. While the expression of Rab3D and Rab3D Q81L had no effect on amylase secretion, Rab3D N135I and T36N functioned as dominant negative mutants and inhibited CCK-induced amylase release by 40-50% at all points on the CCK dose-response curve from 3 to 300 pm. Inhibition was stronger during the first 5 min (71 +/- 5%) than over 30 min (36%+/-5%). Similar inhibition was found using other agonists including bombesin, carbachol, and cAMP. Localization of adenoviral expressed Rab protein showed wild type Rab3D localized to zymogen granules. The two dominant negative mutants did not localize to granules and were primarily in the basolateral region of the cell. Since both dominant negative Rab3D mutants had no effect on intracellular calcium increase induced by CCK, it is unlikely that they acted at receptors or transmembrane signaling. These results suggest that Rab3D plays an important role in regulating the terminal steps of acinar exocytosis and that this effect is greatest on the early phase of amylase release.     

Cyclic AMP-dependent protein kinase and Epac mediate cyclic AMP responses in pancreatic acini

The pancreatic acinar cell has several phenotypic responses to cAMP agonists. At physiological concentrations of the muscarinic agonist carbachol (1 microM) or the CCK analog caerulein (100 pM), ligands that increase cytosolic Ca(2+), cAMP acts synergistically to enhance secretion. Supraphysiological concentrations of carbachol (1 mM) or caerulein (100 nM) suppress secretion and cause intracellular zymogen activation; cAMP enhances both zymogen activation and reverses the suppression of secretion. In addition to stimulating cAMP-dependent protein kinase (PKA), recent studies using cAMP analogs that lack a PKA response have shown that cAMP can also act through the cAMP-binding protein, Epac (exchange protein directly activated by cyclic AMP). The roles of PKA and Epac in cAMP responses were examined in isolated pancreatic acini. The activation of both cAMP-dependent pathways or the selective activation of Epac was found to enhance amylase secretion induced by physiological and supraphysiological concentrations of the muscarinic agonist carbachol. Similarly, activation of both PKA or the specific activation of Epac enhanced carbachol-induced activation of trypsinogen and chymotrypsinogen. Disorganization of the apical actin cytoskeleton has been linked to the decreased secretion observed with supraphysiological concentrations of carbachol and caerulein. Although stimulation of PKA and Epac or Epac alone could largely overcome the decreased secretion observed with either supraphysiological carbachol or caerulein, stimulation of cAMP pathways did not reduce the disorganization of the apical cytoskeleton. These studies demonstrate that PKA and Epac pathways are coupled to both secretion and zymogen activation in the pancreatic acinar cell.     

Effects of growth hormone releasing factor on pancreatic secretion in vivo and in vitro

Growth hormone releasing factor (GRF), a 44-residue peptide originally isolated from human pancreatic tumors, shows structural similarities to the members of the secretin-vasoactive intestinal peptide (VIP) peptides. This study was designed to determine the effects of human GRF (hGRF-(1-44] on pancreatic secretion in vivo in conscious dogs and in vitro in dispersed rat pancreatic acini. GRF given i.v. in graded doses in dogs caused a small but significant stimulation of pancreatic HCO3- and protein outputs and potentiated secretin- and cholecystokinin (CCK)-induced pancreatic HCO3- but not protein secretion. When given together with somatostatin, GRF failed to reverse the inhibitory action of this peptide on HCO3- and protein responses to secretin plus CCK in dogs. Studies in vitro dispersed rat pancreatic acini showed that GRF added to the incubation medium of these acini caused an increase in basal amylase release and shifted to the left the amylase dose-response curve to caerulein and urecholine but failed to affect the amylase response to VIP. This study indicates that GRF in vivo stimulates basal and augments secretin- or CCK-induced pancreatic HCO3- secretion and that this is probably due to direct stimulatory action of the peptide on pancreatic secretory cells.     

Effects of ionophore A23187 on calcium flux and amylase release in isolated mouse pancreatic acini

The divalent cation ionophore A23187 has been used extensively to demonstrate the importance of Ca²⁺ in the control of pancreatic enzyme secretion. The relative importance, however, of the ability of the ionophore to facilitate Ca²⁺ movement across plasma and intracellular membranes in the stimulation of amylase release is not clear. We therefore studied these relationships in isolated pancreatic acini, a preparation in which it is possible to precisely measure both ⁴⁵Ca²⁺ fluxes, Ca²⁺ content and amylase release. A23187 increased the initial rates of both ⁴⁵Ca²⁺ uptake and washout. In addition, the content of both exchangeable ⁴⁵Ca²⁺ and total Ca²⁺ were reduced. These results indicated, therefore, that A23187 increases Ca²⁺ fluxes across both plasma and intracellular membranes. Consistent with this observation, the initial stimulation of amylase release by A23187 was independent of extracellular Ca²⁺. In the absence of extracellular Ca²⁺, however, A23187 caused a rapid fall in acinar Ca²⁺ and subsequent amylase release was abolished. Depletion of intracellular Ca²⁺ by the ionophore also blocked the subsequent stimulation by cholecystokinin (CCK). The results indicate certain similarities in the actions of A23187 and CCK on pancreatic acini; both the agonists have striking effects on intracellular Ca²⁺ which in turn mediates their actions.     

Osmotic stress-induced remodeling of the cortical cytoskeleton

Osmoticstress is known to affect the cytoskeleton; however, this adaptiveresponse has remained poorly characterized, and the underlyingsignaling pathways are unexplored. Here we show that hypertonicityinduces submembranous de novo F-actin assembly concomitant with theperipheral translocation and colocalization of cortactin and theactin-related protein 2/3 (Arp2/3) complex, which are key components ofthe actin nucleation machinery. Additionally, hyperosmolarity promotesthe association of cortactin with Arp2/3 as revealed bycoimmunoprecipitation. Using various truncation orphosphorylation-incompetent mutants, we show that cortactin translocation requires the Arp2/3- or the F-actin binding domain, butthe process is independent of the shrinkage-induced tyrosine phosphorylation of cortactin. Looking for an alternative signaling mechanism, we found that hypertonicity stimulates Rac and Cdc42. Thisappears to be a key event in the osmotically triggered cytoskeletal reorganization, because 1) constitutively active smallGTPases translocate cortactin, 2) Rac and cortactincolocalize at the periphery of hypertonically challenged cells, and3) dominant-negative Rac and Cdc42 inhibit thehypertonicity-provoked cortactin and Arp3 translocation. The Rhofamily-dependent cytoskeleton remodeling may be an importantosmoprotective response that reinforces the cell cortex.

     

Glucagon-like peptide-1 does not mediate amylase release from AR42J cells.

In this study, AR42J pancreatic acinar cells were used to investigate if glucagon-like peptide-1 (GLP-1) or glucagon might influence amylase release and acinar cell function. We first confirmed the presence of GLP-1 receptors on AR42J cells by reverse trasncriptase-polymerase chain reaction (RT-PCR), Western blotting, and partial sequencing analysis. While cholecystokinin (CCK) increased amylase release from AR42J cells, GLP-1, alone or in the presence of CCK, had no effect on amylase release but both CCK and GLP-1 increased intracellular calcium. Similar to GLP-1, glucagon increased both cyclic adenosine monophosphate (cAMP) and intracellular calcium in AR42J cells but it actually decreased CCK-mediated amylase release (n = 20, P < 0.01). CCK stimulation resulted in an increase in tyrosine phosphorylation of several cellular proteins, unlike GLP-1 treatment, where no such increased phosphorylation was seen. Instead, GLP-1 decreased such protein phosphorylations. Genestein blocked CCK-induced phosphorylation events and amylase secretion while vanadate increased amylase secretion. These results provide evidence that tyrosine phosphorylation is necessary for amylase release and that signaling through GLP-1 receptors does not mediate amylase release in AR42J cells. J. Cell. Physiol. 181:470-478, 1999. Published 1999 Wiley-Liss, Inc.     

Dual effects of n-alcohols on fluid secretion from guinea pig pancreatic ducts

Ethanol strongly augments secretin-stimulated, but not acetylcholine (ACh)-stimulated, fluid secretion from pancreatic duct cells. To understand its mechanism of action, we examined the effect of short-chain n-alcohols on fluid secretion and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in guinea pig pancreatic ducts. Fluid secretion was measured by monitoring the luminal volume of isolated interlobular ducts. [Ca²⁺]_i was estimated using fura-2 microfluorometry. Methanol and ethanol at 0.3–10 mM concentrations significantly augmented fluid secretion and induced a transient elevation of [Ca²⁺]_i in secretin- or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP)-stimulated ducts. However, they failed to affect fluid secretion and [Ca²⁺]_i in unstimulated and ACh-stimulated ducts. In contrast, propanol and butanol at 0.3–10 mM concentrations significantly reduced fluid secretion and decreased [Ca²⁺]_i in unstimulated ducts and in ducts stimulated with secretin, DBcAMP, or ACh. Both stimulatory and inhibitory effects of n-alcohols completely disappeared after their removal from the perfusate. Propanol and butanol inhibited the plateau phase, but not the initial peak, of [Ca²⁺]_i response to ACh as well as the [Ca²⁺]_i elevation induced by thapsigargin, suggesting that they inhibit Ca²⁺ influx. Removal of extracellular Ca²⁺ reduced [Ca²⁺]_i in duct cells and completely abolished secretin-stimulated fluid secretion. In conclusion, there is a distinct cutoff point between ethanol (C2) and propanol (C3) in their effects on fluid secretion and [Ca²⁺]_i in duct cells. Short-chain n-alcohols appear to affect pancreatic ductal fluid secretion by activating or inhibiting the plasma membrane Ca²⁺ channel. intracellular calcium; acetylcholine     

Ca2+-sensing receptor induces Rho kinase-mediated actin stress fiber assembly and altered cell morphology, but not in response to aromatic amino acids

The Ca²⁺-sensing receptor (CaR) is a pleiotropic, type III G protein-coupled receptor (GPCR) that associates functionally with the cytoskeletal protein filamin. To investigate the effect of CaR signaling on the cytoskeleton, human embryonic kidney (HEK)-293 cells stably transfected with CaR (CaR-HEK) were incubated with CaR agonists in serum-free medium for up to 3 h. Addition of the calcimimetic NPS R-467 or exposure to high extracellular Ca²⁺ or Mg²⁺ levels elicited actin stress fiber assembly and process retraction in otherwise stellate cells. These responses were ablated by cotreatment with the calcilytic NPS 89636 and were absent in vector-transfected HEK-293 cells. Cotreatment with the Rho kinase inhibitors Y-27632 and H1152 attenuated the CaR-induced morphological change but not intracellular Ca²⁺ (Ca_i²⁺) mobilization or ERK activation, although transfection with a dominant-negative RhoA-binding protein also inhibited calcimimetic-induced actin stress fiber assembly. CaR effects on morphology were unaffected by inhibition of G_q/11 or G_i/o signaling, epidermal growth factor receptor, or the metalloproteinases. In contrast, CaR-induced cytoskeletal changes were not induced by the aromatic amino acids, treatments that also failed to potentiate CaR-induced ERK activation despite inducing Ca_i²⁺ mobilization. Together, these data establish that CaR can elicit Rho-mediated changes in stress fiber assembly and cell morphology, which could contribute to the receptor's physiological actions. In addition, this study provides further evidence that aromatic amino acids elicit differential signaling from other CaR agonists. cytoskeleton; signaling     

Stimulation of pancreatic amylase release is associated with a parallel sustained increase of cytoplasmic calcium

The kinetics of the changes in the cytoplasmic Ca²⁺ concentration (Ca_i²⁺) and amylase release were measured in fura-2-loaded pancreatic acinar cells and perifused pancreatic acini, respectively. Cholecystokinin octapeptide (CCK-8) and its amphibian analogue caerulein induced similar dose-related increases of Ca_i²⁺ and amylase secretion with threshold concentrations of 2–6·10⁻¹² M, and maximal effects at 2·10⁻¹⁰ M. The action of CCK/caerulein on Ca_i²⁺ was complex and similar to that of carbachol and bombesin with a prompt several-fold increase within seconds followed by a gradual decline over more than 5 min to a new sustained suprabasal level. The kinetics of amylase release in response to CCK and carbachol correlated with the changes in Ca_i²⁺. Additions of the antagonists N²,O²-dibutyrylguanosine 3′:5′-cyclic monophosphate and atropine after 30 min of CCK-8 and carbachol stimulation, respectively, were associated with prompt lowerings of Ca_i²⁺ and inhibitions of amylase secretion. The patterns observed with substance P (SP) and eledoisin were different with high concentrations (10⁻⁸–10⁻⁷ M) giving monophasic increases of Ca_i²⁺ and amylase release. An initial stimulation of cells with a high dose of CCK eliminated the Ca_i²⁺ response to further stimulation with CCK, carbachol, bombesin and SP, whereas cells subjected to initial stimulation with SP responded to subsequent exposure to CCK with prolonged elevation of Ca_i²⁺. The data indicate that stimulation with CCK, carbachol and bombesin may be associated with intracellular mobilization of calcium from more than one pool, and that an increase of Ca_i²⁺ is involved even in threshold stimulation of amylase release.