Rat Pancreatic Acini Research Articles

Regulation of translation elongation and phosphorylation of eEF2 in rat pancreatic acini

While pancreatic protein synthesis and the initiation of translation are regulated by hormones and neurotransmiters, whether the elongation process is also regulated is unknown. Stimulatory doses of cholecystokinin (CCK) (100 pM), bombesin (10 nM), and carbachol (10 μM) increased elongation rates (measured as ribosomal half-transit time) in pancreatic acini in vitro. At the same time these secretagogues reduced elongation factor 2 (eEF2) phosphorylation, the main factor known to regulate elongation, and increased the phosphorylation of the eEF2 kinase. The mTOR inhibitor rapamycin reversed the dephosphorylation of eEF2 induced by CCK, as did treatment with the p38 MAPK inhibitor SB202190, the MEK inhibitor PD98059, and the phosphatase inhibitor calyculin A. Neither rapamycin, SB202190, PD98059 nor calyculin A had an effect on CCK mediated eEF2 kinase phosphorylation. Translation elongation in pancreatic acinar cells is likely regulated by eEF2 through the mTOR, p38, and MEK pathways, and modulated through PP2A.

Effects of KCNQ1 Channel Blocker, 293B, on the Acetylcholine-Induced Cl??? Secretion of Rat Pancreatic Acini

In rat pancreatic acini (RPAs), acetylcholine (ACh) typically induces a tonic depolarization of membrane potential (Vm) via increasing cytoplasmic Ca2+ concentration and subsequent activation of Cl- channels. In this study, to investigate the role of K+ channels during the ACh-induced Cl- secretion, the intracellular Cl- concentration ([Cl-]i) of RPAs was monitored using SPQ, a fluorescent dye quenchable by Cl-, and the effects of K+ channel blockers were examined. Also, the secretion of fluid and enzyme from the whole pancreas of rat was measured. The fluorescence of RPAs loaded with SPQ (FSPQ) was slightly increased by the application of ACh (ACh-Delta FSPQ), indicating net secretion of Cl-. However, the relative change of FSPQ normalized to the control fluorescence (F/F0) of RPAs was only about 20% of the effect observed in rat submandibular gland acinus. The ACh-Delta FSPQ of RPAs was not influenced by the pretreatment with 293B (20 micromol/L), a blocker of KCNQ-type K+ channels. Even the cocktail of K+ channel blockers (10 mmol/L TEA, 3 mmol/L Ba2+, 20 micromol/L 293B) exerted only minute inhibitory effects on ACh-Delta FSPQ in RPAs. In the vascularly perfused rat pancreas, the fluid and enzyme secretion induced by ACh was directly measured. 293B and HMR-1556, both specific blockers of KCNQ1 channel, did not block but even enhanced the secretion of fluid and amylase. These results suggest that the role of KCNQ1 channels may not be essential in the Ca2+-mediated Cl- secretion in rat pancreatic acini.

Calcineurin is required for translational control of protein synthesis in rat pancreatic acini.

CCK increases the rate of net protein synthesis in rat pancreatic acini by activating initiation and elongation factors required for translation. The immunosuppressant FK506 inhibits the Ca(2+)-calmodulin-dependent phosphatase calcineurin in pancreatic acinar cells and blocks pancreatic growth induced by chronic CCK treatment. To test a requirement for calcineurin in the activation of the translational machinery stimulated by CCK, we evaluated the effects of FK506 on protein synthesis and on regulatory initiation and elongation factors in rat pancreatic acini in vitro. CCK acutely increased protein synthesis in acini from normal rats with a maximum increase at 100 pM CCK to 170 +/- 11% of control. The immunosuppressant FK506 dose-dependently inhibited CCK-stimulated protein synthesis over the same concentration range that blocked calcineurin activity, as assessed by dephosphorylation of the calcineurin substrate calcium-regulated heat-stable protein of 24 kDa. Another immunosuppressant, cyclosporin A, inhibited protein synthesis, but its effects appeared more complex. FK506 also inhibited protein synthesis stimulated by bombesin and carbachol. FK506 did not significantly affect the activity of the initiation factor-2B, or the phosphorylation of the initiation factor-2alpha, ribosomal protein protein S6, or the mRNA cap binding protein eukaryotic initiation factor (eIF) 4E. Instead, blockade of calcineurin with FK506 reduced the phosphorylation of the eIF4E binding protein, reduced the formation of the eIF4F complex, and increased the phosphorylation of eukaryotic elongation factor 2. From these results, we conclude that calcineurin activity is required for protein synthesis, and this action may be related to an effect on the formation of the mRNA cap binding complex and the elongation processes.

Receptor-operated Ca 2+ influx and its association with the Src family in secretagogue-stimulated pancreatic acini

We investigated signal transduction between receptor-operated Ca 2+ influx (ROCI) and Src-related nonreceptor protein tyrosine kinase (PTK) in rat pancreatic acini. CCK and the Ca 2+ ionophore enhanced the Src-related PTK activity, whereas the high-affinity CCK-A receptor agonists, fibroblast growth factor (FGF), and the protein kinase C (PKC) activator had no or little effect. This increase was abolished by eliminating [Ca 2+] o, loading of the intracellular Ca 2+ chelator, and administering the PTK inhibitor genistein. While genistein inhibited extracellular Ca 2+ or Mn 2+ entry induced by CCK and carbachol, it did not affect intracellular Ca 2+ release and oscillations. CCK dose-dependently increased the Src phosphotransferase activity, which was abolished by inhibitors of G q protein, phospholipase C (PLC), and Src, but not by the calmodulin kinase (CaMK) inhibitor. Intensities of the Src band and amounts of tyrosine phosphorylated Src were enhanced by CCK stimulation. Thus, Src cascades appear to be coupled to the low-affinity CCK-A receptor and utilize G q-PLC pathways for their activation, independent of PKC and CaMK cascades. The low-affinity CCK-A receptor regulates ROCI via mediation of Src-related PTK and activates Src pathways to cause [Ca 2+] o-dependent pancreatic exocytosis.

Nonoxidative ethanol metabolites alter extracellular matrix protein content in rat pancreas

Background & aims : The mechanisms involved in ethanol-induced pancreas fibrosis are poorly understood. Here we show that fatty acid ethyl esters (FAEEs), nonoxidative ethanol metabolites, increase extracellular matrix (ECM) protein levels in pancreas. Methods : Rat pancreatic acini were incubated for 1–4 hours with FAEEs or acetaldehyde. In another set of experiments, rats received an intravenous infusion of FAEEs for 6 hours. Collagens were assessed by a hydroxyproline assay. Laminin and fibronectin were analyzed by Western blotting. Gene expression of ECM proteins was measured by conventional and real-time reverse-transcription polymerase chain reaction (RT-PCR). Matrix metalloproteinase (MMP), plasmin, and urokinase-type plasminogen activator (uPA) activities were determined by zymography and fluorogenic assays. Results : FAEEs increased collagen, laminin, and fibronectin levels in pancreatic acini without affecting messenger RNA (mRNA) expression for these proteins. Actinomycin D, a transcriptional inhibitor, did not block the increase in ECM proteins induced by FAEEs. FAEEs reduced the activity of the serine protease, plasmin, and that of the uPA. Consistent with these results, the serine protease inhibitor aprotinin reproduced the effects of FAEEs and prevented the further increase in ECM proteins induced by FAEEs. In vivo administration of FAEEs reduced plasmin and uPA activities and increased ECM protein levels in pancreas. Acetaldehyde had minor effects on ECM protein levels and did not affect plasmin activity. Conclusions : FAEEs increase ECM protein levels in pancreas. The results suggest that this effect is caused primarily by an inhibition in ECM degradation via serine proteases including the plasminogen system.

Cholecystokinin rapidly stimulates CrkII function in vivo in rat pancreatic acini. Formation of CrkII-protein complexes.

Crk belongs to a family of adapter proteins whose structure allows interaction with tyrosine-phosphorylated proteins and is therefore an important modulator of downstream signals, representing a convergence of the actions of numerous stimuli. Recently, it was demonstrated that cholecystokinin (CCK) induced tyrosine phosphorylation of proteins related to fiber stress formation in rat pancreatic acini. Here, we investigated whether CCK receptor activation signals through CrkII and forms complexes with tyrosine-phosphorylated proteins in rat pancreatic acini. We demonstrated that CCK promoted the transient formation of CrkII-paxillin and CrkII-p130Cas complexes with maximal effect at 1 min. Additionally, CCK decreased the electrophoretic mobility of CrkII. This decrease was time- and concentration-dependent and inversely related with its function. Carbachol and bombesin also decreased CrkII electrophoretic mobility, whereas epidermal growth factor, vasoactive intestinal peptide, secretin or pituitary adenylate cyclase-activating polypeptide had no effect. CCK-induced CrkII electrophoretic shift was dependent on the Src family of tyrosine kinases and occurred in the intact animal, suggesting a physiological role of CrkII mediating CCK actions in the exocrine pancreas in vivo.

Hepatocyte growth factor activates several transduction pathways in rat pancreatic acini

The receptor of hepatocyte growth factor (HGF), c-met induces different physiological responses in several cell types. Little is known about the role of HGF in exocrine pancreas. However, abnormal HGF signaling has been strongly implicated in pancreatic tumorigenesis and association of HGF with pancreatitis has been demonstrated. We have studied the presence of c-met and activation of their intracellular pathways associated in rat pancreatic acini in comparison with cholecystokinin (CCK) and epidermal growth factor (EGF). C-met expression in rat exocrine pancreas was identified by immunohistochemistry and immunoprecipitation followed by Western analysis. Tyrosine phosphorylation of c-met is strongly stimulated as well as kinase pathways leading to ERK1/2 cascade. HGF, but not CCK or EGF, selectively caused a consistent increase in the amount of p85 regulatory subunit of PI3-K present in anti-phosphotyrosine immunoprecipitates. Downstream of PI3-K, HGF increased Ser473 phosphorylation of Akt selectively, as CCK or EGF did not affect it. HGF selectively stimulated tyrosine phosphorylation of phosphatase PTP1D. HGF failed to promote the well-known CCK effects in pancreatic acini such as amylase secretion and intracellular calcium mobilization. Although HGF shares activation of ERK1/2 with CCK, we demonstrate that it promotes the selective activation of intracellular pathways not regulated by CCK or EGF. Our results suggest that HGF is an in vivo stimulus of pancreatic acini and provide novel insight into the transduction pathways and effects of c-met/HGF in normal pancreatic acinar cells.

Cholecystokinin-stimulated Protein Kinase C-δ Kinase Activation, Tyrosine Phosphorylation, and Translocation Are Mediated by Src Tyrosine Kinases in Pancreatic Acinar Cells

Protein kinase C-delta (PKC-delta) is involved in growth, differentiation, tumor suppression, and regulation of other cellular processes. PKC-delta activation causes translocation, tyrosine phosphorylation, and serine-threonine kinase activity. However, little is known about the ability of G protein-coupled receptors to activate these processes or the mediators involved. In the present study, we explored the ability of the neurotransmitter/hormone, CCK, to stimulate these changes in PKC-delta and explored the mechanisms. In rat pancreatic acini under basal conditions, PKC-delta is almost exclusively located in cytosol. CCK and TPA stimulated a rapid PKC-delta translocation to membrane and nuclear fractions, which was transient with CCK. CCK stimulated rapid tyrosine phosphorylation of PKC-delta and increased kinase activity. Using tyrosine kinase (B44) and a tyrosine phosphatase inhibitor (orthovanadate), changes in both CCK- and TPA-stimulated PKC-delta tyrosine phosphorylation were shown to correlate with changes in its kinase activity but not translocation. Both PKC-delta tyrosine phosphorylation and activation occur exclusively in particulate fractions. The Src kinase inhibitors, SU6656 and PP2, but not the inactive related compound, PP3, inhibited CCK- and TPA-stimulated PKC-delta tyrosine phosphorylation and activation. In contrast, PP2 also had a lesser effect on CCK- but not TPA-stimulated PKC-delta translocation. CCK stimulated the association of Src kinases with PKC-delta, demonstrated by co-immunoprecipitation. These results demonstrate that CCKA receptor activation results in rapid translocation, tyrosine phosphorylation, and activation of PKC-delta. Stimulation of PKC-delta translocation precedes tyrosine phosphorylation, which is essential for activation to occur. Activation of Src kinases is essential for the tyrosine phosphorylation and kinase activation to occur and plays a partial role in translocation.

Selective activation by photodynamic action of cholecystokinin receptor in the freshly isolated rat pancreatic acini.

1 Sulphonated aluminium phthalocyanine (SALPC) photodynamic action induces amylase secretion and permanent calcium oscillation in rat pancreatic acinar cells, because of the activation of phospholipase C or signalling proteins upstream. The aim of the present study was to investigate the involvement of muscarinic acetylcholine and cholecystokinin (CCK) receptors. 2 Muscarinic receptor antagonist atropine (10 micro M) blocked amylase secretion induced by bethanechol (100 micro M), and CCK(1) receptor antagonist (S)-N-[1-(2-fluorophenyl)-3,4,6,7-tetrahydor-4-oxo-pyrrolo-[3,2,1-jk][1,4] benzodiazepine-3yl]-1H-indole-2-carboxamide (FK480) (1 micro M) blocked amylase secretion induced by CCK (100 pM). 3 Amylase secretion induced by SALPC photodynamic action was not inhibited when atropine and FK480 were present during photodynamic action. However, addition of FK480 1 micro M after initiation of photodynamic action inhibited photodynamic amylase secretion. Bethanechol (10, 100 micro M) added after photodynamic action resulted in a full secretory response. 4 Atropine (10 nM) abolished calcium oscillation induced by bethanechol (5 micro M), and FK480 (10 nM) blocked calcium oscillation induced by CCK (10 pM). 5 Atropine up to 10 micro M was without effect on Ca(2+) oscillation triggered by photodynamic action, but these oscillations were abolished by FK480 (10 nM). FK480 (10 nM) had no effect on calcium oscillations induced by bethanechol (5 micro M). Bethanechol 5 micro M, added after FK480 blockade of photodynamic calcium oscillation, still triggered regular calcium oscillation. 6 It is concluded that SALPC photodynamic action selectively and permanently activates CCK receptor in rat pancreatic acini. Such permanent and selective modulation of signalling proteins has important implications for the treatment of pancreatitis, prion diseases, and neurodegenerative disorders.

Phosphospecific Site Tyrosine Phosphorylation of p125FAK and Proline-rich Kinase 2 Is Differentially Regulated by Cholecystokinin Receptor Type A Activation in Pancreatic Acini

The focal adhesion kinases, p125FAK and proline-rich kinase 2 (PYK2), are involved in numerous processes as adhesion, cytoskeletal changes, and growth. These kinases have 45% homology and share three tyrosine phosphorylation (TyrP) sites. Little information exists on the ability of stimulants to cause TyrP of each kinase site and the cellular mechanism involved. We explored the ability of the neurotransmitter/hormone, CCK, to stimulate TyrP at each site. In rat pancreatic acini, CCK stimulated TyrP at each site in both kinases. TyrP was rapid except for pY397FAK. The magnitude of TyrP differed with the different FAK and PYK2 sites. The CCK dose-response curve for TyrP for sites in each kinase was similar. CCK-JMV, an agonist of the high affinity receptor state and antagonist of the low affinity receptor state, was less efficacious than CCK at each FAK/PYK2 site and inhibited CCK maximal stimulation. Thapsigargin decreased CCK-stimulated TyrP of pY402PYK2 and pY925FAK but not the other sites. GF109203X reduced TyrP of only the PYK2 sites, pY402 and pY580. GF109203X with thapsigargin decreased TyrP of pY402PYK2 and the three FAK sites more than either inhibitor alone. Basal TyrP of pY397FAK was greater than other sites. These results demonstrate that CCK stimulates tyrosine phosphorylation of each of the three homologous phosphorylation sites in FAK and PYK2. However, CCK-stimulated TyrP at these sites differs in kinetics, magnitude, and participation of the high/low affinity receptor states and by protein kinase C and [Ca2+]i. These results show that phosphorylation of these different sites is differentially regulated and involves different intracellular mechanisms in the same cell.

Limk-2 activation by CCK in rat pancreatic acini

Limk-2 activation by CCK in rat pancreatic acini

Exogenous leptin inhibits the secretion of pancreatic juice via a duodenal CCK 1-vagal-dependent mechanism in anaesthetized rats

Leptin originally described as product of the ob gene has been shown to be expressed in various tissues including the gastrointestinal tract. In this study, we investigated the influence of leptin on the secretion of pancreatic juice in biliary–pancreatic duct cannulated anaesthetised rats and in dispersed rat pancreatic acini in vitro. Exogenous leptin was given in boluses intravenously with or without CCK-8 (12 pmol kg −1 body weight) in the presence or absence pharmacological CCK 1 receptor blockade, cervical vagotomy, and capsaicin pre-treatment. Administration of leptin (0.1, 1 and 10 μg kg −1 body weight) did not affect the volume of bile and pancreatic juice while the protein and trypsin outputs were reduced in a dose-dependent manner. In the rats, leptin inhibited CCK-8 stimulated protein and trypsin outputs stronger than the basal pancreatic secretion. The inhibition by leptin was abolished by the pharmacological CCK 1 receptor blockade, cervical vagotomy, and capsaicin pre-treatment. In contrast, leptin did not affect basal and CCK-8-stimulated amylase release from the dispersed rat pancreatic acini in vitro. In conclusion, the results of the present study suggest that leptin does not act directly on the rat pancreatic acinar cells but inhibits the secretion of pancreatic enzymes acting indirectly via a neurohormonal CCK-vagal-dependent mechanism.

Cyclic AMP agonists sensitize isolated rat pancreatic acini to cerulein induced zymogen activation

Cyclic AMP agonists sensitize isolated rat pancreatic acini to cerulein induced zymogen activation

Cholecystokinin-stimulated tyrosine phosphorylation of PKC-δ in pancreatic acinar cells is regulated bidirectionally by PKC activation

PKC-δ is important in cell growth, apoptosis, and secretion. Recent studies show its stability is regulated by tyrosine phosphorylation (TYR-P), which can be stimulated by a number of agents. Many of these stimuli also activate phospholipase C (PLC) cascades and little is known about the relationship between these cascades and PKC-δ TYR-P. Cholecystokinin (CCK) stimulates PKCs but it is unknown if it causes PKC-δ TYR-P and if so, the relationship between these cascades is unknown. In rat pancreatic acini, CCK-8 stimulated rapid PKC-δ TYR-P by activation of the low affinity CCK A receptor state. TPA had a similar effect. BAPTA did not decrease CCK-stimulated PKC-δ TYR-P but instead, increased it. A23187 did not stimulate PKC-δ TYR-P. Wortmannin and LY 294002 did not alter CCK-stimulated PKC-δ TYR-P. GF 109203X, at low concentrations, increased PKC-δ TYR-P stimulated by CCK or TPA and at higher concentrations, inhibited it. The cPKC inhibitors, Gö 6976 and safingol, caused a similar increase in TPA- and CCK-stimulated PKC-δ TYR-P. These results demonstrate that CCK A receptor activation causes PKC-δ TYR-P through activation of only one of its two receptor affinity states. This PKC-δ TYR-P is not directly influenced by changes in [Ca 2+] i; however, the resultant activation of PKC-α has an inhibitory effect. Therefore, CCK activates both stimulatory and inhibitory PKC cascades regulating PKC-δ TYR-P and, hence, likely plays an important role in regulating PKC-δ degradation and cellular abundance.

Protease-activated receptor-2 (PAR-2) in the pancreas and parotid gland: Immunolocalization and involvement of nitric oxide in the evoked amylase secretion

Protease-activated receptor-2, a G protein-coupled receptor activated by serine proteases such as trypsin, tryptase and coagulation factors VIIa and Xa, modulates pancreatic and salivary exocrine secretion. In the present study, we examined the distribution of PAR-2 in the pancreas and parotid gland, and characterized the PAR-2-mediated secretion of amylase by these tissues in vivo. Immunohistochemical analyses using the polyclonal antibody against rat PAR-2 clearly showed abundant expression of PAR-2 in rat pancreatic and parotid acini. The PAR-2 agonist SLIGRL-NH 2, administered intraperitoneally (i.p.) at 1–10 μmol/kg and 1.5–15 μmol/kg, in combination with amastatin, an aminopeptidase inhibitor, facilitated in vivo secretion of pancreatic and salivary amylase in a dose-dependent manner, respectively, in the mouse. The PAR-2-mediated secretion of pancreatic amylase was abolished by pretreatment with N G-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor. The secretion of salivary amylase in response to the PAR-2 agonist at a large dose, 15 μmol/kg, but not at a smaller dose, 5 μmol/kg, was partially reduced by L-NAME. Pretreatment with capsaicin for ablation of the sensory neurons did not modify the PAR-2-mediated secretion of pancreatic and salivary amylase in the mouse. In conclusion, our study demonstrates expression of PAR-2 in rat pancreatic acini as well as parotid acini and indicates that nitric oxide participates in the PAR-2-mediated in vivo secretion of pancreatic amylase, and, to a certain extent, of salivary amylase, although capsaicin-sensitive sensory neurons, known to be activated by PAR-2, are not involved in the evoked pancreatic or salivary amylase secretion.

Dibutyltin dichloride modifies amylase release from isolated rat pancreatic acini.

Dibutyltin dichloride (DBTC) is widely used as a stabilizer for polyvinylchloride plastics and is of particular toxicologic interest. To examine the effects of DBTC on pancreatic exocrine function in isolated rat pancreatic acini. Isolated rat pancreatic acini were incubated with various secretagogues in the presence or absence of DBTC. We investigated the effects of DBTC on amylase release, receptor binding, and protein kinase C (PKC) enzyme activity. DBTC reduced cholecystokinin octapeptide (CCK-8)-stimulated and carbamylcholine-stimulated amylase release and the binding of [(125)I]CCK-8 to isolated rat pancreatic acini. Conversely, DBTC potentiated secretin-stimulated amylase release, although it slightly inhibited [(125)I]secretin binding to its receptors. In addition, DBTC potentiated amylase release stimulated by vasoactive intestinal peptide, 8-bromoadenosine 3', 5'-monophosphate (8Br-cAMP) or calcium ionophore A23187, whereas it had no influence on amylase release stimulated by 12-O-tetradecanoylphorbol 13-acetate. The protein kinase C (PKC) inhibitor calphostin C abolished the DBTC-induced potentiation of amylase release stimulated by 8Br-cAMP or A23187. Moreover, DBTC caused a significant translocation of PKC enzyme activity from cytosol to membrane fraction. These results indicate that DBTC reduces CCK-8- and carbamylcholine-stimulated amylase release by inhibiting their receptor bindings to pancreatic acini, whereas it potentiates cAMP-mediated amylase release by activating PKC in isolated rat pancreatic acini.

Inhibitory effects of PGE(2) on K(+) currents and Ca(2+) oscillations in rat pancreatic acinar cells.

Prostaglandin E(2) (PGE(2)) inhibits pancreatic enzyme secretion and shows a protective action against pancreatitis. In this study, we tested the effects of PGE(2) on the slowly activating voltage-dependent K(+) channel current ( I(Ks)) and cholecystokinin (CCK)-induced oscillations of cytosolic [Ca(2+)] ([Ca(2+)](i)) in rat pancreatic acini (RPA). I(Ks) in RPA is reportedly augmented by both Ca(2+)- and cAMP-mediated secretagogues. PGE(2) (10(-7) M) decreased the amplitude of I(Ks), an effect that was more prominent following prior stimulation with secretin. The application of the membrane-permeable cAMP analogue 8-Br-cAMP prevented the effect of PGE(2) on I(Ks). The Ca(2+)-mediated augmentation of I(Ks) by ACh was unaffected by pretreatment with PGE(2). Using fura-2 fluorescence ratiometry to assess [Ca(2+)](i), CCK (<or=10(-10) M)-induced Ca(2+) oscillations were observed in RPAs. The amplitude of the Ca(2+) oscillations was decreased by PGE(2), irrespective of the presence of 8-Br-cAMP. RT-PCR analysis showed that RPAs express predominantly the EP3 subtype of the PGE(2) receptor and its splice variants. Enzyme-immunoassay showed that the secretin-induced production of cAMP in RPAs was inhibited by treatment with PGE(2). In summary, PGE(2) acts on the EP3 receptors to antagonize the cAMP-generating effect of secretin, resulting in the decrease of I(Ks). In addition, PGE(2) suppresses CCK-induced Ca(2+) oscillations in a cAMP-independent manner. These effects of PGE(2) may explain the inhibitory action mechanism of PGE(2) in the exocrine pancreas.

Caspase 8-mediated cleavage of plectin precedes F-actin breakdown in acinar cells during pancreatitis

Pancreatic acinar cells depend on the integrity of the cytoskeleton for regulated secretion. Stimulation of isolated rat pancreatic acini with the secretagogue CCK serves as a model for human acute edematous pancreatitis. It induces the breakdown of the actin filament system (F-actin) with the consecutive inhibition of secretion and premature activation of digestive enzymes. However, the mechanisms that regulate F-actin breakdown are largely unknown. Plectin is a versatile cytolinker protein regulating F-actin dynamics in fibroblasts. It was recently demonstrated that plectin is a substrate of caspase 8. In pancreatic acinar cells, plectin strongly colocalizes with apical and basolateral F-actin. Supramaximal secretory stimulation of acini with CCK leads to a rapid redistribution and activation of caspase 8, followed by degradation of plectin that in turn precedes the F-actin breakdown. Inhibition of caspase 8 before CCK hyperstimulation prevents plectin cleavage, stabilizes F-actin morphology, and reverses the inhibition of secretion. Thus we propose that the caspase 8-mediated degradation of plectin represents a critical biochemical event during CCK-induced secretory blockade and cell injury.

Stimulatory effects of bilirubin on amylase release from isolated rat pancreatic acini.

Considered to be an etiologic factor of acute pancreatitis, hypersecretion of pancreatic juice and digestive enzymes is often associated with hyperbilirubinemia. We explored the intracellular mechanisms through which bilirubin affects pancreatic exocrine secretory function by examining the effect of bilirubin on isolated rat pancreatic acini. Bilirubin stimulated amylase release in a concentration- and time-dependent manner, significantly increasing amylase release at concentrations >5 mg/100 ml and after 15 min of incubation. Coincubation of bilirubin with vasoactive intestinal polypeptide, 8-bromo-cAMP, or A-23187 had a synergistic effect on amylase release, whereas coincubation with CCK-8, carbamylcholine, or 12-O-tetradecanoylphorbol 13-acetate had an additive effect. Bilirubin did not affect acinar cAMP content or Ca(2+) efflux. Intracellular Ca(2+) pool depletion had no influence on bilirubin-evoked amylase release. The protein kinase C (PKC) inhibitors staurosporine and calphostin C partially but significantly inhibited bilirubin-stimulated amylase release, whereas the PKA inhibitor H-89 did not. The tyrosine kinase (TK) inhibitor genistein, phospholipase A(2) (PLA(2)) inhibitor indoxam, and PLC inhibitor U-73122 also inhibited amylase release. Bilirubin significantly translocated PKC activity from the cytosol to the membrane fraction and activated TK in cytosol and membrane fractions. These results indicate that bilirubin stimulates amylase release by activating PKC and TK in rat pancreatic acini and that PLC and PLA(2) partly mediate this process.

Characteristics and mechanism of enzyme secretion and increase in [Ca2+]i in Saikosaponin(I) stimulated rat pancreatic acinar cells.

This investigation was to reveal the characteristics and mechanism of enzyme secretion and increase in [Ca2+]i stimulated by saikosaponin(I) (SA(I)) in rat pancreatic acini. Pancreatic acini were prepared from male Wistar rats. Isolated acinar cells were suspended in Eagle's MEM solution. After adding drugs, the incubation was performed at 37 degrees for a set period of time. Amylase of supernatant was assayed using starch-iodide reaction. Isolated acinar single cell was incubated with Fura-2/AM at 37 degrees, then cells were washed and resuspended in fresh solution and attached to the chamber. Cytoplasm [Ca2+]i of a single cell was expressed by fluorescence ratio F340/F380 recorded in a Nikon PI Ca2+ measurement system. Rate course of amylase secretion stimulated by SA(I) in rat pancreatic acini appeared in bell-like shape. The peak amplitude increased depended on SA(I) concentration. The maximum rate responded to 1 x 10(-5)mol/L SA(I) was 13.1-fold of basal and the rate decreased to basal level at 30 min. CCK-8 receptor antagonist Bt(2)-cGMP markedly inhibited amylase secretion stimulated by SA(I) and the dose-effect relationship was similar to that by CCK-8. [Ca2+]i in a single acinar cell rose to the peak at 5 min after adding 5 x 10(-6)mol/L SA(I) and was 5.1-fold of basal level. In addition, there was a secondary increase after the initial peak. GDP could inhibit both the rate of amylase secretion and rising of [Ca2+]i stimulated by SA(I) in a single pancreatic acinar cell. SA(I) is highly efficient in promoting the secretion of enzymes synthesized in rat pancreatic acini and raising intracellular [Ca2+]i. Signaling transduction pathway of SA(I) involves activating special membrane receptor and increase in cytoplasm [Ca2+]i sequentially.