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Endoplasmic reticulum (ER) stress leads to the accumulation of misfolded proteins in the ER lumen and initiates the unfolded protein response (UPR). Components of the UPR are important in pancreatic development, and recent studies have indicated that the UPR is activated in the arginine model of acute pancreatitis. However, the effects of secretagogues on UPR components in the pancreas are unknown. The present study aimed to examine the effects of different types and concentrations of secretagogues on acinar cell function and specific components of the UPR. Rat pancreatic acini were stimulated with the CCK analogs CCK8 (10 pM-10 nM) or JMV- 180 (10 nM-10 [micro]M) or with bombesin (1-100 nM). Components of the UPR, including chaperone BiP expression, PKR-like ER kinase (PERK) phosphorylation, X box-binding protein 1 (XBP1) splicing, and CCAAT/ enhancer binding protein homologous protein (CHOP) expression, were measured, as were effects on amylase secretion and intracellular trypsin activation. CCK8 generated a biphasic secretion dose-response curve, and high concentrations increased intracellular active trypsin levels. In contrast, JMV-180 and bombesin secretion dose-response curves were monophasic, and high concentrations did not increase intracellular trypsin activity. All three secretagogues increased BiP levels and XBP1 splicing. However, only supraphysiological levels of CCK8 associated with inhibited amylase secretion and trypsin activation stimulated PERK phosphorylation and expression of CHOP. The effects of CCK8 on UPR components were rapid, occurring within 5-20 min. In conclusion, ER stress response mechanisms appear to be involved in both pancreatic physiology and pathophysiology, and future efforts should be directed at understanding the roles of these mechanisms in the pancreas. exocrine acini; pancreas doi:10.1152/ajpgi.00078.2007.

To investigate the role of transforming growth factor (TGF)-[beta] family signaling in the adult pancreas, a transgenic mouse (E-dnSmad4) was created that expresses a dominant-negative Smad4 protein driven by a fragment of the elastase promoter. Although E-dnSmad4 mice have normal growth, pancreas weight, and pancreatic exocrine and ductal histology, beginning at 4-6 wk of age, E-dnSmad4 mice show an age-dependent increase in the size of islets. In parallel, an expanded population of replicating cells expressing the E-dnSmad4 transgene is found in the stroma between the enlarged islets and pancreatic ducts. Despite the marked enlargement, E-dnSmad4 islets contain normal ratios and spatial organization of endocrine cell subtypes and have normal glucose homeostasis. Replication of cells derived from primary duct cultures of wild-type mice, but not E-dnSmad4 mice, was inhibited by the addition of TGF-[beta] family proteins, demonstrating a cell-autonomous effect of the transgene. These data show that, in the adult pancreas, TGF-[beta] family signaling plays a role in islet size by regulating the growth of a pluripotent progenitor cell residing in the periductal stroma of the pancreas. islet growth; diabetes; nestin; transgenic mice; elastase; transforming growth factor-[beta]

Endoplasmic reticulum (ER) stress mechanisms have been found to play critical roles in a number of diseases states, such as diabetes mellitus and Alzheimer disease, but whether they are involved in acute pancreatitis is unknown. Here we show for the first time that all major ER stress sensing and signaling mechanisms are present in exocrine acini and are activated early in the arginine model of experimental acute pancreatitis. Pancreatitis was induced in rats by intraperitoneal injection of 4.0 g/kg body wt arginine. Pancreatitis severity was assessed by analysis of serum amylase, pancreatic trypsin activity, water content, and histology. ER stress-related molecules PERK, eIF2[alpha], ATF6, XBP-1, BiP, CHOP, and caspase-12 were analyzed. Arginine treatment induced rapid and severe pancreatitis, as indicated by increased serum amylase, pancreatic tissue edema, and acinar cell damage within 4 h. Arginine treatment also caused an early activation of ER stress, as indicated by phosphorylation of PERK and its downstream target eIF2[alpha], ATF6 translocation into the nucleus (within 1 h), and upregulation of BiP (within 4 h). XBP-1 splicing and CHOP expression were observed within 8 h. After 24 h, increased activation of the ER stress-related proapoptotic molecule caspase-12 was observed along with an increase in caspase-3 activity and TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labeling (TUNEL) staining in exocrine acini. These results indicate that ER stress is an important early acinar cell event that likely contributes to the development of acute pancreatitis in the arginine model. pancreas; apoptosis doi:10.1152/ajpgi.00471.2005

G17 has growth promoting and antiapoptotic effects on the AR4-2J pancreatic acinar cell line. We previously reported that whereas MAPK regulates G17-stimulation of AR4-2J cell proliferation, Akt mediates the antiapoptotic action of G17. We examined the signal-transduction pathways mediating G17 stimulation of AR4-2J cell growth and survival. G17 activated the small GTP binding proteins Ras, Rac, Rho, and Cdc42. Transduction of the cells with adenoviral vectors expressing dominant negative Akt, Ras, Rho, and Cdc42 but not dominant negative Rac inhibited AR4-2J cell proliferation and survival. Both exoenzyme C3 from Clostridium botulinum (C3), a toxin known to inactivate Rho, and PD98059, a MAPK inhibitor, reversed G17 inhibition of AR4-2J cell apoptosis. G17 induction of Akt activation was reduced by >60% by both dominant negative Ras and Rho and by 30% by dominant negative Cdc42. In contrast, G17-stimulated MAPK activation was blocked by >80% by dominant negative Ras but not by dominant negative Rho and Cdc42. Similar results were observed in the presence of C3. Dominant negative Rac failed to affect G17 induction of both Akt and MAPK, whereas it inhibited sorbitol by almost 50% but not G17-stimulated activation of p38 kinase. Thus G17 promotes AR4-2J cell growth and survival through the activation of multiple GTP binding proteins, which, in turn, regulate different protein kinase cascades. Whereas Ras activates Akt and MAPK, Rho and Cdc42 appear to regulate Akt and possibly other as yet unidentified kinases mediating the growth-stimulatory actions of G17. cellular proliferation; protein kinases; phosphatidylinositol 3-kinase; Akt kinase; extracellular signal-related kinase

Research was conducted to examine the role of nuclear factor-kappa-B (NF-kappa-B) in cholecystokinin octapeptide (CCK-8)-induced mob-1 chemokine expression in pancreatic acinar cells in vitro. The relationship between the activation of NF-kappa-B and the expression of mob-1 in vitro was investigated. Results suggest that mob-1 chemokine gene expression is an early and specific response to CCK-8 hyperstimulation and that NF-kappa-B activation is required for the regulation of the chemokine gene in pancreatic acinar cells.

Experimental evidence shows that the gene transfer of the dominant-negative Ras molecule, Ras(super N17), suppresses the effects of cholecystokinin on pancreatic acinar cells. The mechanisms of adenovirus-mediated gene transfer were investigated in an experiment in which isolated acini were infected with different titers of either a control adenovirus or an adenoviral construct expressing Ras(super N17). Results showed that adenoviral infection induces the expression of the dominant-negative mutant Ras, which increases basal cell secretion but has no effect on cholecystokinin activation of extracellular-regulated kinase.

Mechanisms concerning the growth effects of gastrin were studied using model systems in which rat cholecystokinin-B (CCK-B) receptors were transfected into Chinese hamster ovary (CHO) and Swiss 3T3 cells. It was observed that the activation of CCK-B receptors inhibited the growth of CHO cells. On the other hand, the activation of CCK-B receptors stimulated the growth of Swiss 3T3 cells. In the two models, CCK-B receptor activation were significantly influenced by cellular conditions such that differences in growth responses could be attributed to factors other than differences in receptor coupling.

DNA synthesis in the acinar cells of rat pancreas increases due to interactions of cholecystokinin (CCK) with high- and low-affinity receptor states. This is achieved through the activation of two different sets of intracellular messengers with contrasting effects on amylase release. The ability of CCK to react with high- and low-affinity states explains the higher response to CCK octapeptide CCK-8 rather than the analogue JMV-180.

Smad4 mediates activation of mitogen-activated protein kinases by TGF-[Beta] in pancreatic acinar cells. Am J Physiol Cell Physiol 281: C311-C319, 2001.--Transforming growth factor-[Beta] (TGF-[Beta]) inhibits pancreatic acinar cell growth. In many cell types, TGF-[Beta] mediates its growth inhibitory effects by activation of Smad proteins. Recently, it has been reported that Smad proteins may interact with the mitogen-activated protein (MAP) kinase signaling pathways. In this study, we report on the interactions between the TGF-[Beta] and MAP kinase signaling pathways in isolated rat pancreatic acinar cells. TGF-[Beta] activated the MAP kinases extracellular signal-related kinases (ERKs) and p38 in pancreatic acinar cells, but had no effect on c-jun [NH.sub.2]-terminal kinase activity. Activation of MAP kinase by TGF-[Beta] was maximal 4 h after treatment. The ability of TGF-[Beta] to activate ERKs was concentration dependent and dependent on protein synthesis. TGF-[Beta]'s stimulation of ERK activation was blocked by PD-98059, an inhibitor of MAP kinase kinase 1, and by adenoviral transfer of dominant negative [Ras.sup.N17]. Furthermore, adenoviral-mediated expression of dominant negative Smad4 blocked the ability of TGF-[Beta] to activate acinar cell MAP kinase, demonstrating that this activation is downstream of Smads. The biological relevance of ERK activation by TGF-[Beta] was indicated by demonstrating that inhibition of ERK signaling by PD-98059 blocked the ability of TGF-[Beta] to activate the transcription factor activator protein-1. These studies provide new insight into the signaling mechanisms by which TGF-[Beta] mediates biological actions in pancreatic acinar cells. transforming growth factor-[Beta]

Transforming growth factor-[Beta] (TGF-[Beta]) is a potent inhibitor of pancreatic acinar cell growth. Smad4 is a central mediator in the TGF-[Beta] signaling pathway. To study the effect of Smad4 on pancreatic growth, cell cycle protein expression, and the expression of a TGF-[Beta]-responsive promoter in vitro, we constructed an adenovirus containing dominant-negative COOH terminal truncated Smad4 (AddnSmad4) downstream of the rat elastase promoter. Acinar cells expressed dominant-negative Smad4 within 8 h after infection, and expression persisted for 72 h. Mouse pancreatic acini were infected with either AddnSmad4 or control adenovirus expressing green fluorescent protein, and TGF-[Beta] was added 8 h after infection. Acinar cells were then incubated for 1, 2, or 3 days, and [[sup.3]H]thymidine incorporation was determined. AddnSmad4 significantly reduced TGF-[Beta] inhibition of [sup.3H]thymidine incorporation, with maximal effects on day 3. AddnSmad4 also completely blocked TGF-[Beta]-mediated growth inhibition in the presence of basic fibroblast growth factor. We next examined the effects of AddnSmad4 on TGF-[Beta]-induced expression of the cell cycle regulatory proteins [p21.sup.Cip1] and [p27.sup.Kip1]. TGF-[Beta] induced upregulation of [p21.sup.Ccip1], which was completely blocked by AddnSmad4. AddnSmad4 also inhibited TGF-[Beta] induced expression of the TGF-[Beta]-responsive luciferase reporter 3TP-Lux. These results show that Smad4 is essential in TGF-[Beta]-mediated signaling in pancreatic acinar cells. growth regulation; pancreas; cell cycle; Smad proteins

Han, Bing, Baoan Ji, and Craig D. Logsdon. CCK independently activates intracellular trypsinogen and NF-[Kappa]B in rat pancreatic acinar cells. Am J Physiol Cell Physiol 49: C465-C472, 2001.--In the cholecystokinin (CCK) hyperstimulation model of acute pancreatitis, two early intracellular events, activation of trypsinogen and activation of nuclear factor-[Kappa]B (NF-[Kappa]B), are thought to be important in the development of the disease. In this study, the relationship between these two events was investigated. NF-[Kappa]B activity was monitored by using a DNA binding assay and mob-1 chemokine gene expression. Intracellular trypsin activity was measured by using a fluorogenic substrate. Protease inhibitors including FUT-175, Pefabloc, and E-64d prevented CCK stimulation of intracellular trypsinogen and NF-[Kappa]B activation. Likewise, the NF-[Kappa]B inhibitors pyrrolidine dithiocarbamate and N-acetyl-L-cysteine inhibited CCK stimulation of NF-[Kappa]B and intracellular trypsinogen activation. These results suggested a possible codependency of these two events. However, CCK stimulated NF-[Kappa]B activation in Chinese hamster ovary-[CCK.sub.A] cells, which do not express trypsinogen, indicating that trypsin is not necessary for CCK activation of NF-[Kappa]B. Furthermore, adenovirus-mediated expression in acinar cells of active p65 subunits to stimulate NF-[Kappa]B, or of inhibitory [Kappa]B-,[Alpha] molecules to inhibit NF-[Kappa]B, did not affect either basal or CCK-mediated trypsinogen activation. Thus trypsinogen and NF-[Kappa]B activation are independent events stimulated by CCK. pancreatitis; inflammation; cholecystokinin; nuclear factor-[Kappa]B

Han, Bing, and Craig D. Logsdon. CCK stimulates mob-1 expression and NF-[Kappa]B activation via protein kinase C and intracellular [Ca.sup.2+]. Am. J. Physiol. Cell Physiol. 278: C344-C351, 2000.--Supraphysiological concentrations of oholecystokinin (CCK) induce chemokine expression in rat pancreatic acini through the activation of the transcription factor NF-[Kappa]B. In the current study, the intracellular signals involved in these pathophysiological effects of CCK were investigated. CCK induction of mob-1 expression in isolated rat pancreatic acini was blocked by the protein kinase C (PKC) inhibitors GF-109203X and Ro-32-0432 and by the intracellular [Ca.sup.2+] chelator BAPTA. CCK induced NF-[Kappa]B nuclear translocation, and DNA binding was also blocked by GF109203X and BAPTA. Direct activation of PKC with TPA induced mob-1 chemokine expression and activated NF-[Kappa]B DNA binding to a similar extent as did CCK. Increasing intracellular [Ca.sup.2+] using ionomycin had no effect on mob-1 mRNA levels or NF-[Kappa]B activity. Both CCK and TPA treatments decreased inhibitory [Kappa]B-[Alpha] (I[Kappa]B-[Alpha]) levels, whereas ionomycin had no effect. However, the effects of TPA on I[Kappa]B-[Alpha] degradation were less complete than for CCK. In combination, TPA and ionomycin degraded I[Kappa]B-[Alpha] to a similar extent as CCK. Therefore, activation of NF-[Kappa]B and mob-1 expression by supraphysiological CCK is likely mediated by both PKC activation and elevated intracellular [Ca.sup.2+]. pancreas; acinar cells; pancreatitis; chemokine; cholecystokinin; nuclear factor-[Kappa]B