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Background & Aims: Oncogenic Kras G12D induces neoplastic transformation of pancreatic acinar cells through acinar-to-ductal metaplasia (ADM), an actin-based morphogenetic process, and drives pancreatic ductal adenocarcinoma (PDAC). mTOR (mechanistic target of rapamycin kinase) complex 1 (mTORC1) and 2 (mTORC2) contain Rptor and Rictor, respectively, and are activated downstream of Kras G12D , thereby contributing to PDAC. Yet, whether and how mTORC1 and mTORC2 impact on ADM and the identity of the actin nucleator(s) mediating such actin rearrangements remain unknown., Methods: A mouse model of inflammation-accelerated Kras G12D -driven early pancreatic carcinogenesis was used. Rptor, Rictor, and Arpc4 (actin-related protein 2/3 complex subunit 4) were conditionally ablated in acinar cells to deactivate the function of mTORC1, mTORC2 and the actin-related protein (Arp) 2/3 complex, respectively., Results: We found that mTORC1 and mTORC2 are markedly activated in human and mouse ADM lesions, and cooperate to promote Kras G12D -driven ADM in mice and in vitro. They use the Arp2/3 complex as a common downstream effector to induce the remodeling the actin cytoskeleton leading to ADM. In particular, mTORC1 regulates the translation of Rac1 (Rac family small GTPase 1) and the Arp2/3-complex subunit Arp3, whereas mTORC2 activates the Arp2/3 complex by promoting Akt/Rac1 signaling. Consistently, genetic ablation of the Arp2/3 complex prevents Kras G12D -driven ADM in vivo. In acinar cells, the Arp2/3 complex and its actin-nucleation activity mediated the formation of a basolateral actin cortex, which is indispensable for ADM and pre-neoplastic transformation., Conclusions: Here, we show that mTORC1 and mTORC2 attain a dual, yet nonredundant regulatory role in ADM and early pancreatic carcinogenesis by promoting Arp2/3 complex function. The role of Arp2/3 complex as a common effector of mTORC1 and mTORC2 fills the gap between oncogenic signals and actin dynamics underlying PDAC initiation., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Molecular signalling mediated by the phosphatidylinositol-3-kinase (PI3K)-Akt axis is a key regulator of cellular functions. Importantly, alteration of the PI3K-Akt signalling underlies the development of different human diseases, thus prompting the investigation of the pathway as a molecular target for pharmacologic intervention. In this regard, recent studies showed that small molecule inhibitors of PI3K, the upstream regulator of the pathway, reduced the development of inflammation during acute pancreatitis, a highly debilitating and potentially lethal disease. Here we investigated whether a specific reduction of Akt activity, by using either pharmacologic Akt inhibition, or genetic inactivation of the Akt1 isoform selectively in pancreatic acinar cells, is effective in ameliorating the onset and progression of the disease. We discovered that systemic reduction of Akt activity did not protect the pancreas from initial damage and only transiently delayed leukocyte recruitment. However, reduction of Akt activity decreased acinar proliferation and exacerbated acinar-to-ductal metaplasia (ADM) formation, two critical events in the progression of pancreatitis. These phenotypes were recapitulated upon conditional inactivation of Akt1 in acinar cells, which resulted in reduced expression of 4E-BP1, a multifunctional protein of key importance in cell proliferation and metaplasia formation. Collectively, our results highlight the critical role played by Akt1 during the development of acute pancreatitis in the control of acinar cell proliferation and ADM formation. In addition, these results harbour important translational implications as they raise the concern that inhibitors of PI3K-Akt signalling pathways may negatively affect the regeneration of the pancreas. Finally, this work provides the basis for further investigating the potential of Akt1 activators to boost pancreatic regeneration following inflammatory insults. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (© 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Pancreatic cancer remains a daunting foe despite a vast number of accumulating molecular analyses regarding the mutation and expression status of a variety of genes. Indeed, most pancreatic cancer cases uniformly present with a mutation in the KRAS allele leading to enhanced RAS activation. Yet our understanding of the many epigenetic/environmental factors contributing to disease incidence and progression is waning. Epidemiologic data suggest that diet may be a key factor in pancreatic cancer development and potentially a means of chemoprevention at earlier stages. While diets high in ω3 fatty acids are typically associated with tumor suppression, diets high in ω6 fatty acids have been linked to increased tumor development. Thus, to better understand the contribution of these polyunsaturated fatty acids to pancreatic carcinogenesis, we modeled early stage disease by targeting mutant KRAS to the exocrine pancreas and administered diets rich in these fatty acids to assess tumor formation and altered cell-signaling pathways. We discovered that, consistent with previous reports, the ω3-enriched diet led to reduced lesion penetrance via repression of proliferation associated with reduced phosphorylated AKT (pAKT), whereas the ω6-enriched diet accelerated tumor formation. These data provide a plausible mechanism underlying previously observed effects of fatty acids and suggest that administration of ω3 fatty acids can reduce the pro-survival, pro-growth functions of pAKT. Indeed, counseling subjects at risk to increase their intake of foods containing higher amounts of ω3 fatty acids could aid in the prevention of pancreatic cancer.

Background & Aims: Intraductal papillary mucinous neoplasias (IPMNs) are precancerous cystic lesions that can develop into pancreatic ductal adenocarcinomas (PDACs). These large macroscopic lesions are frequently detected during medical imaging, but it is unclear how they form or progress to PDAC. We aimed to identify cells that form IPMNs and mutations that promote IPMN development and progression., Methods: We generated mice with disruption of Pten specifically in ductal cells (Sox9CreER T2 ;Pten flox/flox ;R26R YFP or Pten ΔDuct/ΔDuct mice) and used Pten ΔDuct/+ and Pten +/+ mice as controls. We also generated Kras G12D ;Pten ΔDuct/ΔDuct and Kras G12D ;Pten ΔDuct/+ mice. Pancreata were collected when mice were 28 weeks to 14.5 months old and analyzed by histology, immunohistochemistry, and electron microscopy. We performed multiplexed droplet digital polymerase chain reaction to detect spontaneous Kras mutations in Pten ΔDuct/ΔDuct mice and study the effects of Ras pathway activation on initiation and progression of IPMNs. We obtained 2 pancreatic sections from a patient with an invasive pancreatobiliary IPMN and analyzed the regions with and without the invasive IPMN (control tissue) by immunohistochemistry., Results: Mice with ductal cell-specific disruption of Pten but not control mice developed sporadic, macroscopic, intraductal papillary lesions with histologic and molecular features of human IPMNs. Pten ΔDuct/ΔDuct mice developed IPMNs of several subtypes. In Pten ΔDuct/ΔDuct mice, 31.5% of IPMNs became invasive; invasion was associated with spontaneous mutations in Kras. Kras G12D ;Pten ΔDuct/ΔDuct mice all developed invasive IPMNs within 1 month. In Kras G12D ;Pten ΔDuct/+ mice, 70% developed IPMN, predominately of the pancreatobiliary subtype, and 63.3% developed PDAC. In all models, IPMNs and PDAC expressed the duct-specific lineage tracing marker yellow fluorescent protein. In immunohistochemical analyses, we found that the invasive human pancreatobiliary IPMN tissue had lower levels of PTEN and increased levels of phosphorylated (activated) ERK compared with healthy pancreatic tissue., Conclusions: In analyses of mice with ductal cell-specific disruption of Pten, with or without activated Kras, we found evidence for a ductal cell origin of IPMNs. We also showed that PTEN loss and activated Kras have synergistic effects in promoting development of IPMN and progression to PDAC., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Acinar-to-ductal metaplasia (ADM) is a reversible epithelial transdifferentiation process that occurs in the pancreas in response to acute inflammation. ADM can rapidly progress towards pre-malignant pancreatic intraepithelial neoplasia (PanIN) lesions in the presence of mutant KRas and ultimately pancreatic adenocarcinoma (PDAC). In the present work, we elucidate the role and related mechanism of glycogen synthase kinase-3beta (GSK-3β) in ADM development using in vitro 3D cultures and genetically engineered mouse models. We show that GSK-3β promotes TGF-α-induced ADM in 3D cultured primary acinar cells, whereas deletion of GSK-3β attenuates caerulein-induced ADM formation and PanIN progression in Kras G12D transgenic mice. Furthermore, we demonstrate that GSK-3β ablation influences ADM formation and PanIN progression by suppressing oncogenic KRas-driven cell proliferation. Mechanistically, we show that GSK-3β regulates proliferation by increasing the activation of S6 kinase. Taken together, these results indicate that GSK-3β participates in early pancreatitis-induced ADM and thus could be a target for the treatment of chronic pancreatitis and the prevention of PDAC progression. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

The mechanism by which pancreas secretes high HCO3- has not been fully resolved. This alkaline secretion, formed in pancreatic ducts, can be achieved by transporting HCO3- from serosa to mucosa or by moving H+ in the opposite direction. The aim of the present study was to determine whether H+/K+-ATPases are expressed and functional in human pancreatic ducts and whether proton pump inhibitors (PPIs) have effect on those. Here we show that the gastric HKα1 and HKβ subunits (ATP4A; ATP4B) and non-gastric HKα2 subunits (ATP12A) of H+/K+-ATPases are expressed in human pancreatic cells. Pumps have similar localizations in duct cell monolayers (Capan-1) and human pancreas, and notably the gastric pumps are localized on the luminal membranes. In Capan-1 cells, PPIs inhibited recovery of intracellular pH from acidosis. Furthermore, in rats treated with PPIs, pancreatic secretion was inhibited but concentrations of major ions in secretion follow similar excretory curves in control and PPI treated animals. In addition to HCO3-, pancreas also secretes K+. In conclusion, this study calls for a revision of the basic model for HCO3- secretion. We propose that proton transport is driving secretion, and that in addition it may provide a protective pH buffer zone and K+ recirculation. Furthermore, it seems relevant to re-evaluate whether PPIs should be used in treatment therapies where pancreatic functions are already compromised.

Background/aims: Main pancreatic duct dilatation raises concerns about the possibility of pancreatobiliary malignancy. We evaluated the etiologic yield of endosonography (EUS) for main pancreatic duct dilatation without definite pathology on Ultrasonography (US)., Methodology: A retrospective review was conducted in 54 consecutive patients referred for EUS., Results: No pathological finding (37.0%, 20/54), followed by periampullary cancer (35.2%, 19/54), was the most common finding. Elevated alkaline phosphatase (ALK-P) and marked common bile duct (CBD) dilatation (≥ 12 mm) were the predictors of malignancy (p < 0.05). Among the 37 subjects with available ALK-P and CBD diameter, the probability of malignancy was 84.6% (11/13) for both elevated ALK-P and marked CBD dilatation, 16.7% (1/6) for isolated elevated ALK-P, 18.2% (2/11) for isolated marked CBD dilatation, and none (0/7) was for subjects with neither elevated ALK-P nor marked CBD dilatation, respectively. The overall accuracy of EUS for periampullary carcinomas was 94.7% (18/19) and for choledocholithiasis was 100% (7/7), respectively. EUS had a 100.0% (20/20) sensitivity and a 97.1% (33/34) specificity in the diagnosis of no pathological obstruction., Conclusions: EUS is accurate for main pancreatic duct dilatation without definite pathology on US, and the presence of concomitant elevated ALK-P and CBD dilatation highly suggests malignancy.

Objectives: Cigarette smoking is a major risk factor for pancreatic cancer (PaCa). However, the mechanisms of smoking-induced PaCa remain unknown. Here we investigated the effect of smoking compounds on cell death pathways in pancreatic ductal cells, precursors of PaCa., Methods: Human pancreatic ductal cells (HPDE6-c7) were cultured with cigarette smoke extract (CSE) or smoking compound 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Apoptosis and autophagy were assessed by DNA fragmentation and immunofluorescence, respectively., Results: Exposure to CSE or NNK decreased DNA fragmentation and up-regulated BclXL. Akt kinase was activated by smoking compounds through reactive oxygen species-dependent mechanism. Specifically, Akt activation was prevented by inhibition of nicotinamide adenine dinucleotide oxidase. Molecular or pharmacologic inhibitions of Akt prevented the antiapoptotic effect of smoking compounds. Smoking compounds stimulated rapid (1 hour) and transient activation of 5'-adenosine monophosphate-activated protein kinase and formation of autophagic vacuoles, indicating stimulation of autophagy. Repeated exposure to CSE/NNK (48 hours or longer) abolished the early activation of autophagic markers. Inhibition of Akt prevented the antiautophagic effect of long exposure to smoking compounds, indicating that smoking-induced late activation of Akt prevents autophagy., Conclusions: Long exposure of pancreatic ductal cells to smoking compounds inhibited apoptosis and autophagy. The results revealed a central role for Akt kinase in mediating key procarcinogenic effects of smoking compounds.

Pancreas secretes fluid rich in digestive enzymes and bicarbonate. The alkaline secretion is important in buffering of acid chyme entering duodenum and for activation of enzymes. This secretion is formed in pancreatic ducts, and studies to date show that plasma membranes of duct epithelium express H(+)/HCO(3)(-) transporters, which depend on gradients created by the Na(+)/K(+)-ATPase. However, the model cannot fully account for high-bicarbonate concentrations, and other active transporters, i.e. pumps, have not been explored. Here we show that pancreatic ducts express functional gastric and non-gastric H(+)-K(+)-ATPases. We measured intracellular pH and secretion in small ducts isolated from rat pancreas and showed their sensitivity to H(+)-K(+) pump inhibitors and ion substitutions. Gastric and non-gastric H(+)-K(+) pumps were demonstrated on RNA and protein levels, and pumps were localized to the plasma membranes of pancreatic ducts. Quantitative analysis of H(+)/HCO(3)(-) and fluid transport shows that the H(+)-K(+) pumps can contribute to pancreatic secretion in several species. Our results call for revision of the bicarbonate transport physiology in pancreas, and most likely other epithelia. Furthermore, because pancreatic ducts play a central role in several pancreatic diseases, it is of high relevance to understand the role of H(+)-K(+) pumps in pathophysiology.

Noxious stimuli cause prompt phosphorylation of extracellular signal-regulated kinase (ERK) in the spinal dorsal horn that contributes to facilitation of pain sensation and is often used as an immediate marker for excitation of spinal neurons following somatic and colonic nociception. Here we asked whether two distinct pronociceptive stimuli with proteinase-activated receptor-2 (PAR2) agonists and hydrogen sulfide (H(2)S) in the pancreas cause phosphorylation of ERK in the spinal dorsal horn and also examined involvement of their possible downstream signaling molecules, transient receptor potential vanilloid-1 (TRPV1) and T-type Ca(2+) channels, respectively. Capsaicin (a TRPV1 agonist), trypsin (an endogenous PAR2 agonist), SLIGRL-NH(2) (a PAR2-activating peptide), and NaHS (an H(2)S donor) were infused into the pancreatic duct in anesthetized rats, and phosphorylated ERK in the spinal cord was detected by immunohistochemistry. Intraductal administration of capsaicin and trypsin caused prompt phosphorylation of ERK in the superficial layers of T9, but not T5 or T12, spinal dorsal horn. SLIGRL-NH(2) and NaHS, administered in the same manner, also produced ERK phosphorylation in the corresponding spinal regions. Mibefradil, a T-type Ca(2+) channel blocker, abolished the phosphorylation of ERK caused by intraductal NaHS but not SLIGRL-NH(2). In contrast, capsazepine, an inhibitor of TRPV1, suppressed the phosphorylation of ERK caused by intraductal SLIGRL-NH(2) but not NaHS. Our data thus demonstrate that pancreatic pronociceptive stimuli with PAR2 agonists and H(2)S cause ERK phosphorylation in the spinal dorsal horn, through activation of TRPV1 and T-type Ca(2+) channels, respectively, and that those two pronociceptive pathways are independent of each other.

Tricellulin (TRIC) is a tight junction protein at tricellular contacts where three epithelial cells meet, and it is required for the maintenance of the epithelial barrier. To investigate whether TRIC is regulated via a c-Jun N-terminal kinase (JNK) pathway, human pancreatic HPAC cells, highly expressed at tricellular contacts, were exposed to various stimuli such as the JNK activators anisomycin and 12-O-tetradecanoylphorbol 13-acetate (TPA), and the proinflammatory cytokines IL-1β, TNFα, and IL-1α. TRIC expression and the barrier function were moderated by treatment with the JNK activator anisomycin, and suppressed not only by inhibitors of JNK and PKC but also by siRNAs of TRIC. TRIC expression was induced by treatment with the PKC activator TPA and proinflammatory cytokines IL-1β, TNFα, and IL-1α, whereas the changes were inhibited by a JNK inhibitor. Furthermore, in normal human pancreatic duct epithelial cells using hTERT-transfected primary cultured cells, the responses of TRIC expression to the various stimuli were similar to those in HPAC cells. TRIC expression in tricellular tight junctions is strongly regulated together with the barrier function via the JNK transduction pathway. These findings suggest that JNK may be involved in the regulation of tricellular tight junctions including TRIC expression and the barrier function during normal remodeling of epithelial cells, and prevent disruption of the epithelial barrier in inflammation and other disorders in pancreatic duct epithelial cells., (© 2010 Wiley-Liss, Inc.)

Pancreatic ductal epithelial cells (PDECs) were induced to differentiate into insulin-producing cells by hepatocyte growth factor (HGF) in our previous study, but the mechanism through which this induction occurs is still unknown. HGF is a ligand that activates a tyrosine kinase encoded by the c-Met proto-oncogene. This activation is followed by indirect activation of multiple downstream signal transduction pathways (including MAPKs and the PI3K/AKT signaling pathways) that initiate various biological effects. Therefore, we speculated that the differentiation of PDECs is through either the MAPK signaling pathway or the PI3K/AKT signaling pathway. To test this hypothesis, isolated PDECs from adult rats were stimulated by adding HGF to their medium for 28days. Then, the expression levels of several protein kinases, including MAPKs (ERK1/2, p38, and JNK) and AKT, were determined by Western blotting to determine if specific protein kinases are activated in these pathways. Subsequently, re-isolated from adult rats and cultured PDECs were pre-treated with specific inhibitors of proteins shown to be activated in these signaling pathways; these cells were then induced to differentiate by the addition of HGF. The expression levels of protein kinases were determined by Western blotting, and the differentiation rate of insulin-positive cells was determined by flow cytometry. The change of PDEC differentiation rates were compared between the groups in which cells with or without inhibitors pretreatment to determine the specific signaling pathway(s) that may be involved in HGF-induced differentiation of PDECs. After isolating PDECs and stimulating them with HGF for 28days, the expression levels of phosphorylated ERK1/2 as well as total and phosphorylated AKT of cultured cells were significantly increased compared to the normal control group (p<0.05), suggesting that the signaling pathways involving ERK1/2 and Akt (MEK-ERK and PI3K-AKT) are activated during HGF-induced PDEC differentiation. MEK1/2 or PI3K inhibitors were separately added to the culture medium of PDECs pre-treated with HGF. These results show that compared to the HGF-treated group, the differentiation rate of insulin-positive cells was significantly decreased in the HGF/LY294002 (PI3K inhibitor) group (13.47+/-1.57% vs. 33.47+/-1.34%, p<0.05); however, the differentiation rate of insulin-positive cells was not significantly different in the HGF/PD98059 (MEK1/2 inhibitor) group. These data suggest that HGF induces PDECs to differentiate into insulin-producing cells through the PI3K/AKT signaling pathway., (Copyright 2010 Elsevier Inc. All rights reserved.)

Aim: To perform a comparative analysis of clinicopathological correlations of cyclooxygenase-2 (COX-2) expression in pancreatic cancer, examined by monoclonal and polyclonal antibodies., Methods: The COX-2 expression in 85 resection specimens of pancreatic ductal adenocarcinoma was immunohistochemically examined using both monoclonal and polyclonal antibodies. The final immunoscores were obtained by multiplying the percentage of positive cells with the numeric score reflecting the staining intensity. COX-2 expression levels were classified into three categories (0, 1+, and 2+) and the clinicopathological correlations were statistically evaluated and analyzed., Results: The positive tumor expression rates of COX-2 were 80.5% using monoclonal antibody and 69.4% using polyclonal antibody. In the Kaplan-Meier analysis, no significant correlations were found between levels of COX-2 expression and overall survival (OS), but trends to longer OS were found in COX-2 negative cases using monoclonal antibody. Significantly longer disease free survival was revealed in COX-2 negative cases using monoclonal antibody (P = 0.019). No correlations between COX-2 expression levels and grade (G), tumor (T) status and nodal (N) status were demonstrated. Low histological grade showed a strong association with a longer OS (P < 0.001). Correlation of survival and T status revealed a shorter OS in T3 tumors, but the results reached only marginal statistical significance (P = 0.070). In the multivariate Cox proportional hazards regression model, histological grade, T and N status remained valuable predictors of a worse survival with borderline significance for T [hazards ratio (HR) = 4.18 for G (if G = 3, P < 0.001); HR = 1.64 for T (if T = 3, P = 0.065); HR = 2.53 for N (if N = 1, P = 0.006)]. Higher grade, T or N status was associated with a worse OS., Conclusion: The immunohistochemically assessed level of COX-2 expression does not seem to represent a valuable independent prognostic factor and is not superior to the conventional prognostic factors.

The gelatinases B (MMP9) and A (MMP2) are two members of the matrix metalloproteinase (MMPs) family that are expressed in human cancer, and play a critical role in tumor cell invasion and metastasis. Caveolin-1 (Cav1) has recently been identified as a tumor metastasis modifier gene. However, the effect and mechanism of Cav1 in pancreatic carcinoma cell invasion remain unknown. In this study, we investigated the expression of Cav1, MMP2, and MMP9 in several different pancreatic carcinoma cell lines. We transfected pcDNA3.0-Cav1 plasmid and Cav1 siRNA into SW1990 and Bxpc3 cells, respectively. Using cell invasion assay, we found that overexpression of Cav1 inhibited cell invasion, whereas the knockdown of Cav1 in Bxpc3 cells promoted cell invasion. Moreover, to explore the mechanisms underlying these observations, we further investigated the expression of MMP2, MMP9, phospho-Akt, and phospho-Erk by Western blot, and the activities of MMP2 and MMP9 by gelatin zymography. The results indicated that Cav1 gene could inhibit pancreatic carcinoma cell invasion, at least in part, probably through Erk-MMP signal pathway, suggesting that the endogenous expression or re-expression of Cav1 might help therapeutically reduce their invasive potential in pancreatic carcinoma cells., (Copyright (c) 2010. Published by Elsevier Inc. All rights reserved.)

Matriptase, also known as MT-SP1, is a type II transmembrane serine protease strongly implicated in both the development and progression of a variety of epithelial cancers. Evidence comes from studies of its expression in human cancers and from mouse models of spontaneous cancer. Matriptase is considered to be a major activator of two key stimulators of invasive growth, namely hepatocyte growth factor/scatter factor and urokinase-type plasminogen activator. The aim of this study was to examine the role of matriptase in pancreatic ductal adenocarcinoma by expression analysis and functional assays in vitro. Immunohistochemical analysis of matriptase performed on microtissue arrays and large samples of 55 pancreatic ductal adenocarcinomas and on 31 samples of normal pancreatic ducts revealed that although matriptase expression differed greatly in both malignant and normal ductal pancreatic tissue, matriptase scores were significantly (p=0.02) elevated in pancreatic ductal adenocarcinoma compared to normal pancreatic ducts. To evaluate the role of matriptase during development of pancreatic cancer, we studied the effects of newly designed matriptase inhibitors on the processing of the zymogen of urokinase-type plasminogen activator in the human adenocarcinoma cell lines AsPC-1 and BxPC-3. In both cell lines, at 1 microM, all matriptase inhibitors completely prevented zymogen activation. At lower inhibitor concentrations, the degree of inhibition of zymogen processing correlated with the affinities of the inhibitors towards matriptase indicating that this is a specific result of matriptase inhibition. Furthermore, matriptase inhibitors reduced the phosphorylation of the HGF receptor/cMet and the overall cellular invasiveness of the human pancreatic adenocarcinoma cell line AsPC-1. Our findings demonstrate for the first time that matriptase may be involved in the progression of pancreatic ductal adenocarcinoma and that matriptase inhibition may contribute to preventing the progression of this devastating disease.

The regenerative process in the pancreas is of particular interest because diabetes results from an inadequate number of insulin-producing beta cells and pancreatic cancer may arise from the uncontrolled growth of progenitor/stem cells. Continued and substantial growth of islet tissue occurs after birth in rodents and humans, with additional compensatory growth in response to increased demand. In rodents there is clear evidence of pancreatic regeneration after some types of injury, with proliferation of preexisting differentiated cell types accounting for some replacement. Additionally, neogenesis or the budding of new islet cells from pancreatic ducts has been reported, but the existence and identity of a progenitor cell have been debated. We hypothesized that the progenitor cells are duct epithelial cells that after replication undergo a regression to a less differentiated state and then can form new endocrine and exocrine pancreas. To directly test whether ductal cells serve as pancreatic progenitors after birth and give rise to new islets, we generated transgenic mice expressing human carbonic anhydrase II (CAII) promoter: Cre recombinase (Cre) or inducible CreER(TM) to cross with ROSA26 loxP-Stop-loxP LacZ reporter mice. We show that CAII-expressing cells within the pancreas act as progenitors that give rise to both new islets and acini normally after birth and after injury (ductal ligation). This identification of a differentiated pancreatic cell type as an in vivo progenitor of all differentiated pancreatic cell types has implications for a potential expandable source for new islets for replenishment therapy for diabetes.

During pancreas development, transcription factors play critical roles in exocrine and endocrine differentiation. Transcriptional regulation in eukaryotes occurs within chromatin and is influenced by posttranslational histone modifications (e.g., acetylation) involving histone deacetylases (HDACs). Here, we show that HDAC expression and activity are developmentally regulated in the embryonic rat pancreas. We discovered that pancreatic treatment with different HDAC inhibitors (HDACi) modified the timing and determination of pancreatic cell fate. HDACi modified the exocrine lineage via abolition and enhancement of acinar and ductal differentiation, respectively. Importantly, HDACi treatment promoted the NGN3 proendocrine lineage, leading to an increased pool of endocrine progenitors and modified endocrine subtype lineage choices. Interestingly, treatments with trichostatin A and sodium butyrate, two inhibitors of both class I and class II HDACs, enhanced the pool of beta cells. These results highlight the roles of HDACs at key points in exocrine and endocrine differentiation. They show the powerful use of HDACi to switch pancreatic cell determination and amplify specific cellular subtypes, with potential applications in cell replacement therapies in diabetes.

Objectives: To further explore the oncogenic activity of Aurora A kinase while attempting to develop a useful mouse model for pancreatic cancer, Aurora A kinase was targeted to pancreatic duodenal homeobox gene-1 (Pdx-1)-positive cells., Methods: Aurora A kinase overexpression was targeted to mouse pancreas tissues using the Pdx-1 promoter in a transgenic model. The pancreas tissues of 7- to 11-month-old transgenic animals were evaluated for metastatic adenocarcinomas, preinvasive ductal neoplasia, or other histological anomalies., Results: Examination of pancreatic tissue from Pdx-1-Aurora A transgenic mice revealed abnormalities, such as mild islet cell hyperplasia, lymphocytic infiltration, and general dysplasia between ductal/islet cell interfaces. However, most tissues from these transgenic mice were normal., Conclusions: The overexpression of Aurora A can potentially initiate the development of mild abnormalities in pancreatic tissue; however, neither preinvasive ductal neoplasia nor fully metastatic adenocarcinomas were observed. Combining the Pdx-1-Aurora A transgenic model with other genetic alterations may provide additional insight.

The NBT-PABA test is an established method for diagnosis of pancreatic exocrine insufficiency. In the present study the NBT-PABA test was used to test and compare the efficacy of two multienzyme preparations (product A and B) differing in galenic preparation in minipigs in which pancreatic exocrine insufficiency (PEI) was induced by pancreatic duct ligation. Without enzyme substitution no distinct increase in PABA was found in blood after oral administration of NBT-PABA. Administration of both enzyme preparations led to a clear dose dependent rise in PABA-concentrations in blood. Interestingly, the two preparations showed different time curves of serum PABA concentration, indicating differences in the kinetic of proteolytic enzyme action. It is concluded that the NBT-PABA test can be a very useful test for indirectly evaluating proteolytic enzyme efficacy in vivo, and also gives information about the kinetics of enzyme action, not only the end-result of enzyme action (like digestibility trials which were used traditionally). A single test is performed in a few hours and there is no need for fistulated animals.

We have previously demonstrated that the phosphatidylinositol-3 kinase (PI3K)/Akt signaling is essential for pancreatic regeneration after partial pancreatectomy in mice. In the present study, we examined a role of PI3K/Akt signaling for pancreatic duct cell differentiation into insulin-producing cells. Epithelial-like cells were isolated from mouse pancreas and confirmed to be positive for a duct cell marker cytokeratin-20 (CK-20) but negative for insulin. Incubation of these cells with epidermal growth factor, exhibited a gradual increase in Akt phosphorylation and expression of pancreatic duodenal homeobox-1 (PDX-1), a regulator of beta-cell differentiation. Three weeks later, these CK-20-positive cells were noted to express insulin as determined by immunofluorescent double-staining. Akt phosphorylation, PDX-1 expression, and insulin production were effectively reduced by blocking the PI3K/Akt pathway using siRNA to the p85alpha regulatory subunit of PI3K. Our results demonstrate that PI3K/Akt activation has a critical role for pancreatic duct cell differentiation into insulin-producing cells.

Objective: The purpose of our study was to determine whether the phosphatidylinositol 3-kinase (PI3K)/Akt pathway contributes to expression of pancreatic duodenal homeobox-1 (PDX-1) in duct cells and the cell differentiation during pancreatic regeneration., Methods: The role of PI3K in PDX-1 expression and duct cell differentiation with pancreatic regeneration in mice after partial pancreatectomy (Px) was examined using either wortmannin, a pharmacological PI3K inhibitor, or small-interfering RNA directed to the p85alpha regulatory subunit of PI3K. Akt phosphorylation, a marker of PI3K activation, and PDX-1 expression were assessed by Western blot analysis and immunohistochemistry., Results: Both PDX-1 levels and Akt phosphorylation were concomitantly increased in pancreatic ducts after partial Px and, conversely, blocked by treatment with wortmannin or p85alpha small-interfering RNA. Pancreatic duct cell differentiation, as assessed by appearance of insulin-positive cells 3 days after partial Px, was effectively reduced by wortmannin., Conclusions: The PI3K/Akt activation plays a critical role for both PDX-1 expression and pancreatic duct cell differentiation into insulin-producing cells during pancreatic regeneration.

A stable and reliable infected necrotizing pancreatitis (INP) model in rats was established in order to study the pathophysiological mechanism and pathological development rule of INP and explore the new therapeutic methods for the diseases. Forty-six SD rats were randomly divided into 5 groups. The animals in group A received the injection of 5% sodium taurocholate into the pancreatic duct and those in group B underwent that of E. coli into the pancreatic duct. The rats in groups C, D and E were subjected to the injection of 5% sodium taurocholate in combination with different concentrations of E. coli (10(3), 10(4), 10(5)/mL, respectively) into the pancreatic duct. The dose of injection was 0.1 mL/100 g and the velocity of injection was 0.2 mL/min in all the 5 groups. Eight h after the injection, the survival rate of animals was recorded and the surviving rats were killed to determine the serum content of amylase and perform pathological examination and germ cultivation of the pancreatic tissue. The results showed that acute necrotizing pancreatitis model was induced by injection of 5% sodium taurocholate into the pancreatic duct. The positive rate of germ cultivation in group A was 12.5%. The acute necrotizing pancreatitis model was not induced by injection of E. coli into the pancreatic duct and the positive rate of germ cultivation in group B was 0. The INP model was established in groups C to E. The positive rate of germ cultivation was 60%, 100% and 100% and 8-h survival rate 100%, 100% and 70% in groups C, D and E, respectively. It was concluded that a stable and reliable model of INP was established by injection of 5% sodium taurocholate in combination with 10(4)/mL E. coli into the pancreatic duct with a dose of 0.1 mL/100 g and a velocity of 0.2 mL/min. The pathogenesis of INP might be that the hemorrhage and necrosis of pancreatic tissue induced by sodium taurocholate results in weakness of pancreatic tissue in fighting against the germs. Meanwhile, the necrotic pancreatic tissue provides a good proliferative environment for the germs.

The studies of the mechanisms by which trauma-hemorrhagic shock leads to gut injury and dysfunction have largely ignored the nonbacterial factors contained within the lumen of the intestine. Yet, there is increasing evidence suggesting that intraluminal pancreatic proteases may be involved in this process. Thus, we tested the hypothesis that pancreatic proteases are necessary for the trauma-hemorrhagic shock-induced gut injury and the production of biologically active mesenteric lymph by determining the extent to which pancreatic duct ligation (PDL) would limit gut injury and mesenteric lymph bioactivity. To assess the effect of PDL on gut injury and dysfunction gut morphology, the mucus layer structure and the gut permeability were measured in the following four groups of male rats subjected to laparotomy (trauma) and hemorrhagic shock (pressure, 30 mmHg for 90 min): (1) rats subjected to trauma plus sham-shock (T/SS), (2) T/SS rats undergoing PDL (T/SS + PDL), (3) rats subjected to trauma and hemorrhagic shock (T/HS), and (4) rats subjected to T/HS + PDL. The ability of mesenteric lymph from these four rat groups to kill endothelial cells and activate neutrophils was tested in vitro. The PDL did not affect any of the parameters studied because there were no differences between the T/SS and the T/SS + PDL groups. However, PDL protected the gut from injury and dysfunction because PDL significantly abrogated T/HS-induced mucosal villous injury, loss of the intestinal mucus layer, and gut permeability. Likewise, PDL totally reversed the endothelial cell cytotoxic activity of T/HS lymph and reduced the ability of T/HS lymph to prime naive neutrophils for an augmented respiratory burst. Thus, it seems that intraluminal pancreatic proteases are necessary for the T/HS-induced gut injury and the production of bioactive mesenteric lymph.

Background: Pancreatic ductal adenocarcinoma (PDAC) is among the most common causes of cancer death. Preclinical and clinical studies on the preventive effects of beta-carotene or other retinoids have used dietary supplements that yielded high systemic concentrations (1-50 microM). While some of the preclinical data suggested cancer preventive effects of these agents, they have disappointed in clinical investigations., Materials and Methods: The effects of low concentrations (10 fM-200 nM)of beta-carotene on the proliferation, intracellular cAMP levels, PKA activation status and phosphorylation of EGFR-specific tyrosine kinases and ERK1/2 in immortalized human pancreatic duct epithelial cells was investigated., Results: Our data show significant concentration-dependent and PKA-dependent stimulation of all measured endpoints. Similar responses were achieved with forskolin. Our data indicate that low concentrations of beta-carotene stimulate the proliferation of the putative origin of PDAC, pancreatic duct epithelial cells via cAMP and PKA-dependent transactivation of the EGFR pathway. This could potentially have promoting effects on the development of PDAC.

Objective: To study the role of IIIb isoform of human fibroblast growth factor receptor 1 (FGFR1-IIIb) in proliferation of pancreatic ductal cells and its effects on mitogen-activated protein kinase (MAPK)., Methods: Human pancreatic ductal cells of the line TAKA-1 were cultured. The plasmid of human full-length FGFR1-IIIb isoform, pSVK4/FGFR1-IIIb, was stable transfected into the cultured TAKA-1 pancreatic ductal cells facilitated by lipofectamine. Un-transfected TAKA-1 cells and TAKA-1 ductal cells transfected with blank plasmid were used as controls. The expression, distribution and character of protein of FGFR1-IIIb in the TAKA-1 cells were estimated by Western blotting, Northern blotting, immunofluorescence assay, and glycosylation assay. The function and mechanism of FGFR1-IIIb in the transfected pancreatic ductal cells stimulated by FGF were examined by MTT assay and MAPK assay. Tunicamycin, an inhibitor of N-terminal glycoprotein synthesis, was added into the culture fluid of the FGFR1-IIIb transfected TAKA-1 cells to observe the changes of the FGFR1 bands., Results: FGFR1-IIIb, a glycosylated receptor at various levels at 120 kDa and between 130 - 150 kDa, was localized at moderate levels at the cell membrane and cytoplasm and at higher level in the perinuclear region of the cytoplasm of the pSVK4/FGFR1-IIIb-transfected cells. FGF-1, -2, and -4 significantly increased the growth of FGFR1-IIIb-transfected TAKA-1 cells, and at the same time induced the p44/p42 MAPK phosphorylation., Conclusion: Human FGFR1-IIIb receptor is a functional receptor in pancreatic ductal cells. FGF-1, -2, and -4 can increase the growth of FGFR1-IIIb-transfected pancreatic ductal cells, and the mechanism is that they can induce the p44/p42 MAPK phosphorylation.

More than 100 years ago it was proposed that pancreatitis essentially is a disease in which the pancreas undergoes autodigestion by its own prematurely activated digestive enzymes. Why and how digestive zymogens autoactivate within the pancreas early in the disease process has been a matter of controversy and debate. Some of the mechanisms that are considered to be involved indigestive protease activation are inherited and as of recently can be tested for clinically. Here we review the most recent progress in elucidating the mechanisms involved in the onset of pancreatitis. We specifically focus on serine and cysteine proteases in the autodigestive cascade that precedes acinar cell injury and the biochemical processes involved in their activation.

The inhibitory control of pancreatic ductal HCO(3)(-) secretion may be physiologically important in terms of limiting the hydrostatic pressure developed within the ducts and in terms of switching off pancreatic secretion after a meal. Substance P (SP) inhibits secretin-stimulated HCO(3)(-) secretion by modulating a Cl(-)-dependent HCO(3)(-) efflux step at the apical membrane of the duct cell (Hegyi P, Gray MA, and Argent BE. Am J Physiol Cell Physiol 285: C268-C276, 2003). In the present study, we have shown that SP is present in periductal nerves within the guinea pig pancreas, that PKC mediates the effect of SP, and that SP inhibits an anion exchanger on the luminal membrane of the duct cell. Secretin (10 nM) stimulated HCO(3)(-) secretion by sealed, nonperfused, ducts about threefold, and this effect was totally inhibited by SP (20 nM). Phorbol 12,13-dibutyrate (PDBu; 100 nM), an activator of PKC, reduced basal HCO(3)(-) secretion by approximately 40% and totally blocked secretin-stimulated secretion. In addition, bisindolylmaleimide I (1 nM to 1 microM), an inhibitor of PKC, relieved the inhibitory effect of SP on secretin-stimulated HCO(3)(-) secretion and also reversed the inhibitory effect of PDBu. Western blot analysis revealed that guinea pig pancreatic ducts express the alpha-, beta(I)-, delta-, epsilon-, eta-, theta-, zeta-, and mu-isoforms of PKC. In microperfused ducts, luminal H(2)DIDS (0.5 mM) caused intracellular pH to alkalinize and, like SP, inhibited basal and secretin-stimulated HCO(3)(-) secretion. SP did not inhibit secretion further when H(2)DIDS was present in the lumen, suggesting that SP and H(2)DIDS both inhibit the activity of an anion exchanger on the luminal membrane of the duct cell.

Background: Ki-Ras is well studied in its oncogenic form in relation to pancreatic pathologies. However, the individual contribution of each of the wild-type Ras isoforms (Ha-, Ki-, and N-) in pancreatic cells in health and disease is unknown., Methods: Archival formalin-fixed, paraffin-embedded specimens of normal (n = 6) and malignant pancreas (n = 35) were used for immuno-histochemical detection of Ras isoforms using a modified polymer system. In addition, immunogold labelling for Ras isoforms was done for subcellular localisation under electron microscopy., Results: Pancreatic ductal cells expressed Ha-Ras in the cytoplasm, with Ki-Ras in the apical region and N-Ras (50% of cases) in a supranuclear distribution. Pancreatic acinar cells express all three isoforms with some nuclear expression of Ki-Ras and supranuclear expression of N-Ras. Islets show Ki- and Ha-Ras mainly with differential expression of Ha-Ras (beta cells showing less Ha-Ras and more Ki-Ras than alpha cells). Electron microscopy shows that Ha-Ras is mainly localised in the endoplasmic reticulum and Golgi apparatus of the acinar cells with some plasma membrane localisation of Ki-Ras in the ductal cells. There was no change in any of the Ras isoform expression in the ductal or acinar cells in various malignancies studied (Mann-Whitney U test, p > 0.1)., Conclusions: Ras isoforms have distinct and separate cellular and subcellular distribution that may persist even in the malignantly transformed state. Understanding this distinct functional distribution patterns in detail is an essential step if mutant Ki-Ras is to be targeted in the pancreas by genetic or molecular therapeutic tools.

Human postnatal pancreatic duct cells are a potential source of new beta cells. Factors regulating proliferation of human pancreatic duct cells in vitro are unknown. In several other cell types, this process is influenced by ligands of the ErbB receptor family. The expression and functionality of the ErbB family members and their possible role in duct cell proliferation were determined. In cultured adult human pancreatic duct cells the different members of the ErbB family (ErbB1-4) were present at transcript and protein level. Stimulation of the duct cells with epidermal growth factor (EGF) and betacellulin results in Tyr-phosphorylation of ErbB1 and ErbB2, followed by activation of Shc, MEK1/2 and ERK1/2. Duct cells with activated ErbB signaling changed morphology and motility. EGF induced proliferation of a fraction of the duct cells and treatment with PD98059 prevented Ki67 expression in EGF-supplemented cells. When transduced with recombinant adenovirus expressing constitutively activated MEK1, duct cells proliferate and spread even in the absence of EGF. Importantly, the adult human duct cells retain their capacity to recapitulate ngn3-induced embryonic (neuro)endocrine differentiation after proliferation. Therefore, the present data support a possible role for human adult pancreatic duct cells, following expansion and transdifferentiation, as a source of insulin by transplantation to type I diabetes patients.

ATP is an extracellular regulator in numerous physiological and pathologic processes. Recently, 7 different subtypes of purinoceptors were identified on either the basolateral or the luminal membrane of pancreatic duct cells. However, the in vivo regulatory role of ATP in pancreatic function has not been established. We investigated the possible regulatory role of endogenous ATP in pancreatic function by measuring ATP concentrations and ATPase activity in pancreatic juice obtained from anesthetized rats and guinea pigs and from human patients undergoing endoscopy. Juice was collected from the main pancreatic duct in rats and guinea pigs under basal conditions or during stimulation with CCK, bombesin, or secretin. In guinea pigs, CCK, bombesin, and secretin did not affect ATP output, although they did stimulate fluid secretion. ATPase activity in the juice was evaluated by measuring the rate of hydrolysis of added ATP. Consistent with the low ATP concentrations in rat pancreatic juice, we found high levels of ATPase activity in this species. This was confirmed by HPLC, which also showed the metabolites of ATP hydrolysis. Ecto-ATPase activity was demonstrated by enzyme histochemistry in both the pancreatic acini and ducts in rats, but it was not detectable in guinea pigs and humans. These differences in ATP levels and ATPase expression may indicate significant species differences in the purinergic regulation of pancreatic secretion.

The high concentration of HCO(3)(-) ions (150 mM) in the human pancreatic ducts raises the question of the membrane proteins responsible for their secretion in addition to the Cl(-)/HCO(3)(-) exchanger. In this study, we investigated the expression of carbonic anhydrase IV (CA IV), a possible candidate. Experiments were carried out on specimens of normal human pancreas obtained from brain-dead donors ( n=9) as well as on isolated human ductal cells. Two antibodies were generated: CA IV NH(2) antibody directed against the NH(2) terminal of human glycosyl phosphatidylinositol (GPI)-anchored CA IV and CA IV COOH antibody directed against the COOH terminal of the same protein before its association with a GPI in the rough endoplasmic reticulum. A 35-kDa CA IV was detected in the homogenates of human pancreas. Immunocytochemistry demonstrated the expression of CA IV in centroacinar cells and in intercalated, intralobular, and interlobular ductal cells. The immunoreactivity observed with the CA IV COOH antibody was mainly localized on luminal membranes of ductal cells. Treatment of purified plasma membranes with phosphatidylinositol-phospholipase C indicated that the CA IV expressed in pancreatic ducts was not GPI-anchored. Its detection in the same extracts by the CA IV COOH antibody indicated that it was anchored by a hydrophobic segment at the carboxy terminal. Taken together, these results suggest that normal human pancreatic ductal cells express a 35-kDa CA IV anchored in their luminal plasma membrane by a hydrophobic segment of the COOH terminus. In view of its localization and its mode of anchorage in luminal plasma membranes, this CA IV may participate in the maintenance of luminal pH.

Phenotypic change of adult pancreatic islets has been implicated in the development of certain pancreatic cancers and in islet transplant failure. The aim of this study was to characterize intracellular events that mediate changes in adult islet phenotype. Using an in vitro islet-to-duct transformation model, canine islets were induced to undergo phenotypic transformation to duct-like epithelial structures through a two-stage process. Stage one was characterized by widespread islet cell apoptosis associated with the formation of cavitary spaces within the islets. During this stage, c-Jun N-terminal regulated kinase (JNK) and caspase-3 activities were elevated, while extracellular signal-regulated kinase (ERK) and Akt activities were decreased. The second stage of the process was characterized by an inversion in the balance in activity between these signal transduction pathways and by a concomitant decrease in apoptosis. The transformed islets were no longer immunoreactive for islet cell hormones, but expressed the duct epithelial cell marker CK-AE1/AE3. In contrast to islet cells, these duct epithelial cells were highly proliferative. To clarify the role of the identified changes in signal transduction events, we performed additional studies using pharmacological inhibitors of enzyme activity and demonstrated that inhibition of JNK and caspase-3 activity prevented cystic transformation. Our results indicate that the balance in signaling activity between ERK/Akt and JNK/caspase-3 appears to be an important regulator of islet cell death and differentiation.

Metabolic activation of many toxins, carcinogens, drugs, and anti-cancer agents is governed by the cytochrome P450 (CYP) drug-metabolizing enzyme system. To help elucidate the role of this enzyme system in the pathogenesis of chronic inflammatory and malignant pancreatic diseases, we compared the immunohistochemical expression pattern of 8 CYP-enzymes in 24 normal, 20 chronic pancreatitis, and 21 pancreatic cancer specimens using antibodies to CYP 1A1, 1A2, 2B6, 2C8/9/19, 2D6, 2E1, and 3A4, and the NADPH cytochrome P450 oxido-reductase (NA-OR). Compared to the normal pancreas, a higher frequency of immunopositivity for CYP 1A2, 2B6, 2C8/9/19, 2D6, and NA-OR was found in chronic pancreatitis, and of all CYPs but 1A2 in pancreatic cancer. On the other hand, CYP 1A1 and 2E1 antibody staining was less frequently observed in chronic pancreatitis. In all specimens with pancreatic polypeptide (PP)-rich regions (pancreas head), more islet cells than ductal and acinar cells were immunopositive. Moreover, the immunoreactivity of islet cells from PP-rich specimens with anti-CYP antibodies was consistently more frequent and intense than in islet cells from PP-poor areas (body and tail). Immunoreactivity for xenobiotic-metabolizing enzymes was frequently observed in the normal pancreas, chronic pancreatitis, and pancreatic cancer, and displayed differences of its frequency and intensity between the 3 groups. Considering immunohistochemical evidence of enzyme expression and pancreatic blood supply together, islet cells appear to be an important and possible early site of CYP-enzyme induction in pancreatic diseases.

Enzyme load in pancreas has been considered a risk factor in the development of acute pancreatitis. In order to confirm this hypothesis our aim was to analyze the development and evolution of acute pancreatitis (AP) induced by bile-pancreatic duct obstruction (BPDO) after reducing the pancreatic enzyme content. L-364,718 - a potent CCK-receptor antagonist - was administered (0.1 mg/kg/day) for 7 days before inducing AP by BPDO. The course of AP was evaluated at different times from 1.5-48 h after BPDO. Amylase and trypsinogen contents and cytosolic calcium levels were measured by flow cytometry using specific antisera against pancreatic enzymes labelled with isothiocyanate of fluorescein and Fluo 3, respectively. The severity of the disease at the different stages was evaluated by measurements of amylase activity in ascites and plasma, percentage of pancreatic fluid and haematocrit. Electron microscopy study of the pancreas showed an increased number of zymogen granules spread through the acinar cells of control rats treated with L-364,718 for 7 days, however, total enzyme content in individual acinar cells was significantly (p < 0.01) diminished. AP significantly increased intracellular amylase and trypsinogen load from 3-12 h after BPDO, and prior L-364,718 treatment enhanced the blockade of enzyme secretion. As a result, acinar enzyme content was significantly increased from earlier stages (1.5 h after BPDO). In parallel, increased cytosolic calcium levels observed up to 24 h after BPDO appeared earlier in L-364,718-treated rats than in those not treated. The severity of AP seems to have been higher in rats previously treated with the CCK-receptor antagonist as indicated by the significantly higher pancreatic fluid and amylase activity in ascites and plasma observed at different times after BPDO. Our results indicate that there is no correlation between the severity of pancreatitis and the amount of enzymes accumulated in the pancreas before the disease is induced.

Acute experiments on dogs with separate secretion of the pancreatic enzymes, either stimulated or inhibited, from two lobes of pancreas, and investigation into the character of kinetics of the secretion from 5-7 ductal pools of the pancreas showed the functional specifics, with the device of ductal valves and microdepot of a secret of the pancreas ductal system playing an important role. The conclusion is made that the final secret of pancreas removed to the duodenum is a product of a secret-motor activation of non-equipotentional microregions of the gland, being the components of an intervisceral dynamical mosaic.

Human pancreatic duct cells secrete HCO3- ions mediated by a Cl-/HCO3- exchanger and a HCO3- channel that may be a carbonic anhydrase IV (CA IV) in a channel-like conformation. This secretion is regulated by CFTR (Cystic Fibrosis Transmembrane conductance Regulator). In CF cells homozygous for the deltaF508 mutation, the defect in targeting of CFTR to plasma membranes leads to a disruption in the secretion of Cl- and HCO3 ions along with a defective targeting of other proteins. In this study, we analyzed the targeting of membrane CA IV in the human pancreatic duct cell line CFPAC-1, which expresses a deltaF508 CFTR, and in the same cells transfected with the wild-type CFTR (CFPAC-PLJ-CFTR6) or with the vector alone (CFPAC-PLJ6). The experiments were conducted on cells in the stationary phase the polarized state of which was checked by the distribution of occludin and actin. We show that both cell lines express a 35-kDa CA IV at comparable levels. Analysis of fractions of plasma membranes purified on a Percoll gradient evidenced lower levels of CA IV (8-fold) in the CFPAC-1 than in the CFPAC-PLJ-CFTR6 cells. Quantitative analyses showed that 6- to 10-fold fewer cells in the CFPAC-1 cell line exhibited membrane CA IV-immunoreactivity than in the CFPAC-PLJ-CFTR6 cell line. Taken together, these results suggest that the targeting of CA IV to apical plasma membranes is impaired in CFPAC-1 cells. CA IV/gamma-adaptin double labeling demonstrated the presence of CA IV in the trans-Golgi network (TGN) of numerous CFPAC-1 cells, indicating that trafficking was disrupted on the exit face of the TGN. The retargeting of CA IV observed in CFPAC-PLJ-CFTR6 cells points to a relationship between the traffic of CFTR and CA IV. On the basis of these observations, we propose that the absence of CA IV in apical plasma membranes due to the impairment in targeting in cells expressing a deltaAF508 CFTR largely contributes to the disruption in HCO3- secretion in CF epithelia.

The expression of protein gene product 9.5 (PGP9.5), a known neuron marker, was immunohistochemically investigated in rat pancreas. In fetal pancreas, a cluster of cells expressed PGP9.5 among the initial epithelial buds at embryonic day 11.5 (E 11.5). At E 13.5, PGP9.5 appeared among elongated and branching epithelial cells as well as along nerve fibers in the mesenchyme. On E 17.5, tubular cells became ductal cells with lumen, which strongly expressed PGP9.5. In newborn rats, ductal cells of the common bile duct (CBD) to the centroacinar cells and islet cells expressed PGP9.5. Ten days after birth, the number of the ductal cells expressing PGP9.5 was reduced, and PGP9.5-negative cells appeared in half of the duct cells. On day 21, all centroacinar cells and intercalated ductal cells became PGP9.5-negative, but some CBD and interlobular ductal cells remained positive for PGP9.5. On day 28 and thereafter, PGP9.5 was no longer detected. In a pancreatic duct ligation model, acinar cells changed to cells with duct-like structure after duct ligation. These cells strongly expressed PGP9.5 on the fifth day after duct ligation. Three to four weeks after ligation, the cells with duct-like structure changed to acinar cells, islets of Langerhans and ductal cells, but the ductal cells were PGP9.5-negative at this point. These results suggested that PGP9.5 is expressed in ductal cells that possess a potential for differentiation to pancreatic endocrine cells, and therefore can serve as a marker for the progenitor of pancreatic endocrine cells.

The subcellular distribution of carbonic anhydrase II, either throughout the cytosol or in the cytoplasm close to the apical plasma membrane or vesicular compartments, suggests that this enzyme may have different roles in the regulation of pH in intra- or extracellular compartments. To throw more light on the role of pancreatic carbonic anhydrase II, we examined its expression and subcellular distribution in Capan-1 cells. Immunocytochemical analysis by light, confocal, and electron microscopy, as well as immunoblotting of cell homogenates or purified plasma membranes, was performed. A carbonic anhydrase II of 29 kD associated by weak bonds to the inner leaflet of apical plasma membranes of polarized cells was detected. This enzyme was co-localized with markers of Golgi compartments. Moreover, the defect of its targeting to apical plasma membranes in cells treated with brefeldin A was indicative of its transport by the Golgi apparatus. We show here that a carbonic anhydrase II is associated with the inner leaflet of apical plasma membranes and with the cytosolic side of the endomembranes of human cancerous pancreatic duct cells (Capan-1). These observations point to a role for this enzyme in the regulation of intra- and extracellular pH.

Transcript levels of the human serine/threonine kinase h-sgk have been found to be highest in pancreas. In the present study, localization and regulation of h-sgk transcription in pancreatic tissue were elucidated. As was apparent from radioactive in situ hybridization, most pancreatic acinar cells expressed high levels of h-sgk mRNA. h-sgk mRNA-positive cells were also found in ductal epithelia but not in pancreatic islets. In biopsy specimens from patients with pancreatitis, h-sgk mRNA levels were decreased in acinar cells but abundant in numerous mononuclear interstitial cells within areas of pancreatic necrosis and fibrosis. As shown by Northern blotting, h-sgk transcription in DAN-G pancreatic tumor cells is upregulated by osmotic cell shrinkage, serum, phorbol esters (phorbol 12,13-didecanoate), and Ca(2+) ionophore A-23187 and decreased by staurosporine and cAMP. In conclusion, h-sgk transcription is regulated not only by cell volume but also by serum, protein kinase C stimulation, cAMP, and increase of intracellular Ca(2+) activity. The kinase may participate not only in normal function of exocrine pancreas but also in fibrosing pancreatitis.

Carbonic anhydrase (CA) IX and XII are transmembrane isoenzymes which are expressed in several epithelia and overexpressed in some carcinomas. They have recently been linked to von Hippel-Lindau gene-mediated carcinogenesis in that both isoenzymes are downregulated by the product of the wild-type von Hippel-Lindau tumour suppressor gene. This paper describes the localisation of CA IX and XII in the normal human pancreas and pancreatic tumours. Both isoenzymes showed positive reaction in the basolateral plasma membrane of the normal acinar and ductal epithelia. The hyperplastic ductal epithelium in tumour specimens generally showed an increased staining for CA IX. Of 29 malignant tumours of exocrine pancreas, 10 showed moderate or strong immunoreaction for CA IX. The signal for CA XII remained weak in most malignant lesions. The present results show that both CA IX and XII are unevenly expressed in the ductal and acinar compartments of the human pancreas. The expression of these isoenzymes in a relatively low number of malignant tumour specimens suggests that they have a limited value in diagnostic evaluation of pancreatic carcinoma. However, the increased expression of CA IX in hyperplastic ductal epithelium may contribute to the pancreatic tumourigenesis.

Context: We have isolated five stable clones from a primary culture of Syrian golden hamster pancreatic duct epithelial cells and have designated them as CK1 through CK5., Design: Here we describe the ability of two of these, CK1 and CK5, to metabolize the pancreas carcinogen N-nitrosobis(2-oxopropyl)amine. The metabolism was assessed as the production of mutated V79 cells in a CK cell/V79 co-culture set up., Results: At a dose of 0.1 mM N-nitrosobis(2-oxopropyl)amine, the CK1 cells produced 82.3 +/- 17.2 mutants/1,000,000 survivors while the CK5 cells produced only 33.2 +/- 10.8 mutants/1,000,000 survivors, both are mean +/- SD (n = 8). Furthermore, both cell types responded differently to two inducers of cytochrome P450 activity, namely Arochlor 1254 and EtOH. Arochlor 1254 treatment did not affect the metabolizing ability of CK1 cells while EtOH treatment resulted in a twofold increase in the mutation frequency. Arochlor and EtOH treatment inhibited the ability of CK5 cells to metabolize N-nitrosobis(2-oxopropyl)amine., Conclusions: These data show that the duct epithelium of the pancreas is a multi-cellular tissue and the different cell types within the epithelium have different abilities to metabolize xenobiotic chemicals.

These studies investigated the growth characteristics and functional properties of isolated canine pancreatic ductal epithelial cells. Cells were isolated from the accessory pancreatic duct and cultured by using three conditions: on vitrogen-coated petri dishes with fibroblast conditioned medium (nonpolarized); in vitrogen-coated Transwells above a fibroblast feeder layer (polarized); or as organotypic rafts above a fibroblast-embedded collagen layer (polarized). Growth characteristics, transepithelial resistances, and carbonic anhydrase and cyclic adenosine monophosphate (AMP) responses were evaluated. Under polarized conditions, the cells grew as monolayers with columnar epithelial characteristics. The monolayers developed high transepithelial resistance and became impervious to the passage of horseradish peroxidase. Epithelial growth factor (EGF) (2 ng/ml) stimulated ductal cell growth and accelerated the formation of a high-resistance monolayer. Forskolin (10 microM) rapidly decreased transepithelial resistance. Carbonic anhydrase activity, which was lower in nonpolarized compared with polarized conditions, was stimulated by carbachol (175 microM). Secretin, however, did not stimulate carbonic anhydrase activity in these cells. Although secretin stimulated adenylyl cyclase activity in early-passage cells, this response was lost in later-passage cells. Both vasoactive intestinal polypeptide (VIP; 1 microM) and forskolin (10 microM) consistently increased adenylyl cyclase activity. Isolated canine pancreatic ductal epithelial cells proliferate in vitro, develop high-resistance epithelial monolayers, and respond to stimuli that activate adenylyl cyclase. These cells should provide a useful model for regulatory studies of ductal cell functions.

Background: Brush cytology for the diagnosis of pancreaticobiliary malignancy has an overall sensitivity of 50%. Accurate and specific markers are therefore needed for the optimal evaluation of pancreaticobiliary strictures. Telomerase activity is present in 85% to 90% of all human cancers. We sought to determine the utility of in situ hybridization for telomerase RNA in endoscopic brushings for the diagnosis of pancreaticobiliary malignancy., Methods: Endoscopic brushings from 18 patients with pancreatic or biliary strictures were evaluated by routine cytology and in situ hybridization for telomerase RNA., Results: Eight of 18 strictures were malignant. Cytology was positive in 5 patients, whereas telomerase RNA was positive in 6. All malignancies were diagnosed by either cytology or telomerase RNA; however, both studies were positive in only 3. There were no false-positive results by either technique., Conclusion: The detection of telomerase RNA in endoscopic brushings may be an important adjunct to cytology for cancer diagnosis in pancreaticobiliary strictures.

The present study investigated both HCO-3 and Cl- secretions in a human pancreatic duct cell line, CAPAN-1, using the short-circuit current (Isc) technique. In Cl-/HCO-3-containing solution, secretin (1 microM) or forskolin (10 microM) stimulated a biphasic rise in the Isc which initially reached a peak level at about 3 min and then decayed to a plateau level after 7 min. Removal of external Cl- abolished the initial transient phase in the forskolin-induced Isc while the plateau remained. In HCO-3/CO2-free solution, on the contrary, only the initial transient increase in Isc was prominent. Summation of the current magnitudes observed in Cl--free and HCO-3-free solutions over a time course of 10 min gave rise to a curve which was similar, both in magnitude and kinetics, to the current observed in Cl-/HCO-3-containing solution. Removal of external Na+ greatly reduced the initial transient rise in the forskolin-induced Isc response, and the plateau level observed under this condition was similar to that obtained in Cl--free solution, suggesting that Cl--dependent Isc was also Na+-dependent. Bumetanide (50 microM), an inhibitor of the Na+-K+-2Cl- cotransporter, and Ba2+ (1 mm), a K+ channel blocker, could reduce the forskolin-induced Isc obtained in Cl-/HCO-3-containing or HCO-3-free solution. However, they were found to be ineffective when external Cl- was removed, indicating the involvement of these mechanisms in Cl- secretion. On the contrary, the HCO-3-dependent (in the absence of external Cl-) forskolin-induced Isc could be significantly reduced by carbonic anhydrase inhibitor, acetazolamide (45 microM). Basolateral application of amiloride (100 microM) inhibited the Isc; however, a specific Na+-H+ exchanger blocker, 5-N-methyl-N-isobutylamiloride (MIA, 5-10 microM) was found to be ineffective, excluding the involvement of the Na+-H+ exchanger. However, an inhibitor of H+-ATPase, N-ethylmaleimide did suppress the Isc (IC50 = 22 microM). Immunohistochemical studies also confirmed the presence of a vacuolar type of H+-ATPase in these cells. H2DIDS (100 microM), an inhibitor of Na+-HCO-3 cotransporter, was without effect. Apical addition of Cl- channel blocker, diphenylamine-2,2'-dicarboxylic acid (DPC, 1 mm), but not disulfonic acids, DIDS (100 microM) or SITS (100 microM), exerted an inhibitory effect on both Cl- and HCO-3-dependent forskolin-induced Isc responses. Histochemical studies showed discrete stainings of carbonic anhydrase in the monolayer of CAPAN-1 cells, suggesting that HCO-3 secretion may be specialized to a certain population of cells. The present results suggest that both HCO-3 and Cl- secretion by the human pancreatic duct cells may occur concurrently and independently.

In patients with type I diabetes mellitus, clinical studies have demonstrated decreased secretion of pancreatic juice by the pancreatic excretory duct system. The cause of this decrease is unknown, but could involve changes in initial signal transduction pathways or one or more of the electrolyte transport components that subserve regulated fluid secretion. We have compared responsiveness to secretin in pancreatic ducts isolated from healthy and diabetic Hartley guinea pigs and also have compared the expression of CFTR and Na+, K(+)-ATPase in these two groups, as the activities of these two proteins are essential for secretion of pancreatic juice. The increases in cyclic AMP levels evoked by exposure to either 0.1 nM or 0.1 microM secretin were not significantly different in pancreatic ducts isolated from healthy and diabetic guinea pigs nor were levels of CFTR or Na+, K(+)-ATPase expression. By contrast, Na+, K(+)-ATPase activity in pancreatic ducts isolated from diabetic guinea pigs was decreased by 70%, suggesting a change in the enzyme's catalytic properties in the diabetic tissues. The observed decrease would be expected to seriously compromise the production of pancreatic juice.

Impaired pancreatic duct secretion is frequently observed in insulin-dependent diabetes mellitus (IDDM), although the cellular mechanism(s) of dysfunction remains unknown. Studies in other tissues have suggested that a hyperglycemia-induced decrease in Na, K-ATPase activity could contribute to the metabolic complications of IDDM and that increased polyol metabolism is involved in this response. The present studies examined the effects of glucose on Na, K-ATPase activity and on expression and activity of aldose reductase (AR), a primary enzyme of polyol metabolism, in Capan-1 human pancreatic duct cells. Increasing medium glucose from 5.5 to 22 mM caused a 29% decrease in Na,K-ATPase activity. The decrease was corrected by 100 microM sorbinil, a specific AR inhibitor. Increasing glucose from 5.5 to 110 mM also resulted in concentration-dependent increases in AR mRNA and enzyme activity that could be resolved into two components, one that was glucose specific and observed at pathophysiological concentrations (< 55 mM) and a second that was osmotically induced at high concentrations (> 55 mM) and which was not glucose specific. The present study demonstrates that pathophysiological levels of glucose specifically activate polyol metabolism with a consequent decrease in Na,K-ATPase activity in pancreatic duct epithelial cells, and that this response to hyperglycemia could contribute to decreased pancreatic secretion observed in IDDM. This is the first report of AR regulation in the pancreatic duct epithelium.

The effect of duct pressure on the barrier function of the pancreatic duct mucosa to both activated and nonactivated pancreatic exocrine enzymes was studied in a feline model. The cat main pancreatic duct was perfused from the tail to the head of the gland with rat pancreatic juice at high duct pressure (40 cm H2O). In a first experiment, nonactivated pancreatic juice was perfused. Analysis of the juice for loss of fluid volume (measurement by weight) and for loss of individual proteins (two-dimensional isoelectric focusing/sodium dodecyl sulphate gel electrophoresis, reversed phase-high performance liquid chromatography) after duct passage showed that pancreatic secretions were completely recovered from the duct lumen. In another experiment, applying the same pressure, the duct was perfused with activated pancreatic juice. Morphologic analysis showed a preservation of the pancreatic duct mucosal integrity immediately after duct perfusion and absence of pancreatic inflammatory lesions 24 h after duct passage. We conclude that the pancreatic duct mucosa is impermeable to the leakage of pancreatic exocrine proteins from the duct lumen at duct pressure < or = 40 cm H2O. Flow of activated pancreatic juice along the pancreatic duct at duct pressure < or = 40 cm H2O does not cause acute pancreatitis. These data do not support the hypothesis that leakage of pancreatic juice from the duct lumen caused by high intraductal pressure due to duct obstruction initiates acute pancreatitis.