Murine Pancreatic Acinar Cells Research Articles

Trefoil Factor 2 Expressed by the Murine Pancreatic Acinar Cells Is Required for the Development of Islets and for β-Cell Function During Aging.

Exocrine-to-endocrine crosstalk in the pancreas is crucial to maintain beta cell function. However, the molecular mechanisms underlying this crosstalk are largely undefined. Trefoil factor 2 (Tff2) is a secreted factor known to promote the proliferation of beta cells in vitro, but its physiological role in vivo in the pancreas is unknown. Also, it remains unclear which pancreatic cell type expresses Tff2 protein. We therefore created a mouse model with a conditional knockout of Tff2 in the murine pancreas. We find that the Tff2 protein is preferentially expressed in acinar but not ductal or endocrine cells. Tff2 deficiency in the pancreas reduces beta cell mass on embryonic day 16.5. However, homozygous mutant mice are born without a reduction of beta cells and with acinar Tff3 compensation by day 7. When mice are aged to 1 year, both male and female homozygous and male heterozygous mutants develop impaired glucose tolerance without affected insulin sensitivity. Perifusion analysis reveals that the second phase of glucose-stimulated insulin secretion from islets is reduced in aged homozygous mutant compared to controls. Collectively, these results demonstrate a previously unknown role of Tff2 as an exocrine acinar cell-derived protein required for maintaining functional endocrine beta cells in mice.

O101 Mitochondrial P53 inhibition ameliorates acute pancreatitis through reduction of mitochondrial injury and cell necrosis

Abstract Introduction Persistent opening of the mitochondrial permeability transition pore (MPTP) is central to acute pancreatitis (AP). Mitochondrial translocation of P53 promotes MPTP opening, which induces necrotic cell death. We sought to determine whether and how mitochondrial P53 inhibition is protective in experimental AP. Methods Mitochondrial translocation of P53 was inhibited by pifithrin-mu (PFT-µ, mitochondrial specific P53 inhibitor) or by Trp53 KO. Mitochondrial membrane potential (ΔΨm) and necrosis of murine pancreatic acinar cells (PACs) were evaluated by confocal microscopy with TMRM and propidium iodide, respectively, in the presence of TLCS. Fluo4 was used for calcium imaging. Caerulein AP (Cer-AP) was induced in male (WT, P53−/+, P53−/−; 10-12 week old) mice by 7 hourly i.p. injections of 50 μg/kg caerulein. PFT-µ (5 mg/kg) was injected after the third injection of caerulein; sacrifice was made 12h after the first injection of caerulein. AP was assessed by standard biomarkers and blinded histopathology. Results TLCS-induced collapse of ΔΨm and cell necrosis of wild type PACs were significantly reduced in the presence of PFTµ or in PACs isolated from Trp53 KO mice; calcium overload was also reduced, indicative of improved calcium clearance, and ATP was maintained. Pharmacological or genetic P53 inhibition resulted in significant reductions of biochemical, immunological, and histopathological indices of severity in the Cer-AP model. Conclusion Inhibition of P53 maintained mitochondrial integrity, reduced PACs necrosis, and ameliorated the severity of Cer-AP. These data suggest that inhibition of mitochondrial translocation of P53 could be an effective strategy for the treatment of AP.

IRAG2 Interacts with IP3-Receptor Types 1, 2, and 3 and Regulates Intracellular Ca2+ in Murine Pancreatic Acinar Cells.

The inositol 1,4,5-triphosphate receptor-associated 2 (IRAG2) is also known as Jaw1 or lymphoid-restricted membrane protein (LRMP) and shares homology with the inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate 1 (IRAG1). IRAG1 interacts with inositol trisphosphate receptors (IP3 receptors /IP3R) via its coiled-coil domain and modulates Ca2+ release from intracellular stores. Due to the homology of IRAG1 and IRAG2, especially in its coiled-coil domain, it is possible that IRAG2 has similar interaction partners like IRAG1 and that IRAG2 also modulates intracellular Ca2+ signaling. In our study, we localized IRAG2 in pancreatic acinar cells of the exocrine pancreas, and we investigated the interaction of IRAG2 with IP3 receptors and its impact on intracellular Ca2+ signaling and exocrine pancreatic function, like amylase secretion. We detected the interaction of IRAG2 with different subtypes of IP3R and altered Ca2+ release in pancreatic acinar cells from mice lacking IRAG2. IRAG2 deficiency decreased basal levels of intracellular Ca2+, suggesting that IRAG2 leads to activation of IP3R under unstimulated basal conditions. Moreover, we observed that loss of IRAG2 impacts the secretion of amylase. Our data, therefore, suggest that IRAG2 modulates intracellular Ca2+ signaling, which regulates exocrine pancreatic function.

Slc26a6 is an apical membrane anion exchanger that drives HCO3--dependent fluid secretion in murine pancreatic acinar cells.

The nonselective anion exchanger Slc26a6, also known as putative anion transporter 1 and chloride/formate exchanger, is thought to play a major role in transport in exocrine glands. In this study, Slc26a6 null mice were used to explore the function of Slc26a6 in the exocrine pancreas. Slc26a6 primarily localized to the apical membrane of pancreatic exocrine acinar cells. The volume of stimulated juice secretion by the ex vivo pancreas was significantly reduced ~35% in Slc26a6-/- mice, but no changes occurred in the gross structure or gland weights of Slc26a6 null mice. The secretion of pancreatic juice by Slc26a6+/+ mice was dependent on while, in contrast, fluid secretion by Slc26a6-/- mice was independent of , suggesting that Slc26a6 mediates the -dependent component of fluid secretion. Consistent with these observations, disruption of Slc26a6 also significantly reduced secretion by the pancreas ~35%. Taken together, these results demonstrate that the apical Slc26a6 anion exchanger in acinar cells is involved in -dependent fluid secretion but that another major -independent pathway is the primary driver of the fluid secretion process in the mouse pancreas.

Dihydrodiosgenin protects against experimental acute pancreatitis and associated lung injury through mitochondrial protection and PI3Kγ/Akt inhibition.

Acute pancreatitis (AP) is a painful and distressing disorder of the exocrine pancreas with no specific treatment. Diosgenyl saponins extracted from from Dioscorea zingiberensis C. H. Wright have been reported to protect against experimental models of AP. Diosgenin, or its derivatives are anti-inflammatory in various conditions. However, the effects of diosgenin and its spiroacetal ring opened analogue, dihydrodiosgenin (Dydio), on AP have not been determined. Effects of diosgenin and Dydio on sodium taurocholate hydrate (Tauro)-induced necrosis were tested, using freshly isolated murine pancreatic acinar cells. Effects of Dydio on mitochondrial dysfunction in response to Tauro, cholecystokinin-8 and palmitoleic acid ethyl ester were also assessed. Dydio (5 or 10mg·kg-1 ) was administered after the induction in vivo of Tauro-induced AP (Wistar rats), caerulein-induced AP and palmitoleic acid plus ethanol-induced AP (Balb/c mice). Pancreatitis was assessed biochemically and histologically. Activation of pancreatic PI3Kγ/Akt was measured by immunoblotting. Dydio inhibited Tauro-induced activation of the necrotic cell death pathway and prevented pancreatitis stimuli-induced mitochondrial dysfunction. Therapeutic administration of Dydio ameliorated biochemical and histopathological responses in all three models of AP through pancreatic mitochondrial protection and PI3Kγ/Akt inactivation. Moreover, Dydio improved pancreatitis-associated acute lung injury through preventing excessive inflammatory responses. These data provide in vitro and in vivo mechanistic evidence that the diosgenin analogue, Dydio could be potential treatment for AP. Further medicinal optimization of diosgenin and its analogue might be a useful strategy for identifying lead candidates for inflammatory diseases.

Ethanol suppresses carbamylcholine-induced intracellular calcium oscillation in mouse pancreatic acinar cells.

Oscillation of intracellular calcium levels is closely linked to initiating secretion of digestive enzymes from pancreatic acinar cells. Excessive alcohol consumption is known to relate to a variety of disorders in the digestive system, including the exocrine pancreas. In this study, we have investigated the role and mechanism of ethanol on carbamylcholine (CCh)-induced intracellular calcium oscillation in murine pancreatic acinar cells. Ethanol at concentrations of 30 and 100mM reversibly suppressed CCh-induced Ca2+ oscillation in a dose-dependent manner. Pretreatment of ethanol has no effecton the store-operated calcium entry induced by 10μM of CCh. Ethanol significantly reduced the initial calcium peak induced by low concentrations of CCh and therefore, the CCh-induced dose-response curve of the initial calcium peak was shifted to the right by ethanol pretreatment. Furthermore, ethanol significantly dose-dependently reduced inositol 1,4,5-trisphosphate-induced calcium release from the internal stores in permeabilized acinar cells. These results provide evidence that excessive alcohol intake could impair cytosolic calcium oscillation through inhibiting calcium release from intracellular stores in mouse pancreatic acinar cells.

The MET Receptor Tyrosine Kinase Confers Repair of Murine Pancreatic Acinar Cells following Acute and Chronic Injury.

Acinar cells represent the primary target in necroinflammatory diseases of the pancreas, including pancreatitis. The signaling pathways guiding acinar cell repair and regeneration following injury remain poorly understood. The purpose of this study was to determine the importance of Hepatocyte Growth Factor Receptor/MET signaling as an intrinsic repair mechanism for acinar cells following acute damage and chronic alcohol-associated injury. Here, we generated mice with targeted deletion of MET in adult acinar cells (MET-/-). Acute and repetitive pancreatic injury was induced in MET-/- and control mice with cerulein, and chronic injury by feeding mice Lieber-DeCarli diets containing alcohol with or without enhancement of repetitive pancreatic injury. We examined the exocrine pancreas of these mice histologically for acinar death, edema, inflammation and collagen deposition and changes in the transcriptional program. We show that MET expression is relatively low in normal adult pancreas. However, MET levels were elevated in ductal and acinar cells in human pancreatitis specimens, consistent with a role for MET in an adaptive repair mechanism. We report that genetic deletion of MET in adult murine acinar cells was linked to increased acinar cell death, chronic inflammation and delayed recovery (regeneration) of pancreatic exocrine tissue. Notably, increased pancreatic collagen deposition was detected in MET knockout mice following repetitive injury as well alcohol-associated injury. Finally, we identified specific alterations of the pancreatic transcriptome associated with MET signaling during injury, involved in tissue repair, inflammation and endoplasmic reticulum stress. Together, these data demonstrate the importance of MET signaling for acinar repair and regeneration, a novel finding that could attenuate the symptomology of pancreatic injury.

Small Molecule Inhibitors of Cyclophilin D To Protect Mitochondrial Function as a Potential Treatment for Acute Pancreatitis.

Opening of the mitochondrial permeability transition pore (MPTP) causes mitochondrial dysfunction and necrosis in acute pancreatitis (AP), a condition without specific drug treatment. Cyclophilin D (CypD) is a mitochondrial matrix peptidyl-prolyl isomerase that regulates the MPTP and is a drug target for AP. We have synthesized urea-based small molecule inhibitors of cyclophilins and tested them against CypD using binding and isomerase activity assays. Thermodynamic profiles of the CypD/inhibitor interactions were determined by isothermal titration calorimetry. Seven new high-resolution crystal structures of CypD-inhibitor complexes were obtained to guide compound optimization. Compounds 4, 13, 14, and 19 were tested in freshly isolated murine pancreatic acinar cells (PACs) to determine inhibition of toxin-induced loss of mitochondrial membrane potential (ΔΨm) and necrotic cell death pathway activation. Compound 19 was found to have a Kd of 410 nM and a favorable thermodynamic profile, and it showed significant protection of ΔΨm and reduced necrosis of murine as well as human PACs. Compound 19 holds significant promise for future lead optimization.

Chronic Nicotine Exposure In Vivo and In Vitro Inhibits Vitamin B1 (Thiamin) Uptake by Pancreatic Acinar Cells.

Thiamin (vitamin B1), a member of the water-soluble family of vitamins, is essential for normal cellular functions; its deficiency results in oxidative stress and mitochondrial dysfunction. Pancreatic acinar cells (PAC) obtain thiamin from the circulation using a specific carrier-mediated process mediated by both thiamin transporters -1 and -2 (THTR-1 and THTR-2; encoded by the SLC19A2 and SLC19A3 genes, respectively). The aim of the current study was to examine the effect of chronic exposure of mouse PAC in vivo and human PAC in vitro to nicotine (a major component of cigarette smoke that has been implicated in pancreatic diseases) on thiamin uptake and to delineate the mechanism involved. The results showed that chronic exposure of mice to nicotine significantly inhibits thiamin uptake in murine PAC, and that this inhibition is associated with a marked decrease in expression of THTR-1 and THTR-2 at the protein, mRNA and hnRNAs level. Furthermore, expression of the important thiamin-metabolizing enzyme, thiamin pyrophosphokinase (TPKase), was significantly reduced in PAC of mice exposed to nicotine. Similarly, chronic exposure of cultured human PAC to nicotine (0.5 μM, 48 h) significantly inhibited thiamin uptake, which was also associated with a decrease in expression of THTR-1 and THTR-2 proteins and mRNAs. This study demonstrates that chronic exposure of PAC to nicotine impairs the physiology and the molecular biology of the thiamin uptake process. Furthermore, the study suggests that the effect is, in part, mediated through transcriptional mechanism(s) affecting the SLC19A2 and SLC19A3 genes.

Mechanism of mitochondrial permeability transition pore induction and damage in the pancreas: inhibition prevents acute pancreatitis by protecting production of ATP

ObjectiveAcute pancreatitis is caused by toxins that induce acinar cell calcium overload, zymogen activation, cytokine release and cell death, yet is without specific drug therapy. Mitochondrial dysfunction has been implicated...

Effects of the mitochondria-targeted antioxidant mitoquinone in murine acute pancreatitis.

Although oxidative stress has been strongly implicated in the development of acute pancreatitis (AP), antioxidant therapy in patients has so far been discouraging. The aim of this study was to assess potential protective effects of a mitochondria-targeted antioxidant, MitoQ, in experimental AP using in vitro and in vivo approaches. MitoQ blocked H2O2-induced intracellular ROS responses in murine pancreatic acinar cells, an action not shared by the control analogue dTPP. MitoQ did not reduce mitochondrial depolarisation induced by either cholecystokinin (CCK) or bile acid TLCS, and at 10 µM caused depolarisation per se. Both MitoQ and dTPP increased basal and CCK-induced cell death in a plate-reader assay. In a TLCS-induced AP model MitoQ treatment was not protective. In AP induced by caerulein hyperstimulation (CER-AP), MitoQ exerted mixed effects. Thus, partial amelioration of histopathology scores was observed, actions shared by dTPP, but without reduction of the biochemical markers pancreatic trypsin or serum amylase. Interestingly, lung myeloperoxidase and interleukin-6 were concurrently increased by MitoQ in CER-AP. MitoQ caused biphasic effects on ROS production in isolated polymorphonuclear leukocytes, inhibiting an acute increase but elevating later levels. Our results suggest that MitoQ would be inappropriate for AP therapy, consistent with prior antioxidant evaluations in this disease.

Epiplakin deficiency aggravates murine caerulein-induced acute pancreatitis and favors the formation of acinar keratin granules.

Epiplakin, a member of the plakin protein family, is exclusively expressed in epithelial tissues and was shown to bind to keratins. Epiplakin-deficient (EPPK−/−) mice showed no obvious spontaneous phenotype, however, EPPK−/− keratinocytes displayed faster keratin network breakdown in response to stress. The role of epiplakin in pancreas, a tissue with abundant keratin expression, was not yet known. We analyzed epiplakin’s expression in healthy and inflamed pancreatic tissue and compared wild-type and EPPK−/− mice during caerulein-induced acute pancreatitis. We found that epiplakin was expressed primarily in ductal cells of the pancreas and colocalized with apicolateral keratin bundles in murine pancreatic acinar cells. Epiplakin’s diffuse subcellular localization in keratin filament-free acini of K8-deficient mice indicated that its filament-associated localization in acinar cells completely depends on its binding partner keratin. During acute pancreatitis, epiplakin was upregulated in acinar cells and its redistribution closely paralleled keratin reorganization. EPPK−/− mice suffered from aggravated pancreatitis but showed no obvious regeneration phenotype. At the most severe stage of the disease, EPPK−/− acinar cells displayed more keratin aggregates than those of wild-type mice. Our data propose epiplakin to be a protective protein during acute pancreatitis, and that its loss causes impaired disease-associated keratin reorganization.

Involvement of the MPTP in minocycline-induced mitochondrial dysfunction in murine pancreatic acinar cells

Introduction: Fish Oil Based Lipid Emulsions (FOBLE) can potentially improve outcome in acute inflammatory conditions. However, lipid utilisation can be greatly impaired in these patients resulting in lipid disorders and “fat-overload” syndrome. The aim of this study was to evaluate the safety and tolerability of two lipid emulsions in patients with severe acute pancreatitis (SAP). Methods: A phase II single centre, double blind randomised control trial was conducted between October 2010 and February 2012. Forty-four patients with SAP were randomly assigned to receive a FOBLE (MCT/LCT/ Omega-3)(Lipidem 20%,BBraun)(study group, n1⁄422) or an MCT/LCT emulsion (Lipofundin MCT 20%,BBraun)(control group, n1⁄422). Lipid emulsions were initiated within 72 hours of symptom onset and infused daily at a dose of 2g/10ml per kg per day over 14 hours for a maximum of 7days. A non-fasting serum triglyceride and random cholesterol levels were obtained 6-10 hours post infusion on days 0, 1, 2, 3, 5 and 7. Standard care was provided including enteral or parenteral nutrition. Results: Both emulsions were well tolerated and with no unexpected severe adverse events noted. One critically ill patient developed transient hypertriglyceridemia, which resolved after temporary cessation of the MCT/LCT lipid emulsion. Another patient developed low-grade pyrexia which resolved by halving the MCT/LCT lipid emulsion dose. The mean post-infusion serum triglycerides and random cholesterol levels in the control group were slightly higher than the fish oil group at days 1, 2, 3, 5 and 7 (p1⁄4>0.05). Conclusion: Lipid emulsions can safely be infused in patients with SAP as long as manufacturer recommendations are followed. Take-home message: Lipid emulsions can safely be administered in SAP Abstract previously presented? no () Any disclosures? yes (D. Al-Leswas: Grant/Research Support from B Braun, A. Arshad: Grant/Research Support from B Braun, A. Eltweri: None declared,W.-Y. Chung: None declared, C. Pollard: None declared, O. Al-Taan: Grant/Research Support from B Braun, G. Garcea : None declared, M. Metcalfe: Grant/Research Support from B Braun, A. Dennison: Grant/Research Support from B Braun)

Involvement of MPTP in minocycline – induced mitochondrial dysfunction in murine pancreatic acinar cells

Involvement of MPTP in minocycline – induced mitochondrial dysfunction in murine pancreatic acinar cells

Inhibitory effects of minocycline on mitochondrial function in murine pancreatic acinar cells: involvement of the MPTP

s / Pancreatology 12 (2012) 502–597 516 investigation of thyroid gland: TSH, FT3, FT4, thyroid echography antithyroid antibodies: thyroid antiperoxidase (antiTPO AB) and antithyroglobulin (antiTg AB) antibodies Results: The antiTPO AB were present in high titers in 85.18% cases (n 1⁄4 46) and in normal titers in 14.81% cases (n 1⁄4 8). The mean values of these antibodies were 416.11 319.11 mIU/ml. We noticed significant differences between patients with positive and negative antiTPO antibodies related with the onset age of diabetes (were younger) (4.86 3.94 vs. 8.62 0.51), the duration of diabetes (much longer) (9.23 4.05 vs. 8.12 0.64 years), the values of HbA1c (more elevated) (8.91 2.03 vs.7.55 1.96%) and the TSH values (much lower) (12.00 23.7 vs. 14.77 17.03 mIU/ml) than those with negative antiTPO AB. (p<0.001). The incidence of positive titers antiTg AB was the same as for antiTPO antibodies. In 45 cases both antibodies were increased. We also noticed significant differences related with ATAB between the age groups 10-14 years and 15-17 years (p <0.001), values being higher in the age group 10-14 years (puberty) to age group 15 -17 years (adolescence), but not between another age groups. Conclusions: Because early age of diabetes mellitus was smaller at patients with positive antiTPO antibodies, it is necessary to determine the possibility of association of autoimmune diseases, especially for thyroid diseases (higher risk for those with autoimmune chronic thyroiditis for hypothyroidism) for all patients with type 1 diabetes mellitus Although it is recommended that antithyroid antibodies, particularly those antiTPO positive to be determinate at children and adolescents with type 1 diabetes at the onset of diabetes, or at the latest before puberty. If the antibodies are positive should be tested the thyroid function and due the thyroid ultrasound to minimize the risk of undiagnosed hypothyroidism in young patients with type 1 diabetes.

Activation of neurokinin‐1 receptors up‐regulates substance P and neurokinin‐1 receptor expression in murine pancreatic acinar cells

Acute pancreatitis (AP) has been associated with an up-regulation of substance P (SP) and neurokinin-1 receptor (NK1R) in the pancreas. Increased SP-NK1R interaction was suggested to be pro-inflammatory during AP. Previously, we showed that caerulein treatment increased SP/NK1R expression in mouse pancreatic acinar cells, but the effect of SP treatment was not evaluated. Pancreatic acinar cells were obtained from pancreas of male swiss mice (25–30 g). We measured mRNA expression of preprotachykinin-A (PPTA) and NK1R following treatment of SP (10−6M). SP treatment increased PPTA and NK1R expression in isolated pancreatic acinar cells, which was abolished by pretreatment of a selective NK1R antagonist, CP96,345. SP also time dependently increased protein expression of NK1R. Treatment of cells with a specific NK1R agonist, GR73,632, up-regulated SP protein levels in the cells. Using previously established concentrations, pre-treatment of pancreatic acinar cells with Gö6976 (10 nM), rottlerin (5 μM), PD98059 (30 μM), SP600125 (30 μM) or Bay11-7082 (30 μM) significantly inhibited up-regulation of SP and NK1R. These observations suggested that the PKC-ERK/JNK-NF-κB pathway is necessary for the modulation of expression levels. In comparison, pre-treatment of CP96,345 reversed gene expression in SP-induced cells, but not in caerulein-treated cells. Overall, the findings in this study suggested a possible auto-regulatory mechanism of SP/NK1R expression in mouse pancreatic acinar cells, via activation of NK1R. Elevated SP levels during AP might increase the occurrence of a positive feedback loop that contributes to abnormally high expression of SP and NK1R.

Role of protein kinase C in caerulein induced expression of substance P and neurokinin‐1‐receptors in murine pancreatic acinar cells

Substance P (SP) is involved in the pathophysiology of acute pancreatitis (AP) via binding to its high-affinity receptor, neurokinin-1-receptor (NK1R). An up-regulation of SP and NK1R expression was observed in experimental AP and in caerulein-stimulated pancreatic acinar cells. However, the mechanisms that lead to this up-regulation are not fully understood. In this study, we showed the role of protein kinase C (PKC) in caerulein-induced SP and NK1R production in isolated mouse pancreatic acinar cells. Caerulein (10−7 M) stimulation rapidly activated the conventional PKC-α and novel PKC-δ as observed by the phosphorylation of these molecules. Pre-treatment of pancreatic acinar cells with Gö6976 (1–10 nM) and rottlerin (1–10 μM) inhibited PKC-α and PKC-δ phosphorylation, respectively, but not the other way round. At these concentrations used, PKC-α and PKC-δ inhibition reversed the caerulein-induced up-regulation of SP and NK1R, indicating an important role of PKCs in the modulation of SP and NK1R expression. Further experiments looking into signalling mechanisms showed that treatment of pancreatic acinar cells with both Gö6976 and rottlerin inhibited the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). Inhibition of PKC-α or PKC-δ also affected caerulein-induced transcription factor activation, as represented by nuclear factor-κB and AP-1 DNA-binding activity. The findings in this study suggested that PKC is upstream of the mitogen-activated protein kinases and transcription factors, which then lead to the up-regulation of SP/NK1R expression in caerulein-treated mouse pancreatic acinar cells.

Abstract 1123: Transforming growth factor α signaling in pancreatic acinar-to-ductal metaplasia

Abstract Pancreatic cancer is the most lethal type of cancer due to its high proliferation, metastasis and resistance to chemotherapy. In pancreatic cancer, pancreatic ductal adenocacinoma (PDAC) is the most common form. Current opinion is that PDAC originates from acinar or centroacinar cells though a process called “acinar-to-ductal metaplasia” (ADM). During ADM, acinar cells undergo a transformation to ductal PanIN (pancreatic intraepithelial neoplasia) cells, which is believed as the first step in PDAC formation. Transforming growth factor α (TGFα) is produced by pancreatic cancer cells and promotes tumor growth. In an ex vivo explant culture system, TGFα transforms murine pancreatic acinar cells into ductal cells. Here we describe a cell signaling pathway that TGFα utilizes to induce acinar-to-ductal transformation. We show that K-ras is downstream of TGFα and is sufficient to drive ADM. We further show the mechanisms that K-ras uses to induce cell structural rearrangement of acinar cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1123. doi:10.1158/1538-7445.AM2011-1123

Reactive Oxygen Species Induced by Bile Acid Induce Apoptosis and Protect Against Necrosis in Pancreatic Acinar Cells

Oxidative stress is implicated in the pathogenesis of pancreatitis, but clinical trials of antioxidants have produced conflicting results. We examined the role of intracellular reactive oxygen species (ROS) in pancreatic acinar cell injury. Freshly isolated murine and human pancreatic acinar cells were studied using confocal microscopy to measure changes in intracellular and mitochondrial ROS concentrations ([ROS]I and [ROS]M), cytosolic and mitochondrial calcium concentrations ([Ca2+]C and [Ca2+]M), reduced nicotinamide adenine dinucleotide phosphate levels, and death pathways in response to taurolithocholate acid sulfate (TLC-S) or the oxidant menadione. Ca2+-activated Cl- currents were measured using whole-cell patch clamp, with or without adenosine triphosphate (ATP). TLC-S induced prolonged increases in [Ca2+]C and [Ca2+]M, which led to dose-dependent increases in [ROS]I and [ROS]M, impaired production of ATP, apoptosis, and necrosis. Inhibition of the antioxidant reduced nicotinamide adenine dinucleotide phosphate quinine oxidoreductase by 2,4-dimethoxy-2-methylnaphthalene potentiated the increases in [ROS]I and apoptosis but reduced necrosis, whereas the antioxidant N-acetyl-L-cysteine reduced [ROS]I and apoptosis but increased necrosis. Inhibition of mitochondrial ROS production prevented apoptosis but did not alter necrosis; autophagy had no detectable role. Patched ATP prevented sustained increases in [Ca2+]C and necrosis. Increases in [ROS]M and [ROS]I during bile acid injury of pancreatic acinar cells promote apoptosis but not necrosis. These results indicate that alternative strategies to antioxidants are required for oxidative stress in acute pancreatitis.

Cholecystokinin-58 and cholecystokinin-8 exhibit similar actions on calcium signaling, zymogen secretion, and cell fate in murine pancreatic acinar cells

The gastrointestinal hormone CCK exists in various molecular forms, with differences in bioactivity between the well-characterized CCK-8 and larger CCK-58 previously reported. We have compared the effects of these peptides on cytosolic calcium concentration ([Ca(2+)](c)), mitochondrial metabolism, enzyme secretion, and cell fate in murine isolated pancreatic acinar cells using fluorescence confocal microscopy and patch-clamp electrophysiology. CCK-58 (1-10 pM) induced transient, oscillatory increases of [Ca(2+)](c), which showed apical to basolateral progression and were associated with a rise of mitochondrial NAD(P)H. CCK-58 (10 pM) induced zymogen exocytosis in isolated cells and amylase secretion from isolated cells and whole tissues. Hyperstimulation with supraphysiological CCK-58 (5 nM) induced a single large increase of [Ca(2+)](c) that declined to a plateau, which remained above the basal level 20 min after application and was dependent on external Ca(2+) entry. In cells dispersed from the same tissues, CCK-8 induced similar patterns of responses to those of CCK-58, with oscillatory increases of [Ca(2+)](c) at lower (pM) concentrations and sustained responses at 5 nM. CCK-58 and CCK-8 exhibited similar profiles of action on cell death, with increases in necrosis at high CCK-58 and CCK-8 (10 nM) that were not significantly different between peptides. The present experiments indicate that CCK-8 and CCK-58 have essentially identical actions on the acinar cell at high and low agonist concentrations, suggesting an action via the same receptor and that the differences observed in an intact rat model may result from indirect effects of the peptides. Our data strengthen the argument that CCK-58 is an important physiological form of this gastrointestinal hormone.