c-Jun/AP-1 is required for CCK-induced pancreatic acinar cell dedifferentiation and DNA synthesis in vitro

Endogenous CCK plays an important role in pancreatic regeneration after pancreatitis. We used primary culture of mouse pancreatic acinar cells to evaluate the effect of CCK on acinar cell morphology and gene expression and to determine signaling pathways required for proliferation of acinar cells in vitro. Over 4 days in culture, cells grew out from acini and formed patches of monolayer, which displayed a reduced expression of acinar cell markers including digestive enzymes and Mist1 and an increased expression of ductal and embryonic markers, including cytokeratin 7, β-catenin, E-cadherin, pdx-1, and nestin. There was no appearance of stellate cell markers. CCK enhanced cellular spreading, DNA synthesis, and cyclin D1 expression. When signaling pathways were evaluated, CCK stimulation increased c-Jun expression, JNK and ERK activity, and AP-1 activation. Chemical inhibitors of JNK and ERK pathways, dominant-negative JNK and c-Jun, and c-Jun shRNA significantly inhibited CCK-induced DNA synthesis, CCK-induced AP-1 activation, and cyclin D1 expression. Furthermore, dominant-negative c-Jun reduced the increased expression of β-catenin and the decreased expression of amylase during culture. These results show that MAPK/c-Jun/AP-1 pathway plays an important role in pancreatic acinar cell dedifferentiation and proliferation in culture. Monolayer culture can serve as a model to study acinar cell proliferation similar to regeneration after pancreatitis in vivo.     

Morphometrical and immunocytochemical studies on rat pancreatic acinar cells under control and experimental conditions

Process of amylase and chymotrypsinogen secretion by acinar cells has been studied applying morphological and biochemical approaches. Three conditions were investigated; resting (fed control), cholinergic stimulation and fasting. Morphometrical evaluations have shown that under stimulation, the volume density of zymogen granules decreases drastically while that of the Golgi apparatus increases. This may result from the enhancement in protein processing and the rapid discharge. Quantitation of amylase and chymotrypsinogen immunolabelings present over the cellular compartments has shown that there is no difference in the intensities between tissues from control and stimulated animals. These results imply that total amounts of protein processed by the Golgi apparatus are markedly enhanced primarily because of the increase in size of the organelle, the amounts of protein processed per unit surface remaining unchanged. Under starvation where reduction of secretion occurs, there is a significant decrease in the volume density of the Golgi apparatus but no variation in that of the zymogen granules. However, the morphological aspect of these was markedly altered since many of them present an electron luscent periphery which was devoid of immunolabeling for amylase and chymotrypsinogen. Quantitation of amylase and chymotrypsinogen immunolabelings has shown significant diminution for both enzymes. In both experimental conditions, the volume density of lysosomes was enhanced, however in none of these conditions evidence of crinophagy was observed. The morphometrical and immunocytochemical results were consistent with those obtained from biochemical determination of amylase and chymotrypsinogen contents in tissues. Correlations between results obtained from morphometric and immunocytochemical studies were made leading to a better understanding of the cellular secretory activity during experimental conditions.     

An implication of novel methodology to study pancreatic acinar mitochondria under in situ conditions

Mitochondria maintain numerous energy‐consuming processes in pancreatic acinar cells, yet characteristics of pancreatic mitochondrial oxidative phosphorylation in native conditions are poorly studied. Besides, it is not known which type of solution is most adequate to preserve functions of pancreatic mitochondria in situ. Here we propose a novel experimental protocol suitable for in situ analysis of pancreatic mitochondria metabolic states. Isolated rat pancreatic acini were permeabilized with low doses of digitonin. Different metabolic states of mitochondria were examined in KCl‐ and sucrose‐based solutions using Clark oxygen electrode. Respiration of digitonin‐treated, unlike of intact, acini was substantially intensified by succinate or mixture of pyruvate plus malate. Substrate‐stimulated respiration rate did not depend on solution composition. In sucrose‐based solution, oligomycin inhibited State 3 respiration at succinate oxidation by 65.4% and at pyruvate plus malate oxidation by 60.2%, whereas in KCl‐based solution, by 32.0% and 36.1%, respectively. Apparent respiratory control indices were considerably higher in sucrose‐based solution. Rotenone or thenoyltrifluoroacetone severely inhibited respiration, stimulated by pyruvate plus malate or succinate, respectively. This revealed low levels of non‐mitochondrial oxygen consumption of permeabilized acinar cells. These results suggest a stronger coupling between respiration and oxidative phosphorylation in sucrose‐based solution. Copyright © 2012 John Wiley & Sons, Ltd.     

Folate deficiency and pancreatic acinar cell function

The present study was designed to determine the effect of folate deficiency on pancreatic acinar cell function. In the first series of experiments, three groups of rats were fed ad libitum regular rat feed, folate-deficient diet, or an equivalent amount of folate-sufficient diet. In the second series of experiments, rats were either fed ad libitum or rendered folate deficient by a purified folate-deficient diet; half of the folate-deficient group was replenished with oral folate. Body weight, pancreatic weight, DNA [methyl-14C]thymidine incorporation into DNA, RNA, [8-14C]adenine incorporation into RNA, protein content, synthesis of proteins, amylase content, and basal and bethanechol-stimulated amylase secretion were determined. The parameters were the same in the rats fed a folate-sufficient diet as in those fed a regular rat feed. Feeding a folate-deficient diet resulted in impaired DNA synthesis as evidenced by diminished incorporation of [methyl-14C]thymidine into DNA. There was no change in secretion of amylase. Similar results were obtained in the second series of experiments. These studies indicate that folate deficiency (rather than antibiotic content of the diet) impaired pancreatic function. Folate deficiency may therefore contribute to pancreatic injury in malnutrition and alcoholism.     

Inhibition of P-glycoprotein stimulates cell death under Hypoxia-mimicking conditions

The most common drug resistance mechanism in tumor cells is expression on their surface of the energy-dependent pump like P-glycoprotein (P-gp) that expels chemotherapeutic agents from the interior. An imitation of the hypoxic condition by the iron chelator deferoxamine caused Hypoxia-inducible factor 1-alpha (HIF-1α) stabilization and inhibition of doxorubicin-induced apoptosis in colon cancer НСТ116 cells. P-gp blocker verapamil suppressed doxorubicin accumulation leading to cell death induction. Considering these results, P-gp may be used as a potential target to stimulate chemotherapeutic drugs activity that will contribute to more efficient tumor cells elimination.     

Diurnal pattern of rat pancreatic acinar cell replication

Fully differentiated pancreatic acinar cells can enter the cell cycle under appropriate conditions in the rat. The aim of this study was to analyse the diurnal pattern of acinar cell proliferation as a function of food intake and the release of cholecystokinin (CCK), because the peptide hormone CCK is a major physiological regulator of rat pancreatic acinar cell replication. Pancreatic acinar cell replication was quantitated using an antibody against the S-phase marker proliferating cell nuclear antigen (PCNA). In addition, acinar cells in S-phase were detected after injecting bromodeoxyuridine (BrdU) and subsequent immunohistochemical staining of BrdU-positive nuclei. Rat pancreata were analysed during the day under standard diet conditions, as well as after various schedules of fasting and refeeding and after the application of the CCK receptor antagonist L-364,718. Between 6 a.m. and 6 p.m., the PCNA labeling index was 4.4±0.9%, while between 9 p.m. and 3 a.m. the PCNA labeling index was elevated and reached peak values of 11.4% (mean value: 7.8±2.5%) around midnight. BrdU-positive cells also doubled around midnight, compared to the 9:00 a.m. value. In fasted rats, acinar cell proliferation was completely suppressed and this suppression could be overcome by injection of the CCK analog cerulein. In addition, the CCK antagonist L-364,718 led to the same results as fasting. Here we show for the first time that there is a diurnal pattern of pancreatic acinar cell proliferation in rats, which is dependent on food intake and is mediated by CCK.     

Haematococcus pluvialis cultivation in split-cylinder internal-loop airlift photobioreactor under aeration conditions avoiding cell damage

The effects of superficial gas velocity in the riser (U_Gr) and gas entrance velocity (v) on the growth of Haematococcus pluvialis cultivated in a split-cylinder internal-loop airlift photobioreactor were investigated. Cell growth decreased when U_Gr and v were increased above 12 mm s^–1 and 22.8 m s^–1, respectively. The maximum cell density of H. pluvialis was 110×10⁴ vegetative cells ml^–1 and the chlorophyll-a titer was 7 mg l^–1. The cell damage in the photobioreactor was greater when v was increased by an increase in U_Gr rather than by a decrease in sparger internal diameter. The overall volumetric mass transfer coefficient (k_La) of the photobioreactor was measured at the same U_Gr (6–24 mm s^–1) and v (12–80 m s^–1). The k_La values reached in the airlift photobioreactor were between 10 h^–1 and 32 h^–1.     

Early changes in pancreatic acinar cell calcium signaling after pancreatic duct obstruction

Intracellular Ca(2+)-changes not only participate in important signaling pathways but have also been implicated in a number of disease states including acute pancreatitis. To investigate the underlying mechanisms in an experimental model mimicking human gallstone-induced pancreatitis, we ligated the pancreatic duct of Sprague-Dawley rats and NMRI mice for up to 6 h and studied intrapancreatic changes including the dynamics of [Ca(2+)](i) in isolated acini. In contrast to bile duct ligation, pancreatic duct obstruction induced intra-pancreatic trypsinogen activation, leukocytosis, hyperamylasemia, and pancreatic edema and increased lung myeloperoxidase activity. Although resting [Ca(2+)](i) in isolated acini rose by 45% to 205 +/- 7 nmol, the acetylcholine- and cholecystokinin (CCK)-stimulated calcium peaks as well as the amylase secretion declined, but neither the [Ca(2+)](i)-signaling pattern nor the amylase output in response to the Ca(2+)-ATPase inhibitor thapsigargin nor the secretin-stimulated amylase release were impaired by pancreatic duct ligation. On the single cell level pancreatic duct ligation reduced the percentage of cells in which submaximal secretagogue stimulation was followed by a physiological response (i.e. Ca(2+) oscillations) and increased the percentage of cells with a pathological response (i.e. peak plateau or absent Ca(2+) signal). Moreover, it reduced the frequency and amplitude of Ca(2+) oscillation as well as the capacitative Ca(2+) influx in response to secretagogue stimulation. Serum pancreatic enzyme elevation as well as trypsinogen activation was significantly reduced by pretreatment of animals with the calcium chelator BAPTA-AM. These experiments suggest that pancreatic duct obstruction rapidly changes the physiological response of the exocrine pancreas to a Ca(2+)-signaling pattern that has been associated with premature digestive enzyme activation and the onset of pancreatitis, both of which can be prevented by administration of an intracellular calcium chelator.     

Scanning electron microscopy of dissociated pancreatic acinar cell surfaces

Summary The pancreatic acinar cell surfaces have been studied by SEM with a dissection technique and correlated with results obtained by TEM. The SEM results demonstrate characteristic arrangement of microplicae which in some areas are densely packed.In many areas, the microplicae are distributed in such a manner that they create zones with typical geometrical shapes and show a relatively smooth surface.These smooth areas may coincide, as indicated by correlated TEM results, with the limits of intimate contact between adjacent acinar cells which, in turn, represent part of the junctional complex. Another aspect revealed by these SEM preparations concerns the presence of groups of densely packed microplicae, arranged in regular rows and distributed along some grooves and/or infoldings of the cellular surface. On the basis of SEM and TEM information, it is likely that these structures correspond to intercellular (and possibly, in some cases, intracellular) canaliculi which topographically form a kind of extensive microlabyrinthine arrangement running along all the cell sides.One final point revealed by fractured samples concerns the finding of spherical zymogen droplets within the vesicles of the Golgi complex. Because in many scanning images these vesicles appear connected by small openings, it is suggested that they may represent a system of intercommunicating chambers (vacuoles) through which the zymogen droplets can be continuously accumulated and discharged into the acinar lumen.     

Role of apoptotic and necrotic cell death under physiologic conditions

Apoptosis is considered to be a programmed and controlled mode of cell death, whereas necrosis has long been described as uncontrolled and accidental cell death resulting from extremely harsh conditions. In the following review, we will discuss the features and physiological meanings as well as recent advances in the elucidation of the signaling pathways of both apoptotic cell death and programmed necrotic cell death.     

Zymogen activation in a reconstituted pancreatic acinar cell system

Pathological activation of digestive zymogens within the pancreatic acinar cell initiates acute pancreatitis. Cytosolic events regulate this activation within intracellular compartments of unclear identity. In an in vivo model of acute pancreatitis, zymogen activation was detected in both zymogen granule-enriched and microsomal cellular fractions. To examine the mechanism of this activation in vitro, a reconstituted system was developed using pancreatic cytosol, a zymogen granule-enriched fraction, and a microsomal fraction. Addition of cytosol to either particulate fraction resulted in a prominent increase in both trypsin and chymotrypsin activities. The percentage of the pool of trypsinogen and chymotrypsinogen activated was about twofold and sixfold greater, respectively, in the microsomal than in the zymogen granule-enriched fraction. Activation of chymotrypsinogen but not trypsinogen was significantly enhanced by ATP (5 mM) but not by the inactive ATP analog AMP-PNP. The processing of procarboxypeptidase B to its mature form also demonstrated a requirement for ATP and cytosol. E64d, an inhibitor of cathepsin B, a thiol protease that can activate trypsin, completely inhibited trypsin activity but did not affect chymotrypsin activity or carboxypeptidase B generation. These studies demonstrate that both zymogen granule-enriched and microsomal fractions from the pancreas can support cytosol-dependent zymogen activation. A component of the activation of some zymogens, such as chymotrypsinogen and procarboxypeptidase, may depend on ATP but not on trypsin or cathepsin B.     

Accumulation of dinucleoside polyphosphates in Saccharomyces cerevisiae under stress conditions. High levels are associated with cell death

Adenosine tetraphosphonucleosides (Ap4X) were measured in Saccharomyces cerevisiae by a coupled phosphodiesterase-luciferase assay. After exposure of the cells to cadmium or to hyperthermic treatment (46 degrees C) a marked increase of the cellular pool from 0.08 microM (base level) to 4 microM or higher was observed. The accumulation of Ap4X to high levels is associated with irreversible processes leading to cell death.     

Secretagogues differentially activate endoplasmic reticulum stress responses in pancreatic acinar cells

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

Serine proteases mediate apoptosis-like cell death and phagocytosis under caspase-inhibiting conditions

Effective execution of apoptosis requires the activation of caspases. However, in many cases, broad-range caspase inhibitors such as Z-VAD.fmk do not inhibit cell death because death signaling continues via basal caspase activities or caspase-independent processes. Although death mediators acting under caspase-inhibiting conditions have been identified, it remains unknown whether they trigger a physiologically relevant cell death that shows typical signs of apoptosis, including phosphatidylserine (PS) exposure and the removal of apoptotic cells by phagocytosis. Here we show that cells treated with ER stress drugs or deprived of IL-3 still show hallmarks of apoptosis such as cell shrinkage, membrane blebbing, mitochondrial release of cytochrome c, PS exposure and phagocytosis in the presence of Z-VAD.fmk. Cotreatment of the stressed cells with Z-VAD.fmk and the serine protease inhibitor Pefabloc (AEBSF) inhibited all these events, indicating that serine proteases mediated the apoptosis-like cell death and phagocytosis under these conditions. The serine proteases were found to act upstream of an increase in mitochondrial membrane permeability as opposed to the serine protease Omi/HtrA2 which is released from mitochondria at a later stage. Thus, despite caspase inhibition or basal caspase activities, cells can still be phagocytosed and killed in an apoptosis-like fashion by a serine protease-mediated mechanism that damages the mitochondrial membrane.     

The regulation of exocytosis in the pancreatic acinar cell.

The pancreatic acinar cell synthesises a variety of digestive enzymes. In transit through the secretory pathway, these enzymes are separated from constitutively secreted proteins and packaged into zymogen granules, which are localised in the apical pole of the cell. Stimulation of the cell by secretagogues such as acetylcholine and cholecystokinin, acting at receptors on the basolateral plasma membrane, causes the generation of an intracellular Ca(2+) signal. This signal, in turn, triggers the fusion of the zymogen granules with the apical plasma membrane, leading to the polarised secretion of the enzymes. This review describes recent advances in our understanding of the control of secretion in the acinar cell. In particular, we discuss the mechanisms underlying the sorting of digestive enzymes into the zymogen granules, the molecular components of the exocytotic "membrane fusion machine," the generation and propagation of the Ca(2+ signal and the development of new techniques for the visualisation of single granule fusion events.     

Zinc chelator TPEN induces pancreatic cancer cell death through causing oxidative stress and inhibiting cell autophagy

The essential trace element zinc (Zn) is widely required in cellular functions, and abnormal Zn homeostasis causes a variety of health problems including immunodeficiency and sensory dysfunctions. Previous studies had shown that Zn availability was also important for tumor growth and progression. The aim of the present study was to investigate the potential mechanisms of N,N,N,N-Tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) (a membrane permeable zinc chelator) induced pancreatic cancer cell death. The text of inductively coupled plasma-mass spectrometry (ICP-MS) showed in human pancreatic cancer samples that the zinc content in cancer was higher than that in adjacent tissues. The pancreatic cancer cell lines Panc-1, 8988T, BxPc-3, and L3.6 were used in this study. Our results indicated that TPEN markedly induced cell death, via increasing reactive oxygen species (ROS) and restraining autophagy. Our data also indicated that TPEN-stimulated mitochondrial metabolism produced much ROS. Meanwhile, TPEN reduced the levels of glutathione (GSH) and triggered ROS outbreak, which were the main causes of cell death. In addition, cell autophagy was significantly depressed in Panc-1 cells treated by TPEN, which was due to the ability of disrupting lysosomal by TPEN. Thus, we thought zinc depletion by TPEN was a potential therapeutic strategy for pancreatic cancer.     

Mathematical models of bacterial growth,inhibition and death under combined stress conditions

Summary In this report we review the history of growth theories. We show how classical growth models may be derived as special cases of a generic growth rate equation. We show how growth models may be modified to represent survival data. We use linear combinations of growth and survival models to represent complex growth/survival curves and give practical examples utilizing nonlinear regression analysis. We show that traditional methods of estimating D values are inappropriate for complex, multiphasic growth/survival data. We show how such data may be modeled mathematically and illustrate methods for estimating true D values from such data.