Ethanol induces secretion of oxidized proteins by pancreatic acinar cells

The pancreas is vulnerable to ethanol toxicity, but the pathogenesis of alcoholic pancreatitis is not fully defined. The intracellular oxidative balance and the characteristics of the secretion of isolated rat pancreatic acinar cells stimulated with the cholecystokinin analogue cerulein were assayed after acute oral ethanol (4 g/kg) load. Pancreatic acinar cells from ethanol-treated rats showed a significant (p < 0.02) lower content of total glutathione and protein sulfhydryls, and higher levels of oxidized glutathione (p < 0.03), malondialdehyde, and protein carbonyls (p < 0.05). Ethanol-intoxicated acinar cells showed a lower baseline amylase output compared to controls, with the difference being significantly exacerbated by cerulein stimulation. After cerulein, the release of protein carbonyls by ethanol-treated cells was significantly increased, whereas that of protein sulfhydryls was significantly decreased. In conclusion, ethanol oxidatively damages pancreatic acinar cells; cerulein stimulation is followed by a lower output of amylase and by a higher release of oxidized proteins by pancreatic acinar cells from ethanol-treated rats. These findings may account for the decreased exocrine function, intraductular plug formation, and protein precipitation in alcoholic pancreatitis.     

Enzymic profiles of bovine pancreatic ductal and acinar tissues.

The plasma membrane enzymes, alkaline phosphatase, bicarbonate-dependent adenosine triphosphatase, 5'-nucleotidase, and carbonate dehydratase, were measured in ductal and acinar preparations of bovine pancreas. Epithelial cells were scraped from the main duct and a piece of acinar tissue was dissected from the whole pancreas for homogenization. All enzymes studied demonstrated higher levels in the duct per milligram protein than in the acinus: bicarbonate-dependent adenosine triphosphatase was 2.8 times higher; 5'-nucleotidase, 4.1 times higher; carbonate dehydratase, 16.9 times higher, while alkaline phosphatase showed only a slight increase in the duct compared to acini.     

Comparative pyrimidine- and purine-specific RNAse-gold labeling on pancreatic acinar cells and isolated hepatocytes.

We applied the enzyme-gold approach to investigate the potential of various ribonucleases displaying different affinities for ultrastructural localization of particular RNA molecules. Five specific ribonucleases were used: three from a pancreatic source, RNAses A, B, and S with affinities for pyrimidine bases; and two from Aspergillus oryzae, RNAses T1 and T2 specific for purine bases. Conditions required for preparing each RNAse-gold complex, as well as for obtaining specific labelings, were determined. Application of the probes on thin sections of pancreatic acinar cells yielded labeling patterns that differed according to the enzyme used. Pancreatic RNAses labeled mostly the rough endoplasmic reticulum and the nucleolus, whereas fungal RNAses labeled more intensely the interchromatin space and the nucleolus, the rough endoplasmic reticulum being labeled to a lesser extent. Areas rich in interchromatin granules were intensely labeled by the RNAses T1 and T2. This was confirmed on DRB-treated hepatocytes, which displayed large clusters of interchromatin granules. Perichromatin granules were labeled by the RNAse A- and T1-gold complexes. These results provide a strong indication for the presence of RNA molecules in both types of granules. Nuclear pores were labeled, particularly by the RNAses T1 and T2, thus supporting the hypothesis for the site of RNA transit between nucleus and cytoplasm. The differences in patterns of labeling among the various enzyme-gold complexes could be related to differences in affinities. The use of a panel of specific RNAses, displaying different affinities, could thus allow for the topographical distribution of particular RNA molecules according to their relative content of specific bases.     

Dexamethasone effects on somatostatin receptors in pancreatic acinar AR4-2J cells

The effects of glucocorticoids on somatostatin binding and cAMP response in the rat pancreatic acinar carcinoma AR4-2J cell line were examined. Dexamethasone treatment reduced the number of somatostatin receptors 2.5 fold without any change in receptor affinity. In addition, dexamethasone increased the sensitivity of the cells to somatostatin-inhibited cAMP formation and restored the biphasic pattern of cAMP response to somatostatin previously observed in normal pancreatic acinar cells. Such effect may be associated with the glucocorticoid-promoted cellular pancreatic differentiation of AR4-2J cells.     

Conversion of pancreatic cells to hepatocytes

Transdifferentiation is the name used to describe the conversion of one differentiated cell type to another. During development, the liver and pancreas arise from the same region of the endoderm and cells from the two organs can transdifferentiate in the adult under different experimental procedures. We have produced two in vitro models for the transdifferentiation of pancreatic cells to hepatocytes. The first utilizes a pancreatic exocrine cell line AR42J-B13 and the second comprises cultures of mouse embryonic pancreas. We have analysed the pancreatic hepatocytes and they express a range of liver markers including albumin, transferrin and transthyretin. We also present evidence for (i) the molecular mechanism which regulates the conversion between pancreas and liver and (ii) the cellular basis of the switch in phenotype.     

Commitment and oncogene-induced plasticity of human stem cell-derived pancreatic acinar and ductal organoids

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Crambene induces pancreatic acinar cell apoptosis via the activation of mitochondrial pathway

We investigated the apoptotic pathway activated by crambene (1-cyano-2-hydroxy-3-butene), a plant nitrile, on pancreatic acinar cells. As evidenced by annexin V-FITC staining, crambene treatment for 3 h induced the apoptosis but not necrosis of pancreatic acini. Caspase-3, -8, and -9 activities in acini treated with crambene were significantly higher than in untreated acini. Treatment with caspase-3, -8, and -9 inhibitors inhibited annexin V staining, as well as caspase-3 activity, pointing to an important role of these caspases in crambene-induced acinar cell apoptosis. The mitochondrial membrane potential was collapsed, and cytochrome c was released from the mitochondria in crambene-treated acini. Neither TNF-alpha nor Fas ligand levels were changed in pancreatic acinar cells after crambene treatment. These results provide evidence for the induction of pancreatic acinar cell apoptosis in vitro by crambene and suggest the involvement of mitochondrial pathway in pancreatic acinar cell apoptosis.     

Protein malnutrition and response of pancreatic acinar cells to stimulation by cholecystokinin

Pancreatic lobules were isolated from 2 groups of male Wistar rats after 23 days of diet. A control group (C) fed on a 20% protein diet (16% gluten + 4% casein) and an experimental group (E) on a 5% protein diet (4% gluten + 1% casein). After isolation, lobules were preincubated 10 min with 10 muCi [3H]-leucine, washed, then incubate within Krebs Ringer bicarbonate Hepes. Basal secretion, then stimulated secretion (50 pM of cholecystokinin (CCK] of radioactive and non-radioactive protein and amylase outputs were measured. During basal secretion, in (E) group, lobules secreted more proteins than (C) one, the same outputs of amylase and radioactive protein were observed in both groups. The stimulated secretion by CCK increased the outputs of non-radioactive protein and amylase of lobules (T) (2-3 fold), but was without effect on lobule (E) outputs. Therefore, a low-protein diet involved a decrease of CCK sensibility on acinar cells, this fact might be mediated by a decreasing number and/or affinity of their CCK receptors.     

Calcium-magnesium interactions in pancreatic acinar cells.

Although the role of calcium (Ca2+) in the signal transduction and pathobiology of the exocrine pancreas is firmly established, the role of magnesium (Mg2+) remains unclear. We have characterized the intracellular distribution of Mg2+ in response to hormone stimulation in isolated mouse pancreatic acinar cells and studied the role of Mg2+ in modulating Ca2+ signaling using microspectrofluorometry and digital imaging of Ca2+- or Mg2+-sensitive fluorescent dyes as well as Mg2+-sensitive intracellular microelectrodes. Our results indicate that an increase in intracellular Mg2+ concentrations reduced the cholecystokinin (CCK) -induced Ca2+ oscillations by inhibiting the capacitive Ca2+ influx. An intracellular Ca2+ mobilization, on the other hand, was paralleled by a decrease in [Mg2+]i, which was reversible upon hormone withdrawal independent of the electrochemical gradients for Mg2+, Ca2+, Na+, and K+, and not caused by Mg2+ efflux from acinar cells. In an attempt to characterize possible Mg2+ stores that would explain the reversible, hormone-induced intracellular Mg2+ movements, we ruled out mitochondria or ATP as potential Mg2+ buffers and found that the CCK-induced [Mg2+]i decrease was initiated at the basolateral part of the acinar cells, where most of the endoplasmic reticulum (ER) is located, and progressed from there toward the apical pole of the acinar cells in an antiparallel fashion to Ca2+ waves. These experiments represent the first characterization of intracellular Mg2+ movements in the exocrine pancreas, provide evidence for possible Mg2+ stores in the ER, and indicate that the spatial and temporal distribution of intracellular Mg concentrations profoundly affects acinar cell Ca2+ signaling.     

Non-uniform distribution of mitochondria in pancreatic acinar cells

The distribution of mitochondria in pancreatic acinar cells was investigated using confocal fluorescence microscopy and transmission electron microscopy (EM). Acinar cells were studied either after enzymatic isolation or in small segments of undisassociated pancreatic tissue. Loading of isolated acinar cells with Mito Tracker Green or Red, a fluorescence mitochondrial probe, showed that mitochondria are predominantly situated in the perigranular, subplasmalemmal and perinuclear regions. Subsequent applications of EM fixatives induced a leak of the fluorescent indicator to the cytosol but did not change the distribution of mitochondria. EM was then performed on isolated acinar cells and on acinar cells of pancreatic tissue segments. The intracellular distribution of mitochondria was quantified by calculating the percentage of the cross-sectional area that was occupied by mitochondria. In isolated acinar cells the highest density of mitochondria was seen in the perigranular region, where mitochondria occupied 25.69±1.58% of the area, then the subplasmalemmal region with 12.61±0.77% and the perinuclear region with 9.07±0.97% (n=26). Similar results were obtained from acinar cells of pancreatic tissue segments: the perigranular 22.9±1.95%, subplasmalemmal 12.45±0.78% and perinuclear regions 9.07±0.97% (n=26). The outer mitochondrial membranes were frequently positioned close to membranes of the ER, which followed the outer contour of mitochondria. Mitochondria were never found in direct contact with the nuclear envelope: there were usually layers of ER between the mitochondrial and nuclear membranes. Subplasmalemmal mitochondria were found in a very close proximity to the plasma membrane with no ER layers between the mitochondrial and the corresponding plasma membranes. We conclude that in pancreatic acinar cells mitochondria are preferentially distributed to perigranular, subplasmalemmal and perinuclear regions and this distribution is not affected by isolation or fixation procedures.P.R. Johnson and N.J. Dolman contributed equally to the study. This work was supported by a Medical Research Council programme grant. P.R.J. is a Medical Research Council PhD student and N.J.D. is a Wellcome Trust PhD student.     

Substance P treatment stimulates chemokine synthesis in pancreatic acinar cells via the activation of NF-kappaB

Acinar cell injury early in acute pancreatitis leads to a local inflammatory reaction and to the subsequent systemic inflammatory response, which may result in multiple organ dysfunction and death. Inflammatory mediators, including chemokines and substance P (SP), are known to play a crucial role in the pathogenesis of acute pancreatitis. It has been shown that pancreatic acinar cells produce the chemokine monocyte chemoattractant protein-1 (MCP-1) in response to caerulein hyperstimulation, demonstrating that acinar-derived MCP-1 is an early mediator of inflammation in acute pancreatitis. Similarly, SP levels in the pancreas and pancreatic acinar cell expression of neurokinin-1 receptor, the primary receptor for SP, are both increased during secretagogue-induced experimental pancreatitis. This study aims to examine the functional consequences of exposing mouse pancreatic acinar cells to SP and to determine whether it leads to proinflammatory signaling, such as production of chemokines. Exposure of mouse pancreatic acini to SP significantly increased synthesis of MCP-1, macrophage inflammatory protein-1alpha (MIP-1alpha), as well as MIP-2. Furthermore, SP also increased NF-kappaB activation. The stimulatory effect of SP was specific to chemokine synthesis through the NF-kappaB pathway, since the increase in chemokine production was completely attenuated when pancreatic acini were pretreated with the selective NF-kappaB inhibitor NF-kappaB essential modulator-binding domain peptide. This study shows that SP-induced chemokine synthesis in mouse pancreatic acinar cells is NF-kappaB dependent.     

Transgenic expression of FGF8 and FGF10 induces transdifferentiation of pancreatic islet cells into hepatocytes and exocrine cells

FGF signaling is essential for normal development of pancreatic islets. To examine the effects of overexpressed FGF8 and FGF10 on pancreatic development, we generated FGF8- and FGF10-transgenic mice (Tg mice) under the control of the glucagon promoter. In FGF8-Tg mice, hepatocyte-like cells were observed in the periphery of pancreatic islets, but areas of alpha and beta cells did not decrease, whereas in FGF10-Tg mice, pancreatic ductal and acinar cells were found in islets, concomitantly with disturbed beta-cell differentiation. These results suggest that FGF8 and FGF10 play important roles in development of hepatocytes and exocrine cells, respectively, and explain the absence of FGF8 expression in normal islets and pancreatic hypoplasia in FGF10-deficient mice.     

Pancreatitis severity in mice with impaired CFTR function but pancreatic sufficiency is mediated via ductal and inflammatory cells-Not acinar cells

Mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR) are an established risk factor for cystic fibrosis (CF) and chronic pancreatitis. Whereas patients with CF usually develop complete exocrine pancreatic insufficiency, pancreatitis patients with CFTR mutations have mostly preserved exocrine pancreatic function. We therefore used a strain of transgenic mice with significant residual CFTR function (CFTR^tm1HGU) to induce pancreatitis experimentally by serial caerulein injections. Protease activation and necrosis were investigated in isolated acini, disease severity over 24h, pancreatic function by MRI, isolated duct stimulation and faecal chymotrypsin, and leucocyte function by ex vivo lipopolysaccharide (LPS) stimulation. Pancreatic and lung injury were more severe in CFTR^tm1HGU but intrapancreatic trypsin and serum enzyme activities higher than in wild-type controls only at 8h, a time interval previously attributed to leucocyte infiltration. CCK-induced trypsin activation and necrosis in acini from CFTR^tm1HGU did not differ from controls. Fluid and bicarbonate secretion were greatly impaired, whereas faecal chymotrypsin remained unchanged. LPS stimulation of splenocytes from CFTR^tm1HGU resulted in increased INF-γ and IL-6, but decreased IL-10 secretion. CFTR mutations that preserve residual pancreatic function significantly increase the severity of experimental pancreatitis—mostly via impairing duct cell function and a shift towards a pro-inflammatory phenotype, not by rendering acinar cells more susceptible to pathological stimuli.