Effects of cholecystokinin on cytosolic calcium in human pancreatic cancer cells.

Cholecystokinin (CCK) has been shown to increase cytosolic calcium and stimulate enzyme release from pancreatic acinar cells and a rat acinar cell line, AR42J. CCK is also trophic to normal pancreas and pancreatic cancer; however, the cellular mechanisms which regulate CCK-stimulated growth are unknown. The effect of CCK on intracellular calcium was evaluated in four human pancreatic cancer cell lines known to grow in response to CCK but not secrete enzymes (SW-1990, MIA PaCa-2, BXPC-3 and PANC-1) and a rat acinar cell line (AR42J) shown to secrete enzymes but not grow with CCK. By using single cell fluorescence microscopy in fura-2 loaded cells, intracellular calcium [Ca2+]i was measured. After obtaining baseline fluorescent cell images, synthetic CCK-octapeptide (CCK8) was added to the cells and images of cell fluorescence captured. [Ca2+]i of the rat acinar cells increased (603%) over the baseline within the first minute after the addition of CCK (4.10(-13) M to 4.10(-10) M) in 77% of cells tested. In contrast [Ca2+]i failed to significantly change in the human cancer cells treated with CCK. To further localize the defect in hormone signal transduction in cancer cells, cells were suspended in low calcium media and the plasma membranes were selectively permeabilized with digitonin. Media free calcium concentration was continuously monitored by fura-2 fluorescence. Addition of inositol 1,4,5-trisphosphate (IP3) resulted in a marked increase in medium calcium concentration indicating IP3 was capable of releasing calcium from intracellular stores in both the AR42J rat acinar cell line and in the human pancreas cancer cell lines. In conclusion, CCK does not increase cytosolic calcium in human pancreatic cancer cells in contrast to rat acinar cells although all contain IP3-sensitive intracellular Ca2+ pools. Our results suggest that growth promoting and secretory effects of CCK on pancreatic cells may occur via two independent signalling pathways.     

A microplate binding assay for the somatostatin type-2 receptor (SSTR2)

The clinical importance of somatostatin type-2 receptors (SSTR2) and the study of novel analogues of somatostatin such as OctreoScan or [Tyr3]-octreotide containing DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) as metal chelator led us to develop a methodology to monitor the expression of SSTR2 on tumours of pancreatic origin (e.g. rat AR4-2J cancer cells). Usual binding assay protocols using the commercial [125I][Tyr1]-somatostatin radioligand failed, even in the presence of a cocktail of protease inhibitors with a broad spectrum of activity, possibly due to the high susceptibility of this tracer to proteases expressed in pancreatic cells. We prepared our own radioligand [125I][Tyr2]-octreotide which was shown to be much more resistant to degradation after incubation with AR4-2J plasma membranes. As expected, the increased stability of [125I][Tyr3]-octreotide was associated with good binding to SSTR2. Addition of appropriate protease inhibitors further increased the specific binding of [125I][Tyr3]-octreotide to AR4-2J plasma membranes without affecting the stability of the tracer, suggesting that the protease inhibitors also protect the integrity of SSTR2. Optimal conditions (time, temperature, medium) were developed for a binding assay in 96-well plates using AR4-2J plasma membranes in order to make the assay suitable for high-throughput analysis. This protocol was the basis for studying the in vivo regulation of SSTR2 expression in AR4-2J cells implanted into scid mice after exposure to different compounds.     

Glucagon-like peptide-1 does not mediate amylase release from AR42J cells.

In this study, AR42J pancreatic acinar cells were used to investigate if glucagon-like peptide-1 (GLP-1) or glucagon might influence amylase release and acinar cell function. We first confirmed the presence of GLP-1 receptors on AR42J cells by reverse trasncriptase-polymerase chain reaction (RT-PCR), Western blotting, and partial sequencing analysis. While cholecystokinin (CCK) increased amylase release from AR42J cells, GLP-1, alone or in the presence of CCK, had no effect on amylase release but both CCK and GLP-1 increased intracellular calcium. Similar to GLP-1, glucagon increased both cyclic adenosine monophosphate (cAMP) and intracellular calcium in AR42J cells but it actually decreased CCK-mediated amylase release (n = 20, P < 0.01). CCK stimulation resulted in an increase in tyrosine phosphorylation of several cellular proteins, unlike GLP-1 treatment, where no such increased phosphorylation was seen. Instead, GLP-1 decreased such protein phosphorylations. Genestein blocked CCK-induced phosphorylation events and amylase secretion while vanadate increased amylase secretion. These results provide evidence that tyrosine phosphorylation is necessary for amylase release and that signaling through GLP-1 receptors does not mediate amylase release in AR42J cells. J. Cell. Physiol. 181:470-478, 1999. Published 1999 Wiley-Liss, Inc.     

Differentially expressed proteins in cerulein-stimulated pancreatic acinar cells: implication for acute pancreatitis

The proteins expressed in pancreatic acinar cells during the initiation of acute pancreatitis may determine the severity of the disease. Cerulein pancreatitis is one of the best characterized models for acute pancreatitis. Present study aims to determine the differentially expressed proteins in cerulein-stimulated pancreatic acinar cells as an in vitro model for acute pancreatitis. Rat pancreatic acinar AR42J cells were treated with 10(-8)M cerulein for 12h. The protein patterns separated by two-dimensional electrophoresis using pH gradients of 5-8 were compared between the cells treated without cerulein and those with cerulein. The changed proteins were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. As a result, 10 proteins (Orp150 protein, protein disulfide isomerase related protein, dnaK-type molecular chaperone hsp72-ps1, mitochondrial glutamate dehydrogenase, similar to chaperonin containing TCP-1 beta subunit, RuvB-like protein 1, heterogeneous nuclear ribonucleoprotein H1, aldehyde reductase 1, triosephosphate isomerase 1, peroxiredoxin 2) were up-regulated while four proteins (vasolin-containing protein, 78 kDa glucose-regulated protein precursor, heat shock protein 8, adenosylhomocysteinase) were down-regulated by cerulein in pancreatic acinar AR42J cells. These proteins are related to chaperone, cell defense mechanism against oxidative stress or DNA damage, anti-apoptosis and energy generation. The differentially expressed proteins by ceruein share their functional roles in pancreatic acinar cells, suggesting the possible involvement of oxidative stress, DNA damage, and anti-apoptosis in pathogenesis of acute pancreatitis. Proteins involved in cellular defense mechanism and energy production may protect pancreatic acinar cells during the development of pancreatitis.     

Increase of heat shock protein gene expression by melatonin in AR42J cells.

Heat shock proteins (HSPs) have been reported to protect the pancreatic cells from the acute damage produced by caerulein overstimulation. However the effects of caerulein, melatonin or hyperthermia preconditioning on mRNA signal for HSP60 in the pancreatic acinar cells has not been examined yet. The aims of this study were: 1. To investigate the gene expression for HSP60 in the pancreatic AR42J cells stimulated by melatonin, caerulein or combination of both these substances. 2. To compare above changes with mRNA signal for HSP60 in pancreatic AR42J cells subjected to hyperthermia preconditioning. AR42J cells were incubated in standard medium at 37 degrees C for: 0, 1, 3, 5, 12 or 24 h, under basal conditions. Above cells were then subjected to heat shock (42 degrees C) for 0, 1 or 3 h. In the next part of the study AR42J cells were incubated in presence of caerulein (10(-11), 10(-9) or 10( -7) M), melatonin (10(-8) or 10(-6) M), or combination of above under basal conditions or following heat shock pretreatment. Gene expression for HSP60 was determined by RT-PCR. The mRNA signal for HSP60 has been observed in AR42J cells under basal conditions, and this signal was markedly and time-dependently increased in these cells subjected to hyperthermia preconditioning. Incubation of AR42J cells in presence of melatonin (10(-8) or 10(-6) M) resulted in the significant and dose-dependent increase of gene expression for HSP60 in both groups of AR42J cells: preconditioned and in those, which were not subjected to hyperthermia. Caerulein stimulation reduced mRNA signal for HSP60. The strongest signal has been observed after the exposition of AR42J cells to hyperthermia preconditioning, combined with melatonin and caerulein. We conclude that: 1. Gene expression for HSP60 has been detected in pancreatic AR42J cells under basal conditions. 2. Hyperthermia preconditioning resulted in a significant and time-dependent increase of HSP60 signal in pancreatic AR42J cells. 3. HSP60 gene expression was significantly increased in pancreatic AR42J cells stimulated by melatonin whereas caerulein reduced this signal. 4. The strongest gene expression for HSP60 has been found in the cells subjected to the combination of hyperthermia preconditioning, caerulein and melatonin.     

Coupled induction of exocrine proteins and intracellular compartments involved in the secretory pathway in AR4-2J cells by glucocorticoids

Treatment of AR4-2J cells with dexamethasone at 10 nM for 96 h inhibited cell replication by 75% and increased cell size (30%), protein content (1.6-fold) and protein synthesis (2-fold). The increase in protein synthesis was largely due to a 5 to 10-fold increase in the synthesis of secretory proteins. Amylase activity increased 20 to 30-fold in cellular homogenates and 10 to 20-fold in culture medium. Both in the presence and absence of dexamethasone AR4-2J cells release their secretory proteins by constitutive secretion. The proportion of newly synthesized amylase retained by the cells over the 14 h labeling period increased from 15 to 30% with hormone treatment. As judged by comigration on polyacrylamide gels and Western blots analyzed by immunospecific sera, AR4-2J cells synthesize and secrete the majority of known pancreatic secretory proteins. Dexamethasone increased the synthesis of trypsinogen 12 to 16-fold, chymotrypsinogen 4.5 to 6-fold, the group of procarboxypeptidases 6-fold, and amylase 7 to 10-fold. Messenger RNA levels for trypsinogen, amylase and lipase were each increased 4 to 5-fold. At the ultrastructural level dexamethasone led to significant increases in rough endoplasmic reticulum (RER) (30-fold) and Golgi elements (1.5-fold) and to the de novo appearance of electron-opaque granules (0.1-0.5 microns) which were shown to contain amylase by immunolocalization techniques employing protein A-gold. Dexamethasone also led to the formation of gap junctions between AR4-2J cells. These findings indicate that AR4-2J cells provide a model for differentiation of pancreatic acinar cells which should also be studied for the differentiation markers for the regulated secretory pathway.     

Bombesin binding and biological effects on pancreatic acinar AR42J cells

The effects of bombesin on amylase release and the receptor binding of 125I-[Tyr4]bombesin in the rat pancreatic acinar carcinoma cell line AR42J were examined. Bombesin-like peptides stimulated amylase release from AR42J cells in a dose-dependent manner; a maximal 2-fold stimulation occurred at a bombesin concentration of 300 pM. Binding of 125I-[Tyr4]-bombesin to AR42J cells was specific, saturable and temperature dependent. The relative potencies with which various structurally related peptides stimulated amylase release correlated well with their relative abilities to compete for the bombesin receptor.     

Role of vesicular nucleotide transporter VNUT (SLC17A9) in release of ATP from AR42J cells and mouse pancreatic acinar cells

ATP is released from cells in response to various stimuli. Our previous studies on pancreas indicated that pancreatic acini could be major stores of secreted ATP. In the present study, our aim was to establish the role of the vesicular nucleotide transporter (VNUT), SLC17A9, in storage and release of ATP. Freshly prepared acini from mice and AR42J rat acinar cells were used in this study. We illustrate that in AR42J cells, quinacrine (an ATP store marker) and Bodipy ATP (a fluorescent ATP analog) co-localized with VNUT-mCherry to vesicles/granules. Furthermore, in acini and AR42J cells, a marker of the zymogen granule membranes, Rab3D, and VNUT co-localized. Dexamethasone treatment of AR42J cells promoted formation of acinar structures, paralleled by increased amylase and VNUT expression, and increased ATP release in response to cholinergic stimulation. Mechanical stimulus (pressure) and cell swelling also induced ATP release, but this was not influenced by dexamethasone, most likely indicating different non-zymogen-related release mechanism. In conclusion, we propose that VNUT-dependent ATP release pathway is associated with agonist-induced secretion process and downstream purinergic signalling in pancreatic ducts.     

Membrane lipid composition of pancreatic AR42J cells: modification by exposure to different fatty acids

Dietary fat type influences fatty acids in rat pancreatic membranes, in association with modulation of secretory activity and cell signalling in viable acini. We aimed to confirm whether AR42J cells are a valid model to study the interactions between lipids and pancreatic acinar cell function. For this purpose we have (i) compared the baseline fatty acid composition of AR42J cells with that of pancreatic membranes from rats fed a standard chow; (ii) investigated if fatty acids in AR42J membranes can be modified in culture; and (iii) studied if similar compositional variations that can be evoked in rats when dietary fat type is altered occur in AR42J cells. Weaning Wistar rats were fed for 8 weeks either a commercial chow (C) or semi-purified diets containing virgin olive oil (VOO) or sunflower oil (SO) as fat source. AR42J cells were incubated for 72 hrs in medium containing unmodified fetal calf serum (FCS, AR42J-C cells), FCS enriched with 18:1 n-9 (AR42J-O cells), or FCS enriched with 18:2 n-6 (AR42J-L cells). Fatty acids in crude membranes from rat pancreas and AR42J cells were determined by gas-liquid chromatography. Differences in membrane fatty acids between C rats and AR42J-C cells can be explained in part by variations in the amount of fatty acids in the extracellular environment. Supplementation of FCS with 18:1 n-9 or 18:2 n-6 changed the fatty acid spectrum of AR42J cells in a manner that resembles the pattern found, respectively, in VOO and SO rats, although AR42J-L cells were unable to accumulate 20:4 n-6. The AR42J cell line can be a useful tool to assess the effect of membrane compositional changes on acinar cell function. However, differences in baseline characteristics, and perhaps fatty acid metabolism, indicate that results obtained in AR42J cells should be confirmed with experiments in the whole animal.     

An amphicrine pancreatic cell line: AR42J cells combine exocrine and neuroendocrine properties.

The permanent cell culture line AR42J, derived from a rat pancreatic acinar carcinoma, is widely used for functional studies of exocrine pancreatic acinar cells. We now present evidence that these cells are amphicrine in that they contain zymogen granules as well as small (40-80 nm) neuroendocrine (NE) vesicles and typical neurotransmitters. Using the small NE vesicle-specific markers synaptophysin and "protein S.V.2", including synaptophysin cDNA probes, we have found that AR42J cells synthesize these proteins and contain vesicles harboring these proteins with biophysical properties similar to those of small NE vesicles. NE properties of these cells are further indicated by the presence of considerable amounts of stored amino acids (gamma-aminobutyric acid (GABA), glycine, glutamate) and by the presence of the GABA-synthesizing enzyme, glutamic acid decarboxylase. Finally, intermediate filament (IF) protein typing showed only cytokeratins 8 and 18, indicating that AR42J cells possess an IF protein complement indistinguishable from that of acinar and islet cells. Our results document the unusual case of a permanent cell line with combined exocrine and neuroendocrine properties that may be indicative of a derivation from a cell with multipotential character.     

Pancreatic acinar cells utilize tyrosine to synthesize L-dihydroxyphenylalanine

The pancreatic β cells can synthesize dopamine by taking L-dihydroxyphenylalanine, but whether pancreatic acinar cells synthesize dopamine has not been confirmed. By means of immunofluorescence, the tyrosine hydroxylase -immunoreactivity and aromatic amino acid decarboxylase (AADC)- immunoreactivity were respectively observed in pancreatic acinar cells and islet β cells. Treatment with L-dihydroxyphenylalanine, not tyrosine, caused the production of dopamine in the incubation of INS-1 cells (rat islet β cell line) and primary isolated islets, which was blocked by AADC inhibitor NSD-1015. However, only L-dihydroxyphenylalanine, but not dopamine, was detected when AR42J cells (rat pancreatic acinar cell line) were treated with tyrosine, which was blocked by tyrosine hydroxylase inhibitor AMPT. Dopamine was detected in the coculture of INS-1 cells with AR42J cells after treatment with tyrosine. In an in vivo study, pancreatic juice contained high levels of L-dihydroxyphenylalanine and dopamine. Both L-dihydroxyphenylalanine and dopamine accompanied with pancreatic enzymes and insulin in the pancreatic juice were all significantly increased after intraperitoneal injection of bethanechol chloride and their increases were all blocked by atropine. Inhibiting TH with AMPT blocked bethanechol chloride-induced increases in L-dihydroxyphenylalanine and dopamine, while inhibiting AADC with NSD-1015 only blocked the dopamine increase. Bilateral subdiaphragmatic vagotomy of rats leads to significant decreases of L-dihydroxyphenylalanine and dopamine in pancreatic juice. These results suggested that pancreatic acinar cells could utilize tyrosine to synthesize L-dihydroxyphenylalanine, not dopamine. Islet β cells only used L-dihydroxyphenylalanine, not tyrosine, to synthesize dopamine. Both L-dihydroxyphenylalanine and dopamine were respectively released into the pancreatic duct, which was regulated by the vagal cholinergic pathway. The present study provides important evidences for the source of L-dihydroxyphenylalanine and dopamine in the pancreas.     

Synthesis and sst(2) binding profiles of new [Tyr(3)]octreotate analogs.

One of the main objectives of our current work is the development of new somatostatin analogs that would retain the general characteristics of [Tyr(3)]octreotate (Tate) while showing potential for clinical application. In this respect, study of their interaction with the sst(2) is crucial in providing preliminary structure-activity relationships data. In the present work we report on the synthesis and the preliminary biological evaluation of a total of 15 new structurally modified [Tyr(3)]octreotate analogs. The binding affinities were determined during competition binding assays in sst(2)-positive rat acinar pancreatic AR4-2J cell membranes using [(125)I-Tyr(3)]octreotide as the radioligand.     

Prostaglandin E2 modulates TNF-alpha-induced MCP-1 synthesis in pancreatic acinar cells in a PKA-dependent manner

Cyclooxygenase (COX)-2 is increased in human chronic pancreatitis. We recently demonstrated in a model of chronic pancreatitis (WBN/Kob rat) that inhibition of COX-2 activity reduces and delays pancreatic inflammation and fibrosis. Monocyte chemoattractant protein (MCP)-1 mRNA and PGE(2) were significantly reduced, correlating with a decreased infiltration of macrophages. MCP-1 plays an important role in the recruitment of macrophages to the site of tissue injury. The aim of our study is to identify mechanisms by which macrophages and acinar cells maintain an inflammatory reaction. The expression profile of E prostanoid receptors EP(1-4) and MCP-1 was analyzed by RT-PCR from pancreatic specimens and AR42J cells. MCP-1 secretion was detected by ELISA from rat pancreatic lobuli. We determined EP(1-4) mRNA levels in WBN/Kob rats with chronic pancreatic inflammation. Individual isoforms were highly increased in rat pancreas, concurrent with MCP-1 mRNA expression. In supernatants of pancreatic lobuli and AR42J cells, MCP-1 was detectable by ELISA. In the presence of TNF-alpha, MCP-1 was upregulated. Coincubation with PGE(2) enhanced the TNF-alpha-induced MCP-1 synthesis significantly. Similarly, TNF-alpha mRNA was synergistically upregulated by TNF-alpha and PGE(2). Furthermore, the synergistic effect of TNF-alpha and PGE(2) was abolished by inhibition of PKA but not of PKC. We conclude that EP receptors are upregulated during chronic pancreatic inflammation. PGE(2) modulates the TNF-alpha-induced MCP-1 synthesis and secretion from acinar cells. This synergistic effect is controlled by PKA. This mechanism might explain the COX-2-dependent propagation of pancreatic inflammation.     

Mechanisms of activated Ca2+ entry in the rat pancreatoma cell line, AR4-2J.

The characteristics of Ca2+ entry activated by surface receptor agonists and membrane depolarization were studied in the rat pancreatoma cell line, AR4-2J. Ca2+ mobilization activated by substance P, bombesin, or muscarinic receptor stimulation was found to involve both Ca2+ release and entry. In addition, depolarization of the surface membrane of AR4-2J cells with elevated concentrations of K+ activated Ca2+ entry. Ca2+ entry induced by membrane depolarization was inhibited by the L-channel antagonist, nimodipine, while that due to surface receptor agonists was not inhibited by this agent. The microsomal Ca(2+)-ATPase inhibitor, thapsigargin, caused both depletion of the agonist-sensitive intracellular Ca2+ pool and sustained Ca2+ influx indistinguishable from that produced by bombesin or methacholine. These results confirm that, unlike the pancreatic acinar cells from which they are presumably derived, AR4-2J cells express voltage-sensitive, dihydropyridine-inhibitable Ca2+ channels. However, in contrast to previous reports with this cell line, in the AR4-2J cells in use in our laboratory, and under our experimental conditions, surface receptor agonists (including substance P) do not cause Ca2+ influx through voltage-sensitive Ca2+ channels. Instead, we conclude that agonist-activated Ca2+ mobilization is initiated by (1,4,5)IP3-mediated intracellular Ca2+ release and that Ca2+ influx is regulated primarily, if not exclusively, by the state of depletion of the (1,4,5)IP3-sensitive intracellular Ca2+ pool.     

Lipopolysaccharides induce p8 mRNA expression in vivo and in vitro.

Systemic LPS endotoxin is associated with acute pancreatic damage. Whether damage results from direct interaction of LPS with pancreatic cells is unknown. We addressed that question by monitoring p8 expression in reponse to LPS, in vivo and in vitro, because overexpression of the p8 protein is a sensitive marker of pancreatic agression. For in vivo studies, rats were sacrificed at different times after a single intraperitoneal injection of LPS, and pancreas, liver, kidney, lung, brain, and intestine were processed for RNA preparation. In vitro, pancreatic acinar AR4-2J cells were cultivated with 0.1, 1, or 10 micrograms/ml LPS for 6, 12, or 24 h. p8 mRNA expression was monitored by Northern blotting. In vivo, it was strongly increased in the pancreas after 12 h of treatment and remained elevated after 24 h. It was also induced in kidney and liver, with a maximum at 6 and 12 h, respectively, but not in lung, brain, or intestine. In AR4-2J cells, basal p8 mRNA expression was very low and increased in a time- and dose-dependent manner after treatment with LPS. LPS-induced overexpression of p8 mRNA in vivo confirmed the adverse effect of endotoxemia on pancreas and its overexpression in vitro demonstrated a direct interaction of LPS with pancreatic cells.