Rat Pancreatic AR42J Cells Research Articles

Rat pancreatic AR42J cells. Amphicrine cells as an in vitro model to study peptide hormone receptor regulation.

The rat pancreatic acinar tumor cell line, AR42J, is widely used to study pancreatic acinar cell biology and biochemistry. In addition to the well-documented exocrine cell features, we have identified by immunofluorescence and by electron microscopy the co-expression of small neuroendocrine (NE) vesicles using the NE vesicle-specific markers synaptophysin and "protein S.V.2." AR24J cells store [3H]GABA, which is secreted upon potassium depolarization in a calcium-dependent manner. In addition, we found the expression of the receptor for the neurotransmitter substance P by using a receptor-specific cDNA probe. Glucocorticoid treatment, which profoundly inhibits cellular growth and induces differentiation, results in a rapid decrease of substance P receptor (SPR) gene expression as assessed by Northern blot analysis. Intracellular Ca2+ mobilization was then determined in response to substance P in control cells and glucocorticoid-pretreated cells by dual wavelength spectrophotometry using fura-2 in single cells. Glucocorticoid-mediated down-regulation of substance P receptors resulted in a dose- and time-dependent decrease of the intracellular Ca2+ mobilization stimulated by substance P. In summary, these data indicate that AR42J cells display an amphicrine phenotype with two differentially regulated secretory pathways; during glucocorticoid-induced differentiation, the cells become less sensitive to substance P stimulation as a consequence of reduced gene expression of the substance P receptor.

Stimulation of rat pancreatic tumoral AR4-2J cell proliferation by Pituitary Adenylate cyclase-activating peptide

In the present work the effects of the novel neuropeptide Pituitary Adenylate Cyclase Activating Peptide (PACAP) on both AR4-2J cell growth and the modulation of ornithine decarboxylase activity were investigated. Both PACAP38 and the amidated form PACAP27 caused a concentration-dependent stimulation of AR4-2J cell growth; the maximal increase was seen at 1 nmol/L (30% above control, P < 0.01) with a half-maximal effect at 0.01 nmol/L. Ornithine decarboxylase activity was also increased by PACAP in a dose-dependent manner, reaching half-maximal stimulation at 0.5 nmol/L. The addition of 1 nmol/L of somatostatin analog SMS 201–995 totally suppressed PACAP-stimulated AR4-2J cell growth. Vasoactive intestinal polypeptide (3 μmol/L) and 8-bromo-cyclic adenosine monophosphate (1 mmol/L) had no effect on cell proliferation. Treatment of cells by pertussis toxin (25 ng · mL−1 · day−1) suppressed PACAP-stimulated AR4-2J cell growth but enhanced PACAP-induced stimulation of adenylate cyclase activity. It was concluded that PACAP stimulates AR4-2J cell proliferation by a mechanism that seems independent of cyclic adenosine monophosphate production. The mitogenic effect of PACAP depends on a pertussis toxin-sensitive G protein and is associated with an increase of ornithine decarboxylase activity.

Glucocorticoids stimulate ornithine decarboxylase gene expression in pancreatic AR42J cells

The effects of dexamethasone on ornithine decarboxylase gene expression were examined in rat pancreatic AR42J cells. Dexamethasone increased ornithine decarboxylase activity and messenger RNA (mRNA) concentrations in a time-dependent manner, with a maximal effect at 12 hours (207% ± 63% and 327% ± 34% of control, respectively; n = 5). Ornithine decarboxylase mRNA levels returned to control values at 48 hours, whereas ornithine decarboxylase activity was decreased to 41% ± 8% of control (n = 3). Dexamethasone induction of ornithine decarboxylase mRNA was dose dependent, with half-maximal effects at 10−8mol/L (210% ± 20% of control; n = 4) and maximal effects at 10−7 mol/L (327% ± 26% of control; n = 4). The glucocorticoid antagonist RU 38486 blocked the dexamethasone effects in a dose-dependent manner, with maximal effects occurring at 10−7 mol/L (120% ± 18% of control; n = 3). When protein synthesis was blocked by addition of cycloheximide, ornithine decarboxylase mRNA levels remained unchanged in response to glucocorticoids, indicating a primary effect of dexamethasone. Furthermore, cycloheximide by itself had no significant effect on ornithine decarboxylase mRNA levels. Inhibition of transcription with actinomycin D showed a half-life for ornithine decarboxylase mRNA of approximately 240 minutes. Ornithine decarboxylase mRNA stability was not affected by dexamethasone pretreatment for 12 hours. Therefore, these data suggest that dexamethasone regulates ODC gene expression via glucocorticoid receptor-mediated gene transcription. Furthermore, translational mechanisms seem to be involved in glucocorticoid-regulated ornithine decarboxylase induction.

Vasoactive intestinal peptide receptors on AR42J rat pancreatic acinar cells

VIP receptors on AR42J rat pancreatic cells were analyzed by competition binding, affinity labeling and by N-glycanase digestion analyses. These studies revealed the presence of specific, high affinity (K d ∼ 1 nM) VIP receptors with a mass of 68 kDa or 59 kDa under reducing or non-reducing conditions, respectively. N-glycanase digestion of affinity labeled membranes generated a core receptor protein of ∼44 kDa and evidence for at least two N-linked glycans on the mature receptor. The receptor lacked O-linked oligosaccharides but contained terminal sialic acid residues of its N-linked glycan(s) based on digestions with O-glycanase and neuraminidase. The similarity of the AR42J VIP receptor to the recently cloned cDNA for human VIP receptors makes this cell line an attractive model for further analysis of VIP receptor signal transduction events.

Cholesterol esterase biosynthesis in rat pancreatic AR42J cells. Post-transcriptional activation by gastric hormones.

This study used the rat pancreatoma AR42J as a model system to investigate the possible regulation of cholesterol esterase biosynthesis in pancreas. Initial experiments were performed to verify the synthesis of pancreatic cholesterol esterase by the AR42J cells. Results indicated that this pancreatoma cell line synthesized two forms of cholesterol esterase (Mr = 71,000 and 74,000). The 74-kDa protein is most likely the precursor protein, and the 71-kDa protein is the matured enzyme secreted by the AR42J cells. The synthesis and secretion of cholesterol esterase were stimulated 2-5-fold by incubating the cells with 0.5-4 nM of cholecystokinin or 0.5-2 nM of secretin. Analysis of the RNA isolated from the hormone-stimulated cells revealed that stimulation of cholesterol esterase biosynthesis was not due to an increase in cholesterol esterase mRNA. Furthermore, inhibition of transcription with actinomycin D has no effect on the hormone-induced cholesterol esterase biosynthesis. Therefore, the mechanism of hormone stimulation was not dependent on de novo RNA synthesis. In vitro translation studies showed that the cholesterol esterase mRNA isolated from stimulated AR42J cells were translated more efficiently than those from control cells. Thus, the increased cholesterol esterase biosynthesis induced by gastric hormones was most likely mediated by posttranscriptional modification of the cholesterol esterase mRNA.

Ca2+ Dynamics in Rat Pancreatic AR-42J and AR-IP Cells.

Spatiotemporal change of the cytosolic free Ca2+ concentration ([Ca2+]i) in response to a variety of secretagogues was examined in rat pancreatoma AR-42J and AR-IP cells by microspectroflurometry and digital imaging microscopy after loading with fura-2. In the presence of external Ca2+, carbachol, CCK-OP (cholecystokinin-octapeptide), gastrin, norepinephrine or high K+ evoked a large transient increase in [Ca2+]i in AR-42J cells which declined to a sustained level before slowly declining towards the resting level. In the absence of external Ca2+, a transient increase in [Ca2+]i were evoked by all the ligands except for high K+ stimulation, which declined rapidly towards the resting level. The [Ca2+]i increase caused by carbachol and high K+ treatment was inhibited by muscarinic receptor antagonist, atropine, and by L-type Ca2+ channel blocker, nifedipine, respectively. The transient [Ca2+]i increase induced by gastrin stimulation was not blocked by Ca2+ channel blocker, lanthanum. In the AR-IP cells, which are non-differentiated pancreatoma cell line, all stimulations including high K+ treatment have failed to evoke [Ca2+]i response. These intracellular Ca2+ mobilizations in response to ligands in AR-42J cells were displayed by digital imaging microscopy. From these results we conclude that AR-42J cells has an alpha-adrenergic receptor, in addition to muscarinic acetylcholine receptor, CCK-OP receptor, gastrin receptor and voltage dependent Ca2+ channel. In marked contrast, AR-IP cells have neither any hormone receptor for the above ligands nor voltage dependent Ca2+ channel.

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.