Glucocorticoids increase cholecystokinin receptors and amylase secretion in pancreatic acinar AR42J cells

We recently reported in AR42J pancreatic acinar cells that glucocorticoids increased the synthesis, cell content, and mRNA levels for amylase (Logsdon, C.D., Moessner, A., Williams, J.A., and Goldfine, I.D. (1985) J. Cell Biol. 100, 1200-1208). In addition, in these cells glucocorticoids increased the volume density of secretory granules and rough endoplasmic reticulum. In the present study we investigate the effects of glucocorticoids on the receptor binding and biological effects of cholecystokinin (CCK) on AR42J cells. Treatment with 10 nM dexamethasone for 48 h increased the specific binding of 125I-CCK. This increase in binding was time-dependent, with maximal effects occurring after 48 h, and dose-dependent, with a one-half maximal effect elicited by 1 nM dexamethasone. Other steroid analogs were also effective and their potencies paralleled their relative effectiveness as glucocorticoids. Analyses of competitive binding experiments conducted at 4 degrees C to minimize hormone internalization and degradation revealed the presence of a single class of CCK binding sites with a Kd of approximately 6 nM and indicated that dexamethasone treatment nearly tripled the number of CCK receptors/cell with little change in receptor affinity. Treatment with 10 nM dexamethasone increased both basal amylase secretion and the amylase released in response to CCK stimulation. In addition, dexamethasone increased the sensitivity of the cells to CCK. The glucocorticoid decreased the concentration of CCK required for one half-maximal stimulation of amylase secretion from 35 +/- 6 to 8 +/- 1 pM. These data indicate, therefore, that glucocorticoids induce an increase in the number of CCK receptors in AR42J cells, and this increase leads to enhanced sensitivity to CCK.     

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.     

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.     

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.     

Glucocorticoids modulate the in vitro development of the embryonic rat pancreas

The effect of the glucocorticoid analogue, dexamethasone, on the development of the embryonic pancreas was studied in tissue culture. It specifically enhances the accumulation of exocrine enzymes without altering the level of general cell proteins. The enhancement, however, is not symmetrical: the cellular levels of the two major exocrine products, amylase and chymotrypsinogen, are increased about 10- and 2- fold, respectively. Two other zymogens that are present in minor quantities, procarboxypeptidases A and B, are also increased, whereas no effect is seen on lipase A. Coordinate with these effects on synthesis, there is a dramatic change in the morphology of dexamethasone-stimulated acinar cells. Their number of zymogen granules is higher and crystalline arrays are found in the rough endoplasmic reticulum. Dexamethasone also inhibits cell replication, perhaps by selectively inhibiting the last cell divisions of the culture period. At the same time, there is a disproportionate reduction in the insulin content of cultured rudiments. We find that pancreatic development is normal in the absence of dexamethasone and that this glucocorticoid does not precociously induce the appearance of the specific secretory products, but rather enhances by a constant degree their synthesis and accumulation. Therefore, we conclude that glucocorticoids may play a modulatory but not an inductive role in pancreatic development.     

Rab3D regulates amylase levels,not agonist-induced amylase release,in AR42J cells

Rab3D is a low molecular weight GTP-binding protein that associates with secretory granules in exocrine cells. AR42J cells are derived from rat pancreatic exocrine tumor cells and develop an acinar cell-like phenotype when treated with dexamethasone (Dex). In the present study, we examined the role of Rab3D in Dex-treated AR42J cells. Rab3D expression and localization were analyzed by subcellular fractionation and immunoblotting. The role of Rab3D was examined by overexpressing myc-labeled wild-type-Rab3D and a constitutively active form of Rab3D (Rab3D-Q81L) in AR42J cells. We found that Rab3D is predominantly membrane-associated in AR42J cells and co-localizes with zymogen granules (ZG). Following CCK-8-induced exocytosis, amylase-positive ZGs appeared to move towards the periphery of the cell and co-localization between Rab3D and amylase was less complete when compared to basal conditions. Overexpression of WT, but not mutant Rab3D, resulted in an increase in cellular amylase levels. Overexpression of mutant and WT Rab3D did not affect granule morphology, CCK-8-induced secretion, long-term (48 hr) basal amylase release or granule density. We conclude that Rab3D is not involved in agonist-induced exocytosis in AR42J cells. Instead, Rab3D may regulate amylase content in these cells.     

Dynamics of pancreatic tissue cells in the rat exposed to long-term caerulein treatment: 1. Biochemical, morphological and morphometrical evaluations

This study was undertaken to evaluate whether hypertrophy and hyperplasia of the pancreatic acinar cells induced by caerulein remained after termination of the hormonal treatment. Rats received subcutaneous injections of saline or caerulein for 4 days and were killed immediately after termination of treatment or 2, 15 and 50 days later. Caerulein treatment induced significant increases in pancreatic weight and contents of DNA, RNA, protein, amylase and chymotrypsinogen along with an increased number of acinar cells per acinus and zymogen granules per acinar cells. During the post-treatment period, the caerulein-treated pancreas reverted to control values for their contents in proteins, enzymes and RNA and number of zymogen granules per acinar cell while the number of pancreatic cells remained constant as indicated by the absence of modification in total DNA content and acinar cells per acinus. During that same period, saline-treated pancreas exhibited constant growth. These morphological and biochemical data indicate that the already present and newly formed acinar cells can remain in place once the trophic stimulus is withdrawn and that they can adjust their cellular components and thus their digestive capacity to the circulating levels of endogenous cholecystokinin released in response to normal meals.     

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.     

Glucocorticoids decrease the synthesis of type I procollagen mRNAs

Glucocorticoids selectively decrease procollagen synthesis in animal and human skin fibroblasts. beta-Actin content and beta-actin mRNA are not affected by glucocorticoid treatment of chick skin fibroblasts. The inhibitory effect of glucocorticoids on procollagen synthesis is associated with a decrease in total cellular type I procollagen mRNAs in chick skin fibroblasts. These effects of dexamethasone are receptor mediated as determined by pretreatment with the glucocorticoid antagonists progesterone and RU-486 and with the agonist beta-dihydrocortisol. Dexamethasone has a small but significant inhibitory effect on cell growth of chick skin fibroblasts. The ability of this corticosteroid to decrease the steady-state levels of type I procollagen mRNAs in nuclei, cytoplasm, and polysomes varies. The largest decrease of type I procollagen mRNAs is observed in the nuclear and cytoplasmic subcellular fractions 24 h after dexamethasone treatment. Type I procollagen hnRNAs are also decreased as determined by Northern blot analysis of total nuclear RNA. The synthesis of total cellular type I procollagen mRNAs is reversibly decreased by dexamethasone treatment. In addition the synthesis of total nuclear type I procollagen mRNA sequences is decreased at 2, 4, and 24 h following the addition of radioactive nucleoside and dexamethasone to cell cultures. Although the synthesis of pro alpha 1(I) and pro alpha 2(I) mRNAs is decreased in dexamethasone-treated chick skin fibroblasts, the degradation of the total cellular procollagen mRNAs is not altered while the degradation of total cellular RNA is stabilized. These data indicate that the dexamethasone-mediated decrease of procollagen synthesis in embryonic chick skin fibroblasts results from the regulation of procollagen gene expression.     

Ultrastructural and functional changes in pancreatic acinar cells during autolysis.

Effects of anoxemic cell injury on rat pancreatic acinar cells were studied in a preparation where tissue samples were incubated at temperature between 18-20 degrees C in a moist atmosphere for 0, 0.5, 1, 3, 6, 12, and 24 h in vitro. Electron microscopy revealed that disintegration of acinar cells began by swelling of various cell compartments and gradual breakdown of cell membranes. Zymogen granules remained morphologically intact for at least 3 h. There were no signs of increased autophagic activity during the period of observation. Myelin figures and other membranous remnants of disintegrated cells, together with individual cells and cell organelles whose morphology was relatively well preserved were seen even after w4 h incubation. The secretory response of acinar cells to pancreozymin stimulation, as measured by amylase release into the incubation medium in vitro, decreased progressively closer to zero during 12 h autolysis. No active trypsin could be detected in the tissue samples during the 24 h observation time. It was concluded that during hypoxic autolysis at room temperature between 18-20 degrees C in vitro: 1. Acinar cell disintegration results from breakdown of cellular membranes, 2. autophagocytosis is not involved, 3. most of zymogen granules remain morphologically intact even at the time when cell membranes show evidence of damage, 4. there is no trypsin activation taking place in the tissue, and 5. the acinar cells are capable of responding to secretory stimulation for 3 to 6 h after removal of the tissue from the experimental animal.     

Glucocorticoids have opposite effects on ornithine decarboxylase and cell growth in pancreatic acinar AR42J cells.

This paper reviews the relationships between the effects of glucocorticoids on rat pancreatic acinar AR42J cell polyamine levels and cellular growth and differentiation. Glucocorticoids inhibit the growth of AR42J cells. Glucocorticoids either stimulate or inhibit the formation of polyamines in a variety of cell types. Cells require polyamines for normal growth. Therefore, we tested the hypothesis that polyamines mediate the effects of glucocorticoids on AR42J cells. First, to confirm that AR42J cells required polyamines for growth we examined the effects of inhibiting ornithine decarboxylase (ODC). ODC is the most important and generally rate-limiting enzyme in the synthesis of the polyamines. As expected, the ODC inhibitor difluoromethylornithine (DFMO) inhibited AR42J cell DNA synthesis, and the addition of exogenous putrescine reversed this effect. The levels of growth inhibition by glucocorticoids and DFMO treatment were similar. Second, we examined the effects of glucocorticoids on ODC. Surprisingly, glucocorticoids increased levels of AR42J cell ODC mRNA, ODC activity, and putrescine. Glucocorticoids increased these parameters over a similar time-course as they decreased DNA synthesis. Analog specificity studies indicated that a glucocorticoid receptor mediated both the growth inhibitory and ODC stimulatory effects. Dose-response studies indicated, however, that growth inhibition was more sensitive to dexamethasone (DEX) than were ODC levels. Therefore, polyamines do not account for the effects of glucocorticoids on AR42J cell growth. In these cells, glucocorticoids have opposite and independent effects on ODC and growth.     

Ultrastructural changes in the rat exocrine pancreas after jejunoileal bypass

The influence of a 90% jejunoileal bypass on the rat exocrine pancreas was studied by morphometrical procedures. In sham-operated animals exocrine acinar cells accounted for 80.3% of the pancreas volume. These cells are composed of 9.9% nuclei, 8.4% mitochondria, 12.2% zymogen granules, 0.3% lipid droplets and 69.2% of a compartment ("ERGLS") composed of endoplasmic reticulum, ribosomes, Golgi areas, lysosomes and the cytoplasmic ground substance. Intestinal bypass did not change the volume density of exocrine cells nor that of nuclei in the cells during the three postoperative months. The means nuclear diameter was approximately the same in both groups. However, the volume density of secretory granules diminished by 50%. This was accompanied by a decrease in mean granular diameter, but not in their numerical density. The volume density of lipid droplets increased 10 fold, that of mitochondria increased slightly from the 15th postoperative day but significantly from the 45th day. The remaining cellular compartment composed of "ERGLS" was not modified by intestinal bypass. These findings suggest that a 90% jejunoileal bypass induces major changes in the composition of pancreatic acinar cells but not in their size.     

Reduced sensitivity to dexamethasone of pancreatic islets from obese (fa/fa) rats.

The direct effects of dexamethasone exposure on insulin secretion from islets of fa/fa rats and their lean littermates (Fa/?) were compared. After 72 h culture in 1 nM dexamethasone, glucose (27.5 mM)-stimulated insulin secretion over 90 min from islets of lean rats was significantly decreased compared with islets cultured without dexamethasone (12.9 +/- 1.4 vs. 5.7 +/- 1.0% of total islet content, p < 0.05). Higher doses of dexamethasone for 24-48 h culture produced similar effects. For islets of fa/fa rats, the minimum inhibitory concentration of dexamethasone was 10-fold higher, and islets required at least 48 h exposure for inhibitory effects to be observed. Dexamethasone also decreased the insulin response by islets to glybenclamide, indicating that dexamethasone effects were not specific to glucose transport or metabolism. The results suggest that islets of fa/fa rats may be less sensitive to direct inhibitory effects of glucocorticoids on glucose-stimulated insulin release than islets of lean animals.     

A soluble form of human nectin-2 impairs exocrine secretion of pancreas and formation of zymogen granules in transgenic mice

Transgenic mouse lines expressing a soluble form of human nectin-2 (hNectin-2Ig Tg) exhibited distinctive elevation of amylase and lipase levels in the sera. In this study, we aimed to clarify the histopathology and to propose the transgenic mouse lines as new animal model for characteristic pancreatic exocrine defects. The significant increase of amylase and lipase levels in sera of the transgenic lines approximately peaked at 8 weeks old and thereafter, plateaued or gradually decreased. The histopathology in transgenic acinar cells was characterized by intracytoplasmic accumulation of abnormal proteins with decrease of normal zymogen granules. The hNectin-2Ig expression was observed in the cytoplasm of pancreatic acinar cells, which was consistent with zymogen granules. However, signals of hNectin-2Ig were very weak in the transgenic acinar cells with the abnormal cytoplasmic accumulaion. The PCNA-positive cells increased in the transgenic pancreas, which suggested the affected acinar cells were regenerated. Acinar cells of hNectin-2Ig Tg had markedly small number of zymogen granules with remarkable dilation of the endoplasmic reticulum (ER) lumen containing abundant abnormal proteins. In conclusion, hNectin-2Ig Tg is proposed as a new animal model for characteristic pancreatic exocrine defects, which are due to the ER stress induced by expression of mutated cell adhesion molecule that is a soluble form of human nectin-2.     

Insulin-like growth factors decrease catecholamine content in PC12 rat pheochromocytoma cells.

Insulin-like growth factors (IGFs) stimulate proliferation and differentiation of PC12 rat pheochromocytoma cells and modulate catecholamine release in bovine adrenal medullary cells. Dexamethasone increases catecholamine synthesis in PC12 cells. We therefore studied the effects of IGFs and dexamethasone on catecholamine content in PC12 cells. Dopamine (DA) and norepinephrine (NE) content of PC12 cells were measured after incubation for 72 h with IGFs (100 ng/ml) and/or dexamethasone (500 nM). IGF-I (100 ng/ml) and IGF-II (100 ng/ml) decreased DA and NE content to approximately 35% and approximately 25% of control, respectively. [Leu27]IGF-II, which binds to the IGF-I receptor with markedly decreased affinity, did not reduce catecholamine levels, indicating that the effect is likely to be mediated by the IGF-I receptor. Dexamethasone (500 nM) increased levels of DA and NE to 173 +/- 20% and 331 +/- 48% of controls, respectively. Coincubation with IGFs did not significantly affect the stimulation of DA by dexamethasone, but abolished the rise in NE. Levels of tyrosine hydroxylase mRNA, protein and activity were increased following incubation with dexamethasone, but were unchanged by IGFs. These results indicate that IGFs decrease catecholamine content in PC12 cells via the IGF-I receptor. Complex regulation involving multiple synthetic and/or degradative steps is implicated in this process.     

Glucocorticoids effects on exocrine pancreatic secretion in caerulein-induced acute pancreatitis in the rat.

The present work reports on exocrine pancreatic secretion in control rats, adrenalectomized rats and hydrocortisone-treated (10 mg/Kg/d) rats during 7 days, under normal conditions and after induction of acute pancreatitis with caerulein (20 micrograms/Kg) by 4 subcutaneous injections at hourly intervals. Pancreatic secretion was seen to be affected by the procedure of adrenalectomy, which led to a marked reduction in the secretion of proteins and amylase with respect to control values. This was probably due to the decrease occurring in the zymogen granules in the acinar cells of the exocrine pancreas, a phenomenon which also led to a decrease in pancreatic weight observed in these animals. Treatment with hydrocortisone induced a decrease in the secretion of proteins and amylase, as well as an increase in pancreatic weight. This agrees with the accepted hypothesis that large amounts glucocorticoids stimulate the synthesis and storage of proteins in the exocrine pancreas, reducing the secretory phase. The administration of high doses of caerulein under these conditions led to acute pancreatitis in the three groups of animals. This was paralleled by a dramatic decrease in protein and amylase secretion and by severe interstitial edema of the pancreas and by increases in serum amylase values. In the case of the animals treated previously with hydrocortisone, the latter were tripled with respect to the control animals. The conclusion is offered that since the storage of enzyme proteins is governed by glucocorticoids, which furthermore increase the sensitivity of the acinar cells to stimulation by secretagogues, the administration of these substances during the development of pancreatic lesions such as acute pancreatitis is highly compromising to the organism.     

Transpancreatic transport of digestive enzyme

When porcine α-amylase or bovine chymotrypsinogen A was added to the medium bathing the rabbit pancreas in short-term organ culture, the secretion of these enzymes collected via the duct system increased greatly. To determine if it was indeed the amylase added to the bath that was recovered in secretion, endogenous enzyme stores were prelabeled during a 4 h incubation with [³H]- leucine and the specific radioactivity of amylase in secretion followed. The addition of unlabeled exogenous amylase to the bathing medium reduced the specific radioactivity of secreted amylase of about 90% suggesting that the response was due to the transpancreatic transport of the added enzyme. This inhibition was maintained over time, and was a result, not only of the increased secretion of unlabeled enzyme, but also of a 72% steady-state inhibition in the secretion of endogenous (labeled) amylase. This latter observation indicates that the exogenous enzyme crosses the acinar cell and mixes with endogenous cellular stores. A cellular route is also suggested by the observation that the addition of amylase to the bath increased the amylase concentration in ductal fluid relative to that in the bath by about 20 times; it did not reduce it as would be expected if paracellular shunts were involved. In addition, a cellular pathway is suggested by the observation that a 2 h prior incubation in bovine chymotrypsinogen resulted in a greatly augmented chymotrpsinogen response to a maximal cholinergic stimulus. In all, the data support the prediction of the equilibrium theory of digestive enzyme secretion that enzyme secretion should be responsive to mass action, and the prediction of the enteropancreatic circulation hypothesis that a capacity exists for a substantial transpancreatic flux of digestive enzyme.