Rat Pancreatic Cell Line AR42J Research Articles

Expression of pro-Muclin in pancreatic AR42J cells induces functional regulated secretory granules

It is not clear how protein cargo is sorted to and retained in forming regulated secretory granules (RSG). Here, the sulfated mucin-type glycoprotein pro-Muclin was tested for its ability to induce RSG in the poorly differentiated rat pancreatic cell line AR42J. AR42J cells express RSG content proteins, but they fail to make granules. Adenovirus-pro-Muclin-infected AR42J cells store amylase, accumulate RSG, and respond to hormonal stimulation by secreting the stored protein. Expression of pro-Muclin combined with the inducing effect of dexamethasone resulted in a significant enhancement of the efficiency of regulated secretion. The effect of pro-Muclin was a strong decrease in constitutive secretion compared with dexamethasone-induction alone. A pro-Muclin construct missing the cytosolic tail domain was less effective at improving the efficiency of regulated secretion compared with the full-length construct. Increased expression of cargo (using adenovirus amylase) also modestly enhanced regulated secretion, indicating that part of pro-Muclin's effect may be due to increased expression of cargo protein. Overall, the data show that pro-Muclin acts as a sorting receptor that can induce RSG, and that its cytosolic tail is important in this process.

Effects of GP2 expression on secretion and endocytosis in pancreatic AR4-2J cells

GP2 is the major membrane protein present in secretory granules of the exocrine pancreas. GP2’s function is unknown, but a role in digestive enzyme packaging or secretion from secretory granules has been proposed. In addition, GP2 has been proposed to influence endocytosis and membrane recycling following stimulated secretion. Adenovirus-mediated GP2 overexpression in the rat pancreatic cell line AR4-2J was used to study its impact on digestive enzyme secretion and membrane recycling. Immunoelectron microscopy showed that GP2 and amylase co-localized in secretory granules in infected AR4-2J cells. CCK-8 stimulation resulted in a fourfold increase in amylase secretion with or without GP2 expression. GP2 expression also did not influence endocytosis following CCK-8 stimulation. Thus, GP2 expression in AR4-2J cells does not affect amylase packaging in secretory granules or stimulated secretion. GP2 expression also does not influence membrane recycling in response to stimulated stimulation in AR4-2J cells.

Effects of lectins on CCK-8-stimulated enzyme secretion and differentiation of the rat pancreatic cell line AR42J.

The peptide hormone cholecystokinin (CCK) plays an important role in the gastrointestinal tract. The rat pancreatic CCK receptor is a highly glycosylated membrane receptor that is able to bind to plant lectins such as wheat germ agglutinin (WGA) and Ulex europaeus agglutinin (UEA-I). We used both lectins to block this receptor for studying the pathophysiologic relevance of its oligosaccharide side chains. In the present study we investigated the influence of WGA and UEA-I on CCK-8-induced alpha-amylase secretion of the rat pancreatic tumor cell line AR42J, which expresses both CCK-A and CCK-B receptors. Under the influence of WGA (25 microg/mL), the alpha-amylase release was reduced by 25% after 30 minutes compared with the hormone-stimulated controls. UEA-I (25 microg/mL) caused a reduction of 20%. The simultaneous application of the lectins with CCK antagonists L 364,718 or L 365,260 led to a reduction of secretion, but the assignment to CCK-A or CCK-B receptors was not possible. In long-term studies, both lectins revealed no toxic or apoptosis-inducing effects. On the contrary, WGA showed an inhibitory effect on cell proliferation and led to improved differentiation of cells.

Effects of ethanol on the expression and secretion of bile salt-dependent lipase by pancreatic AR4-2J cells

The mechanisms by which ethanol administration alters pancreatic function are unknown. We have evaluated the effects of chronic ethanol treatment on secretion of a digestive enzyme: the bile salt-dependent lipase (BSDL), by the rat pancreatic cell line AR4-2J (as a model). We report that ethanol (50–300 mM) in culture medium induced a rise, in secreted and intracellular BSDL, that was a function of the duration of treatment and of the ethanol concentration. This effect was not abolished by pyrazole, which suggests a direct effect of ethanol. We have further established that the increase of BSDL activity was due to an enhanced biosynthesis of the enzyme consecutive to a major steady-state level of mRNA encoding BSDL. Also, the subcellular localization showed a specific accumulation of BSDL in the cytosolic fraction of cells chronically treated with ethanol. Given the enzymatic properties of BSDL, all these data could have some physiological consequences regarding the digestive function, plasma lipid metabolism and intracellular cholesterol homeostasis.

PACAP-38 causes phospholipase C-dependent calcium signaling in rat acinar cell line

Background. Pituitary adenylate cyclase activating peptide (PACAP-38), a neuropeptide of the vasoactive intestinal peptide/secretin family, localizes to intrapancreatic neurons and stimulates exocrine secretion from the pancreas. PACAP-38 stimulates calcium signaling in the rat pancreatic cell line AR42J. The purpose of this study was to elucidate the mechanisms of PACAP-evoked calcium signaling in these cells. Methods. Continuous measurements of intracellular calcium were taken by fluorescent digital microscopy with the dye fura-2. Mechanisms of PACAP-38—evoked calcium signals were determined by a panel of inhibitors. Inositol phosphates production in response to PACAP-38 was measured. The ability of PACAP-38 to stimulate amylase release was used to determine a relevant dose range for these studies. Results. We have shown that (1) AR42J cells respond to PACAP-38 with biphasic increases in [Ca 2+] i in a dose-dependent fashion; (2) PACAP-38 acts through phospholipase C to release inositol triphosphate (IP 3)-sensitive Ca 2+ stores with (3) a subsequent influx of extracellular Ca 2+. Conclusions. PACAP-38 activates calcium signaling through phospholipase C at concentrations that stimulate amylase release in AR42J cells.

Inhibitory effects of the gastrin receptor antagonist CR2093 on basal, gastrin-stimulated and growth factor-stimulated growth of the rat pancreatic cell line AR42J

AR42J, a rat pancreatic cell line expressing receptors for both gastrin and epidermal growth factor (EGF), has been used to examine the effect of the gastrin receptor antagonist CR2093 on basal, gastrin-17 (G17), EGF and transforming growth factor (TGF)-alpha stimulated growth in vitro. In serum-free conditions, CR2093 reduced the basal growth of AR42J at a concentration known to displace physiological levels of G17 from gastrin receptors and this effect was reversed by G17 at 1 x 10(-9) M. Alone, G17 had little effect on growth, but EGF and TGF-alpha stimulated growth to 181 and 176% of control values, respectively, and marked synergy was observed when G17 was used in combination with both EGF (212%) and TGF-alpha (259%). When CR2093 was added, the synergistic effects of the G17/EGF and G17/TGF-alpha combinations were reduced to basal levels. In addition, CR2093 inhibited the growth stimulation induced by EGF and TGF-alpha alone. When the ability of CR2093 to bind to EGF receptors was assessed in a ligand binding assay, it was found that the antagonist displaced up to 23% of labeled EGF. Thus CR2093 has potent inhibitory effects on the basal growth of AR42J which can be reversed by G17. It can also inhibit type 1 growth factor-stimulated growth, but although this action may in part be related to the antagonist's ability to inhibit binding to the EGF receptor, other mechanisms may be involved.