Rat Pancreatic Acinar AR42J Cells Research Articles

Algal Oil Mitigates Sodium Taurocholate-Induced Pancreatitis by Alleviating Calcium Overload, Oxidative Stress, and NF-κB Activation in Pancreatic Acinar Cells.

Acute pancreatitis (AP) is characterized by elevated intracellular Ca2+ concentrations, mitochondrial dysfunction, and oxidative stress in pancreatic acinar cells. Algal oil (AO) has demonstrated antioxidant and anti-inflammatory properties. This study aims to explore the effects of algal oil on the microenvironment of AP. Rat pancreatic acinar AR42J cells were pretreated with AO containing 0, 50, 100, or 150 μM of docosahexaenoic acid (DHA) 2 h prior to AP induction using sodium taurocholate (STC). After 1 h of STC treatment, AR42J cells exhibited a significant increase in intracellular Ca2+ concentration and the production of amylase, lipase, reactive oxygen species, and pro-inflammatory mediators, including tumor necrosis factor-α and interleukin-6. These STC-induced increases were markedly reduced in cells pretreated with AO. In comparison to cells without AO, those treated with a high dose of AO before STC exposure demonstrated a significant increase in mitochondrial membrane potential and a decrease in lipid peroxidation. Furthermore, STC-activated nuclear factor kappa-B (NF-κB) was attenuated in AO-pretreated cells, as evidenced by a significant decrease in activated NF-κB. In conclusion, AO may prevent damage to pancreatic acinar cells by alleviating intracellular Ca2+ overload, mitigating mitochondrial dysfunction, reducing oxidative stress, and attenuating NF-κB-targeted inflammation.

Circ_0000284 Promoted Acute Pancreatitis Progression through the Regulation of miR-10a-5p/Wnt/β-Catenin Pathway.

Circular RNAs (circRNAs) have been found to be involved in the progression of acute pancreatitis (AP). The objective of our study was to investigate the effects of circ_0000284 on caerulein-induced AR42J cell injury. To mimic AP in vitro, rat pancreatic acinar AR42J cells were treated with caerulein. The expression of circ_0000284 and miR-10a-5p was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Enzyme-linked immunosorbent assay (ELISA) was employed to determine the content of inflammatory cytokines interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor α (TNF-α). Western blotting was applied to analyze the levels of Wnt/β-catenin pathway-related and apoptosis-related proteins. Cell viability and apoptosis were monitored by Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. The target connection between circ_0000284 and miR-10a-5p was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. AP induced inflammation in patients, and caerulein treatment increased apoptosis and inflammation in AR42J cells. Circ_0000284 was upregulated in serum of AP patients and caerulein-induced AR42J cells, while Wnt/β-catenin pathway was inactivated. Knockdown of circ_0000284 could decrease apoptosis and inflammation in caerulein-induced AR42J cells, which was attenuated by miR-10a-5p inhibition or Wnt signaling pathway antagonist Dickkopf-related protein 1 (DKK1). MiR-10a-5p was sponged by circ_000028 and was downregulated in caerulein-induced AR42J cells. Circ_0000284 depletion could protect caerulein-induced AR42J cells from apoptosis and inflammation by upregulating miR-10a-5p expression and activating Wnt/β-catenin pathway, underscoring a potential target for AP therapy.

N-glycosylation of somatostatin receptor type 2 protects rats from acute pancreatitis.

BackgroundThe growth hormone inhibitor somatostatin and its analogs are promising therapeutic agents for acute pancreatitis. However, the therapeutic effects remain controversial. Somatostatin analogs preferentially bind to somatostatin receptor 2 (SSTR2), and this study aimed to investigate whether N-glycosylation affects SSTR2 stability, membrane trafficking, and signal transduction.MethodsWestern blot analysis following PNGase F digestion was performed to confirm N-glycosylation of SSTR2 in rat pancreatic acinar AR42J cells. Rats were subjected to 4 hourly intraperitoneal injections of cerulein (50 µg/kg) plus LPS (5 µg/mL) to induce acute pancreatitis. Mass spectrometry was conducted to identify the glycosylation sites of SSTR2, and immunofluorescent staining was carried out to examine the localization of wild-type and asparagine 9 (N9)Q-mutant SSTR2. Proteasome inhibitor MG132 was employed to assess the stability of SSTR2, and overexpression of wild-type or N9Q-mutant SSTR2 was used to examine the role of N-glycosylation in SSTR2 signal transduction in vitro and its therapeutic effect on pancreatitis in rats.ResultsSSTR2 protein in AR42J cells was N-glycosylated at the N9 residue. Wild-type SSTR2 localized in the plasma membrane whereas N9Q-mutant SSTR2 was retained in the endoplasmic reticulum (ER). MG132 rapidly restored the ectopic expression of mutant but not wild-type SSTR2 protein in AR42J cells. Overexpression of wild-type but not N9Q-mutant SSTR2 activated NF-κB signaling and facilitated nuclear transportation of p65 in AR42J cells upon cerulein plus LPS stimulation. Furthermore, pretreatment with wild-type but not N9Q-mutant SSTR2-overexpressing vectors significantly ameliorated biochemical parameters and pancreatic histological impairments in rats with pancreatitis.ConclusionsN-glycosylation of SSTR2 is essential for membrane localization, stability, and signal transduction of SSTR2 in pancreatic cells, playing a protective role in experimental acute pancreatitis.

Circ_ZFP644 attenuates caerulein-induced inflammatory injury in rat pancreatic acinar cells by modulating miR-106b/Pias3 axis

(AP) is a kind of inflammatory misorder existing in pancreas. Non-coding RNAs (ncRNAs) have been reported to play important roles in development of AP. The current study was designed to explore the role of circular RNA zinc finger protein 644 (circRNA circ_ZFP644) in caerulein-induced AR42J cells. AP model in vitro was established by exposure of rat pancreatic acinar AR42J cells to caerulein. Amylase activity was measured using a kit. Enzyme-linked immunosorbent assay (ELISA) was performed to examine the levels of several inflammatory factors. The expression of circ_ZFP644, microRNA (miR)-106b and protein inhibitor of activated STAT 3 (Pias3) was detected by quantitative real-time PCR (qRT-PCR) or western blot assay. And flow cytometry was employed to monitor cell apoptosis. Western blot assay was also conducted to analyze the expression of apoptosis-related proteins. The association among circ_ZFP644, miR-106b and Pias3 was validated by dual-luciferase reporter assay. Caerulein treatment activated amylase activity and promoted the secretion of inflammatory cytokines in AR42J cells. Circ_ZFP644 and Pias3 were downregulated, but miR-106b was upregulated in caerulein-induced AR42J cells. Enforced expression of circ_ZFP644 or miR-106b inhibition could reduce amylase activity and inflammatory cytokine secretion, while promote apoptosis in caerulein-induced AR42J cells, which was almost reversed by Pias3 knockdown. Circ_ZFP644 targeted miR-106b to upregulate Pias3 expression. Circ_ZFP644 might exert its anti-inflammation and pro-apoptosis roles in caerulein-induced AR42J cells by regulating miR-106b/Pias3 axis.

A chloride channel in rat pancreatic acinar AR42J cells is sensitive to extracellular acidification and dependent on ROS

Extracellular acidification, playing a promoting role in the process of acute pancreatitis, has been reported to activate Cl− channels in several types of cells. However, whether extracellular acidification aggravates acute pancreatitis via activating Cl− channels remains unclear. Here, we investigated the effects of extracellular acidification on Cl− channels in rat pancreatic acinar AR42J cells using whole-cell patch-clamp recordings. We found that extracellular acidification induced a moderately outward-rectified Cl− current, with a selectivity sequence of I− > Br− ≥ Cl− > gluconate−, while intracellular acidification failed to induce the currents. The acid-sensitive currents were inhibited by Cl− channel blockers, 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate and 5-Nitro-2-(3-phenylpropylamino) benzoic acid. After ClC-3 was silenced by ClC-3 shRNA, the acid-sensitive Cl− currents were attenuated significantly, indicating that ClC-3 plays a vital role in the induction of acid-sensitive Cl− currents. Extracellular acid elevated the intracellular level of reactive oxygen species (ROS) significantly, prior to inducing Cl− currents. When ROS production was scavenged, the acid-sensitive Cl− currents were abolished. Whereas, the level of acid-induced ROS was unaffected with silence of ClC-3. Our findings above demonstrate that extracellular acidification induces a Cl− current in pancreatic acinar cells via promoting ROS generation, implying an underlying mechanism that extracellular acidification might aggravate acute pancreatitis through Cl− channels.

Pharmacological inhibition of mTORC1 increases CCKBR-specific tumor uptake of radiolabeled minigastrin analogue [177Lu]Lu-PP-F11N.

Rationale: A high tumor-to-healthy-tissue uptake ratio of radiolabeled ligands is an essential prerequisite for safe and effective peptide receptor radionuclide therapy (PRRT). In the present study, we searched for novel opportunities to increase tumor-specific uptake of the radiolabeled minigastrin analogue [177Lu]Lu-DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2 ([177Lu]Lu-PP-F11N), that targets the cholecystokinin B receptor (CCKBR) in human cancers.Methods: A kinase inhibitor library screen followed by proliferation and internalization assays were employed to identify compounds which can increase uptake of [177Lu]Lu-PP-F11N in CCKBR-transfected human epidermoid carcinoma A431 cells and natural CCKBR-expressing rat pancreatic acinar AR42J cells. Western blot (WB) analysis verified the inhibition of the signaling pathways and the CCKBR level, whereas the cell-based assay analyzed arrestin recruitment. Biodistribution and SPECT imaging of the A431/CCKBR xenograft mouse model as well as histological analysis of the dissected tumors were used for in vivo validation.Results: Our screen identified the inhibitors of mammalian target of rapamycin complex 1 (mTORC1), which increased cell uptake of [177Lu]Lu-PP-F11N. Pharmacological mTORC1 inhibition by RAD001 and metformin increased internalization of [177Lu]Lu-PP-F11N in A431/CCKBR and in AR42J cells. Analysis of protein lysates from RAD001-treated cells revealed increased levels of CCKBR (2.2-fold) and inhibition of S6 phosphorylation. PP-F11N induced recruitment of β-arrestin1/2 and ERK1/2 phosphorylation. In A431/CCKBR-tumor bearing nude mice, 3 or 5 days of RAD001 pretreatment significantly enhanced tumor-specific uptake of [177Lu]Lu-PP-F11N (ratio [RAD001/Control] of 1.56 or 1.79, respectively), whereas metformin treatment did not show a significant difference. Quantification of SPECT/CT images confirmed higher uptake of [177Lu]Lu-PP-F11N in RAD001-treated tumors with ratios [RAD001/Control] of average and maximum concentration reaching 3.11 and 3.17, respectively. HE staining and IHC of RAD001-treated tumors showed a significant increase in necrosis (1.4% control vs.10.6% of necrotic area) and the reduction of proliferative (80% control vs. 61% of Ki67 positive cells) and mitotically active cells (1.08% control vs. 0.75% of mitotic figures). No significant difference in the tumor vascularization was observed after five-day RAD001 or metformin treatment.Conclusions: Our data demonstrates, that increased CCKBR protein level by RAD001 pretreatment has the potential to improve tumor uptake of [177Lu]Lu-PP-F11N and provides proof-of-concept for the development of molecular strategies aimed at enhancing the level of the targeted receptor, to increase the efficacy of PRRT and nuclear imaging.

Pim-3 alleviates lipopolysaccharide-stimulated AR42J pancreatic acinar cell injury via improving the inflammatory microenvironment.

Acute pancreatitis (AP) is a common acute abdominal disease characterized by pancreatic aseptic inflammation, with ~20% of patients progressing to severe AP (SAP) with a high mortality rate. The aim of this study was to explore the protective effects of Pim-3 proto-oncogene, serine/threonine kinase (Pim-3) on rat pancreatic acinar AR4-2J cells damaged by lipopolysaccharide (LPS). The recombinant plasmid p-enhanced green fluorescent protein (pEGFP)-N2/Pim-3 was transiently transfected into AR42J cells and the AR42J cells were then treated with 2 µg/ml LPS. Subsequently, the proliferation of AR42J cells was detected using MTT assay. The cell cycle progression and apoptosis rate of the AR42J cells were examined using flow cytometry. AR42J cell migration was assessed using wound healing assays. Additionally, RT-semi quantitative PCR and western blot analyses were used to detect the mRNA and protein expression levels, respectively, of Pim-3, interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF)-α, intercellular adhesion molecule (ICAM)-1 and Occludin in AR42J cells. The results revealed that proliferation of AR42J cells was significantly enhanced and cell apoptosis was markedly reduced in the pEGFP-N2/Pim-3 + LPS group. The proportion of AR42J cells in G1 phase in the pEGFP-N2/Pim-3 + LPS group was decreased, whereas the proportion of cells in the G2 and S phases was increased. The wound healing assays demonstrated that AR42J cell migration was significantly increased in the pEGFP-N2/Pim-3 + LPS group. Finally, the expression levels of IL-6, IL-1β, TNF-α and ICAM-1 were significantly decreased in the pEGFP-N2/Pim-3 + LPS group, whereas the expression of Occludin was significantly increased. The present study demonstrated that raised expression levels of Pim-3 can protect AR42J cells from LPS-induced injury by modifying the inflammatory microenvironment, suggesting that Pim-3 may be a potential target for AP or SAP therapy.

The JAK2/STAT3 signaling pathway is required for inflammation and cell death induced by cerulein in AR42J cells.

The study was designed to investigate the JAK2/STAT3 signaling pathway in pancreatitis and its association with inflammation and cell death to provide a potential treatment method for pancreatitis. The rat pancreatic acinar AR42J cells were used for the study, and they were transfected with JAK2 and STAT3 siRNAs to mimic knockdown condition. Cerulein was used to treat AR42J cells. Western blot and ELISA were employed to detect the expression of related proteins. Flow cytometry was done to analysis the necrosis of AR42J cells. In this study, we found that cell death and the secretion of IL-6 and TGF-β1 were significantly increased, and the JAK2/STAT3 signaling pathway was activated in cerulein-induced AP. To determine the role of JAK2 and STAT3, JAK2 siRNA and STAT3 siRNA were used to block JAK2 and STAT3, respectively. The levels of IL-6 and TGF-β1 levels in the medium were lower in JAK2 siRNA and STAT3 siRNA-treated cells compared with controls. Flow cytometry analysis showed that the level of cell death, expression of cleaved caspase-3, and the release of LDH were decreased following JAK2 siRNA and STAT3 siRNA treatment. These findings point to a novel role for the JAK2/STAT3 signaling pathway in the progression of cerulein-induced AP.

Screening and validation of differentially expressed extracellular miRNAs in acute pancreatitis.

The present study aimed to screen for differentially expressed extracellular microRNAs (miRNAs) during the development of acute pancreatitis (AP) and validate the miRNA expression in the plasma of patients with AP. The culture medium of taurolithocholic acid‑3 sulfate‑treated rat pancreatic acinar AR42J cells was collected to extract total RNA for miRNA microarray analysis. Compared with the miRNA test results of the AP rats in the GEO databases, the differentially expressed extracellular miRNAs were screened. The TargetScan, miRanda, and PicTar programs were used for target gene prediction of the identified miRNAs, and gene ontology‑biological processes (GO‑BP) functional annotation was performed. Finally, the results from the combined microarray analyses (invitro cell line and invivo rat samples) were validated using plasma samples from patients with mild and moderately severe AP by reverse transcription‑polymerase chain reaction. The results demonstrated that extracellular miR‑24 was differentially expressed by microarray and bioinformatics analysis in both the cell line and the animal model of AP. Bioinformatics prediction analysis revealed that downstream target genes of miR‑24 included Vav2, Syk, Lhcgr, Slc9a3r1, Cacnb1, Cacna1b, Bcl10, and Fgd3. Functional enrichment analysis revealed that the main GO‑BP predicted functional presentations were positive regulation of calcium‑mediated signaling, activation of c‑Jun N‑terminal kinase activity, calcium ion transport, regulation of Rho protein signal transduction, negative regulation of the protein kinase B signaling cascade, and the T cell receptor signaling pathway. Validation analysis for the plasma miR‑24 expression in humans revealed a significant upregulation of miR‑24 in the plasma samples of AP patients compared with the healthy controls, while no significant difference was observed in the miR‑24 expression between the mild and the moderately severe AP groups. The present study confirmed the high expression of miR‑24 in peripheral blood during AP, suggesting that miR‑24 might have an intercellular communication role contributing to the AP‑associated distant organ injury.

Transforming growth factor (TGF)-β-induced microRNA-216a promotes acute pancreatitis via Akt and TGF-β pathway in mice.

Both transforming growth factor β (TGF-β) and MicroRNA-216a (miR-216a) were reported to be upregulated during acute pancreatitis (AP). Moreover, miR-216a can be induced by TGF-β. This study aimed to investigate how TGF-β and miR-216a involved in the pathogenesis of AP both in a mouse model and in rat pancreatic acinar AR42J cells. Cerulein-induced AP mouse model was established and pretreated with a TGF-β inhibitor, SB431542. Serum amylase, lipase, tumor necrosis factor (TNF)-α, interleukin 6 (IL-6), TGF-β and histopathological changes of pancreas were determined. Expression of miR-216a was detected by quantitative real-time RT-PCR. Bioinformatics was utilized to predict the targets of miR-216a. Expression levels of phosphatase and tensin homolog (PTEN), mothers against decapentaplegic homolog 7 (Smad7), TGF-β receptor I, total Akt and pAkt were detected by Western blot. SB431542 significantly decreased serum amylase, lipase, TNF-α, IL-6, TGF-β, histopathological changes of pancreas and expression of miR-216a in cerulein-induced mouse (P < 0.05). TGF-β induced miR-216a in AR42J cells. PTEN and Smad7 were identified to be the possible targets of miR-216a. Transfection of miR-216a mimics (or inhibitors) in AR42J cells downregulated (or upregulated) the expression of PTEN and Smad7, thus affected the expression of downstream pAkt and TGF-β receptor I. The expression changes of these protein caused by miR-216a can be regulated by SB431542 both in mouse model and AR42J cells. TGF-β promotes AP by inducing miR-216a targeting PTEN and Smad7, thus through PI3K/Akt and TGF-β feedback pathway.

Alcohol oxidizing enzymes and ethanol-induced cytotoxicity in rat pancreatic acinar AR42J cells

Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disease causing significant morbidity and mortality. Due to lack of a suitable animal model, the underlying mechanism of ACP is poorly understood. Chronic alcohol abuse inhibits alcohol dehydrogenase (ADH) and facilitates nonoxidative metabolism of ethanol to fatty acid ethyl esters (FAEEs) in the pancreas frequently damaged during chronic ethanol abuse. Earlier, we reported a concentration-dependent formation of FAEEs and cytotoxicity in ethanol-treated rat pancreatic tumor (AR42J) cells, which express high FAEE synthase activity as compared to ADH and cytochrome P450 2E1. Therefore, the present study was undertaken to investigate the role of various ethanol oxidizing enzymes in ethanol-induced pancreatic acinar cell injury. Confluent AR42J cells were pre-treated with inhibitors of ADH class I and II [4-methylpyrazole (MP)] or class I, II, and III [1,10-phenanthroline (PT)], cytochrome P450 2E1 (trans-1,2-dichloroethylene) or catalase (sodium azide) followed by incubation with 800 mg% ethanol at 37°C for 6 h. Ethanol metabolism, cell viability, cytotoxicity (apoptosis and necrosis), cell proliferation status, and formation of FAEEs in AR42J cells were measured. The cell viability and cell proliferation rate were significantly reduced in cells pretreated with 1,10-PT + ethanol followed by those with 4-MP + ethanol. In situ formation of FAEEs was twofold greater in cells incubated with 1,10-PT + ethanol and ∼1.5-fold in those treated with 4-MP + ethanol vs. respective controls. However, cells treated with inhibitors of cytochrome P450 2E1 or catalase in combination of ethanol showed no significant changes either for FAEE formation, cell death or proliferation rate. Therefore, an impaired ADH class I-III catalyzed oxidation of ethanol appears to be a key contributing factor in ethanol-induced pancreatic injury via formation of nonoxidative metabolites of ethanol.

The adipocytokine resistin stimulates the production of proinflammatory cytokines TNF-α and IL-6 in pancreatic acinar cells via NF-κB activation.

Resistin, an adipocytokine secreted by fat tissues, has been associated with the inflammatory response, though its role in inflammation during acute pancreatitis (AP) remains unclear. The proinflammatory response following acinar cell injury impacts pancreatitis severity, necessitating better understanding of functional consequences associated with pancreatic acinar cell resistin exposure and resultant effects on proinflammatory signaling. Amylase-secreting rat pancreatic acinar AR42J cells were subjected to 1, 10, or 100 ng/ml recombinant rat resistin treatments. Cytotoxicity was evaluated by amylase secretion and lactate dehydrogenase (LDH) release. Tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6) mRNA and protein expressions were determined by real-time real time-PCR and enzyme-linked immunosorbent assay, respectively. Nuclear NF-κB p65 subunit protein level was measured by western blotting. Significantly increased amylase secretion and LDH release was observed in the 100 ng/ml resistin treatment (p<0.01). Both TNF-α and IL-6 protein expression levels increased in a concentration-dependent manner when treated with resistin. Pretreatment of resistin- treated AR42J cells with the NF-κB inhibitor PDTC, which decreases the NF-κB p65 subunit protein expression levels in the nuclei, produced significantly lower mRNA expression levels for both TNF-α and IL-6 compared with those produced by resistin-treated cells (p<0.01). Resistin exhibits some cytotoxic activity in rat pancreatic acinar AR42J cells and stimulates proinflammatory cytokine TNF-α and IL-6 production via NF-κB activation. Thus, overproduction of obesity-related circulating resistin and associated lowgrade inflammation may result in mild injury to pancreatic acini, increasing AP severity and risk.

Early proteome analysis of rat pancreatic acinar AR42J cells treated with taurolithocholic acid 3-sulfate

BackgroundBile acids are the initiating factors of biliary acute pancreatitis. Bile acids can induce the activation of intracellular zymogen, thus leading injury in pancreatic acinar cells. Pathological zymogen activation in pancreatic acinar cells is a common feature of all types of acute pancreatitis. The proteins expressed in pancreatic acinar cells during the activation of zymogen may determine the severity of acute pancreatitis. The present study aims to determine the differentially expressed proteins in taurolithocholic acid 3-sulfate-stimulated pancreatic acinar cells as an in vitro model for acute pancreatitis. MethodsRat pancreatic acinar AR42J cells were treated with taurolithocholic acid 3-sulfate for 20 min. Laser confocal scanning microscopy and flow cytometry were used to detect activated trypsinogen in pancreatic acinar AR42J cells. After the determination of trypsinogen activation, proteome analysis was performed to identify the proteins differentially expressed in taurolithocholic acid 3-sulfate-treated cells and non-treated cells. ResultsAfter treatment with taurolithocholic acid 3-sulfate for 20 min, the activation of trypsinogen in AR42J cells was concurrent with changes in the protein expression profile. Thirty-nine differentially expressed proteins were detected; among these, 23 proteins were up-regulated and 16 proteins were down-regulated. KEGG analysis indicated that these proteins are involved in cellular metabolic pathways, cellular defensive mechanisms, intracellular calcium regulation and cytoskeletal changes. ConclusionThe expression of proteins in the pancreatic acinar cell changes at the early stage of biliary acute pancreatitis. These differentially expressed proteins will provide valuable information to understand the pathophysiologic mechanism biliary acute pancreatitis and may be useful for prognostic indices of acute pancreatitis.

Role of intracellular calcium on hydrogen peroxide-induced apoptosis in rat pancreatic acinar AR42J cells

The authors investigated whether cytosolic free calcium concentration ([Ca2+]c) plays a role in hydrogen peroxide-induced pancreatic acinar AR42J cells apoptosis. We analysed mitochondrial depolarization, [Ca2+]c determination and caspase-3 activity by fluorimetric methods, and cytochrome c release by subcellular fractionation and western blotting. The data shown that hydrogen peroxide, which causes a sustained [Ca2+]c increase, induces mitochondrial depolarization and cytochrome c release, and activation of caspase-3. Dimethyl-BAPTA loading did not affect hydrogen peroxide-evoked mitochondrial apoptosis, suggesting that these responses are independent of increases in [Ca2+]c. Treatment with thapsigargin, to induce extensive calcium store depletion and subsequent increases in [Ca2+]c, also stimulates mitochondrial depolarization cytochrome c release, and caspase-3 activation. Similar results were observed in AR42J cells loaded with dimethyl-BAPTA, suggesting that activation of apoptosis by thapsigargin does not require rises in [Ca2+]c. However, the blockade of mitochondrial calcium entry by pretreating with Ru360 showed protection against hydrogen peroxide- and thapsigargin-induced mitochondrial apoptosis. These results indicate that the apoptosis evoked by hydrogen peroxide and thapsigargin is mediated by mitochondrial calcium uptake.

Ethanol-induced cytotoxicity in rat pancreatic acinar AR42J cells: Role of fatty acid ethyl esters

To understand the mechanism(s) of alcoholic pancreatitis and role of fatty acid ethyl esters (FAEEs, non-oxidative metabolites of ethanol) in ethanol-induced pancreatic injury. A time- and concentration-dependent synthesis of FAEEs and the cytotoxicity of ethanol and its predominant fatty acid esters were studied in rat pancreatic tumour (AR42J) cells in cultures. Role of FAEEs in ethanol-induced cytotoxicity was investigated by measuring the synthesis of FAEEs, injury markers and apoptosis in cells incubated simultaneously with ethanol and FAEE synthase inhibitor, 3-benzyl-6-chloro-2-pyrone. The cells were pre-incubated with caspase-3 inhibitor (N-acetyl-DEVD-CHO) to measure the effect of caspase-3 inhibition on ethanol-induced apoptosis. The levels of FAEEs synthesized in cell cultures incubated with 800 mg% ethanol for 6 h were approximately 10-fold higher (60 nmol/25 x 10(6) cells) than those in cells incubated with 100 mg% ethanol (5.4 nmol/25 x 10(6) cells). Ethanol exposure resulted in a concentration-dependent apoptosis (10, 12 and 13% at 200, 400 and 800 mg% ethanol, respectively, vs 5% in controls). A similar concentration-dependent apoptosis was also found in the cells incubated with ethyl oleate (one of the predominant FAEEs reported in alcoholic patients). Inhibition of FAEE synthesis and resultant apoptosis was found in the cells incubated simultaneously with pancreatic FAEE synthase inhibitor and ethanol. Ethanol-induced apoptosis was significantly inhibited in cells pre-incubated with caspase-3 inhibitor. These results support our hypothesis that ethanol-induced cytotoxicity in AR42J cells is mediated by the non-oxidative metabolite(s) of ethanol, and caspase-3 mediated apoptosis could be one of the mechanisms involved in ethanol-induced pancreatic injury.

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 12 h. 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.

Hydrogen peroxide-induced activation of defense mechanisms against oxidative stress in rat pancreatic acinar AR42J cells

Oxidative stress has been implicated in the pathogenesis of acute pancreatitis. Generally, cells respond to oxidative stress with adaptive changes in gene expression aimed at preventing cellular damage and increasing their survival. However, the overall extent of these genetic changes remains poorly defined. This issue was, therefore, examined in the current study. Following exposure of rat pancreatic AR42J cells to 0.08 mM hydrogen peroxide (H 2O 2), a concentration failing to induce necrotic cell death, the expression of 96 stress-related genes was monitored by cDNA microarray analysis. H 2O 2 provoked a time-dependent reorientation of 54 genes. In particular, at 6 and 24 h, 27 and 11 genes were induced, whereas 10 and 6 genes were suppressed, respectively, showing that the degree of change was stronger at the early time point, and that the number of up-regulated genes was obviously larger than the number of down-regulated genes. Reverse transcription-PCR for selected genes confirmed the gene expression pattern. Many of the differentially up-regulated genes can be related to the antioxidant enzymatic defense system, to cell cycle arrest, to repair and/or replacement of damaged DNA, to repair of damaged protein, and to activation of the NF-κB pathway. The results suggest that AR42J cells respond to sublethal oxidative stress with transient transcriptional activation of multiple defense mechanisms that may be an indication for a complex adaptation process. An understanding of the cellular stress responses may lead to new insights into the pathogenesis of oxidative stress-related diseases including acute pancreatitis.

Phosphatidic acid production, required for cholecystokinin octapeptide-stimulated amylase secretion from pancreatic acinar AR42J cells, is regulated by a wortmannin-sensitive process

To investigate the role of phospholipids in exocytotic secretory events, we utilized rat pancreatic acinar AR42J cells that secreted amylase in response to cholecystokinin octapeptide (CCK-8). Wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), was found to inhibit the secretion in a dose-dependent manner. When changes in cell membrane phospholipids were investigated before and after CCK-8 stimulation using [ 32P]orthophosphoric acid-labeled AR42J cells, we observed a rapid increase in phosphatidic acid (PtdOH) levels right after stimulation, which was not observed in non-stimulated cells. The increase, however, was suppressed by wortmannin pre-treatment, which also inhibited amylase secretion. Changes in other major phospholipids were not significant. These results indicate that CCK-8 induces amylase secretion through PI3K-regulated production of PtdOH in cell membranes.

Transient Agonist-Induced Regulation of the Cholecystokinin-A and Cholecystokinin-B Receptor mRNA Levels in Rat Pancreatic Acinar AR42J Cells

Background: CCK-8 and gastrin exert multiple effects in the gastrointestinal tract and the nervous system. Their actions are mediated via the G-protein coupled CCK-A and CCK-B receptors. Methods: Rat pancreatic acinar tumor AR42J cells express both CCK receptor subtypes. This cell line was used to characterize the agonist-dependent regulation of CCK-A and CCK-B receptor gene expression. Results: CCK-8 (10 nM) or gastrin (10 nM) reduced CCK-A receptor mRNA expression to 56% and 53%, respectively 2 h after hormonal exposure. In contrast, the level of CCK-B receptor gene expression was upregulated to 157% and 153%, respectively. These effects are most probably linked to the CCK-B receptor in AR42J cells. The phorbolester PMA (100 nM), a protein kinase C activator, downregulated CCK-A receptor expression but did not affect CCK-B receptor gene transcription. Activation of protein kinase A by forskolin (10 µM) or Bt₂cAMP (100 µM) is not involved in the transient regulation of CCK receptor mRNA expression. Both elevated CCK-B and decreased CCK-A receptor mRNA expression returned to basal levels 6 h after continuous stimulation. Conclusion: These results demonstrate that CCK-A and CCK-B receptor mRNA levels are differentially regulated by their agonists via distinct signal transduction mechanisms in AR42J cells.

Induction of chemokines in rat pancreatic acinar cell injury.

The pathogenesis of chronic pancreatitis (CP), especially of acinar cell injury, is still unclear. Interleukin (IL)-8 is a chemokine that is involved in various inflammatory diseases. To examine whether IL-8 and other chemokines are expressed in experimental acinar cell injury. IL-8 expression was analyzed in spontaneous CP in the WBN/Kob rat and in rat pancreatic acinar AR4-2J cells treated with various stimuli using reverse transcription-polymerase chain reaction (semiquantitative) and immunohistochemistry. Chronic pancreatitis developed at 12 weeks in the WBN/Kob rats. IL-8, macrophage chemoattractant protein-1, and macrophage inflammatory protein-2 mRNA was expressed from 4 weeks and peaked at 12 weeks. Immunohistochemistry showed a strong expression of IL-8 in acinar cells, proliferating ductular cells, and interstitial infiltrating cells. In contrast, normal pancreatic tissues lacked IL-8 expression. Further, IL-8 mRNA and protein were detectable in AR4-2J cells treated with the various stimuli, such as menadione, tumor necrosis factor-alpha and transforming growth factor beta1. These results suggest that IL-8 is expressed in the pancreatic parenchyma and infiltrates in CP and that it plays a role in the initial pathogenesis of CP together with other chemokines and cytokines.