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.     

Pivotal role of reactive oxygen species as intracellular mediators of hyperthermia-induced apoptosis

The effects of cellular antioxidant capacity on hyperthermia (HT)-induced apoptosis and production of antiapoptotic heat shock proteins (HSPs) were investigated in HL-60 cells and in HL-60AR cells that are characterized by an elevated endogenous catalase activity. Exposure of both cell lines to 43 degrees C for 1 h initiated apoptosis. Apoptosis peaked at 3-6 h after heat exposure in the HL-60 cells. Whereas HL-60AR cells were partially protected against HT-induced apoptosis at these early time points, maximal levels of apoptosis were detected later, i.e. 12-18 h after heat exposure. This differential induction of apoptosis was directly correlated to the induction of the antiapoptotic HSP27 and HSP70. In particular, in the HL-60 cells HSP27 was significantly induced at 12-18 h after exposure to 43 degrees C when apoptosis dropped. In contrast, coinciding with the late onset of apoptosis in HL-60AR cells at that time HL-60AR cells lacked a similar HSP response. In line with the higher antioxidant capacity HL-60AR cells accumulated reactive oxygen species to a lesser degree than HL-60 cells after heat treatment. Protection from HT-induced apoptosis as well as diminished heat-induced HSP27 expression was also observed after cotreatment of HL-60 cells with 43 degrees C and catalase but not with superoxide dismutase. These data emphasize the pivotal role of reactive oxygen species for HT induced pro- and antiapoptotic pathways.     

Modulating zymogen granule formation in pancreatic AR42J cells

Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. To investigate ZG biogenesis, cargo sorting and packaging, suitable cellular model systems are required. Here, we demonstrate that granule formation in pancreatic AR42J cells, an acinar model system, can be modulated by altering the growth conditions in cell culture. We find that cultivation of AR42J cells in Panserin^? 401, a serum-free medium, enhances the induction of granule formation in the presence or absence of dexamethasone when compared to standard conditions including serum. Biochemical and morphological studies revealed an increase in ZG markers on the mRNA and protein level, as well as in granule size compared to standard conditions. Our data indicate that this effect is related to pronounced differentiation of AR42J cells. To address if enhanced expression of ZG proteins promotes granule formation, we expressed several zymogens and ZG membrane proteins in unstimulated AR42J cells and in constitutively secreting COS-7 cells. Neither single expression nor co-expression was sufficient to initiate granule formation in AR42J cells or the formation of granule-like structures in COS-7 cells as described for neuroendocrine cargo proteins. The importance of our findings for granule formation in exocrine cells is discussed.     

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.     

Enhanced expression of heat shock protein and mRNA synthesis by type I interferon in human HL-60 leukemic cells

The effects of IFN and mild hyperthermia on the responses of human promyelocytic HL-60 cells were investigated. Cells subjected to an elevated culture temperature (39.5 degrees-40.5 degrees C instead of 37 degrees C, herein referred to as heat-treated cells) showed an increase in heat shock proteins (HSPs) and corresponding mRNA synthesis, which were additionally potentiated by the presence of IFN. With cells cultured at 37 degrees C, IFN had no effect on HSP expression. The observed inhibition (40-70%) of RNA polymerase II-directed RNA synthesis (based on alpha-amanitin sensitivity) in isolated nuclei of heat-treated cells was also significantly reversed by the simultaneous addition of IFN. These data suggest that the IFN-amplified HSP gene expression may be involved in preventing irreversible damage or in fine tuning the recovery of mammalian cells from heat stress.     

PSD-95 protects the pancreas against pathological damage through p38 MAPK signaling pathway in acute pancreatitis

Acute pancreatitis is one of the leading causes of gastrointestinal disorder-related hospitalizations, yet its pathogenesis remains to be fully elucidated. Postsynaptic density protein-95 (PSD-95) is closely associated with tissue inflammation and injury. We aimed to investigate the expression of PSD-95 in pancreatic acinar cells, and its function in regulating the inflammatory response and pancreatic pathological damage in acute pancreatitis. A mouse model of edematous acute pancreatitis was induced with caerulein and lipopolysaccharide in C57BL/6 mice. Tat-N-dimer was injected to inhibit the PSD-95 activity separately, or simultaneously with SB203580, inhibitor of p38 MAPK phosphorylation. Rat pancreatic acinar cells AR42J were cultured with 1 μM caerulein to build a cell model of acute pancreatitis. PSD-95-knockdown and negative control cell lines were constructed by lentiviral transfection of AR42J cells. Paraffin-embedded pancreatic tissue samples were processed for routine HE staining to evaluate the pathological changes of human and mouse pancreatic tissues. Serum amylase and inflammatory cytokine levels were detected with specific ELISA kits. Immunofluorescence, immunohistochemical, Western-blot, and qRT-PCR were used to detect the expression levels of PSD-95, p38, and phosphorylated p38. Our findings showed that PSD-95 is expressed in the pancreatic tissues of humans, C57BL/6 mice, and AR42J cells, primarily in the cytoplasm. PSD-95 expression increased at 2 h, reaching the peak at 6 h in mice and 12 h in AR42J cells. IL-6, IL-8, and TNF-α increased within 2 h of disease induction. The pancreatic histopathologic score was greater in the PSD-95 inhibition group compared with the control (P < 0.05), while it was lesser when phosphorylation of p38 MAPK was inhibited compared with the PSD-95 inhibition group (P < 0.05). Moreover, phosphorylation of p38 MAPK increased statistically after PSD-95 knocked-down. In conclusion, PSD-95 effectively influences the pathological damage of the pancreas in acute pancreatitis by affecting the phosphorylation of p38 MAPK.     

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.     

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.     

Sensory nerves in central and peripheral control of pancreatic integrity by leptin and melatonin.

Central nervous system affects pancreatic secretion of enzymes however, the neural modulation of acute pancreatitis has not been investigated. Leptin and melatonin have been recently reported to affect the inflammatory response of various tissues. The identification of specific receptors for both peptides in the pancreas suggests that leptin and melatonin could contribute to the pancreatic protection against inflammation. The aim of this study was: 1/ to compare the effect of intracerebroventricular (i.c.v.) or intraperitoneal (i.p.) administration of leptin or melatonin on the course of caerulein-induced pancreatitis (CIP) in the rat, 2/ to examine the involvement of sensory nerves (SN) and calcitonin gene-related peptide (CGRP) in pancreatic protection afforded by leptin or melatonin, 3/ to assess the effect of tested peptides on lipid peroxidation products (MDA + 4-HNE) in the pancreas of CIP rats, 4/ to investigate the influence of leptin or melatonin on nitric oxide (NO) release from isolated pancreatic acini and 5/ to determine the effects of caerulein and leptin on leptin receptor gene expression in these acini by RT-PCR. CIP was induced by subcutaneous (s.c.) infusion of caerulein (25 microg/kg) to the conscious rats, confirmed by the significant increases of pancreatic weight and plasma amylase and by histological examination. This was accompanied in marked reduction of pancreatic blood flow and significant rise of MDA + 4-HNE in the pancreas. Leptin or melatonin were administered i.p. or i.c.v. 30 min prior to the start of CIP. Deactivation of SN was produced by s.c. capsaicin (100 mg/kg). An antagonist of CGRP, CGRP 8-37 (100 microg/kg i.p.), was given together with leptin or melatonin to the CIP rats. MDA + 4-HNE was measured using LPO commercial kit. NO was determined using the Griess reaction. Pretreatment of CIP rats with i.p. leptin (2 or 10 microg/kg) or melatonin (10 or 50 mg/kg) significantly attenuated the severity of CIP. Similar protective effects were observed following i.c.v. application of leptin (0.4 or 2 microg/rat) but not melatonin (10 or 40 microg/rat) to the CIP rats. Capsaicin deactivation of SN oradministration of CGRP 8-37 abolished above beneficial effects of leptin on CIP, whereas melatonin-induced protection of pancreas was unaffected. Pretreatment with i.p. melatonin (10 or 50 mg/kg), but not leptin, significantly reduced MDA + 4-HNE in the pancreas of CIP rats. Leptin (10(-10) - 10(-6) M) but not melatonin (10(-8) - 10(-5) M) significantly stimulated NO release from isolated pancreatic acini. Leptin receptor gene expression in these acini was significantly increased by caerulein and leptin. We conclude that 1/ central or peripheral pretreatment with leptin protects the pancreas against its damage induced by CIP, whereas melatonin exerts its protective effect only when given i.p., but not following its i.c.v. adminstration, 2/ activation of leptin receptor in the pancreatic acini appears to be involved in the beneficial effects of leptin on acute pancreatitis, 3/ the protective effects of leptin involve sensory nerves, CGRP and increased generation of NO whereas melatonin-induced protection of the pancreas depends mainly on the antioxidant local effect of this indole, and scavenging of the radical oxygen species in the pancreatic tissue.     

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.     

The role of neutral endopeptidase in caerulein-induced acute pancreatitis

Substance P (SP) is well known to promote inflammation in acute pancreatitis (AP) by interacting with neurokinin-1 receptor. However, mechanisms that terminate SP-mediated responses are unclear. Neutral endopeptidase (NEP) is a cell-surface enzyme that degrades SP in the extracellular fluid. In this study, we examined the expression and the role of NEP in caerulein-induced AP. Male BALB/c mice (20-25 g) subjected to 3-10 hourly injections of caerulein (50 μg/kg) exhibited reduced NEP activity and protein expression in the pancreas and lungs. Additionally, caerulein (10(-7) M) also downregulated NEP activity and mRNA expression in isolated pancreatic acinar cells. The role of NEP in AP was examined in two opposite ways: inhibition of NEP (phosphoramidon [5 mg/kg] or thiorphan [10 mg/kg]) followed by 6 hourly caerulein injections) or supplementation with exogenous NEP (10 hourly caerulein injections, treatment of recombinant mouse NEP [1 mg/kg] during second caerulein injection). Inhibition of NEP raised SP levels and exacerbated inflammatory conditions in mice. Meanwhile, the severity of AP, determined by histological examination, tissue water content, myeloperoxidase activity, and plasma amylase activity, was markedly better in mice that received exogenous NEP treatment. Our results suggest that NEP is anti-inflammatory in caerulein-induced AP. Acute inhibition of NEP contributes to increased SP levels in caerulein-induced AP, which leads to augmented inflammatory responses in the pancreas and associated lung injury.     

Proteomic analysis of apoptotic and oncotic pancreatic acinar AR42J cells treated with caerulein

This study aims to determine the differentially expressed proteins in the pancreatic acinar cells undergoing apoptosis and oncosis stimulated with caerulein to explore different cell death process of the acinar cell. AR42J cells were treated with caerulein to induce cell model of acute pancreatitis. Cells that were undergoing apoptosis and oncosis were separated by flow cytometry. Then differentially expressed proteins in the two groups of separated cells were detected by shotgun liquid chromatography-tandem mass spectrometry. The results showed that 11 proteins were detected in both apoptosis group and oncosis group, 17 proteins were detected only in apoptosis group and 29 proteins were detected only in oncosis group. KEGG analysis showed that proteins detected only in apoptosis group were significantly enriched in 10 pathways, including ECM-receptor interaction, cell adhesion molecules, and proteins detected only in oncosis group were significantly enriched in three pathways, including endocytosis, base excision repair, and RNA degradation. These proteins we detected are helpful for us to understand the process of cell death in acute pancreatitis and may be useful for changing the death mode of pancreatic acinar cells, thus attenuating the severity of pancreatitis.