Primary Pancreatic Acinar Cells Research Articles

Large triglyceride-rich lipoproteins in hypertriglyceridemia are associated with the severity of acute pancreatitis in experimental mice

Hypertriglyceridemia severity is linked to acute pancreatitis prognosis, but it remains unknown why a portion of severe hypertriglyceridemia patients do not develop severe acute pancreatitis. To investigate whether hypertriglyceridemia subtypes affect acute pancreatitis progression, we analyzed two genetically modified hypertriglyceridemia mouse models—namely, glycosylphosphatidylinositol high-density lipoprotein binding protein 1 knockout (Gpihbp1−/−) and apolipoprotein C3 transgenic (ApoC3-tg) mice. Acute pancreatitis was induced by 10 intraperitoneal caerulein injections. Biochemical assays and pathological analysis were performed for the severity evaluation of acute pancreatitis. Plasma triglyceride-rich lipoproteins (TRLs), including chylomicrons and very low-density lipoprotein (VLDL), were collected via ultracentrifugation to evaluate their cytotoxic effects on primary pancreatic acinar cells (PACs). We found that the particle sizes of Gpihbp1−/− TRLs were larger than ApoC3-tg TRLs. Severe pancreatic injury with large areas of pancreatic necrosis in the entire lobule was induced in Gpihbp1−/− mice when plasma triglyceride levels were greater than 2000 mg/dL. However, ApoC3-tg mice with the same triglyceride levels did not develop large areas of pancreatic necrosis, even upon the administration of poloxamer 407 to further increase triglyceride levels. Meanwhile, in the acute pancreatitis model, free fatty acids (FFAs) in the pancreas of Gpihbp1−/− mice were greater than in ApoC3-tg mice. TRLs from Gpihbp1−/− mice released more FFAs and were more toxic to PACs than those from ApoC3-tg mice. Chylomicrons from patients showed the same effects on PACs as TRLs from Gpihbp1−/− mice. Gpihbp1−/− mice with triglyceride levels below 2000 mg/dL had milder pancreatic injury and less incidence of pancreatic necrosis than those with triglyceride levels above 2000 mg/dL, similar to Gpihbp1−/−mice with triglyceride levels above 2000 mg/dL but with fenofibrate administration. These findings demonstrated that hypertriglyceridemia subtypes with large TRL particles could affect acute pancreatitis progression and that chylomicrons showed more cytotoxicity than VLDL by releasing more FFAs.

Pyridoxine and pancreatic acinar cells: transport physiology and effect on gene expression profile

Pyridoxine (vitamin B6), an essential micronutrient for normal cell physiology, plays an important role in the function of the exocrine pancreas. Pancreatic acinar cells (PACs) obtain vitamin B6 from circulation, but little is known about the mechanism involved in the uptake process; limited information also exists on the effect of pyridoxine availability on the gene expression profile in these cells. We addressed both these issues in the current investigation using mouse-derived pancreatic acinar 266-6 cells (PAC 266-6) and human primary PACs (hPACs; obtained from organ donors), together with appropriate physiological and molecular (RNA-Seq) approaches. The results showed [3H]pyridoxine uptake to be 1) pH and temperature (but not Na+) dependent, 2) saturable as a function of concentration, 3) cis-inhibited by unlabeled pyridoxine and its close structural analogs, 4) trans-stimulated by unlabeled pyridoxine, 5) regulated by an intracellular Ca2+/calmodulin-mediated pathway, 6) adaptively-regulated by extracellular substrate (pyridoxine) availability, and 7) negatively impacted by exposure to cigarette smoke extract. Vitamin B6 availability was found (by means of RNA-Seq) to significantly (FDR < 0.05) modulate the expression profile of many genes in PAC 266-6 cells (including those that are relevant to pancreatic health and development). These studies demonstrate, for the first time, the involvement of a regulatable and specific carrier-mediated mechanism for pyridoxine uptake by PACs; the results also show that pyridoxine availability exerts profound effects on the gene expression profile in mammalian PACs.

Effect of bacterial flagellin on thiamin uptake by human and mouse pancreatic acinar cells: inhibition mediated at the level of transcription of thiamin transporters 1 and 2.

Thiamin (vitamin B1) is essential for normal cellular metabolism and function. Pancreatic acinar cells (PACs) obtain thiamin from the circulation via a specific carrier-mediated process that involves the plasma membrane thiamin transporters 1 and 2 (THTR-1 and THTR-2; products of SLC19A2 and SLC19A3 genes, respectively). There is nothing known about the effect of bacterial products/toxins on thiamin uptake by PACs. We addressed this issue in the present investigation by examining the effect of bacterial flagellin on physiological and molecular parameters of thiamin uptake by PACs. We used human primary PACs, mice in vivo, and cultured mouse-derived pancreatic acinar 266-6 cells in our investigation. The results showed that exposure of human primary PACs to flagellin led to a significant inhibition in thiamin uptake; this inhibition was associated with a significant decrease in expression of THTR-1 and -2 at the protein and mRNA levels. These findings were confirmed in mice in vivo as well as in cultured 266-6 cells. Subsequent studies showed that flagellin exposure markedly suppressed the activity of the SLC19A2 and SLC19A3 promoters and that this effect involved the Sp1 regulatory factor. Finally, knocking down Toll-like receptor 5 by use of gene-specific siRNA was found to lead to abrogation in the inhibitory effect of flagellin on PAC thiamin uptake. These results show, for the first time, that exposure of PACs to flagellin negatively impacts the physiological and molecular parameters of thiamin uptake and that this effect is mediated at the level of transcription of the SLC19A2 and SLC19A3 genes. NEW & NOTEWORTHY The present study demonstrates, for the first time, that prolonged exposure of pancreatic acinar cells to flagellin inhibits uptake of vitamin B1, a micronutrient that is essential for energy metabolism and ATP production. This effect is mediated at the level of transcription of the SLC19A2 and SLC19A3 genes and involves the Sp1 transcription factor.

PYK2 Is Involved in Premalignant Acinar Cell Reprogramming and Pancreatic Ductal Adenocarcinoma Maintenance by Phosphorylating β-CateninY654.

Background & AimsIdentification and validation of new functionally relevant and pharmacologically actionable targets for pancreatic ductal adenocarcinoma (PDAC) remains a great challenge. Premalignant acinar cell reprogramming (acinar-to-ductal metaplasia [ADM]) is a precursor of pancreatic intraepithelial neoplasia (PanIN) lesions that can progress to PDAC. This study investigated the role of proline-rich tyrosine kinase 2 (PYK2) in mutant Kras-induced and pancreatitis-associated ADM and PanIN formation, as well as in PDAC maintenance.MethodsGenetically engineered mouse models of mutant Kras (glycine 12 to aspartic acid) and Pyk2 deletion were used for investigating the role of PYK2 in PDAC genesis in mice. In vitro ADM assays were conducted using primary pancreatic acinar cells isolated from mice. Immunohistochemistry, immunofluorescence, and a series of biochemical experiments were used to investigate upstream regulators/downstream targets of PYK2 in pancreatic carcinogenesis. PDAC cell line xenograft experiments were performed to study the role of PYK2 and its downstream target in PDAC maintenance.ResultsPYK2 was increased substantially in ADM lesions induced by mutant Kras or inflammatory injury. Pyk2 deletion remarkably suppressed ADM and PanIN formation in a mutant Kras-driven and pancreatitis-associated PDAC model, whereas PYK2 knockdown substantially inhibited PDAC cell growth in vitro and in nude mice. This study uncovered a novel yes-associated protein 1/transcriptional co-activator with PDZ binding motif/signal transducer and activator of transcription 3/PYK2/β-catenin regulation axis in PDAC. Our results suggest that PYK2 contributes to PDAC genesis and maintenance by activating the Wnt/β-catenin pathway through directly phosphorylating β-cateninY654.ConclusionsThe current study uncovers PYK2 as a novel downstream effector of mutant KRAS signaling, a previously unrecognized mediator of pancreatitis-induced ADM and a novel intervention target for PDAC.

Lineage Tracing of Primary Human Pancreatic Acinar and Ductal Cells for Studying Acinar-to-Ductal Metaplasia.

Acinar-to-ductal metaplasia may play important roles in the development of various pancreatic diseases. Here, we describe a method to induce ADM in primary human cells in 3D culture. We developed a flow cytometry lineage tracing strategy to identify and sort viable acinar, ductal, and acinar-derived ductal-like cells for further molecular and functional analysis.

Induction of Pancreatic Inflammation Accelerates Pancreatic Tumorigenesis in Mice.

Pancreatitis is a major risk factor for the development of pancreatic cancer. In genetically engineered mouse models, induction of pancreatic inflammation dramatically accelerates oncogenic KRas-induced fibrosis, precancerous PanIN formation, and tumorigenesis. Here we describe simple methods of secretagogue-induced experimental acute and chronic pancreatitis, the most commonly used pancreatitis models, and their applications in pancreatic cancer research. Additionally, the preparation of primary pancreatic acinar cells is introduced. Primary acinar cells can be used to study the early events of pancreatic inflammation and pancreatic acinar-to-ductal (ADM) metaplasia.

Blocking β₁/β₂-Adrenergic Signaling Reduces Dietary Fat Absorption by Suppressing Expression of Pancreatic Lipase in High Fat-Fed Mice.

We investigated whether β-adrenergic antagonists attenuates dietary fat absorption through the regulation of pancreatic lipase (PNLIP) expression in pancreatic acinar cells in the context of high fat diet feeding. Male six-week-old C57BL/6 mice were assigned into an ad libitum fed control diet (CON) and a high fat diet (HIGH). Within each diet group, subgroups of mice were treated with vehicle (VEH) or propranolol, a β-adrenergic antagonist (BB). Over 12 weeks, body weight gain observed in HIGHVEH was mitigated in HIGHBB (+103% vs. +72%). Increase in fecal fat amount observed in HIGHVEH was further increased in HIGHBB. Increase in PNLIP expressions observed in HIGHVEH pancreatic tissues was abolished in HIGHBB. PNLIP expression in mouse primary pancreatic acinar cells and 266-6 cell lines increased with isoproterenol treatment, which was blocked by propranolol. Isoproterenol increased PNLIP expression in a cAMP/protein kinase A/ cyclic AMP response element binding protein (CREB)-dependent manner. CREB directly bound to the CRE on the mouse PNLIP promoter and transactivated PNLIP expression. These results suggest that sympathetic activation increases dietary fat absorption through the upregulation of PNLIP expression and that a β-adrenergic antagonist attenuates obesity development partly through the downregulation of PNLIP expression and inhibition of dietary fat absorption in the context of high fat diet feeding.

Abstract 2805: Protein oxidation in pancreatic neoplasia and cancer

Abstract Rationale- Peroxiredoxin-1 (Prdx1) is overexpressed in pancreatic cancer patient serum and correlates with worse prognosis. Prdx1 is an important antioxidant protein whose deletion causes the formation of malignant lesions in mice. Prdx1's redox function depends on its reduction by two other Thioredoxin system members, Thioredoxin (Txn) and Sulfiredoxin (Srxn). Prdx1 may also contribute to tumorigenesis by oligomerizing and becoming a nuclear chaperone when it's overoxidized and its redox function is inhibited. Objective- Our objective was to investigate disruptions in the Txn system during pancreatic neoplasia and to determine how that affects phosphorylation of Kras effectors. Findings- Prdx1 expression was elevated and Srxn expression was reduced in pancreatic lesions of El-Kras, Pdx1-Cre/LSL-Kras (KC-Pdx1), and P48-Cre/LSL-Kras (KC-P48) mice. Txn was absent in lesions of KC-Pdx1 and KC-P48 mice but present and differentially located in EL-Kras lesions. In primary pancreatic acinar cells, oxidation of the Txn system with auranofin increased ERK and AKT phosphorylation in EL-Kras mice, but not in KC-P48 mice, indicating differential regulation of the Txn system in KC-P48 mice. This could be due to the presence of a higher molecular weight Prdx1 being present in KC-p48 mice and not in EL-Kras mice. This suggests that the effect of mutant Kras on the thioredoxin system in these two models may be different. Prdx1 and (p)ERK also interacted in vitro and inhibition of ERK and AKT phosphorylation modified the expression of Txn and Srxn in pancreatic cell lines. We also found that in human pancreatic cancer patient tissue, Prdx1 was overexpressed in pancreatic cancer as compared to adjacent normal tissue. Conclusions- The Txn system is disrupted during pancreatic neoplasia and may be associated with modified ERK and AKT regulation in Kras mutant pancreatic tissue. Citation Format: Michelle Schultz, Andrew Diaz, Sharon Smite, Brian DeCant, David Bentrem, Paul Grippo. Protein oxidation in pancreatic neoplasia and cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2805.

Chronic alcohol exposure affects pancreatic acinar mitochondrial thiamin pyrophosphate uptake: studies with mouse 266-6 cell line and primary cells

Thiamin is essential for normal metabolic activity of all mammalian cells, including those of the pancreas. Cells obtain thiamin from their surroundings and enzymatically convert it into thiamin pyrophosphate (TPP) in the cytoplasm; TPP is then taken up by mitochondria via a specific carrier the mitochondrial TPP transporter (MTPPT; product of the SLC25A19 gene). Chronic alcohol exposure negatively impacts the health of pancreatic acinar cells (PAC), but its effect on physiological/molecular parameters of MTPPT is not known. We addressed this issue using mouse pancreatic acinar tumor cell line 266-6 and primary PAC of wild-type and transgenic mice carrying the SLC25A19 promoter that were fed alcohol chronically. Chronic alcohol exposure of 266-6 cells (but not to its nonoxidative metabolites ethyl palmitate and ethyl oleate) led to a significant inhibition in mitochondrial TPP uptake, which was associated with a decreased expression of MTPPT protein, mRNA, and activity of the SLC25A19 promoter. Similarly, chronic alcohol feeding of mice led to a significant inhibition in expression of MTPPT protein, mRNA, heterogeneous nuclear RNA, as well as in activity of SLC25A19 promoter in PAC. While chronic alcohol exposure did not affect DNA methylation of the Slc25a19 promoter, a significant decrease in histone H3 euchromatin markers and an increase in H3 heterochromatin marker were observed. These findings show, for the first time, that chronic alcohol exposure negatively impacts pancreatic MTPPT, and that this effect is exerted, at least in part, at the level of Slc25a19 transcription and appears to involve epigenetic mechanism(s).

Protein kinase D1 drives pancreatic acinar cell reprogramming and progression to intraepithelial neoplasia

The transdifferentiation of pancreatic acinar cells to a ductal phenotype (acinar-to-ductal metaplasia, ADM) occurs after injury or inflammation of the pancreas and is a reversible process. However, in the presence of activating Kras mutations or persistent epidermal growth factor receptor (EGF-R) signaling, cells that underwent ADM can progress to pancreatic intraepithelial lesions (PanINs) and eventually pancreatic cancer. In transgenic animal models, ADM and PanINs are initiated by high-affinity ligands for EGF-R or activating Kras mutations, but the underlying signaling mechanisms are not well understood. Here, using a conditional knockout approach, we show that Protein Kinase D1 (PKD1) is sufficient to drive the reprogramming process to a ductal phenotype and progression to PanINs. Moreover, using 3D explant culture of primary pancreatic acinar cells, we show that PKD1 acts downstream of TGFα and Kras to mediate formation of ductal structures through activation of the Notch pathway.

Nicotine Promotes Initiation and Progression of KRAS-Induced Pancreatic Cancer via Gata6-Dependent Dedifferentiation of Acinar Cells in Mice

Although smoking is a leading risk factor for pancreatic ductal adenocarcinoma (PDAC), little is known about the mechanisms by which smoking promotes initiation or progression of PDAC. We studied the effects of nicotine administration on pancreatic cancer development in Kras(+/LSLG12Vgeo);Elas-tTA/tetO-Cre (Ela-KRAS) mice, Kras(+/LSLG12D);Trp53+/LSLR172H;Pdx-1-Cre (KPC) mice (which express constitutively active forms of KRAS), and C57/B6 mice. Mice were given nicotine for up to 86 weeks to produce blood levels comparable with those of intermediate smokers. Pancreatic tissues were collected and analyzed by immunohistochemistry and reverse transcriptase polymerase chain reaction; cells were isolated and assayed for colony and sphere formation and gene expression. The effects of nicotine were also evaluated in primary pancreatic acinar cells isolated from wild-type, nAChR7a(-/-), Trp53(-/-), and Gata6(-/-);Trp53(-/-) mice. We also analyzed primary PDAC cells that overexpressed GATA6 from lentiviral expression vectors. Administration of nicotine accelerated transformation of pancreatic cells and tumor formation in Ela-KRAS and KPC mice. Nicotine induced dedifferentiation of acinar cells by activating AKT-ERK-MYC signaling; this led to inhibition of Gata6 promoter activity, loss of GATA6 protein, and subsequent loss of acinar differentiation and hyperactivation of oncogenic KRAS. Nicotine also promoted aggressiveness of established tumors as well as the epithelial-mesenchymal transition, increasing numbers of circulating cancer cells and their dissemination to the liver, compared with mice not exposed to nicotine. Nicotine induced pancreatic cells to acquire gene expression patterns and functional characteristics of cancer stem cells. These effects were markedly attenuated in K-Ras(+/LSL-G12D);Trp53(+/LSLR172H);Pdx-1-Cre mice given metformin. Metformin prevented nicotine-induced pancreatic carcinogenesis and tumor growth by up-regulating GATA6 and promoting differentiation toward an acinar cell program. In mice, nicotine promotes pancreatic carcinogenesis and tumor development via down-regulation of Gata6 to induce acinar cell dedifferentiation.

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 10(6) acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

Effect of nicotine on exocytotic pancreatic secretory response: role of calcium signaling

BackgroundNicotine is a risk factor for pancreatitis resulting in loss of pancreatic enzyme secretion. The aim of this study was to evaluate the mechanisms of nicotine-induced secretory response measured in primary pancreatic acinar cells isolated from Male Sprague Dawley rats. The study examines the role of calcium signaling in the mechanism of the enhanced secretory response observed with nicotine exposure.MethodsIsolated and purified pancreatic acinar cells were subjected to a nicotine exposure at a dose of 100 μM for 6 minutes and then stimulated with cholecystokinin (CCK) for 30 min. The cell’s secretory response was measured by the percent of amylase released from the cells in the incubation medium Calcium receptor antagonists, inositol trisphosphate (IP3) receptor blockers, mitogen activated protein kinase inhibitors and specific nicotinic receptor antagonists were used to confirm the involvement of calcium in this process.ResultsNicotine exposure induced enhanced secretory response in primary cells. These responses remained unaffected by mitogen activated protein kinases (MAPK’s) inhibitors. The effects, however, have been completely abolished by nicotinic receptor antagonist, calcium channel receptor antagonists and inositol trisphosphate (IP3) receptor blockers.ConclusionsThe data suggest that calcium activated events regulating the exocytotic secretion are affected by nicotine as shown by enhanced functional response which is inhibited by specific antagonists… The results implicate the role of nicotine in the mobilization of both intra- and extracellular calcium in the regulation of stimulus-secretory response of enzyme secretion in this cell system. We conclude that nicotine plays an important role in promoting enhanced calcium levels inside the acinar cell.

Age‐dependent reduction of the PI3K regulatory subunit p85α suppresses pancreatic acinar cell proliferation

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway is important for tissue proliferation. Previously, we found that tissue regeneration after partial pancreatic resection was markedly attenuated in aged mice as compared to young mice and that this attenuation was because of an age-dependent reduction of PI3K/Akt signaling in the pancreatic acini; however, the mechanisms for the age-associated decline of pancreatic PI3K/Akt signaling remained unknown. To better delineate the mechanisms for the decreased PI3K/Akt activation with aging, age-associated changes in cell proliferation and PI3K/Akt signaling were investigated in the present study using in vitro primary pancreatic acinar cell cultures derived from young and aged mice. In response to treatment with insulin-like growth factor 1 (IGF-1), acinar cells from young but not aged mice showed increased activation of PI3K/Akt signaling and cell proliferation, indicating that intrinsic cellular mechanisms cause the age-associated changes in pancreatic acinar cells. We also found that the expression of PI3K p85α subunit, but not IGF-1 receptor or other PI3K subunits, was significantly reduced in pancreatic acinar cells from aged mice; this age-associated reduction of p85α was confirmed in both mouse and human pancreatic tissues. Finally, small interfering RNA (siRNA)-mediated knockdown of p85α expression in acinar cells from young mice resulted in markedly attenuated activation of PI3K/Akt downstream signaling in response to IGF-1. From these results, we conclude that exocrine pancreatic expression of PI3K p85α subunit is attenuated by aging, which is likely responsible for the age-associated decrease in activation of pancreatic PI3K signaling and acinar cell proliferation in response to growth-promoting stimuli.

Relative contribution of THTR-1 and THTR-2 in thiamin uptake by pancreatic acinar cells: studies utilizing Slc19a2 and Slc19a3 knockout mouse models.

Thiamin is essential for normal function of pancreatic acinar cells, and its deficiency leads to a reduction in pancreatic digestive enzymes. We have recently shown that thiamin uptake by rat pancreatic acinar cells is carrier-mediated and that both thiamin transporter (THTR)-1 and THTR-2 are expressed in these cells; little, however, is known about the relative contribution of these transporters toward total carrier-mediated thiamin uptake by these cells. We addressed this issue using a gene-specific silencing approach (siRNA) in mouse-derived pancreatic acinar 266-6 cells and Slc19a2 and Slc19a3 knockout mouse models. First we established that thiamin uptake by mouse pancreatic acinar cells is via a carrier-mediated process. We also established that these cells as well as native human pancreas express THTR-1 and THTR-2, with expression of the former (and activity of its promoter) being significantly higher than that of the latter. Using gene-specific siRNA against mouse THTR-1 and THTR-2, we observed a significant inhibition in carrier-mediated thiamin uptake by 266-6 cells in both cases. Similarly, thiamin uptake by freshly isolated primary pancreatic acinar cells of the Slc19a2 and Slc19a3 knockout mice was significantly lower than uptake by acinar cells of the respective littermates; the degree of inhibition observed in the former knockout model was greater than that of the latter. These findings demonstrate, for the first time, that both mTHTR-1 and mTHTR-2 are involved in carrier-mediated thiamin uptake by pancreatic acinar cells.

Expression of claudin-4 molecule in canine exocrine pancreatic acinar cell carcinomas.

Claudins, integral membrane proteins are components of the tight junction structures between epithelial and endothelial cells. These transmembrane proteins create a primary barrier to prevent paracellular transport of solutes, and also restrict the lateral diffusion of membrane lipids and proteins to maintain the cellular polarity. The aim of the present study was to characterise the expression pattern of claudin-4 tight junction molecule in canine normal pancreatic tissues and in the well-differentiated and poorly-differentiated pancreatic acinar cell carcinomas in canines. The necropsy samples included canine intact pancreatic tissues, and canine well-differentiated and poorly-differentiated pancreatic acinar cell carcinomas samples. Claudin-4 was detected as an intense lateral membrane labelling of acinar cells in all intact pancreatic tissues. The intact epithelial cells of the different ducts were negative for the claudin-4 molecule. All primary and secondary canine well-differentiated exocrine pancreatic acinar cell carcinoma tissues showed intense apical lateral positivity for the claudin-4 molecule. All primary and secondary poorly-differentiated pancreatic acinar cell carcinoma tissues showed diffusely the loss of claudin-4 expression. Consequently, we hypothesize that the loss of expression of claudin-4 plays a role in the progression of canine pancreatic acinar cell carcinoma and may lead to cellular detachment, disorientation and invasion of these pancreatic cancers. Furthermore, claudin-4 can be used as an immunohistochemical marker to distinguish canine well-differentiated and undifferentiated exocrine pancreatic acinar cell carcinomas.

The Ecto-enzyme CD38 Is a Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) Synthase That Couples Receptor Activation to Ca2+ Mobilization from Lysosomes in Pancreatic Acinar Cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca(2+)-mobilizing intracellular messenger and is linked to a variety of stimuli and cell surface receptors. However, the enzyme responsible for endogenous NAADP synthesis in vivo is unknown, and it has been proposed that another enzyme differing from ADP-ribosyl cyclase family members may exist. The ecto-enzyme CD38, involved in many functions as diverse as cell proliferation and social behavior, represents an important alternative. In pancreatic acinar cells, the hormone cholecystokinin (CCK) stimulates NAADP production evoking Ca(2+) signals by discharging acidic Ca(2+) stores and leading to digestive enzyme secretion. From cells derived from CD38(-/-) mice, we provide the first physiological evidence that CD38 is required for endogenous NAADP generation in response to CCK stimulation. Furthermore, CD38 expression in CD38-deficient pancreatic AR42J cells remodels Ca(2+)-signaling pathways in these cells by restoring Ca(2+) mobilization from lysosomes during CCK-induced Ca(2+) signaling. In agreement with an intracellular site for messenger synthesis, we found that CD38 is expressed in endosomes. These CD38-containing vesicles, likely of endosomal origin, appear to be proximal to lysosomes but not co-localized with them. We propose that CD38 is an NAADP synthase required for coupling receptor activation to NAADP-mediated Ca(2+) release from lysosomal stores in pancreatic acinar cells.

Potential role of CXCL10 in the induction of cell injury and mitochondrial dysfunction

Chemokines have been known to play a critical role in pathogenesis of chronic pancreatitis and acinar cell death. However, the role played by one of the CXC chemokines: CXCL10 in regulation of acinar cell death has remained unexplored. Hence, this study was designed to assess the role of CXCL10 promoting apoptosis in ex vivo cultured acinar cells. Primary human pancreatic acinar cell cultures were established and exposed to varying doses of CXCL10 for different time intervals. Apoptotic induction was evaluated by both qualitative as well as quantitative analyses. Various mediators of apoptosis were also studied by Western blotting, membrane potential (Psim) and ATP depletion in acinar cells. Analysis of apoptosis via DNA ladder and cell death detection - ELISA demonstrated that CXCL10 induced 3.9-fold apoptosis when administrated at an optimal dose of 0.1 mug of recombinant CXCL10 for 8 h. Quantitative analysis using FACS and dual staining by PI-annexin showed increased apoptosis (48.98 and 53.78% respectively). The involvement of upstream apoptotic regulators like pJNK, p38 and Bax was established on the basis of their increased expression of CXCL10. The change of Psim by 50% was observed in the presence of CXCL10 in treated acinar cells along with enhanced expression of Cytochrome C, apaf-1 and caspase 9/3 activation. In addition, ATP depletion was also noticed in CXCL10 stimulated acinar cells. CXCL10 induces cell death in human cultured pancreatic cells leading to apoptosis and DNA fragmentation via CXCR3 signalling. These signalling mechanisms may play an important role in parenchymal cell loss and injury in pancreatitis.

Mechanism and regulation of folate uptake by pancreatic acinar cells: effect of chronic alcohol consumption

Folate plays an essential role in one-carbon metabolism, and a relationship exists between methyl group metabolism and pancreatic exocrine function. Little, however, is known about the mechanism(s) and regulation of folate uptake by pancreatic acinar cells and the effect of chronic alcohol use on the process. We addressed these issues using the rat-derived pancreatic acinar cell line AR42J and freshly isolated primary rat pancreatic acinar cells as models. We found [(3)H]folic acid uptake to be 1) temperature and pH dependent with a higher uptake at acidic than at neutral/alkaline pH; 2) saturable as a function of substrate concentration at both buffer pH 7.4 and 6.0; 3) inhibited by folate structural analogs and by anion transport inhibitors at both buffer pH 7.4 and 6.0; 4) trans-stimulated by unlabeled folate; 5) adaptively regulated by the prevailing extracellular folate level, and 6) inhibited by modulators of the cAMP/PKA-mediated pathway. Both the reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT) were found to be expressed in AR42J and in primary pancreatic acinar cells, as well as in native human pancreas with expression of RFC being higher than PCFT. Chronic alcohol feeding of rats (4 wk; 36% of calories from ethanol) led to a significant decrease in folate uptake by freshly isolated primary pancreatic acinar cells compared with cells from pair-fed controls; this effect was associated with a parallel decrease in the level of expression of RFC and PCFT. These studies reveal that folate uptake by pancreatic acinar cells is via a regulated carrier-mediated process which may involve RFC and PCFT. In addition, chronic alcohol feeding leads to a marked inhibition in folate uptake by pancreatic acinar cells, an effect that is associated with reduction in level of expression of RFC and PCFT.