SULFATE METABOLISM IN PANCREATIC ACINAR CELLS

The metabolism of inorganic sulfate in pancreatic acinar cells was studied by electron microscope radioautography in mice injected with sulfate-³⁵S. Labeled sulfate was concentrated in the Golgi complex at 10 min. Within 30 min, much of the radioactive material had been transferred to condensing vacuoles. These were subsequently transformed into zymogen granules. By 4 hr after injection, some of the zymogen granules with radioactive contents were undergoing secretion, and labeled material was present in the pancreatic duct system. The Golgi complex in pancreatic acinar cells is known to be responsible for concentrating and packaging digestive enzymes delivered to it from the endoplasmic reticulum. Our work demonstrates that the Golgi complex in these cells is also engaged in the manufacture of sulfated materials, probably sulfated mucopolysaccharides, which are packaged along with the enzymes in zymogen granules and released with them into the pancreatic secretion.     

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.     

Raf-1 Kinase Inhibitory Protein (RKIP) Mediates Ethanol-induced Sensitization of Secretagogue Signaling in Pancreatic Acinar Cells

Excessive alcohol consumption is associated with most cases of chronic pancreatitis, a progressive necrotizing inflammatory disease that can result in pancreatic insufficiency due to acinar atrophy and fibrosis and an increased risk of pancreatic cancer. At a cellular level acute alcohol exposure can sensitize pancreatic acinar cells to secretagogue stimulation, resulting in dysregulation of intracellular Ca²⁺ homeostasis and premature digestive enzyme activation; however, the molecular mechanisms by which ethanol exerts these toxic effects have remained undefined. In this study we identify Raf-1 kinase inhibitory protein as an essential mediator of ethanol-induced sensitization of cholecystokinin- and carbachol-regulated Ca²⁺ signaling in pancreatic acinar cells. We show that exposure of rodent acinar cells to ethanol induces protein kinase C-dependent Raf-1 kinase inhibitory protein phosphorylation, sensitization of cholecystokinin-stimulated Ca²⁺ signaling, and potentiation of both basal and cholecystokinin-stimulated extracellular signal-regulated kinase activation. Furthermore, we show that either suppression of Raf-1 kinase inhibitory protein expression using short hairpin RNA or gene ablation prevented the sensitizing effects of ethanol on cholecystokinin- and carbachol-stimulated Ca²⁺ signaling and intracellular chymotrypsin activation in pancreatic acinar cells, suggesting that the modulation of Raf-1 inhibitory protein expression may have future therapeutic utility in the prevention or treatment of alcohol-associated pancreatitis.     

Critical role for NHE1 in intracellular pH regulation in pancreatic acinar cells

The primary function of pancreatic acinar cells is to secrete digestive enzymes together with a NaCl-rich primary fluid which is later greatly supplemented and modified by the pancreatic duct. A Na+/H+ exchanger(s) [NHE(s)] is proposed to be integral in the process of fluid secretion both in terms of the transcellular flux of Na+ and intracellular pH (pHi) regulation. Multiple NHE isoforms have been identified in pancreatic tissue, but little is known about their individual functions in acinar cells. The Na+/H+ exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride completely blocked pHi recovery after an NH4Cl-induced acid challenge, confirming a general role for NHE in pHi regulation. The targeted disruption of the Nhe1 gene also completely abolished pHi recovery from an acid load in pancreatic acini in both HCO3--containing and HCO3--free solutions. In contrast, the disruption of either Nhe2 or Nhe3 had no effect on pHi recovery. In addition, NHE1 activity was upregulated in response to muscarinic stimulation in wild-type mice but not in NHE1-deficient mice. Fluctuations in pHi could potentially have major effects on Ca2+ signaling following secretagogue stimulation; however, the targeted disruption of Nhe1 was found to have no significant effect on intracellular Ca2+ homeostasis. These data demonstrate that NHE1 is the major regulator of pHi in both resting and muscarinic agonist-stimulated pancreatic acinar cells.     

Absence of the major zymogen granule membrane protein, GP2, does not affect pancreatic morphology or secretion

The majority of digestive enzymes in humans are produced in the pancreas where they are stored in zymogen granules before secretion into the intestine. GP2 is the major membrane protein present in zymogen granules of the exocrine pancreas. Numerous studies have shown that GP2 binds digestive enzymes such as amylase, thereby supporting a role in protein sorting to the zymogen granule. Other studies have suggested that GP2 is important in the formation of zymogen granules. A knock-out mouse was generated for GP2 to study the impact of the protein on pancreatic function. GP2-deficient mice displayed no gross signs of nutrient malab-sorption such as weight loss, growth retardation, or diarrhea. Zymogen granules in the GP2 knock-out mice appeared normal on electron microscopy and contained the normal complement of proteins excluding GP2. Primary cultures of pancreatic acini appropriately responded to secretagogue stimulation with the secretion of digestive enzymes. The course of experimentally induced pancreatitis was also examined in the knock-out mice because proteins known to associate with GP2 have been found to possess a protective role. When GP2 knock-out mice were subjected to two different models of pancreatitis, no major differences were detected. In conclusion, GP2 is not essential for pancreatic exocrine secretion or zymogen granule formation. It is unlikely that GP2 serves a major intracellular role within the pancreatic acinar cell and may be functionally active after it is secreted from the pancreas.     

自噬在胰腺炎中的研究及进展

自噬是广泛存在于真核细胞内的一种溶酶体依赖性降解途径,作为细胞生存的一种机制,在很多生理过程如清除损伤、衰老细胞器以及冗余蛋白上发挥重要作用。自噬在人类胰腺炎的研究最早由Helin等人早在1980年提出,随着不断深入研究,发现自噬在胰腺炎发生发展过程中起主导作用。急性胰腺炎是一种发病率和死亡率极高的疾病,目前表明这种疾病始于胰腺腺泡细胞,主要诊断指标为高淀粉酶血症,胰腺腺泡细胞内消化酶的激活、液泡的大量堆积和炎症因子的聚集,最终胰腺炎症细胞侵润及引起的全身炎症反应导致腺泡细胞的凋亡和坏死,在其发病机制和治疗方面仍需进一步研究探讨。本文综述近年最新研究成果,深入探讨自噬在胰腺炎中的研究及进展。     

The homeodomain protein PDX1 is required at mid-pancreatic development for the formation of the exocrine pancreas

The homeoprotein PDX1 is expressed throughout pancreatic development and is thought to play important roles at multiple stages. We describe the properties of a tet-off regulatory scheme to manage the expression of Pdx1 in utero. Cessation of Pdx1 expression at increasingly later gestational times blocked pancreatic development at progressive and morphologically distinct stages and provided the opportunity to assess the requirement for Pdx1 at each stage. Embryonic PDX1 is depleted below effective levels within 1 day of the initiation of doxycycline treatment of pregnant mice. We show that PDX1, which is necessary for early pancreatic development, is also required later for the genesis of acinar tissue, the compartment of the pancreas that produces digestive enzymes. Without PDX1, acini do not form; the precursor epithelium continues to grow and branch, creating a truncated ductal tree comprising immature duct-like cells. The bHLH factor PTF1a, a critical regulator of acinar development, is not expressed and cells producing digestive enzymes are rare. This approach should be generally applicable to study the in vivo functions of other developmental regulators with multiple, temporally distinct roles.     

Membrane Dipeptidase and Glutathione Are Major Components of Pig Pancreatic Zymogen Granules

Membrane proteins of highly purified porcine zymogen granules were separated by two-dimensional gel electrophoresis in order to isolate proteins which are involved in intracellular trafficking of digestive enzymes in the exocrine pancreas. A 48-kDa glycoprotein was a major component in membrane preparations washed with 0.1 M Na₂CO₃and 0.5 M NaCl. By N-terminal amino acid sequencing this protein was identified as membrane dipeptidase (MDP; EC 3.4.13.19). MDP mRNA levels in rat pancreas were increased threefold by feeding rats with FOY-305, which is a known stimulus of endogenous cholecystokinin release from the gut. Cholecystokinin then stimulates secretion in pancreatic acinar cells. In another set of experiments treatment of the rat pancreatic acinar tumor cell line AR42J with dexamethasone led to an eightfold increase in the expression of MDP. Thus, the expression pattern of the MDP gene in response to hormonal stimulationin vivoandin vitroresembles those found for most of the enzymes and proteins which are involved in secretion. Since MDP has been thought to have a role in glutathione (GSH) metabolism, we also measured GSH concentration in zymogen granules and found high levels of GSH. Based on our data we propose a working model for the function of MDP. According to this model, MDP might play a pivotal role in maintaining the oxidizing conditions in the ER, which are required for the correct folding of secretory proteins.     

Alleles of the major histocompatibility complex play a role in the pathogenesis of pancreatic acinar atrophy in dogs

Exocrine pancreatic insufficiency (EPI) is a disease wherein pancreatic acinar cells fail to synthesize and secrete sufficient amounts of digestive enzymes for normal digestion of food. EPI affects many dog breeds, with a dramatically higher prevalence in the German shepherd dog (GSD) population. In this breed and perhaps others, EPI most often results from degeneration of the acinar cells of the pancreas, a hereditary disorder termed pancreatic acinar atrophy (PAA). Evidence of lymphocytic infiltration indicates that PAA is an autoimmune disease, but the genetic etiology remains unclear. Data from global gene expression and single nucleotide polymorphism profiles in the GSD suggest the involvement of the major histocompatibility complex [MHC; dog leukocyte antigen (DLA)]. To determine if alleles of the MHC influence development of EPI, genotyping of polymorphic class I (DLA-88) and II loci (DLA-DRB1, DLA-DQA1, and DLA-DQB1) was carried out for 70 affected and 63 control GSDs, and four-locus haplotypes were determined. One haplotype containing a novel allele of DLA-88 is very highly associated with EPI (OR?>?17; P?=?0.000125), while two haplotypes were found to confer protection from EPI (P?=?0.00087 and 0.0115). Described herein is the genotyping of MHC class I and II loci in a GSD cohort, establishment of four-locus haplotypes, and association of alleles/haplotypes with EPI.     

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

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

Expression of human cationic trypsinogen (PRSS1) in murine acinar cells promotes pancreatitis and apoptotic cell death

Hereditary pancreatitis (HP) is an autosomal dominant disease that displays the features of both acute and chronic pancreatitis. Mutations in human cationic trypsinogen (PRSS1) are associated with HP and have provided some insight into the pathogenesis of pancreatitis, but mechanisms responsible for the initiation of pancreatitis have not been elucidated and the role of apoptosis and necrosis has been much debated. However, it has been generally accepted that trypsinogen, prematurely activated within the pancreatic acinar cell, has a major role in the initiation process. Functional studies of HP have been limited by the absence of an experimental system that authentically mimics disease development. We therefore developed a novel transgenic murine model system using wild-type (WT) human PRSS1 or two HP-associated mutants (R122H and N29I) to determine whether expression of human cationic trypsinogen in murine acinar cells promotes pancreatitis. The rat elastase promoter was used to target transgene expression to pancreatic acinar cells in three transgenic strains that were generated: Tg(Ela-PRSS1)NV, Tg(Ela-PRSS1*R122H)NV and Tg(Ela-PRSS1*N29I)NV. Mice were analysed histologically, immunohistochemically and biochemically. We found that transgene expression is restricted to pancreatic acinar cells and transgenic PRSS1 proteins are targeted to the pancreatic secretory pathway. Animals from all transgenic strains developed pancreatitis characterised by acinar cell vacuolisation, inflammatory infiltrates and fibrosis. Transgenic animals also developed more severe pancreatitis upon treatment with low-dose cerulein than controls, displaying significantly higher scores for oedema, inflammation and overall histopathology. Expression of PRSS1, WT or mutant, in acinar cells increased apoptosis in pancreatic tissues and isolated acinar cells. Moreover, studies of isolated acinar cells demonstrated that transgene expression promotes apoptosis rather than necrosis. We therefore conclude that expression of WT or mutant human PRSS1 in murine acinar cells induces apoptosis and is sufficient to promote spontaneous pancreatitis, which is enhanced in response to cellular insult.     

Pancreatic acinar cell: New insights into the control of secretion

Pancreatic acinar cells secrete fluid and digestive enzymes. Both types of secretion are activated by a rise in intracellular calcium but how the stimulus-secretion cascade actually regulates secretory output is not well understood. It has long been known that the calcium response of acinar cells to physiological stimulation is complex. Dependent on the type and concentration of agonist, it consists of either local or global calcium increases as well as spreading waves of calcium across the cell. In the past it has been speculated that these different calcium signals drive different secretory responses. Now, recent employment of two-photon microscopy has enabled the simultaneous recording of both enzyme secretion and calcium signals and is beginning to resolve this issue. The data shows that local calcium responses exclusively drive fluid secretion. Where-as, global calcium responses drive both fluid and enzyme secretion. This differential control of secretory output is likely central to controlling the physiological responses of pancreatic acinar cells.     

Pancreas-specific protein disulfide isomerase has a cell type-specific expression in various mouse tissues and is absent in human pancreatic adenocarcinoma cells: implications for its functions

Members of the protein disulfide isomerase (PDI) family play a critical role in catalyzing the formation of disulfide bonds in secretory proteins, and most of these enzymes have a wide tissue distribution. However, the pancreas-specific PDI homolog was previously suggested to be exclusively expressed in the pancreas (thus commonly referred to as PDIp). In the present study, we found that PDIp was also highly expressed in several other tissues in mice, including the stomach, cecum, ileum, adrenal glands, epididymis, and prostate. Notably, in the digestive organs, such as the stomach and pancreas, very high levels of PDIp were selectively expressed in the digestive enzyme-secreting cells (e.g., gastric chief cells and pancreatic acinar cells). This observation suggests that PDIp may function as a protein-folding catalyst for secretory digestive enzymes. In ileum, PDIp was exclusively expressed in Paneth cells. In addition, high levels of PDIp expression were also detected in normal human pancreas, but its expression was mostly absent in human pancreatic duct adenocarcinoma and pancreatic cancer cell lines. The absence of PDIp expression in pancreatic adenocarcinoma may serve as an additional biomarker for pancreatic cancer.     

Pancreatic acinar cell necrosis with intact storage of digestive enzymes in selenomethionine treated rats

Morphological and biochemical changes were observed in the pancreas and serum of rats after the intraperitoneal administration of selenomethionine, sodium selenite and methionine. Selenomethionine caused rapidly developing acinar cell necrosis. The first pathological changes were mitochondrial swelling and flocculent densities, and dilatation of cisternae of the endoplasmic reticulum. Zymogen granules appeared disrupted only in disintegrated acinar cells. Signs of autodigestive pancreatic inflammation with fat necrosis, elevation of pancreatic phospholipase A2 and serum amylase activities, as well as pulmonary oedema were present. Sodium selenite caused similar histologic changes to those produced by selenomethionine, but no changes were seen after methionine administration. Destruction of pancreatic acinar cells by an intraductal oleic acid injection that resulted in exocrine atrophy did not prevent systemic selenomethionine toxicity. Our results show that selenomethionine causes pancreatic acinar cell necrosis and that intracellular transport and storage of digestive enzymes is not primarily altered by this chemical.     

Exocrine pancreatic secretion of phospholipid, menaquinone-4, and caveolin-1 in vivo

The exocrine pancreas releases secretory products essential for nutrient assimilation. In addition to digestive enzymes, the release of lipoprotein-like particles containing the membrane trafficking protein caveolin-1 from isolated pancreatic explants has been reported. The present study examined: (1) if gastrointestinal hormones induce the apical secretion of phospholipid in vivo and (2) a potential association of caveolin-1 and the lipid-soluble vitamin K analog menaquinone-4 (MK-4) with these structures. Analysis of isolated acinar cells, purified zymogen granules, and pancreatic juice collected in vivo indicated the presence a caveolin-1 immunoreactive protein that was acutely released in response hormone stimulation. Chloroform-extracted fractions of pancreatic juice also contained high concentrations of MK-4 that was secreted in parallel to protein and phospholipid. The presence of caveolin-1 and MK-4 in the phospholipid fraction of pancreatic juice places these molecules in the secretory pathway of exocrine cells and suggests a physiological role in digestive enzyme synthesis and/or processing.     

Apoptosis versus necrosis in acute pancreatitis

Acute pancreatitis is a disease of variable severity in which some patients experience mild, self-limited attacks, whereas others manifest a severe, highly morbid, and frequently lethal attack. The events that regulate the severity of acute pancreatitis are, for the most part, unknown. It is generally believed that the earliest events in acute pancreatitis occur within acinar cells and result in acinar cell injury. Other processes, such as recruitment of inflammatory cells and generation of inflammatory mediators, are believed to occur subsequent to acinar cell injury, and these "downstream" events are believed to influence the severity of the disease. Several recently reported studies, however, have suggested that the acinar cell response to injury may, itself, be an important determinant of disease severity. In these studies, mild acute pancreatitis was found to be associated with extensive apoptotic acinar cell death, whereas severe acute pancreatitis was found to involve extensive acinar cell necrosis but very little acinar cell apoptosis. These observations led to the hypothesis that apoptosis could be a favorable response to acinar cells and that interventions that favor induction of apoptotic, as opposed to necrotic, acinar cell death might reduce the severity of an attack of acute pancreatitis. Indeed, in an experimental setting, the induction of pancreatic acinar cell apoptosis protects mice against acute pancreatitis. Little is known about the mechanism of apoptosis in the pancreatic acinar cell, although some early attempts have been made in that direction. Also, clinical relevance of these experimental studies remains to be investigated.     

Autophagy and acute pancreatitis: a novel autophagy theory for trypsinogen activation

Autodigestion of the pancreas by its own prematurely activated digestive proteases is thought to be an important event in the onset of acute pancreatitis. Although lysosomal hydrolases, such as cathepsin B, play a key role in intrapancreatic trypsinogen activation, it remains unclear where and how trypsinogen meets these lysosomal enzymes. Autophagy is an intracellular bulk degradation system in which cytoplasmic components are directed to the lysosome/vacuole by a membrane-mediated process. To analyze the role of autophagy in acute pancreatitis, we produced a conditional knockout mouse that lacks the autophagy-related (Atg) gene Atg5 in the pancreatic acinar cells. The severity of acute pancreatitis induced by cerulein is greatly reduced in these mice. In addition, Atg5-deficient acinar cells show a significantly decreased level of trypsinogen activation. These data suggest that autophagy exerts a detrimental effect in pancreatic acinar cells by activation of trypsinogen to trypsin. We propose a theory in which autophagy accelerates trypsinogen activation by lysosomal hydrolases under acidic conditions, thus triggering acute pancreatitis in its early stage.     

Robust acinar cell transgene expression of CreErT via BAC recombineering

Pancreatic acinar cells are critical in gastrointestinal physiology and pancreatitis and may be involved in pancreatic cancer. Previously, a short rat pancreatic elastase promoter has been widely utilized to control acinar cell transgene expression. However, this partial sequence does not confer robust and stable expression. In this study, we tested the hypothesis that a transgene employing bacterial-artificial-chromosome (BAC) technology to express a tamoxifen-regulated Cre recombinase from a full-length mouse elastase gene (BAC-Ela-CreErT) would be more robust and stable. When founders were crossed with Rosa26 reporter mice nearly 100% of acini expressed beta-galactosidase after tamoxifen treatment. The expression was specific for pancreatic acinar cells and these characteristics have remained stable for 2 years. However, because of high levels of expression in differentiated acinar cells, this construct is tamoxifen independent in approximately 50% of adult acinar cells. This model of pancreatic acinar specific Cre expression is a powerful tool for future transgenic and knockout studies.