Pancreatic gene expression during recovery after pancreatitis reveals unique transcriptome profiles.

It is well known that pancreatic recovery after a single episode of injury such as an isolated bout of pancreatitis occurs rapidly. It is unclear, however, what changes are inflicted in such conditions to the molecular landscape of the pancreas. In the caerulein hyperstimulation model of pancreatitis, the murine pancreas has the ability to recover within one week based on histological appearance. In this study, we sought to characterize by RNA-sequencing (RNA-seq) the transcriptional profile of the recovering pancreas up to two weeks post-injury. We found that one week after injury there were 319 differentially expressed genes (DEGs) compared with baseline and that after two weeks there were 53 DEGs. Forty (12.5%) of the DEGs persisted from week one to week two, and another 13 DEGs newly emerged in the second week. Amongst the top up-regulated DEGs were several trypsinogen genes (trypsinogen 4, 5, 12, 15, and 16). To our knowledge, this is the first characterization of the transcriptome during pancreatic recovery by deep sequencing, and it reveals on a molecular basis that there is an ongoing recovery of the pancreas even after apparent histological resolution. The findings also raise the possibility of an emerging novel transcriptome upon pancreatic recovery.

Pancreatitis-Induced Depletion of Syntaxin 2 Promotes Autophagy and Increases Basolateral Exocytosis.

BACKGROUND & AIMS: Pancreatic acinar cells are polarized epithelial cells that store enzymes required for digestion as inactive zymogens, tightly packed at the cell apex. Stimulation of acinar cells causes the zymogen granules to fuse with the apical membrane, and the cells undergo exocytosis to release proteases into the intestinal lumen. Autophagy maintains homeostasis of pancreatic acini. Syntaxin 2 (STX2), an abundant soluble N-ethyl maleimide sensitive factor attachment protein receptor in pancreatic acini, has been reported to mediate apical exocytosis. Using human pancreatic tissues and STX2-knockout (KO) mice, we investigated the functions of STX2 in zymogen granule-mediated exocytosis and autophagy. METHODS: We obtained pancreatic tissues from 5 patients undergoing surgery for pancreatic cancer and prepared 80-µm slices; tissues were exposed to supramaximal cholecystokinin octapeptide (CCK-8) or ethanol and a low concentration of CCK-8 and analyzed by immunoblot and immunofluorescence analyses. STX2-KO mice and syntaxin 2+/+ C57BL6 mice (controls) were given intraperitoneal injections of supramaximal caerulein (a CCK-8 analogue) or fed ethanol and then given a low dose of caerulein to induce acute pancreatitis, or saline (controls); pancreata were isolated and analyzed by histology and immunohistochemistry. Acini were isolated from mice, incubated with CCK-8, and analyzed by immunofluorescence microscopy or used in immunoprecipitation experiments. Exocytosis was quantified using live-cell exocytosis and Ca2+ imaging analyses and based on formation of exocytotic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes. Dysregulations in autophagy were identified using markers, electron and immunofluorescence microscopy, and protease activation assays. RESULTS: Human pancreatic tissues and dispersed pancreatic acini from control mice exposed to CCK-8 or ethanol plus CCK-8 were depleted of STX2. STX2-KO developed more severe pancreatitis after administration of supramaximal caerulein or a 6-week ethanol diet compared with control. Acini from STX2-KO mice had increased apical exocytosis after exposure to CCK-8, as well as increased basolateral exocytosis, which led to ectopic release of proteases. These increases in apical and basolateral exocytosis required increased formation of fusogenic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes, mediated by STX3 and STX4. STX2 bound ATG16L1 and prevented it from binding clathrin. Deletion of STX2 from acini increased binding of AT16L1 to clathrin, increasing formation of pre-autophagosomes and inducing autophagy. Induction of autophagy promoted the CCK-8-induced increase in autolysosome formation and the activation of trypsinogen. CONCLUSIONS: In studies of human pancreatic tissues and pancreata from STX2-KO and control mice, we found STX2 to block STX3- and STX4-mediated fusion of zymogen granules with the plasma membrane and exocytosis and prevent binding of ATG16L1 to clathrin, which contributes to induction of autophagy. Exposure of pancreatic tissues to CCK-8 or ethanol depletes acinar cells of STX2, increasing basolateral exocytosis and promoting autophagy induction, leading to activation of trypsinogen.

Depletion of the membrane-fusion regulator Munc18c attenuates caerulein hyperstimulation-induced pancreatitis.

Epithelial pancreatic acinar cells perform crucial functions in food digestion, and acinar cell homeostasis required for secretion of digestive enzymes relies on SNARE-mediated exocytosis. The ubiquitously expressed Sec1/Munc18 protein mammalian uncoordinated-18c (Munc18c) regulates membrane fusion by activating syntaxin-4 (STX-4) to bind cognate SNARE proteins to form a SNARE complex that mediates exocytosis in many cell types. However, in the acinar cell, Munc18c's functions in exocytosis and homeostasis remain inconclusive. Here, we found that pancreatic acini from Munc18c-depleted mice (Munc18c+/-) and human pancreas (lenti-Munc18c-shRNA-treated) exhibit normal apical exocytosis of zymogen granules (ZGs) in response to physiologic stimulation with the intestinal hormone cholecystokinin (CCK-8). However, when stimulated with supraphysiologic CCK-8 levels to mimic pancreatitis, Munc18c-depleted (Munc18c+/-) mouse acini exhibited a reduction in pathological basolateral exocytosis of ZGs resulting from a decrease in fusogenic STX-4 SNARE complexes. This reduced basolateral exocytosis in part explained the less severe pancreatitis observed in Munc18c+/- mice after hyperstimulation with the CCK-8 analog caerulein. Likely as a result of this secretory blockade, Munc18c-depleted acini unexpectedly activated a component of the endoplasmic reticulum (ER) stress response that contributed to autophagy induction, resulting in downstream accumulation of autophagic vacuoles and autolysosomes. We conclude that Munc18c's role in mediating ectopic basolateral membrane fusion of ZGs contributes to the initiation of CCK-induced pancreatic injury, and that blockade of this secretory process could increase autophagy induction.

Risk Factors for Asparaginase-associated Pancreatitis: A Systematic Review.

GOALS: To evaluate potential risk factors for the development of asparaginase-associated pancreatitis (AAP), we performed a systematic review of the current literature from January 1946 through May 2015. BACKGROUND: Asparaginase, a primary treatment for the most common childhood cancer, acute lymphoblastic leukemia (ALL), is a well-described cause of pancreatitis. Further, pancreatitis is among the most burdensome and common complications of asparaginase treatment and represents a major reason for early-drug termination and inferior outcomes. The literature lacks clarity about the risk factors for AAP, and this knowledge gap has hampered the ability to reliably predict which patients are likely to develop AAP. STUDY: In an expansive screen, 1842 citations were funneled into a review of 59 full articles, of which 10 were deemed eligible based on predetermined inclusion criteria. RESULTS: Of the 10 identified studies, only 2 studies showed that children above 10 years of age had a >2-fold risk of AAP compared with younger children. Patients placed in high-risk ALL categories had a greater incidence of pancreatitis in 2 studies. In addition, use of pegylated asparaginase resulted in a higher incidence of AAP in 1 study. CONCLUSIONS: In this systematic review, older age, asparaginase formulation, higher ALL risk stratification, and higher asparaginase dosing appear to play a limited role in the development of AAP. Further studies are needed to probe the underlying mechanisms contributing to the development of pancreatitis in patients receiving asparaginase.

Ex vivo human pancreatic slice preparations offer a valuable model for studying pancreatic exocrine biology.

A genuine understanding of human exocrine pancreas biology and pathobiology has been hampered by a lack of suitable preparations and reliance on rodent models employing dispersed acini preparations. We have developed an organotypic slice preparation of the normal portions of human pancreas obtained from cancer resections. The preparation was assessed for physiologic and pathologic responses to the cholinergic agonist carbachol (Cch) and cholecystokinin (CCK-8), including 1) amylase secretion, 2) exocytosis, 3) intracellular Ca2+ responses, 4) cytoplasmic autophagic vacuole formation, and 5) protease activation. Cch and CCK-8 both dose-dependently stimulated secretory responses from human pancreas slices similar to those previously observed in dispersed rodent acini. Confocal microscopy imaging showed that these responses were accounted for by efficient apical exocytosis at physiologic doses of both agonists and by apical blockade and redirection of exocytosis to the basolateral plasma membrane at supramaximal doses. The secretory responses and exocytotic events evoked by CCK-8 were mediated by CCK-A and not CCK-B receptors. Physiologic agonist doses evoked oscillatory Ca2+ increases across the acini. Supraphysiologic doses induced formation of cytoplasmic autophagic vacuoles and activation of proteases (trypsin, chymotrypsin). Maximal atropine pretreatment that completely blocked all the Cch-evoked responses did not affect any of the CCK-8-evoked responses, indicating that rather than acting on the nerves within the pancreas slice, CCK cellular actions directly affected human acinar cells. Human pancreas slices represent excellent preparations to examine pancreatic cell biology and pathobiology and could help screen for potential treatments for human pancreatitis.

Targeted inhibition of pancreatic acinar cell calcineurin is a novel strategy to prevent post-ERCP pancreatitis.

BACKGROUND AND AIMS: There is a pressing need to develop effective preventative therapies for post-ERCP pancreatitis (PEP). We demonstrated that early PEP events are induced through the calcium-activated phosphatase calcineurin and that global calcineurin deletion abolishes PEP in mice. A crucial question is whether acinar cell calcineurin controls the initiation of PEP in vivo. METHODS: We used a mouse model of PEP and examined the effects of in vivo acinar cell-specific calcineurin deletion by either generating a conditional knockout line or infusing a novel AAV-Ela-iCre into the pancreatic duct of a calcineurin floxed line. RESULTS: We found that PEP is dependent on acinar cell calcineurin in vivo, and this led us to determine that calcineurin inhibitors, infused within the radiocontrast, can largely prevent PEP. CONCLUSIONS: These results provide impetus for launching clinical trials to test the efficacy of intraductal calcineurin inhibitors to prevent PEP.

Valproic Acid Limits Pancreatic Recovery after Pancreatitis by Inhibiting Histone Deacetylases and Preventing Acinar Redifferentiation Programs.

The mechanisms by which drugs induce pancreatitis are unknown. A definite cause of pancreatitis is due to the antiepileptic drug valproic acid (VPA). On the basis of three crucial observations-that VPA inhibits histone deacetylases (HDACs), HDACs mediate pancreas development, and aspects of pancreas development are recapitulated during recovery of the pancreas after injury-we hypothesized that VPA does not cause injury on its own, but it predisposes patients to pancreatitis by inhibiting HDACs and provoking an imbalance in pancreatic recovery. In an experimental model of pancreatic injury, we found that VPA delayed recovery of the pancreas and reduced acinar cell proliferation. In addition, pancreatic expression of class I HDACs (which are the primary VPA targets) increased in the midphase of pancreatic recovery. VPA administration inhibited pancreatic HDAC activity and led to the persistence of acinar-to-ductal metaplastic complexes, with prolonged Sox9 expression and sustained ß-catenin nuclear activation, findings that characterize a delay in regenerative reprogramming. These effects were not observed with valpromide, an analog of VPA that lacks HDAC inhibition. This is the first report, to our knowledge, that VPA shifts the balance toward pancreatic injury and pancreatitis through HDAC inhibition. The work also identifies a new paradigm for therapies that could exploit epigenetic reprogramming to enhance pancreatic recovery and disorders of pancreatic injury.

Exposure to Radiocontrast Agents Induces Pancreatic Inflammation by Activation of Nuclear Factor-κB, Calcium Signaling, and Calcineurin.

BACKGROUND & AIMS: Radiocontrast agents are required for radiographic procedures, but these agents can injure tissues by unknown mechanisms. We investigated whether exposure of pancreatic tissues to radiocontrast agents during endoscopic retrograde cholangiopancreatography (ERCP) causes pancreatic inflammation, and studied the effects of these agents on human cell lines and in mice. METHODS: We exposed mouse and human acinar cells to the radiocontrast agent iohexol (Omnipaque; GE Healthcare, Princeton, NJ) and measured intracellular release of Ca(2+), calcineurin activation (using a luciferase reporter), activation of nuclear factor-κB (NF-κB, using a luciferase reporter), and cell necrosis (via propidium iodide uptake). We infused the radiocontrast agent into the pancreatic ducts of wild-type mice (C57BL/6) to create a mouse model of post-ERCP pancreatitis; some mice were given intraperitoneal injections of the calcineurin inhibitor FK506 before and after infusion of the radiocontrast agent. CnAß(-/-) mice also were used. This experiment also was performed in mice given infusions of adeno-associated virus 6-NF-κB-luciferase, to assess activation of this transcription factor in vivo. RESULTS: Incubation of mouse and human acinar cells, but not HEK293 or COS7 cells, with iohexol led to a peak and then plateau in Ca(2+) signaling, along with activation of the transcription factors NF-κB and nuclear factor of activated T cells. Suppressing Ca(2+) signaling or calcineurin with BAPTA, cyclosporine A, or FK506 prevented activation of NF-κB and acinar cell injury. Calcineurin Aß-deficient mice were protected against induction of pancreatic inflammation by iohexol. The calcineurin inhibitor FK506 prevented contrast-induced activation of NF-κB in pancreata of mice, this was observed by live imaging of mice given infusions of adeno-associated virus 6-NF-κB-luciferase. CONCLUSIONS: Radiocontrast agents cause pancreatic inflammation in mice, via activation of NF-κB, Ca(2+) signaling, and calcineurin. Calcineurin inhibitors might be developed to prevent post-ERCP pancreatitis in patients.

ESPGHAN and NASPGHAN Report on the Assessment of Exocrine Pancreatic Function and Pancreatitis in Children.

The purpose of this clinical report is to discuss several recent advances in assessing exocrine pancreatic insufficiency (EPI) and pancreatitis in children, to review the array of pancreatic function tests, to provide an update on the inherited causes of EPI, with special emphasis on newly available genetic testing, and to review newer methods for evaluating pancreatitis.

Dynamic imaging of pancreatic nuclear factor κB (NF-κB) activation in live mice using adeno-associated virus (AAV) infusion and bioluminescence.

Nuclear factor κB (NF-κB) is an important signaling molecule that plays a critical role in the development of acute pancreatitis. Current methods for examining NF-κB activation involve infection of an adenoviral NF-κB-luciferase reporter into cell lines or electrophoretic mobility shift assay of lysate. The use of adeno-associated viruses (AAVs) has proven to be an effective method of transfecting whole organs in live animals. We examined whether intrapancreatic duct infusion of AAV containing an NF-κB-luciferase reporter (AAV-NF-κB-luciferase) can reliably measure pancreatic NF-κB activation. We confirmed the infectivity of the AAV-NF-κB-luciferase reporter in HEK293 cells using a traditional luciferase readout. Mice were infused with AAV-NF-κB-luciferase 5 weeks before induction of pancreatitis (caerulein, 50 µg/kg). Unlike transgenic mice that globally express NF-κB-luciferase, AAV-infused mice showed a 15-fold increase in pancreas-specific NF-κB bioluminescence following 12 h of caerulein compared with baseline luminescence (p < 0.05). The specificity of the NF-κB-luciferase signal to the pancreas was confirmed by isolating the pancreas and adjacent organs and observing a predominant bioluminescent signal in the pancreas compared with liver, spleen, and stomach. A complementary mouse model of post-ERCP-pancreatitis also induced pancreatic NF-κB signals. Taken together these data provide the first demonstration that NF-κB activation can be examined in a live, dynamic fashion during pancreatic inflammation. We believe this technique offers a valuable tool to study real-time activation of NF-κB in vivo.

Intraductal infusion of taurocholate followed by distal common bile duct ligation leads to a severe necrotic model of pancreatitis in mice.

OBJECTIVE: The most common etiology of acute pancreatitis results from the impaction of gallstones or sludge in the distal common bile duct (CBD). The result is pancreatic duct obstruction, diversion of bile into the pancreas, or cholestasis. In the current study, we examined whether combining both aspects, that is, infusion of the bile acid taurocholate (TC) followed by bile duct ligation (BDL), could yield a more severe form of pancreatitis that mimics biliary pancreatitis. METHODS: In mice, after laparotomy, the CBD was infused with either normal saline (NS) or TC. Subsequently, the CBD was ligated at the ampulla. RESULTS: Mice receiving TC infusion followed by BDL (TC + BDL) had higher mortality compared with animals receiving intraductal NS with BDL (NS + BDL). The TC + BDL arm developed more severe and diffuse pancreatic necrosis. In addition, serum amylase, IL-6, and bilirubin were significantly higher. However, pancreatic edema as well as lung and liver injury were unchanged between TC + BDL and NS + BDL. CONCLUSIONS: In summary, the combination of bile infusion into the pancreas followed by BDL causes a more severe, necrotizing pancreatitis. We believe that this novel model of pancreatitis is useful because it can be used in transgenic mice and recapitulates several aspects of biliary pancreatitis.

The ryanodine receptor is expressed in human pancreatic acinar cells and contributes to acinar cell injury.

Physiological calcium (Ca(2+)) signals within the pancreatic acinar cell regulate enzyme secretion, whereas aberrant Ca(2+) signals are associated with acinar cell injury. We have previously identified the ryanodine receptor (RyR), a Ca(2+) release channel on the endoplasmic reticulum, as a modulator of these pathological signals. In the present study, we establish that the RyR is expressed in human acinar cells and mediates acinar cell injury. We obtained pancreatic tissue from cadaveric donors and identified isoforms of RyR1 and RyR2 by qPCR. Immunofluorescence staining of the pancreas showed that the RyR is localized to the basal region of the acinar cell. Furthermore, the presence of RyR was confirmed from isolated human acinar cells by tritiated ryanodine binding. To determine whether the RyR is functionally active, mouse or human acinar cells were loaded with the high-affinity Ca(2+) dye (Fluo-4 AM) and stimulated with taurolithocholic acid 3-sulfate (TLCS) (500 µM) or carbachol (1 mM). Ryanodine (100 µM) pretreatment reduced the magnitude of the Ca(2+) signal and the area under the curve. To determine the effect of RyR blockade on injury, human acinar cells were stimulated with pathological stimuli, the bile acid TLCS (500 µM) or the muscarinic agonist carbachol (1 mM) in the presence or absence of the RyR inhibitor ryanodine. Ryanodine (100 µM) caused an 81% and 47% reduction in acinar cell injury, respectively, as measured by lactate dehydrogenase leakage (P < 0.05). Taken together, these data establish that the RyR is expressed in human acinar cells and that it modulates acinar Ca(2+) signals and cell injury.

A non-invasive method of quantifying pancreatic volume in mice using micro-MRI.

In experimental models of pancreatic growth and recovery, changes in pancreatic size are assessed by euthanizing a large cohort of animals at varying time points and measuring organ mass. However, to ascertain this information in clinical practice, patients with pancreatic disorders routinely undergo non-invasive cross-sectional imaging of the pancreas using magnetic resonance imaging (MRI) or computed tomography (CT). The aim of the current study was to develop a thin-sliced, optimized sequence protocol using a high field MRI to accurately calculate pancreatic volumes in the most common experimental animal, the mouse. Using a 7 Telsa Bruker micro-MRI system, we performed abdominal imaging in whole-fixed mice in three standard planes: axial, sagittal, and coronal. The contour of the pancreas was traced using Vitrea software and then transformed into a 3-dimensional (3D) reconstruction, from which volumetric measurements were calculated. Images were optimized using heart perfusion-fixation, T1 sequence analysis, and 0.2 to 0.4 mm thick slices. As proof of principle, increases in pancreatic volume among mice of different ages correlated tightly with increasing body weight. In summary, this is the first study to measure pancreatic volumes in mice, using a high field 7 Tesla micro-MRI and a thin-sliced, optimized sequence protocol. We anticipate that micro-MRI will improve the ability to non-invasively quantify changes in pancreatic size and will dramatically reduce the number of animals required to serially assess pancreatic growth and recovery.

Cluster of differentiation 38 (CD38) mediates bile acid-induced acinar cell injury and pancreatitis through cyclic ADP-ribose and intracellular calcium release.

Aberrant Ca(2+) signals within pancreatic acinar cells are an early and critical feature in acute pancreatitis, yet it is unclear how these signals are generated. An important mediator of the aberrant Ca(2+) signals due to bile acid exposure is the intracellular Ca(2+) channel ryanodine receptor. One putative activator of the ryanodine receptor is the nucleotide second messenger cyclic ADP-ribose (cADPR), which is generated by an ectoenzyme ADP-ribosyl cyclase, CD38. In this study, we examined the role of CD38 and cADPR in acinar cell Ca(2+) signals and acinar injury due to bile acids using pharmacologic inhibitors of CD38 and cADPR as well as mice deficient in Cd38 (Cd38(-/-)). Cytosolic Ca(2+) signals were imaged using live time-lapse confocal microscopy in freshly isolated mouse acinar cells during perifusion with the bile acid taurolithocholic acid 3-sulfate (TLCS; 500 µM). To focus on intracellular Ca(2+) release and to specifically exclude Ca(2+) influx, cells were perifused in Ca(2+)-free medium. Cell injury was assessed by lactate dehydrogenase leakage and propidium iodide uptake. Pretreatment with either nicotinamide (20 mM) or the cADPR antagonist 8-Br-cADPR (30 µM) abrogated TLCS-induced Ca(2+) signals and cell injury. TLCS-induced Ca(2+) release and cell injury were reduced by 30 and 95%, respectively, in Cd38-deficient acinar cells compared with wild-type cells (p < 0.05). Cd38-deficient mice were protected against a model of bile acid infusion pancreatitis. In summary, these data indicate that CD38-cADPR mediates bile acid-induced pancreatitis and acinar cell injury through aberrant intracellular Ca(2+) signaling.

Preparation of pancreatic acinar cells for the purpose of calcium imaging, cell injury measurements, and adenoviral infection.

The pancreatic acinar cell is the main parenchymal cell of the exocrine pancreas and plays a primary role in the secretion of pancreatic enzymes into the pancreatic duct. It is also the site for the initiation of pancreatitis. Here we describe how acinar cells are isolated from whole pancreas tissue and intracellular calcium signals are measured. In addition, we describe the techniques of transfecting these cells with adenoviral constructs, and subsequently measuring the leakage of lactate dehydrogenase, a marker of cell injury, during conditions that induce acinar cell injury in vitro. These techniques provide a powerful tool to characterize acinar cell physiology and pathology.

Pancreatic acinar cell nuclear factor κB activation because of bile acid exposure is dependent on calcineurin.

Biliary pancreatitis is the most common etiology of acute pancreatitis, accounting for 30-60% of cases. A dominant theory for the development of biliary pancreatitis is the reflux of bile into the pancreatic duct and subsequent exposure to pancreatic acinar cells. Bile acids are known to induce aberrant Ca(2+) signals in acinar cells as well as nuclear translocation of NF-κB. In this study, we examined the role of the downstream Ca(2+) target calcineurin on NF-κB translocation. Freshly isolated mouse acinar cells were infected for 24 h with an adenovirus expressing an NF-κB luciferase reporter. The bile acid taurolithocholic acid-3-sulfate caused NF-κB activation at concentrations (500 µm) that were associated with cell injury. We show that the NF-κB inhibitor Bay 11-7082 (1 µm) blocked translocation and injury. Pretreatment with the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, the calcineurin inhibitors FK506 and cyclosporine A, or use of acinar cells from calcineurin Aß-deficient mice each led to reduced NF-κB activation with taurolithocholic acid-3-sulfate. Importantly, these manipulations did not affect LPS-induced NF-κB activation. A critical upstream regulator of NF-κB activation is protein kinase C, which translocates to the membranes of various organelles in the active state. We demonstrate that pharmacologic and genetic inhibition of calcineurin blocks translocation of the PKC-δ isoform. In summary, bile-induced NF-κB activation and acinar cell injury are mediated by calcineurin, and a mechanism for this important early inflammatory response appears to be upstream at the level of PKC translocation.

Bile acids induce pancreatic acinar cell injury and pancreatitis by activating calcineurin.

Biliary pancreatitis is the leading cause of acute pancreatitis in both children and adults. A proposed mechanism is the reflux of bile into the pancreatic duct. Bile acid exposure causes pancreatic acinar cell injury through a sustained rise in cytosolic Ca(2+). Thus, it would be clinically relevant to know the targets of this aberrant Ca(2+) signal. We hypothesized that the Ca(2+)-activated phosphatase calcineurin is such a Ca(2+) target. To examine calcineurin activation, we infected primary acinar cells from mice with an adenovirus expressing the promoter for a downstream calcineurin effector, nuclear factor of activated T-cells (NFAT). The bile acid taurolithocholic acid-3-sulfate (TLCS) was primarily used to examine bile acid responses. TLCS caused calcineurin activation only at concentrations that cause acinar cell injury. The activation of calcineurin by TLCS was abolished by chelating intracellular Ca(2+). Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl ester) (BAPTA-AM) or the three specific calcineurin inhibitors FK506, cyclosporine A, or calcineurin inhibitory peptide prevented bile acid-induced acinar cell injury as measured by lactate dehydrogenase leakage and propidium iodide uptake. The calcineurin inhibitors reduced the intra-acinar activation of chymotrypsinogen within 30 min of TLCS administration, and they also prevented NF-κB activation. In vivo, mice that received FK506 or were deficient in the calcineurin isoform Aß (CnAß) subunit had reduced pancreatitis severity after infusion of TLCS or taurocholic acid into the pancreatic duct. In summary, we demonstrate that acinar cell calcineurin is activated in response to Ca(2+) generated by bile acid exposure, bile acid-induced pancreatic injury is dependent on calcineurin activation, and calcineurin inhibitors may provide an adjunctive therapy for biliary pancreatitis.

IP3 receptor type 2 deficiency is associated with a secretory defect in the pancreatic acinar cell and an accumulation of zymogen granules.

Acute pancreatitis is a painful, life-threatening disorder of the pancreas whose etiology is often multi-factorial. It is of great importance to understand the interplay between factors that predispose patients to develop the disease. One such factor is an excessive elevation in pancreatic acinar cell Ca(2+). These aberrant Ca(2+) elevations are triggered by release of Ca(2+) from apical Ca(2+) pools that are gated by the inositol 1,4,5-trisphosphate receptor (IP3R) types 2 and 3. In this study, we examined the role of IP3R type 2 (IP3R2) using mice deficient in this Ca(2+) release channel (IP3R2(-/-)). Using live acinar cell Ca(2+) imaging we found that loss of IP3R2 reduced the amplitude of the apical Ca(2+) signal and caused a delay in its initiation. This was associated with a reduction in carbachol-stimulated amylase release and an accumulation of zymogen granules (ZGs). Specifically, there was a 2-fold increase in the number of ZGs (P<0.05) and an expansion of the ZG pool area within the cell. There was also a 1.6- and 2.6-fold increase in cellular amylase and trypsinogen, respectively. However, the mice did not have evidence of pancreatic injury at baseline, other than an elevated serum amylase level. Further, pancreatitis outcomes using a mild caerulein hyperstimulation model were similar between IP3R2(-/-) and wild type mice. In summary, IP3R2 modulates apical acinar cell Ca(2+) signals and pancreatic enzyme secretion. IP3R-deficient acinar cells accumulate ZGs, but the mice do not succumb to pancreatic damage or worse pancreatitis outcomes.

The association of primary hyperparathyroidism with pancreatitis.

The association between primary hyperparathyroidism (PHPT) and acute or chronic pancreatitis is controversial. For this reason, we conducted a review of the literature over the past 30 years to explore the relationship between these 2 disorders. Ten retrospective studies each with >50 patients diagnosed with PHPT were identified. With the notable exception of 2 studies, the rate of pancreatitis among patients with PHPT was higher than that reported in general among hospitalized patients without PHPT. A higher serum calcium level may contribute to pancreatitis in these cases, along with additional genetic or environmental insults. Hypercalcemia may predispose the pancreatic acinar cell to abnormal, sustained calcium levels, lead to premature pancreatic protease activation, and pancreatitis. Although there was only short-term follow-up, most reports cited that definitive treatment of PHPT by parathyroidectomy led to the resolution of pancreatitis attacks. The published cohorts of patients with PHPT and pancreatitis are subject to bias, because serum calcium screening was not universally performed among all control nonpancreatitis patients to evaluate for PHPT. However, the pooled clinical and experimental data suggest an association between PHPT and pancreatitis and implicate hypercalcemia. For clinicians, it is important to recognize pancreatitis in patients with PHPT and, conversely, to consider PHPT by checking serum calcium levels in patients, who present with an unexplained pancreatitis.