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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Ethanol enhances carbachol-induced protease activation and accelerates Ca2+ waves in isolated rat pancreatic acini.

Alcohol abuse is a leading cause of pancreatitis, accounting for 30% of acute cases and 70-90% of chronic cases, yet the mechanisms leading to alcohol-associated pancreatic injury are unclear. An early and critical feature of pancreatitis is the aberrant signaling of Ca(2+) within the pancreatic acinar cell. An important conductor of this Ca(2+) is the basolaterally localized, intracellular Ca(2+) channel ryanodine receptor (RYR). In this study, we examined the effect of ethanol on mediating both pathologic intra-acinar protease activation, a precursor to pancreatitis, as well as RYR Ca(2+) signals. We hypothesized that ethanol sensitizes the acinar cell to protease activation by modulating RYR Ca(2+). Acinar cells were freshly isolated from rat, pretreated with ethanol, and stimulated with the muscarinic agonist carbachol (1 µM). Ethanol caused a doubling in the carbachol-induced activation of the proteases trypsin and chymotrypsin (p < 0.02). The RYR inhibitor dantrolene abrogated the enhancement of trypsin and chymotrypsin activity by ethanol (p < 0.005 for both proteases). Further, ethanol accelerated the speed of the apical to basolateral Ca(2+) wave from 9 to 18 µm/s (p < 0.0005; n = 18-22 cells/group); an increase in Ca(2+) wave speed was also observed with a change from physiologic concentrations of carbachol (1 µM) to a supraphysiologic concentration (1 mM) that leads to protease activation. Dantrolene abrogated the ethanol-induced acceleration of wave speed (p < 0.05; n = 10-16 cells/group). Our results suggest that the enhancement of pathologic protease activation by ethanol is dependent on the RYR and that a novel mechanism for this enhancement may involve RYR-mediated acceleration of Ca(2+) waves.

Pharmacological and genetic inhibition of calcineurin protects against carbachol-induced pathological zymogen activation and acinar cell injury.

Acute pancreatitis is a major health burden for which there are currently no targeted therapies. Premature activation of digestive proenzymes, or zymogens, within the pancreatic acinar cell is an early and critical event in this disease. A high-amplitude, sustained rise in acinar cell Ca(2+) is required for zymogen activation. We previously showed in a cholecystokinin-induced pancreatitis model that a potential target of this aberrant Ca(2+) signaling is the Ca(2+)-activated phosphatase calcineurin (Cn). However, in this study, we examined the role of Cn on both zymogen activation and injury, in the clinically relevant condition of neurogenic stimulation (by giving the acetylcholine analog carbachol) using three different Cn inhibitors or Cn-deficient acinar cells. In freshly isolated mouse acinar cells, pretreatment with FK506, calcineurin inhibitory peptide (CiP), or cyclosporine (CsA) blocked intra-acinar zymogen activation (n = 3; P < 0.05). The Cn inhibitors also reduced leakage of lactate dehydrogenase (LDH) by 79%, 62%, and 63%, respectively (n = 3; P < 0.05). Of the various Cn isoforms, the ß-isoform of the catalytic A subunit (CnAß) was strongly expressed in mouse acinar cells. For this reason, we obtained acinar cells from CnAß-deficient mice (CnAß-/-) and observed an 84% and 50% reduction in trypsin and chymotrypsin activation, respectively, compared with wild-type controls (n = 3; P < 0.05). LDH release in the CnAß-deficient cells was reduced by 50% (n = 2; P < 0.05). The CnAß-deficient cells were also protected against zymogen activation and cell injury induced by the cholecystokinin analog caerulein. Importantly, amylase secretion was generally not affected by either the Cn inhibitors or Cn deficiency. These data provide both pharmacological and genetic evidence that implicates Cn in intra-acinar zymogen activation and cell injury during pancreatitis.

Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice.

Biliary pancreatitis is the most common etiology for acute pancreatitis, yet its pathophysiological mechanism remains unclear. Ca(2+) signals generated within the pancreatic acinar cell initiate the early phase of pancreatitis, and bile acids can elicit anomalous acinar cell intracellular Ca(2+) release. We previously demonstrated that Ca(2+) released via the intracellular Ca(2+) channel, the ryanodine receptor (RyR), contributes to the aberrant Ca(2+) signal. In this study, we examined whether RyR inhibition protects against pathological Ca(2+) signals, acinar cell injury, and pancreatitis from bile acid exposure. The bile acid tauro-lithocholic acid-3-sulfate (TLCS) induced intracellular Ca(2+) oscillations at 50 µM and a peak-plateau signal at 500 µM, and only the latter induced acinar cell injury, as determined by lactate dehydrogenase (LDH) leakage. Pretreatment with the RyR inhibitors dantrolene or ryanodine converted the peak-plateau signal to a mostly oscillatory pattern (P < 0.05). They also reduced acinar cell LDH leakage, basolateral blebbing, and propidium iodide uptake (P < 0.05). In vivo, a single dose of dantrolene (5 mg/kg), given either 1 h before or 2 h after intraductal TLCS infusion, reduced the severity of pancreatitis down to the level of the control (P < 0.05). These results suggest that the severity of biliary pancreatitis may be ameliorated by the clinical use of RyR inhibitors.

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.

Novel characterization of drug-associated pancreatitis in children.

BACKGROUND AND OBJECTIVES: Medications are a major cause of acute pancreatitis; however, little is known about their influence in children. Our primary aims were to identify common comorbidities and concomitant pancreatitis etiologies in children with drug-associated pancreatitis. Our secondary aims were to identify the most commonly associated drugs in the different age groups, evaluate management practices, and compare drug-associated cases with non-drug-associated cases. PATIENTS AND METHODS: In the present study, we examined children (ages 0-20 years) admitted to Yale-New Haven Children's Hospital with pancreatitis between 1994 and 2007. RESULTS: Of a total of 271 pediatric cases, drugs were associated with pancreatitis in 25.6% (55). The 3 most common comorbidities in children with drug-associated pancreatitis were seizure disorders, acute lymphocytic leukemia, and Crohn disease. One third of drug-associated cases had an additional pancreatitis etiology. The most commonly associated drugs were valproic acid and corticosteroids. Compared with non-drug-associated cases, children with drug-associated cases were more likely to undergo CT scanning (54.5% vs 28.4%; P < 0.001), stay in the hospital longer (10 vs 4 days; P < 0.001), and transition to parenteral nutrition from a nil per os status (37.5% vs 21.2%; P < 0.05). There was a higher frequency of valproic acid-associated cases in children younger than 11 years (29.4% vs 9.5% in the 11- to 20-year-old age group). CONCLUSIONS: Our study underscores the importance of considering drugs as a cause and a contributor to pancreatitis in children, particularly valproic acid in young children.

Dantrolene mitigates caerulein-induced pancreatitis in vivo in mice.

Acute pancreatitis is a painful, inflammatory disorder for which adequate treatments are lacking. An early, critical step in its development is the aberrant signaling of Ca(2+) within the pancreatic acinar cell. This Ca(2+) release is modulated by the intracellular Ca(2+) channel the ryanodine receptor (RYR). We have previously shown that RYR inhibition reduces pathological intra-acinar protease activation, an early marker of pancreatitis. In this study, we examined whether pretreatment with the RYR inhibitor dantrolene attenuates the severity of caerulein-induced pancreatitis in mice. Immunofluorescent labeling for RYR from mouse pancreatic sections showed localization to the basolateral region of the acinar cell. After 1 h of caerulein hyperstimulation in vivo, dantrolene 1) reduced pancreatic trypsin activity by 59% (P < 0.05) and 2) mitigated early ultrastructural derangements within the acinar cell. Eight hours after pancreatitis induction, dantrolene reduced pancreatic trypsin activity and serum amylase by 61 and 32%, respectively (P < 0.05). At this later time point, overall histological severity of pancreatitis was reduced by 63% with dantrolene pretreatment (P < 0.05). TUNEL-positive cells were reduced by 58% (P < 0.05). These data suggest that the RYR plays an important role in mediating early acinar cell events during in vivo pancreatitis and contributes to disease severity. Blockade of Ca(2+) signals and particularly RYR-Ca(2+) may be useful as prophylactic treatment for this disease in high-risk settings for pancreatitis.

A comparison of presentation and management trends in acute pancreatitis between infants/toddlers and older children.

OBJECTIVES: Acute pancreatitis is a necroinflammatory disease that leads to 210,000 hospitalizations in the United States annually. Recent reports suggest that there may be important differences in clinical features between infants/toddlers and older children. Thus, in this study we make a direct comparison between the pediatric age groups in presentation and management trends of acute pancreatitis. PATIENTS AND METHODS: We examined all children (ages 0 to 20 years) admitted to Yale-New Haven Children's Hospital with pancreatitis between 1994 and 2007. RESULTS: Two hundred seventy-one cases met inclusion criteria for acute pancreatitis. Infants and toddlers manifested fewer signs and symptoms of abdominal pain, epigastric tenderness, and nausea compared with older children (43% vs 93%; 57% vs 90%; and 29% vs 76%, respectively; P < 0.05 for all comparisons). They were more likely to be diagnosed by serum lipase than by amylase and to undergo radiographic evaluation (P < 0.05). They had a longer hospital stay (19.5 vs 4 days; P < 0.05) and were less likely to be directly transitioned to oral nutrition (14% vs 71%; P < 0.05). CONCLUSIONS: Infants and toddlers with acute pancreatitis present with fewer classical symptoms and are managed differently from older children. We believe these data will be helpful in evaluating and understanding treatment practices in this age group.