Inhibition of pancreatic acinar mitochondrial thiamin pyrophosphate uptake by the cigarette smoke component 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Thiamin is essential for normal metabolism in pancreatic acinar cells (PAC) and is obtained from their microenvironment through specific plasma-membrane transporters, converted to thiamin pyrophosphate (TPP) in the cytoplasm, followed by uptake of TPP by mitochondria through the mitochondrial TPP (MTPP) transporter (MTPPT; product of SLC25A19 gene). TPP is essential for normal mitochondrial function. We examined the effect of long-term/chronic exposure of PAC in vitro (pancreatic acinar 266-6 cells) and in vivo (wild-type or transgenic mice carrying the SLC25A19 promoter) of the cigarette smoke toxin, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), on the MTPP uptake process. Our in vitro and in vivo findings demonstrate that NNK negatively affects MTPP uptake and reduced expression of MTPPT protein, MTPPT mRNA, and heterogenous nuclear RNA, as well as SLC25A19 promoter activity. The effect of NNK on Slc25a19 transcription was neither mediated by changes in expression of transcriptional factor NFY-1 (known to drive SLC25A19 transcription), nor due to changes in methylation profile of the Slc25a19 promoter. Rather, it appears to be due to changes in histone modifications that involve significant decreases in histone H3K4-trimethylation and H3K9-acetylation (activation markers). The effect of NNK on MTPPT function is mediated through the nonneuronal α7-nicotinic acetylcholine receptor (α7-nAChR), as indicated by both in vitro (using the nAChR antagonist mecamylamine) and in vivo (using an α7-nAchR(-/-) mouse model) studies. These findings demonstrate that chronic exposure of PAC to NNK negatively impacts PAC MTPP uptake. This effect appears to be exerted at the level of Slc25a19 transcription, involve epigenetic mechanism(s), and is mediated through the α7-nAchR.

Low extracellular pH induces damage in the pancreatic acinar cell by enhancing calcium signaling.

Low extracellular pH (pHe) occurs in a number of clinical conditions and sensitizes to the development of pancreatitis. The mechanisms responsible for this sensitization are unknown. Because abnormal Ca(2+) signaling underlies many of the early steps in the pathogenesis of pancreatitis, we evaluated the effect of decreasing pHe from 7.4 to 7.0 on Ca(2+) signals in the acinar cell. Low pHe significantly increased the amplitude of cerulein-induced Ca(2+) signals. The enhancement in amplitude was localized to the basolateral region of the acinar cell and was reduced by pretreatment with ryanodine receptor (RYR) inhibitors. Because basolateral RYRs also have been implicated in the pathogenesis of pancreatitis, we evaluated the effects of RYR inhibitors on pancreatitis responses in acidic conditions. RYR inhibitors significantly reduced the sensitizing effects of low pHe on zymogen activation and cellular injury. These findings suggest that enhanced RYR-mediated Ca(2+) signaling in the basolateral region of the acinar cell is responsible for the injurious effects of low pHe on the exocrine pancreas.

Cerulein hyperstimulation decreases AMP-activated protein kinase levels at the site of maximal zymogen activation.

The premature activation of digestive enzyme zymogens in the pancreatic acinar cell is an important initiating event in acute pancreatitis. We have previously demonstrated that vacuolar ATPase (vATPase) activity is required for zymogen activation. Adenosine monophosphate-activated protein kinase (AMPK) regulates vATPase function in kidney and epididymal clear cells. To determine whether AMPK could affect pancreatitis responses, its effects were first examined in a cellular model of pancreatitis, cerulein-hyperstimulated (100 nM) pancreatic acini. This treatment caused a prominent increase in trypsin and chymotrypsin activities. Pretreatment with AICAR or metformin (AMPK activators) or compound C (an AMPK inhibitor) reduced or increased cerulein-induced zymogen activation, respectively. The association of the vATPase E subunit with membranes, a marker of its activation, tended to be inversely related to AMPK activity (assessed by AICAR and compound C treatments). Cerulein treatment did not change AMPK (α and ß) levels but did lead to an increase in its activation (phosphorylation of Thr172) and induced the time-dependent translocation of the enzyme to a Triton-insoluble compartment. Basal in vivo studies showed that AMPK was widely distributed between membrane and soluble fractions generated by differential centrifugation. After cerulein hyperstimulation, AMPK levels selectively decreased in fractions containing the highest levels of active zymogens. These studies suggest that AMPK activity has a protective role in the pancreatic acinar cell that inhibits zymogen activation in the basal state, and this AMPK effect is reduced during pancreatitis. Therapies that prevent the selective reduction of AMPK in compartments that support zymogen activation could reduce injury during pancreatitis.

A novel protein kinase D inhibitor attenuates early events of experimental pancreatitis in isolated rat acini.

Novel protein kinase C isoforms (PKC δ and ε) mediate early events in acute pancreatitis. Protein kinase D (PKD/PKD1) is a convergent point of PKC δ and ε in the signaling pathways triggered through CCK or cholinergic receptors and has been shown to activate the transcription factor NF-κB in acute pancreatitis. For the present study we hypothesized that a newly developed PKD/PKD1 inhibitor, CRT0066101, would prevent the initial events leading to pancreatitis. We pretreated isolated rat pancreatic acinar cells with CRT0066101 and a commercially available inhibitor Gö6976 (10 µM). This was followed by stimulation for 60 min with high concentrations of cholecystokinin (CCK, 0.1 µM), carbachol (CCh, 1 mM), or bombesin (10 µM) to induce initial events of pancreatitis. PKD/PKD1 phosphorylation and activity were measured as well as zymogen activation, amylase secretion, cell injury and NF-κB activation. CRT0066101 dose dependently inhibited secretagogue-induced PKD/PKD1 activation and autophosphorylation at Ser-916 with an IC(50) ∼3.75-5 µM but had no effect on PKC-dependent phosphorylation of the PKD/PKD1 activation loop (Ser-744/748). Furthermore, CRT0066101 reduced secretagogue-induced zymogen activation and amylase secretion. Gö6976 reduced zymogen activation but not amylase secretion. Neither inhibitor affected basal zymogen activation or secretion. CRT0066101 did not affect secretagogue-induced cell injury or changes in cell morphology, but it reduced NF-κB activation by 75% of maximal for CCK- and CCh-stimulated acinar cells. In conclusion, CRT0066101 is a potent and specific PKD family inhibitor. Furthermore, PKD/PKD1 is a potential mediator of zymogen activation, amylase secretion, and NF-κB activation induced by a range of secretagogues in pancreatic acinar cells.

The emerging role of smoking in the development of pancreatitis.

BACKGROUND/AIMS: Cigarette smoking has been linked to many diseases, including pancreatic cancer and more recently, pancreatitis. METHODS: Electronic searches of primarily PubMed from 1990 to August 2011 were conducted and only articles published in English were reviewed. Original articles and reviews were selected based on screening of article abstracts and their relevance to tobacco smoking, its components, nicotine and its metabolites, and their effects particularly on the pancreas. RESULTS: Smoking may affect the risk of developing chronic pancreatitis or its progression. Smoking may also affect the risk for developing acute pancreatitis. Its effects in pancreatitis appear to be dose dependent and its effects may be alcohol independent but synergize with alcohol. CONCLUSION: Specific constituents of cigarette smoke, including nicotine and its metabolites, could mediate effects on the pancreas.

Reducing extracellular pH sensitizes the acinar cell to secretagogue-induced pancreatitis responses in rats.

BACKGROUND & AIMS: Protease activation within the pancreatic acinar cell is a key early event in acute pancreatitis and may require low pH intracellular compartments. Clinical studies suggest that acidosis may affect the risk for developing pancreatitis. We hypothesized that exposure to an acid load might sensitize the acinar cell to secretagogue-induced pancreatitis. METHODS: Secretagogues (cerulein, carbachol, and bombesin) can induce protease activation in acinar cells at high (100 nmol/L, 1 mmol/L, and 10 micromol/L, respectively) but not at physiologically relevant concentrations. The effects of decreasing extracellular pH (pHe) in early secretagogue-induced pancreatitis (zymogen activation and injury) were examined in rats (1) in vitro with isolated acini and (2) in vivo with an acid challenge. RESULTS: In acini, lowering pHe from 7.6 to 6.8 enhanced secretagogue-induced zymogen activation and injury, but did not affect secretion. For cerulein, this sensitization was seen over a range of concentrations (0.01-100.00 nmol/L). However, reduced pHe alone had no effect on zymogen activation, amylase secretion, or cell injury. We have reported that zymogen activation is mediated by the vacuolar ATPase (vATPase), a proton transporter. vATPase inhibition, using concanamycin (100 nmol/L), blocked the low pHe effects on zymogen activation. An acute acid load given in vivo enhanced cerulein-induced (50 microg/kg) trypsinogen activation and pancreatic edema. CONCLUSION: These studies suggest that acid challenge sensitizes the pancreatic acinar cell to secretagogue-induced zymogen activation and injury and may increase the risk for the development and severity of acute pancreatitis.

Molecular and cellular mechanisms of pancreatic injury.

PURPOSE OF REVIEW: This review focuses on studies from the past year that highlight molecular and cellular mechanisms of pancreatic injury arising from acute and chronic pancreatitis. RECENT FINDINGS: Factors that induce or ameliorate injury as well as cellular pathways involved have been examined. Causative or sensitizing factors include refluxed bile acids, hypercalcemia, ethanol, hypertriglyceridemia, and acidosis. In addition, the diabetes drug exendin-4 has been associated with pancreatitis, whereas other drugs may reduce pancreatic injury. The intracellular events that influence disease severity are better understood. Cathepsin-L promotes injury through an antiapoptotic effect, rather than by trypsinogen activation. In addition, specific trypsinogen mutations lead to trypsinogen misfolding, endoplasmic reticulum stress, and injury. Endogenous trypsin inhibitors and upregulation of proteins including Bcl-2, fibroblast growth factor 21, and activated protein C can reduce injury. Immune cells, however, have been shown to increase injury via an antiapoptotic effect. SUMMARY: The current findings are critical to understanding how causative factors initiate downstream cellular events resulting in pancreatic injury. Such knowledge will aid in the development of targeted treatments for pancreatitis. This review will first discuss factors influencing pancreatic injury, and then conclude with studies detailing the cellular mechanisms involved.

The acinar cell and early pancreatitis responses.

Pathologic responses arising from the pancreatic acinar cell appear to have a central role in initiating acute pancreatitis. Environmental factors that sensitize the acinar cell to harmful stimuli likely have a critical role in many forms of pancreatitis, including that induced by alcohol abuse. Activation of zymogens within the acinar cell and an inhibition of secretion are critical, but poorly understood, early pancreatitis events. While there is firm evidence relating trypsinogen activation to pancreatitis, the importance of other zymogens has been less studied. Preliminary studies suggest that trypsin may be activated by mechanisms that are distinct from other zymogens. Further, unlike the small intestine, it may not catalyze the activation of other zymogens. These features could affect strategies aimed at inhibiting proteases to treat pancreatitis. Specific intracellular signals are required to activate pancreatitis pathways in the acinar cell. The most important is calcium. Recent studies have suggested that calcium release through specific calcium channels in the endoplasmic reticulum is the means by which pathological elevations in cytosolic calcium occur. Although the targets of abnormal calcium signaling are unknown, calcineurin, a calcium-dependent phosphatase, may serve such a role. Finally, recent work suggests that an acute acid load might sensitize the acinar cell to pancreatitis responses. Therapies aimed at preventing or reversing the effects of an acid load on the pancreas may be important for treatment.

Molecular and cellular regulation of pancreatic acinar cell function.

PURPOSE OF REVIEW: This review focuses on studies from the past year that have greatly advanced our understanding of molecular and cellular regulation of pancreatic acinar cell function. RECENT FINDINGS: Recent advances focus on signals dictating pancreatic development, acinar cell fate, pancreatic growth, and secretion. Regeneration of acinar cells after pancreatitis depends on expression of embryonic signals in mature acinar cells. In this setting, acinar cells can also transdifferentiate into adipose cells. With the forced induction of certain early and endocrine-driving transcription factors, acinar cells can also transdifferentiate into beta-cells. There has also been an increased understanding of acinar-to-ductal metaplasia and the subsequent formation of pancreatic intraepithelial neoplasia lesions. Multiple proteins involved in secretion have been characterized, including small guanosine triphosphate-binding proteins, soluble N-ethylmaleimide-sensitive factor attachment proteins, and ion channels. SUMMARY: These findings demonstrate the regenerative potential of the acinar cell to mitigate injurious states such as pancreatitis. The ability of acinar cells to transdifferentiate into beta-cells could potentially provide a treatment for diabetes. Finally, the results might be helpful in preventing malignant transformation events arising from the acinar cell. Developments in proteomics and computer modeling could expand our view of proteins mediating acinar cell function.

Molecular basis for pancreatitis.

PURPOSE OF REVIEW: This timely review will focus on clinical and basic science studies that have greatly advanced our knowledge of the molecular mechanisms of both acute pancreatitis and chronic pancreatitis over the last year. RECENT FINDINGS: Animal models of both severe acute pancreatitis and chronic pancreatitis have recently been developed. Several unexpected protective mechanisms, mediated by the protease activated receptor 2 and heat shock protein 70, have been described. A genetic study suggested that polymorphisms in toll-like receptor-4 might affect the risk of developing infections in acute pancreatitis. Studies of chronic pancreatitis have shown that specific neural receptors, transient receptor potential vanilloid subtype 1, mediate pain responses in a model of chronic pancreatitis. The pancreatic zymogen, chymotrypsin C, can degrade pathologically activated trypsin in the acinar cell. Inactivating mutations in chymotrypsin C have been reported to predispose to the development of chronic pancreatitis, especially in those who are prone to alcohol abuse. SUMMARY: The implications of the last year's findings are widespread. Improved animal models of acute pancreatitis and chronic pancreatitis will be critical for performing pilot studies of therapy. A greater understanding of genetic factors and pain responses could lead to potential treatments. This review will first discuss issues related to acute pancreatitis, and then conclude with studies most relevant to chronic disease.

Protein kinase C in the pancreatic acinar cell.

Pathological activation of selective signaling molecules within the pancreatic acinar cell mediates the development of acute pancreatitis. Some of the key early acinar cell events include activation of proteases, inhibition of apical secretion, and elaboration of inflammatory mediators. Previous studies have shown that supraphysiological concentrations of cholecystokinin (CCK) that can cause pancreatitis in vivo, also initiate these pathological responses in dispersed groups of acinar cells (acini). Protein kinase C (PKC) regulates many cellular events and a role for this family of signaling molecules has been described in some of the pathological responses of pancreatitis. Notably, ethanol can activate specific PKC isoforms and sensitize the acinar cells to the pathological effects of CCK. Our preliminary studies in isolated pancreatic acini and a cell-free reconstitution system suggest that PKC can mediate protease activation in the acinar cell. These findings may be relevant to the pathogenesis of pancreatitis from alcohol and other etiologies.

Pathologic cellular events in smoking-related pancreatitis.

Pancreatitis, a debilitating inflammatory disorder, results from pancreatic injury. Alcohol abuse is the foremost cause, although cigarette smoking has recently surfaced as a distinct risk factor. The mechanisms by which cigarette smoke and its toxins initiate pathological cellular events leading to pancreatitis, have not been clearly defined. Although cigarette smoke is composed of more than 4000 compounds, it is mainly nicotine and the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which have been extensively studied with respect to pancreatic diseases. This review summarizes these research findings and highlights cellular pathways which may be of relevance in initiation and progression of smoking-related pancreatitis.

Epidemiologic and Mechanistic Associations Between Smoking and Pancreatitis.

OPINION STATEMENT: Alcohol has long been associated with pancreatitis. Although first described more than three decades ago, smoking has been widely accepted as an important risk factor for all forms of pancreatitis only in the past few years. Empiric data has confirmed smoking as an independent and dose-dependent risk for both acute and chronic pancreatitis. Smoking also increases the risk of recurrences and progression of established chronic pancreatitis. The effects of smoking are enhanced in the presence of alcohol consumption. Indirect evidence suggests that smoking cessation may be beneficial in preventing disease progression. Smoking cessation can therefore be an important strategy for primary as well as secondary prevention of pancreatitis. Therefore, in addition to alcohol, physicians should routinely counsel patients for the benefits of smoking cessation. The mechanisms through which cigarette smoke triggers pathological cellular events, resulting in pancreatitis, are unresolved. Although cigarette smoke contains greater than 4000 compounds, principally nicotine and the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) have been broadly studied with regard to pancreatic diseases. Both nicotine and NNK have been shown to induce morphological changes in the pancreas consistent with those seen in pancreatitis. Furthermore, nicotine affects pancreatic secretion and NNK induces premature zymogen activation, two well-known features of pancreatitis. These cigarette toxins may mediate both pro- and anti-inflammatory pathways and can induce changes in pancreatic acinar cell function at the level of transcription, leading to conditions such as thiamin deficiency and mitochondrial dysfunction. Such circumstances could leave the pancreas prone to the development of pancreatitis. This review summarizes relevant research findings and focuses on the epidemiologic links between smoking and pancreatitis, and the cellular pathways that may be significant in induction and evolution of smoking-related pancreatitis.

Chronic Nicotine Exposure In Vivo and In Vitro Inhibits Vitamin B1 (Thiamin) Uptake by Pancreatic Acinar Cells.

Thiamin (vitamin B1), a member of the water-soluble family of vitamins, is essential for normal cellular functions; its deficiency results in oxidative stress and mitochondrial dysfunction. Pancreatic acinar cells (PAC) obtain thiamin from the circulation using a specific carrier-mediated process mediated by both thiamin transporters -1 and -2 (THTR-1 and THTR-2; encoded by the SLC19A2 and SLC19A3 genes, respectively). The aim of the current study was to examine the effect of chronic exposure of mouse PAC in vivo and human PAC in vitro to nicotine (a major component of cigarette smoke that has been implicated in pancreatic diseases) on thiamin uptake and to delineate the mechanism involved. The results showed that chronic exposure of mice to nicotine significantly inhibits thiamin uptake in murine PAC, and that this inhibition is associated with a marked decrease in expression of THTR-1 and THTR-2 at the protein, mRNA and hnRNAs level. Furthermore, expression of the important thiamin-metabolizing enzyme, thiamin pyrophosphokinase (TPKase), was significantly reduced in PAC of mice exposed to nicotine. Similarly, chronic exposure of cultured human PAC to nicotine (0.5 µM, 48 h) significantly inhibited thiamin uptake, which was also associated with a decrease in expression of THTR-1 and THTR-2 proteins and mRNAs. This study demonstrates that chronic exposure of PAC to nicotine impairs the physiology and the molecular biology of the thiamin uptake process. Furthermore, the study suggests that the effect is, in part, mediated through transcriptional mechanism(s) affecting the SLC19A2 and SLC19A3 genes.

Tissue Pharmacology of Da-Cheng-Qi Decoction in Experimental Acute Pancreatitis in Rats.

Objectives. The Chinese herbal medicine Da-Cheng-Qi Decoction (DCQD) can ameliorate the severity of acute pancreatitis (AP). However, the potential pharmacological mechanism remains unclear. This study explored the potential effective components and the pharmacokinetic characteristics of DCQD in target tissue in experimental acute pancreatitis in rats. Methods. Acute pancreatitis-like symptoms were first induced in rats and then they were given different doses of DCQD (6 g/kg, 12 g/kg, and 24 g/kg body weight) orally. Tissue drug concentration, tissue pathological score, and inflammatory mediators in pancreas, intestine, and lung tissues of rats were examined after 24 hours, respectively. Results. Major components of DCQD could be found in target tissues and their concentrations increased in conjunction with the intake dose of DCQD. The high-dose compounds showed maximal effect on altering levels of anti-inflammatory (interleukin-4 and interleukin-10) and proinflammatory markers (tumor necrosis factor α and interleukin-6) and ameliorating the pathological damage in target tissues (P < 0.05). Conclusions. DCQD could alleviate pancreatic, intestinal, and lung injury by altering levels of inflammatory cytokines in AP rats with tissue distribution of its components.

Smoking and Pancreatic Disease.

Smoking is a major risk factor for chronic pancreatitis and pancreatic cancer. However, the mechanisms through which it causes the diseases remain unknown. In the present manuscript we reviewed the latest knowledge gained on the effect of cigarette smoke and smoking compounds on cell signaling pathways mediating both diseases. We also reviewed the effect of smoking on the pancreatic cell microenvironment including inflammatory cells and stellate cells.

Risk factors for pancreatic cancer: underlying mechanisms and potential targets.

PURPOSE OF THE REVIEW: Pancreatic cancer is extremely aggressive, forming highly chemo-resistant tumors, and has one of the worst prognoses. The evolution of this cancer is multi-factorial. Repeated acute pancreatic injury and inflammation are important contributing factors in the development of pancreatic cancer. This article attempts to understand the common pathways linking pancreatitis to pancreatic cancer. RECENT FINDINGS: Intracellular activation of both pancreatic enzymes and the transcription factor NF-κB are important mechanisms that induce acute pancreatitis (AP). Recurrent pancreatic injury due to genetic susceptibility, environmental factors such as smoking, alcohol intake, and conditions such as obesity lead to increases in oxidative stress, impaired autophagy and constitutive activation of inflammatory pathways. These processes can stimulate pancreatic stellate cells, thereby increasing fibrosis and encouraging chronic disease development. Activation of oncogenic Kras mutations through inflammation, coupled with altered levels of tumor suppressor proteins (p53 and p16) can ultimately lead to development of pancreatic cancer. SUMMARY: Although our understanding of pancreatitis and pancreatic cancer has tremendously increased over many years, much remains to be elucidated in terms of common pathways linking these conditions.

Activation of soluble adenylyl cyclase protects against secretagogue stimulated zymogen activation in rat pancreaic acinar cells.

An early feature of acute pancreatitis is activation of zymogens, such as trypsinogen, within the pancreatic acinar cell. Supraphysiologic concentrations of the hormone cholecystokinin (CCK; 100 nM), or its orthologue cerulein (CER), induce zymogen activation and elevate levels of cAMP in pancreatic acinar cells. The two classes of adenylyl cyclase, trans-membrane (tmAC) and soluble (sAC), are activated by distinct mechanisms, localize to specific subcellular domains, and can produce locally high concentrations of cAMP. We hypothesized that sAC activity might selectively modulate acinar cell zymogen activation. sAC was identified in acinar cells by PCR and immunoblot. It localized to the apical region of the cell under resting conditions and redistributed intracellularly after treatment with supraphysiologic concentrations of cerulein. In cerulein-treated cells, pre-incubation with a trans-membrane adenylyl cyclase inhibitor did not affect zymogen activation or amylase secretion. However, treatment with a sAC inhibitor (KH7), or inhibition of a downstream target of cAMP, protein kinase A (PKA), significantly enhanced secretagogue-stimulated zymogen activation and amylase secretion. Activation of sAC with bicarbonate significantly inhibited secretagogue-stimulated zymogen activation; this response was decreased by inhibition of sAC or PKA. Bicarbonate also enhanced secretagogue-stimulated cAMP accumulation; this effect was inhibited by KH7. Bicarbonate treatment reduced secretagogue-stimulated acinar cell vacuolization, an early marker of pancreatitis. These data suggest that activation of sAC in the pancreatic acinar cell has a protective effect and reduces the pathologic activation of proteases during 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.

GENETIC AND PHARMACOLOGIC MANIPULATION OF VACUOLAR ATPASE; EFFECTS ON ZYMOGEN ACTIVATION IN PANCREATIC ACINI.

Premature activation of inactive digestive enzymes (or zymogens) within the pancreatic acinar cell is an initiating event in acute pancreatitis (AP). We have found that this response depends on the assembly and activation of an ATP-dependent proton pump, the vacuolar ATPase (vATPase). Previously, we have shown that the classic vATPase inhibitors concanamycin and bafilomycin can inhibit zymogen activation induced experimentally by high doses of the cholecystokinin orthologue, cerulein (CER) in isolated acinar cells. Recent studies have questioned the specificity of these inhibitors. In the current study we examine the role of the vATPase in pancreatitis using the newly developed novel vATPase inhibitors lobatomide-B and salicylihalamide-A as well as a genetic approach using siRNA. Both lobatomide-B and salicylihalamide-A inhibited CER stimulated zymogen (trypsinogen and chymotrypsinogen) activation but had no effect on amylase secretion. Lobatomide-B (0.1µM) was more potent, reducing activation to baseline levels. Treatment of cells with siRNA specific for the vATPase E-subunit (V1E) significantly decreased V1E expression. V1E siRNA also significantly decreased chymotrypsinogen activation, but not amylase secretion. These studies confirm a role for the vATPase in zymogen activation and demonstrate that the novel and specific inhibitors lobatomide-B and salicylihalamide-A reduce early pancreatitis responses.