Pharmacological inhibition of PAR2 with the pepducin P2pal-18S protects mice against acute experimental biliary pancreatitis

Pancreatic acinar cells express proteinase-activated receptor-2 (PAR2) that is activated by trypsin-like serine proteases and has been shown to exert model-specific effects on the severity of experimental pancreatitis, i.e., PAR2−/− mice are protected from experimental acute biliary pancreatitis but develop more severe secretagogue-induced pancreatitis. P2pal-18S is a novel pepducin lipopeptide that targets and inhibits PAR2. In studies monitoring PAR2-stimulated intracellular Ca2+ concentration changes, we show that P2pal-18S is a full PAR2 inhibitor in acinar cells. Our in vivo studies show that P2pal-18S significantly reduces the severity of experimental biliary pancreatitis induced by retrograde intraductal bile acid infusion, which mimics injury induced by endoscopic retrograde cholangiopancreatography (ERCP). This reduction in pancreatitis severity is observed when the pepducin is given before or 2 h after bile acid infusion but not when it is given 5 h after bile acid infusion. Conversely, P2pal-18S increases the severity of secretagogue-induced pancreatitis. In vitro studies indicate that P2pal-18S protects acinar cells against bile acid-induced injury/death, but it does not alter bile acid-induced intracellular zymogen activation. These studies are the first to report the effects of an effective PAR2 pharmacological inhibitor on pancreatic acinar cells and on the severity of experimental pancreatitis. They raise the possibility that a pepducin such as P2pal-18S might prove useful in the clinical management of patients at risk for developing severe biliary pancreatitis such as occurs following ERCP.

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Pancreatic Acinar Cell Nuclear Factor κB Activation Because of Bile Acid Exposure Is Dependent on Calcineurin

Journal of Biological Chemistry ◽

10.1074/jbc.m113.471425 ◽

2013 ◽

Vol 288 (29) ◽

pp. 21065-21073 ◽

Cited By ~ 20

Author(s):

Kamaldeen A. Muili ◽

Shunqian Jin ◽

Abrahim I. Orabi ◽

John F. Eisses ◽

Tanveer A. Javed ◽

...

Keyword(s):

Bile Acid ◽

Acinar Cell ◽

Cell Injury ◽

Nuclear Translocation ◽

Calcineurin Inhibitors ◽

Acinar Cells ◽

Pancreatic Acinar Cell ◽

Pancreatic Acinar Cells ◽

Luciferase Reporter ◽

Biliary Pancreatitis

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 Ca2+ signals in acinar cells as well as nuclear translocation of NF-κB. In this study, we examined the role of the downstream Ca2+ 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 Ca2+ 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 Aβ-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.

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The role of intracellular Ca2+ signaling in the exocrine pancreatic damage during acute pancreatitis

10.14232/phd.10488 ◽

2020 ◽

Author(s):

Júlia Fanczal

Keyword(s):

Oxidative Stress ◽

Acute Pancreatitis ◽

Bile Acid ◽

Bile Acids ◽

Digestive Enzyme ◽

Acinar Cells ◽

Mitochondrial Damage ◽

Enzyme Activation ◽

Pancreatic Acinar Cells ◽

Biliary Pancreatitis

Acute biliary pancreatitis poses a significant clinical challenge as currently no specific pharmaceutical treatment exists. Disturbed intracellular Ca2+ signalling caused by bile acids is a hallmark of the disease, which induces increased reactive oxygen species (ROS) production, mitochondrial damage, intra-acinar digestive enzyme activation and cell death. Because of this mechanism of action, prevention of toxic cellular Ca2+ overload is a promising therapeutic target. Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel that has recently emerged as an important contributor to oxidative-stress-induced cellular Ca2+ overload across different diseases. However, the expression and possible functions of TRPM2 in the exocrine pancreas remain unknown. Here we found that TRPM2 is expressed in the plasma membrane of mouse pancreatic acinar, which can be activated by increased oxidative stress induced by H2O2 treatment. TRPM2 activity was found to contribute to bile acid-induced extracellular Ca2+ influx in acinar cells. The generation of intracellular ROS in response to bile acids was remarkably higher in pancreatic acinar cells. This activity promoted acinar cell necrosis in vitro independently from mitochondrial damage or mitochondrial fragmentation. In addition, bile-acid-induced experimental pancreatitis was less severe in TRPM2 knockout mice, whereas the lack of TRPM2 had no protective effect in cerulein-induced acute pancreatitis. Our results suggest that the inhibition of TRPM2 may be a potential treatment option for biliary pancreatitis.

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Pancreatogenic Diabetes: Triggering Effects of Alcohol and HIV

Biology ◽

10.3390/biology10020108 ◽

2021 ◽

Vol 10 (2) ◽

pp. 108

Author(s):

Moses New-Aaron ◽

Murali Ganesan ◽

Raghubendra Singh Dagur ◽

Kusum K. Kharbanda ◽

Larisa Y. Poluektova ◽

...

Keyword(s):

Multiorgan Failure ◽

Acinar Cells ◽

Pancreatic Stellate Cells ◽

Pancreatic Acinar Cells ◽

Alcoholic Pancreatitis ◽

People Living With Hiv ◽

Zymogen Activation ◽

Pancreatogenic Diabetes ◽

Hiv Entry ◽

Living With Hiv

Multiorgan failure may not be completely resolved among people living with HIV despite HAART use. Although the chances of organ dysfunction may be relatively low, alcohol may potentiate HIV-induced toxic effects in the organs of alcohol-abusing, HIV-infected individuals. The pancreas is one of the most implicated organs, which is manifested as diabetes mellitus or pancreatic cancer. Both alcohol and HIV may trigger pancreatitis, but the combined effects have not been explored. The aim of this review is to explore the literature for understanding the mechanisms of HIV and alcohol-induced pancreatotoxicity. We found that while premature alcohol-inducing zymogen activation is a known trigger of alcoholic pancreatitis, HIV entry through C-C chemokine receptor type 5 (CCR5) into pancreatic acinar cells may also contribute to pancreatitis in people living with HIV (PLWH). HIV proteins induce oxidative and ER stresses, causing necrosis. Furthermore, infiltrative immune cells induce necrosis on HIV-containing acinar cells. When necrotic products interact with pancreatic stellate cells, they become activated, leading to the release of both inflammatory and profibrotic cytokines and resulting in pancreatitis. Effective therapeutic strategies should block CCR5 and ameliorate alcohol’s effects on acinar cells.

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Absence of the neutrophil serine protease cathepsin G decreases neutrophil granulocyte infiltration but does not change the severity of acute pancreatitis

Scientific Reports ◽

10.1038/s41598-019-53293-0 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Ali A. Aghdassi ◽

Daniel S. John ◽

Matthias Sendler ◽

Christian Storck ◽

Cindy van den Brandt ◽

...

Keyword(s):

Acute Pancreatitis ◽

Serine Protease ◽

Acinar Cells ◽

Neutrophil Infiltration ◽

Cathepsin G ◽

Pancreatic Acinar Cells ◽

Neutrophil Granulocyte ◽

Zymogen Activation ◽

Trypsinogen Activation ◽

Extracellular Matrix Components

AbstractAcute pancreatitis is characterized by an early intracellular protease activation and invasion of leukocytes into the pancreas. Cathepsins constitute a large group of lysosomal enzymes, that have been shown to modulate trypsinogen activation and neutrophil infiltration. Cathepsin G (CTSG) is a neutrophil serine protease of the chymotrypsin C family known to degrade extracellular matrix components and to have regulatory functions in inflammatory disorders. The aim of this study was to investigate the role of CTSG in pancreatitis. Isolated acinar cells were exposed to recombinant CTSG and supramaximal cholezystokinin stimulation. In CTSG−/− mice and corresponding controls acute experimental pancreatitis was induced by serial caerulein injections. Severity was assessed by histology, serum enzyme levels and zymogen activation. Neutrophil infiltration was quantified by chloro-acetate ersterase staining and myeloperoxidase measurement. CTSG was expessed in inflammatory cells but not in pancreatic acinar cells. CTSG had no effect on intra-acinar-cell trypsinogen activation. In CTSG−/− mice a transient decrease of neutrophil infiltration into the pancreas and lungs was found during acute pancreatitis while the disease severity remained largely unchanged. CTSG is involved in pancreatic neutrophil infiltration during pancreatitis, albeit to a lesser degree than the related neutrophil (PMN) elastase. Its absence therefore leaves pancreatitis severity essentially unaffected.

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Inhibition of phosphatidylinositol 3-kinase inhibits intracellular zymogen activation but not enzyme secretion in pancreatic acinar cells

Journal of the American College of Surgeons ◽

10.1016/j.jamcollsurg.2006.05.029 ◽

2006 ◽

Vol 203 (3) ◽

pp. S13

Author(s):

L. Andy Chen ◽

Hiroki Watanabe ◽

B. Mark Evers

Keyword(s):

Acinar Cells ◽

Enzyme Secretion ◽

Pancreatic Acinar Cells ◽

Zymogen Activation ◽

Phosphatidylinositol 3 Kinase ◽

Phosphatidylinositol 3

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Docosahexaenoic acid inhibits zymogen activation by suppressing vacuolar ATPase activation in cerulein-stimulated pancreatic acinar cells

Genes & Nutrition ◽

10.1186/s12263-020-00664-2 ◽

2020 ◽

Vol 15 (1) ◽

Author(s):

Yeeun Park ◽

Leeyeon Ku ◽

Joo Weon Lim ◽

Hyeyoung Kim

Keyword(s):

Docosahexaenoic Acid ◽

Acinar Cells ◽

Vacuolar Atpase ◽

Pancreatic Acinar Cells ◽

Zymogen Activation

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Sorafenib-induced loss of polarity of zymogen granules in pancreatic acinar cells.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.4_suppl.236 ◽

2012 ◽

Vol 30 (4_suppl) ◽

pp. 236-236

Author(s):

Tatsuo Ito ◽

Ryuichiro Doi ◽

Shinji Uemoto

Keyword(s):

Acute Pancreatitis ◽

Kinase Inhibitor ◽

Pancreatic Enzyme ◽

Acinar Cells ◽

In Vivo Studies ◽

Pancreatic Acinar Cells ◽

Control Group ◽

Amylase Level ◽

Zymogen Granules ◽

Tissue Samples

236 Background: Sorafenib is an oral multi-kinase inhibitor which is regarded as a key drug for HCC and RCC. It has been unexpectedly found that the compound causes an increase of serum pancreatic enzyme levels without clinically recognized pancreatitis. The reason for this event is not well understood yet. The aim of this study was to clarify the mechanisms involved in this phenomenon. Methods: Eight-week old BALB/cA male mice were used in in vivo studies. Sorafenib tosylate was administered per os once daily at a dose of 150 mg/kg body weight. Control mice were given vehicle alone. Mice were sacrificed 24 hr after 1-, 2-, 3- and 7-day administration of the compound, and blood samples and pancreatic tissue samples were obtained (n=5 for each group). The tissue samples were used for hematoxylin and eosin (HE) staining, immunohistochemistry, electron microscopy (EM), western blot and RT-quantitative PCR studies. Results: Serum amylase levels were elevated after sorafenib administration. The amylase level hit the peak after 2-day administration, and then gradually decreased. By HE staining, the control group without sorafenib showed a basophilic stained area in the baso-lateral site of the acinar cells. In contrast, the acinar cytoplasm after 2-day administration of sorafenib was totally eosinophilic. The typical findings of acute pancreatitis were not seen in the both group. By EM examination, zymogen granules (ZGs) of the sorafenib group spread into basal site of the acinar cells. ZGs mounted up on both of apical and baso-lateral plasma membrane and showed exocytosis. The levels of amylase mRNA were not elevated by sorafenib. In addition the expression of N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) proteins was not changed. Conclusions: The results suggest that the amount of acinar amylase production was not changed but the distribution of ZGs was altered by sorafenib. Sorafenib seemed to cause temporary loss of polarity of ZGs secretion in acinar cells by blocking apical exocytosis. Acute pancreatitis was not evident; thus the current model was not similar to the pancreatitis model caused by the supra-maximal secretagogue stimulation which blocks the apical exocytosis.

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Supramaximal caerulein stimulation and ultrastructure of rat pancreatic acinar cell: early morphological changes during development of experimental pancreatitis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1984.246.4.g457 ◽

1984 ◽

Vol 246 (4) ◽

pp. G457-G467 ◽

Cited By ~ 22

Author(s):

O. Watanabe ◽

F. M. Baccino ◽

M. L. Steer ◽

J. Meldolesi

Keyword(s):

Morphological Changes ◽

Freeze Fracture ◽

Acinar Cells ◽

Pancreatic Acinar Cell ◽

Experimental Pancreatitis ◽

Pancreatic Acinar Cells ◽

Morphological Evidence ◽

Lysosomal Hydrolases ◽

Thin Section Electron Microscopy ◽

Section Electron

Rats infused with a supramaximally stimulating dose of the cholecystokinin-pancreozymin analogue caerulein develop acute interstitial pancreatitis (M. Lampel and H.F. Kern. Virchows Arch. A 373: 97-117, 1977). We have studied the early (30-180 min) morphological changes in pancreatic acinar cells induced by infusing caerulein (2.5 micrograms X kg-1 X h-1). The techniques of thin-section electron microscopy, freeze fracture, and enzyme and immunocytochemistry were employed. Shortly (30 min) after the onset of caerulein infusion, large vacuoles appeared in the Golgi area. After longer periods of infusion, these vacuoles further enlarged (probably by fusion with other such vacuoles as well as autophagic vacuoles) and became more widely distributed in the cytoplasm. These large vacuoles were found to be acid phosphatase positive and to be labeled by antibodies directed against digestive zymogens as well as the lysosomal enzyme cathepsin D. These observations indicate that the large vacuoles contain both digestive zymogens and lysosomal hydrolases. During caerulein infusion, morphological evidence of exocytosis at the luminal plasmalemma was reduced or absent, and evidence of basolateral exocytosis was not noted. These studies suggest that secretagogue hyperstimulation with caerulein interferes with the processes involved in condensing vacuole maturation, which normally lead to the separation of digestive zymogens and lysosomal hydrolases. As a result, both types of enzymes remain within the same compartment. This may lead to the intracellular activation of digestive enzymes by lysosomal hydrolases and be an important step in the development of acute pancreatitis.

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