S1817 Protein Kinase D Modulates Secretagogue-Induced Zymogen Activation and Amylase Secretion in Rat Pancreatic Acinar Cells

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 IC50 ∼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.

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Genetic inhibition of protein kinase Cε attenuates necrosis in experimental pancreatitis

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00432.2013 ◽

2014 ◽

Vol 307 (5) ◽

pp. G550-G563 ◽

Cited By ~ 10

Author(s):

Yannan Liu ◽

Jingzhen Yuan ◽

Tanya Tan ◽

Wenzhuo Jia ◽

Aurelia Lugea ◽

...

Keyword(s):

Protein Kinase ◽

Inflammatory Responses ◽

Acinar Cells ◽

Pancreatic Acinar Cells ◽

Caspase Activation ◽

Zymogen Activation ◽

Cytochrome C Release ◽

Interacting Protein ◽

Regulated Necrosis

Understanding the regulation of death pathways, necrosis and apoptosis, in pancreatitis is important for developing therapies directed to the molecular pathogenesis of the disease. Protein kinase Cε (PKCε) has been previously shown to regulate inflammatory responses and zymogen activation in pancreatitis. Furthermore, we demonstrated that ethanol specifically activated PKCε in pancreatic acinar cells and that PKCε mediated the sensitizing effects of ethanol on inflammatory response in pancreatitis. Here we investigated the role of PKCε in the regulation of death pathways in pancreatitis. We found that genetic deletion of PKCε resulted in decreased necrosis and severity in the in vivo cerulein-induced pancreatitis and that inhibition of PKCε protected the acinar cells from CCK-8 hyperstimulation-induced necrosis and ATP reduction. These findings were associated with upregulation of mitochondrial Bak and Bcl-2/Bcl-xL, proapoptotic and prosurvival members in the Bcl-2 family, respectively, as well as increased mitochondrial cytochrome c release, caspase activation, and apoptosis in pancreatitis in PKCε knockout mice. We further confirmed that cerulein pancreatitis induced a dramatic mitochondrial translocation of PKCε, suggesting that PKCε regulated necrosis in pancreatitis via mechanisms involving mitochondria. Finally, we showed that PKCε deletion downregulated inhibitors of apoptosis proteins, c-IAP2, survivin, and c-FLIPs while promoting cleavage/inactivation of receptor-interacting protein kinase (RIP). Taken together, our findings provide evidence that PKCε activation during pancreatitis promotes necrosis through mechanisms involving mitochondrial proapoptotic and prosurvival Bcl-2 family proteins and upregulation of nonmitochondrial pathways that inhibit caspase activation and RIP cleavage/inactivation. Thus PKCε is a potential target for prevention and/or treatment of acute pancreatitis.

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W1821 Protein Kinase D Mediates Nf-κB Activation Induced By Cholecystokinin and Cholinergic Signaling in Pancreatic Acinar Cells

Gastroenterology ◽

10.1016/s0016-5085(08)63375-2 ◽

2008 ◽

Vol 134 (4) ◽

pp. A-722

Author(s):

Jingzhen Yuan ◽

Aurelia Lugea ◽

Ling Zheng ◽

Ilya Gukovsky ◽

Mouad Edderkaoui ◽

...

Keyword(s):

Protein Kinase ◽

Acinar Cells ◽

Pancreatic Acinar Cells ◽

Protein Kinase D ◽

Cholinergic Signaling

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The novel protein kinase C isoforms -δ and -ε modulate caerulein-induced zymogen activation in pancreatic acinar cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00020.2008 ◽

2008 ◽

Vol 294 (6) ◽

pp. G1344-G1353 ◽

Cited By ~ 28

Author(s):

Edwin C. Thrower ◽

Sara Osgood ◽

Christine A. Shugrue ◽

Thomas R. Kolodecik ◽

Anamika M. Chaudhuri ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Acinar Cell ◽

Acinar Cells ◽

Pancreatic Acinar Cells ◽

Cell Fractionation ◽

Zymogen Activation ◽

Cellular Models ◽

Kinase C

Isoforms of protein kinase C (PKC) have been shown to modulate some cellular responses such as pathological secretion and generation of inflammatory mediators during acute pancreatitis (AP). We propose that PKC also participates in premature zymogen activation within the pancreatic acinar cell, a key event in the initiation of AP. This hypothesis was examined in in vivo and cellular models of caerulein-induced AP using PKC activators and inhibitors. Phorbol ester, 12- O-tetradecanoylphorbol-13-acetate (TPA, 200 nM), a known activator of PKC, enhanced zymogen activation at both 0.1 nM and 100 nM caerulein, concentrations which mimic physiological and supraphysiological effects of the hormone cholecystokinin, respectively, in preparations of pancreatic acinar cells. Isoform-specific PKC inhibitors for PKC-δ and PKC-ε reduced supraphysiological caerulein-induced zymogen activation. Using a cell-free reconstitution system, we showed that inhibition of PKC-δ and -ε, reduced zymogen activation in both zymogen granule-enriched and microsomal fractions. In dispersed acinar cells, 100 nM caerulein stimulation caused PKC-δ and -ε isoform translocation to microsomal membranes using cell fractionation and immunoblot analysis. PKC translocation was confirmed with in vivo studies and immunofluorescence microscopy in pancreatic tissues from rats treated with or without 100 nM caerulein. PKC-ε redistributed from an apical to a supranuclear region following caerulein administration. The signal for PKC-ε overlapped with granule membrane protein, GRAMP-92, an endosomal/lysosomal marker, in a supranuclear region where zymogen activation takes place. These results indicate that PKC-δ and -ε isoforms translocate to specific acinar cell compartments and modulate zymogen activation.

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Linoleic acid is a potent activator of protein kinase C type III- α isoform in pancreatic acinar cells; its role in amylase secretion

Biochemical and Biophysical Research Communications ◽

10.1016/s0006-291x(88)81190-2 ◽

1988 ◽

Vol 153 (1) ◽

pp. 67-73 ◽

Cited By ~ 20

Author(s):

Marie W. Wooten ◽

Robert W. Wrenn

Keyword(s):

Protein Kinase C ◽

Linoleic Acid ◽

Protein Kinase ◽

Acinar Cells ◽

Pancreatic Acinar Cells ◽

Amylase Secretion ◽

Type Iii ◽

Kinase C

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Effect of protein kinase C on amylase secretion and cyclic AMP production in rat pancreatic acinar cells

Gastroenterologia Japonica ◽

10.1007/bf02782819 ◽

1991 ◽

Vol 26 (4) ◽

pp. 489-496 ◽

Cited By ~ 1

Author(s):

Tatsuo Ishizuka ◽

Yasufumi Ito ◽

Kiyoshi Miura ◽

Sciji Nagao ◽

Yoshinori Nozawa

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Cyclic Amp ◽

Acinar Cells ◽

Pancreatic Acinar Cells ◽

Amylase Secretion ◽

Kinase C

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Caerulein-induced intracellular pancreatic zymogen activation is dependent on calcineurin

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00500.2006 ◽

2007 ◽

Vol 292 (6) ◽

pp. G1594-G1599 ◽

Cited By ~ 35

Author(s):

Sohail Z. Husain ◽

Wayne M. Grant ◽

Fred S. Gorelick ◽

Michael H. Nathanson ◽

Ahsan U. Shah

Keyword(s):

Calcineurin Inhibitors ◽

Acinar Cells ◽

Enzyme Secretion ◽

Pancreatic Acinar Cells ◽

Amylase Secretion ◽

Zymogen Activation ◽

Inhibitory Peptide ◽

Protein Phosphatase 2B ◽

Chymotrypsin Activity ◽

Downstream Effectors

Aberrant cytosolic Ca2+ flux in pancreatic acinar cells is critical to the pathological pancreatic zymogen activation observed in acute pancreatitis, but the downstream effectors are not known. In this study, we examined the role of Ca2+-activated protein phosphatase 2B (or calcineurin) in zymogen activation. Isolated pancreatic acinar cells were stimulated with supraphysiological caerulein (100 nM) with or without the calcineurin inhibitors FK506 or cell-permeable calcineurin inhibitory peptide (CiP). Chymotrypsin activity was measured as a marker of zymogen activation, and the percent amylase secretion was used as a measure of enzyme secretion. Cytosolic Ca2+ changes were recorded in acinar cells loaded with the intermediate Ca2+-affinity dye fluo-5F using a scanning confocal microscope. A 50% reduction in chymotrypsin activity was observed after pretreatment with 1 μM FK506 or 10 μM CiP. These pretreatments did not affect amylase secretion or the rise in cytosolic Ca2+ after caerulein stimulation. These findings suggest that calcineurin mediates caerulein-induced intra-acinar zymogen activation but not enzyme secretion or the initial caerulein-induced cytosolic Ca2+ signal.

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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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