preparation-pancreatic-acinar-cells-for-purpose-calcium-imaging-cell

Preparation of Pancreatic Acinar Cells for the Purpose of Calcium Imaging, Cell Injury Measurements, and Adenoviral Infection

The pancreatic acinar  cell is the main parenchymal cell of the exocrine pancreas and plays a primary  role in the secretion of pancreatic enzymes into ...

The mechanisms by which reflux of bile acids into the pancreas induces pancreatitis are unknown. We reasoned that key events responsible for this phenomenon might be mediated by Gpbar1, a recently identified and widely expressed G-protein-coupled, cell surface bile acid receptor.

Biliary acute pancreatitis in mice is mediated by the G-protein-coupled cell surface bile acid receptor Gpbar1.

Arthritis is one of the hallmarks of late-stage Lyme disease. Previous studies have shown that infection with Borrelia burgdorferi, the causative agent of Lyme disease, results in degradation of proteoglycans and collagen in cartilage. B burgdorferi do not appear to produce any exported proteases capable of digesting proteoglycans and collagen, but instead, induce and activate host proteases, such as matrix metalloproteinases (MMPs), which results in cartilage degradation. The role of aggrecanases in Lyme arthritis has not yet been determined. We therefore sought to delineate the contribution of aggrecanases to joint destruction in Lyme arthritis.

Role of aggrecanase 1 in Lyme arthritis.

We have hypothesized that the colocalization of digestive zymogens with lysosomal hydrolases, which occurs during the early stages of every experimental pancreatitis model, facilitates activation of those zymogens by lysosomal hydrolases such as cathepsin B and that this activation triggers acute pancreatitis by leading to acinar cell injury. Some, however, have argued that the colocalization phenomenon may be the result, rather than the cause, of zymogen activation during pancreatitis. To resolve this controversy and explore the causal relationships between zymogen activation and other early pancreatitis events, we induced pancreatitis in mice by repeated supramaximal secretagogue stimulation with caerulein. Some animals were pretreated with the cathepsin B inhibitor CA-074 me to inhibit cathepsin B, prevent intrapancreatic activation of digestive zymogens, and reduce the severity of pancreatitis. We show that inhibition of cathepsin B by pretreatment with CA-074 me prevents intrapancreatic zymogen activation and reduces organellar fragility, but it does not alter the caerulein-induced colocalization phenomenon or subcellular F-actin redistribution or prevent caerulein-induced activation of NF-kappaB, ERK1/2, and JNK or upregulated expression of cytochemokines. We conclude 1) that the colocalization phenomenon, F-actin redistribution, activation of proinflammatory transcription factors, and upregulated expression of cytochemokines are not the results of zymogen activation, and 2) that these early events in pancreatitis are not dependent on cathepsin B activity. In contrast, zymogen activation and increased subcellular organellar fragility during caerulein-induced pancreatitis are dependent on cathepsin B activity.

Cause-effect relationships between zymogen activation and other early events in secretagogue-induced acute pancreatitis.

Pancreatic and lung inflammation during acute pancreatitis is a poorly understood, but clinically important, phenomenon. The proto-oncogene Tpl2 (tumor progression locus-2) has recently been shown to have important immunomodulatory effects on some inflammatory processes, but its importance to pancreatitis has not been previously examined. Our studies were designed to (a) define the effects of Tpl2 on pancreatic and lung inflammation during pancreatitis and (b) identify mechanisms and cell types responsible for those effects. We examined pancreatitis-associated Tpl2 effects in wild type and Tpl2(-/-) mice subjected to either secretagogue-induced or bile salt-induced pancreatitis. To determine the myeloid or non-myeloid lineage of cells responsible for the Tpl2 effects, we used Tpl2(-/-) chimeric mice generated by lethal irradiation followed by bone marrow transplantation. Mechanisms responsible for the effects of Tpl2 ablation on caerulein-induced proinflammatory events were evaluated under in vivo and in vitro conditions using the techniques of electrophoretic mobility shift assay, immunoblot analysis, and quantitative reverse transcription-PCR. We found that Tpl2 ablation markedly reduced pancreatic and lung inflammation in these two dissimilar models of pancreatitis, but it did not alter pancreatic injury/necrosis in either model. The reduction in caerulein-induced pancreatic inflammation is dependent upon Tpl2 ablation in non-myeloid cells and is associated with both in vivo and in vitro inhibition of MEK, JNK, and AP-1 activation and the expression of MCP-1, MIP-2, and interleukin-6. Non-myeloid cell expression of Tpl2 regulates pancreatic inflammation during pancreatitis by mediating proinflammatory signals and the generation of neutrophil chemoattracting factors.

Tumor progression locus-2 is a critical regulator of pancreatic and lung inflammation during acute pancreatitis.

Most mechanistic studies of pancreatitis in mice employ the secretagogue-induced model. The currently reported studies were designed to develop an alternative, and possibly more clinically relevant, mouse model of pancreatitis.

A mouse model of acute biliary pancreatitis induced by retrograde pancreatic duct infusion of Na-taurocholate.

Sepsis is a deadly disease characterized by considerable derangement of the proinflammatory, anti-inflammatory and coagulation responses. Protease-activated receptor 1 (PAR1), an important regulator of endothelial barrier function and blood coagulation, has been proposed to be involved in the lethal sequelae of sepsis, but it is unknown whether activation of PAR1 is beneficial or harmful. Using a cell-penetrating peptide (pepducin) approach, we provide evidence that PAR1 switched from being a vascular-disruptive receptor to a vascular-protective receptor during the progression of sepsis in mice. Unexpectedly, we found that the protective effects of PAR1 required transactivation of PAR2 signaling pathways. Our results suggest therapeutics that selectively activate PAR1-PAR2 complexes may be beneficial in the treatment of sepsis.

'Role reversal' for the receptor PAR1 in sepsis-induced vascular damage.

Protease-activated receptor-2 (PAR2) is a 7-transmembrane G-protein-coupled tethered ligand receptor that is expressed by pancreatic acinar and ductal cells. It can be physiologically activated by trypsin. Previously reported studies (Namkung, W., Han, W., Luo, X., Muallem, S., Cho, K. H., Kim, K. H., and Lee, M. G. (2004) Gastroenterology 126, 1844-1859; Sharma, A., Tao, X., Gopal, A., Ligon, B., Andrade-Gordon, P., Steer, M. L., and Perides, G. (2005) Am. J. Physiol. 288, G388-G395) have shown that PAR2 activation exerts a protective effect on the experimental model of pancreatitis induced by supramaximal secretagogue (caerulein) stimulation. We now show that PAR2 exerts a worsening effect on a different model of experimental pancreatitis, i.e. one induced by retrograde pancreatic ductal infusion of bile salts. In vitro studies using freshly prepared pancreatic acini show that genetic deletion of PAR2 reduces bile salt-induced pathological calcium transients, acinar cell injury, and activation of c-Jun N-terminal kinase, whereas genetic deletion of PAR2 has the opposite or no effect on these pancreatitis-related events when they are elicited, in vitro, by caerulein stimulation. Studies employing a combination of trypsin inhibition and activation of PAR2 with the activating peptide SLIGRL show that all these differences indeed depend on the activation of PAR2. These studies are the first to report that a single perturbation can have model-specific and opposite effects on pancreatitis, and they underscore the importance of performing mechanistic pancreatitis studies using two dissimilar models of the disease to detect idiosyncratic, model-specific events. We suggest PAR2 activation exerts a worsening effect on the severity of clinical pancreatitis and that interventions interfering with PAR2 activation may be of benefit in the treatment of patients with severe pancreatitis.

Protease-activated receptor-2 exerts contrasting model-specific effects on acute experimental pancreatitis.

Borrelia burgdorferi, the causative agent of Lyme arthritis, does not produce any exported proteases capable of degrading extracellular matrix despite the fact that it is able to disseminate from a skin insertion site to infect multiple organs. Prior studies have shown that B. burgdorferi induces the host protease, matrix metalloproteinase 9 (MMP-9), and suggested that the induction of MMP-9 may allow the organism to disseminate and produce local tissue destruction. We examined the role of MMP-9 in dissemination of B. burgdorferi and pathogenesis of Lyme arthritis. In a MMP-9(-/-) mouse model, MMP-9 was not required for the dissemination of the spirochete to distant sites. However, MMP-9(-/-) exhibited significantly decreased arthritis compared to wild-type mice. The decrease in arthritis was not due to an inability to control infection since the spirochete numbers in the joints were identical. Levels of inflammatory chemokines and cytokines were also similar in MMP-9(-/-) and wild-type mice. We examined whether decreased inflammation in MMP-9(-/-) mice may be the result of decreased production of neoattractants by MMP-9-dependent cleavage of collagen. MMP-9 cleavage of type I collagen results in increased monocyte chemoattraction. MMP-9 plays an important role in regulating inflammation in Lyme arthritis, potentially through the cleavage of type I collagen.

Matrix metalloproteinase 9 plays a key role in lyme arthritis but not in dissemination of Borrelia burgdorferi.

Mechanistic studies of acute pancreatitis require animal models because clinical material is generally not available during the early phases of the disease. Here we describe a protocol to induce biliary pancreatitis by retrogradely infusing bile acids into the pancreatic duct of anesthetized mice. The resulting model replicates events believed to be responsible for the onset of clinical biliary (i.e., gallstone) pancreatitis and creates highly reproducible pancreatitis with a severity that depends on the concentration of infused bile acid. Pancreatitis reaches its maximal level of severity within 24 h of induction, and it resolves over the subsequent week. This protocol enables the investigator to use genetically modified strains of mice, and it requires only relatively simple and easily learned techniques of small animal surgery. With practice and gentle technique, the surgery (from induction of anesthesia to completion of the infusion) can be completed within 25 min per animal.

Experimental acute biliary pancreatitis induced by retrograde infusion of bile acids into the mouse pancreatic duct.

The roles of monocytes/macrophages and their mechanisms of action in the regulation of pancreatitis are poorly understood. To address these issues, we have employed genetically altered mouse strains that either express the human diphtheria toxin receptor (DTR) coupled to the CD11b promoter or have global deletion of TNF-Î±. Targeted, conditional depletion of monocytes/macrophages was achieved by administration of diphtheria toxin (DT) to CD11b-DTR mice. We show that in the absence of DT administration, pancreatitis is associated with an increase in pancreatic content of Ly-6C(hi) monocytes/macrophages but that this response is prevented by prior administration of DT to CD11b-DTR mice. DT administration also reduces pancreatic edema and acinar cell injury/necrosis in two dissimilar experimental models of acute pancreatitis (a secretagogue-induced model and a model elicited by retrograde pancreatic duct infusion of sodium taurocholate). In the secretagogue-elicited model, the DT-induced decrease in pancreatitis severity is reversed by adoptive transfer of purified Ly-6C(hi) monocytes harvested from non-DT-treated CD11b-DTR mice or by the transfer of purified Ly-6C(hi) monocytes harvested from TNF-Î±(+/+) donor mice, but it is not reversed by the transfer of Ly-6C(hi) monocytes harvested from TNF-Î±(-/-) donors. Our studies indicate that the Ly-6C(hi) monocyte subset regulates the severity of pancreatitis by promoting pancreatic edema and acinar cell injury/necrosis and that this phenomenon is dependent upon the expression of TNF-Î± by those cells. They suggest that therapies targeting Ly-6C(hi) monocytes and/or TNF-Î± expression by Ly-6C(hi) monocytes might prove beneficial in the prevention or treatment of acute pancreatitis.

TNF-alpha-dependent regulation of acute pancreatitis severity by Ly-6C(hi) monocytes in mice.

Protease-activated receptor-2 (PAR2), a cell surface receptor for trypsin-like proteases, plays a key role in a number of acute and chronic inflammatory diseases of the joints, lungs, brain, gastrointestinal tract, and vascular systems. Despite considerable effort by the pharmaceutical industry, PAR2 has proven recalcitrant to targeting by small molecule inhibitors, which have been unable to effectively prevent the interaction of the protease-generated tethered ligand with the body of the receptor. Here, we report the development of first-in-class cell-penetrating lipopeptide "pepducin" antagonists of PAR2. The design of the third intracellular (i3) loop pepducins were based on a structural model of a PAR2 dimer and by mutating key pharmacophores in the receptor intracellular loops and analogous pepducins. Individual pharmacophores were identified, which controlled constitutive, agonist, and antagonist activities. This approach culminated in the identification of the P2pal-18S pepducin which completely suppressed trypsin and mast cell tryptase signaling through PAR2 in neutrophils and colon cancer cells. The PAR2 pepducin was highly efficacious in blocking PAR2-dependent inflammatory responses in mouse models. These effects were lost in PAR2-deficient and mast-cell-deficient mice, thereby validating the specificity of the pepducin in vivo. These data provide proof of concept that PAR2 pepducin antagonists may afford effective treatments of potentially debilitating inflammatory diseases and serve as a blueprint for developing highly potent and specific i3-loop-based pepducins for other G protein-coupled receptors (GPCRs).

Interdicting protease-activated receptor-2-driven inflammation with cell-penetrating pepducins.

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 Î¼M and a peak-plateau signal at 500 Î¼M, 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.

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

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-ÎºB activation. In vivo, mice that received FK506 or were deficient in the calcineurin isoform AÎ² (CnAÎ²) 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.

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

BACKGROUND: Ischemia-reperfusion injury worsens infarct size, a major determinant of morbidity and mortality after acute myocardial infarction (MI). We tested the hypothesis that reducing left ventricular (LV) wall stress with a percutaneous left atrial-to-femoral artery centrifugal (pLA-FA) bypass system, while delaying coronary reperfusion limits myocardial injury in a model of acute MI. METHODS AND RESULTS: MI was induced by balloon occlusion of the left anterior descending artery (LAD) in adult, male swine. In the MI group(n=4), 120 minutes of LAD occlusion was followed by 120 minutes of reperfusion without mechanical support. In the mechanically supported group (MI+Unload)(n=4), pLA-FA bypass was initiated after 120 minutes of ischemia and LAD occlusion prolonged for an additional 30 minutes, followed by 120 minutes of reperfusion with device support. All animals were sacrificed after reperfusion and infarct size quantified by triphenyltetrazolium chloride staining. Compared to baseline, mean LV wall stress and stroke work were not changed at any point in the MI group, but were decreased after reperfusion in the MI+Unload group (mean LV wall stress: 44658 vs 22963 dynes/cm(2); stroke work: 2823 vs 655 mmHg x mL, MI vs MI+Unload). Phosphorylation of reperfusion injury salvage kinase (RISK) pathway proteins from non-infarcted LV tissue was unchanged in the MI group, but increased in the MI+Unload group. Compared to MI alone, total infarct size was reduced in the MI+Unload group (49% vs 28%, MI vs MI+Unload). CONCLUSIONS: These data support that first unloading the LV despite delaying coronary reperfusion during an acute MI reduces myocardial injury.

Article	Year
Biliary acute pancreatitis in mice is mediated by the G-protein-coupled cell surface bile acid receptor Gpbar1. Gastroenterology\| PubMed ID: 19900448	2010
Role of aggrecanase 1 in Lyme arthritis. Arthritis and rheumatism\| PubMed ID: 17009305	2006
Cause-effect relationships between zymogen activation and other early events in secretagogue-induced acute pancreatitis. American journal of physiology. Gastrointestinal and liver physiology\| PubMed ID: 17332471	2007
Tumor progression locus-2 is a critical regulator of pancreatic and lung inflammation during acute pancreatitis. The Journal of biological chemistry\| PubMed ID: 17537724	2007
A mouse model of acute biliary pancreatitis induced by retrograde pancreatic duct infusion of Na-taurocholate. Gut\| PubMed ID: 17591621	2007
'Role reversal' for the receptor PAR1 in sepsis-induced vascular damage. Nature immunology\| PubMed ID: 17965715	2007
Protease-activated receptor-2 exerts contrasting model-specific effects on acute experimental pancreatitis. The Journal of biological chemistry\| PubMed ID: 18511423	2008
Matrix metalloproteinase 9 plays a key role in lyme arthritis but not in dissemination of Borrelia burgdorferi. Infection and immunity\| PubMed ID: 19364840	2009
Experimental acute biliary pancreatitis induced by retrograde infusion of bile acids into the mouse pancreatic duct. Nature protocols\| PubMed ID: 20134432	2010
TNF-alpha-dependent regulation of acute pancreatitis severity by Ly-6C(hi) monocytes in mice. The Journal of biological chemistry\| PubMed ID: 21343291	2011
Interdicting protease-activated receptor-2-driven inflammation with cell-penetrating pepducins. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 21536878	2011
Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice. American journal of physiology. Gastrointestinal and liver physiology\| PubMed ID: 22517774	2012
Bile acids induce pancreatic acinar cell injury and pancreatitis by activating calcineurin. The Journal of biological chemistry\| PubMed ID: 23148215	2013
Mechanically Unloading the Left Ventricle Before Coronary Reperfusion Reduces Left Ventricular Wall Stress and Myocardial Infarct Size. Circulation\| PubMed ID: 23766351	2013

George Perides

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