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892 PKD3 Is the Predominant PKD Isoform Expressed in the Exocrine Pancreas and Regulates Hormone-Induced Exocrine Enzyme Secretion
years of this long-term study, both continuous esomeprazole treatment and laparoscopic fundoplication caused a highly significant and similar improvement in microscopic esophagitis by the end of the first year that was maintained at 3 years.
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864
The activation of trypsinogen is an early event in experimental pancreatitis and trypsin can initiate further zymogen activation. In the mouse pancreas, at least five trypsinogen isoforms are expressed. Here we demonstrate that the trypsin activity generated in response to supramaximal caerulein stimulation is largely insensitive towards trypsin inhibitors. In order to study the enzymatic properties of this atypical trypsin activity we compared it to recombinantly expressed isoforms of mouse trypsin(ogen). In NMRI mice acute pancreatitis was induced by 7 hourly injections of caerulein (50 µg/h, i.p.). Control mice received i. p. saline. Trypsin activity was fluorometrically measured with BOC-QAR-MCA in homogenates and via zymography after 1D and 2D-PAGE. Affinity chromatography and gel filtration were used to separate trypsin activities. Recombinant mouse trypsinogen isoforms 4, 7, 8, 9, and 20 were expressed as untagged or His-tagged SUMO fusion proteins in Escherichia coli. Trypsinogens were subsequently isolated and purified to homogeneity by ecotin or metal chelate affinity chromatography. After supramaximal caerulein stimulation trypsin activity typically peaked at 3 and at 8 hours. This activity could be inhibited using soybean trypsin inhibitor (SBTI) or recombinant mouse SPINK3 by only about 10%. The inhibitor-insensitive activity was found in zymograms and in gel filtration in the M.W.-range of 23 kDa. Zymography of fractions from SBTI-affinity chromatography indicate trypsin 4 as the inhibitorinsensitive trypsin isoform. Inhibitor titration of recombinant trypsins indicated a strong sensitivity against SBTI or SPINK of trypsins 7, 8, 9, and 20, whereas trypsin 4 was barely inhibited. Our results show that the trypsin activity characteristically detected in experimental pancreatitis cannot be attributed to major (cationic, anionic) trypsin isoforms. The inhibitorinsensitive trypsin activity resembles a mesotrypsin-like enzyme and corresponds to the mouse trypsin 4 isoform. Considering the restricted proteolytic potential of mesotrypsin (PRSS3) the pathophysiological role of trypsinogen activation during pancreatitis needs to be reconsidered - particularly in view of its predominant, inhibitor-insensitive component.
Effect of Proton Pump Inhibitor On the Gastric Volume: Assessed By Magnetic Resonance Imaging Arash Babaei, Valmik Bhargava, Sohrab Aalam, Miriam Scadeng, Ravinder K. Mittal Background: Aim of this study was to determine the effect of proton pump inhibitor (PPI) therapy on gastric volume after a standard meal. It is hypothesized that by reducing acid secretion, PPI therapy will reduce the gastric content available for reflux into the esophagus. Methods: 9 healthy subjects (33 ± 10 yrs, 6 males) were studied at baseline, before and after a 474 ml, 950Kcal high fat liquid meal (Nepro®). Meal was homogenously mixed with 6ml of Magnevist® to help demarcate the gastric region. Immediately after the ingestion of the meal, magnetic resonance images (MRI) were acquired on a 3 Tesla GE scanner every 5 minutes for a total of 90 minutes. Subjects then took Esomeprazole (Nexium®) 40 mg twice daily for 7 days and MRI of the stomach was repeated at the end of the therapy period. Images then were analyzed using a commercial software package (Amira 4.1®). The gastric area of interest was manually outlined and total gastric volumes were calculated. All data are presented as mean ± standard deviation and compared using two-way analysis of variance with repeated measures and Bonferroni post hoc test. Intra and inter-observer variability was also examined in 3 subjects by reanalysis of data at least two weeks apart. Results: Fasting stomach volume was 85 ± 29 ml and did not differ significantly on the PPI therapy 77 ± 25 ml. Stomach volume, 15 minutes after the meal peaks to 607 ± 35 ml at baseline and 539 ± 30 ml following seven days of PPI therapy (p < 0.001). Average gastric volume remained significantly lower (56 ± 8 ml, p < 0.05) on PPI therapy throughout the 5 to 65 minutes interval after the meal. Correlation values for intra and inter observers were 0.98 and 0.96 with average regression equations of y = 0.98x +18 (n=106) and y = 1.04x -14 (n=54) showing a high degree of reproducibility in our laboratory. Conclusion: PPI therapy causes a significant reduction of gastric volume during the first hour after the meal. PPI therapy, in addition to raising stomach pH, decreases gastric volume that will likely decrease the frequency of gastro-esophageal reflux.
892 PKD3 Is the Predominant PKD Isoform Expressed in the Exocrine Pancreas and Regulates Hormone-Induced Exocrine Enzyme Secretion L. A. Chen, Jing Li, Scott R. Silva, Lindsey N. Jackson, Hiroaki Watanabe, Kirk L. Ives, Mark R. Hellmich, B. M. Evers The protein kinase D (PKD) family of serine / threonine kinases, which can be activated by gastrointestinal (GI) hormones, consists of three distinct isoforms that modulate a variety of cellular processes including exocytic trafficking and protein secretion. Previously, we demonstrated that PKD1 mediates neurotensin peptide secretion from a pancreas-derived neuroendocrine cell line, BON; PKD1 and 2 isoforms are highly expressed in this endocrine cell line with little to no PKD3 expression. PKD3, the least well-characterized isoform, appears to have a functional role distinct from that of PKD1 and PKD2. The purpose of this study was (i) to characterize the expression pattern of PKD in the pancreas, (ii) to delineate the mechanisms of activation of PKD in the pancreas, and (iii) to determine whether PKD3 has a functional role in acinar cell secretion. METHODS. Primary pancreas specimens and isolated pancreatic acini were obtained from human and mouse tissues. (i) Expression patterns of PKD1, PKD2, and PKD3 were investigated in the pancreas and isolated acini using RT-PCR, immunohistochemistry, and Western blotting. (ii) Acini isolated from human or mouse pancreas were stimulated with various GI hormones; PKD activation was determined by translocation, phosphorylation, and kinase activity assays. Pharmacologic inhibitors were used to investigate the roles of Ca2+, diacylglycerol, and upstream protein kinases involved in PKD activation. (iii) The role of PKD3 in hormone-induced amylase secretion was assessed using inhibitors of PKD and adenoviral-mediated expression of PKD3 in pancreatic acini. RESULTS. (i) In both human and mouse pancreas, PKD3 is the predominant isoform expressed in exocrine acini, whereas PKD1 and PKD2 are more specifically expressed in the islets. PKD3 partially co-localizes with zymogen granules in isolated acinar cells and is undetectable in isolated human islet cells. (ii) Upon GI hormone stimulation, PKD3 undergoes membrane translocation, trans-activating phosphorylation, and catalytic activation. PKD3 phosphorylation in exocrine acini occurs via a Ca2+-independent, diacylglycerol- and PKCdependent mechanism. (iii) Inhibition of PKD activity with Gö6976 attenuates CCK-induced amylase secretion, while overexpression of PKD3 potentiates MEK/ERK/RSK signaling and significantly enhances CCK-induced amylase secretion in pancreatic acini. CONCLUSIONS. Our findings reveal a novel distinction between exocrine and endocrine cells in the pancreas and, moreover, identify a critical role for the PKD3 isoform in mediating GI hormoneregulated secretion in the exocrine pancreas.
890 Intracellular Trypsinogen Activation Induces Pancreatic Acinar Cell Apoptosis and Necrosis While Extracellular Trypsin Activates Nf-κB Baoan Ji, Sebastian Gaiser, Craig D. Logsdon Background and Aims: Premature intracellular activation of the digestive enzyme trypsinogen is widely believed to be the initiating event in pancreatitis. However, the consequences of intracellular trypsin activation have not previously been directly tested. Methods: In the current study, a mutant trypsinogen, which can be activated intracellularly by the endogenous protease PACE, was utilized to determine for the first time the direct effects of intracellular trypsinogen activation on pancreatic acinar cell function. Protein expression was detected by Western blot. Trypsinogen localization was performed by immunofluorescent confocal microscopy. Cell morphology was monitored by both light and electronic microscopy. NFκB activity was measured by target gene expression and EMSA. Apoptosis was detected by FACS, PARP cleavage and DNA laddering. LDH release was used as a marker for Necrosis. Results: The mutant trypsinogen was expressed and activated in acinar cells. In pancreatic acinar cells, it was localized in the secretory pathway. Activation of this trypsinogen construct caused cell death. Within hours of expression, this construct induced pancreatic acinar cell apoptosis through caspase dependent (cleavage of caspase 3 and PARP) and caspase independent but trypsin dependent (Pefabloc sensitive) pathways. At later time, intracellular trypsin also led to acinar cell necrosis. Interestingly, intracellular trypsinogen activation had no significant effect in the activity of the transcription factor NF-κB, a mediator of the inflammatory response that is a central early event in pancreatitis. However, extracellular trypsin activity caused dramatic increases of NF-κB activity and Src activation. Conclusion: These results suggest that intracellular trypsinogen activation may have a protective effect by stimulating apoptosis of damaged acinar cells. Extracellular trypsin leaked from necrotic cells may account for the pathological inflammatory responses through activating NF-κB in nearby intact cells during the initiation of acute pancreatitis. These studies provide important insights into the mechanisms of initiation of pancreatitis.
893 Pro-Survival BCL-2 Proteins Regulate Ca2+ Transport in Pancreatic Acinar Cells Julia Gerasimenko, Irina V. Odinokova, Kai-Feng Sung, Olga A. Mareninova, Moses A. Lee, Lars Fischer, Stephen J. Pandol, Anna S. Gukovskaya Background and Aims: Bcl-2 proteins are key regulators of cell death. In response to death stimuli they redistribute to mitochondria, where they regulate the mitochondrial membrane potential (∆Ψm) and release into the cytosol of cytochrome c, a key pro-apoptotic factor. Bcl-2 proteins are also located in the endoplasmic reticulum (ER), and have been found to play an important role in Ca2+ homeostasis. The recently introduced small-molecule inhibitors of Bcl-xL and Bcl-2, e.g. HA14-1 and BH3I-2', became a major tool in elucidating the roles of these pro-survival Bcl-2 proteins. In the present study we measured the effects of these inhibitors on pancreatic mitochondria permeability (∆Ψm and cytochrome c release); ER Ca2+ content; Ca2+-dependent physiological (i.e. amylase secretion) and pathological (i.e. trypsinogen activation) responses of the pancreatic acinar cell; and acinar cell apoptosis and necrosis. Methods: The effects of HA14-1 and BH3I-2' (1-50 µM) on ∆Ψm and cytochrome
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AGA Abstracts
AGA Abstracts
Trypsin Activity in the Mouse Pancreas After Supramaximal Caerulein Stimulation Represents An Inhibitor-Insensitive Isoform Walter Halangk, Milada Butueva, Markus M. Lerch, Miklós Sahin-Tóth, Thomas Wartmann
891
864
The activation of trypsinogen is an early event in experimental pancreatitis and trypsin can initiate further zymogen activation. In the mouse pancreas, at least five trypsinogen isoforms are expressed. Here we demonstrate that the trypsin activity generated in response to supramaximal caerulein stimulation is largely insensitive towards trypsin inhibitors. In order to study the enzymatic properties of this atypical trypsin activity we compared it to recombinantly expressed isoforms of mouse trypsin(ogen). In NMRI mice acute pancreatitis was induced by 7 hourly injections of caerulein (50 µg/h, i.p.). Control mice received i. p. saline. Trypsin activity was fluorometrically measured with BOC-QAR-MCA in homogenates and via zymography after 1D and 2D-PAGE. Affinity chromatography and gel filtration were used to separate trypsin activities. Recombinant mouse trypsinogen isoforms 4, 7, 8, 9, and 20 were expressed as untagged or His-tagged SUMO fusion proteins in Escherichia coli. Trypsinogens were subsequently isolated and purified to homogeneity by ecotin or metal chelate affinity chromatography. After supramaximal caerulein stimulation trypsin activity typically peaked at 3 and at 8 hours. This activity could be inhibited using soybean trypsin inhibitor (SBTI) or recombinant mouse SPINK3 by only about 10%. The inhibitor-insensitive activity was found in zymograms and in gel filtration in the M.W.-range of 23 kDa. Zymography of fractions from SBTI-affinity chromatography indicate trypsin 4 as the inhibitorinsensitive trypsin isoform. Inhibitor titration of recombinant trypsins indicated a strong sensitivity against SBTI or SPINK of trypsins 7, 8, 9, and 20, whereas trypsin 4 was barely inhibited. Our results show that the trypsin activity characteristically detected in experimental pancreatitis cannot be attributed to major (cationic, anionic) trypsin isoforms. The inhibitorinsensitive trypsin activity resembles a mesotrypsin-like enzyme and corresponds to the mouse trypsin 4 isoform. Considering the restricted proteolytic potential of mesotrypsin (PRSS3) the pathophysiological role of trypsinogen activation during pancreatitis needs to be reconsidered - particularly in view of its predominant, inhibitor-insensitive component.
Effect of Proton Pump Inhibitor On the Gastric Volume: Assessed By Magnetic Resonance Imaging Arash Babaei, Valmik Bhargava, Sohrab Aalam, Miriam Scadeng, Ravinder K. Mittal Background: Aim of this study was to determine the effect of proton pump inhibitor (PPI) therapy on gastric volume after a standard meal. It is hypothesized that by reducing acid secretion, PPI therapy will reduce the gastric content available for reflux into the esophagus. Methods: 9 healthy subjects (33 ± 10 yrs, 6 males) were studied at baseline, before and after a 474 ml, 950Kcal high fat liquid meal (Nepro®). Meal was homogenously mixed with 6ml of Magnevist® to help demarcate the gastric region. Immediately after the ingestion of the meal, magnetic resonance images (MRI) were acquired on a 3 Tesla GE scanner every 5 minutes for a total of 90 minutes. Subjects then took Esomeprazole (Nexium®) 40 mg twice daily for 7 days and MRI of the stomach was repeated at the end of the therapy period. Images then were analyzed using a commercial software package (Amira 4.1®). The gastric area of interest was manually outlined and total gastric volumes were calculated. All data are presented as mean ± standard deviation and compared using two-way analysis of variance with repeated measures and Bonferroni post hoc test. Intra and inter-observer variability was also examined in 3 subjects by reanalysis of data at least two weeks apart. Results: Fasting stomach volume was 85 ± 29 ml and did not differ significantly on the PPI therapy 77 ± 25 ml. Stomach volume, 15 minutes after the meal peaks to 607 ± 35 ml at baseline and 539 ± 30 ml following seven days of PPI therapy (p < 0.001). Average gastric volume remained significantly lower (56 ± 8 ml, p < 0.05) on PPI therapy throughout the 5 to 65 minutes interval after the meal. Correlation values for intra and inter observers were 0.98 and 0.96 with average regression equations of y = 0.98x +18 (n=106) and y = 1.04x -14 (n=54) showing a high degree of reproducibility in our laboratory. Conclusion: PPI therapy causes a significant reduction of gastric volume during the first hour after the meal. PPI therapy, in addition to raising stomach pH, decreases gastric volume that will likely decrease the frequency of gastro-esophageal reflux.
892 PKD3 Is the Predominant PKD Isoform Expressed in the Exocrine Pancreas and Regulates Hormone-Induced Exocrine Enzyme Secretion L. A. Chen, Jing Li, Scott R. Silva, Lindsey N. Jackson, Hiroaki Watanabe, Kirk L. Ives, Mark R. Hellmich, B. M. Evers The protein kinase D (PKD) family of serine / threonine kinases, which can be activated by gastrointestinal (GI) hormones, consists of three distinct isoforms that modulate a variety of cellular processes including exocytic trafficking and protein secretion. Previously, we demonstrated that PKD1 mediates neurotensin peptide secretion from a pancreas-derived neuroendocrine cell line, BON; PKD1 and 2 isoforms are highly expressed in this endocrine cell line with little to no PKD3 expression. PKD3, the least well-characterized isoform, appears to have a functional role distinct from that of PKD1 and PKD2. The purpose of this study was (i) to characterize the expression pattern of PKD in the pancreas, (ii) to delineate the mechanisms of activation of PKD in the pancreas, and (iii) to determine whether PKD3 has a functional role in acinar cell secretion. METHODS. Primary pancreas specimens and isolated pancreatic acini were obtained from human and mouse tissues. (i) Expression patterns of PKD1, PKD2, and PKD3 were investigated in the pancreas and isolated acini using RT-PCR, immunohistochemistry, and Western blotting. (ii) Acini isolated from human or mouse pancreas were stimulated with various GI hormones; PKD activation was determined by translocation, phosphorylation, and kinase activity assays. Pharmacologic inhibitors were used to investigate the roles of Ca2+, diacylglycerol, and upstream protein kinases involved in PKD activation. (iii) The role of PKD3 in hormone-induced amylase secretion was assessed using inhibitors of PKD and adenoviral-mediated expression of PKD3 in pancreatic acini. RESULTS. (i) In both human and mouse pancreas, PKD3 is the predominant isoform expressed in exocrine acini, whereas PKD1 and PKD2 are more specifically expressed in the islets. PKD3 partially co-localizes with zymogen granules in isolated acinar cells and is undetectable in isolated human islet cells. (ii) Upon GI hormone stimulation, PKD3 undergoes membrane translocation, trans-activating phosphorylation, and catalytic activation. PKD3 phosphorylation in exocrine acini occurs via a Ca2+-independent, diacylglycerol- and PKCdependent mechanism. (iii) Inhibition of PKD activity with Gö6976 attenuates CCK-induced amylase secretion, while overexpression of PKD3 potentiates MEK/ERK/RSK signaling and significantly enhances CCK-induced amylase secretion in pancreatic acini. CONCLUSIONS. Our findings reveal a novel distinction between exocrine and endocrine cells in the pancreas and, moreover, identify a critical role for the PKD3 isoform in mediating GI hormoneregulated secretion in the exocrine pancreas.
890 Intracellular Trypsinogen Activation Induces Pancreatic Acinar Cell Apoptosis and Necrosis While Extracellular Trypsin Activates Nf-κB Baoan Ji, Sebastian Gaiser, Craig D. Logsdon Background and Aims: Premature intracellular activation of the digestive enzyme trypsinogen is widely believed to be the initiating event in pancreatitis. However, the consequences of intracellular trypsin activation have not previously been directly tested. Methods: In the current study, a mutant trypsinogen, which can be activated intracellularly by the endogenous protease PACE, was utilized to determine for the first time the direct effects of intracellular trypsinogen activation on pancreatic acinar cell function. Protein expression was detected by Western blot. Trypsinogen localization was performed by immunofluorescent confocal microscopy. Cell morphology was monitored by both light and electronic microscopy. NFκB activity was measured by target gene expression and EMSA. Apoptosis was detected by FACS, PARP cleavage and DNA laddering. LDH release was used as a marker for Necrosis. Results: The mutant trypsinogen was expressed and activated in acinar cells. In pancreatic acinar cells, it was localized in the secretory pathway. Activation of this trypsinogen construct caused cell death. Within hours of expression, this construct induced pancreatic acinar cell apoptosis through caspase dependent (cleavage of caspase 3 and PARP) and caspase independent but trypsin dependent (Pefabloc sensitive) pathways. At later time, intracellular trypsin also led to acinar cell necrosis. Interestingly, intracellular trypsinogen activation had no significant effect in the activity of the transcription factor NF-κB, a mediator of the inflammatory response that is a central early event in pancreatitis. However, extracellular trypsin activity caused dramatic increases of NF-κB activity and Src activation. Conclusion: These results suggest that intracellular trypsinogen activation may have a protective effect by stimulating apoptosis of damaged acinar cells. Extracellular trypsin leaked from necrotic cells may account for the pathological inflammatory responses through activating NF-κB in nearby intact cells during the initiation of acute pancreatitis. These studies provide important insights into the mechanisms of initiation of pancreatitis.
893 Pro-Survival BCL-2 Proteins Regulate Ca2+ Transport in Pancreatic Acinar Cells Julia Gerasimenko, Irina V. Odinokova, Kai-Feng Sung, Olga A. Mareninova, Moses A. Lee, Lars Fischer, Stephen J. Pandol, Anna S. Gukovskaya Background and Aims: Bcl-2 proteins are key regulators of cell death. In response to death stimuli they redistribute to mitochondria, where they regulate the mitochondrial membrane potential (∆Ψm) and release into the cytosol of cytochrome c, a key pro-apoptotic factor. Bcl-2 proteins are also located in the endoplasmic reticulum (ER), and have been found to play an important role in Ca2+ homeostasis. The recently introduced small-molecule inhibitors of Bcl-xL and Bcl-2, e.g. HA14-1 and BH3I-2', became a major tool in elucidating the roles of these pro-survival Bcl-2 proteins. In the present study we measured the effects of these inhibitors on pancreatic mitochondria permeability (∆Ψm and cytochrome c release); ER Ca2+ content; Ca2+-dependent physiological (i.e. amylase secretion) and pathological (i.e. trypsinogen activation) responses of the pancreatic acinar cell; and acinar cell apoptosis and necrosis. Methods: The effects of HA14-1 and BH3I-2' (1-50 µM) on ∆Ψm and cytochrome
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AGA Abstracts
AGA Abstracts
Trypsin Activity in the Mouse Pancreas After Supramaximal Caerulein Stimulation Represents An Inhibitor-Insensitive Isoform Walter Halangk, Milada Butueva, Markus M. Lerch, Miklós Sahin-Tóth, Thomas Wartmann