Sa1862 A Two-Phase Strategy for Long-Term In Vitro Maintenance of Functionally Competent Human...
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AGA Abstracts staining and western blot respectively. Acute pancreatitis was induced by ten injections of cerulein (50 μg/kg, i.p.) at hourly intervals. Mice were monitored for up to 7 days after induction of pancreatitis. Pancreatic injury was determined by measuring serum levels of amylase and examining pathologic changes of pancreas. For quantifying severity of tissue inflammation, we measured mRNA levels of myeloperoxidase (MPO, a marker of neutrophil infiltration) with real-time RT-PCR. Results: We found that MFG-E8 was constitutively expressed in murine pancreas. The protein was revealed to localize in pancreatic ductal epithelial cells. As expected, administration of cerulein induced acute pancreatitis ranging from the acute phase (i.e. 1 h after the final injection of cerulein) to recovery phase (i.e. up to 7 days after cerulein treatment) in WT mice. Pancreatic MFG-E8 was markedly increased 24 hours after induction of pancreatitis. The protein expression was maintained at high levels during the recovery phase. Using MFG-E8 KO mice, we found that MFG-E8 deficiency did not affect the severity of acute phase of cerulein-induced pancreatitis. However, pancreatic recovery following cerulein-induced pancreatitis was markedly delayed in the KO mice comparing to WT controls. Conclusions: Together, the data suggested that MFG- E8 is a critical protein that protects against progression of acute pancreatitis. MFG-E8 deficiency impairs pancreatic recovery and it is needed in pancreatic recovery from pancreati- tis. This novel finding may benefits the development of a potential novel molecular agent for pancreatitis therapy. Sa1859 Decreased P85α Expression Contributes to the Loss of Sensitivity to ER Stress in Aged Pancreatic Acinar Cells Jing Li, Jun Song, Katherine E. Campbell, Ji Tae Kim, Daiki Okamura, Hiroshi Saito, B. Mark Evers We have recently reported that the expression of the PI3K regulatory subunit p85 α is significantly reduced in pancreatic acinar cells from aged mice and humans. However, the precise mechanisms for this specific and age-dependent decrease of p85 α expression and the downstream functional alterations of this decreased expression are not known. Endoplasmic reticulum (ER) stress induces unfolded protein response (UPR), an adaptive signaling pathway which maintains ER homeostasis and is crucial for the survival and function of all cells. UPR is mediated initially by three molecules: PERK, ATF6, and IRE-1. During aging, UPR signaling and stress resistance clearly declines, and this loss accelerates the aging process. p85α and p85β have been reported to be required for UPR following induction of ER stress. The purpose of the present study was to investigate the role of p85 α in the regulation of ER stress in aged pancreatic acinar cells. METHODS. i) Acinar cells were isolated from young and aged mice; mouse 266-6 and rat AR42J acinar cell lines were also used; ER stress was induced by thapsigargin (Tg) treatment. ii) To monitor translocation of XBP-1, a downstream effector of IRE-1 α, nuclear and cytoplasmic fractionation was performed. iii) To inhibit endogenous p85α expression, acinar cell lines were transfected with either mouse or rat p85α siRNA. RESULTS. i) In acinar cells isolated from young mice and treated with different doses of Tg, the UPR response was easily detected by Western blot as noted by the increased phosphorylation of PERK, IRE-1 α and expression of BIP, a chaperon protein. In contrast, aged acinar cells lost the UPR response in the presence of Tg, as noted by decreased PERK, IRE-1α and BIP. ii) Inhibition of PI3K in AR42J cells by wortmannin, a general PI3K inhibitor, increased Tg-mediated activation of PERK/eIF2 α signaling, whereas BIP induction was blocked. Consistently, the apoptotic markers, caspase-3 and -12, were increased in cells treated with a combination of Tg and wortmannin. Transfection of p85 α siRNA in 266-6 or AR42J cells decreased translocation of XBP-1s, a downstream effector of IRE-1α, into the nucleus. Moreover, transfection of p85 α siRNA further decreased 266-6 and AR42J cell numbers after Tg treatment, further supporting the notion of p85 α as a protective factor in response to ER stress. CONCLUSIONS. These results demonstrate that aged acinar cells lost sensitivity to ER stress, suggesting the possible contributions of the decreased p85α expression associated with aging. Sa1860 SRC-Dependent Role of CCK in the Pancreatic Acinar Cell Signaling and Chemokine Release Bernardo Nuche-Berenguer, Taichi Nakamura, Paola Moreno, Robert T. Jensen Introduction: In pancreatic acinar cells, the Src Family of kinases (SFK) play a central role in secretion, endocytosis, growth, cytoskeletal integrity and apoptosis. However, the signaling cascades of Src mediating these effects are not fully understood. Aim: To investigate the signaling cascades of SFK in rat pancreatic acinar cells and also its possible role in chemokine release using an in vitro model of pancreatitis. Methods: Dispersed rat pancreatic acini were prepared. Src modulation of signaling cascades was assessed using three different approaches: #1: The general Src antagonist PP2, with the inactive analogue PP3 as a control; #2: Adenovirus-induced expression of an inactive dominant-negative version of CSK (Dn-CSK- Advirus) which results in Src activation and #3: Adenovirus-induced expression of Wild- Type CSK (Wt-CSK-Advirus), which inhibits Src. Phospho-specific antibodies were used to investigate activation with Western blotting. To assess the importance of Src-mediated chemokine release, an in vitro model of pancreatitis was used. Cells were incubated with 100nM CCK for 2 hours and the release of MCP-1 and MIP-1 α were measured in the cell supernatant by ELISA. Results: CCK-8 (0.3, 100nM) activated Src, Pyk2, Cas, FAK, paxillin, ERK1/2, JNK, SHC, PKD and phosphorylated MARCKS but not P38 and GSK. The CCK activation of Src, Pyk2, Cas, FAK, paxillin and Shc was inhibited by PP2. In contrast, the basal activation of p44/42, MARCKS and JNK was inhibited by PP2 but not its CCK- induced activation. Neither basal nor CCK-mediated PKD activation was affected by PP2. Pre-incubation with Dn-CSK-Advirus, which activates Src, caused the reverse effects in each cellular cascade. Furthermore, the pre-incubation with WT-CSK-Ad virus, which also inhibits Src, confirmed the results obtained with PP2. CCK increased the release of the chemokine MCP-1 (97.1±20.8% over control, p ,0.01). With PP2 this CCK-increase was significantly reduced by 60% (40.4.±3.4 % over PP2-control, p ,0.05 vs CCK-control). PP3 was inactive. A similar result was observed with the chemokine MIP-1- α where the CCK-induced release (49.6.±7.3 % over control, p ,0.01) was completely inhibited by PP2 (4.5.±8.9 % over PP2 control, p,0.01 vs CCK-control), but not by PP3. Conclusions: In rat pancreatic acini, S-322 AGA Abstracts Src inhibition selectively affects CCK-mediated activation of a number of important key signaling cascades including Pyk2, Cas, FAK, paxillin and Shc whereas it does not affect others (PKC, MAPKs, JNK). These results show SFK has a central role in activating a number of important cellular signaling cascades, which are important in mediating numerous cellular responses (growth, apoptosis, cytoskeletal integrity). In addition to Src reported importance in secretion and growth, it is also important in mediating chemokine release in an in vitro model of pancreatitis. Sa1861 Pancreatitis-Associated Alterations of Pancreatic Acinar Cells in Response to Tobacco Compared With Alcohol Maria Luaces-Regueira, Margarita Castineira, Enrique Dominguez-Munoz Alterations in amylase secretion, reactive oxygen species (ROS) production and acinar cell death are involved in the pathogenesis of chronic pancreatitis. The effect of the main toxic risk factors alcohol and, even more, tobacco in these events is poorly understood, despite being well accepted risk factors for chronic pancreatitis (CP). Aim: To evaluate the role of tobacco compared with alcohol in the intracellular pathophysiologic events associated with pancreatitis. Methods: Acinar cells were isolated from pancreas of Swiss mice by enzymatic and mechanic degradation, and stimulated with increasing concentrations of alcohol (from 10 to 100mM), tobacco (from 0.001 to 0.5mg/ml), and CCK as positive control. ROS production was evaluated by fluorescence using DCFDA substrate. Amylase secretion was evaluated by using p-nitrophenyl-maltohexaoside. Cytotoxicity was measured by ilactate dehydrogenase production and apoptosis by caspase 3 activation by western blot. Statistic analysis was performed by ANOVA test. Results: Tobacco but not alcohol induced a supra- physiological pancreatic secretion of amylase (21%, 0.4mg/ml). Tobacco, but not alcohol, increased significantly ROS production (p ,0.05) in a dose-dependent manner. Moreover, tobacco (0.3 mg/ml and 0.4 mg/ml), but not alcohol, produced a significant cytotoxicity (p,0.05); and dosis of 0.01 and 0.1 mg/ml induced caspase 3 activation 2.5 fold more than negative control (p,0.05). Conclusions: In pancreatic acinar cell culture, tobacco, but not alcohol, stimulates pancreatic secretion of amylase, induces ROS production, cytotoxicity and apoptosis. These results support the role of smoking in the pathogenesis of CP. Sa1862 A Two-Phase Strategy for Long-Term In Vitro Maintenance of Functionally Competent Human Pancreatic Acinar Cells Merja Blauer, Juhani Sand, Isto Nordback, Johanna Laukkarinen Objectives: In vitro culture models allowing long-term experimentation on human pancreatic acinar cells are currently lacking. We recently reported a long-term culture strategy for mouse pancreatic acinar cells. The aim of this study was to develop new approaches for long-term maintenance of functionally competent human pancreatic acinar cells in vitro. Methods: Tissue samples were obtained from patients undergoing pancreatic surgery. The samples were digested with collagenase and the resulting acinar clusters were collected, embedded in soft Matrigel and placed in tissue culture inserts for primary culture. After three days the clusters were dissociated and the cells were passaged onto standard cell culture plastic. The responsiveness of the cells to stimulation with 0.1 nM and 10 nM caerulein and 0.1 mM carbachol was determined after four days. Results: Human pancreatic acinar cells could be maintained for a minimum of seven days in vitro. Acinar clusters showed excellent morphology throughout the three-day period in primary culture and little decline in their basal amylase secretion was observed. In secondary culture acinar cells arranged into monolayers. Both carbachol and caerulein stimulated amylase secretion in secondary cultures. Conlusions: The secretory phenotype of human pancreatic acinar cells can be maintained functionally competent for a minimum of seven days in the present culture conditions. This novel two-phase culture system permits long-term studies on acinar clusters and isolated acinar cells in vitro. Sa1863 Cd38/ADP Ribosyl Cyclase Mediates Bile Acid-Induced Ca2+ Signals and Acinar Cell Injury Kamaldeen A. Muili, Abrahim I. Orabi, Shunqian Jin, Tanveer A. Javed, John F. Eisses, Tianming Le, Sohail Z. Husain Aberrant Ca2+ signals within pancreatic acinar cells are an early and critical feature in acute pancreatitis, yet it is unclear how these signals are generated. An important mediator of the aberrant Ca2+ signals due to bile acid exposure is the intracellular Ca2+ channel the ryanodine receptor (RyR). A putative activator of the RyR is a small nucleotide messenger cADP ribose, which is generated by an ectoenzyme ADP ribosyl cyclase called CD38. In this study, we examined the role of CD38 in acinar cell Ca2+ signals and injury using both pharmacologic inhibitors of CD38 and cADP ribose as well as mice deficient in CD38 (CD38-/-). Freshly isolated mouse acinar cells were imaged using live time lapse confocal microscopy while being perifused with the bile acid taurolithocholic acid 3-sulfate (TLCS; 500 uM). In addition, cell injury was assessed by LDH leakage and propidium iodide uptake. Pretreatment with either the CD38 inhibitor nicotinamide (20 mM) or the specific cADP ribose inhibitor 8- Br-cADPR (30 uM) abrogated TLCS-induced Ca2+ signals and cell injury (p ,0.05). In CD38-/- acinar cells compared to wild type cells, the shape of the TLCS-induced Ca2+ signal shifted from that of a single spike to oscillations, which is a more physiologic response. In addition, cell injury was reduced by 95% in CD38-/- acinar cells following TLCS stimula- tion (p,0.05). In summary, these data demonstrate that CD38 regulates TLCS-induced acinar cell Ca2+ signals and cell injury, and they suggest that CD38 plays a critical role in aberrant Ca2+ in pancreatitis.
Transcript of Sa1862 A Two-Phase Strategy for Long-Term In Vitro Maintenance of Functionally Competent Human...
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sstaining and western blot respectively. Acute pancreatitis was induced by ten injections ofcerulein (50 μg/kg, i.p.) at hourly intervals. Mice were monitored for up to 7 days afterinduction of pancreatitis. Pancreatic injury was determined by measuring serum levels ofamylase and examining pathologic changes of pancreas. For quantifying severity of tissueinflammation, we measured mRNA levels of myeloperoxidase (MPO, a marker of neutrophilinfiltration) with real-time RT-PCR. Results: We found that MFG-E8 was constitutivelyexpressed in murine pancreas. The protein was revealed to localize in pancreatic ductalepithelial cells. As expected, administration of cerulein induced acute pancreatitis rangingfrom the acute phase (i.e. 1 h after the final injection of cerulein) to recovery phase (i.e.up to 7 days after cerulein treatment) in WT mice. Pancreatic MFG-E8 was markedlyincreased 24 hours after induction of pancreatitis. The protein expression was maintainedat high levels during the recovery phase. Using MFG-E8 KO mice, we found that MFG-E8deficiency did not affect the severity of acute phase of cerulein-induced pancreatitis. However,pancreatic recovery following cerulein-induced pancreatitis was markedly delayed in theKO mice comparing to WT controls. Conclusions: Together, the data suggested that MFG-E8 is a critical protein that protects against progression of acute pancreatitis. MFG-E8deficiency impairs pancreatic recovery and it is needed in pancreatic recovery from pancreati-tis. This novel finding may benefits the development of a potential novel molecular agentfor pancreatitis therapy.
Sa1859
Decreased P85α Expression Contributes to the Loss of Sensitivity to ER Stressin Aged Pancreatic Acinar CellsJing Li, Jun Song, Katherine E. Campbell, Ji Tae Kim, Daiki Okamura, Hiroshi Saito, B.Mark Evers
We have recently reported that the expression of the PI3K regulatory subunit p85 α issignificantly reduced in pancreatic acinar cells from aged mice and humans. However, theprecise mechanisms for this specific and age-dependent decrease of p85α expression and thedownstream functional alterations of this decreased expression are not known. Endoplasmicreticulum (ER) stress induces unfolded protein response (UPR), an adaptive signaling pathwaywhich maintains ER homeostasis and is crucial for the survival and function of all cells.UPR is mediated initially by three molecules: PERK, ATF6, and IRE-1. During aging, UPRsignaling and stress resistance clearly declines, and this loss accelerates the aging process.p85α and p85β have been reported to be required for UPR following induction of ER stress.The purpose of the present study was to investigate the role of p85 α in the regulation ofER stress in aged pancreatic acinar cells. METHODS. i) Acinar cells were isolated fromyoung and aged mice; mouse 266-6 and rat AR42J acinar cell lines were also used; ERstress was induced by thapsigargin (Tg) treatment. ii) To monitor translocation of XBP-1,a downstream effector of IRE-1α, nuclear and cytoplasmic fractionation was performed. iii)To inhibit endogenous p85α expression, acinar cell lines were transfected with either mouseor rat p85α siRNA. RESULTS. i) In acinar cells isolated from young mice and treated withdifferent doses of Tg, the UPR response was easily detected by Western blot as noted bythe increased phosphorylation of PERK, IRE-1α and expression of BIP, a chaperon protein.In contrast, aged acinar cells lost the UPR response in the presence of Tg, as noted bydecreased PERK, IRE-1α and BIP. ii) Inhibition of PI3K in AR42J cells by wortmannin, ageneral PI3K inhibitor, increased Tg-mediated activation of PERK/eIF2α signaling, whereasBIP induction was blocked. Consistently, the apoptotic markers, caspase-3 and -12, wereincreased in cells treated with a combination of Tg and wortmannin. Transfection of p85 αsiRNA in 266-6 or AR42J cells decreased translocation of XBP-1s, a downstream effector ofIRE-1α, into the nucleus. Moreover, transfection of p85 α siRNA further decreased 266-6and AR42J cell numbers after Tg treatment, further supporting the notion of p85 α as aprotective factor in response to ER stress. CONCLUSIONS. These results demonstrate thataged acinar cells lost sensitivity to ER stress, suggesting the possible contributions of thedecreased p85α expression associated with aging.
Sa1860
SRC-Dependent Role of CCK in the Pancreatic Acinar Cell Signaling andChemokine ReleaseBernardo Nuche-Berenguer, Taichi Nakamura, Paola Moreno, Robert T. Jensen
Introduction: In pancreatic acinar cells, the Src Family of kinases (SFK) play a central rolein secretion, endocytosis, growth, cytoskeletal integrity and apoptosis. However, the signalingcascades of Src mediating these effects are not fully understood. Aim: To investigate thesignaling cascades of SFK in rat pancreatic acinar cells and also its possible role in chemokinerelease using an in vitro model of pancreatitis. Methods: Dispersed rat pancreatic acini wereprepared. Src modulation of signaling cascades was assessed using three different approaches:#1: The general Src antagonist PP2, with the inactive analogue PP3 as a control; #2:Adenovirus-induced expression of an inactive dominant-negative version of CSK (Dn-CSK-Advirus) which results in Src activation and #3: Adenovirus-induced expression of Wild-Type CSK (Wt-CSK-Advirus), which inhibits Src. Phospho-specific antibodies were used toinvestigate activation with Western blotting. To assess the importance of Src-mediatedchemokine release, an in vitro model of pancreatitis was used. Cells were incubated with100nM CCK for 2 hours and the release of MCP-1 and MIP-1 α were measured in the cellsupernatant by ELISA. Results: CCK-8 (0.3, 100nM) activated Src, Pyk2, Cas, FAK, paxillin,ERK1/2, JNK, SHC, PKD and phosphorylated MARCKS but not P38 and GSK. The CCKactivation of Src, Pyk2, Cas, FAK, paxillin and Shc was inhibited by PP2. In contrast, thebasal activation of p44/42, MARCKS and JNK was inhibited by PP2 but not its CCK-induced activation. Neither basal nor CCK-mediated PKD activation was affected by PP2.Pre-incubation with Dn-CSK-Advirus, which activates Src, caused the reverse effects in eachcellular cascade. Furthermore, the pre-incubation withWT-CSK-Ad virus, which also inhibitsSrc, confirmed the results obtained with PP2. CCK increased the release of the chemokineMCP-1 (97.1±20.8% over control, p,0.01). With PP2 this CCK-increase was significantlyreduced by 60% (40.4.±3.4 % over PP2-control, p,0.05 vs CCK-control). PP3 was inactive.A similar result was observed with the chemokine MIP-1-α where the CCK-induced release(49.6.±7.3 % over control, p,0.01) was completely inhibited by PP2 (4.5.±8.9 % over PP2control, p,0.01 vs CCK-control), but not by PP3. Conclusions: In rat pancreatic acini,
S-322AGA Abstracts
Src inhibition selectively affects CCK-mediated activation of a number of important keysignaling cascades including Pyk2, Cas, FAK, paxillin and Shc whereas it does not affectothers (PKC, MAPKs, JNK). These results show SFK has a central role in activating a numberof important cellular signaling cascades, which are important in mediating numerous cellularresponses (growth, apoptosis, cytoskeletal integrity). In addition to Src reported importancein secretion and growth, it is also important in mediating chemokine release in an in vitromodel of pancreatitis.
Sa1861
Pancreatitis-Associated Alterations of Pancreatic Acinar Cells in Response toTobacco Compared With AlcoholMaria Luaces-Regueira, Margarita Castineira, Enrique Dominguez-Munoz
Alterations in amylase secretion, reactive oxygen species (ROS) production and acinar celldeath are involved in the pathogenesis of chronic pancreatitis. The effect of the main toxicrisk factors alcohol and, even more, tobacco in these events is poorly understood, despitebeing well accepted risk factors for chronic pancreatitis (CP). Aim: To evaluate the role oftobacco compared with alcohol in the intracellular pathophysiologic events associated withpancreatitis. Methods: Acinar cells were isolated from pancreas of Swiss mice by enzymaticand mechanic degradation, and stimulated with increasing concentrations of alcohol (from10 to 100mM), tobacco (from 0.001 to 0.5mg/ml), and CCK as positive control. ROSproduction was evaluated by fluorescence using DCFDA substrate. Amylase secretion wasevaluated by using p-nitrophenyl-maltohexaoside. Cytotoxicity was measured by ilactatedehydrogenase production and apoptosis by caspase 3 activation by western blot. Statisticanalysis was performed by ANOVA test. Results: Tobacco but not alcohol induced a supra-physiological pancreatic secretion of amylase (21%, 0.4mg/ml). Tobacco, but not alcohol,increased significantly ROS production (p,0.05) in a dose-dependent manner. Moreover,tobacco (0.3 mg/ml and 0.4 mg/ml), but not alcohol, produced a significant cytotoxicity(p,0.05); and dosis of 0.01 and 0.1 mg/ml induced caspase 3 activation 2.5 fold morethan negative control (p,0.05). Conclusions: In pancreatic acinar cell culture, tobacco, butnot alcohol, stimulates pancreatic secretion of amylase, induces ROS production, cytotoxicityand apoptosis. These results support the role of smoking in the pathogenesis of CP.
Sa1862
A Two-Phase Strategy for Long-Term In Vitro Maintenance of FunctionallyCompetent Human Pancreatic Acinar CellsMerja Blauer, Juhani Sand, Isto Nordback, Johanna Laukkarinen
Objectives: In vitro culture models allowing long-term experimentation on human pancreaticacinar cells are currently lacking. We recently reported a long-term culture strategy formouse pancreatic acinar cells. The aim of this study was to develop new approaches forlong-term maintenance of functionally competent human pancreatic acinar cells in vitro.Methods: Tissue samples were obtained from patients undergoing pancreatic surgery. Thesamples were digested with collagenase and the resulting acinar clusters were collected,embedded in soft Matrigel and placed in tissue culture inserts for primary culture. Afterthree days the clusters were dissociated and the cells were passaged onto standard cellculture plastic. The responsiveness of the cells to stimulation with 0.1 nM and 10 nMcaerulein and 0.1 mM carbachol was determined after four days. Results: Human pancreaticacinar cells could be maintained for a minimum of seven days in vitro. Acinar clustersshowed excellent morphology throughout the three-day period in primary culture and littledecline in their basal amylase secretion was observed. In secondary culture acinar cellsarranged into monolayers. Both carbachol and caerulein stimulated amylase secretion insecondary cultures. Conlusions: The secretory phenotype of human pancreatic acinar cellscan be maintained functionally competent for a minimum of seven days in the presentculture conditions. This novel two-phase culture system permits long-term studies on acinarclusters and isolated acinar cells in vitro.
Sa1863
Cd38/ADP Ribosyl Cyclase Mediates Bile Acid-Induced Ca2+ Signals andAcinar Cell InjuryKamaldeen A. Muili, Abrahim I. Orabi, Shunqian Jin, Tanveer A. Javed, John F. Eisses,Tianming Le, Sohail Z. Husain
Aberrant Ca2+ signals within pancreatic acinar cells are an early and critical feature in acutepancreatitis, yet it is unclear how these signals are generated. An important mediator of theaberrant Ca2+ signals due to bile acid exposure is the intracellular Ca2+ channel the ryanodinereceptor (RyR). A putative activator of the RyR is a small nucleotide messenger cADP ribose,which is generated by an ectoenzyme ADP ribosyl cyclase called CD38. In this study, weexamined the role of CD38 in acinar cell Ca2+ signals and injury using both pharmacologicinhibitors of CD38 and cADP ribose as well as mice deficient in CD38 (CD38-/-). Freshlyisolated mouse acinar cells were imaged using live time lapse confocal microscopy whilebeing perifused with the bile acid taurolithocholic acid 3-sulfate (TLCS; 500 uM). In addition,cell injury was assessed by LDH leakage and propidium iodide uptake. Pretreatment witheither the CD38 inhibitor nicotinamide (20 mM) or the specific cADP ribose inhibitor 8-Br-cADPR (30 uM) abrogated TLCS-induced Ca2+ signals and cell injury (p ,0.05). InCD38-/- acinar cells compared to wild type cells, the shape of the TLCS-induced Ca2+signal shifted from that of a single spike to oscillations, which is a more physiologic response.In addition, cell injury was reduced by 95% in CD38-/- acinar cells following TLCS stimula-tion (p,0.05). In summary, these data demonstrate that CD38 regulates TLCS-inducedacinar cell Ca2+ signals and cell injury, and they suggest that CD38 plays a critical role inaberrant Ca2+ in pancreatitis.
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