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Caerulein modulates the ghrelin system in the pancreatic acini
Abstracts / Pancreatology 12 (2012) 502–597
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P26. Intracellular calcium levels and enzyme secretion in response to tobacco vs ethanol in pancreatic acinar cells M. Luaces-Regueira 1, M. Castiñeira-Alvariño 1, M. Castro-Manzanares 2, A. Carrascal-Miniño 2, M. Campos-Toimil 2, J.E. Domínguez-Muñoz 1. 1 Foundation for Research in Digestive Diseases, Department of Gastroenterology, University Hospital of Santiago de Compostela, Spain 2 Farmacology Department, University of Santiago de Compostela, Spain
Alterations in intracellular calcium levels and enzyme secretion in acinar cells are involved in the pathophysiology of chronic pancreatitis (CP). It has been previously shown that alcohol induces an increase of intracellular calcium, but its effect in enzyme secretion is controversial. The effect of tobacco in this context is unknown. Hypothesis: Tobacco, similarly to alcohol, promotes the development of CP by altering intracellular calcium levels and enzyme secretion in acinar cells. Aim: To evaluate the effect of tobacco compared with alcohol in intracellular calcium levels and amylase secretion in pancreatic acinar cells. Methods: Experimental in-vitro study on primary acinar cell culture. Acinar cells were isolated by enzymatic and mechanic degradation from Swiss mice pancreas. Cells were stimulated by increasing concentrations of alcohol (10-100 mM) and tobacco (0.1-0.5 mg/ml), and with CCK as positive control. Intracellular calcium levels were measured by fluorescence and amylase secretion by using p-nitrophenyl-maltohexaoside as substrate. Data were analysed by ANOVA. Results: Tobacco induced a dose-dependent increase of intracellular calcium levels from 11.384.89% (0.1 mg/ml) to 56.2613.14% (0.5 mg/ml). A similar effect was observed with alcohol [14.402.06% (10 mM) to 59.82.57% (75mM)]. This was associated with an amylase hypersecretion with tobacco (up to 21% at 0.4 mg/ml), but not with alcohol. Conclusions: Both tobacco and alcohol induce a similar increase of intracellular calcium levels in pancreatic acinar cell, but this is associated with an increased amylase secretion only with tobacco. Alcohol and tobacco have a different effect in pancreatic acinar cells and should play a different role in the pathophysiology of CP.
P27. Caerulein modulates the ghrelin system in the pancreatic acini J. Bonior 1, J. Jaworek 1, M. Kot 1, S.J. Konturek 2. 1
Department of Medical Physiology, Faculty of Health Sciences, School of Medicine Jagiellonian University, Krakow, Poland 2 Chair of Physiology Medical Faculty, School of Medicine Jagiellonian University, Krakow, Poland Introduction: Ghrelin, an endogenous ligand for the growth hormone (GH) secretagogue receptor (GHS-R) was originally isolated from the stomach and identified in the pancreas. Ghrelin protects the pancreas from the damage caused by caerulein-induced pancreatitis, but the implication of GHS-R and its endogenous ligand in the pancreatic protection is unclear. Aim: To determine the effect of ghrelin and caerulein on mRNA and protein levels of GHS-R1a subtype and of acylated ghrelin in isolated pancreatic acini. Material and methods: Wistar rats were injected with ghrelin (12,5; 25,0 or 50,0 mg/kg i.p.) or with physiological saline. 48 h later pancreatic acini were isolated and subjected to caerulein stimulation (10–12, 10–10 or 10–8 M) for: 0 h, 20, 1, 3 or 5h at 37 C. The most effective time of incubation was 3 h. High doses of ghrelin and caerulein were selected for further experiments. RT-PCR and Western blot methods were used to determine mRNA and protein levels. Results: GHS-R1a and acylated ghrelin were identified in the pancreatic acini isolated from control rats. Pretreatment of the rats with ghrelin resulted in the significant and dose-dependent upregulation of both
investigated parameters. On the contrary, application of caerulein to the acini significantly and dose-dependently downregulated GHS-R1a, but failed to affect the signal for ghrelin. Pretreatment of the rats with ghrelin prevented from caerulein-induced downregulation of GHS-R1a. Conclusions: Caerulein is able to modify the GHS-R1a subtype in the pancreatic acini. This effect could be prevented by pretreatment with ghrelin and perhaps might be implicated in the mechanism of pancreatic damage induced by caerulein overstimulation.
P28. Changes of hemorheological laboratory parameters and course of medication in experimental acute pancreatitis R. Kotan 1, N. Nemeth 2, F. Kiss 2, J. Posan 1, K. Miszti-Blasius 3, L. Toth 4, I. Furka 2, I. Miko 2, P. Sapy 1, Zs. Szentkereszty 1. 1
Institute of Surgery, Medical and Health Science Center, University of Debrecen, Hungary 2 Department of Operative Techniques and Surgical Research, Medical and Health Science Center, University of Debrecen, Hungary 3 Department of Clinical Biochemistry and Molecular Pathology, Medical and Health Science Center, University of Debrecen, Hungary 4 Department of Pathology, Medical and Health Science Center, University of Debrecen, Hungary Introduction: The role of microcirculatory disturbance in severe acute pancreatitis is generally accepted. Objectives: Effectiveness of Pentoxifyllin, Enoxaparine and Flunixin was analysed in experimental cerulein pancreatitis. Methods: Fifty female Sprague-Dawley rats divided into 5 groups (n¼10). In general anaesthesia 2 hours after acute pancreatitis was induced tissue microcirculation was examined, cupping, was performed in all group. Control group (C): no pancreatitis. Acute pancreatitis group (AP): 10 mg/kg cerulein was injected s.c. AP+Flunixin group (AP+Fl): 2,5 mg/kg Flunixin was also administered. AP+Pentoxifyllin group (AP+Pe): 50 mg/kg Pentoxifyllin i.p. was also administered. AP+Clexan group (AP+E): 2 mg/kg Enoxaparine s.c. was also administered. Tissue blood flow unit (BFU), rectal temperature, blood acidity and hemorheological parameters were measured. Results: The rectal temperature was significantly elevated in AP, it was moderate in AP+Fl and AP+E and high in AP+Pe. The decrease of BFU was lower in AP+Pe and AP+Fl. The blood pH level decrease was moderate in all treated groups. The elevation of hematocrit level was moderate in AP+Fl. The maximal elongation index values decrease in AP+Fl was not noticed. The osmoscan confirmed the best result also in AP+Fl. The M and M1 indices were elevated in all AP, it was moderate in AP+Fl and significant in AP+Pe. The changes of the RBC aggregation parameters were resemble. Conclusion: The measured drugs had good effects on microcirculation, pH levels and microrheologic parameters in cerulein induced acute pancreatitis in rats.
P29. Potential role of KLF10 in the regulation of gemcitabine resistance in pancreatic cancer cells Mei-Ren Pan, Li-Tzong Chen, Wen-Chun Hung. National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan, ROC Krueppel-like factor 10 (also named as transforming growth factor-betainduced early gene 1, TIEG1) has been shown to play different roles in various types of cells. It is also involved in the steps of tumorigenesis and can induce apoptosis in different cancer cell lines. However, whether it plays a role in the control of drug resistance is not clear. By using a gemcitabine selection system, we isolated a gemcitabine-resistant cell line from the parental PANC1 pancreatic cell line. We found that KLF10 is reduced in drug-resistant cell line. Ectopic expression of KLF10 could modulate drug sensitivity. In addition, we also identified a number of downstream target genes and some of them are involved in epigenetic regulation. Our data suggest that KLF10 may modulate
510
P26. Intracellular calcium levels and enzyme secretion in response to tobacco vs ethanol in pancreatic acinar cells M. Luaces-Regueira 1, M. Castiñeira-Alvariño 1, M. Castro-Manzanares 2, A. Carrascal-Miniño 2, M. Campos-Toimil 2, J.E. Domínguez-Muñoz 1. 1 Foundation for Research in Digestive Diseases, Department of Gastroenterology, University Hospital of Santiago de Compostela, Spain 2 Farmacology Department, University of Santiago de Compostela, Spain
Alterations in intracellular calcium levels and enzyme secretion in acinar cells are involved in the pathophysiology of chronic pancreatitis (CP). It has been previously shown that alcohol induces an increase of intracellular calcium, but its effect in enzyme secretion is controversial. The effect of tobacco in this context is unknown. Hypothesis: Tobacco, similarly to alcohol, promotes the development of CP by altering intracellular calcium levels and enzyme secretion in acinar cells. Aim: To evaluate the effect of tobacco compared with alcohol in intracellular calcium levels and amylase secretion in pancreatic acinar cells. Methods: Experimental in-vitro study on primary acinar cell culture. Acinar cells were isolated by enzymatic and mechanic degradation from Swiss mice pancreas. Cells were stimulated by increasing concentrations of alcohol (10-100 mM) and tobacco (0.1-0.5 mg/ml), and with CCK as positive control. Intracellular calcium levels were measured by fluorescence and amylase secretion by using p-nitrophenyl-maltohexaoside as substrate. Data were analysed by ANOVA. Results: Tobacco induced a dose-dependent increase of intracellular calcium levels from 11.384.89% (0.1 mg/ml) to 56.2613.14% (0.5 mg/ml). A similar effect was observed with alcohol [14.402.06% (10 mM) to 59.82.57% (75mM)]. This was associated with an amylase hypersecretion with tobacco (up to 21% at 0.4 mg/ml), but not with alcohol. Conclusions: Both tobacco and alcohol induce a similar increase of intracellular calcium levels in pancreatic acinar cell, but this is associated with an increased amylase secretion only with tobacco. Alcohol and tobacco have a different effect in pancreatic acinar cells and should play a different role in the pathophysiology of CP.
P27. Caerulein modulates the ghrelin system in the pancreatic acini J. Bonior 1, J. Jaworek 1, M. Kot 1, S.J. Konturek 2. 1
Department of Medical Physiology, Faculty of Health Sciences, School of Medicine Jagiellonian University, Krakow, Poland 2 Chair of Physiology Medical Faculty, School of Medicine Jagiellonian University, Krakow, Poland Introduction: Ghrelin, an endogenous ligand for the growth hormone (GH) secretagogue receptor (GHS-R) was originally isolated from the stomach and identified in the pancreas. Ghrelin protects the pancreas from the damage caused by caerulein-induced pancreatitis, but the implication of GHS-R and its endogenous ligand in the pancreatic protection is unclear. Aim: To determine the effect of ghrelin and caerulein on mRNA and protein levels of GHS-R1a subtype and of acylated ghrelin in isolated pancreatic acini. Material and methods: Wistar rats were injected with ghrelin (12,5; 25,0 or 50,0 mg/kg i.p.) or with physiological saline. 48 h later pancreatic acini were isolated and subjected to caerulein stimulation (10–12, 10–10 or 10–8 M) for: 0 h, 20, 1, 3 or 5h at 37 C. The most effective time of incubation was 3 h. High doses of ghrelin and caerulein were selected for further experiments. RT-PCR and Western blot methods were used to determine mRNA and protein levels. Results: GHS-R1a and acylated ghrelin were identified in the pancreatic acini isolated from control rats. Pretreatment of the rats with ghrelin resulted in the significant and dose-dependent upregulation of both
investigated parameters. On the contrary, application of caerulein to the acini significantly and dose-dependently downregulated GHS-R1a, but failed to affect the signal for ghrelin. Pretreatment of the rats with ghrelin prevented from caerulein-induced downregulation of GHS-R1a. Conclusions: Caerulein is able to modify the GHS-R1a subtype in the pancreatic acini. This effect could be prevented by pretreatment with ghrelin and perhaps might be implicated in the mechanism of pancreatic damage induced by caerulein overstimulation.
P28. Changes of hemorheological laboratory parameters and course of medication in experimental acute pancreatitis R. Kotan 1, N. Nemeth 2, F. Kiss 2, J. Posan 1, K. Miszti-Blasius 3, L. Toth 4, I. Furka 2, I. Miko 2, P. Sapy 1, Zs. Szentkereszty 1. 1
Institute of Surgery, Medical and Health Science Center, University of Debrecen, Hungary 2 Department of Operative Techniques and Surgical Research, Medical and Health Science Center, University of Debrecen, Hungary 3 Department of Clinical Biochemistry and Molecular Pathology, Medical and Health Science Center, University of Debrecen, Hungary 4 Department of Pathology, Medical and Health Science Center, University of Debrecen, Hungary Introduction: The role of microcirculatory disturbance in severe acute pancreatitis is generally accepted. Objectives: Effectiveness of Pentoxifyllin, Enoxaparine and Flunixin was analysed in experimental cerulein pancreatitis. Methods: Fifty female Sprague-Dawley rats divided into 5 groups (n¼10). In general anaesthesia 2 hours after acute pancreatitis was induced tissue microcirculation was examined, cupping, was performed in all group. Control group (C): no pancreatitis. Acute pancreatitis group (AP): 10 mg/kg cerulein was injected s.c. AP+Flunixin group (AP+Fl): 2,5 mg/kg Flunixin was also administered. AP+Pentoxifyllin group (AP+Pe): 50 mg/kg Pentoxifyllin i.p. was also administered. AP+Clexan group (AP+E): 2 mg/kg Enoxaparine s.c. was also administered. Tissue blood flow unit (BFU), rectal temperature, blood acidity and hemorheological parameters were measured. Results: The rectal temperature was significantly elevated in AP, it was moderate in AP+Fl and AP+E and high in AP+Pe. The decrease of BFU was lower in AP+Pe and AP+Fl. The blood pH level decrease was moderate in all treated groups. The elevation of hematocrit level was moderate in AP+Fl. The maximal elongation index values decrease in AP+Fl was not noticed. The osmoscan confirmed the best result also in AP+Fl. The M and M1 indices were elevated in all AP, it was moderate in AP+Fl and significant in AP+Pe. The changes of the RBC aggregation parameters were resemble. Conclusion: The measured drugs had good effects on microcirculation, pH levels and microrheologic parameters in cerulein induced acute pancreatitis in rats.
P29. Potential role of KLF10 in the regulation of gemcitabine resistance in pancreatic cancer cells Mei-Ren Pan, Li-Tzong Chen, Wen-Chun Hung. National Institute of Cancer Research, National Health Research Institutes, Tainan, Taiwan, ROC Krueppel-like factor 10 (also named as transforming growth factor-betainduced early gene 1, TIEG1) has been shown to play different roles in various types of cells. It is also involved in the steps of tumorigenesis and can induce apoptosis in different cancer cell lines. However, whether it plays a role in the control of drug resistance is not clear. By using a gemcitabine selection system, we isolated a gemcitabine-resistant cell line from the parental PANC1 pancreatic cell line. We found that KLF10 is reduced in drug-resistant cell line. Ectopic expression of KLF10 could modulate drug sensitivity. In addition, we also identified a number of downstream target genes and some of them are involved in epigenetic regulation. Our data suggest that KLF10 may modulate