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Expression of aquaporins in pancreatic ductal cells
Abstracts / Pancreatology 14 (2014) S1eS129
Results: Sequencing analysis of the discovery cohort revealed four common mutations: intronic mutations c.23+71_23+103del, c.183-4C>A and c.1134+32C>A; and exonic missense mutation p.V206M. . These four mutations were found in linkage disequilibrium indicating a conserved haplotype. We found this haplotype in 18 heterozygous and 2 homozygous cases, and in 24 heterozygous and 2 homozygous controls (allele frequency 11.4% and 14.1% respectively). A synonymous mutation p.P397P was also detected in a single case. Conclusion: We found a novel, common haplotype in the SLC26A6 gene, which did not show association with CP. Supported by TAMOP and OTKA.
T-101. Expression of aquaporins in pancreatic ductal cells n Rakonczay, Jr. b, Viktoria Venglovecz a, Lajos V. Kem eny b, Zolta szlo s c, P Puska eter Hegyi b Agnes Zvara c, La a University of Szeged, Department of Pharmacology and Pharmacotherapy, Hungary b University of Szeged, First Department of Medicine, Hungary c Biological Research Centre, Laboratory of Functional Genomics, Hungary
Background: Acute pancreatitis (AP) is a multicellular disease in which pancreatic ductal cells play an important role. Toxic agents inducing AP inhibit pancreatic ductal bicarbonate secretion, however, no information is available concerning their effects on the regulation of aquaporins (AQPs). Aims: The aim of this study was to investigate the effects of bile acids, ethanol and its metabolites on the expression of AQPs. Materials & methods: CAPAN-1 cells were treated with ethanol (EtOH; 1-100 mM), chenodeoxycholate (CDC; 0.1-0.5 mM), glycochenodeoxycholate (GCDC; 0.1-0.5 mM) palmitoleic acid (POA; 10, 100 and 200 uM) and palmitoleic acid ethyl ester (POAEE; 10, 100 and 200 uM) for 6, 12, 24 and 48 hours and the expression of AQP isoforms (AQP1-12) was examined by real-time RT-PCR and immunocytochemistry. Results: All 12 AQPs were expressed in the CAPAN-1 cell line to a certain degree. AQP1, 3, 5, 6 and 11 were expressed at the highest levels while AQP2 and 4 were hardly detectable. In almost all treated group, the expression of AQPs decreased both at mRNA and protein levels dose- and time-dependently. Notably, a 72-hour incubation in culture media restored the expression of AQPs in the 6- and 12-hour CDC- and GCDC-treated groups and in the 24-hour EtOH-treated group. Conclusion: The role of AQP in the pathogenesis of AP needs further investigations. Our research is supported by Hungarian National Development Agency grants (TAMOP-4.2.2.A-11/1/KONV-2012-0035, -0052, -0073, TAMOP 4.2.4.A/2-11-1-2012-0001 ‘National Excellence Program) and the Hungarian Scientific Research Fund (OTKA NF105758, NF100677, K109756).
T-102. A human ex vivo system for the study of pancreatic injury and regeneration ndez Moro a, Sougat Misra a, Soledad Pouso a, Marita Carlos Ferna €hr c, Mikael Bjo €rnstedt a, Marco Wallenberg a, Rainer Heuchel b, Matthias Lo Del Chiaro c, Caroline Verbeke a a Division of Pathology, Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden, Sweden b Department of Clinical Science, Intervention and Technology (CLINTEC), Center of Biosciences. Karolinska Institute, Stockholm, Sweden c Division of Surgery, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institute, Stockholm, Sweden
S79
Background: Animal experiments have shown that injury induces a regenerative response in pancreatic tissue, which is driven by the acinar compartment in the cerulein-induced pancreatitis model and by ductal epithelium in the ‘px model’. However, little is known about the response to injury in the human pancreas. Aims: To investigate regenerative responses in the human pancreas using an ex vivo culture system. Materials & methods: Fresh normal pancreatic tissue was sampled by knife-excision from surgical specimens (n¼10). The samples were sliced (350 mm thickness) and cultured for at least 4 days in organotypic culture under mildly hypoxic conditions (21% O2, no insert). Every 24 hours, two slices were processed for light microscopy, and sections were examined by two independent pathologists (CV, CFM) for signs of regeneration in the acinar and/or ductal compartment. Immunohistochemistry for epithelial (CK19, trypsin, Mist1), stromal (vimentin, aSMA), proliferative (Ki-67) and progenitor (PDX1, Hes1, SOX9) markers was performed. Results: From day 1, a single layer of epithelial cells was seen to grow out onto the surface of the tissue slice and appeared to originate from transected small branch ducts. During subsequent days, the cells extended progressively along the surface and acquired a duct-epithelial appearance (CK19+, vimentin). The acinar compartment showed acinar-ductal metaplasia, which was not connected with the cellular outgrowth on the tissue surface. Conclusion: The organotypic culture system shows evidence of injuryinduced duct-epithelial regeneration and acinar-ductal metaplasia.
T-103. Cigarette smoke extract inhibits fluid secretion and CFTR activity in guinea pig pancreatic ductal cells th a, Andrea Schnúr a, Jo zsef Mal Krisztina To eth a, Dezs Csupor b, c a eter Hegyi a Viktoria Venglovecz , Eleonora Gal , Zoltan Rakonczay, Jr. a, P a First Department of Medicine, University of Szeged, Szeged, Hungary, Hungary b Department of Pharmacognosy, University of Szeged, Szeged, Hungary, Hungary c Department of Pharmacology and Pharmacotherapy, University of Szeged, Szeged, Hungary, Hungary
Background: Smoking represents an independent risk factor for the development of chronic pancreatitis, however, the pathomechanism remains unknown. Secretion of fluid and bicarbonate plays a crucial role in maintaining the integrity of the gland. Aims: The aim of this study was to investigate the effects of cigarette smoke extract (CSE) on pancreatic ductal fluid secretion and on cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel activity. Materials & methods: Intra/interlobular pancreatic ducts were isolated from guinea pig pancreas. Basal and forskolin stimulated fluid secretion were measured by videomicroscopy, whereas, CFTR currents were detected by whole cell configuration of the patch clamp technique. CSE was prepared by smoking of 3 cigarettes into 40ml distilled water by a smoking machine and 10x (21mg/ml), 40x (5.25mg/ml) and 400x (0.5mg/ml) dilution of the extract were studied. Results: Administration of 5mM forskolin activated CFTR currents by 10-15-fold in magnitude. 15 min administration of 0.5, 5.25 and 21 mg/ml CSE inhibited the currents by 44%, 64.6% and 79.4%, respectively (n¼2-4). Concerning the fluid secretion, the basal volume of isolated intact pancreatic ducts in bicarbonate-free solution was considered to be 1.0. Administration of 25mM bicarbonate increased the relative luminal volume up to 1.57±0.02 (n¼7). Administration of 5 mM forskolin further increased the luminal volume to 1.87±0.1 (n¼16). Simultaneous administration of 21mg/ml CSE decreased fluid secretion by 24% (1.42±0.06; n¼12).
Results: Sequencing analysis of the discovery cohort revealed four common mutations: intronic mutations c.23+71_23+103del, c.183-4C>A and c.1134+32C>A; and exonic missense mutation p.V206M. . These four mutations were found in linkage disequilibrium indicating a conserved haplotype. We found this haplotype in 18 heterozygous and 2 homozygous cases, and in 24 heterozygous and 2 homozygous controls (allele frequency 11.4% and 14.1% respectively). A synonymous mutation p.P397P was also detected in a single case. Conclusion: We found a novel, common haplotype in the SLC26A6 gene, which did not show association with CP. Supported by TAMOP and OTKA.
T-101. Expression of aquaporins in pancreatic ductal cells n Rakonczay, Jr. b, Viktoria Venglovecz a, Lajos V. Kem eny b, Zolta szlo s c, P Puska eter Hegyi b Agnes Zvara c, La a University of Szeged, Department of Pharmacology and Pharmacotherapy, Hungary b University of Szeged, First Department of Medicine, Hungary c Biological Research Centre, Laboratory of Functional Genomics, Hungary
Background: Acute pancreatitis (AP) is a multicellular disease in which pancreatic ductal cells play an important role. Toxic agents inducing AP inhibit pancreatic ductal bicarbonate secretion, however, no information is available concerning their effects on the regulation of aquaporins (AQPs). Aims: The aim of this study was to investigate the effects of bile acids, ethanol and its metabolites on the expression of AQPs. Materials & methods: CAPAN-1 cells were treated with ethanol (EtOH; 1-100 mM), chenodeoxycholate (CDC; 0.1-0.5 mM), glycochenodeoxycholate (GCDC; 0.1-0.5 mM) palmitoleic acid (POA; 10, 100 and 200 uM) and palmitoleic acid ethyl ester (POAEE; 10, 100 and 200 uM) for 6, 12, 24 and 48 hours and the expression of AQP isoforms (AQP1-12) was examined by real-time RT-PCR and immunocytochemistry. Results: All 12 AQPs were expressed in the CAPAN-1 cell line to a certain degree. AQP1, 3, 5, 6 and 11 were expressed at the highest levels while AQP2 and 4 were hardly detectable. In almost all treated group, the expression of AQPs decreased both at mRNA and protein levels dose- and time-dependently. Notably, a 72-hour incubation in culture media restored the expression of AQPs in the 6- and 12-hour CDC- and GCDC-treated groups and in the 24-hour EtOH-treated group. Conclusion: The role of AQP in the pathogenesis of AP needs further investigations. Our research is supported by Hungarian National Development Agency grants (TAMOP-4.2.2.A-11/1/KONV-2012-0035, -0052, -0073, TAMOP 4.2.4.A/2-11-1-2012-0001 ‘National Excellence Program) and the Hungarian Scientific Research Fund (OTKA NF105758, NF100677, K109756).
T-102. A human ex vivo system for the study of pancreatic injury and regeneration ndez Moro a, Sougat Misra a, Soledad Pouso a, Marita Carlos Ferna €hr c, Mikael Bjo €rnstedt a, Marco Wallenberg a, Rainer Heuchel b, Matthias Lo Del Chiaro c, Caroline Verbeke a a Division of Pathology, Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden, Sweden b Department of Clinical Science, Intervention and Technology (CLINTEC), Center of Biosciences. Karolinska Institute, Stockholm, Sweden c Division of Surgery, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institute, Stockholm, Sweden
S79
Background: Animal experiments have shown that injury induces a regenerative response in pancreatic tissue, which is driven by the acinar compartment in the cerulein-induced pancreatitis model and by ductal epithelium in the ‘px model’. However, little is known about the response to injury in the human pancreas. Aims: To investigate regenerative responses in the human pancreas using an ex vivo culture system. Materials & methods: Fresh normal pancreatic tissue was sampled by knife-excision from surgical specimens (n¼10). The samples were sliced (350 mm thickness) and cultured for at least 4 days in organotypic culture under mildly hypoxic conditions (21% O2, no insert). Every 24 hours, two slices were processed for light microscopy, and sections were examined by two independent pathologists (CV, CFM) for signs of regeneration in the acinar and/or ductal compartment. Immunohistochemistry for epithelial (CK19, trypsin, Mist1), stromal (vimentin, aSMA), proliferative (Ki-67) and progenitor (PDX1, Hes1, SOX9) markers was performed. Results: From day 1, a single layer of epithelial cells was seen to grow out onto the surface of the tissue slice and appeared to originate from transected small branch ducts. During subsequent days, the cells extended progressively along the surface and acquired a duct-epithelial appearance (CK19+, vimentin). The acinar compartment showed acinar-ductal metaplasia, which was not connected with the cellular outgrowth on the tissue surface. Conclusion: The organotypic culture system shows evidence of injuryinduced duct-epithelial regeneration and acinar-ductal metaplasia.
T-103. Cigarette smoke extract inhibits fluid secretion and CFTR activity in guinea pig pancreatic ductal cells th a, Andrea Schnúr a, Jo zsef Mal Krisztina To eth a, Dezs Csupor b, c a eter Hegyi a Viktoria Venglovecz , Eleonora Gal , Zoltan Rakonczay, Jr. a, P a First Department of Medicine, University of Szeged, Szeged, Hungary, Hungary b Department of Pharmacognosy, University of Szeged, Szeged, Hungary, Hungary c Department of Pharmacology and Pharmacotherapy, University of Szeged, Szeged, Hungary, Hungary
Background: Smoking represents an independent risk factor for the development of chronic pancreatitis, however, the pathomechanism remains unknown. Secretion of fluid and bicarbonate plays a crucial role in maintaining the integrity of the gland. Aims: The aim of this study was to investigate the effects of cigarette smoke extract (CSE) on pancreatic ductal fluid secretion and on cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel activity. Materials & methods: Intra/interlobular pancreatic ducts were isolated from guinea pig pancreas. Basal and forskolin stimulated fluid secretion were measured by videomicroscopy, whereas, CFTR currents were detected by whole cell configuration of the patch clamp technique. CSE was prepared by smoking of 3 cigarettes into 40ml distilled water by a smoking machine and 10x (21mg/ml), 40x (5.25mg/ml) and 400x (0.5mg/ml) dilution of the extract were studied. Results: Administration of 5mM forskolin activated CFTR currents by 10-15-fold in magnitude. 15 min administration of 0.5, 5.25 and 21 mg/ml CSE inhibited the currents by 44%, 64.6% and 79.4%, respectively (n¼2-4). Concerning the fluid secretion, the basal volume of isolated intact pancreatic ducts in bicarbonate-free solution was considered to be 1.0. Administration of 25mM bicarbonate increased the relative luminal volume up to 1.57±0.02 (n¼7). Administration of 5 mM forskolin further increased the luminal volume to 1.87±0.1 (n¼16). Simultaneous administration of 21mg/ml CSE decreased fluid secretion by 24% (1.42±0.06; n¼12).