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Balazs A.,Boston University | Balazs A.,University of Szeged | Hegyi P.,University of Szeged | Hegyi P.,MTA SZTE Translational Gastroenterology Research Group | And 2 more authors.
American Journal of Physiology - Gastrointestinal and Liver Physiology | Year: 2016

Mutations in the PRSS1 gene encoding human cationic trypsinogen are associated with hereditary and sporadic chronic pancreatitis. Highpenetrance PRSS1 mutations found in hereditary pancreatitis alter activation and/or degradation of cationic trypsinogen, thereby promoting intrapancreatic trypsinogen activation. In contrast, a number of rare PRSS1 variants identified in subjects with sporadic chronic pancreatitis cause misfolding and endoplasmic reticulum (ER) stress. Mutation p.L104P is unique among natural PRSS1 variants, since it affects the substrate binding site of trypsin. The aim of the present study was to establish the clinical significance of variant p.L104P through functional analysis. We found that p.L104P trypsin exhibited decreased activity on peptide and protein substrates; however, autoactivation was slightly accelerated. Remarkably, binding of the physiological trypsin inhibitor serine protease inhibitor Kazal type 1 (SPINK1) was decreased by 70-fold. In the presence of the trypsinogen- degrading enzyme chymotrypsin C, mutant p.L104P autoactivated to higher trypsin levels than wild-type trypsinogen. This apparent resistance to degradation was due to slower cleavage at Arg122 rather than Leu81. Finally, secretion of mutant p.L104P from transfected cells was markedly reduced due to intracellular retention and aggregation with concomitant elevation of ER stress markers. We conclude that PRSS1 variant p.L104P exhibits a variety of phenotypic changes that can increase risk for chronic pancreatitis. Mutationinduced misfolding and associated ER stress are the dominant effects that support a direct pathogenic role in chronic pancreatitis. © 2016 The American Physiological Society.


Hegyi E.,University of Szeged | Hegyi E.,Comenius University | Geisz A.,University of Szeged | Geisz A.,Boston University | And 13 more authors.
Pancreas | Year: 2016

Objectives Serine protease inhibitor Kazal type 1 (SPINK1) provides an important line of defense against premature trypsinogen activation within the pancreas. Our aim was to identify pathogenic SPINK1 promoter variants associated with chronic pancreatitis (CP). Methods One hundred CP patients (cases) and 100 controls with no pancreatic disease from the Hungarian National Pancreas Registry were enrolled. Direct sequencing of SPINK1 promoter region was performed. Functional characterization of variants was carried out using luciferase reporter gene assay. Results Two common polymorphisms (c.-253T>C and c.-807C>T) were found in both cases and controls. Variant c.253T>C was enriched in cases relative to controls (odds ratio, 2.1; 95% confidence interval, 1.2-3.8; P = 0.015). Variant c.-215G>A was detected in 3 of 100 cases; always linked with the pathogenic variant c.194+2T>C. Novel promoter variants c.-14G>A, c.-108G>T, and c.-246A>G were identified in 1 case each. Functional analysis showed decreased promoter activity for variants c.-14G>A (80%), c.-108G>T (31%), and c.-246A>G (47%) whereas activity of variant c.-215G>A was increased (201%) and variant c.-253T>C was unchanged compared with wild type. Conclusions The common promoter variant c.-253T>C was associated with CP in this cohort. Two of 3 newly identified SPINK1 promoter variants seem to exhibit significant functional defects and should be considered potential risk factors for CP. Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.


Balazs A.,University of Szeged | Ruffert C.,University of Leipzig | Hegyi E.,University of Szeged | Hegyi E.,Comenius University | And 33 more authors.
Pancreatology | Year: 2015

Background Pancreatic ductal HCO3 - secretion is critically dependent on the cystic fibrosis transmembrane conductance regulator chloride channel (CFTR) and the solute-linked carrier 26 member 6 anion transporter (SLC26A6). Deterioration of HCO3 - secretion is observed in chronic pancreatitis (CP), and CFTR mutations increase CP risk. Therefore, SLC26A6 is a reasonable candidate for a CP susceptibility gene, which has not been investigated in CP patients so far. Methods As a first screening cohort, 106 subjects with CP and 99 control subjects with no pancreatic disease were recruited from the Hungarian National Pancreas Registry. In 60 non-alcoholic CP cases the entire SLC26A6 coding region was sequenced. In the Hungarian cohort variants c.616G > A (p.V206M) and c.1191C > A (p.P397=) were further genotyped by restriction fragment length polymorphism analysis. In a German replication cohort all exons were sequenced in 40 non-alcoholic CP cases and variant c.616G > A (p.V206M) was further analyzed by sequencing in 321 CP cases and 171 controls. Results Sequencing of the entire coding region revealed four common variants: intronic variants c.23 + 78-110del, c.183-4C > A, c.1134 + 32C > A, and missense variant c.616G > A (p.V206M) which were found in linkage disequilibrium indicating a conserved haplotype. The distribution of the haplotype did not show a significant difference between patients and controls in the two cohorts. A synonymous variant c.1191C > A (p.P397=) and two intronic variants c.1248 + 9-20del and c.-10C > T were detected in single cases. Conclusion Our data show that SLC26A6 variants do not alter the risk for the development of CP. Copyright © 2015, IAP and EPC. Published by Elsevier India, a division of Reed Elsevier India Pvt. Ltd. All rights reserved.


Pallagi-Kunstar E.,University of Szeged | Farkas K.,University of Szeged | Maleth J.,University of Szeged | Rakonczay Z.,University of Szeged | And 9 more authors.
Pflugers Archiv European Journal of Physiology | Year: 2015

Bile acids play important physiological role in the solubilisation and absorption of dietary lipids. However, under pathophysiological conditions, such as short bowel syndrome, they can reach the colon in high concentrations inducing diarrhoea. In this study, our aim was to characterise the cellular pathomechanism of bile-induced diarrhoea using human samples. Colonic crypts were isolated from biopsies of patients (controls with negative colonoscopic findings) and of cholecystectomised/ileum-resected patients with or without diarrhoea. In vitro measurement of the transporter activities revealed impaired Na+/H+ exchanger (NHE) and Cl−/HCO3 − exchanger (CBE) activities in cholecystectomised/ileum-resected patients suffering from diarrhoea, compared to control patients. Acute treatment of colonic crypts with 0.3 mM chenodeoxycholate caused dose-dependent intracellular acidosis; moreover, the activities of acid/base transporters (NHE and CBE) were strongly impaired. This concentration of chenodeoxycholate did not cause morphological changes in colonic epithelial cells, although significantly reduced the intracellular ATP level, decreased mitochondrial transmembrane potential and caused sustained intracellular Ca2+ elevation. We also showed that chenodeoxycholate induced Ca2+ release from the endoplasmic reticulum and extracellular Ca2+ influx contributing to the Ca2+ elevation. Importantly, our results suggest that the chenodeoxycholate-induced inhibition of NHE activities was ATP-dependent, whereas the inhibition of CBE activity was mediated by the sustained Ca2+ elevation. We suggest that bile acids inhibit the function of ion transporters via cellular energy breakdown and Ca2+ overload in human colonic epithelial cells, which can reduce fluid and electrolyte absorption in the colon and promote the development of diarrhoea. © 2014, Springer-Verlag Berlin Heidelberg.


Schnur A.,McGill University | Hegyi P.,University of Pécs | Hegyi P.,MTA SZTE Translational Gastroenterology Research Group | Rousseau S.,McGill University | And 2 more authors.
Mediators of Inflammation | Year: 2016

The pivotal role of epithelial cells is to secrete and absorb ions and water in order to allow the formation of a luminal fluid compartment that is fundamental for the epithelial function as a barrier against environmental factors. Importantly, epithelial cells also take part in the innate immune system. As a first line of defense they detect pathogens and react by secreting and responding to chemokines and cytokines, thus aggravating immune responses or resolving inflammatory states. Loss of epithelial anion transport is well documented in a variety of diseases including cystic fibrosis, chronic obstructive pulmonary disease, asthma, pancreatitis, and cholestatic liver disease. Here we review the effect of aberrant anion secretion with focus on the release of inflammatory mediators by epithelial cells and discuss putative mechanisms linking these transport defects to the augmented epithelial release of chemokines and cytokines. These mechanisms may contribute to the excessive and persistent inflammation in many respiratory and gastrointestinal diseases. © 2016 Andrea Schnúr et al.


Venglovecz V.,University of Szeged | Rakonczay Z.,University of Szeged | Gray M.A.,Northumbria University | Hegyi P.,University of Szeged | Hegyi P.,MTA SZTE Translational Gastroenterology Research Group
Pflugers Archiv European Journal of Physiology | Year: 2015

Pancreatic ductal epithelial cells play a fundamental role in HCO3 − secretion, a process which is essential for maintaining the integrity of the pancreas. Although several studies have implicated impaired HCO3 − and fluid secretion as a triggering factor in the development of pancreatitis, the mechanism and regulation of HCO3 − secretion is still not completely understood. To date, most studies on the ion transporters that orchestrate ductal HCO3 − secretion have focussed on the role of Cl−/HCO3 − exchangers and Cl− channels, whereas much less is known about the role of K+ channels. However, there is growing evidence that many types of K+ channels are present in ductal cells where they have an essential role in establishing and maintaining the electrochemical driving force for anion secretion. For this reason, strategies that increase K+ channel function may help to restore impaired HCO3 − and fluid secretion, such as in pancreatitis, and therefore provide novel directions for future pancreatic therapy. In this review, our aims are to summarize the types of K+ channels found in pancreatic ductal cells and to discuss their individual roles in ductal HCO3 − secretion. We will also describe how K+ channels are involved in pathophysiological conditions and discuss how they could act as new molecular targets for the development of therapeutic approaches to treat pancreatic diseases. © 2014, Springer-Verlag Berlin Heidelberg.

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