Bile acid- and ethanol-mediated activation of Orai1 damages pancreatic ductal secretion in acute pancreatitis

Sustained intracellular Ca2+ overload in pancreatic acinar and ductal cells is a hallmark of biliary and alcohol-induced acute pancreatitis, which leads to impaired ductal ion and fluid secretion. Orai1 is a plasma membrane Ca2+ channel that mediates extracellular Ca2+ influx upon endoplasmic reticulum Ca2+ depletion. Our results showed that Orai1 is expressed on the luminal plasma membrane of the ductal cells and selective Orai1 inhibition impaired Stim1-dependent extracellular Ca2+ influx evoked by bile acids or ethanol combined with non-oxidative ethanol metabolites. The prevention of sustained extracellular Ca2+ influx protected ductal cell secretory functions in in vitro models and maintained exocrine pancreatic secretion in in vivo AP models. Orai1 inhibition prevents the bile acid-, and alcohol-induced damage of the pancreatic ductal secretion and holds the potential of improving the outcome of acute pancreatitis.Regardless of its etiology, sustained intracellular Ca2+ overload is a well-known hallmark of acute pancreatitis (AP). Toxic Ca2+ elevation induces pancreatic ductal cell damage characterized by impaired ion- and fluid secretion -essential to wash out the protein-rich fluid secreted by acinar cells while maintaining the alkaline intra-ductal pH under physiological conditions- and mitochondrial dysfunction. While prevention of ductal cell injury decreases the severity of AP, no specific drug target has yet been identified in the ductal cells. Although Orai1 -a store operated Ca2+ influx channel- is known to contribute to sustained Ca2+ overload in acinar cells, details concerning its expression and function in ductal cells are currently lacking. In this study, we demonstrate that functionally active Orai1 channels reside dominantly in the apical plasma membrane of pancreatic ductal cells. Selective CM5480-mediated Orai1 inhibition impairs Stim1-dependent extracellular Ca2+ influx evoked by bile acids or ethanol combined with non-oxidative ethanol metabolites. Furthermore, prevention of sustained extracellular Ca2+ influx protects ductal cell secretory function in vitro and decrease pancreatic ductal cell death. Finally, Orai1-inhibition partially restores and maintains proper exocrine pancreatic secretion in in vivo AP models. In conclusion, our results indicate that Orai1 inhibition prevents AP-related ductal cell function impairment and holds the potential of improving disease outcome. Abstract figure legend This article is protected by copyright. All rights reserved

Thiopurines impair the apical plasma membrane expression of CFTR in pancreatic ductal cells via RAC1 inhibition

Thiopurine-induced acute pancreatitis (TIP) is one of the most common adverse events among inflammatory bowel disease patients treated with azathioprine (AZA), representing a significant clinical burden. Previous studies focused on immune-mediated processes, however, the exact pathomechanism of TIP is essentially unclear.To model TIP in vivo, we triggered cerulein-induced experimental pancreatitis in mice receiving a daily oral dose of 1.5 mg/kg AZA. Also, freshly isolated mouse pancreatic cells were exposed to AZA ex vivo, and acinar cell viability, ductal and acinar Ca2+ signaling, ductal Cl- and HCO3- secretion, as well as cystic fibrosis transmembrane conductance regulator (CFTR) expression were assessed using microscopy techniques. Ras-related C3 botulinum toxin substrate (RAC1) activity was measured with a G-LISA assay. Super-resolution microscopy was used to determine protein colocalization.We demonstrated that AZA treatment increases tissue damage in the early phase of cerulein-induced pancreatitis in vivo. Also, both per os and ex vivo AZA exposure impaired pancreatic fluid and ductal HCO3- and Cl- secretion, but did not affect acinar cells. Furthermore, ex vivo AZA exposure also inhibited RAC1 activity in ductal cells leading to decreased co-localization of CFTR and the anchor protein ezrin, resulting in impaired plasma membrane localization of CFTR.AZA impaired the ductal HCO3- and Cl- secretion through the inhibition of RAC1 activity leading to diminished ezrin-CFTR interaction and disturbed apical plasma membrane expression of CFTR. We report a novel direct toxic effect of AZA on pancreatic ductal cells and suggest that the restoration of ductal function might help to prevent TIP in the future

Thiopurines Impair the Apical Plasma Membrane Expression of CFTR in Pancreatic Ductal Cells via RAC1 Inhibition

Background and aims Thiopurine-induced acute pancreatitis (TIP) is one of the most common adverse events among infammatory bowel disease patients treated with azathioprine (AZA), representing a signifcant clinical burden. Previous studies focused on immune-mediated processes, however, the exact pathomechanism of TIP is essentially unclear. Methods To model TIP in vivo, we triggered cerulein-induced experimental pancreatitis in mice receiving a daily oral dose of 1.5 mg/kg AZA. Also, freshly isolated mouse pancreatic cells were exposed to AZA ex vivo, and acinar cell viability, ductal and acinar Ca2+ signaling, ductal Cl–and HCO3–secretion, as well as cystic fbrosis transmembrane conductance regulator (CFTR) expression were assessed using microscopy techniques. Ras-related C3 botulinum toxin substrate (RAC1) activity was measured with a G-LISA assay. Super-resolution microscopy was used to determine protein colocalization. Results We demonstrated that AZA treatment increases tissue damage in the early phase of cerulein-induced pancreatitis in vivo. Also, both per os and ex vivo AZA exposure impaired pancreatic fuid and ductal HCO3– and Cl–secretion, but did not afect acinar cells. Furthermore, ex vivo AZA exposure also inhibited RAC1 activity in ductal cells leading to decreased co-localization of CFTR and the anchor protein ezrin, resulting in impaired plasma membrane localization of CFTR. Conclusions AZA impaired the ductal HCO3– and Cl–secretion through the inhibition of RAC1 activity leading to diminished ezrin-CFTR interaction and disturbed apical plasma membrane expression of CFTR. We report a novel direct toxic efect of AZA on pancreatic ductal cells and suggest that the restoration of ductal function might help to prevent TIP in the future

The role of Ca2+ signaling in the physiology and pathophysiology of the pancreatic ductal cells

Sustained intracellular Ca2+ overload is a well-known hallmark of acute pancreatitis (AP). Toxic Ca2+ elevations in the pancreatic ductal cells damage the mitochondrial function and impair ductal ion and fluid secretion which –under physiological conditions– maintains the alkaline intra-ductal pH and washes out the protein-rich fluid secreted by acinar cells. While prevention of ductal cell injury decreases severity of acute pancreatitis, there is however no specific drug target identified yet in the ductal cells. Although Orai1 –a store operated Ca2+ influx channel– is known to contribute to sustained Ca2+ overload in acinar cells, details concerning its expression and function in ductal cells are currently lacking. Firstly, we demonstrated that functionally active Orai1 channels reside in the apical plasma membrane of pancreatic ductal cells. Next, selective CM-5480-mediated Orai1 inhibition impaired Stim1-dependent extracellular Ca2+ influx evoked by bile acids or ethanol combined with non-oxidative ethanol metabolites. Furthermore, prevention of sustained extracellular Ca2+ influx protected ductal cell secretory function in vitro. Orai1-inhibition maintained exocrine pancreatic secretion in in vivo AP models. Finally, bile acids markedly inhibited the HCO3- secretion both in TRPM2 KO and WT ductal cells, the genetic knockout of TRPM2 had no protective effect, suggesting that bile acids affect ductal cells via a TRPM2-independent mechanism. In conclusion, our results indicate that Orai1 inhibition prevents acute pancreatitis-related ductal cell function impairment and holds the potential of improving disease outcome

Mouse pancreatic ductal organoid culture as a relevant model to study exocrine pancreatic ion secretion

Pancreatic exocrine secretory processes are challenging to investigate on primary epithelial cells. Pancreatic organoid cultures may help to overcome shortcomings of the current models, however the ion secretory processes in pancreatic organoids-and therefore their physiological relevance or their utility in disease modeling-are not known. To answer these questions, we provide side-by-side comparison of gene expression, morphology, and function of epithelial cells in primary isolated pancreatic ducts and organoids. We used mouse pancreatic ductal fragments for experiments or were grown in Matrigel to obtain organoid cultures. Using PCR analysis we showed that gene expression of ion channels and transporters remarkably overlap in primary ductal cells and organoids. Morphological analysis with scanning electron microscopy revealed that pancreatic organoids form polarized monolayers with brush border on the apical membrane. Whereas the expression and localization of key proteins involved in ductal secretion (cystic fibrosis transmembrane conductance regulator, Na+/H+ exchanger 1 and electrogenic Na+/HCO3- cotransporter 1) are equivalent to the primary ductal fragments. Measurements of intracellular pH and Cl- levels revealed no significant difference in the activities of the apical Cl-/HCO3- exchange, or in the basolateral Na+ dependent HCO3- uptake. In summary we found that ion transport activities in the mouse pancreatic organoids are remarkably similar to those observed in freshly isolated primary ductal fragments. These results suggest that organoids can be suitable and robust model to study pancreatic ductal epithelial ion transport in health and diseases and facilitate drug development for secretory pancreatic disorders like cystic fibrosis, or chronic pancreatitis

TRPM2-mediated extracellular Ca2+ entry promotes acinar cell necrosis in biliary acute pancreatitis

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