Key points Acute biliary pancreatitis is a significant clinical challenge as currently no specific pharmaceutical treatment exists. Intracellular Ca2+ overload, increased reactive oxygen species (ROS) production, mitochondrial damage and intra‐acinar digestive enzyme activation caused by bile acids are hallmarks of acute biliary pancreatitis. Transient receptor potential melastatin 2 (TRPM2) is a non‐selective cation channel that has recently emerged as an important contributor to oxidative‐stress‐induced cellular Ca2+ overload across different diseases. We demonstrated that TRPM2 is expressed in the plasma membrane of mouse pancreatic acinar and ductal cells, which can be activated by increased oxidative stress induced by H2O2 treatment and contributed to bile acid‐induced extracellular Ca2+ influx in acinar cells, which promoted acinar cell necrosis in vitro and in vivo. These results suggest that the inhibition of TRPM2 may be a potential treatment option for biliary pancreatitis. Abstract Acute biliary pancreatitis poses a significant clinical challenge as currently no specific pharmaceutical treatment exists. Disturbed intracellular Ca2+ signalling caused by bile acids is a hallmark of the disease, which induces increased reactive oxygen species (ROS) production, mitochondrial damage, intra‐acinar digestive enzyme activation and cell death. Because of this mechanism of action, prevention of toxic cellular Ca2+ overload is a promising therapeutic target. Transient receptor potential melastatin 2 (TRPM2) is a non‐selective cation channel that has recently emerged as an important contributor to oxidative‐stress‐induced cellular Ca2+ overload across different diseases. However, the expression and possible functions of TRPM2 in the exocrine pancreas remain unknown. Here we found that TRPM2 is expressed in the plasma membrane of mouse pancreatic acinar and ductal cells, which can be activated by increased oxidative stress induced by H2O2 treatment. TRPM2 activity was found to contribute to bile acid‐induced extracellular Ca2+ influx in acinar cells, but did not have the same effect in ductal cells. The generation of intracellular ROS in response to bile acids was remarkably higher in pancreatic acinar cells compared to isolated ducts, which can explain the difference between acinar and ductal cells. This activity promoted acinar cell necrosis in vitro independently from mitochondrial damage or mitochondrial fragmentation. In addition, bile‐acid‐induced experimental pancreatitis was less severe in TRPM2 knockout mice, whereas the lack of TRPM2 had no protective effect in cerulein‐induced acute pancreatitis. Our results suggest that the inhibition of TRPM2 may be a potential treatment option for biliary pancreatitis.
Dantrolene is a ryanodine receptor (RyR) inhibitor, which is used to relax muscles in malignant hyperthermia syndrome. Although dantrolene binds to the RyR protein, its mechanism of action is unknown, mainly because of the controversial results showing that dantrolene inhibited Ca 21 release from intact fibers and sarcoplasmic reticulum (SR) vesicles, but failed to inhibit single RyR channel currents in bilayers. Accordingly, it was concluded that an important factor for dantrolene's action was lost during the purification procedure of RyR. Recently, Mg 21 was demonstrated to be the essential factor for dantrolene to inhibit Ca 21 release in skinned muscle fibers. The aim of the present study was to confirm these results in Ca 21 release and bilayer experiments, using SR vesicles and solubilized channels, respectively. Our Ca 21 release experiments demonstrated that the effect of dantrolene and Mg 21 was cooperative and that ATP enhanced the inhibiting effect of dantrolene. Namely, 10 mM dantrolene reduced RyR channel open probability by ∼50% in the presence of 3 mM free Mg 21 and 1 mM ATP, whereas channel activity further decreased to ∼20% of control when [ATP] was increased to 2 mM. Our data provide important complementary information that supports the direct, Mg 21-dependent mechanism of dantrolene's action and suggests that dantrolene also requires ATP to inhibit RyR.
a b s t r a c tThe earliest critical event of pancreatitis is a long lasting high amplitude rise of intracellular Ca 2+ concentration of the acinar cell, which can be triggered by high concentration of bile acids. Although, Ca 2+ -release through ryanodine receptors (RyR) is involved in the process, the significance and the exact mechanism of bile acid's action on RyR has not been fully elucidated yet. Therefore, we aimed to test with various techniques and aspects whether bile acids exert a direct effect on RyR and SERCA pump.Our data show that taurocholic acid (TCA)-induced Ca 2+ release in pancreatic acinar cells was significantly reduced by the RyR antagonist dantrolene. Further, we show that TCA enhanced RyR's 3 H-ryanodine binding and triggered robust Ca 2+ -release from RyR-enriched vesicles in the pathologically relevant concentration range. RyR single channel current analysis demonstrated that 200 M TCA induced a 5-fold increase in the channel's open probability and caused a significant lengthening of the mean open time. TCA also suppressed Ca 2+ -uptake rate and ATP-ase activity of SERCA-enriched vesicles, but interestingly, failed to decrease Ca 2+ elimination rate in intact cells. Overall, our results strongly suggest that TCA opens RyR by an allosteric mechanism, which contribute significantly to bile acid-induced pathologic Ca 2+ -leak from the endoplasmic reticulum in pancreatic acinar cells.
Chondrogenic progenitor cells (CPCs) may be used as an alternative source of cells with potentially superior chondrogenic potential compared to mesenchymal stem cells (MSCs), and could be exploited for future regenerative therapies targeting articular cartilage in degenerative diseases such as osteoarthritis (OA). In this study,This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
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