It is generally believed that the pancreas is challenged by two harmful insults during acute pancreatitis, pathologically activated trypsin, released from acinar cells, and bile acids flowing into pancreatic ducts from adjacent bile ducts. Recent research suggests that pancreatic duct epithelial cells (PDEC) react actively to the insults. We have demonstrated that protease- activated receptor-2 (PAR-2) in PDEC, activated by trypsin, promotes two secretory products of PDEC, mucin and bicarbonate with fluid. In this proposal we seek to understand the cellular mechanisms of modulation by PAR-2 and bile acids. We will start by examining the effect of the PAR-2-generated intracellular Ca2+ signal on the trafficking of secretory vesicles (SV) that carry mucin or membrane transporters responsible for bicarbonate secretion. We will label the SV with fluorescent forms of both mucin and transporters. Tracking the SV in real-time with fluorescence microscopy will reveal how PAR-2 activation controls the SV in the cytoplasm and near the plasma membrane. We will monitor optically the final fusion of the SV to the membrane during the release of mucin and the delivery of the transporters to the membrane. The rate of bicarbonate secretion will be detected by measuring a specific fluorescent dye BCECF in the cells as they reside in our recently developed microchamber. Next, we will test whether sticky and protease-resistant mucin proteins coat the cell surface to protect PDEC from the activated trypsin, while pancreatic ducts deliver trypsin from acinar cells to the duodenum. We hypothesize that a high volume of bicarbonate and fluid, secreted from PDEC into the ductal lumen, dilutes and flushes away the harmful trypsin and bile acids.
Our aim i s to elevate mucin and bicarbonate secretion by manipulating PAR-2 and to test whether the remedy reduces the damage to the monolayer and PDEC. In summary our study will reveal cellular mechanisms responsible for the recently found PAR-2 effect ameliorating pancreatitis symptoms in our animal models. In addition, our results will identify cellular responses of pancreatic cells at the early stage of acute pancreatitis that may lead to therapeutic intervention and will be broadly relevant to treatment of disorders of the digestive system.
We seek to understand the cellular mechanisms of modulation of pancreatic duct epithelial cells by PAR-2 and bile acids. Our results will identify cellular responses of pancreatic cells at the early stage of acute pancreatitis and potentially could lead to therapeutic intervention. The experiments are broadly relevant to treatment of disorders of the digestive system.
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|Park, Yongsoo; Seo, Jong Bae; Fraind, Alicia et al. (2015) Synaptotagmin-1 binds to PIP(2)-containing membrane but not to SNAREs at physiological ionic strength. Nat Struct Mol Biol 22:815-23|
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