Trafficking mechanisms for secretory vesicles in pancreatic duct epithelial cells

描述（由申请人提供）：一般认为，在急性胰腺炎期间，胰腺受到两种有害的损害：从腺泡细胞释放的病理激活的胰蛋白酶和从邻近胆管流入胰管的胆汁酸。最近的研究表明，胰管上皮细胞（PDEC）对损伤反应积极。我们已经证明，蛋白酶激活受体-2 （PAR-2）在胰蛋白酶的激活下，促进PDEC的两种分泌产物，黏液蛋白和碳酸氢盐。在这个建议中，我们试图了解PAR-2和胆汁酸调节的细胞机制。我们将从研究par -2产生的细胞内Ca2+信号对分泌囊泡（SV）运输的影响开始，SV携带负责碳酸氢盐分泌的粘蛋白或膜转运蛋白。我们将用粘蛋白和转运蛋白的荧光形式标记SV。利用荧光显微镜实时跟踪SV将揭示PAR-2激活如何控制细胞质和质膜附近的SV。我们将通过光学监测黏液蛋白释放和转运蛋白向膜的传递过程中SV与膜的最终融合。在我们最近开发的微室中，将通过测量细胞中特定的荧光染料BCECF来检测碳酸氢盐的分泌率。接下来，我们将测试当胰管将胰蛋白酶从腺泡细胞运送到十二指肠时，黏性和蛋白酶抗性粘蛋白是否覆盖在细胞表面以保护PDEC免受激活胰蛋白酶的影响。我们推测，从PDEC分泌的大量碳酸氢盐和液体进入导管管腔，稀释并冲走了有害的胰蛋白酶和胆汁酸。我们的目的是通过控制PAR-2来提高粘蛋白和碳酸氢盐的分泌，并测试该疗法是否减少了对单层和PDEC的损伤。总之，我们的研究将揭示最近在我们的动物模型中发现的PAR-2改善胰腺炎症状的细胞机制。此外，我们的研究结果将确定急性胰腺炎早期胰腺细胞的细胞反应，这可能导致治疗干预，并将与消化系统疾病的治疗广泛相关。

DESCRIPTION (provided by applicant): 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 is 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. PUBLIC HEALTH RELEVANCE: 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.