Trafficking mechanisms for secretory vesicles in pancreatic duct epithelial cells

描述(申请人提供)：通常认为，在急性胰腺炎期间，胰腺受到两种有害侮辱的挑战，一种是腺泡细胞释放的病理激活的胰酶，另一种是胆汁酸从邻近的胆管流入胰管。最近的研究表明，胰腺管上皮细胞(PDEC)对这种侮辱有积极的反应。我们已经证明，PDEC中的蛋白酶激活受体-2(PAR-2)被胰酶激活后，促进了PDEC的两种分泌产物，粘蛋白和液体中的碳酸氢盐。在这个提案中，我们试图了解PAR-2和胆汁酸调节的细胞机制。我们将首先研究PAR-2产生的细胞内钙信号对携带粘蛋白或负责碳酸氢盐分泌的膜转运体的分泌小泡(SV)运输的影响。我们将用粘蛋白和转运蛋白的荧光形式标记SV。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中和质膜附近的SV。我们将在粘蛋白释放和转运体输送到膜上的过程中，光学监测SV最终与膜的融合。小苏打的分泌速度将通过测量细胞中一种特定的荧光染料BCECF来检测，因为它们驻留在我们最近开发的微室中。接下来，我们将测试粘性和抗蛋白酶粘蛋白是否覆盖在细胞表面以保护PDEC免受激活的胰酶的影响，同时胰腺导管将胰蛋白酶从腺泡细胞输送到十二指肠。我们假设，大量的碳酸氢盐和液体从PDEC分泌到导管管腔，稀释并冲走有害的胰酶和胆汁酸。我们的目的是通过操纵PAR-2来增加粘蛋白和碳酸氢盐的分泌，并测试该药物是否减少了对单层和PDEC的损害。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，并将广泛相关的消化系统疾病的治疗。

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.