Interaction of endocytic vacuoles with cellular organelles as a trigger for the cell damage in acute pancreatitis.

急性胰腺炎是一种常见的危及生命的炎症性疾病。过量饮酒和胆结石是导致这种衰弱状况的重要危险因素。受这种疾病影响的人口比例正在迅速增加。在急性胰腺炎的情况下，通常帮助消化肠道中食物的消化酶在胰腺内变得活跃，这个器官本身就被“消化”了。胰酶是参与这一过程的关键消化酶。在正常的胰腺健康细胞中，胰酶的前体(胰酶原)被包裹在小颗粒(分泌颗粒)中。这种前体不能分解蛋白质或对细胞造成损害。当消化过程被刺激时，由于含有胰酶原的分泌颗粒与细胞表面膜的分泌-融合，胰酶原从细胞中释放出来。通常情况下，胰酶原只有在到达肠道时才被激活，在那里胰酶完成了分解(消化)饮食蛋白质的功能。然而，当我们重新创造导致急性胰腺炎的条件时，胰蛋白酶原在胰腺细胞内被不适当地激活。我们认为，这种情况的发生是因为并不是所有的胰酶原都在分泌过程中从细胞中释放出来，其中一些被带回细胞内，形成被称为内胞空泡(以某种方式获得激活胰酶原并形成活性的、潜在的破坏性胰酶的能力)的囊泡。我们最近发现，内吞液泡可以破裂，并将其内容物释放到细胞内部。我们还发现，液泡可以与细胞表面融合，并将其内容物输送到细胞外空间。这些重要的未发表的观察结果构成了本申请的基础。我们推测，空泡的破裂和/或空泡与细胞表面的融合是引发急性胰腺炎的细胞损伤的关键(因为这些过程应该允许胰酶破坏重要的细胞内和细胞外蛋白)。我们想要检验这一假设。另一个重要且悬而未决的问题是“胰蛋白酶原是如何在内吞液泡中被激活的？”我们认为，这是因为含有胰酶原的空泡与负责细胞内消化的细胞室(细胞器)相互作用。细胞内消化是细胞各部分和蛋白质循环所必需的。在负责细胞内消化的隔室中发现了能够分解蛋白质的分子，原则上它们可以激活胰蛋白酶原。因此，我们正计划研究将胰酶原回收到细胞内的小泡与负责细胞消化的细胞器(内体、溶酶体和自噬体)的相互作用。我们将试图确定这些细胞器中的哪些有助于激活胰蛋白酶原，以及细胞器相互作用的过程是如何启动的。胰酶原激活并不是细胞损伤的唯一标志。其他指标包括负责产生能量的细胞室(线粒体)和维持细胞形状的结构(细胞骨架)的损害。我们将研究胰蛋白酶原激活与细胞损伤的其他指标，以确定导致急性胰腺炎细胞死亡的事件顺序。最后，我们将研究胰酶在一个细胞中产生的损害如何传播并影响器官中的其他细胞。对消化酶激活过程的了解将有助于急性胰腺炎治疗的发展。为了加快治疗方法的开发，项目过程中产生的所有信息、分子探针和方法创新将直接提供给利物浦NIHR胰腺生物医学研究单位进行的药物发现转化研究计划。

Acute pancreatitis is a frequent and life-threatening inflammatory disease. Excessive alcohol intake and gallstones are important risk factors for this debilitating condition. The proportion of the population affected by this disease is increasing rapidly. In conditions of acute pancreatitis, digestive enzymes, which normally help to digest food in the intestine, become active inside the pancreas and this organ "digests" itself. Trypsin is a key digestive enzyme involved in this process. In normal healthy cells of the pancreas, a precursor of trypsin (trypsinogen) is packaged inside small granules (secretory granules). This precursor cannot breakdown proteins or cause damage to the cells. When the process of digestion is stimulated, trypsinogen is released from the cells as a result of secretion - fusion of the secretory granules containing trypsinogen with the surface membrane of the cell. Normally trypsinogen becomes activated only when it reaches intestine where trypsin fulfils its function of breaking down (digesting) dietary proteins. However when we recreated conditions that lead to acute pancreatitis, trypsinogen was activated inappropriately inside the cells of the pancreas. We think that this happens because not all trypsinogen is released from the cell during secretion and some of it is taken back into the cell in vesicles termed endocytic vacuoles (which somehow acquire the ability to activate trypsinogen and to form the active potentially damaging trypsin). We recently discovered that the endocytic vacuoles can rupture and release their contents into the interior of the cell. We also found that the vacuoles can fuse with the cell surface and deliver their contents into the extracellular space. These important unpublished observations form the basis of this application. We hypothesise that the rupture of the vacuoles and/or the fusion of vacuoles with the cell surface are essential for the cell damage that initiates acute pancreatitis (because these processes should allow trypsin to destroy vital intracellular and extracellular proteins). We would like to test this hypothesis. Another important and unresolved question is "How the trypsinogen is activated inside the endocytic vacuoles?" We propose that this happens because the trypsinogen-containing vacuoles interact with cell compartments (organelles) that are responsible for intracellular digestion. Intracellular digestion is needed to recycle the cell parts and proteins. Molecules that can break proteins are found in the compartments responsible for intracellular digestion and they can in principle activate trypsinogen. We are therefore planning to study the interaction of vesicles that retrieve trypsinogen into the cell with organelles responsible for cellular digestion (endosomes, lysosomes and autophagosomes). We will try to determine which of these organelles helps to activate trypsinogen and how the process of organelle interaction is initiated. Trypsinogen activation is not the only hallmark of cell injury. Other indicators include damage to the cellular compartment responsible for energy production (mitochondria) and damage to structures maintaining cell shape (cytoskeleton). We will investigate trypsinogen activation together with the other indicators of cell damage to determine the sequence of events leading to cell death in acute pancreatitis. Finally we will study how damage produced by trypsin in one cell can propagate and affect other cells in the organ. The understanding of the processes leading to activation of digestive enzymes will facilitate the development of the treatment against acute pancreatitis. To accelerate the development of the treatment all information, molecular probes and methodological innovations generated in the course of the project will feed directly into the translational research programme of drug discovery undertaken by the Liverpool NIHR Pancreas Biomedical Research Unit.