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

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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 These important unpublished observations form the basis of this application.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.