Ca2+ Signalling, Organelle Dysfunction and Pancreatitis

Pancreatitis is a very common and serious illness caused by gallstones, which can make bile go into the pancreas, or by drinking excess alcohol, which is turned into dangerous chemicals in the pancreas. One in five people with pancreatitis have severe disease that may need intensive care and treatment in hospital for six months, and that can lead to chronic illness or death. The pancreas makes digestive juice containing enzymes, which break down food to be absorbed, but in pancreatitis the enzymes attack the pancreas itself. Unfortunately there are few treatments for pancreatitis, and no useful drugs, so this disease must be understood better. We are a leading team of scientists and hospital doctors who have discovered that calcium is the most important signal in the release of enzymes from the cells of the pancreas, and that when the calcium signals go wrong, the cells are damaged and pancreatitis develops. We have also found out how the calcium signals work in these cells, and have discovered that the calcium signals go wrong when bile or chemicals from alcohol get into the pancreas. Building on this work, we aim to find out how alcohol and bile damage the pancreas and how such damage and its devastating effects can be prevented or limited. We will carry out many experiments using the most modern equipment with high-powered microscopes that show changes in living cells, including human cells given freely and willingly by pancreas patients. We will research changes in each part of each cell that can cause pancreatitis, or make it worse. Our research will include testing substances that could be used to prevent or treat pancreatitis, and so could improve the outcome of the many patients who suffer from this disease.

The acinar cells of the exocrine pancreas secrete a battery of zymogens essential for digestion, but premature intracellular digestive enzyme activation, mostly from cholelithiasis or excessive alcohol intake, leads to the common, devastating disease pancreatitis. The lack of specific treatment of pancreatitis, with over 200 almost entirely negative randomized clinical trials to date, demonstrates the need to fully understand the pathogenesis. We have discovered that local apical increases (repetitive spikes) in the cytosolic Ca2+ concentration ([Ca2+]i), elicited by acetylcholine or cholecystokinin, regulate normal acinar secretion, whereas sustained and global [Ca2+]i elevations, which can be induced by bile acids or non-oxidative ethanol metabolites, result in intracellular digestive enzyme activation, vacuole formation and cell death. Our aim is to understand how alcohol and bile damage the pancreas and how such damage and its consequent impact can be prevented or limited. We will use our combined strengths as internationally recognized investigators of Ca2+ signalling and leaders within the UK?s foremost centre for clinical pancreatology to investigate the mechanisms underlying toxic Ca2+ release from the various subcellular pools, the roles of mitochondrial inhibition, of reactive oxygen and nitrogen species (ROS and RNS), vacuole formation and the pathological digestive enzyme activation that causes the disease. We will employ patch clamp technology with confocal, two-photon and TIRF microscopy using isolated acinar cells and cell clusters including our two-photon permeabilized preparations, as well as intact pancreata, isolated or in vivo. Novel methods include those for measuring and changing intracellular ATP concentrations ([ATP]i), nucleofection and assessing vacuole formation in vivo. Various pancreatitis models will be used, including transgenics and KOs. We will examine the effects of bile and ethanol metabolites on changes in [Ca2+]i, ROS/RNS, mitochondrial membrane potential and [ATP]i, as well as the balance of oxidative and non-oxidative ethanol metabolism in the switch from apoptosis to necrosis. We will monitor Ca2+ changes simultaneously with premature intracellular enzyme activation in zymogen granules, lysosomes, endosomes and vacuoles within intact or permeabilised cells, or isolated organelles. We will determine the contribution of failure of exocytic pore expansion and of the formation, transport and rupture of endocytic vacuoles, including release of intravacuolar digestive enzymes. Key findings made in rodent cells will be explored in human cells. Specific interventions in the pathological signalling cascade will be tested, such as inhibition of Ca2+ release or Ca2+ entry, changes in FAEE metabolism and/or oxidant defence, to pave the way for eventual rational treatment.