Premature activation of pancreatic digestive zymogens within the acinar cell is a pivotal early step in initiation of acute pancreatitis, but the mechanisms underlying this phenomenon are largely unknown. Cytosolic events, particularly an increase in intracellular Ca2+, regulate the activation of zymogens within intracellular compartments, although the specific identity of these compartments remains unclear. To examine the identity of the compartment(s) and the molecular mechanism of this activation, we have performed the following preliminary studies. First, using a caerulein-hyperstimulation model of acute pancreatitis, we find that activated zymogens are found in both zymogen granule and microsomal fractions. Next, to directly examine pathways that regulate the activation, we have developed a reconstituted system using isolated pancreatic cytosol and a 100,000xg particulate fraction. Using this system we find that zymogen activation requires the presence of both cytosol and the particulate fraction. This activation is prominently enhanced by ATP (5 mM) but not by the inactive ATP analogue, AMP-PNP. The ATP-dependent activation is further enhanced by the addition of Ca2+ to the incubation medium and reduced by calcium chelation. When the particulate fraction was separated into zymogen granule and microsomal fractions, ATP and Ca2+ -dependent zymogen activation was observed in both. These studies are the first to report reconstitution of zymogen activation using acinar cell fractions. They demonstrate that activation occurs in relevant compartments, requires cytosolic factors and ATP and is enhanced by Ca2+. We plan to use this system to identify the compartment(s) responsible for zymogen processing and to examine the ATP and calcium-dependent mechanism of activation. ? ? ?
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