The objective of this proposal is to define the molecular mechanisms involved in both normal and abnormal pancreatic enzyme secretion. Our focus is on the pancreatic acinar cell and the mechanisms that support the agonist-induced formation and transport of zymogen granules from the trans-Golgi network (TGN) to the apical membrane for subsequent enzyme release. Using both primary acini preparations and a novel cultured pancreatic acinar cell model that we developed, we have made the following novel observations. (i), CCK-stimulation induces vesiculation of the Golgi apparatus while increasing zymogen granule transport, (ii), specific vesicle-coat and cytoskeletal proteins are recruited to the acinar cell TGN following agonist stimulation, and (iii), the microtubule (Mt) associated motor enzyme kinesin is associated with zymogen granules and, along with Mts, is required for granule movement. This study will utilize state-of-the-art microscopic imaging techniques of living and fixed acinar cells expressing GFP-protein constructs, combined with biochemical and molecular methods to test the following CENTRAL HYPOTHESIS: specific coat and motor proteins in the acinar cell are recruited and activated during a secretory stimulus to mediate the appropriate formation and transport of zymogen granules from the TGN to the apical lumen. We predict that disruption of this zymogen transport machinery will lead to missorting of nascent proteases in the acinar cell and subsequent pancreatitis. Thus, we propose three SPECIFIC AIMS. First, we will define the structure of the acinar cell TGN and observe how it changes during stimulated secretion. Mechanistic studies will be conducted to inhibit motor and coat function and subsequently assess the effects on TGN structure and zymogen granule formation. Second, we will define which motor proteins support the transport of zymogen granules to the apical lumen and test whether these motors are upregulated during secretion. Third, we will directly test if a disruption of coat and motor proteins, or suprastimulation, leads to an aberrant missorting and subsequent release of zymogens into the cytoplasm. To our knowledge this will be one of the first studies to manipulate and view vesicle formation and transport in living acinar cells as they secrete. We are confident that this study will make important contributions essential to understanding pancreatic acinar cell function and disease.
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