PANCREATIC ACINAR SECRETION--RECONSTITUTED EXOCYTOSIS

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Pancreatic acinar cells synthesize and secrete by exocytosis, a variety of enzymes important in the digestion of all major classes of food. Exocytosis is a ubiquitous cellular process which is understood only in the most general terms. In the pancreatic acinar cell, exocytosis involves binding and fusion of the zymogen granule membrane with the apical plasma membrane domain, releasing digestive enzymes into the acinar lumen. The long term goal is to understand exocytosis at a molecular level by developing a model system reconstituted from subcellular fractions of the pancreatic acinar cell. The specific aims are: 1) to generate monoclonal antibodies (mAb) as specific markers for the apical and basolateral domains of the plasma membrane and the zymogen granule membrane of the pancreatic acinar cell; 2) to purify the apical plasma membrane domain by using domain specific mAbs either in immuno-affinity techniques or as immunochemical markers for the different membrane domains in density gradient fractionation; 3) to determine the necessary conditions for granule-apical plasma membrane binding (e.g., Ca2+, Mg-ATP and nonhydrolyzable analogues, GTP and analogues, electrolytes, or cytosolic components), using immunochemical techniques to follow the interaction; 4) to determine further factors necessary for fusion of bound apical plasma membranes and zymogen granules (e.g., free fatty acids plus those to be tested for effects on binding) monitored by release of granule content, electron microscopy, and immunochemically as an irreversible interaction between the two membranes; and 5) to use mAbs to identify the molecules that act as receptors between zymogen granule membranes and apical plasma membranes in the pancreatic acinar cell.