EXCEED THE SPACE PROVIDED. Understanding pancreatic secretion is important both as it relates to the function of this digestive organ and in understandingbasic cellular mechanisms. Secretagogues such as cholecystokinin (CCK) and acetylcholine (ACh) act on pancreatic acinar cells to increase cytoplasmic Ca2+ and to release digestive enzymes. There is good evidence that the increase in Ca2+ is both necessary and sufficient to drive enzyme secretion. However, little is known of how Ca2+serves as an intracellular messenger to control digestive enzyme secretion. This requires better understanding of Ca2+ regulated kinases and phosphatases in acini, their target molecules, and the molecules on the zymogen granule involved in movement to the apical membrane and exocytosis.
Specific aims of this proposal include: 1) identifying the role in secretion of the calcium sensitive phosphatase, calcineurin by use of inhibitors and autoinhibitory domain peptides as well as study of mice with targeted deletion. 2) We have recently identified Rab3D on the zymogen granule and shown that overexpression in transgenic mice enhances early secretion. We will carry out transgenic and gene knockout studies to evaluate the effect of Rab3D deletion on secretion. Initial studies will use adenoviral vectors to express RabSD with altered guanine nucleotide binding to test the hypothesis that Rab3D-GTP regulates exocytosis. We will also evaluate how CCK and ACh regulate Rab3D GTP binding and hydrolysis and identify downstream target proteins with which Rab3D interacts. 3) We will evaluate the role of other proteins on the secretory granule in exocytosis with focus on the function of the SNARE protein VAMP and the heterotrimeric G protein Gq/n. In all these studies we will utilize molecular techniques to characterize and alter the molecules involved in secretion. Permeabilized cells, microinjection, adenoviral vectors and transgenic animal techniques will be used to introduce dominant negative molecules, inhibitory domains, or competing fragments. Secretion will be measured in populations of cells by biochemical assays and in single cells by optical or patch clamp technique. A new technique to visualize secretion at the single cell level using a green fluorescent protein-ribonuclease chimera, is being developed. These studies on the mechanism of acinar cell secretion are also relevant to understanding of the abnormal secretion seen in pancreatic diseases such as pancreatitis. PERFORMANCE SITE ========================================Section End===========================================
Showing the most recent 10 out of 61 publications
