Role of NOS and NOX-4 on pancreatic signal transduction
Cowell, Jessica M.
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Nitric oxide is a signaling molecule which has been shown to be a necessary component for secretion. The actual process of exocytosis that leads to secretion is of yet unknown. This project attempted to understand the protein complex NOX-4 as well as NO, a product of NOS, in their signaling roles that lead to the exocytosis and secretion of digestive enzymes from pancreatic acinar cells. There were several objectives of this project which included determining the location of NOX-4, how hormonal stimulation affected recruitment to the zymogen granules and if NOX-4 was necessary for secretion to occur. Also investigated was the presence of nitrosylated proteins on zymogen granules, an activation strategy which utilizes the product of NOS and NOX-4. This project was completed using lysed pancreatic cells which had been stimulated with CCK. The liberated zymogen granules were collected, lysed and subsequently subjected to SDS-PAGE and western blotting procedures. Antibodies specific for the protein in question, NOX-4, were applied, allowing us to definitively conclude the presence and location of NOX-4. Western blotting procedures also assayed for nitrosylated zymogen granule proteins. The results of these procedures could be quantified using Scion software to identify how CCK stimulation affected NOX-4 recruitment. These experiments allowed us to conclude that NOX-4 resides on the zymogen granule and that there is an increased recruitment for a limited period of time in vitro when subjected to CCK. Also discovered was that, upon inhibition of NOX-4, secretion decreases. This leads to the conclusion that NOX-4 is in fact a vital component in the process of secretion. Finally, western blotting revealed the presence of nitrosylated proteins on the zymogen granule, allowing us to hypothesize how the actual event of exocytosis occurs.