MECHANISMS OF PEPSINOGEN SECRETION FROM CHIEF CELLS

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The long-range goals of this project are to elucidate the cellular mechanisms of pepsinogen secretion. For this purpose, the Principal Investigator has developed methods for preparing a nearly homogeneous population of dispersed chief cells that secrete pepsinogen in response to various stimuli. These cells have been used to determine that changes in cAMP mediate the actions of secretin, vasoactive intestinal peptide, prostaglandins, and cholera toxin, whereas changes in cell calcium concentration play a role in mediating the actions of carbachol, cholecystokinin, and calcium ionophores. Although it has been known for several years that potentiation of pepsinogen secretion occurs when agents whose actions involve changes in cAMP are combined with agents whose actions involve changes in cell calcium, the cellular mechanisms mediating this phenomonon were unknown. However, during the previous period of funding, the Principal Investigator discovered that agents whose actions are mediated by changes in cell calcium can potentiate cAMP-mediated enzyme secretion by means of a calcium/calmodulin-dependent activation of the adenylate cyclase system. In other tissues, similar effects appear to be caused by a protein kinase C-mediated phosphorylation of components of adenylate cyclase. Therefore, in the present application, we propose to test the following hypothesis: In chief cells, protein kinase C, activated by secretagogue-induced stimulation of the calcium/phosphoinositide messenger system, has calcium-calmodulin- dependent actions on the adenylate cyclase system that result in augmentation of cellular levels of cAMP, thereby causing potentiation of pepsinogen secretion. This hypothesis will be tested by using various inhibitors of calmodulin and activators and inhibitors of protein kinase C, and by modulating intra- and extra- cellular calcium concentration to determine the role of these cellular mediators in the interaction between second messenger systems. We will also determine whether phosphorylation of chief cell proteins, such as components of the adenylate cyclase system, occurs when cells are stimulated by secretagogues that activate protein kinase C. This new direction for our laboratory will require consultation with faculty in the Dept. of Biochemistry who agree to lend their support to this project. These experiments will increase our understanding of so-called "cross-talk" between second messenger systems in chief cells. Moreover, the new information gained from these studies should increase our understanding of signal transduction in secretory cells in general.