In the exocrine pancreas the major intracellular transduction pathway for hormone and neurotransmitter stimulated enzyme secretion is the calcium-phosphatidylinositol (PI) system. Receptor occupation causes the hydrolysis of phosphatidylinositol 4,5 bisphosphate to inositol 1,4,S-trisphosphate (IP3) and 1,2- diacylglycerol (DAG). IP3 serves to mobilize intracellular calcium and DAG activates protein kinase C (PKC). The production of these compounds has been proposed to mediate pancreatic amylase secretion. Three phases of pancreatic amylase secretion have been described: initial, sustained, and postmaximal. However, the specific roles for the components of the PI system in regulating the three phases of pancreatic amylase secretion remain unclear. We propose that PKC plays a pivotal role in regulating each of the phases of pancreatic amylase secretion. We will demonstrate that translocation of PKC from cytosol to the cell membrane is an essential step for the initial release of amylase and that this is mediated by increased intracellular calcium induced by IP3. We will establish the time course of translocation of PKC upon stimulation and determine the contribution of PKC to the initial phase of amylase secretion. To evaluate the requirement for intracellular calcium to mediate translocation of PKC we will utilize drugs which either selectively block intracellular mobilization of calcium or antagonize calcium flux across the cell membrane. We propose that continued activation of PKC by DAG is essential for the sustained secretion of amylase. The magnitude and temporal relationship between DAG and PKC during sustained amylase secretion will be examined using activators and inhibitors of PKC. We hypothesize that during the sustained release phase that DAG acts in concert with calcium flux across the cell membrane to maintain PKC activity. The role of calcium flux will be examined using Ca++-channel blockers, measurement of 45Ca flux, and stimulation of Ca++ flux with calcium channel agonists. Lastly, we suggest that the postmaximal diminution in amylase secretion observed in the presence of supramaximal concentrations of secretagogues is regulated by high membrane PKC activity mediating internalization of agonist receptors. To test this hypothesis the magnitude of PKC activity and amylase release after supramaximal stimulation with secretagogues will be evaluated in the presence and absence of PKC inhibitors. In addition, changes in PI turnover and receptor binding studies to known secretagogues will be performed under various experimental conditions which influence PKC activity. These studies should increase our understanding of intracellular stimulus-secretion coupling events which regulate the initial, sustained and postmaximal phases of pancreatic secretion.
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