Recently, cholecystokinin (CCK) receptors in pancreatic acinar cells have been found to exist in three different affinity states for CCK (high affinity, low affinity and very low affinity) and certain actions of CCK on acinar cells appear to be produced by individual receptor states. Protein kinase C (PKC) enzyme activity has also been found in pancreatic acinar cells; however. there is no information available concerning isoforms of PKC in pancreatic acinar cells. The long-term goal of the present study is to characterize two fundamental regulatory mechanisms of pancreatic acinar cell function; namely, the interaction of CCK with its receptors and the activation of PKC by various agonists, including CCK. To achieve this goal we propose to address four specific questions.
AIM 1. CHARACTERIZE THE THREE STATES OF THE CCK RECEPTOR IN PANCREATIC ACINI. We will test the hypothesis that ligands can be identified that show selective interaction with the different states of the CCK receptor, that ligand occupation of one receptor state can influence the affinities of other receptor states for the same or different ligands and that each receptor state is under separate biochemical regulation. We will use intact acini, permeabilized acini and acinar cell membranes to identify different CCK receptor states by measuring binding of 125I-CCK-8 and [3H]L-364,718 (a CCK receptor antagonist).
AIM 2. RELATE ACTIVATION OF A PARTICULAR STATE OF THE CCK RECEPTOR TO CHANGES IN SIGNAL TRANSDUCTION MECHANISMS AND OTHER ACINAR CELL FUNCTIONS. We will test the hypothesis that occupation of each different state of the CCK receptor in pancreatic acinar cells produces a unique, characteristic repertoire of changes in acinar cell function. We will measure individual CCK receptor states plus a variety of functions including enzyme secretion, cytosolic calcium, IP3(1,4,5), diacylglycerol, cyclic AMP, protein synthesis, uptake of sugars and amino acids. and release of lactate dehydrogenase.
AIM 3. Measure the effects of different pancreatic secretagogues on activation of different isoforms of PKC in pancreatic acini. We will test the hypothesis that agonists cause translocation of PKC-epsilon to zymogen granules and translocation of PKC-zeta to the plasma membrane, and that agonist-induced translocation is always accompanied by phosphorylation of the PKC isoform. We will measure isoforms of PKC using quantitative immunoblot analysis and selective immunoprecipitation of phosphorylated isoforms of PKC.
AIM 4. Examine the relationship of changes in different isoforms of PKC in pancreatic acini to other changes in acinar cell function. We will test the hypothesis that activation of a particular isoform of PKC produces unique, characteristic changes in different acinar cell functions including secretion of digestive enzymes. We will use fresh acini and acini that have been cultured for 24 hours to measure isoforms of PKC and changes in signal transduction mechanisms, digestive enzyme secretion and other cellular functions. These studies should increase our understanding of the regulatory biology of the pancreatic acinar cell, which could lead to new approaches to diseases of the pancreas like pancreatitis and possibly pancreatic cancer.
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