Effects of cholecystokinin (CCK) and other secretagogues on isoforms of protein kinase C (PKC) in pancreatic acini

Abstract

We used rat pancreatic acini and measured the effects of various agents on digestive enzyme secretion, diacylglycerol (DAG) and the cellular distribution of protein kinase C (PKC) enzyme activity as well as isoforms of PKC determined by quantitative immunoblot analysis. TPA, but not CCK-8, caused translocation of PKC enzyme activity from the cytosol fraction to the membrane fraction. Immunoblot analysis detected PKC-α, PKC-δ, PKC-ϵ and PKC-ζ. PKC-β, PKC-γ and PKC-η were not detected. TPA caused translocation of all isoforms from cytosol to membrane, whereas CCK-8 caused translocation of PKC-δ and PKC-ϵ, carbachol caused translocation of PKC-ϵ, and bombesin and secretin caused no detectable translocation of any isoform. Specific receptor antagonists could prevent, as well as reverse completely, the translocation of PKC isoforms caused by CCK-8 or carbachol. Agonists added in sequence with an interposed addition of a specific receptor antagonist caused cycling of PKC-ϵ between cytosol and membrane fractions. Each receptor-mediated agonist that caused translocation of PKC also increased DAG, and with CCK-8 and carbachol cycling of PKC-ϵ between cytosol and membrane was accompanied by corresponding cyclic changes in cellular DAG. CCK-JMV-180, bombesin and secretin increased DAG but did not cause translocation of any PKC isoform. Translocation of a PKC isoform could be accounted for by whether the increased DAG originated from PIP 2 (accompanied by translocation) or from phosphatidylcholine (no accompanying translocation). Thus it appeared that DAG, in pancreatic acini, is functionally compartmentalized depending on the source of the lipid. Studies using CCK-8 and CCK-JMV-180 indicated that occupation of the low affinity state of the CCK receptor by either peptide increased DAG from phosphatidylcholine, whereas occupation of the very low affinity state by CCK-8 increased DAG from PIP 2 and caused translocation of PKC-δ and PKC-ϵ. TPA stimulated amylase secretion, indicating that activation of PKC can stimulate enzyme secretion; however, with the various receptor-mediated secretagogues there was no consistent, unequivocal correlation between translocation of an isoform of PKC and accompanying changes in enzyme secretion.