The acinar cell of the exocrine pancreas synthesizes and secretes approximately 20 digestive enzymes. Both dietary and hormonal factors have been implicated in the regulation of the synthesis of these enzymes. The mechanisms whereby this regulation is effected are not known. Further, it is not clear whether pancreatic enzyme synthesis and secretion are linked via intracellular feedback mechanisms, coupled to respond to the same stimuli, or independently regulated. The divalent cation manganese (Mn) exerts direct effects on pancreatic protein synthesis in rat pancreatic acini, enhancing synthesis at low concentrations, and inhibiting synthesis at high concentrations. Mn also enhances pancreatic digestive enzyme release. Cholecystokinin (CCK), a pancreatic secretagogue that mobilizes intracellular Ca2+ by activating the phosphatidylinositol cascade, also exerts biphasic effects on pancreatic protein synthesis. The actions of Mn on protein synthesis, like the actions of CCK, are modulated by Ca2+ and are greatly enhanced in diabetic rat acini. Further, Mn and CCK alter protein phosphorylation and enhance Ca2+ efflux in the pancreatic acinar cell. Inasmuch as the pancreas is rich in Mn, these observations suggest that Mn may coordinate the coupling of pancreatic digestive enzyme synthesis with enzyme secretion, and act as an intracellular mediator that modulates acinar cell responsiveness to second messengers and their effector pathways. This possibility is strengthened by our recent observation that Mn-mediated phosphorylation of a cytosolic substrate is calmodulin-dependent and hormonally regulated. To test the hypothesis that Mn participates in the physiological regulation of pancreatic exocrine function, the proposed studies will focus on two aspects of Mn action. First, the effects of Mn on protein phosphorylation in isolated rat pancreatic acini and in homogenates of whole pancreas will be determined, in order to characterize the effects of Mn on the activities of cytosolic and particulate kinases that are endogenous to the exocrine pancreas. Specific substrates that are phosphorylated in the presence of Mn will be identified. The actions of any Mn-dependent kinase in the pancreatic acinar cell will be characterized with respect to substrate specificity, and compared with the effects of other divalent cations and with previously described kinases. Polyclonal antibodies will be raised against a protein whose phosphorylation by Mn is modulated by insulin and calmodulin in order to study its developmental expression. These antibodies and the appropriate synthetic oligonucleotides will be used to screen cDNA libraries in order to isolate the cDNA corresponding to the mRNA coding for the protein. Second, the importance of Mn in the regulation of cellular Ca2+ homeostasis will be assessed by comparing its effects on cellular calcium fluxes with its actions on phospholipid hydrolysis in both basal and CCK-stimulated states, and by studying the actions of Mn and CCK on a putative acinar cell calcium channel using the patch-clamp technique.
