Phorbol Ester, Kinase C & Exocrine Pancreatic Secretion

Wrenn, Robert

Abstract

The proposed project seeks to provide new information concerning the molecular mechanism of Ca2+ as a regulator of pancreatic exocrine secretion. A well-established regulatory pathway is the protein kinase-catalyzed phosphorylation of cellular proteins, with subsequent alterations in the functional properties of those proteins. Ca2+ activates various protein kinases, among them one requiring phospholipid and Ca2+ for stimulation (phospholipid/Ca2+ dependent protein kinase; PL/Ca-PK; C kinase). Activation of PL/Ca-PK is also linked to membrane phospholipid turnover, which is increased during stimulation of exocrine secretion. PL/Ca-PK and its substrate proteins have been demonstrated in pacreatic acinar cells. Our preliminary studies indicate a differential distribution of PL/Ca-PK and its substrates within subcellular compartments, including components of the secretory apparatus. Tumor promoting phorbol esters have recently been linked closely with PL/Ca-PK in studies using tissue culture cell lines, appearing to cause a redistribution of the kinase within the cell. The proposed studies will examine the effects of phorbol ester treatment of pancreatic acinar cells upon subcellular distribution of PL/Ca-PK and its substrates, and correlate these effects with secretion. Comparative studies will be carried out using cells stimulated with cholinergic agonists or hormones. Using acinar cells with 32P-labelled ATP pools, an attempt will be made to compare proteins phosphorylated in the intact cell in response to secretogogue or phorbol ester treatment with those substrate proteins noted in defined subcellular fractions. The possible existence and nature of a phorbol ester receptor on acinar cells will also be investigated. Pharmacologic inhibitors specific for PL/Ca-PK will also be utilized to examine this kinase as a regulator of secretion. The project will therefore utilize newly-discovered chemical probes to attempt to define a role for PL/Ca-PK in the modulation of pancreatic exocrine function. Hopefully, knowledge obtained will contribute to the understanding of control pathways in the acinar cell, providing some basis for suggesting points of pathologic lesion in diseases such as cystic fibrosis.