Membrane active agents exert their actions by modifying calcium (Ca++) availability and the composition of membrane phospholipids. Our previous investigations suggest that a pivotal biochemical event in membrane perturbations involves the Ca++ dependent activation of membranous phospholipase A2 which cleaves arachidonic acid from position-2 of phospholipids, particularly phosphatidylinositol (PI). Thus, the overal aim of this present study is to develop a clearer picture of the events associated with stimulus secretion coupling in the exocrine pancreas by comprehensive investigation of the interactions between Ca++, phospholipid metabolism, and arachidonic acid metabolism. These studies will be performed by incubating isolated rat pancreatic acini prepared by proteolytic digestion with 14C-arachidonate to label phospholipids and arachidonate metabolites, and/or 35S-glutathione to label leukotrienes C, D, or E. Phospholipid turnover and arachidonic acid metabolism will be monitored in intact acini and in homogenates and subcellular fractions by high performance liquid chromatography. The ability of various stimuli and inhibitors to modify arachidonate metabolism and/or amylase release will be explored. Since our previous studies implicate the deacylation-reacylation pathway in arachidonate metabolism of secretory cells, we will also probe the subcellular distribution of acylation of lysophospholipids in purified subcellular fractions of acini. Finally, we will examine the possible association between diacyglycerol derived from the receptor-linked breaddown of PI through a phospholipase C-mediated reaction and amylase secretion. We will utilize phorbol esters as a pharmacological tool to directly activate protein kinase C, and thus act as a substitute for diacylglycerol. Levels of free cellular Ca++ will be monitored with quin2 during stimulation of acini with phorbol esters and other pancreatic secretagogues. These studies may render support for the concept that synergistic - but independent - actions of Ca++ and the diaylglycerol-protein kinase C system exist to evoke pancreatic secretion. Our findings should not only help to elucidate the nature of calcium's action in the regulation of amylase release, but they should also provide additional clues toward expanding our knowledge of the pathophysiological processes associated with diseases of exocrine secretion such as cystic fibrosis.
