Arachidonic Acid Release and Catecholamine Secretion from Bovine Chromaffin Cells: Possible Involvement of Phospholipase A(,2) in Exocytosis.

Abstract

During exocytosis a rise in cytosolic Ca('2+) triggers fusion of the secretory vesicle membrane with the plasma membrane via an unknown biochemical mechanism. Many agents which activate exocytosis, such as Ca('2+)-ionophores, phorbol esters, and agonists of Ca('2+)-mobilizing receptors, also activate intracellular phospholipase A(,2). Phospholipase A(,2) is activated by a rise in cytosolic Ca('2+) to catalyze the release from membrane phospholipid of free cis-unsaturated fatty acids which are known to be potent membrane fusion-inducing agents. Because phospholipase A(,2) is activated as a result of increased cytosolic Ca('2+) and because it produces membrane fusion-inducing substances, it is hypothesized that phospholipase A(,2) action constitutes an integral step in the mechanism that links elevated cytosolic Ca('2+) to the vesicle fusion event of exocytosis. This hypothesis was investigated using a well known model system for exocytosis, the cultured bovine adrenal chromaffin cell. Experiments utilizing commonly-used inhibitors of phospholipase A(,2) were inconclusive because of the lack of specificity and unexpected side effects (inhibition of Ca('2+) uptake) associated with these putative inhibitors. However, experiments comparing exocytosis (catecholamine secretion) with arachidonic acid release from intact and digitonin-permeabilized chromaffin cells showed that: (1) both exocytosis and arachidonic acid release are activated by cytosolic Ca('2+) in the low micromolar range, (2) both exocytosis and arachidonic acid release occur with the same time course, (3) both exocytosis and arachidonic acid release are inhibited by the protein alkylating agents p-bromophenacylbromide and N-ethylmaleimide, and (4) both exocytosis and arachidonic acid release are activated by 12-O-tetradecanoylphorbol-13-acetate, a phorbol ester that activates protein kinase C. These findings demonstrate that arachidonic acid release is closely associated with catecholamine secretion and are consistent with a role for phospholipase A(,2) in exocytosis.