Phospholipase Activation and Arachidonic Acid Release in Isolated Intestinal Epithelial Cells

Abstract

A novel method for studying the mobilization of free arachidonic acid (AA) in isolated intestinal epithelial cells is described. The method is based on labeling the cellular phospholipids with ¹⁴C-AA and studying the release of this ¹⁴C-AA on subsequent phospholipase activation. Cells of high viability were isolated from the small intestine of guinea pigs and incubated with ¹⁴C-AA for 2 h; most of the incorporated ¹⁴C-AA was then esterified into phosphatidylethanolamine and phosphatidylcholine. When the labeled cells were stimulated with the calcium ionophore A23187 in the presence of external calcium, they released significant amounts of AA. In contrast, the cells released no AA when stimulated with A23187 in the absence of external calcium or in the presence of chlorpromazine or 4-bromophenacyl bromide, both of which are known to inhibit phospholipase A₂ activity. On the other hand, the cells released significant AA in response to exogenous phospholipase C from Clostridium perfringens. These findings indicate that AA release in intestinal cells may be caused by calcium-mediated phospholipase A₂ activation or by products of microbial phospholipase C activity. They also suggest the further use of ¹⁴C-AA-labeled cells for studying agents and mechanisms that may influence the release of AA in the gastrointestinal tract.

Key Words:

• Arachidonic acid release
• intestinal mucosa
• isolated epithelial cells
• phospholipase A₂
• phospholipase C