Phospholipase Activation and Arachidonic Acid Release in Cultured Intestinal Epithelial Cells (INT 407)

Abstract

The release of free arachidonic acid (AA) in cultured intestinal epithelial cells (INT 407) was investigated. INT-407 cells were first incubated overnight with radio-labeled ¹⁴C-AA, and most of the incorporated ¹⁴C-AA esterified into phos-phatidylethanolamine, phosphatidylcholine, and phosphatidylinositol. Labeled cells were then exposed to different stimulating agents and the release of free ¹⁴C-AA determined. The calcium ionophore A23187 caused a dose-dependent AA release that was preceded by a rapid uptake and a subsequent efflux of ⁴⁵Ca²⁺. By contrast, phospholipase C from Clostridium perfringens caused a great AA release that was accompanied by an apparent uptake and a sustained intracellular accumulation of ⁴⁵Ca²⁺. The cells also released AA when exposed to the protein kinase C activator, 4β-phorbol-12-myristate-13-acetate (PMA), and this agent, like the diacylglycerol l-oleoyl-2-acetyl-rac-glycerol, significantly potentiated the AA release caused by A23187. Not only A23187-mediated but also phospholipase C- and PMA-mediated AA release was inhibited by 4-bromophenacyl bromide, a known phospholipase A₂ inhibitor. These findings, taken together, indicate that AA release in intestinal epithelial cells can be caused by (i) Ca²⁺-mediated phospholipase activation, (ii) products of phospholipase C activity, and (iii) stimulation of protein kinase C. It is suggested, therefore, that AA release in intestinal epithelial cells is governed by intracellular Ca²⁺, protein kinase C-mediated protein phosphorylation, and activation of phospholipase A₂.

Key Words:

• Arachidonic acid release
• intestinal epithelial cells
• phospholipase A₂
• phospholipase C
• protein kinase C activators