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

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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.

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