Phospholipase activation and arachidonic acid release in cultured intestinal epithelial cells (INT 407).
The release of free arachidonic acid (AA) in cultured intestinal epithelial cells (INT 407) was investigated. INT-407 cells were first incubated overnight with radiolabeled 14C-AA, and most of the incorporated 14C-AA esterified into phosphatidylethanolamine, 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 alos released AA when exposed to the protein kinase C activator, 4 beta-phorbol-12-myristate-13-acetate (PMA), and this agent, like the diacylglycerol 1-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.
Gustafson
C
C
Clinical Research Center, Linköping University, Sweden.
Tagesson
C
C
D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000493:pharmacokinetics*; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D010740:Phospholipases / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000494:pharmacology
D001095:Arachidonic Acids; D004075:Diglycerides; D016718:Arachidonic Acid; D000001:Calcimycin; C038237:1-oleoyl-2-acetylglycerol; D010740:Phospholipases; D010738:Type C Phospholipases; D013755:Tetradecanoylphorbol Acetate