Phospholipase C from Clostridium perfringens Stimulates Phospholipase A₂-Mediated Arachidonic Acid Release in Cultured Intestinal Epithelial Cells (INT 407)

Abstract

The mechanisms by which phospholipase C from Clostridium perfringens stimulates release of arachidonic acid (AA) in cultured intestinal epithelial cells (INT-407) were investigated. INT-407 cells were first allowed to incorporate ¹⁴C-labeled AA into their phospholipids; the labeled cells were then exposed to phospholipase C, and the release of free ^l4C-AA was determined. Phospholipase C caused a rapid (3 min) intracellular rise of free ^l4C-AA, followed by a considerable, dose- and time-dependent release of ^l4C-AA into the extracellular medium. For comparison, the calcium ionophore A23187 also caused a rapid mobilization of free ¹⁴C-AA, but a much lower extracellular ¹⁴C-AA release than phospholipase C during longer (1 h) incubation. The ¹⁴C-AA release was accompanied by a degradation of ^l4C-myo-inositol-labeled phosphatidylinositols and was reduced by the protein kinase C inhibitor l-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). Both phospholipase C- and A23187-stimulated ¹⁴C-AA release was associated with degradation of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol and was reduced by nor-dihydroguaiaretic acid and 4-bromophenacyl bromide, two known phospholipase A₂ inhibitors. In addition, the ¹⁴C-AA release was reduced by the calmodulin inhibitors trifluoperazine, compound 48/80, and A'-(6-aminohexyl)-5-chloro-l-naphthalene-sulfonamide (W-7). These findings indicate that phospholipase C from C. perfringens stimulates phospholipase A₂-mediated AA release from human intestinal epithelial cells and suggest that this stimulation is brought about via processes involving phosphatidylinositol breakdown and activation of calmodulin and protein kinase C. It is possible that this phospholipase C-evoked AA release may contribute to the mucosal pathologic condition in diseases with altered intestinal microbial flora.

Key Words:

• Arachidonic acid
• bacterial phospholipase C
• intestinal epithelial cells
• phospholipase A₂ activation