Phospholipase D activity in the intestinal mitochondria: activation by oxygen free radicals

Abstract

A prominent feature of cell damage caused by oxidative stress is morphological and functional changes in the mitochondria. The present study looked at the effect of free radical exposure on intestinal mitochondrial lipids. Free radical exposure did not alter neutral lipids, but among the phospholipids, phosphatidylethanolamine (PE) content was decreased on exposure to superoxide anion, generated by xanthine-xanthine oxidase or menadione with a concomitant increase in the level of phosphatidic acid (PA), suggesting activation of phospholipase D (PLD). This enzyme did not show transphosphatidylation activity in the presence of ethanol or butanol, and the product formed was phosphatidic acid (PA). This was confirmed by separation of reaction products by HPLC. This alteration in mitochondrial phospholipid was abolished by the presence of superoxide dismutase. Exposure to H₂O₂ did not have any significant effect. Activation of PLD by free radicals was further confirmed by quantitation of ethanolamine released from PE. Absence of any change in the content of lysophospholipid or diglyceride following exposure of mitochondria to superoxide ruled out the involvement of phospholipase A2 or C in the altered lipid composition. Moreover, inclusion of phospholipase A₂ inhibitors, chlorpromazine, or p-bromophenacyl bromide did not prevent the generation of PA on exposure to free radicals. These findings suggest that superoxide anion stimulates intestinal mitochondrial PLD resulting in PE degradation and PA formation. These alterations in mitochondrial lipids may play a role in causing the functional alteration seen in oxidative stress.