Role of Membrane Oxidation in Controlling the Activity of Secretory Phospholipase A2 Toward Apoptotic Lymphoma Cells

Abstract

The membranes of healthy lymphocytes normally resist hydrolysis by extracellular phospholipase A2 (sPLA2). However, they become susceptible during the process of apoptosis. Previous experiments have demonstrated the importance of certain physical changes to the membrane during cell death such as a reduction in membrane lipid order and exposure of phosphatidylserine on the membrane surface. Nevertheless, those investigations also showed that at least one additional factor was required for rapid hydrolysis by the human group IIA sPLA2 isozyme (hGIIa). This study was designed to test the possibility that oxidation of membrane lipids is the additional factor. Flow cytometry and confocal microscopy with a fluorescent probe of oxidative potential, BODIPY, suggested that oxidation of the plasma membrane occurs during apoptosis stimulated by glucocorticoid or thapsigargin. When oxidative potential was high, the activity of hGIIa sPLA2 was enhanced more than 10-fold compared to any other condition favorable to hydrolysis by other sPLA2 isoforms. Moreover, confocal microscopy with BODIPY and the vital stain propidium iodide verified that sPLA2 preferentially attacked oxidized cells. Direct oxidation of cell membranes with either of two oxidizing agents (TBHP and AAPH) also stimulated hydrolysis by sPLA2. Both agents induced externalization of phosphatidylserine, although only TBHP caused a reduction in the apparent order of membrane lipids. These results demonstrated that membrane oxidation strongly stimulates sPLA2 activity, especially that of the hGIIa isoform. Interestingly, the change in membrane order, previously thought to be imperative for high rates of hydrolysis, was not required when membrane lipids were oxidized. Whether phosphatidylserine exposure is still necessary with oxidation remains unresolved since the two events could not be deconvoluted.