Characterisation of cytosolic phospholipase A 2 as mediator of the enhanced arachidonic acid release from dimethyl sulphoxide differentiated U937 cells

Abstract

Studies were performed to characterise the phospholipase A 2 (PLA 2) responsible for the greatly increased capacity to release arachidonic acid (AA) of dimethyl sulphoxide (DMSO) differentiated U937 monocytic cells compared to undifferentiated cells (18-fold increase in response to Ca 2+ ionophore A23187). Cytosolic PLA 2 (cPLA 2) activity could be measured in homogenates of differentiated cells, and the highly selective cPLA 2 inhibitor arachidonic acid trifluoromethyl ketone reduced A23187 induced [ 3H]AA release from pre-labelled cells by at least 80%, with an IC 50 (12.7 ± 1.4 μM) not significantly different from that for inhibiting authentic cPLA 2 (9.3 ± 2.0 μM). On the other hand, type II PLA 2 activity was not detected in cell homogenates, and [ 3H]AA release was not inhibited by heparin (1 mg/mL), which binds secreted type II PLA 2 and reduces its ability to degrade membrane phospholipids. Stimulation of intact cells with A23187 plus phorbol myristate acetate (PMA) under conditions that released [ 3H]AA did not increase cPLA 2 activity of the cell homogenate, and there was little difference between DMSO differentiated and undifferentiated cells in cPLA 2 protein content, cPLA 2 specific activity of homogenates, or distribution of cPLA 2 between membrane and cytosol in the resting cell. Following stimulation with A23187 plus PMA, no increase in [ 33P] labelling of cPLA 2 immunoprecipitates was seen in cells pre-labelled with [ 33P] orthophosphate, nor a change in electrophoretic mobility of cPLA 2. It was concluded that cPLA 2 releases the bulk of AA from stimulated, DMSO differentiated U937 cells. The failure to observe increased cPLA 2 specific activity following cellular stimulation could be explained by increased [ 3H]AA release requiring the activation of only a small proportion of the cell pool of cPLA 2 or, alternatively, by increased release reflecting greater Ca 2+-dependent association of cPLA 2 with membrane substrate rather than increased specific activity per se. There was no evidence that any such increased membrane association resulted from cPLA 2 phosphorylation. The relative inability of undifferentiated cells to release AA was not due to the absence of cPLA 2 or an altered distribution between membrane and cytosol, but suggested the presence of a repressor mechanism that prevents elevated Ca 2+ from functionally activating the enzyme intracellularly.