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

被引：17

作者：

Withnall, MT

Pennington, A

Wiseman, D

机构：

[1] Discovery Biology, Rhône-Poulenc Rorer Ltd., Dagenham, Essex RM10 7XS, Rainham Road South

来源：

BIOCHEMICAL PHARMACOLOGY | 1995年 / 50卷 / 11期

关键词：

cytosolic phospholipase A(2); arachidonate mobilisation; differentiated U937 cells;

D O I：

10.1016/0006-2952(95)02084-5

中图分类号：

R9 [药学];

学科分类号：

1007 ;

摘要：

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 Ca2+ 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 [H-3]AA release from pre-labelled cells by at least 80%, with an IC50 (12.7 +/- 1.4 mu M) not significantly different from that for inhibiting authentic cPLA(2) (9.3 +/- 2.0 mu M). On the other hand, type II PLA(2) activity was not detected in cell homogenates, and [H-3]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 [H-3]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 [P-33] labelling of cPLA(2) immunoprecipitates was seen in cells pre-labelled with [P-33] 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 [H-3]AA release requiring the activation of only a small proportion of the cell pool of cPLA(2) or, alternatively, by increased release reflecting greater Ca2+-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 Ca2+ from functionally activating the enzyme intracellularly.

引用

页码：1893 / 1902

页数：10

共 47 条

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