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Characterisation of cytosolic phospholipase A2 as mediator of the enhanced arachidonic acid release from dimethyl sulphoxide differentiated U937 cells.

Abstract：

:Studies were performed to characterise the phospholipase A2 (PLA2) 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 PLA2 (cPLA2) activity could be measured in homogenates of differentiated cells, and the highly selective cPLA2 inhibitor arachidonic acid trifluoromethyl ketone reduced A23187 induced [3H]AA release from pre-labelled cells by at least 80%, with an IC50 (12.7 +/- 1.4 microM) not significantly different from that for inhibiting authentic cPLA2 (9.3 +/- 2.0 microM). On the other hand, type II PLA2 activity was not detected in cell homogenates, and [3H]AA release was not inhibited by heparin (1 mg/mL), which binds secreted type II PLA2 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 cPLA2 activity of the cell homogenate, and there was little difference between DMSO differentiated and undifferentiated cells in cPLA2 protein content, cPLA2 specific activity of homogenates, or distribution of cPLA2 between membrane and cytosol in the resting cell. Following stimulation with A23187 plus PMA, no increase in [33P] labelling of cPLA2 immunoprecipitates was seen in cells pre-labelled with [33P] orthophosphate, nor a change in electrophoretic mobility of cPLA2. It was concluded that cPLA2 releases the bulk of AA from stimulated, DMSO differentiated U937 cells. The failure to observe increased cPLA2 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 cPLA2 or, alternatively, by increased release reflecting greater Ca(2+)-dependent association of cPLA2 with membrane substrate rather than increased specific activity per se. There was no evidence that any such increased membrane association resulted from cPLA2 phosphorylation. The relative inability of undifferentiated cells to release AA was not due to the absence of cPLA2 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.

journal_name

Biochem Pharmacol

journal_title

Biochemical pharmacology

authors

Withnall MT,Pennington A,Wiseman D

doi

10.1016/0006-2952(95)02084-5

subject

Has Abstract

pub_date

1995-11-27 00:00:00

pages

1893-902

issue

eissn

0006-2952

issn

1873-2968

pii

0006-2952(95)02084-5

journal_volume

pub_type

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Characterisation of cytosolic phospholipase A2 as mediator of the enhanced arachidonic acid release from dimethyl sulphoxide differentiated U937 cells.