Characterization of hormonally regulated and particulate-associated phospholipase A2 from bovine endothelial cells

Hormonally regulated and particulate-associated phospholipase A2 (PLA2) was detected in endothelial cells from bovine pulmonary artery. The enzyme was solubilized and subjected to initial characterization. PLA2 activity was determined in subcellular fractions from bradykinin (BK)-stimulated and nonstimulated cells by following the release of arachidonic acid (AA) from exogenously added 1-palmitoyl-2-[1-14C]arachidonoyl-phosphatidylcholine. Stimulation of cells with BK led to increased PLA2 activity in a particulate fraction (the 92,000 x g pellet of the postnuclear supernatant). The activity in the cytosolic fractions from BK-stimulated and nonstimulated cells was the same. The association of the hormonally regulated PLA2 (HR-PLA2) activity with the particulate fraction was not affected by decreasing the Ca2+ concentrations in the homogenate from 7 microM to 33 nM and therefore was not induced during homogenization by the presence of Ca2+ in the homogenate. The HR-PLA2 activity was Ca(2+)-dependent and was maximal at submicromolar concentrations of Ca2+. Incubation of the particulate fraction obtained from BK-stimulated and nonstimulated cells with 10 mM n-octyl-beta-D-glucopyranoside resulted in a differential solubilization of the HR-PLA2 activity. Its isoelectric point was determined to be 5.7. HR-PLA2 activity in the octyl glucoside extract of the particulate fraction from stimulated cells co-sedimented in sucrose gradients with the cytosolic PLA2. Their molecular mass was estimated to be 103,000 Da. The extracted enzyme from BK-stimulated cells retained its increased activity toward 1-palmitoyl-2-[1-14C]arachidonoyl-phosphatidylcholine. However, its activity toward 1-palmitoyl-2-[1-14C]oleoyl-phosphatidylcholine was equal to the PLA2 activity extracted from nonstimulated cells. Treatment of the cells with 100 nM 12-O-tetradecanoylphorbol-13-acetate resulted in a 20 +/- 1.2% (mean +/- S.E., p < 0.01, n = 4) increase in the PLA2 activity in the cytosol but failed to increase PLA2 activity in the particulate fraction. In contrast, addition of 7 microM Ca2+ ionophore A23187 resulted in a 21 +/- 0.55% (mean +/- S.E., p < 0.01, n = 5) decrease in the cytosolic activity and a concomitant increase of 68 +/- 9.6% (mean +/- S.E., p < 0.05, n = 5) in the particulate-associated activity. We conclude that stimulation of endothelial cells with BK increases the activity of a Ca(2+)-sensitive high molecular weight isoform of PLA2 which is associated with the particulate fraction. Possible mechanisms of activation are discussed.