CHARACTERIZATION OF HORMONALLY REGULATED AND PARTICULATE-ASSOCIATED PHOSPHOLIPASE-A(2) 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 arachi donic acid (AA) from exogenously added almitoyl-2-[C-14]arachidonoyl-p hosphatidylcholine. Stimulation of cells with BK led to increased PLA2 activity in a particulate fraction (the 92,000 x g pellet of the post nuclear supernatant). The activity in the cytosolic fractions from BK- stimulated and nonstimulated cells was the same. The association of th e hormonally regulated PLA2 (HR-PLA2) activity with the particulate fr action was not affected by decreasing the Ca2+ concentrations in the h omogenate from 7 muM to 33 nM and therefore was not induced during hom ogenization by the presence of Ca2+ in the homogenate. The HR-PLA2 act ivity was Ca2+-dependent and was maximal at submicromolar concentratio ns of Ca2+. Incubation of the particulate fraction obtained from BK-st imulated and nonstimulated cells with 10 mM n-octyl-beta-D-glucopyrano side 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 stimulate d cells co-sedimented in sucrose gradients with the cytosolic PLA2. Th eir molecular mass was estimated to be 103,000 Da. The extracted enzym e from BK-stimulated cells retained its increased activity toward mito yl-2-[1-C-14]arachidonoyl-phosphatidylcholine. However, its activity t oward 1-palmitoyl-2-[1-C-14]oleoyl-phosphatidylcholine was equal to th e PLA2 activity extracted from nonstimulated cells. Treatment of the c ells 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 activit y in the cytosol but failed to increase PLA2 activity in the particula te fraction. In contrast, addition of 7 muM Ca2+ ionophore A23187 resu lted 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 con clude that stimulation of endothelial cells with BK increases the acti vity of a Ca2+-sensitive high molecular weight isoform of PLA2 which i s associated with the particulate fraction. Possible mechanisms of act ivation are discussed.