ROLE OF A MEMBRANE-ASSOCIATED SERINE ESTERASE IN THE OXIDANT ACTIVATION OF PHOSPHOLIPASE-A2 BY T-BUTYL HYDROPEROXIDE

Exposure of bovine pulmonary-arterial endothelial cells to the oxidant lipid t-butyl hydroperoxide (t-Bu-OOH) increases cell-membrane-associ ated phospholipase A2 (PLA2) activity and stimulates arachidonic acid (AA) release. To test the hypothesis that a membrane-associated serine esterase plays an important role in activating PLA2, the present stud y was undertaken. In addition to increasing PLA2 activity and AA relea se, t-Bu-OOH also enhances the activity of a membrane-associated serin e esterase that cleaves the synthetic substrate Nalpha-p-tosyl-L-argin ine methyl ester (TAME). Changes in the activity of this membrane-boun d serine esterase correlate directly with changes in the activity of P LA2. Serine esterase inhibitors such as phenylmethanesulphonyl fluorid e, di-isopropyl fluorophosphate and alpha-proteinase inhibitor, and TA ME, a synthetic substrate for serine esterase, prevent the increase in serine esterase activity. PLA2 activity and AA release caused by t-Bu -OOH. Pretreatment of the endothelial cells with the antioxidant vitam in E prevents t-Bu-OOH-induced stimulation of AA release and the cell- membrane-associated serine esterase and PLA2 activities. Adding t-Bu-O OH or the serine esterase trypsin to the endothelial-cell membrane fra ction also significantly augments PLA2 activity, implying that these t reatments activate latent PLA2. These results suggest that t-Bu-OOH st imulates a membrane-associated serine esterase that plays a crucial ro le in activating PLA2, and releasing AA.