Inactivation of endoplasmic reticulum bound Ca2+-independent phospholipase A2 in renal cells during oxidative stress.

Abstract

The purpose of this study was to determine the actions of oxidants on endoplasmic reticulum bound Ca(2+)-independent phospholipase A(2) (ER-iPLA(2)) and phospholipids in renal cells. Exposure of renal proximal tubule cells (RPTC) to the oxidants tert-butyl hydroperoxide (TBHP), cumene hydroperoxide, and cisplatin resulted in time- and concentration-dependent decreases in the activity of ER-iPLA(2). TBHP-induced ER-iPLA(2) inactivation was reversed by the addition of dithiothreitol to microsomes isolated from treated RPTC. TBHP also directly inactivated ER-iPLA(2) in microsomes isolated from untreated RPTC. Similar to RPTC, dithiothreitol prevented TBHP-induced ER-iPLA(2) inactivation in microsomes as did the reactive oxygen scavengers butylated hydroxytoluene and N,N'-diphenyl-p-phenylenediamine and the iron chelator deferoxamine. Electron paramagnetic resonance spin trapping demonstrated that TBHP initiated a carbon-centered radical after 1 min of exposure in microsomes, preceding ER-iPLA(2) inactivation, and further studies suggested that the formation of the carbon-centered radical species occurred after or in concert with the formation of oxygen-centered radicals. Phospholipid content was determined after TBHP exposure in the presence and absence of the ER-iPLA(2) inhibitor bromoenol lactone. Treatment of RPTC with TBHP resulted in 35% decreases in (16:0, 20:4)-phosphatidylethanolamine (PtdEtn), (18:0, 18:1)-plasmenylethanolamine (PlsEtn), a 30% decrease in (16:0, 18:3)-phosphatidylcholine (PtdCho), and a 25% decrease in (16:0, 20:4)-phosphatidylcholine (PtdCho). In contrast, treatment of RPTC with bromoenol lactone before TBHP exposure decreased the content of 11 phospholipids, decreasing a majority of PlsEtn phospholipids 60%, and 4 of the 8 PlsCho phospholipids 40%, while PtdCho and PtdEtn were marginally affected compared with TBHP. These data demonstrate that ER-iPLA(2) is inactivated by oxidants, that the mechanism of inactivation involves the oxidation of ER-iPLA(2) sulfhydryl groups, and that ER-iPLA(2) inhibition increases oxidant-induced RPTC phospholipid loss.