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

Brian S. Cummings, Andrew K. Gelasco, Gilbert R. Kinsey, Jane McHowat, Rick G. Schnellmann

Research output: Contribution to journalArticlepeer-review

19 Scopus citations


The purpose of this study was to determine the actions of oxidants on endoplasmic reticulum bound Ca2+-independent phospholipase A 2 (ER-iPLA2) 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-iPLA2. TBHP-induced ER-iPLA2 inactivation was reversed by the addition of dithiothreitol to microsomes isolated from treated RPTC. TBHP also directly inactivated ER-iPLA2 in microsomes isolated from untreated RPTC. Similar to RPTC, dithiothreitol prevented TBHP-induced ER-iPLA2 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-iPLA2 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-iPLA2 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-iPLA2 is inactivated by oxidants, that the mechanism of inactivation involves the oxidation of ER-iPLA2 sulfhydryl groups, and that ER-iPLA 2 inhibition increases oxidant-induced RPTC phospholipid loss.

Original languageEnglish (US)
Pages (from-to)1441-1451
Number of pages11
JournalJournal of the American Society of Nephrology
Issue number6
StatePublished - Jun 2004
Externally publishedYes

ASJC Scopus subject areas

  • Nephrology


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