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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 and Rick G. Schnellmann^*

*Department of Pharmaceutical Sciences and Department of Medicine: Division of Nephrology, Medical University of South Carolina and Research Service, Ralph H. Johnson VAMC, Charleston, South Carolina; and Department of Pathology, Saint Louis University, St. Louis, Missouri

Correspondence to Rick G. Schnellmann, Department of Pharmaceutical Sciences, 280 Calhoun St., POB 250140, Medical University of South Carolina, Charleston, SC 29425. Phone: 843-792-3754; Fax: 843-792-2620; E-mail: schnell{at}musc.edu

ABSTRACT. The purpose of this study was to determine the actions of oxidants on endoplasmic reticulum bound Ca²⁺-independent phospholipase A₂ (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-iPLA₂ inhibition increases oxidant-induced RPTC phospholipid loss.

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Copyright © 2008 by the American Society of Nephrology. Online ISSN: 1533-3450 Print ISSN: 1046-6673