Inactivation of endoplasmic reticulum bound Ca2+-independent phospholipase A2 in renal cells during oxidative stress.
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.
Cummings
Brian S
BS
Department of Pharmaceutical Sciences, Division of Nephrology, Medical University of South Carolina, 280 Calhoun Street, Charleston, SC 29425, USA.
Gelasco
Andrew K
AK
Kinsey
Gilbert R
GR
McHowat
Jane
J
Schnellmann
Rick G
RG
D000818:Animals; D017304:Annexin A5 / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D002084:Butylated Hydroxytoluene / Q000494:pharmacology; D002244:Carbon / Q000737:chemistry; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D003676:Deferoxamine / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D005260:Female; D005609:Free Radicals; D054522:Group VI Phospholipases A2; D007502:Iron Chelating Agents / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology*; D007684:Kidney Tubules / Q000378:metabolism; D008280:Magnetics; D008861:Microsomes / Q000378:metabolism; D009281:Naphthalenes / Q000494:pharmacology; D016877:Oxidants / Q000378:metabolism / Q000494:pharmacology; D018384:Oxidative Stress; D010655:Phenylenediamines / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D011753:Pyrones / Q000494:pharmacology; D011817:Rabbits; D017382:Reactive Oxygen Species; D013997:Time Factors; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D017304:Annexin A5; D000975:Antioxidants; D002614:Chelating Agents; D005609:Free Radicals; D007502:Iron Chelating Agents; D009281:Naphthalenes; D016877:Oxidants; D010655:Phenylenediamines; D010726:Phosphodiesterase Inhibitors; D010743:Phospholipids; D011753:Pyrones; D017382:Reactive Oxygen Species; D002084:Butylated Hydroxytoluene; D002244:Carbon; C068314:6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one; D020122:tert-Butylhydroperoxide; C004845:N,N'-diphenyl-4-phenylenediamine; D010741:Phospholipases A; D054522:Group VI Phospholipases A2; D054467:Phospholipases A2; D003676:Deferoxamine; D004229:Dithiothreitol