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 actionsof oxidants on endoplasmic reticulum bound Ca2+-independentphospholipase A2 (ER-iPLA2) and phospholipids in renal cells.Exposure of renal proximal tubule cells (RPTC) to the oxidantstert-butyl hydroperoxide (TBHP), cumene hydroperoxide, and cisplatinresulted in time- and concentration-dependent decreases in theactivity of ER-iPLA2. TBHP-induced ER-iPLA2 inactivation wasreversed by the addition of dithiothreitol to microsomes isolatedfrom treated RPTC. TBHP also directly inactivated ER-iPLA2 inmicrosomes isolated from untreated RPTC. Similar to RPTC, dithiothreitolprevented TBHP-induced ER-iPLA2 inactivation in microsomes asdid the reactive oxygen scavengers butylated hydroxytolueneand N,N’-diphenyl-p-phenylenediamine and the iron chelatordeferoxamine. Electron paramagnetic resonance spin trappingdemonstrated that TBHP initiated a carbon-centered radical after1 min of exposure in microsomes, preceding ER-iPLA2 inactivation,and further studies suggested that the formation of the carbon-centeredradical species occurred after or in concert with the formationof oxygen-centered radicals. Phospholipid content was determinedafter TBHP exposure in the presence and absence of the ER-iPLA2inhibitor bromoenol lactone. Treatment of RPTC with TBHP resultedin 35% decreases in (16:0, 20:4)-phosphatidylethanolamine (PtdEtn),(18:0, 18:1)-plasmenylethanolamine (PlsEtn), a 30% decreasein (16:0, 18:3)-phosphatidylcholine (PtdCho), and a 25% decreasein (16:0, 20:4)-phosphatidylcholine (PtdCho). In contrast, treatmentof RPTC with bromoenol lactone before TBHP exposure decreasedthe content of 11 phospholipids, decreasing a majority of PlsEtnphospholipids 60%, and 4 of the 8 PlsCho phospholipids 40%,while PtdCho and PtdEtn were marginally affected compared withTBHP. These data demonstrate that ER-iPLA2 is inactivated byoxidants, that the mechanism of inactivation involves the oxidationof ER-iPLA2 sulfhydryl groups, and that ER-iPLA2 inhibitionincreases oxidant-induced RPTC phospholipid loss.
Phospholipase A2 (PLA2) are esterases that cleave glycerophospholipidsat the sn-2 position, resulting in the release of a fatty acidand a lysophospholipid (1). To date, over 19 different typesof PLA2 exist, differing in size, localization, and Ca2+ requirement(2,3). Within the last 4 yr, a number of novel Ca2+-independentPLA2 (iPLA2) isoforms have been identified. Ma et al. (4,5)described a cytosolic 85-kD iPLA2 (Group VIA PLA2 using thenewer classification) that exists as two splice variants fromthe same gene (Group VIA-1 and Group VIA-2 PLA2). A human microsomal-bound85-kD iPLA2 (Group VIB PLA2) has also been expressed in insectand bacterial cells (6) and is hypothesized to protect againstoxidant-induced cell death in renal cells (7). Recent data alsodemonstrate that an 85-kD, Ca2+-independent cPLA2 (Group IVCPLA2 or cPLA2) is expressed in the endoplasmic reticulum membrane(8,9). Unlike the Group VI PLA2, Group IVC PLA2 is inhibitedby methylarachidonylfluorophosphonate (MAFP) at 1 µM butnot by bromoenol lactone (BEL) at concentrations has high as10 µM (10). BEL inhibits Groups VIA and VIB with equalpotency (11). Despite similarities in size and location, theseiPLA2 are derived from different gene products and share littleamino acid sequence identity to one another with the exceptionof their catalytic and ATP-binding sites.
We recently demonstrated that rabbit renal proximal tubule cells(RPTC) express an endoplasmic reticulum iPLA2 (ER-iPLA2) thatshares homology with human Group VIB PLA2 (7,12). Like humanGroup VIB PLA2, RPTC ER-iPLA2 is similar in size, inhibitedby BEL, and prefers plasmalogen phospholipids. In contrast toGroup VIB PLA2, RT-PCR, immunoblot analysis, and activity assayssuggested that RPTC do not express Group VIA-1 or VIA-2 PLA2(12).
The roles of iPLA2, such as Groups VIA and VIB PLA2, in cellphysiology are beginning to be understood. Cytosolic iPLA2 (GroupVIA-1 and VIA-2 PLA2) are suggested to mediate ischemia-inducedmyocardial dysfunction (13), cAMP release in rat mesangial cells(14), arginine vasopressin-induced arachidonic acid releasein A-10 smooth muscle cells (15), and nuclear phospholipid massdecreases after myocardial ischemia (16). However, the exactmechanisms controlling cytosolic iPLA2, such as Group VIA-1and VIA-2 PLA2, in these processes and the exact phospholipidsinvolved are not known. Identifying the phospholipids substratesmetabolized by iPLA2 during cellular signaling events and celldeath will be key to further understanding the role of iPLA2in these processes.
Compared with Group VIA PLA2, little is known about the roleof microsomal bound iPLA2 in oxidant-induced cell death. However,we demonstrated that inhibition of ER-iPLA2 in RPTC increasedoxidant-oncosis but had no affect on oncosis induced by non-oxidants(7). This effect was not a result of inhibition of cytosoliciPLA2 (Group IV PLA2) as neither MAFP nor arachidonyltrifluoromethylketone, altered oxidant-induced oncosis, or inhibited ER-iPLA2at concentrations less than 10 µM. However the specificphospholipids targeted by oxidants during oncosis are not known.Furthermore, unlike other forms of PLA2, very little is knownabout the regulation of ER-iPLA2 during oncosis. Finally, nothingis known concerning the physiologic role of ER-iPLA2 in cells.Thus the goals of this study were to determine the regulationof ER-iPLA2 during oxidant exposure in RPTC, identify the phospholipidstargeted by oxidants, and determine the role of ER-iPLA2 onthe maintenance of these phospholipids.
Materials
Female New Zealand White rabbits (1.5 to 2.0 kg) were purchasedfrom Myrtle’s Rabbitry (Thompson Station, TN). L-Ascorbicacid-2-phosphate (magnesium salt) was obtained from Wako ChemicalsUSA (Richmond, VA). Annexin-FITC was obtained from R&D Systems(San Diego, CA). 2-Ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide(EMPO) was purchased from Alexis (San Diego, CA). Butylatedhydroxytoluene (BHT), N,N’-diphenyl-p-phenylenediamine(DPPD), deferoxamine, cisplatin, propidium iodide (PI), menadione,cumene hydroperoxide, dithiothreitol, tert-butyl hydroperoxide(TBHP), and all other chemicals and materials were obtainedfrom Sigma Chemical (St. Louis, MO).
Isolation of Proximal Tubules and Culture Conditions
Rabbit renal proximal tubules were isolated using the iron oxideperfusion method and grown in 35-mm tissue culture dishes underimproved conditions as described previously (17,18). The cellculture medium was a 1:1 mixture of DMEM/Ham’s F-12 (withoutD-glucose, phenol red, or sodium pyruvate) supplemented with15 mM HEPES buffer, 2.5 mM L-glutamine, 1 µM pyridoxineHCL, 15 mM sodium bicarbonate, and 6 mM lactate. Hydrocortisone(50 nM), selenium (5 ng/ml), human transferrin (5 µg/ml),bovine insulin (10 nM), and L-ascorbic acid-2-phosphate (50µM) were added to fresh culture medium immediately beforedaily media change. In general, confluent RPTC were treatedwith inhibitors or diluent control (DMSO at <0.1% [vol/vol])for 30 min before treatment with oxidants.
Measurement of iPLA2 Activity
PLA2 activity was determined under linear reaction conditionsin microsomes and cytosol as described previously (19). Activitywas measured using synthetic (16:0, [3H]18:1) plasmenylcholineand phosphatidylcholine substrates (100 µM, 150 dpm/pmol)in the absence of Ca2+ (presence of 4 mM EGTA). These radiolabeledsubstrates were synthesized in the laboratory as described previously(20). For iPLA2 activity inhibition studies, confluent RPTCwere exposed to either diluent or 5 µM BEL for 30 min(7).
Measurement of Annexin V and PI Staining
Annexin V and PI staining were determined using flow cytometryas described previously (21–23) with modifications (24).Briefly, media were removed, RPTC were washed twice with PBSand incubated in binding buffer (10 mM HEPES, 140 mM NaCl, 5mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, pH = 7.4) containing annexinV-FITC (25 µg/ml) and PI (25 µg/ml) for 10 min.Cells were washed three times in binding buffer and releasedfrom the monolayers using a rubber policeman, and staining wasquantified using a Becton Dickinson FacsCalibur flow cytometer.For each measurement, 10,000 events were counted.
Electron Paramagnetic Resonance (EPR) Spectroscopy and Spin-Trapping
EMPO was added to microsome suspensions immediately before TBHPaddition. Microsomes, EMPO, and TBHP mixtures were incubatedfor 1 to 15 min at 37°C. After incubation, the 300-µlmixture was transferred to a quartz flat cell. The cell wasfitted into a Brüker 9908 SHQ cavity, and EPR spectra werecollected at room temperature on a Brüker ELEXSYS E500CW-EPR spectrometer. Typical spectral conditions were: microwavepower, 20 mW; microwave frequency, 9.719 Ghz; receiver gain,70 dB; sampling time, 40.96 s; time constant, 164 ms, modulationfrequency, 100 kHz; modulation amplitude, 1 G; sweep width,100 G. EPR spectra were simulated using the WinSim 2000 program(25). Based on simulation, the percent radical derivatives weredetermined to be 6% EMPO-OH (hydroxyl radical, tow diastereomers)and 94% EMPO-C·HR (methyl-centered radical). The hyperfineparameters for EMPO-C·HR were AN at 15.59 G and AH at21.02 G. For EMPO-OH the hyperfine parameters were AN 13.27G, AH = 13.24 G and AH = 1.2 G (diastereomer 1), and AN = 14.3G and AH = 14.0 G (diastereomer 2).
Characterization of Cellular Phospholipids
Cellular phospholipids were extracted using chloroform and methanolaccording to the method of Bligh and Dyer (26) at 4°C. Chloroformextracts were dried under N2 and resuspended in a 1:1 mixtureof chloroform:methanol (vol/vol), and phospholipids classeswere separated on the basis of differences in polar head groupcomposition using an Ultra-Si HPLC column and gradient elutionwith hexane/isopropanol/water (46.5/46.5/7.0) (19). Flow ratewas held constant at 1.5 ml/min throughout the separation, andphospholipids eluting from the column were detected by monitoringUV absorbance at 203 nm. Fractions were collected, correspondingto the following order of elution of phospholipids: phosphatidylethanolamine,cardiolipin, phosphatidylinositol, phosphatidylserine, phosphatidylcholine,and sphingomyelin.
Individual choline and ethanolamine glycerophospholipids molecularspecies were isolated by reverse phase HPLC using a ProdigyODS (5 µm, C-18) column (Phenomenex). Individual molecularspecies were separated by gradient elution with methanol/water/acetonitrile(87:6:7) with 20 mM choline chloride for 30 min, followed bylinear increase over 60 min to 76:4:20 methanol/water/acetonitrile(27). The molecular identity of individual species has beenpreviously established by GC characterization of the FAME andDMA derivatives produced after acid-catalyzed methanolysis ofphospholipids recovered by comparison of absolute and relativeretention times, order of elution of each species, and internalstandards of known composition and quantity. Elution from theHPLC column was detected by monitoring UV absorbance at 203nm.
Individual phospholipid molecular species were quantified bymeasurement of lipid phosphorus by microphosphate assays infractions from reverse phase HPLC separation (28). Fractionswere dried under N2, heated to 150°C for 2 h with 0.4 mlperchloric acid, and cooled to room temperature, and excessperchloric acid was neutralized with 1 ml of 4.5 M KOH. Sampleswere centrifuged at 2000 x g for 10 min to pellet the potassiumperchlorate precipitate, and the supernatant was removed foranalysis of lipid phosphorous (29).
Protein Determination
Protein determination was performed using the bicinchonic acidassay method as described by Sigma.
Statistical Analyses
RPTC isolated from one rabbit represented one experiment (n= 1). Microsomes isolated from RPTC or from rabbit kidney cortexrepresented one experiment (n = 1). The appropriate ANOVA wasperformed for each data set using SigmaStat statistical software.Individual means were compared using Fisher protected leastsignificant difference test, with P 0.05 being considered indicativeof a statistically significant difference between mean values.
Effect of TBHP on ER-iPLA2 activity in RPTC
Treatment of RPTC with the model oxidant TBHP (0 to 400 µM)for 1 h decreased ER-iPLA2 activity as determined by the hydrolysisof plasmenylcholine and phosphatidylcholine (Figure 1A). Thedecrease in iPLA2 activity was greater when plasmenylcholinewas used as the substrate. TBHP (400 µM) also inducedtime-dependent decreases in ER-iPLA2 activity (Figure 1B). Analysisof annexin V and PI staining in RPTC exposed to similar concentrationsand duration of TBHP demonstrated that RPTC were not undergoingeither apoptosis or oncosis at the time of decreased iPLA2 activity(data not shown). These data demonstrate that exposure of RPTCto TBHP results in inactivation of ER-iPLA2.
Figure 1. Effect of tert-butyl hydroperoxide (TBHP) on renal proximal tubule cell (RPTC) endoplasmic reticulum bound Ca2+-independent phospholipase A2 (ER-iPLA2) activity. Primary cultures of RPTC were treated with either solvent control (DMSO) or TBHP for 1 h before isolation and fractionation into microsomes. iPLA2 activity was measured using the indicated (16:0, [3H]18:1) phospholipid substrates (100 µM) in the presence of 4 mM EGTA. (A) Concentration-dependent effect of TBHP on RPTC ER-iPLA2 activity. (B) Time-dependent effect of TBHP (400 µM) on ER-iPLA2 activity in RPTC. Values are means ± SEM of at least four separate experiments. Means with different subscripts are significantly different from each other, P < 0.05.
The ability of DTT to reverse TBHP-induced inactivation or ER-iPLA2was determined to test the hypothesis that TBHP was inactivatingER-iPLA2 through protein thiol oxidation (Figure 2). Microsomeswere isolated from RPTC that were exposed to TBHP (0 to 400µM) for 1 h. The addition of 1 mM DTT to the PLA2 assaybuffer resulted in recovery of the iPLA2 activity in the microsomesprepared from TBHP-treated RPTC (Figure 2A). Interestingly,the addition of DTT to microsomes isolated from TBHP-treatedRPTC had no effect on ER-iPLA2 activity when phosphatidylcholinewas used a substrate (Figure 2B).
Figure 2. Effect of dithiothreitol (DTT) on TBHP-induced inactivation of RPTC ER-iPLA2. Primary cultures of RPTC were treated with either solvent control (DMSO) or TBHP before isolation and fractionation into microsomes. Microsomes were then exposed to solvent control or DTT (1 mM), and iPLA2 activity was measured using the indicated (16:0, [3H]18:1) phospholipid substrates (100 µM) in the presence of 4 mM EGTA. (A and B) Effect of DTT on concentration-dependent TBHP-induced (1-h exposure) RPTC ER-iPLA2 inactivation as measured by plasmenylcholine and phosphatidylcholine cleavage. Values are means ± SEM of at least four separate experiments. Means with different subscripts are significantly different from each other, P < 0.05.
Effect of Oxidants on ER-iPLA2 Activity in RPTC
Previous studies demonstrated that cumene hydroperoxide, cisplatin,and menadione all induce oxidative stress and oncosis in RPTC(7). Furthermore, treatment of RPTC with BEL increased oncosisinduced by these oxidants. To test the hypothesis that diverseoxidants inactivate ER-iPLA2, RPTC were exposed to the aboveoxidants under conditions known to induce oxidative stress (7).Exposure of RPTC to 200 µM cumene hydroperoxide for 1h or 400 µM cisplatin for 4 h resulted in 50% decreasesin microsomal ER-iPLA2 activity (Figure 3). In contrast, exposureof RPTC to 5 µM menadione for 30 min significantly increasedER-iPLA2 activity. Alterations in ER-iPLA2 activity in RPTCexposed to oxidants occurred in absence of cell death as determinedby the lack of annexin V and PI staining in concurrently treatedcells (data not shown). These data demonstrate that oxidantsother than TBHP, but not all oxidants, decrease ER-iPLA2 activityin RPTC.
Figure 3. Effect of oxidants on RPTC ER-iPLA2 activity. Primary cultures of RPTC were treated with solvent control (DMSO), cumene hydroperoxide (200 µM for 1 h), cisplatin (400 µM for 4 h), or menadione (5 µM for 0.5 h) before isolation and fractionation into microsomes. iPLA2 activity was measured using 16:0, [3H]18:1-plasmenylcholine (100 µM) in the presence of 4 mM EGTA. Values are means ± SEM of at least six separate experiments. Means with different subscripts are significantly different from each other, P < 0.05.
Effect of TBHP on ER-iPLA2 Activity in RPTC Microsomes
To test the hypothesis that oxidants directly inactivate ER-iPLA2,microsomes were first isolated from untreated RPTC and thenexposed to different concentrations of TBHP in iPLA2 assay buffer(Figure 4). The addition of TBHP to the iPLA2 assay system resultedin both time- and concentration-dependent decreases in bothplasmenylcholine and phosphatidylcholine hydrolysis. Decreasesin ER-iPLA2 activity occurred with concentrations of TBHP aslow as 100 µM (Figure 4A) and at time points as earlyas 10 min after exposure to 200 µM TBHP (Figure 4B). Aswith TBHP treated RPTC, greater decreases in ER-iPLA2 activitywere seen when plasmenylcholine was used as the substrate.
Figure 4. Effect of TBHP on ER-iPLA2 activity in microsomes isolated from untreated RPTC. Microsomes were isolated from primary cultures of RPTC and exposed to either solvent control (DMSO) or TBHP. iPLA2 activity was measured using the indicated (16:0, [3H]18:1) phospholipid substrates (100 µM) in the presence of 4 mM EGTA. (A) Concentration-dependent inactivation of ER-iPLA2 activity in microsomes by TBHP. (B) Time-dependent inactivation of ER-iPLA2 activity in microsomes by TBHP. Values are means ± SEM of at least four separate experiments. Means with different subscripts are significantly different from each other, P < 0.05.
Pretreatment of RPTC microsomes with DTT for 30 min preventedTBHP-induced decreases in ER-iPLA2 activity in a concentration-dependentmanner (Figure 5A). Treatment of microsomes with the reactiveoxygen scavengers BHT and DPPD, and the iron chelator deferoxaminefor 30 min before exposure to TBHP also prevented decreasesin ER-iPLA2 activity (Figures 5, B and C). BHT, DPPD, or deferoxaminealone did not affect ER-iPLA2 activity (data not shown). Finally,treatment of RPTC microsomes with the P450 inhibitor SKF525Adid not alter TBHP-induced decreases in ER-iPLA2 activity (datanot shown).
Figure 5. Effect of dithiothreitol (DTT) and reactive oxygen scavengers on TBHP-induced ER-iPLA2 inactivation in microsomes. Microsomes were isolated from primary cultures of RPTC and exposed to solvent control (DMSO), DTT, BHT, DPPD, or deferoxamine before exposure to TBHP for 30 min. iPLA2 activity was measured using 16:0, [3H]18:1-plasmenylcholine (100 µM) in the presence of 4 mM EGTA. (A) Effect of DTT on TBHP-induced ER-iPLA2 inactivation. (B) Effect of BHT on TBHP-induced ER-iPLA2 inactivation. (C) Effect of DPPD on TBHP-induced ER-iPLA2 inactivation. (D) Effect of deferoxamine on TBHP-induced ER-iPLA2 inactivation. Values are means ± SEM of at least four separate experiments. Means with different subscripts are significantly different from each other, P < 0.05.
TBHP-Mediated Formation of Reactive Oxygen Species in RPTC Microsomes
The above data suggest that TBHP-induced inactivation of RPTCmicrosomal ER-iPLA2 is mediated by the formation of reactiveoxygen species (ROS). EPR spectra derived from both RPTC microsomesand microsomes isolated from rabbit kidney cortex exposed toTBHP (200 µM) demonstrated the formation of a carbon-centeredradical, after or in concert with the formation of oxygen-centeredradicals, after just 1 min of exposure (Figure 6A). The signalintensity of these spectra continued to increase, peaking at5 min (Figure 6B). The formation of this radical species wasprotein-dependent with the optimal protein concentration forEPR studies being 18 mg/ml. Simulation of the spectra obtainedin Figure 6B using the parameters listed in the methods resultedin a spectra shown in Figure 6C. This spectrum is generatedprimarily by the formation of a methyl-centered radical anda smaller quantity of a hydroxyl radical.
Figure 6. TBHP induces a radical species in kidney microsomes. The addition of TBHP to a microsome suspension induced a C-centered radical species as detected by trapping with the spin trap 2-Ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO). EPR spectra was the measured at room temperature as described in methods section. (A) Microsomes, 25 µM EMPO, 200 µM TBHP incubated for 1 min at 37°C. (B) Microsomes, 25 µM EMPO, 200 µM TBHP incubated for 5 min at 37°C. (C) Computer simulation of the spectrum in panel B, using the parameters in Table 1. (D) As in panel B, with DTT in the buffer. (E) As in panel B, with 3% DMSO pretreatment for 30 min at 37°C before EMPO/TBHP addition. (F) As in panel B, with 500 µM desferoximine/3% DMSO pretreatment for 30 min at 37°C before EMPO/TBHP addition. (G) As in panel B, except no TBHP added. (H) As in panel B, with no microsomes present.
Table 1. Phospholipid molecular species analyzed in RPTC
Similar to its effect on TBHP-induced ER-iPLA2 inactivation,pre-incubation with DTT (300 µM) blocked radical formationin TBHP-exposed microsomes (Figure 6D). However, 500 µMdeferoxamine only had a slight affect on TBHP radical formation,and this affect was not attributed to the addition of deferoxaminein DMSO (Figure 6, E and F). Further controls demonstrated thatradical formation required both TBHP and microsomes, as no radicalspecies were detected in spectra obtained using mixtures whereeither was absent (Figure 6, G and H).
Effect of TBHP and ER-iPLA2 Inhibition on RPTC Phospholipids
To identify the phospholipids associated with ER-iPLA2 activityand TBHP exposure in RPTC, we investigated the effect of bromoenollactone (BEL) pretreatment (an inhibitor of ER-iPLA2) and/orTBHP on the hydrolysis of 28 different phospholipid molecularspecies. These phospholipids differed in terms of the polarhead group, the type of glycerol-fatty acid linkage at the sn-1position, length of the fatty acid carbon chains at the sn-1and sn-2 positions of the glycerol backbone, and the numberof double bonds present in the fatty acids (Table 1). Treatmentof RPTC for 1 h with 5 µM BEL (a concentration previouslydemonstrated to inhibit greater than 90% of RPTC iPLA2 activity[7]) decreased only two phospholipids, 18:0, 18:1-plasmenylcholine(PlsCho) and 18:2, 20:4-PlsCho (Figure 7A). In contrast, treatmentof RPTC with 200 µM TBHP for 1 h resulted in decreasesin four phospholipids, 16:0, 20:4-phosphatidylethanolamine (PtdEtn),18:0, 18:1-plasmenylethanolamine (PlsEtn), 16:0, 18:3-phosphatidylcholine(PtdCho), and 16:0, 20:4 PtdCho (Figure 7B). Treatment of RPTCwith BEL before exposure to TBHP resulted in decreases in anadditional ten phospholipid molecular species (Figure 8). Exposureof RPTC to BEL before TBHP treatment resulted in significantdecreases in four of the eight PlsCho studied and five of thesix PlsEtn studied (Figure 8, A and B). Finally treatment ofRPTC with BEL before TBHP exposure resulted in decreases inthree of the seven PtdCho and PtdEtn phospholipids studied (Figure 8, C and D).
Figure 7. Effect of ER-iPLA2 inactivation and TBHP on RPTC phospholipids. Primary cultures of RPTC were exposed to solvent control (DMSO), BEL (5 µM), or TBHP (200 µM) for 1 h before isolation of phospholipids. Individual phospholipids were separated and quantified as described in the methods. (A) Effect of BEL on RPTC phospholipids. (B) Effect of TBHP on RPTC phospholipids. Values are means ± SEM of at least four separate experiments. Means with different subscripts are significantly different from each other, P < 0.05.
Figure 8. Effect of ER-iPLA2 inactivation on TBHP-induced alteration of RPTC phospholipids. Primary cultures of RPTC were exposed to solvent control (DMSO) or to BEL (5 µM) and TBHP (200 µM) for 1 h before isolation of phospholipids. Individual phospholipids were separated and quantified as described in Materials and Methods. (A) Effect of BEL and TBHP on plasmenylcholine phospholipids, (B) plasmenylethanolamine phospholipids, (C) phosphatidylcholine phospholipids, (D) phosphatidylethanolamine phospholipids in RPTC. Values are means ± SEM of at least four separate experiments. Means with different subscripts are significantly different from each other, P < 0.05.
Recently a number of novel ER-iPLA2 have been identified andcharacterized, including those expressed in the endoplasmicreticulum of proximal tubule cells, HEK293 cells, and insectand bacterial cells expressing human Group VIB PLA2 (6–10,30).The functions of these ER-iPLA2 in cell physiology and deathare not well described, but it has been suggested that theyparticipate in the protection against oxidant-induced cell death(7), maintenance of membrane integrity (16), and ischemia-inducedinjury to myocytes (13). Despite these data, little is knownconcerning the effect of oxidants on ER-iPLA2 function and thephospholipids targeted by ER-iPLA2 during oxidative stress.
Exposure of RPTC to diverse oxidants (TBHP, cumene hydroperoxide,and cisplatin) resulted in inactivation of ER-iPLA2, an iPLA2similar to human Group VIB PLA2 (6,30). Inactivation of ER-iPLA2occurred in the absence of cell death, indicating that decreasesin activity precede cell death as opposed to being a resultof cell death. However, not all oxidants tested inactivatedRPTC ER-iPLA2, as menadione treatment actually increased activity.The ability of menadione to increase RPTC ER-iPLA2 activitymay be related to its ability to produce superoxide (31–33)and directly activate PKC (34). In support of this hypothesis,we recently demonstrated that PKC activation in RPTC and myocytesincreases microsomal iPLA2 activity (12,35). In contrast, otherinvestigators have reported that short-term exposures of renalcells and kidney slices to TBHP and cisplatin decrease PKC activity(36,37), while cumene hydroperoxide has no effect on PKC activityin vascular endothelial cells (38). Thus, the ability of menadione,but not TBHP, cumene hydroperoxide, or cisplatin, to induceER-iPLA2 may be linked to its ability to activate PKC.
Oxidant exposure to RPTC resulted in greater decreases in plasmenylcholinecleavage than phosphatidylcholine. The exact reasons for thesedifferences remain unclear, but ER-iPLA2 does prefer plasmenylcholineover phosphatidylcholine (7). The possibility also exists thatother enzymes may be cleaving phosphatidylcholine. Group IVCPLA2 can cleave phospholipids, is Ca2+-independent, and is expressedin the ER. However, preliminary data from our laboratory didnot reveal any evidence of Group IVC PLA2 activity or expressionin RPTC (data not shown). Another possibility is that oxidant-inducedER-iPLA2 inactivation is not a result of direct interactionsat the active site of the enzyme; rather, oxidants may alterthe environment surrounding the active site. For example, oxidationof protein sulfhydryl groups upstream or downstream of the activesite may restrict access of plasmenyl substrates while not affectingthe access of diacyl substrates. In support of this hypothesis,neither the lipase nor the ATP-binding motifs contain cysteineresidues. However, two cysteine residues can be found within20 amino acids of both the lipase and ATP-binding motif of GroupVIB PLA2, and one cysteine is one amino acid upstream of theGVSTG lipase-binding domain. Thus the presence of DTT may aidin the maintenance of ER-iPLA2 conformation in its active stateby reversing the oxidation of the sulfhydryl groups at thesecysteines.
DTT can prevent cisplatin-induced PKC inactivation in renalslices (37) and may therefore be reversing oxidant-induced ER-iPLA2inactivation by maintaining PKC activity. However, this is notlikely the case in our studies because oxidants can directlyinactivate ER-iPLA2 in microsomes isolated from untreated RPTC.The kinetics of oxidant-induced ER-iPLA2 inactivation and thereversal by DTT in microsomes were similar to those seen inRPTC, suggesting that similar mechanisms mediate ER-iPLA2 inactivationin both models. Furthermore, similar to their ability to preventER-iPLA2 inactivation in microsomes, BHT, DPPD, and deferoxamineprevent oxidant-induced renal cell death (39,40).
The ability of antioxidants BHT and DPPD and the iron chelatordeferoxamine to block oxidant-induced ER-iPLA2 inactivationin RPTC microsomes and TBHP-induced cell death in RPTC (39,40)illustrate that oxidant-induced ER-iPLA2 inactivation is mediatedby iron-dependent formation of ROS. This hypothesis is supportedby the EPR studies, which demonstrated that a ROS form in conjunctionwith a methyl-centered radical upstream of ER-iPLA2 inactivation.Simulation of EPR spectra provides evidence that the identificationof the oxygen radical species is a hydroxyl radical. While littlework has been published studying the use of EPR to study theformation of TBHP-induced radicals in microsomes, EPR studiesperformed in rat liver mitochondria using TBHP reported theformation of multiple radicals: one being a methyl-centeredradical and the others determined to contain oxygen centerssimilar to those detected above (41).
The ER-iPLA2 inactivation studies in microsomes suggest thatinactivation is a result of iron-mediated formation of ROS,which results in the oxidation of sulfhydryl groups in ER-iPLA2or at the ER membrane. The source of this iron may be from heme-containingenzymes such as cytochromes P450 and flavin-containing monooxygenases,or thioreductases, which are abundantly expressed in the ER.However, the inability of deferoxamine to completely inhibitradical formation in microsomes during the EPR studies suggeststhat oxidation of sulfhydryl groups alone may indirectly resultin ER-iPLA2 inactivation. The ability of BHT and DPPD to inhibitformation of the radicals in the EPR studies could not be addresseddue to interactions of these scavengers directly with EMPO,the spin trap used for these studies.
Inhibition of ER-iPLA2 with BEL resulted in a decrease in onlytwo phospholipids, both of which were plasmenylcholines, suggestinga physiologic role for ER-iPLA2 in RPTC. The data agree withthose demonstrating that inhibition of cytosolic iPLA2 (GroupVIA PLA2) decreases the percentage of arachidonic acid-containingand plasmenylcholine phospholipids in P388D1 macrophages (42,43)and with studies demonstrating that cytosolic iPLA2 has a rolein the maintenance of phospholipids in heart during ischemia(13,16). The released lysophospholipids serve as limiting substratesfor reacylation and subsequent reesterfication of fatty acidsto the sn-2 position before reinsertion. In the absence of cytosoliciPLA2 activity, lysophospholipids were not released, resultingin a decrease in the affected phospholipids (42,43). As RPTCdo not express cytosolic iPLA2 (7), ER-iPLA2 may serve a similarrole in the ER. RPTC do express a smaller 28-kD cytosolic iPLA2,but this PLA2 isoform is inhibited by concentrations of BELtwofold higher than those used for ER-iPLA2 (7,44–46).It is also unlikely that the decrease in PlsCho phospholipidsin RPTC treated with BEL alone are a result of phosphatidicacid phosphohydrolase-1 (PAPH-1) inhibition by BEL, as PAPH-1is only inhibited 50% by 50 µM BEL (47). In contrast,BEL inhibits ER-iPLA2 greater than 90% at a concentration of5 µM (7). These data suggest the ER-iPLA2 plays a physiologicrole in the maintenance of cellular or ER phospholipids.
Treatment of RPTC with TBHP, at concentrations and time pointsshown to inactivate ER-iPLA2, resulted in significant decreasesin several phospholipids. These decreases occurred before celldeath (7). Three of the four phospholipids decreased by TBHPcontained more than three double bonds at the sn-2 fatty acid,coinciding with the fact that multiple unsaturated phospholipidsdouble bonds are more susceptible to oxidative attack than fattyacids with no double bonds. These data are the first to reporton the specific phospholipid molecular species targeted by anoxidant in renal cells. The identification of these phospholipidspecies may lead to the development of more sensitive markersof renal injury. Furthermore, synthesis of these specific phospholipidswith radiolabels or fluorescence probes will allow for the trackingof their metabolism and identification of the signaling pathwaysinvolved in the mechanisms of oxidant-induced cell death inthe kidney.
Concurrent with the hypothesis that ER-iPLA2 inhibition potentiatesoxidant-induced oncosis, inhibition of ER-iPLA2 increased theloss of phospholipids from RPTC treated with TBHP. The greatestloss in phospholipids, comparatively, were those belonging tothe PlsEtn family, followed by PlsCho. The increased loss ofphospholipids in RPTC in the presence of TBHP and BEL is consistentwith the increases in lipid peroxidation in the presence ofBEL and oxidants (7). The increased loss of these phospholipidsin the presence of BEL may reflect the inability of ER-iPLA2to cleave these lipids, which would prepare the phospholipidsfor reacylation and reinsertion. Furthermore, enhanced phospholipidloss also may be the result of both TBHP and BEL inhibitingER-iPLA2 while TBHP continues to oxidize phospholipids.
Taken together, these data further support the hypothesis thatER-iPLA2 protects RPTC from oxidant-induced oncosis by inhibitingthe nonenzymatic degradation of phospholipids induced by oxidants.We propose that during oxidative stress free radical speciesoxidize phospholipids, which ER-iPLA2 recognizes, cleaves, andremoves from the membrane. The cleavage reaction may set upthe lysophospholipid for reacylation and reinsertion into themembrane. In the absence of ER-iPLA2, oxidized phospholipidsmay not be removed from the membrane and become subjected tofurther oxidation and degradation. Furthermore, in the absenceof ER-iPLA2, other phospholipid pools in the cell may be targetedfor degradation, further enhancing the loss of cell functionand death. These data further support the hypothesis that ER-iPLA2play critical roles not only in the mediation of cell deathbut also in maintenance of cellular membrane integrity.
Acknowledgments
This work was supported by a National Research Service Award(DK-10079) to BSC, a Gateway Research Scholarship from the AmericanFoundation for Pharmaceutical Education, and a Medical Universityof South Carolina Summer Health Professionals Research Grantto GRK, and by a National Institutes of Health Grant (DK-62028)to RGS and JM. A National Institute of Health Grant (K25-DK59950)to AKG and the Research Service of the Department of Veterans’Affairs also supported part of this work. The EPR spectrometeris a shared MUSC resource obtained with a National Institutesof Health Grant (S10RR13656).
Footnotes
Dr. Cummings’ current affiliation: Department of Pharmaceuticaland Biomedical Sciences, University of Georgia, Athens, Georgia.
Cummings BS, McHowat J, Schnellmann RG: Phospholipase A(2)s in cell injury and death. J Pharmacol Exp Ther 294: 793–799, 2000[Abstract/Free Full Text]
Balsinde J, Balboa MA, Insel PA, Dennis EA: Regulation and inhibition of phospholipase A2. Annu Rev Pharmacol Toxicol 39: 175–189, 1999[CrossRef][Medline]
Dennis EA: The growing phospholipase A2 superfamily of signal transduction enzymes. Trends Biochem Sci 22: 1–2, 1997[CrossRef][Medline]
Ma Z, Ramanadham S, Hu Z, Turk J: Cloning and expression of a group IV cytosolic Ca2+-dependent phospholipase A2 from rat pancreatic islets. Comparison of the expressed activity with that of an islet group VI cytosolic Ca2+-independent phospholipase A2. Biochim Biophys Acta 1391: 384–400, 1998[Medline]
Ma Z, Wang X, Nowatzke W, Ramanadham S, Turk J: Human pancreatic islets express mRNA species encoding two distinct catalytically active isoforms of group VI phospholipase A2 (iPLA2) that arise from an exon-skipping mechanism of alternative splicing of the transcript from the iPLA2 gene on chromosome 22q13.1. J Biol Chem 274: 9607–9616, 1999[Abstract/Free Full Text]
Mancuso DJ, Jenkins CM, Gross RW: The genomic organization, complete mRNA sequence, cloning, and expression of a novel human intracellular membrane-associated calcium-independent phospholipase A(2). J Biol Chem 275: 9937–9945, 2000[Abstract/Free Full Text]
Cummings BS, McHowat J, Schnellmann RG: Role of an endoplasmic reticulum Ca(2+)-independent phospholipase A(2) in oxidant-induced renal cell death. Am J Physiol Renal Physiol 283: F492–F498, 2002[Abstract/Free Full Text]
Underwood KW, Song C, Kriz RW, Chang XJ, Knopf JL, Lin LL: A novel calcium-independent phospholipase A2, cPLA2-gamma, that is prenylated and contains homology to cPLA2. J Biol Chem 273: 21926–21932, 1998[Abstract/Free Full Text]
Asai K, Hirabayashi T, Houjou T, Uozumi N, Taguchi R, Shimizu T: Human group IVC phospholipase A2 (cPLA2gamma). Roles in the membrane remodeling and activation induced by oxidative stress. J Biol Chem 278: 8809–8814, 2003[Abstract/Free Full Text]
Stewart A, Ghosh M, Spencer DM, Leslie CC: Enzymatic properties of human cytosolic phospholipase A(2)gamma. J Biol Chem 277: 29526–29536, 2002[Abstract/Free Full Text]
Ackermann EJ, Conde-Frieboes K, Dennis EA: Inhibition of macrophage Ca(2+)-independent phospholipase A2 by bromoenol lactone and trifluoromethyl ketones. J Biol Chem 270: 445–450, 1995[Abstract/Free Full Text]
Cummings BS, McHowat J, Schnellmann RG: Role of an endoplasmic reticulum Ca2+-independent phospholipase A2 in Cisplatin-induced renal cell apoptosis. J Pharmacol Exp Ther 308: 921–928, 2004[Abstract/Free Full Text]
Williams SD, Gottlieb RA: Inhibition of mitochondrial calcium-independent phospholipase A2 (iPLA2) attenuates mitochondrial phospholipid loss and is cardioprotective. Biochem J 362: 23–32, 2002[CrossRef][Medline]
Akiba S, Hayama M, Sato T: Inhibition of Ca2+-independent phospholipase A2 by bromoenol lactone attenuates prostaglandin generation induced by interleukin-1 beta and dibutyryl cAMP in rat mesangial cells. FEBS Lett 437: 225–228, 1998[CrossRef][Medline]
Jenkins CM, Han X, Mancuso DJ, Gross RW: Identification of calcium-independent phospholipase A2 (iPLA2) beta, and not iPLA2gamma, as the mediator of arginine vasopressin-induced arachidonic acid release in A-10 smooth muscle cells. Enantioselective mechanism-based discrimination of mammalian iPLA2s. J Biol Chem 277: 32807–32814, 2002[Abstract/Free Full Text]
Williams SD, Hsu FF, Ford DA: Electrospray ionization mass spectrometry analyses of nuclear membrane phospholipid loss after reperfusion of ischemic myocardium. J Lipid Res 41: 1585–1595, 2000[Abstract/Free Full Text]
Nowak G, Schnellmann RG: L-ascorbic acid regulates growth and metabolism of renal cells: Improvements in cell culture. Am J Physiol 271: C2072–C2080, 1996
Nowak G, Schnellmann RG: Integrative effects of EGF on metabolism and proliferation in renal proximal tubular cells. Am J Physiol 269: C1317–C1325, 1995
McHowat J, Liu S, Creer MH: Selective hydrolysis of plasmalogen phospholipids by Ca2+-independent PLA2 in hypoxic ventricular myocytes. Am J Physiol 274: C1727–C1737, 1998
Creer MH, McHowat J: Selective hydrolysis of plasmalogens in endothelial cells following thrombin stimulation. Am J Physiol 275: C1498–C1507, 1998
Schutte B, Nuydens R, Geerts H, Ramaekers F: Annexin V binding assay as a tool to measure apoptosis in differentiated neuronal cells. J Neurosci Methods 86: 63–69, 1998[CrossRef][Medline]
Goldberg JE, Sherwood SW, Clayberger C: A novel method for measuring CTL and NK cell-mediated cytotoxicity using annexin V and two-color flow cytometry. J Immunol Methods 224: 1–9, 1999[CrossRef][Medline]
Meijerman I, Blom WM, de Bont HJ, Mulder GJ, Nagelkerke JF: Induction of apoptosis and changes in nuclear G-actin are mediated by different pathways: the effect of inhibitors of protein and RNA synthesis in isolated rat hepatocytes. Toxicol Appl Pharmacol 156: 46–55, 1999[CrossRef][Medline]
Duling DR: Simulation of multiple isotropic spin-trap EPR spectra. J Magn Reson B 104: 105–110, 1994[CrossRef][Medline]
Bligh EG, Dyer WJ: A rapid method of total lipid extraction and purification. J Biochem Phsyiol 37: 911–917, 1959
McHowat J, Jones JH, Creer MH: Gradient elution reversed-phase chromatographic isolation of individual glycerophospholipid molecular species. J Chromatogr B Biomed Sci Appl 702: 21–32, 1997[CrossRef][Medline]
Chen PSJ, Toribara TY, Warner H: Microdetermination of phosphorus. Anal Chem 28: 1756–1758, 1956[CrossRef]
Itaya K, Ui M: A new micromethod for the colorimetric determination of inorganic phosphate. Clin Chim Acta 14: 361–366, 1966[CrossRef][Medline]
Tanaka H, Takeya R, Sumimoto H: A novel intracellular membrane-bound calcium-independent phospholipase A(2). Biochem Biophys Res Commun 272: 320–326, 2000[CrossRef][Medline]
Goldberg B, Stern A: Production of superoxide anion during the oxidation of hemoglobin by menadione. Biochim Biophys Acta 437: 628–632, 1976[Medline]
Goldberg B, Stern A: Superoxide anion as a mediator of drug-induced oxidative hemolysis. J Biol Chem 251: 6468–6470, 1976[Abstract/Free Full Text]
Mishin V, Pokrovsky A, Lyakhovich VV: Interactions of some acceptors with superoxide anion radicals formed by the NADPH-specific flavoprotein in rat liver microsomal fractions. Biochem J 154: 307–310, 1976[Medline]
Kass GE, Duddy SK, Orrenius S: Activation of hepatocyte protein kinase C by redox-cycling quinones. Biochem J 260: 499–507, 1989[Medline]
Steer SA, Wirsig KC, Creer MH, Ford DA, McHowat J: Regulation of membrane-associated iPLA2 activity by a novel PKC isoform in ventricular myocytes. Am J Physiol Cell Physiol 283: C1621–1626, 2002[Abstract/Free Full Text]
Nowak G: Protein kinase C mediates repair of mitochondrial and transport functions after toxicant-induced injury in renal cells. J Pharmacol Exp Ther 306: 157–165, 2003[Abstract/Free Full Text]
Zhang JG, Lindup WE: Cisplatin-induced nephrotoxicity in vitro: increases in cytosolic calcium concentration and the inhibition of cytosolic and mitochondrial protein kinase C. Toxicol Lett 89: 11–17, 1996[CrossRef][Medline]
Taher MM, Garcia JG, Natarajan V: Hydroperoxide-induced diacylglycerol formation and protein kinase C activation in vascular endothelial cells. Arch Biochem Biophys 303: 260–266, 1993[CrossRef][Medline]
Schnellmann RG: Mechanisms of t-butyl hydroperoxide-induced toxicity to rabbit renal proximal tubules. Am J Physiol 255: C28–C33, 1988
Groves CE, Lock EA, Schnellmann RG: Role of lipid peroxidation in renal proximal tubule cell death induced by haloalkene cysteine conjugates. Toxicol Appl Pharmacol 107: 54–62, 1991[CrossRef][Medline]
Kennedy CH, Church DF, Winston GW, Pryor WA: tert-Butyl hydroperoxide-induced radical production in rat liver mitochondria. Free Radic Biol Med 12: 381–387, 1992[CrossRef][Medline]
Balsinde J, Bianco ID, Ackermann EJ, Conde-Frieboes K, Dennis EA: Inhibition of calcium-independent phospholipase A2 prevents arachidonic acid incorporation and phospholipid remodeling in P388D1 macrophages. Proc Natl Acad Sci U S A 92: 8527–8531, 1995[Abstract/Free Full Text]
Balsinde J, Balboa MA, Dennis EA: Antisense inhibition of group VI Ca2+-independent phospholipase A2 blocks phospholipid fatty acid remodeling in murine P388D1 macrophages. J Biol Chem 272: 29317–29321, 1997[Abstract/Free Full Text]
Portilla D, Dai G: Purification of a novel calcium-independent phospholipase A2 from rabbit kidney. J Biol Chem 271: 15451–15457, 1996[Abstract/Free Full Text]
Portilla D, Crew MD, Grant D, Serrero G, Bates LM, Dai G, Sasner M, Cheng J, Buonanno A: cDNA cloning and expression of a novel family of enzymes with calcium-independent phospholipase A2 and lysophospholipase activities. J Am Soc Nephrol 9: 1178–1186, 1998[Abstract]
Portilla D, Shah SV, Lehman PA, Creer MH: Role of cytosolic calcium-independent plasmalogen-selective phospholipase A2 in hypoxic injury to rabbit proximal tubules. J Clin Invest 93: 1609–1615, 1994
Balsinde J, Dennis EA: Bromoenol lactone inhibits magnesium-dependent phosphatidate phosphohydrolase and blocks triacylglycerol biosynthesis in mouse P388D1 macrophages. J Biol Chem 271: 31937–31941, 1996[Abstract/Free Full Text]
Received for publication January 15, 2004.
Accepted for publication March 31, 2004.
This article has been cited by other articles:
K. A. Poulsen, S. F. Pedersen, M. Kolko, and I. H. Lambert Induction of group VIA phospholipase A2 activity during in vitro ischemia in C2C12 myotubes is associated with changes in the level of its splice variants
Am J Physiol Cell Physiol,
November 1, 2007;
293(5):
C1605 - C1615.
[Abstract][Full Text][PDF]
G. R. Kinsey, J. McHowat, C. S. Beckett, and R. G. Schnellmann Identification of calcium-independent phospholipase A2{gamma} in mitochondria and its role in mitochondrial oxidative stress
Am J Physiol Renal Physiol,
February 1, 2007;
292(2):
F853 - F860.
[Abstract][Full Text][PDF]
G. Saavedra, W. Zhang, B. Peterson, and B. S. Cummings Differential Roles for Cytosolic and Microsomal Ca2+-Independent Phospholipase A2 in Cell Growth and Maintenance of Phospholipids
J. Pharmacol. Exp. Ther.,
September 1, 2006;
318(3):
1211 - 1219.
[Abstract][Full Text][PDF]
Top
Abstract
Introduction
) is expressed in the endoplasmic reticulum membrane (8,9). Unlike the Group VI PLA2, Group IVC PLA2 is inhibited by methylarachidonylfluorophosphonate (MAFP) at 1 µM but not by bromoenol lactone (BEL) at concentrations has high as 10 µM (10). BEL inhibits Groups VIA and VIB with equal potency (11). Despite similarities in size and location, these iPLA2 are derived from different gene products and share little amino acid sequence identity to one another with the exception of their catalytic and ATP-binding sites. 
at 21.02 G. For EMPO-OH the hyperfine parameters were AN 13.27 G, AH
0.05 being considered indicative of a statistically significant difference between mean values. 
