Involvement of Ca2+-independent phospholipase A2 isoforms in oxidant-induced neural cell death

NeuroToxicology 28 (2007) 150–160

Involvement of Ca2+-independent phospholipase A2 isoforms in oxidant-induced neural cell death Brianna Peterson, Taylor Knotts, Brian S. Cummings * Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, Athens, GA 30602-2352, United States Received 7 July 2006; accepted 8 September 2006 Available online 16 September 2006

Abstract This study determined the roles of Ca2+-independent PLA2 (iPLA2) in phospholipid chemistry and oxidant-induced cell death in human astrocytes. A172 cells expressed both cytosolic Group VIA (iPLA2b) and microsomal Group VIB (iPLA2g) PLA2 as determined by activity assays and immunoblot analysis. Inhibition of total iPLA2 activity using racemic bromoenol lactone (BEL, 2.5 mM) decreased the expression of 14:0– 16:0 phosphatidylcholine (PtdCho) 15% and increased 18:0–18:1-PtdCho expression 15%. Treatment of cells with the iPLA2g specific inhibitor RBEL decreased 14:0–16:0-PtdCho 35%, 16:0–16:0-PtdCho 15% and induced a 35% increase in 18:0–18:1-PtdCho. In contrast, treatment of cells with the iPLA2b inhibitor S-BEL did not alter any phospholipid studied. To determine the roles of iPLA2 in oxidant-induced cell death, A172 cells were exposed to hydrogen peroxide (H2O2) or tert-butylhydroperoxide (TBHP); both induced time- and concentration-dependent increases in cell death as assessed by annexin V and propidium iodide staining. Treatment of cells with racemic-BEL alone did not induce cell death. However, pretreatment with BEL prior to H2O2 (500 mM) or TBHP (200 mM) significantly increased necrosis as determined by increases in propidium iodide staining. Treatment with BEL prior to exposure to oxidants accelerated the loss of ATP levels, but not the formation of reactive oxygen species. These data support the hypothesis that iPLA2 mediates oxidant-induced neural cell death and demonstrates differential roles of iPLA2 isoforms in physiological and pathological events. # 2006 Elsevier Inc. All rights reserved. Keywords: Phospholipase A2; Phospholipids; Neural cells; Necrosis; Electrospray ionization-mass spectrometry

1. Introduction Phospholipases (PLA2) are esterases that hydrolyze the sn-2 ester bond in phospholipids releasing a free fatty acid and lysophospholipid (Cummings et al., 2000). The released free fatty acid is commonly arachidonic acid (AA), which can be synthesized into eicosanoids such as leukotrienes or prostaglandins, while the lysophospholipids are precursors for platelet activating factor (PAF), causing PLA2 to play an important role in the mechanism of inflammation. PLA2 have also been shown to play integral roles in the Lands cycle, the regulatory process of deacylation–reacylation of fatty acids to maintain cell membranes (Lands, 1965). When PLA2 acts on a phospholipid, the released lysophospholipid is acylated by

* Corresponding author at: 371 College of Pharmacy, University of Georgia, Athens, GA 30602, United States. Tel.: +1 706 542 3792; fax: +1 706 542 5358. E-mail address: [email protected] (B.S. Cummings). 0161-813X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2006.09.006

CoA-dependent acyltransferase with a free fatty acid and reintegrated into the cell membrane. Historically, PLA2 have been classified based on whether they were secreted from the cell (sPLA2), Ca2+-dependent and located in the cytosol (cPLA2), or Ca2+-independent (iPLA2) (Cummings et al., 2000). iPLA2, also referred to as Group VI PLA2, consists of two major isoforms, iPLA2b (Group VIA) and iPLA2g (Group VIB) (Six and Dennis, 2000). iPLA2 has been shown to comprise 70% of the PLA2 activity in rat brain homogenates (Yang et al., 1999a,b). In addition, our laboratory recently demonstrated that tissue homogenates isolated from rabbit and rat brain express iPLA2g and contain high amounts of microsomal activity (Kinsey et al., 2005). However, the expression of individual iPLA2 isoforms in human neural cells or their role in phospholipid chemistry has never been determined. Recent studies have demonstrated that both iPLA2b and g mediate phospholipid chemistry in several non-neural cell models including cells isolated from the pancreas, heart and kidney (Cummings et al., 2002). Many of these studies focused

B. Peterson et al. / NeuroToxicology 28 (2007) 150–160

on the role of iPLA2b on phospholipid chemistry and cell death. This isoform is primarily found in the cytosol and is suggested to have a ‘‘housekeeping’’ role in maintaining the cell membrane, but may also play a key role in cell death (Balsinde and Balboa, 2005). In contrast, less is known about the role of iPLA2g in maintaining phospholipid chemistry, especially in neural cells where no studies exist. Further, the differential expression of these isoforms in neural cells, as well as their contribution to phospholipid expression and toxicity has never been determined. The high lipid content of the brain, the majority of which are phospholipids (Farooqui et al., 2000), makes it especially susceptible to alterations in lipid profiles. One of the physiological functions of lipids in the brain is to act as a barrier to the outside environment while maintaining fluidity and permeability to ions. This function is inherently linked to the type of phospholipids present in the cellular membrane, and their interactions with each other. Alterations in these phospholipids’ composition or conformation may serve to make the brain more susceptible to oxidative injury. Further, neural cells also have the highest levels of oxygen consumption, a high content of iron, and the lowest levels of anti-oxidants in the body, making them vulnerable to oxidative stress (Iwashita et al., 2003). While studies demonstrate that oxidative stress correlates to lipid peroxidation in neural cells, the exact phospholipid targeted or the mechanisms involved in the alterations in these phospholipids has not been determined (Brand et al., 2001). Our previous work demonstrated that inhibition of iPLA2 activity in non-neural cells altered the phospholipid profile of these cells and correlated to increases in lipid peroxidation, loss of phospholipids and increased cell death in rabbit renal proximal tubular cells (RPTC), an epithelial cell model (Cummings et al., 2002). Further, inhibition of iPLA2 potentiated oxidant-induced cell death. These studies suggest the hypothesis that iPLA2 mediates oxidant-induced cell death by regulating the metabolism of glycerophospholipids. The high lipid content of the brain coupled with the high level of iPLA2 activity and the susceptibility of brain tissue to oxidative injury, suggest the hypothesis that iPLA2 may play a similar role in neural cells. Astrocytes represent the most abundant type of cell in the brain (Nedergaard et al., 2003). They are epithelial in nature, provide nutrients and structural support to other types of neural cells, regulate cellular communication, and have also been identified as the first line of defense following neuronal injury (Sun et al., 2005). In addition, astrocytes are integral in the formation of the blood–brain barrier (BBB); they form a cohesive layer of cells that encompass the capillary epithelial cells of the BBB (Haseloff et al., 2005). However, the differential expression of iPLA2 isoforms in astrocytes is not known, nor is the role of iPLA2 in phospholipid metabolism and oxidant-induced injury. Recent studies demonstrated differential roles for iPLA2b and g in alpha-amino-3-hydroxy-5-methylisoxazole-propionate receptor regulation during long-term potentiation in brain slices (Martel et al., 2006). However, the toxicological roles of these isoforms were not addressed. Thus, this study tested the

151

hypothesis that iPLA2 isoforms play differential roles in maintaining neural cell phospholipids and mediate oxidantinduced cell death. 2. Materials and methods 2.1. Materials Dulbecco’s Modified Eagle’s Medium, and fetal bovine serum were purchased from American Type Culture Collection (Manassas, VA). Annexin V-FITC was obtained from R&D Systems (San Diego, CA). Cellstripper was obtained from Mediatech, Inc. (Herndon, VA). Propidium iodide (PI), cPLA2 assay kit, and bromoenol lactone (BEL) were obtained from Cayman Chemical Co. (Ann Arbor, MI). Dichlorofluorescein assay was obtained from Invitrogen (Carlsbad, CA). Bioluminescent Somatic Cell Assay kit and all other chemicals were obtained from Sigma Chemical (St. Louis, MO). 2.2. Cell cultures A172 cells (human astrocytic) were obtained from American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, and 1% penicillin and streptomycin. A172 cells grown in 48well plates were exposed to oxidants (H2O2 and TBHP), and BEL at approximately 80% confluence. Cells were treated with BEL for 30 min prior to treatment with oxidants. 2.3. Immunoblot analysis Protein concentration was determined as described prior to immunoblot analysis and 20 mg was mixed with SDS–sample buffer, heated to 70 8C for 10 min, separated under reducing conditions on a 12% SDS–polyacrylamide gel, and transferred to a nitrocellulose membrane. Non-specific binding was blocked by incubating the membrane in 3% bovine serum albumin in TBS overnight at room temperature. The membrane was incubated with a polyclonal rabbit antiiPLA2b antibody against 85 kDa iPLA2 (Cayman Chemical Co.) for 2 h. The membrane was washed, incubated with horse-radish peroxidase-linked secondary antibody for 1 h at room temperature, washed and bands detected using enhanced chemiluminescence (Amersham Biosciences, Inc., Piscataway, NJ). The membrane was stripped and re-probed using an anti-peptide antibody (CZSKYIERNEHKMKKVAK) (rabbit anti-chicken) against iPLA2g as previously described (Saavedra et al., 2006). 2.4. Reverse transcriptase-PCR RNA was isolated from A172 cells using Trizol Reagent (Life Technologies). RT-PCR using total RNA isolated from cells was performed using the following primers designed against the sequence of Group VIA and VIB iPLA2 (Group VIB = sense: 50 -ATTGATGGTGGAGGAACAAGG30 , anti-sense: 50 -ATGGCCTGCCACATTTTATAC-30 ; Group

152

B. Peterson et al. / NeuroToxicology 28 (2007) 150–160

VIA = sense: 50 -GCAAGGGTGATGGGGAGA-30 , anti-sense: 30 -GGGTGTTGCCGTGCTCTC-50 ). The RT step was performed at 50 8C for 30 min followed by 2 min at 94 8C to inactivate the RT. PCR was then performed with 40 cycles of 30 s at 72 8C, 90 s at 56.8 8C, and 40 s at 92 8C followed by a final extension step of 5 min at 72 8C. Negative controls included the lack of TAQ polymerase. PCR products were separated and visualized using agarose gel electrophoresis and ethidium bromide staining.

(Cummings and Schnellmann, 2002). Briefly, media was removed, cells washed twice with PBS and incubated in binding buffer (10 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, pH 7.4) containing annexin V-FITC (25 mg/mL) and PI (25 mg/mL) for 10 min. Cells were washed three times in binding buffer, released from monolayers using a rubber policeman and staining quantified using a Becton Dickinson FacsCalibur flow cytometer. For each measurement 1000 events were counted.

2.5. Measurement of iPLA2 activity

2.8. Measurement of reactive oxygen species

A172 cells were collected and subcellular fractions isolated using differential centrifugation. Arachidonoyl thio-PtdCho was used as a synthetic substrate to detect PLA2 activity. Hydrolysis of the arachidonoyl thioester bond at the sn-2 bond by PLA2 releases a free thiol which can be detected by 5,50 dithiobis(2-nitrobenzoic acid) (DTNB). PLA2 activity was measured in cytosolic and microsomal fractions in the absence and presence of 2.5 mM racemic, R- or S-BEL and 4 mM EGTA. Samples were treated for 30 min at room temperature prior to the assay. Activity was calculated by measuring the absorbance of DTNB (e = 10.66) at 404 nm and normalized with the protein content of each sample.

The generation of reactive oxygen species (ROS) was measured using the fluorescent dye 20 ,70 -dichlorofluorescein diacetate (DCFH-DA). Cells were grown to 80% confluence in 12-well plates and preloaded with 10 mM DCFH-DA for 30 min at 37 8C. After incubation, the media was aspirated and replaced with fresh media. Cells were pretreated with 2.5 mM BEL for 30 min and then dosed with H2O2 and TBHP for 2 or 4 h. After treatment, media was aspirated and cells detached using 0.01 M Tris–HCl with 0.5% Triton X-100 (pH 7.4). A 100 mL aliquot was used to measure fluorescence at excitation 485 nm and emission 520 nm. Measurements were normalized using protein content as determined with the BCA assay.

2.6. Characterization and quantitation of cellular phospholipids using electrospray ionization-mass spectrometry (ESI-MS)

2.9. Measurement of ATP levels

Cellular phospholipids were extracted using chloroform and methanol according to the method of Bligh and Dyer (1959) at 4 8C and extracts dried down under argon. Lipid phosphorus was quantified using malachite green (Zhou and Arthur, 1992) and extract samples (500 pmol/mL) were prepared by reconstituting in chloroform:methanol (2:1, v:v). Mass spectrometry was performed as described previously (Taguchi et al., 2000). Samples were analyzed using a LCT Premier time of flight mass spectrometer (Waters, Milford, MA) equipped with an electrospray ion source. Five microliters of sample was introduced by means of a flow injector into the ESI chamber at a rate of 0.2 ml/ min. The elution solvent was acetonitrile:methanol:water (2:3:1, v:v:v) containing 0.1% (w/v) ammonium formate (pH 6.4). The mass spectrometer was operated in the positive scanning mode. The flow rate of nitrogen drying gas was 10 L/min at 80 8C. The capillary and cone voltages were set at 2.5 kV and 30 V, respectively. As previously described (Taguchi et al., 2000), qualitative identification of individual phospholipid molecular species was based on their calculated theoretical mono-isotopic mass values and quantification was done by comparison to the most abundant phospholipid in each sample, which corresponded to m/z ratio of 760 [(34:1) (16:0–18:1)]PtdCho. 2.7. Measurement of annexin V and propidium iodide staining Annexin V and propidium iodide staining were determined using flow cytometry as previously described with modifications

ATP levels were measured using a bioluminescent luciferase based assay according to manufacturer’s instructions. Briefly, after oxidant exposure, cells were lysed using somatic cell ATP releasing reagent provided by the kit. Fifty microliters of cell lysate was added to the luciferase substrates and bioluminescence was measured. The cellular ATP level was reported as percentage of control cells. 2.10. Measurement of cell morphology Media was removed, filtered through 70 mM mesh into 5 mL snap cap tubes and stored at 4 8C. Five hundred microliters Cellstripper was added to each well and plates were incubated for 60 min at 37 8C. Cells were released by pipetting, filtered through 70 mM mesh and added to the appropriate 5 mL snap cap tube. Analysis of forward and side scatter was measured using a Becton Dickinson FacsCalibur flow cytometer as previously described (Bortner and Cidlowski, 1999). For each measurement 1000 events were counted. Cell shrinkage was indicated by decreases in forward scatter only. Simultaneous decreases in both forward scatter and side scatter indicated cell lysis and necrosis. 2.11. Assessment of cell morphology After treatment, A172 cells were washed twice with PBS and fixed for 10 min using 10% buffered formalin. After fixing, cells were washed three times with PBS and coverslips applied. Images were taken using a Nikon TE300 inverted microscope (Nikon, Melville, NY) equipped with a CCD camera (MicroMAX,

B. Peterson et al. / NeuroToxicology 28 (2007) 150–160

153

Fig. 1. Expression of iPLA2 isoforms in A172 cells. Immunoblot analysis (A and C) and RT-PCR (B and D) for iPLA2g (A and B) and iPLA2b (C and D). Immunoblot panels (A and C) M, marker; 1, microsomes; 2, cytosol. RT-PCR panels (B and D): M, marker; 1, positive control; 2, A172 sample.

Princeton Instruments, Trenton, NJ) using Metamorph software (Universal Imaging Corporation, Westchester, PA). 2.12. Protein determination Protein concentration was determined using the bicinchonic acid (BCA) assay method as described by Sigma. 2.13. Statistical analysis Cells isolated from a distinct passage of A172 cells represent one experiment (n = 1). Data are represented as the average  S.E. of at least three separate experiments (n = 3). The appropriate analysis of variance (ANOVA) was performed for each data set using GraphPad Prism software. Individual means were compared using the Tukey’s test with P < 0.05 being considered indicative of a statistically significant difference between mean values. 3. Results 3.1. Expression of iPLA2b and iPLA2g in A172 cells Microsomal and cytosolic fractions from confluent A172 cells were subjected to immunoblot analysis using antibodies that recognize iPLA2b and iPLA2g. The iPLA2g antibody recognized a single band in the microsomal fraction at 70 kDa (Fig. 1A). The iPLA2b antibody recognized a single band in the cytosolic fraction at 85 kDa (Fig. 1C). In addition, RT-PCR using primers designed against the sequences for human iPLA2g (Fig. 1B) and iPLA2b (Fig. 1D) demonstrated the expression of cDNA products for each isoform. Collectively these data demonstrate that iPLA2b and g are both expressed in A172 cells. 3.2. Activity of iPLA2b and iPLA2g in A172 cells Cytosolic and microsomal iPLA2 activity was detected in A172 cells (Fig. 2). Activity in the cytosolic fraction of control

Fig. 2. Effect of R/S-BEL, R-BEL and S-BEL on iPLA2 activity in A172 cells. A172 cells were exposed to either racemic-BEL (R/S-BEL), R-BEL, or S-BEL for 30 min prior to isolation and subcellular fractionation into microsomes and cytosol. Cytosol (A) and microsomal (B) fractions were assessed for iPLA2 activity based on the hydrolysis of the sn-2 bond of arachidonoyl thio-phosphatidylcholine substrate. Data are represented as the average  S.E. of at least three separate experiments. Means with different subscripts are significantly different from each other (P < 0.05).

154

B. Peterson et al. / NeuroToxicology 28 (2007) 150–160

Fig. 3. Lipidomic analysis of the effect of iPLA2 inactivation in A172 cells. Confluent cells were treated with either solvent control or 2.5 mM racemic, R- or S-BEL for 12 h. Individual phospholipids were then separated and quantified as described in Section 2. The effect on 14:0–16:0-PtdCho (A), 16:0–16:0-PtdCho (B), and 18:0–18:1-PtdCho (C) is shown as percent change from control. Data are represented as the average  S.E. of at least three separate experiments. Means with different subscripts are significantly different from each other (P < 0.05).

cells was 0.8713 mmol min1 mg protein1. Treatment with 2.5 mM racemic, R-, or S-BEL, doses demonstrated to inhibit iPLA2 activity in several cells models (Cummings et al., 2002; Kinsey et al., 2005; Zhang et al., 2005), decreased iPLA2 activity to 0.1041, 0.4690, and 0.1073 mmol min1 mg protein1, respectively (Fig. 2A). Activity in the microsomal fraction of control cells was 0.9920 mmol min1 mg protein1. Treatment with 2.5 mM racemic, R-, or S-BEL decreased iPLA2 activity to 0.3826, 0.0201, and 0.5087 mmol min1 mg protein1, respectively (Fig. 2B). These data demonstrate that A172 cells display activities correlating to iPLA2b and iPLA2g, and that iPLA2b is preferentially inhibited by S-BEL, while iPLA2g is preferentially inhibited by R-BEL.

PtdCho in A172 cells, the effect of iPLA2 inhibition on this profile was determined. To determine the specific roles of iPLA2b and iPLA2g on A172 phospholipid chemistry, A172 cells were exposed to either 2.5 mM racemic, R- or S-BEL for 12 h. Inhibition of total iPLA2 activity using racemic-BEL resulted in significant decreases of 14:0–16:0-PtdCho (Fig. 3A) and increases in 18:0–18:1-PtdCho (Fig. 3C). Treatment with 2.5 mM R-BEL alone decreased 14:0–16:0 and 16:0–16:0-PtdCho (Fig. 3B) and increased 18:0–18:1-PtdCho. However, treatment with 2.5 mM S-BEL did not significantly change any PtdCho studied.

3.3. Lipidomic analysis of the effect of iPLA2 inactivation in A172 cells

Exposure of A172 cells to the model oxidants hydrogen peroxide (H2O2; 0–500 mM) and tert-butylhydroperoxide (TBHP; 0–200 mM) for 12 h resulted in concentrationdependent increases in cell death as assessed by annexin V (apoptotic marker) and propidium iodide (necrosis marker) staining using flow cytometry. These compounds also induced time-dependent increases in cell death (data not shown). H2O2 induced time- and concentration-dependent increases in propidium iodide staining, without an increase in annexin V staining (Fig. 4A). Treatment with TBHP induced time- and concentration-dependent increases in annexin V staining, without an increase in propidium iodide staining (Fig. 4B). Similar findings were seen when cell morphology was assessed (data not shown). Based on these data, 500 mM H2O2 and 200 mM TBHP were chosen for further study.

The basic phospholipid profile of A172 cells was determined using electrospray ionization mass-spectrometry (ESI-MS). ESI-MS offers several advantages over HPLC, including the fact that a lower amount of sample is needed for analysis and multiple head groups can be analyzed in one run. Further, the increased sensitivity of ESI-MS allows for the analysis of phospholipids whose abundance is as low as 1% of total cellular phospholipids (Taguchi et al., 2000). Neural membranes are mainly composed of phosphatidylcholine (PtdCho) in addition to plasmenyl– and phosphatidyl– ethanolamine (PlsEtn and PtdEtn) (Farooqui et al., 2004). Following establishment of the phospholipid profile for

3.4. Effect of oxidants on A172 cell death

B. Peterson et al. / NeuroToxicology 28 (2007) 150–160

155

Table 1 Effect of BEL on propidium iodide staining in A172 cells Treatment

Solvent control

+2.5 mM BELa

Control 500 mM H2O2 200 mM TBHP

6.24  2.17 19.4  4.62b 8.54  4.92

11.23  4.88 26.70  5.70c 23.33  1.84c

a b c

Treated for 30 min prior to exposure to either H2O2 or TBHP for 12 h. Significantly different (P < 0.05) from solvent control. Significantly different (P < 0.05) from oxidant treated only cells.

Table 2 Effect of BEL on DCFH-DA staining in A172 cellsa Treatment

Solvent control

+2.5 mM BELb

Control 500 mM H2O2 200 mM TBHP

21.88  9.8 41.90  11.14 72.67  12.79c

14.82  6.88 38.64  21.41 60.30  10.61c

a b c

As reported in Arbitrary Fluorescence Units. Treated for 30 min prior to exposure to either H2O2 or TBHP for 4 h. Significantly different (P < 0.05) from solvent control.

were seen when TBHP was used in place of H2O2 and propidium iodide staining was assessed (Table 1). Annexin V binding did not increase in the presence of BEL and oxidants (data not shown), indicating the method of cell death was primarily necrosis. These data demonstrate the novel finding that inhibition of total iPLA2 activity increases oxidant-induced A172 cell death. 3.6. ROS accumulation

Fig. 4. Oxidant-induced A172 cell death. Confluent cells were treated with either H2O2 (0–1000 mM) (A) or TBHP (0–200 mM) (B) and annexin V-FITC and propidium iodide staining were determined by flow cytometry. Cells staining positive only for annexin V-FITC indicate cells undergoing apoptosis only. Cells staining positive for both annexin V-FITC and propidium iodide indicate cells undergoing late apoptosis. Cells staining for propidium iodide only indicate cells undergoing necrosis. Data are represented as the average  S.E. of at least three separate experiments. Means with different subscripts are significantly different from each other (P < 0.05).

3.5. Effect of iPLA2 inhibition on oxidant-induced cell death Inhibition of iPLA2 in renal cells increased oxidant-induced necrosis suggesting that iPLA2 protects against oxidantinduced cell death (Cummings et al., 2002). To test the hypothesis that iPLA2 has a similar role in neural cells, A172 cells were exposed to either solvent control or 2.5 mM racemicBEL for 30 min prior to being dosed with either 500 mM H2O2 or 200 mM TBHP for 12 h. BEL treatment alone did not induce cell death itself at the concentration used as determined by cell morphology and propidium iodide staining (necrotic cell marker) (Table 1). However, treatment with BEL prior to exposure to H2O2 significantly increased cell death compared to H2O2-only treated cells based on cell morphology and propidium iodide staining (Table 1 and Fig. 5). Similar results

Exposure of cells to H2O2 and TBHP for 4 h caused concentration-dependent increases in the formation of ROS in A172 cells as indicated by increases in DCFH-DA fluorescence (Table 2). Interestingly, treatment with racemic-BEL prior to oxidant exposure did not alter DCFH-DA fluorescence either in the presence or absence of oxidants (Table 2). 3.7. Intracellular ATP levels Treatment of A172 cells with H2O2 and TBHP induced concentration-dependent decreases in intracellular ATP levels after 4 h (Fig. 6A and B). Pretreatment with 2.5 mM racemicBEL followed by exposure to 500 mM H2O2 or 200 mM TBHP accelerated the decline in ATP levels (Fig. 6C and D). To determine if a specific iPLA2 isoform was responsible for this effect, cells were treated for 30 min with either 2.5 mM R- or SBEL prior to exposure to 500 mM H2O2 or 200 mM TBHP (Fig. 6E and F). Treatment of cells with S-BEL prior to oxidant exposure reduced ATP levels to that comparable to those exposed to racemic-BEL and oxidants. In contrast, R-BEL had no effect. 4. Discussion While numerous studies observed the presence of iPLA2 activity in the brain (Balboa et al., 2002; Farooqui and

156

B. Peterson et al. / NeuroToxicology 28 (2007) 150–160

Fig. 5. Morphological changes in A172 cells undergoing oxidant-induced cell death. Confluent cells were treated with either solvent control (A), 2.5 mM BEL (B), 500 mM H2O2 (C), or pretreated with BEL 30 min prior to exposure of H2O2 (D). After treatment, cells were washed, fixed, and phase contrast images taken using a Nikon TE300 inverted microscope at 20 magnification.

Horrocks, 2004; Farooqui et al., 1997; Larsson Forsell et al., 1999; Molloy et al., 1998; St-Gelais et al., 2004), few studies have examined the differential expression of individual iPLA2 isoforms. Even less have studied physiological or toxicological roles for these isoforms in single cells. We report herein the novel finding that both iPLA2b and iPLA2g are expressed in a human astrocytic cell line (A172) and differentially regulate both cellular phospholipids and oxidant-induced cell death. A direct comparison of iPLA2b to g expression cannot be made due to differences in antibody specificity, primer efficiency, and the fact that the activity assays do not directly measure specific activity. However, comparison of the expression and activity of iPLA2b to tissue isolated from rat and rabbit brain (Kinsey et al., 2005) suggest that A172 cells express comparable levels of iPLA2b. Similar findings were found for iPLA2g. Identification of iPLA2b and g expression in neural cells raises questions concerning their physiological roles. Previous studies in neural tissues suggest roles for PLA2 in maintenance of phospholipids (Farooqui and Horrocks, 2006; Farooqui et al., 1999), and some have even suggested roles for iPLA2 in general. (Muralikrishna Adibhatla and Hatcher, 2006). These studies are supported by data derived in macrophages (Balsinde and Dennis, 1997), pancreatic islets (Ramanadham et al., 1999), and renal cells (Cummings et al., 2004). Although studies have established a housekeeping role for iPLA2 in regards to cellular phospholipids and lipid remodeling (Balsinde and Balboa, 2005), the exact phospholipids involved have not been fully identified.

Analysis of phospholipids expressed in A172 cells suggests that these cells express high levels of PtdCho, a finding supported by one other study (Farooqui et al., 2000). Thus, we focused on the physiological role of iPLA2 in regulation of the expression of PtdCho phospholipids. The ability of racemicBEL to alter the expression of 14:0–16:0 and 18:0–18:1PtdCho suggests that iPLA2 mediates the expression of these phospholipids. This hypothesis is supported by a recent study from our laboratory demonstrating that iPLA2 inhibition decreases 14:0–16:0-PtdCho expression in renal epithelial cells (Zhang et al., 2005). To our knowledge, these data represent the first to identify the specific phospholipids altered in neural cells during iPLA2 inhibition. The exact mechanisms involved are still under investigation, but may involve the interaction of iPLA2 in the Lands cycle (Das et al., 2001). Further, the decrease in these phospholipids may explain why inhibition of iPLA2 alters membrane permeability in rat hippocampal slices (St-Gelais et al., 2004). Experiments using R- and S-BEL demonstrated a differential effect of these inhibitors on A172 cellular phospholipids. The negative effect of S-BEL, compared to R-BEL, supports the hypothesis that iPLA2g plays a greater role than iPLA2b in the maintenance of PtdCho phospholipids in A172 cells under physiological conditions, and support the hypothesis that iPLA2 isoforms have differential roles in astrocyte cell physiology. Treatment of cells with R-BEL resulted in greater decreases in 14:0–16:0-PtdCho, and greater increases in 18:0–18:1-PtdCho compared to racemic-BEL. Further, R-BEL decreased 16:0– 16:0-PtdCho, while racemic-BEL did not. These results are

B. Peterson et al. / NeuroToxicology 28 (2007) 150–160

157

Fig. 6. Effect of iPLA2 inactivation and oxidant treatment on intracellular ATP levels in A172 cells. Confluent cells were dosed with H2O2 (A: 0–1000 mM) or TBHP (B: 0–800 mM) for 4 h. Pretreatment with 2.5 mM racemic-BEL for 30 min prior to exposure to 500 mM H2O2 (C) for 2 h or 200 mM TBHP (D) for 4 h. Differential effect of BEL enantiomers on levels of intracellular ATP. Pretreatment with either 2.5 mM racemic, R-, or S-BEL for 30 min prior to exposure to 500 mM H2O2 (E) or 200 mM TBHP (F) for 2 h. Data are represented as the average  S.E. of at least three separate experiments. Means with different subscripts are significantly different from each other (P < 0.05).

possibly due to the increased efficacy of R-BEL towards iPLA2g compared to racemic-BEL (Kinsey et al., 2005). Data reported above supports the hypothesis that iPLA2g has a larger role than iPLA2b in the maintenance of PtdCho phospholipids. The physiological significance of this finding is currently unclear. However, iPLA2g activity is required for theta burst stimulated long-term potentiation in the hippocampal CA1 area in male rats, and iPLA2g, but not iPLA2b, inhibition affects the insertion of AMPA receptors at the cell surface after N-methyl-D-aspartate-induced potentiation (Martel et al., 2006). The mechanisms involved in these processes are under study, but the ability of iPLA2g, but not iPLA2b, to alter choline phospholipids correlates to decreases in AMPA receptor insertion. It is also possible that iPLA2b and g may differentially regulate other neural cell processes including cell growth (Saavedra et al., 2006) and membrane permeability (Martel et al., 2006; St-Gelais et al., 2004).

This study also addressed the differential roles of iPLA2 isoforms in neurotoxicity induced by oxidants in astrocytes. Cerebral injury resulting from trauma, ischemic events, or glucose deprivation can lead to decreases in ATP, increases in free radicals and reactive oxygen species, which in turn can result in damage to phospholipids and compromised cellular membranes. Recent reviews suggest roles for PLA2 in these events, including iPLA2, but few of these reviews specifically discuss the differential role of iPLA2 isoforms (Farooqui and Horrocks, 2006; Farooqui et al., 1999). Several studies also suggest that PLA2 play critical roles in cell death and oxidative stress in astrocytes (Clemens et al., 1996; Kramer et al., 1996; Lin et al., 2004; Xu et al., 2002). However, these studies primarily addressed cPLA2 (Xu et al., 2002), or sPLA2 (Lin et al., 2004) and not iPLA2. Data reported above support the hypothesis that iPLA2 mediates oxidant-induced death in astrocytes. The finding that

158

B. Peterson et al. / NeuroToxicology 28 (2007) 150–160

compounds such as TBHP induce oxidative stress in astrocytes is not novel (Robb and Connor, 2002), however the ability of iPLA2 inhibitors to increase oxidative stress is. Increases in cell death were not a result of increased apoptosis, as annexin V staining did not increase in tandem with propidium iodide staining (data not shown). The ability of BEL to increase oxidant-induced cell death is probably not due to inhibition of phosphatidic acid phosphohydrolase-1 (PAPH-1) as PAPH-1 is only inhibited 50% by 25 mM BEL (Balsinde and Dennis, 1996), which is 10-fold higher than the concentrations used in this study. Further, it is unlikely that inhibition of cell death is a result of inhibition of cPLA2g (Group IVC) as BEL, at the concentration used, does not significantly decrease the activity of this protein (Stewart et al., 2002). The hypothesis that iPLA2 inhibition increases oxidantinduced A172 cell death is supported by similar findings in U937 epithelial cells exposed to H2O2 (Balboa and Balsinde, 2002), and our own studies in renal cells (Cummings et al., 2002). Similar to renal cells, our current research suggests that iPLA2 also plays a protective role during oxidant-induced neural cell death. These findings, combined with the fact that iPLA2 mediates astrocyte phospholipid physiology, could have a significant impact on several neurological disorders. For example, Alzheimer disease, Huntington disease, ischemic stroke, and schizophrenia are known to induce alterations in the phospholipid composition and increase PLA2 activities (Farooqui et al., 2000; Ross et al., 1999; Smesny et al., 2005). Inhibition of individual iPLA2 isoforms had differential effects on cell death in astrocytes as determined by the ability of S-BEL, but not R-BEL, to decrease ATP levels after 2 h of exposure. Decreases in ATP occurred as early as 2 h and in tandem with increases in ROS formation. However, neither racemic-BEL, nor S-BEL altered ROS formation. Further, increases in ROS and decreases in ATP occurred prior to increases in annexin V and PI staining. Increases in ROS and decreases in ATP also occurred prior to alterations in cellular phospholipids (data not shown). These data suggest the hypothesis that iPLA2 functions downstream of the initial formation of ROS but prior to decreases in ATP levels. The ability of S-BEL, but not R-BEL, to accelerate the loss of ATP in neural cell exposed to oxidants suggests that iPLA2b plays a greater role in oxidant-induced neural cell death than iPLA2g. The complete mechanisms involved in increased death in A172 cells during iPLA2 inhibition remain to be fully determined, and was beyond the scope of this study. However, based on studies in non-neural tissues (Cummings et al., 2004; Cummings et al., 2002), a lack of iPLA2 activity may result in decreased cleavage, reacylation, and reinsertion of the peroxidized phospholipid into membranes. This would lead to an increase in lipid peroxidation and accelerated propagation. Increased lipid peroxidation could lead to decreased mitochondrial function and decreased ATP levels (Fig. 7). The decrease in ATP may also lead to increased cellular permeability and cell death. Such a role for iPLA2 has been demonstrated in macrophages and pancreatic islets (Balsinde and Dennis, 1997; Ramanadham et al., 1999), but never in astrocyte cells.

Fig. 7. Proposed role of iPLA2b in the mechanisms of oxidant-induced A172 cell death. Oxidant exposure results in the formation of ROS, which can lead to increases in lipid peroxidation or mitochondrial dysfunction, or both. Decreased mitochondrial function can result in decreases in cellular ATP, which would facilitate cell death. A role for iPLA2b is proposed downstream of the initial formation of ROS, but upstream of decreases in ATP levels. It is also possible that iPLA2b may mediate lipid peroxidation via its role in phospholipid maintenance.

The ability of iPLA2 to mediate oxidant-induced death in astrocytes begs the question about what role this might play in other models of neurotoxicology. Loss of astrocytes is correlated to neurodegeneration after ischemia in the forebrain of rats (Clemens et al., 1996). Further, cPLA2 activity in astrocytes is suggested to be a key mediator of inflammation in astrocytes (Kramer et al., 1996). It may be possible that iPLA2 plays a similar role in neurotoxicity. However, in contrast to cPLA2, the ability of iPLA2 ability to participate in the Lands Cycle may serve to inhibit neurotoxicity by cleaving oxidized phospholipids and generating lysophospholipid acceptors, which then could be reacylated and re-inserted into the membrane. These processes would decrease the amount of lipid peroxidation and cell death. A role for iPLA2 in mediation of lipid peroxidation and phospholipid remodeling has already been proposed in renal cells (Cummings et al., 2002, 2004). The above studies demonstrate the differential effect of iPLA2 isoforms in oxidant-induced cell death induced by model oxidants. Recent studies in renal cells demonstrate that inhibition of iPLA2 increased oxidative stress induced by multiple oxidants (Cummings et al., 2002). ROS formation and lipid peroxidation has been reported to occur in more clinical models of neurotoxicity, including ischemia, spinal cord injury, Alzheimer disease, Parkinson disease, and head injury (Farooqui and Horrocks, 2006). Further, increased lipid peroxidation has been hypothesized to be a major mechanism by PLA2-generated lipid mediators are involved in these events (Farooqui and Horrocks, 2006). Thus, future studies will focus on the role of lipid peroxidation and iPLA2 in these models of neurotoxicology. In summary, we demonstrate the expression of both iPLA2b and iPLA2g in human astrocyte cells. The phospholipid profile of A172 cells was also established and specific phospholipids

B. Peterson et al. / NeuroToxicology 28 (2007) 150–160

altered during iPLA2 inhibition were identified, as well as the specific iPLA2 isoform involved. Inhibition of total iPLA2 activity increased oxidant-induced cell death and increased the loss of select phospholipids. Selective inhibition of iPLA2b, but not g, differentially altered the loss of ATP induced by oxidants, but had no effect on ROS formation. These data suggest that iPLA2g plays a more important role in maintenance of membrane phospholipids in neural cells during normal physiological functions, whereas iPLA2b activity may be more important in pathological events such as oxidant-induced neural cell death. Acknowledgements This work was supported by a Georgia Cancer Coalition Distinguished Scholar and University of Georgia Junior Faculty grant to B.S.C. and an American Foundation for Pharmaceutical Education and University of Georgia Fellowships to B.P. Thank you Drs. Rick G. Schnellmann and Jane Mchowat for their gift of the iPLA2g antibody. References Balboa MA, Balsinde J. Involvement of calcium-independent phospholipase A2 in hydrogen peroxide-induced accumulation of free fatty acids in human U937 cells. J Biol Chem 2002;277(43):40384–9. Balboa MA, Varela-Nieto I, Killermann Lucas K, Dennis EA. Expression and function of phospholipase A(2) in brain. FEBS Lett 2002;531(1):12–7. Balsinde J, Balboa MA. Cellular regulation and proposed biological functions of group VIA calcium-independent phospholipase A2 in activated cells. Cell Signal 2005;17(9):1052–62. Balsinde J, Dennis EA. Bromoenol lactone inhibits magnesium-dependent phosphatidate phosphohydrolase and blocks triacylglycerol biosynthesis in mouse P388D1 macrophages. J Biol Chem 1996;271(50):31937–41. Balsinde J, Dennis EA. Function and inhibition of intracellular calciumindependent phospholipase A2. J Biol Chem 1997;272(26):16069–72. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 1959;37(8):911–7. Bortner CD, Cidlowski JA. Caspase independent/dependent regulation of K(+), cell shrinkage, and mitochondrial membrane potential during lymphocyte apoptosis. J Biol Chem 1999;274(31):21953–62. Brand A, Gil S, Seger R, Yavin E. Lipid constituents in oligodendroglial cells alter susceptibility to H2O2-induced apoptotic cell death via ERK activation. J Neurochem 2001;76(3):910–8. Clemens JA, Stephenson DT, Smalstig EB, Roberts EF, Johnstone EM, Sharp JD, et al. Reactive glia express cytosolic phospholipase A2 after transient global forebrain ischemia in the rat. Stroke 1996;27(3):527–35. Cummings BS, Gelasco AK, Kinsey GR, McHowat J, Schnellmann RG. Inactivation of endoplasmic reticulum bound Ca2+-independent phospholipase A2 in renal cells during oxidative stress. J Am Soc Nephrol 2004; 15(6):1441–51. Cummings BS, McHowat J, Schnellmann RG. Phospholipase A(2)s in cell injury and death. J Pharmacol Exp Ther 2000;294(3):793–9. 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 2002;283(3):F492–8. Cummings BS, Schnellmann RG. Cisplatin-induced renal cell apoptosis: caspase 3-dependent and -independent pathways. J Pharmacol Exp Ther 2002;302(1):8–17. Das S, Castillo C, Stevens T. Phospholipid remodeling/generation in Giardia: the role of the Lands cycle. Trends Parasitol 2001;17(7):316–9. Farooqui AA, Horrocks LA. Brain phospholipases A2: a perspective on the history. Prostaglandins Leukot Essent Fatty Acids 2004;71(3):161–9.

159

Farooqui AA, Horrocks LA. Phospholipase A2-generated lipid mediators in the brain: the good, the bad, and the ugly. Neuroscientist 2006;12(3):245–60. 10.1177/1073858405285923. Farooqui AA, Horrocks LA, Farooqui T. Glycerophospholipids in brain: their metabolism, incorporation into membranes, functions, and involvement in neurological disorders. Chem Phys Lipids 2000;106(1):1–29. Farooqui AA, Litsky ML, Farooqui T, Horrocks LA. Inhibitors of intracellular phospholipase A2 activity: their neurochemical effects and therapeutical importance for neurological disorders. Brain Res Bull 1999; 49(3):139–53. Farooqui AA, Ong WY, Horrocks LA. Biochemical aspects of neurodegeneration in human brain: involvement of neural membrane phospholipids and phospholipases A2. Neurochem Res 2004;29(11):1961–77. Farooqui AA, Yang HC, Rosenberger TA, Horrocks LA. Phospholipase A2 and its role in brain tissue. J Neurochem 1997;69(3):889–901. Haseloff RF, Blasig IE, Bauer HC, Bauer H. In search of the astrocytic factor(s) modulating blood–brain barrier functions in brain capillary endothelial cells in vitro. Cell Mol Neurobiol 2005;25(1):25–39. Iwashita A, Maemoto T, Nakada H, Shima I, Matsuoka N, Hisajima H. A novel potent radical scavenger, 8-(4-fluorophenyl)-2-((2E)-3-phenyl-2-propenoyl)-1,2,3,4-tetrahydropyrazol o[5,1-c] [1,2,4]triazine (FR210575), prevents neuronal cell death in cultured primary neurons and attenuates brain injury after focal ischemia in rats. J Pharmacol Exp Ther 2003;307(3): 961–8. Kinsey GR, Cummings BS, Beckett CS, Saavedra G, Zhang W, McHowat J, et al. Identification and distribution of endoplasmic reticulum iPLA2. Biochem Biophys Res Commun 2005;327(1):287–93. Kramer RM, Stephenson DT, Roberts EF, Clemens JA. Cytosolic phospholipase A2 (cPLA2) and lipid mediator release in the brain. J Lipid Mediat Cell Signal 1996;14(1–3):3–7. Lands WE. Lipid metabolism. Annu Rev Biochem 1965;34:313–46. Larsson Forsell PK, Kennedy BP, Claesson HE. The human calcium-independent phospholipase A2 gene multiple enzymes with distinct properties from a single gene. Eur J Biochem 1999;262(2):575–85. Lin TN, Wang Q, Simonyi A, Chen JJ, Cheung WM, He YY, et al. Induction of secretory phospholipase A2 in reactive astrocytes in response to transient focal cerebral ischemia in the rat brain. J Neurochem 2004; 90(3):637–45. Martel MA, Patenaude C, Menard C, Alaux S, Cummings BS, Massicotte G. A novel role for calcium-independent phospholipase A in alpha-amino-3hydroxy-5-methylisoxazole-propionate receptor regulation during longterm potentiation. Eur J Neurosci 2006;23(2):505–13. Molloy GY, Rattray M, Williams RJ. Genes encoding multiple forms of phospholipase A2 are expressed in rat brain. Neurosci Lett 1998;258(3): 139–42. Muralikrishna Adibhatla R, Hatcher JF. Phospholipase A2, reactive oxygen species, and lipid peroxidation in cerebral ischemia. Free Radic Biol Med 2006;40(3):376–87. Nedergaard M, Ransom B, Goldman SA. New roles for astrocytes: redefining the functional architecture of the brain. Trends Neurosci 2003;26(10): 523–30. Ramanadham S, Hsu FF, Bohrer A, Ma Z, Turk J. Studies of the role of group VI phospholipase A2 in fatty acid incorporation, phospholipid remodeling, lysophosphatidylcholine generation, and secretagogue-induced arachidonic acid release in pancreatic islets and insulinoma cells. J Biol Chem 1999; 274(20):13915–27. Robb SJ, Connor JR. Nitric oxide protects astrocytes from oxidative stress. Ann N Y Acad Sci 2002;962:93–102. Ross BM, Turenne S, Moszczynska A, Warsh JJ, Kish SJ. Differential alteration of phospholipase A2 activities in brain of patients with schizophrenia. Brain Res 1999;821(2):407–13. Saavedra G, Zhang W, Peterson B, Cummings BS. Differential roles for cytosolic and microsomal Ca2+-independent phospholipase A2 in cell growth and maintenance of phospholipids. J Pharmacol Exp Ther 2006;318(3):1211– 9. 10.1124/jpet.106.105650. Six DA, Dennis EA. The expanding superfamily of phospholipase A(2) enzymes: classification and characterization. Biochim Biophys Acta 2000;1488 (1–2):1–19.

160

B. Peterson et al. / NeuroToxicology 28 (2007) 150–160

Smesny S, Kinder D, Willhardt I, Rosburg T, Lasch J, Berger G, et al. Increased calcium-independent phospholipase A2 activity in first but not in multiepisode chronic schizophrenia. Biol Psychiatry 2005;57(4):399–405. Stewart A, Ghosh M, Spencer DM, Leslie CC. Enzymatic properties of human cytosolic phospholipase A2gamma. J Biol Chem 2002;277(33):29526–36. St-Gelais F, Menard C, Congar P, Trudeau LE, Massicotte G. Postsynaptic injection of calcium-independent phospholipase A2 inhibitors selectively increases AMPA receptor-mediated synaptic transmission. Hippocampus 2004;14(3):319–25. Sun GY, Xu J, Jensen MD, Yu S, Wood WG, Gonzalez FA, et al. Phospholipase A2 in astrocytes: responses to oxidative stress, inflammation, and G proteincoupled receptor agonists. Mol Neurobiol 2005;31(1–3):27–41. Taguchi R, Hayakawa J, Takeuchi Y, Ishida M. Two-dimensional analysis of phospholipids by capillary liquid chromatography/electrospray ionization mass spectrometry. J Mass Spectrom 2000;35(8):953–66.

Xu J, Weng YI, Simonyi A, Krugh BW, Liao Z, Weisman GA, et al. Role of PKC and MAPK in cytosolic PLA2 phosphorylation and arachadonic acid release in primary murine astrocytes. J Neurochem 2002;83(2):259–70. Yang HC, Mosior M, Johnson CA, Chen Y, Dennis EA. Group-specific assays that distinguish between the four major types of mammalian phospholipase A2. Anal Biochem 1999a;269(2):278–88. Yang HC, Mosior M, Ni B, Dennis EA. Regional distribution, ontogeny, purification, and characterization of the Ca2+-independent phospholipase A2 from rat brain. J Neurochem 1999b;73(3):1278–87. Zhang L, Peterson BL, Cummings BS. The effect of inhibition of Ca2+independent phospholipase A2 on chemotherapeutic-induced death and phospholipid profiles in renal cells. Biochem Pharmacol 2005;70(11): 1697–706. Zhou X, Arthur G. Improved procedures for the determination of lipid phosphorus by malachite green. J Lipid Res 1992;33(8):1233–6.