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. Author manuscript; available in PMC: 2009 Aug 1.
Published in final edited form as: J Neurochem. 2008 Jun 28;106(4):1828–1840. doi: 10.1111/j.1471-4159.2008.05527.x

Cytosolic Phospholipase A2α Inhibition Prevents Neuronal NMDA Receptor-Stimulated Arachidonic Acid Mobilization and Prostaglandin Production but not Subsequent Cell Death

Ava L Taylor 1, Joseph V Bonventre 2, Tracy F Uliasz 1, James A Hewett 1, Sandra J Hewett 1
PMCID: PMC2582587  NIHMSID: NIHMS56864  PMID: 18564366

Abstract

Phospholipase A2 (PLA2) enzymes encompass a superfamily of at least 13 extracellular and intracellular esterases that hydrolyze the sn-2 fatty acyl bonds of phospholipids to yield fatty acids and lysophospholipids. The purpose of this study was to characterize which phospholipase paralog regulates NMDA receptor-mediated arachidonic acid (AA) release. Using mixed cortical cell cultures containing both neurons and astrocytes, we found that [3H]-AA released into the extracellular medium following NMDA receptor stimulation (100 µM) increased with time and was completely prevented by the addition of the NMDA receptor antagonist MK-801 (10µM) or by removal of extracellular Ca2+. Neither DAG lipase inhibition (RHC-80267; 10µM) nor selective inhibition of iPLA2 (BEL; 10µM) alone had an effect on NMDA receptor-stimulated release of [3H]-AA. Release was prevented by MAFP (5µM) and AACOCF3 (1µM), inhibitors of both cPLA2 and iPLA2 isozymes. This inhibition effectively translated to block of NMDA-induced prostaglandin (PG) production. An inhibitor of p38MAPK, SB 203580 (7.5µM), also significantly reduced NMDA-induced PG production providing suggestive evidence for the role of cPLA2α. Its involvement in release was confirmed using cultures derived from mice deficient in cPLA2α, which failed to produce PGs in response to NMDA receptor stimulation. Interestingly, neither MAFP, AACOCF3 nor cultures derived from cPLA2α null mutant animals showed any protection against NMDA-mediated neurotoxicity, indicating that inhibition of this enzyme may not be a viable protective strategy in disorders of the cortex involving over-activation of the NMDA receptor.

Keywords: cytosolic phospholipase A2, prostaglandins, arachidonic acid, NMDA, cortical cell culture, mouse

Introduction

Phospholipases are phospholipid hydrolyzing enzymes. Phospholipase A2 (PLA2) comprises a superfamily of extracellular and intracellular esterases that catalyze the hydrolysis of sn-2 fatty acyl bonds of phospholipids to yield fatty acids and lysophospholipids. The PLA2 reaction is the primary pathway through which arachidonic acid (5,8,11,14-eicosatetraenoic acid), a C20 polyunsaturated fatty acid, is liberated from phospholipids (Kudo and Murakami 2002). Unesterified arachidonic acid is a substrate for cyclooxygenases (COXs) and lipoxygenases (LOXs), which are the initial steps in the catalysis of prostaglandins (PGs), thromboxanes (TXs), and leukotrienes (LTs). While arachidonic acid itself may contribute to processes associated with neuronal plasticity (Williams et al. 1989), it is known that unregulated fatty acid release can be directly cytotoxic (Cummings et al. 2000). Additionally, the aforementioned metabolites of arachidonic acid may also contribute to the progression of tissue injury following traumatic or ischemic insults [For reviews please see: (Kim et al. 1999; Sapirstein and Bonventre 2000; Sun et al. 2004; Hoozemans and O'Banion 2005; Hewett et al. 2006a)]. Hence, understanding the mechanism of arachidonic acid release under pathological conditions is of great import. The major PLA2 groups differ with regard to structure, tissue distribution, Ca2+ dependency, and substrate specificity (Mukherjee et al. 1994; Murakami et al. 1998; Balsinde et al. 1999; Kudo and Murakami 2002). Since 1997, PLA2s have been classified according to their nucleotide sequence (Balsinde et al. 1999), with at least 15 separate groups, many of which are further divided into subgroups (Schaloske and Dennis 2006). The previous classification system, which still has utility, is based on whether PLA2 is Ca2+-dependent and cytosolic (cPLA2), is Ca2+-dependent and secreted from the cell (sPLA2), or is cytosolic and Ca2+-independent (iPLA2). All three subclasses have been found in the brain (Molloy et al. 1998; Yang et al. 1999).

Disturbances of glutamatergic transmission leading to excitotoxic cell death, most frequently via over-activation of the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor, contribute to neuronal loss in both acute neurological injuries and chronic neurodegenerative diseases (Meldrum et al. 1985; Choi 1988; Meldrum and Garthwaite 1990; Coyle and Puttfarcken 1993; Ossowska 1994; Nakao and Brundin 1998). Activation of NMDA receptors have been linked to arachidonic acid release (Dumuis et al. 1988; Sanfeliu et al. 1990; Lazarewicz et al. 1992; Tapia-Arancibia et al. 1992; Stella et al. 1994; Taylor and Hewett 2002). Use of broad-spectrum chemical inhibitors that lack specificity for any individual isoform suggests that neuronal NMDA-stimulated release of arachidonic acid occurs via a PLA2-dependent mechanism (Bradford et al. 1983; Lazarewicz et al. 1988; Sanfeliu et al. 1990; Tapia-Arancibia et al. 1992). Taking both a pharmacological and genetic approach, we now report that inhibition of cPLA2α prevents NMDA receptor-stimulated arachidonic acid mobilization and prostaglandin production but not neuronal injury in mouse mixed cortical cell cultures. Preliminary results have appeared in abstract form (Taylor and Hewett 2003).

Materials and Methods

Materials

MK-801 (dizocilpine maleate) and CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) were purchased from Research Biochemicals Inc. (Natick, MA). CdCl2 was obtained from Acros Organics (Morris Plains, NJ). Fatty acid-free bovine serum albumin (BSA), cytosine β-D-arabinofuranoside (Ara-C), NMDA (N-methyl-D-aspartate), kainate, flurbiprofen, and leucine methyl ester (LME) were purchased from Sigma (St. Louis, MO). AMPA [(S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid] was obtained from Tocris (Ellisville, MO). [3H]-arachidonic acid (AA) was purchased from PerkinElmer Life Sciences (Boston, MA) whereas unlabeled arachidonic acid was purchased from Biomol Research Laboratories (Plymouth Meeting, PA). Modified Eagle’s medium was obtained from MediaTech (Washington, D.C.). All sera came from Hyclone (Logan, UT). MAFP (methyl arachidonyl fluorophosphonate), SB-203580 [4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl0-5-(4-pyridyl) 1H-imidazole], RHC-80267 [1,6-bis (cyclohexyloximinocarbonyl-amino) hexane], Bromoenol lactone (BEL), and OOPC (oleyloxyethyl-phosphorylcholine) were all purchased from Biomol (Plymouth Meeting, PA). AACOCF3 (arachidonyltrifluoromethyl ketone) was obtained from Calbiochem (San Diego, CA).

Cell Culture

Purified primary astrocyte cultures were prepared from postnatal CD1 mice (1–3 days) as described in detail (Hamby et al. 2006). Once the astrocytes reached confluence, cultures were treated once with 8µM cytosine β-D-arabinofuranoside for a duration of 5–6 days, which substantially reduces but does not eliminate microglial contamination (Hamby et al. 2006). Thereafter, cultures were maintained in growth medium consisting of MS plus 10% CS, 50IU/ml penicillin, 50µg/ml streptomycin and 2mM L-glutamine. Purified astrocyte cultures were generated by eradicating residual microglia with a 75mM LME treatment (60–90min), one day prior to experimentation as described (Hamby et al. 2006). Depletion of c-fms mRNA – a transcript that encodes the receptor for macrophage colony stimulating factor that is expressed by microglia but not astrocytes (Hao et al. 1990; Krady et al. 2002) – was confirmed in sister cultures (data not shown).

Pure neuronal cultures were prepared from cortices of E15 mouse and plated at 6 hemispheres/plate in Neurobasal medium supplemented with B27, 2 mM glutamine, and antibiotics (Invitrogen, Carlsbad, CA). While Neurobasal medium discourages glia growth, we additionally treated cultures two days after plating with 1 µM cytosine β-D-arabinofuranoside for a duration of two days. Media was partially replenished in cultures twice weekly.

Primary mixed cortical cultures containing both astrocytes and neurons were prepared from postnatal (1–3 days) and fetal (15 day gestation) mice, respectively, as described in detail (Trackey et al. 2001). For most experiments, CD-1 mice (Charles River Labs, Wilmington, MA) were utilized. cPLA2α wild-type and null mutant neurons were cultured from cerebral cortices of single embryos derived from (+/−) × (+/−) breeding of animals maintained congenic on the BALB/c background. Absence of cPLA2α was confirmed by western blotting (data not shown). Following dissection of cortices, dissociated cells were plated on top of an already established bed of astrocytes derived from wild type BALB/c animals. All cultures were kept at 37°C in a humidified 6% CO2-containing atmosphere. Experiments were performed on cultures after 13–14 days in vitro.

Experimental Media/Buffers

HEPES balance salt solution (HBSS) contained in (mM): 120 NaCl, 5.4 KCl, 0.8 MgCl2, 1.8 CaCl2, 20 HEPES, 15 glucose and 0.01 glycine (pH=7.4). Ca2+-free HBSS was made by omitting CaCl2 and adding 100µM EGTA to the aforementioned buffer. Media Stock (MS) was composed of modified Eagle's medium (Earle's salts; Mediatech) supplemented with L-glutamine, glucose, and sodium bicarbonate to a final concentration of 2.0, 25.7, and 28.2 mM, respectively. MS/gly was composed of MS supplemented with 10 µM glycine.

Arachidonic Acid Release

Mixed cortical cell cultures were incubated overnight with [3H]-arachidonic acid (AA; 0.1µCi/well) in MS containing bovine serum albumin (BSA; 0.1%). During this period, approximately 60% of the 3H-AA was incorporated. Next, cultures were washed two to four times (750µl each) with a HEPES Balanced Salt Solution (HBSS) containing 0.1% BSA to remove any extracellular [3H]-AA. Cultures were then exposed to HBSS containing 0.1% BSA alone (basal) or HBSS/0.1% BSA containing NMDA (100µM), AMPA (100µM) or kainate (100µM) with or without other pharmacological agents as indicated in each figure legend. BSA was used to prevent arachidonic acid from being reincorporated into cell membranes once it has been released into the extracellular medium (Peters-Golden et al. 1996). A two-hundred µl sample of the cell culture supernatants was collected and transferred to scintillation vials for β-counting. We previously determined that greater than 80% of the radioactivity released into the extracellular space was arachidonic acid and not a metabolite (Taylor and Hewett 2002). Thereafter, cultures were washed with HBSS and cells solubilized with 400 µl of warm 0.2% SDS, half of which was transferred to scintillation vials for β-counting. Data are expressed as total cpm/well (body of manuscript) or % of total incorporated [3H]-AA [released [3H]-AA/(released + retained [3H]-AA)*100)] (supplemental figure 1).

Measurement of COX metabolites

Mixed cortical cell cultures were washed twice (750µl each) with MS/gly and treated with inhibitors as indicated in each individual figure or table legend. Supernatants were collected 30–40min after NMDA exposure and frozen at −80°C. Prostaglandins (ng/ml) accumulated in the cell culture supernatants were measured at room temperature via enzyme immunoassay according to the manufacturer’s instructions (Prostaglandin Screening EIA kit, Cayman Chemical; Ann Arbor, MI). Values are expressed as mean PGs released + S.E.M normalized to the mean PGs released by wild-type cells treated with NMDA alone (= 100%). In the experiments involving single embryo dissections, this normalization followed correction to the mean LDH value for each individual culture determined following the addition of 250µM NMDA to each well for the ensuing 24 hr. This correction controlled for the variability in plating density between culture wells that unavoidably occurs with single embryo dissections.

For direct measurement of COX activity, cultures were treated as indicated in the table legend, washed and then exogenous arachidonic acid (30 µM) added for 30 min (37°C). The supernatants were removed for PG measurement. Values are expressed as mean PGs released + S.E.M normalized to the mean PGs released by cells treated with arachidonic acid alone (= 100%).

45Ca2+ accumulation studies

Cultures were treated as indicated in the table legend, washed twice (750 µl each) with HBSS then stimulated with NMDA (100µM) containing a trace amount of 45CaCl2 (0.5 µCi/400 µl/well). After 15 min, cultures were washed three times (750 µl each) to remove any residual extracellular 45CaCl2, aspirated dry, and then lysed using 400 µl of warm 0.2% sodium dodecyl sulfate (SDS). The amount of radioactivity in 200 µl of the cell lysate was estimated via liquid scintillation counting. Readings of cpm were corrected back to the original volume of lysates. Values are normalized to accumulation that occurred by cells treated with NMDA alone (= 100%).

Reverse Transcription-Polymerase Chain Reaction (PCR) Analysis

Total RNA was extracted from purified astrocyte or neuronal cultures using TRIZOL reagent (Invitrogen; Carlsbad, CA). Four wells were combined and RNA was resuspended in 20µl of water. One µg of each RNA sample was subjected to first-strand cDNA synthesis using Moloney murine leukemia virus reverse transcriptase (400U; Invitrogen) as previously described (Hewett et al. 1999). Reactions were performed in 20µl volumes at 40–42°C in a water bath for 1h. The other half of each RNA sample was incubated similarly in the absence of reverse transcriptase to test for genomic DNA contamination (none detected). The PCR amplimers for analysis of cPLA2α were 5’-CAGTATCCCAATCAAGCATTCA-3’ (sense) and 5’-TCCCAGCACAGAAATTACACAG-3’ (antisense). cPLA2β cDNA amplimers were 5’-ACGGCTGTATTGCTGTGAATTT-3’ (sense) and 5’-CCTCCAACTAAATTCCAAGTTCC-3’ (antisense). cPLA2γ cDNA amplimers were 5’-AGACAGAGTAAAGGATCCCCAAG-3’ (sense) and 5’-TTATGAGTGGCAGAGAACATCAG-3’ (antisense). β-Actin cDNA amplimers were 5’-GTGGGCCGCTCTAGGCACCAA-3’ (sense) and 5’-CTCTTTGATGTCACGCACGATTTC-3’ (antisense). β-Actin mRNA was assessed to control for the amount and integrity of the RNA sample. Each PCR was performed with 1µl of cDNA sample using Taq DNA polymerase (1U; Invitrogen) in a total of 25µl in a Bio-Rad iCycler (Hercules, CA) DNA thermal cycler. Each cycle consisted of a denaturation step (94°C, 30sec), an annealing step (45 sec) and a primer extension step (72 °C, 1min). Annealing temperatures and cycle number were as follows: cPLA2α (60°C, 31 cycles), cPLA2β (60°C, 29 cycleṣ). cPLA2γ (64°C, 35 cycleṣ). and β-actin (63°C, 23 cycles). PCR products were separated on a 2% agarose gel and detected by ethidium bromide staining using a UV transilluminator (UVP, Kodak, Rochester, NY) and the Kodak Electrophoresis Documentation and Analysis System 120. Images were processed using Adobe Photoshop.

Immunohistochemistry

cPLA2 protein was detected by indirect immunofluorescence. First, cultures were fixed for 15 min with a freshly prepared mix of acetone/methanol (1:1). Non-specific binding sites were blocked by exposure to 10% normal donkey serum (NDS in PBS (1 h, 25°C), followed by the addition of 22µg/ml sheep anti-cytosolic phospholipase A2 polyclonal antibody (4°C, overnight) raised against a 24 amino-acid synthetic peptide sequence from the carboxy-terminus of human cPLA2 (Abcam, Cambridge, MA). The peptide immunogen sequence used as a query line in a BLAST search against mouse sequences demonstrated 95% identity to mouse cPLA2 group IVA (cPLA2α) with no sequence homology to any other protein detected. Unbound antibody was washed out with PBS and cultures incubated in a dark enclosure for 1 h (25°C) with 10µg/ml Alexa 488-conjugated donkey anti-sheep IgG antibody (Molecular Probes, Eugene, OR). Primary and secondary antibodies were diluted in 5% NDS(PBS) containing 0.025% Triton-X-100. Fluorescent images (40x magnification) were acquired using a CRX digital camera (Digital Video Camera Co, Austin, TX) mounted on an Olympus IX50 inverted microscope outfitted with epifluorescence and processed using Adobe Photoshop software. Results were confirmed using a rabbit polyclonal antibody raised against amino acids 1–216 of the amino-terminus of cPLA2 of human origin (Santa Cruz Biotechnology, Santa Cruz, California) (data not shown).

Toxicity Experiments

Exposure to NMDA (Sigma Chemical, St. Louis, MO) was carried out at room temperature in HBSS. After 5 min, the exposure solution was washed away and replaced by MS supplemented with glycine (0.01 mM). The cells were transferred to a 6% CO2-containing incubator for 20–24hr; cell injury was assessed by spectrophotometric measurement of lactate dehydrogenase (LDH) activity (see below).

Quantification of Neuronal Cell Death

Lactate dehydrogenase (LDH) activity in cell culture medium was quantified by measuring the rate of pyruvate-dependent oxidation of NADH as described in detail (Uliasz and Hewett 2000). Activity was expressed as the percentage of total neuronal LDH activity (defined as 100%), which was determined in each experiment by assaying the supernatant of parallel cultures exposed for 20–24 hr to 200–300 µM NMDA. This produces a neuron-selective injury as primary cortical astrocytes lack NMDA receptors (Backus et al., 1989; Chan et al., 1990; Janssens and Lesage, 2001; B. Fogal and SJH unpublished observations) and are not injured following NMDA exposure (Choi et al., 1987; SJH unpublished observations).

Results

Murine mixed cortical cell cultures were incubated overnight (18–24 hr) with [3H]-AA to allow for its incorporation into membrane phospholipids. Exogenous addition of NMDA (100µM), but not HBSS alone, resulted in a time-dependent increase in the release of [3H]-AA that reached a plateau between 13 and 19 min (data not shown). Thus, measurements of 3H-AA release were made 15 min following stimulation in all subsequent experiments. NMDA-stimulated [3H]-AA release– representing approximately 4% of total incorporated (Supplemental Figure 1) – was blocked by MK-801 (10µM) but not CNQX (30µM) (Figure 1). In keeping with the latter observation, neither AMPA (100µM) nor kainate (100µM) were effective inducers of [3H-AA] release (Figure 2). Removal of [Ca2+]e prevented NMDA-induced [3H]-AA release (Figure 3A), whereas addition of CdCl2 (100µM), to block voltage-gated calcium channels, was relatively ineffective (Figure 3B). This concentration of CdCl2 effectively prevents high K+-elicited arachidonic acid release (Taylor and Hewett 2002). Finally, NMDA did not stimulate release of [3H]-AA from pure astrocyte cultures [basal 1230.68 ± 70.99 cpm/well vs. NMDA 1219.98 ± 55.52 cpm/well; (n = 8–9) ]. Hence, these data demonstrate that the lipolysis occurring herein is dependent on calcium entry via neuronal NMDA receptors.

Figure 1. Effect of ionotropic glutamate receptor antagonism on NMDA-stimulated [3H]-AA release.

Figure 1

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Cortical cell cultures were incubated with [3H]-AA overnight, washed, and then exposed to 100 µM NMDA in the absence or presence of MK-801 (10 µM) or CNQX (30 µM) for 15 min. Thereafter, cell supernatants were collected and assayed for [3H]-AA content. Data are expressed as the mean total cpm/well + SEM (*) indicates a value significantly greater than basal [3H]-AA release, whereas (#) represents a significant diminution of NMDA-stimulated [3H]-AA release as determined by one-way ANOVA followed by the Student Newman Keul's t-test for multiple comparisons. Significance was assessed at p < 0.05.

Figure 2. Characterization of the ability of ionotropic glutamate receptor agonists to stimulate [3H]-AA release.

Figure 2

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Mixed cortical cell cultures were labeled with [3H]-AA overnight, washed, and stimulated with (A,B) NMDA (100 µM), (A) AMPA (100 µM), or (B) kainate (100 µM) for 15 min. with or without drugs [MK-801, 10 µM and/or CNQX, 30 µM] as indicated. NB: MK-801 was included in the AMPA and kainate conditions, while CNQX was added to the NMDA conditions to isolate the various receptor responses (Karschin et al. 1988). CNQX and MK801 added alone or in combination had no effect on basal [3H]-AA release (data not shown). Immediately following exposure, samples of supernatant were collected and the amount of radioactivity in each was estimated via liquid scintillation counting. Data are expressed as mean cpm/well + S.E.M.; n= 6 cultures per condition from two separate experiments. (*) denotes values that are significantly increased over non-stimulated release, whereas (#) represents a significant diminution of NMDA-stimulated [3H]-AA release, as determined by one-way ANOVA followed by a Student Newman-Keul’s t-test for multiple comparisons (p < 0.001).

Figure 3. NMDA stimulated arachidonic acid release is dependent on receptor-mediated calcium entry.

Figure 3

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A) Mixed cortical cell cultures were incubated overnight with [3H]-AA, washed, and incubated in HCSS ± Ca2+, 10 min prior to the addition of NMDA (100 µM; 15 min). The [3H]-AA released into the cell culture supernatant is expressed as the mean cpm /well + S.E.M.; n=10–12 cultures from three separate experiments. B). Cultures were incubated overnight with [3H]-AA, washed and then exposed to HCSS alone (basal) or HCSS containing CdCl2 (100 µM), NMDA (100 µM) or both. Fifteen min later cell culture supernatants were collected and assayed for [3H]-AA content using a scintillation counter. Data are expressed as the mean cpm/well + S.E.M; n=6 cultures from two separate experiments. (*) denotes a significant with-in group difference whereas (#) represents a significant between-group (± Ca2+ or CdCl2) difference as determined by two-way ANOVA followed by a Bonferroni’s t-test for multiple comparisons. Significance was assessed at p < 0.05.

Numerous phospholipase enzymes are calcium-dependent and most PLA2 drugs available, with the exception of the sPLA2 inhibitors, target more than one isoform. Hence, to determine which isoform may be involved, a comparative pharmacological approach was first taken (Glaser 1995; Cummings et al. 2000). NMDA receptor-stimulated arachidonic acid release was inhibited by MAFP, a potent and irreversible inhibitor of both cPLA2 (IC50 = 3 µM) and iPLA2 enzymes (IC50 = 0.5 µM) (Balsinde and Dennis 1996; Lio et al. 1996), whereas BEL, a selective iPLA2 inhibitor (IC50 = 1µM)(Hazen and Gross 1993; Balsinde and Dennis 1996), was ineffective (Figure 4). RHC-80267, an inhibitor of DAG lipase (IC50 4µM), or OOPC, a selective sPLA2 inhibitor (IC50 =1µM), respectively, (Sutherland and Amin 1982; Plesniak et al. 1993; Schevitz et al. 1995; Chen and Dennis 1998) were also without effect (Figure 4). Taken in toto, these data suggest a role for a cytosolic PLA2 (cPLA2) in NMDA-stimulated arachidonic acid release.

Figure 4. Effect of inhibitors that target various arachidonic acid release pathways on NMDA-stimulated [3H]-AA release.

Figure 4

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Cortical cell cultures were washed into MS/gly containing MAFP ([final]=6µM), Bromoenol lactone (BEL;10µM), RHC-80267 (RHC;10µM) Oleyloxyethylphosphorylcholine (OOPC; 3µM) or vehicle (DMSO or EtOH), in a volume of 250 µl. Total well volume equaled 400 µl. Ten to thirty min later, cultures were exposed to NMDA (100 µM, 15min) ± inhibitor. Samples of supernatant were collected and [3H]-AA content was quantified. Data are presented as mean cpm /well + S.E.M.; n=7–13. (*) indicates a significant diminution of NMDA-stimulated [3H]-AA release in each inhibitor’s experiment, as determined by one-way ANOVA followed by Student-Newman Keul’s t-test for multiple comparisons. Significance was assessed at p < 0.05.

Several cPLA2 isoforms have now been described (Ghosh et al. 2006). Interestingly, RT-PCR analyses indicate that cultured astrocytes and neurons express at least three isoforms of cPLA2 (α, β, and γ) (Supplemental Figure 2). While MAFP can effectively inhibit both α and γ, the former is calcium-dependent while the latter is not (Pickard et al. 1999; Asai et al. 2003), perhaps ruling out its contribution. However, cPLA2γ can be activated by reactive oxygen species (Asai et al. 2003), which are known to occur subsequent to NMDA receptor activation (Lafon-Cazal et al. 1993; Dugan et al. 1995; Reynolds and Hastings 1995). cPLA2β is also calcium-dependent, but its role in arachidonic acid release in brain is unclear as it exists in an unspliced and putatively inactive form (Pickard et al. 1999). What is clear is that NMDA-stimulated arachidonic acid release by MAFP translated to a decrease in NMDA-stimulated PG production, the effect of which was mimicked by another cPLA2 inhibitor, AACOCF3 (Street et al. 1993) (Figure 5). Additionally, the p38MAP kinase inhibitor, SB 203580 (Davies et al. 2000), significantly attenuated NMDA-induced PG release (Figure 5). This latter finding points to a role for cPLA2α as its activity– but not that of β or γ – is regulated by phosphorylation [for review see (Leslie 1997)] (Lin et al. 1993; Song et al. 1999). Importantly, neither MAFP, AACOCF3, nor SB 203580 affected NMDA-stimulated calcium entry indicating that they were not NMDA receptor antagonists (Table 1). Additionally, none prevented PG production elicited by exogenous addition of arachidonic acid, indicating that they did not inhibit cyclooxygenase activity directly (Table 1). This contrasts with flurbiprofen, the non-selective cyclooxygenase inhibitor (Gierse et al. 1995), used here as a positive control (Figure 5, Table 1).

Figure 5. Effect of inhibitors that target various PLA2 pathways on NMDA-stimulated prostaglandin (PG) production.

Figure 5

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Cortical cell cultures were washed into MS/gly followed by the addition of MAFP ([final]= 5µM), SB 203580 (SB; 7.5µM), flurbiprofen (FBI; 300µM) or vehicle (DMSO) in a volume of 3 µl. Twenty min (MAFP) to two hr later (SB; FBI), cultures were washed into HCSS ± NMDA (75 µM; 5 min). The exposure solution was then washed away and replaced with MS/gly containing the inhibitors. The cells were returned to the incubator and 30–40 min later samples of supernatant were collected and PGs released into the bathing medium were measured via enzyme immunoassay. To facilitate comparisons between compounds, values are expressed as mean PGs released + S.E.M (n=4–9 from four separate experiments) normalized to the mean PGs released by cells treated with NMDA alone (set at 100%). Inset: Effect of AACOCF3 on NMDA-stimulated PG production. Cultures were treated essentially as described above except that cells were pretreated with AACOCF3 (1µM) 24 hr prior to NMDA exposure (n=4, from two separate experiments). (*) indicates a values significantly different from basal, whereas (#) represents a significant diminution of NMDA-stimulated PG production, as determined by one-way ANOVA followed by Student-Newman Keul's t-test for multiple comparisons. Significance was assessed at p < 0.05.

Table 1. Arachidonic acid release modifying drugs do not affect NMDA receptor and/or cyclooxygenase activity.

Cortical cell cultures were washed into MS/gly followed by the addition of vehicle (DMSO), AACOCF3 ([final] = 1µM), MAFP (5µM), SB 203580 (7.5µM), or flurbiprofen (300µM) in a volume of 3µL. For measurement of NMDA receptor activity, cultures were washed into NMDA (100 µM) containing a trace amount of 45Ca2+ ± drugs following a twenty min (MAFP; n = 6), two hr (SB; n = 3) or a 24 hr (AACOCF3; n = 3) exposure. Flurbiprofen was given at the time of NMDA exposure only (n = 8). After 15 min, cell lysates were collected for measurement of 45Ca2+ accumulation. Values are normalized to accumulation that occurred by cells treated with NMDA alone (= 100%). For measurement of COX activity, 30µM arachidonic acid was spiked into culture wells 30 min (MAFP; n = 4), 90 min (SB; Flurbiprofen; n = 6 each) or 24 hr (AACOCF3; n = 4) following addition of drugs. The cells were returned to the incubator and 30 min later, supernatants were collected and PGs released into the bathing medium were measured via enzyme immunoassay. Values are expressed as mean PGs released + S.E.M normalized to the mean PGs released by cells treated with arachidonic acid alone (= 100%).

Inhibitor 45Ca2+ Uptake

(% of NMDA)		 AA-induced PGs

(% of AA)
AACOCF3 90.5 ± 4.1 123.5 ± 12.6
MAFP 103.7 ± 5.4 123.9 ± 15.3
SB 203580 96.8 ± 4.0 99.1 ± 11.7
Flurbiprofen 96.2 ± 4.8 24.0 ± 4.2*
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Significance, denoted by the asterisk, was assessed at p < 0.05 via one way ANOVA followed by Student-Newman-Keul’s test for multiple comparisons.

Immunocytochemical analysis of mixed cortical cell cultures demonstrated that both neurons and astrocytes express cPLA2α protein (Figure 6). Because the staining of the astrocyte monolayer in the mixed cultures is obscured by the neuronal cell bodies (phase bright cells) and the dense neuropil network (Figure 6A,B), and, additionally is out of the plane of focus, constitutive expression of cPLA2α protein was confirmed and is shown in a pure astrocyte culture (Figure 6 C,D). Finally, mixed cultures – containing neurons derived from cPLA2α null mutant animals plated on top of wild-type astrocytes – failed to produce PGs in response to NMDA receptor stimulation, lending further credence to our conclusion that NMDA-stimulated arachidonic acid mobilization is mediated via cPLA2α (Figure 7).

Figure 6. Neurons and astrocytes constitutively express cPLA2α.

Figure 6

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Protein levels of cPLA2α were determined in untreated mixed cortical cell cultures and pure astrocyte cultures by indirect immunofluorescence as described in methods. [A,C] Phase contrast micrographs of mixed cortical cell cultures (A) and pure astrocyte (C) cultures, respectively. [B,D] Immunoreactivity for cPLA2α in neurons (B) and astrocytes (D), respectively.

Figure 7. NMDA-induced prostaglandin production in cortical cell cultures derived from cPLA2α null mutants.

Figure 7

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Cortical neurons were cultured from the cerebral cortices of E15 animals. Following single embryo dissection, dissociated cells were plated on top of an already established bed of astrocytes derived from wild type BALB/c animals. The remainder of the brain was utilized for genotyping. Experiments were performed on wild type (+/+) or cultures containing null mutant neurons (−/−) after 14 days in vitro. Cultures were washed into HCSS ± NMDA (100µM; 5min). The exposure solution was washed away and replaced with MS/gly and the cells returned to the incubator. Thirty-40 min later, samples of supernatant were collected and PGs released into the bathing medium were measured via enzyme immunoassay. Values are expressed as mean PGs released + S.E.M normalized to the mean PGs released by wild-type cells treated with NMDA alone (set at 100%) following correction to total LDH values of each well to control for well-to-well plating density variability. [n=5–6 culture wells per genotype from five-six separate embryos]. (*) denotes a significant with-in group statistical difference while a (#) represents a significant between-group difference as determined by two-way ANOVA followed by a Bonferroni’s t-test for multiple comparisons following appropriate transformation of the percentage data. Significance was assessed at p < 0.05.

We and others have demonstrated that non-steroidal anti-inflammatory drugs (NSAIDs) – which inhibit arachidonic acid metabolism – protect against excitotoxic injury both in vitro and in vivo (Candelario-Jalil et al. 2000; Hewett et al. 2000; Iadecola et al. 2001; Strauss and Marini 2002; Carlson 2003; McCullough et al. 2004; Silakova et al. 2004; Hewett et al. 2006b). As such, we next assessed whether pharmacological inhibition or genetic loss of cPLA2α would limit NMDA-mediated neurotoxicity in our culture preparation. Neither MAFP nor AACOCF3 – at concentrations that significantly or completely blocked arachidonic acid release and/or PG production – ameliorated NMDA-induced neuronal injury (Figure 8). In fact, MAFP slightly, but significantly, enhanced neuronal cell death (Figure 8A). Additionally, neurons derived from cPLA2α null mutant animals demonstrated no greater survival advantage against NMDA than those derived from their wild-type littermate controls (Figure 9).

Figure 8. NMDA-mediated neurotoxicity is not ameliorated by pharmacological inhibition of cPLA2α.

Figure 8

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Cultures were exposed to (A) 6µM MAFP for 30 min prior or (B) 1 µM AACOCF3 for 24 hr prior to the addition of increasing concentrations of NMDA (5 min). The exposure solution was washed away and replaced with MS/gly containing the antagonists and the cells returned to the incubator. Supernatants were collected 20–24 hr later and the activity of LDH quantified spectrophotometrically. Values represent the mean LDH+SEM expressed as a percentage of total neuronal LDH (= 100%), which was determined in each experiment by assaying the supernatant of sister cultures following exposure to 300 µM NMDA for 20–24 hr. Between-group differences (*) were assessed via two-way ANOVA following appropriate transformation of the percentage data. (for each drug, n = 4 culture wells from two independent experiments). Significance was assessed at p <0.05.

Figure 9. NMDA-mediated neurotoxicity is unaffected in cultures derived from cPLA2α null mutant animals.

Figure 9

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Cultures were exposed for 5 min to increasing concentrations of NMDA. The exposure solution was washed away and replaced with MS/gly and the cells returned to the incubator. Cell culture supernatants were collected 20–24 hr later and the activity of LDH quantified spectrophotometrically. Values represent the mean LDH+SEM (n = 11–12 culture wells from six separate embryos of each genotype) expressed as a percentage of total neuronal LDH (= 100%), which was determined in each experiment by assaying the supernatant of sister cultures following exposure to 300 µM NMDA for 20–24 hr. No between-group statistical differences were found as assessed by two-way ANOVA following appropriate transformation of the percentage data.

Discussion

Brain cells in situ contain low concentrations of free polyunsaturated fatty acids, such as arachidonic acid, that are released following pathological insults in vivo (Bazan 1970; Bazan and Rakowski 1970; Yoshida et al. 1983; Westerberg et al. 1987) as well as following stimulation with glutamate agonists in vitro (Lazarewicz et al. 1990; Sanfeliu et al. 1990; Tapia-Arancibia et al. 1992; Dumuis et al. 1993; Stella et al. 1995; Taylor and Hewett 2002). Thus, it may not be surprising that pharmacological inhibition of arachidonic acid metabolism has proven effective in ameliorating brain damage mediated by direct excitotoxin injection (Candelario-Jalil et al. 2000; Iadecola et al. 2001; Kunz and Oliw 2001; Silakova et al. 2004; Hewett et al. 2006b), as well as, cerebral ischemia (Sasaki et al. 1988; Nakagomi et al. 1989; Cole et al. 1993; Patel et al. 1993; Nogawa et al. 1997; Nakayama et al. 1998; Antezana et al. 2003; Candelario-Jalil et al. 2003). Additionally, administration of NSAIDs – particularly those that target cyclooxygenase-2 (COX-2) – has been shown to enhance functional outcomes in an animal model of amyotrophic lateral sclerosis (Drachman et al. 2002; Klivenyi et al. 2003) and to improve histological findings in an animal model of Parkinson’s disease (Teismann and Ferger 2001; Teismann et al. 2003). However, arachidonic acid itself can have effects independent from its metabolites; some of which can have deleterious consequences for a cell (Wieloch and Siesjo 1982; Yu et al. 1986; Barbour et al. 1989; Williams et al. 1989; Miller et al. 1992; Vazquez et al. 1994; Volterra 1994). Given this, we reasoned that inhibition of arachidonic acid release from cellular membranes may be a more effective neuroprotective strategy than inhibition of its metabolism alone, making it of great import to understand the mechanism by which arachidonic acid is released following excitotoxic insults. The results obtained herein demonstrate that in a mixed cortical cell culture preparation, NMDA-stimulated arachidonic acid release is mediated via activation of the calcium-dependent cytosolic group IV cPLA2α, but that inhibition of this enzyme - either pharmacologically or genetically - does not afford protection against subsequent excitotoxicity in this model system.

In most mammalian cells, the release of arachidonic acid from membrane phospholipids can be facilitated directly by numerous extracellular and intracellular esterases, collectively termed phospholipase A2s, some of which are Ca2+-dependent [for review see (Mukherjee et al. 1994; Murakami et al. 1997; Balsinde et al. 1999)]. NMDA receptor-stimulated [3H]-AA release in our experimental paradigm is Ca2+-dependent, as evidenced by the fact that removal of Ca2+ from the HBSS completely suppresses release (Figure 3). Theoretically, Ca2+ entering via voltage-gated calcium channels, which would open secondary to NMDA-mediated depolarization, could lead to lipolysis directly. However, addition of CdCl2 – at a concentration that completely inhibits high K+-induced arachidonic acid release (Taylor and Hewett 2002)– had only a slight effect on release (Figure 3). The comparative pharmacological approach taken herein suggests a role for a cPLA2 paralog (Figure 4, 5). Group IV cytosolic cPLA2s require Ca2+ for translocation to membrane phospholipids (Clark et al. 1991; Evans et al. 2001), are relatively specific for arachidonylated phospholipids (Clark et al. 1991), and their message is ubiquitously expressed throughout brain (Molloy et al. 1998), particularly in neurons (Kishimoto et al. 1999). While several cPLA2 paralogs have now been described (Ghosh et al. 2006), the modulation of NMDA-stimulated PG production by MAP kinase inhibition (SB 203580; Figure 5) points to a role for cPLA2α as it requires both Ca2+ and phosphorylation for full activation (Lin et al. 1993). Unlike Borsch-Haubold and colleagues –who report that SB 203580 can inhibit purified COX-1 and -2 (Borsch-Haubold et al. 1998) –we found no effect of this compound on prostaglandin production stimulated via direct application of arachidonic acid to cultures in situ (Table 1), indicating that block of COX activity directly could not account for the change in PGs produced. Importantly, data derived from studies utilizing cultures derived from cPLA2α null mutant mice confirm the pharmacological results (Figure 7).

Although both astrocytes and neurons can express cPLA2α (Figure 6, Supplemental data Figure 2) and both cell-types are labeled with [3H]-AA and hence could contribute to the changes in observed in arachidonic acid release, we favor the interpretation that release following stimulation with NMDA in our system occurs from neurons. First, we don’t detect NR1 protein expression in cultured cortical astrocytes (B. Fogal and SJH; unpublished observations), which is consistent with the work of others, demonstrating a lack of NMDA receptor expression in primary astrocytes (Kettenmann and Schachner 1985) (Backus et al., 1989; Chan et al., 1990; Janssens and Lesage, 2001). Additionally, NMDA fails to evoke arachidonic acid release from cortical astrocyte cultures (see results section). This is in agreement with the findings of Sanfeliu and colleagues, who reported that astrocytes derived from striatal tissue do not respond to NMDA by releasing arachidonic acid, whereas neurons do (Sanfeliu et al. 1990). Addtionally, Bonventre and colleagues demonstrated that exposure to glutamate enhances the specific activity of cPLA2 (the paralog in question not yet defined in this study) in cell free extracts derived from rat mixed cortical cell cultures, but not from pure astrocytes (Kim et al. 1995). Finally, while glutamate can evoke arachidonic acid liberation from striatal astrocytes in culture (Stella et al. 1995), the concentration of glutamate that was applied exogenously was twenty times that which elicited a maximal [3H]-AA release response from our mixed cultures [100 µM (Taylor and Hewett 2002)]. Indeed, high K+-induced glutamate release from our cultures reaches only 25 µM in 15 min – this in the face of glutamate uptake blockade – arguing that secondary release of glutamate, which necessarily follows neuronal NMDA receptor stimulation, is unlikely to reach levels sufficient to trigger astrocytic release (Fogal et al. 2005). Perhaps most directly, we demonstrate that NMDA-mediated prostaglandin formation is lost from cultures derived from cPLA2α null neurons even when plated on wild-type astrocytes (Figure 7). Nonetheless, we cannot completely rule out the possibility that an astrocyte cPLA2 is activated by the cPLA2α-dependent response of the neurons to NMDA.

Our results conclusively indicate that activation of cPLA2α is responsible for NMDA receptor-stimulated arachidonic acid release in our mixed cortical cell culture system. However, does it contribute to NMDA-mediated excitotoxic neuronal injury? Previous reports suggest a specific role for cPLA2α in neuronal injury: cPLA2α knockout mice are less susceptible than wild-type littermate controls to cerebral ischemic brain damage induced via middle cerebral artery occlusion (Bonventre et al. 1997), as well as to MPTP-induced dopamine depletion (Klivenyi et al. 1998) – an animal model of Parkinson’s disease. While excitotoxicity has been demonstrated to contribute to the pathogenesis of both of these disorders, our results demonstrating a lack of protective effect of cPLA2α inhibition against NMDA-mediated neuronal injury in our cortical cell culture system (Figure 8) would suggest that the positive effects demonstrated in the cPLA2α null animals in vivo, might not simply be due to alterations in glutamate neurotoxicity. While it could be argued that lack of specificity of both MAFP and AACOCF3 for cPLA2α might account for their ineffectiveness, similar negative results in the cPLA2α nulls makes this an unlikely explanation (Figure 9). These results may seem at odds with a recent paper by Sapirstein and colleagues, who demonstrate that the CA1 region of organotypic hippocampal cultures derived from these same cPLA2α null mutant mice or control cultures exposed to AACOCF3 show less neurotoxicity following NMDA exposure than cultures derived from wild-type controls or untreated cultures (Brady et al. 2006). However, differences in experimental preparations (e.g., dispersed cortical vs. organotypic hippocampal culture) and paradigms [e.g., 100µM NMDA for 5 min (fast toxicity) vs. 5–10 µM for 1 hr (slow toxicity)] could account for this discrepancy. Additionally, it is possible that expression of cPLA2α in cells other than neurons –which could be become activated after the initial injury and would be better represented in the organotypic slice culture vs. the culture system used herein– may be an important contributing factor to subsequent neuronal injury. Indeed, while the expression of cPLA2 has been demonstrated to be markedly increased in dentate granule cells post-ischemia (Owada et al. 1994), increased expression in activated microglia and astrocytes in the CA1 hippocampus has also been reported (Clemens et al. 1996; Walton et al. 1997). A more recent study using an acute slice preparation demonstrates potentially favorable changes in CA1 pyramidal neuron NMDA receptor activity in cPLA2α null mutant animals, however (Shen et al. 2007).

Additionally, these results might appear dichotomous with our own work and that of others demonstrating a positive protective effect against excitotoxic neuronal injury upon COX-2 inhibition in both dispersed cultures as well as organotypic slices (Hewett et al. 2000; Silakova et al. 2004; Hewett et al. 2006b) (Kelley et al. 1999; Strauss and Marini 2002; Carlson 2003; McCullough et al. 2004). However, while activation of PGE2 EP1 receptors likely underlie COX-2 neurotoxicity in the setting of experimental cerebral ischemia in vivo and oxygen-glucose deprivation in vitro (Kawano et al. 2006), numerous other studies, describe a neuroprotective function for prostaglandins (Cazevieille et al. 1993; Akaike et al. 1994; Cazevieille et al. 1994; McCullough et al. 2004). Thus, whether prostanoids are injurious or protective may relate to the type of PGs produced, the concentrations achieved, and the signaling receptors that they activate; hence arguing that global removal of substrate may not necessarily be advantageous. Indeed, numerous studies demonstrate a homeostatic or even anti-inflammatory role for cPLA2α in certain cells and tissues [for review see (Yedgar et al. 2006)]. Additionally, it is possible that a component of the neuroprotective potential of COX-2 inhibitors is independent of COX-2 inhibition. For instance, certain NSAIDs have been demonstrated to be good activators of the peroxisome proliferator-activated receptors (PPARs) (Jaradat et al. 2001) and PPAR agonists can modulate inflammatory responses in brain (Heneka et al. 2000). Additionally, loss of the metabolizing ability of COX-2 could lead to a shunting of arachidonic acid to enzymatic pathways that shift lipid mediator production toward an anti-inflammatory, actively regulated program of resolution (Levy et al. 2001). Whether this can occur in the CNS requires experimental confirmation. Finally, loss of COX-2 activity could result in the buildup of protective endocannabinoids [for review see (van der Stelt et al. 2002)] as notably COX-2, but not COX-1, metabolizes both anandamide and 2-arachidonoylglycerol (Yu et al. 1997; Kozak et al. 2000). These latter purported positive protective pathways would necessarily be lost when arachidonic acid is prevented from being released.

In sum, present results demonstrate that inhibition of cPLA2α prevents NMDA receptor-stimulated arachidonic acid release and prostaglandin production but not neuronal injury in mouse cortical cell culture. This – coupled with our previous data (Hewett et al. 2000; Silakova et al. 2004; Hewett et al. 2006b) and that of others [for a comprehensive review see (Hewett et al. 2006a)] demonstrating the effectiveness of NSAIDs as protective agents – could suggest that alterations in metabolism might be a better neuroprotective strategy against CNS diseases associated with over-activation of NMDA receptors than altering mobilization. Nevertheless, more detailed in vivo studies designed to assess the effectiveness of cPLA2α inhibition in various neurological disease models warrant further consideration.

Supplementary Material

Supp Fig s1

Supplemental Figure 1. Effect of ionotropic glutamate receptor antagonism on NMDA-stimulated [3H]-AA release. Cortical cell cultures were incubated with [3H]-AA overnight, washed, and then exposed to 100 µM NMDA in the absence or presence of MK-801 (10 µM) or CNQX (30 µM) for 15 min. Thereafter, cell supernatants were collected and assayed for [3H]-AA content. Cultures were then washed and solubilized with warm 0.2% SDS. Cell lysates were collected and assayed for [3H]-AA content. Data are expressed as the % of total incorporated. (*) indicates a value significantly greater than basal [3H]-AA release, whereas (#) represents a significant diminution of NMDA-stimulated [3H]-AA release as determined by one-way ANOVA followed by the Student Newman Keul's t-test for multiple comparisons. Significance was assessed at p < 0.05.

Supp Fig s2

Supplemental Figure 2: Expression cPLA2 paralogs. RNA was isolated from pure astrocyte (Lanes 1, 2) or pure neuronal (Lanes 3,4) cultures. First-strand cDNA was synthesized and cPLA2α̣, cPLA2β, and cPLA2γ expressions were assessed by PCR as described in the text. β-actin mRNA assessed in all RNA samples as an internal control for the amount of RNA in each sample.

Acknowledgements

This work was supported by NINDS: NS036812-10 to SJH and JAH.

References

  1. Akaike A, Kaneko S, Tamura Y, Nakata N, Shiomi H, Ushikubi F, Narumiya S. Prostaglandin E2 protects cultured cortical neurons against N-methyl-D-aspartate receptor-mediated glutamate cytotoxicity. Brain Res. 1994;663:237–243. doi: 10.1016/0006-8993(94)91268-8. [DOI] [PubMed] [Google Scholar]
  2. Antezana DF, Clatterbuck RE, Alkayed NJ, Murphy SJ, Anderson LG, Frazier J, Hurn PD, Traystman RJ, Tamargo RJ. High-dose ibuprofen for reduction of striatal infarcts during middle cerebral artery occlusion in rats. J Neurosurg. 2003;98:860–866. doi: 10.3171/jns.2003.98.4.0860. [DOI] [PubMed] [Google Scholar]
  3. 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. 2003;278:8809–8814. doi: 10.1074/jbc.M212117200. [DOI] [PubMed] [Google Scholar]
  4. Balsinde J, Dennis EA. Distinct roles in signal transduction for each of the phospholipase A2 enzymes present in P388D1 macrophages. J Biol Chem. 1996;271:6758–6765. doi: 10.1074/jbc.271.12.6758. [DOI] [PubMed] [Google Scholar]
  5. Balsinde J, Balboa MA, Insel PA, Dennis EA. Regulation and inhibition of phospholipase A2. Annu Rev Pharmacol Toxicol. 1999;39:175–189. doi: 10.1146/annurev.pharmtox.39.1.175. [DOI] [PubMed] [Google Scholar]
  6. Barbour B, Szatkowski M, Ingledew N, Attwell D. Arachidonic acid induces a prolonged inhibition of glutamate uptake into glial cells. Nature. 1989;342:918–920. doi: 10.1038/342918a0. [DOI] [PubMed] [Google Scholar]
  7. Bazan NG., Jr Effects of ischemia and electroconvulsive shock on free fatty acid pool in the brain. Biochim Biophys Acta. 1970;218:1–10. doi: 10.1016/0005-2760(70)90086-x. [DOI] [PubMed] [Google Scholar]
  8. Bazan NG, Jr, Rakowski H. Increased levels of brain free fatty acids after electroconvulsive shock. Life Sci. 1970;9:501–507. doi: 10.1016/0024-3205(70)90205-5. [DOI] [PubMed] [Google Scholar]
  9. Bonventre JV, Huang Z, Taheri MR, O'Leary E, Li E, Moskowitz MA, Sapirstein A. Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2. Nature. 1997;390:622–625. doi: 10.1038/37635. [DOI] [PubMed] [Google Scholar]
  10. Borsch-Haubold AG, Pasquet S, Watson SP. Direct inhibition of cyclooxygenase-1 and -2 by the kinase inhibitors SB 203580 and PD 98059. SB 203580 also inhibits thromboxane synthase. J Biol Chem. 1998;273:28766–28772. doi: 10.1074/jbc.273.44.28766. [DOI] [PubMed] [Google Scholar]
  11. Bradford PG, Marinetti GV, Abood LG. Stimulation of phospholipase A2 and secretion of catecholamines from brain synaptosomes by potassium and A23187. J Neurochem. 1983;41:1684–1693. doi: 10.1111/j.1471-4159.1983.tb00881.x. [DOI] [PubMed] [Google Scholar]
  12. Brady KM, Texel SJ, Kishimoto K, Koehler RC, Sapirstein A. Cytosolic phospholipase A alpha modulates NMDA neurotoxicity in mouse hippocampal cultures. Eur J Neurosci. 2006;24:3381–3386. doi: 10.1111/j.1460-9568.2006.05237.x. [DOI] [PubMed] [Google Scholar]
  13. Candelario-Jalil E, Ajamieh HH, Sam S, Martinez G, Leon Fernandez OS. Nimesulide limits kainate-induced oxidative damage in the rat hippocampus. Eur J Pharmacol. 2000;390:295–298. doi: 10.1016/s0014-2999(99)00908-5. [DOI] [PubMed] [Google Scholar]
  14. Candelario-Jalil E, Gonzalez-Falcon A, Garcia-Cabrera M, Alvarez D, Al-Dalain S, Martinez G, Leon OS, Springer JE. Assessment of the relative contribution of COX-1 and COX-2 isoforms to ischemia-induced oxidative damage and neurodegeneration following transient global cerebral ischemia. J Neurochem. 2003;86:545–555. doi: 10.1046/j.1471-4159.2003.01812.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Carlson NG. Neuroprotection of cultured cortical neurons mediated by the cyclooxygenase-2 inhibitor APHS can be reversed by a prostanoid. J Neurosci Res. 2003;71:79–88. doi: 10.1002/jnr.10465. [DOI] [PubMed] [Google Scholar]
  16. Cazevieille C, Muller A, Bonne C. Prostacyclin (PGI2) protects rat cortical neurons in culture against hypoxia/reoxygenation and glutamate-induced injury. Neurosci Lett. 1993;160:106–108. doi: 10.1016/0304-3940(93)90924-a. [DOI] [PubMed] [Google Scholar]
  17. Cazevieille C, Muller A, Meynier F, Dutrait N, Bonne C. Protection by prostaglandins from glutamate toxicity in cortical neurons. Neurochem Int. 1994;24:395–398. doi: 10.1016/0197-0186(94)90118-x. [DOI] [PubMed] [Google Scholar]
  18. Chen Y, Dennis EA. Expression and characterization of human group V phospholipase A2. Biochim Biophys Acta. 1998;1394:57–64. doi: 10.1016/s0005-2760(98)00098-8. [DOI] [PubMed] [Google Scholar]
  19. Choi DW. Glutamate neurotoxicity and diseases of the nervous system. Neuron. 1988;1:623–634. doi: 10.1016/0896-6273(88)90162-6. [DOI] [PubMed] [Google Scholar]
  20. Clemens JA, Stephenson DT, Smalstig EB, Roberts EF, Johnstone EM, Sharp JD, Little SP, Kramer RM. Reactive glia express cytosolic phospholipase A2 after transient global forebrain ischemia in the rat. Stroke. 1996;27:527–535. doi: 10.1161/01.str.27.3.527. [DOI] [PubMed] [Google Scholar]
  21. Cole DJ, Patel PM, Reynolds L, Drummond JC, Marcantonio S. Temporary focal cerebral ischemia in spontaneously hypertensive rats: the effect of ibuprofen on infarct volume. J Pharmacol Exp Ther. 1993;266:1713–1717. [PubMed] [Google Scholar]
  22. Coyle JT, Puttfarcken P. Oxidative stress, glutamate, and neurodegenerative disorders. Science. 1993;262:689–695. doi: 10.1126/science.7901908. [DOI] [PubMed] [Google Scholar]
  23. Cummings BS, McHowat J, Schnellmann RG. Phospholipase A(2)s in cell injury and death. J Pharmacol Exp Ther. 2000;294:793–799. [PubMed] [Google Scholar]
  24. Davies SP, Reddy H, Caivano M, Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J. 2000;351:95–105. doi: 10.1042/0264-6021:3510095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Drachman DB, Frank K, Dykes-Hoberg M, Teismann P, Almer G, Przedborski S, Rothstein JD. Cyclooxygenase 2 inhibition protects motor neurons and prolongs survival in a transgenic mouse model of ALS. Ann Neurol. 2002;52:771–778. doi: 10.1002/ana.10374. [DOI] [PubMed] [Google Scholar]
  26. Dugan LL, Sensi SL, Canzoniero LM, Handran SD, Rothman SM, Lin TS, Goldberg MP, Choi DW. Mitochondrial production of reactive oxygen species in cortical neurons following exposure to N-methyl-D-aspartate. J Neurosci. 1995;15:6377–6388. doi: 10.1523/JNEUROSCI.15-10-06377.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Dumuis A, Sebben M, Haynes L, Pin JP, Bockaert J. NMDA receptors activate the arachidonic acid cascade system in striatal neurons. Nature. 1988;336:68–70. doi: 10.1038/336068a0. [DOI] [PubMed] [Google Scholar]
  28. Dumuis A, Sebben M, Fagni L, Prezeau L, Manzoni O, Cragoe EJ, Jr, Bockaert J. Stimulation by glutamate receptors of arachidonic acid release depends on the Na+/Ca2+ exchanger in neuronal cells. Mol Pharmacol. 1993;43:976–981. [PubMed] [Google Scholar]
  29. Fogal B, Trettel J, Uliasz TF, Levine ES, Hewett SJ. Changes in secondary glutamate release underlie the developmental regulation of excitotoxic neuronal cell death. Neuroscience. 2005;132:929–942. doi: 10.1016/j.neuroscience.2005.01.036. [DOI] [PubMed] [Google Scholar]
  30. Ghosh M, Tucker DE, Burchett SA, Leslie CC. Properties of the Group IV phospholipase A2 family. Progress in Lipid Research. 2006;45:487. doi: 10.1016/j.plipres.2006.05.003. [DOI] [PubMed] [Google Scholar]
  31. Gierse JK, Hauser SD, Creely DP, Koboldt C, Rangwala SH, Isakson PC, Seibert K. Expression and selective inhibition of the constitutive and inducible forms of human cyclo-oxygenase. Biochem J. 1995;305(Pt 2):479–484. doi: 10.1042/bj3050479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Glaser KB. Regulation of phospholipase A2 enzymes: selective inhibitors and their pharmacological potential. Adv Pharmacol. 1995;32:31–66. doi: 10.1016/s1054-3589(08)61011-x. [DOI] [PubMed] [Google Scholar]
  33. Hamby ME, Uliasz TF, Hewett SJ, Hewett JA. Characterization of an improved procedure for the removal of microglia from confluent monolayers of primary astrocytes. J Neurosci Methods. 2006;150:128–137. doi: 10.1016/j.jneumeth.2005.06.016. [DOI] [PubMed] [Google Scholar]
  34. Hao C, Guilbert LJ, Fedoroff S. Production of colony-stimulating factor-1 (CSF-1) by mouse astroglia in vitro. J Neurosci Res. 1990;27:314–323. doi: 10.1002/jnr.490270310. [DOI] [PubMed] [Google Scholar]
  35. Hazen SL, Gross RW. The specific association of a phosphofructokinase isoform with myocardial calcium-independent phospholipase A2. Implications for the coordinated regulation of phospholipolysis and glycolysis. J Biol Chem. 1993;268:9892–9900. [PubMed] [Google Scholar]
  36. Heneka MT, Klockgether T, Feinstein DL. Peroxisome proliferator-activated receptor-gamma ligands reduce neuronal inducible nitric oxide synthase expression and cell death in vivo. J Neurosci. 2000;20:6862–6867. doi: 10.1523/JNEUROSCI.20-18-06862.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Hewett JA, Hewett SJ, Winkler S, Pfeiffer SE. Inducible nitric oxide synthase expression in cultures enriched for mature oligodendrocytes is due to microglia. J Neurosci Res. 1999;56:189–198. doi: 10.1002/(sici)1097-4547(19990415)56:2<189::aid-jnr8>3.0.co;2-b. [DOI] [PubMed] [Google Scholar]
  38. Hewett SJ, Bell SC, Hewett JA. Contributions of cyclooxygenase-2 to neuroplasticity and neuropathology of the central nervous system. Pharmacol Ther. 2006a;112:335–357. doi: 10.1016/j.pharmthera.2005.04.011. [DOI] [PubMed] [Google Scholar]
  39. Hewett SJ, Silakova JM, Hewett JA. Oral treatment with rofecoxib reduces hippocampal excitotoxic neurodegeneration. J Pharmacol Exp Ther. 2006b;319:1219–1224. doi: 10.1124/jpet.106.109876. [DOI] [PubMed] [Google Scholar]
  40. Hewett SJ, Uliasz TF, Vidwans AS, Hewett JA. Cyclooxygenase-2 contributes to N-methyl-D-aspartate-mediated neuronal cell death in primary cortical cell culture. J Pharmacol Exp Ther. 2000;293:417–425. [PubMed] [Google Scholar]
  41. Hoozemans JJ, O'Banion MK. The role of COX-1 and COX-2 in Alzheimer's disease pathology and the therapeutic potentials of non-steroidal anti-inflammatory drugs. Curr Drug Target CNS Neurol Disord. 2005;4:307–315. doi: 10.2174/1568007054038201. [DOI] [PubMed] [Google Scholar]
  42. Iadecola C, Niwa K, Nogawa S, Zhao X, Nagayama M, Araki E, Morham S, Ross ME. Reduced susceptibility to ischemic brain injury and N-methyl-D-aspartate-mediated neurotoxicity in cyclooxygenase-2-deficient mice. Proc Natl Acad Sci U S A. 2001;98:1294–1299. doi: 10.1073/pnas.98.3.1294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Jaradat MS, Wongsud B, Phornchirasilp S, Rangwala SM, Shams G, Sutton M, Romstedt KJ, Noonan DJ, Feller DR. Activation of peroxisome proliferator-activated receptor isoforms and inhibition of prostaglandin H(2) synthases by ibuprofen, naproxen, and indomethacin. Biochem Pharmacol. 2001;62:1587–1595. doi: 10.1016/s0006-2952(01)00822-x. [DOI] [PubMed] [Google Scholar]
  44. Karschin A, Aizenman E, Lipton SA. The interaction of agonists and noncompetitive antagonists at the excitatory amino acid receptors in rat retinal ganglion cells in vitro. J Neurosci. 1988;8:2895–2906. doi: 10.1523/JNEUROSCI.08-08-02895.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Kawano T, Anrather J, Zhou P, Park L, Wang G, Frys KA, Kunz A, Cho S, Orio M, Iadecola C. Prostaglandin E2 EP1 receptors: downstream effectors of COX-2 neurotoxicity. Nat Med. 2006;12:225. doi: 10.1038/nm1362. [DOI] [PubMed] [Google Scholar]
  46. Kelley KA, Ho L, Winger D, Freire-Moar J, Borelli CB, Aisen PS, Pasinetti GM. Potentiation of excitotoxicity in transgenic mice overexpressing neuronal cyclooxygenase-2. Am J Pathol. 1999;155:995–1004. doi: 10.1016/S0002-9440(10)65199-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Kettenmann H, Schachner M. Pharmacological properties of gamma-aminobutyric acid-, glutamate-, and aspartate-induced depolarizations in cultured astrocytes. J Neurosci. 1985;5:3295–3301. doi: 10.1523/JNEUROSCI.05-12-03295.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Kim DK, Rordorf G, Nemenoff RA, Koroshetz WJ, Bonventre JV. Glutamate stably enhances the activity of two cytosolic forms of phospholipase A2 in brain cortical cultures. Biochem J. 1995;310(Pt 1):83–90. doi: 10.1042/bj3100083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Kim HY, Edsall L, Garcia M, Zhang H. The release of polyunsaturated fatty acids and their lipoxygenation in the brain. Advances in Experimental Medicine & Biology. 1999;447:75–85. doi: 10.1007/978-1-4615-4861-4_7. [DOI] [PubMed] [Google Scholar]
  50. Klivenyi P, Gardian G, Calingasan NY, Yang L, Beal MF. Additive neuroprotective effects of creatine and a cyclooxygenase 2 inhibitor against dopamine depletion in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. J Mol Neurosci. 2003;21:191–198. doi: 10.1385/jmn:21:3:191. [DOI] [PubMed] [Google Scholar]
  51. Klivenyi P, Beal MF, Ferrante RJ, Andreassen OA, Wermer M, Chin MR, Bonventre JV. Mice deficient in group IV cytosolic phospholipase A2 are resistant to MPTP neurotoxicity. J Neurochem. 1998;71:2634–2637. doi: 10.1046/j.1471-4159.1998.71062634.x. [DOI] [PubMed] [Google Scholar]
  52. Kozak KR, Rowlinson SW, Marnett LJ. Oxygenation of the endocannabinoid, 2-arachidonylglycerol, to glyceryl prostaglandins by cyclooxygenase-2. J Biol Chem. 2000;275:33744–33749. doi: 10.1074/jbc.M007088200. [DOI] [PubMed] [Google Scholar]
  53. Krady JK, Basu A, Levison SW, Milner RJ. Differential expression of protein tyrosine kinase genes during microglial activation. Glia. 2002;40:11–24. doi: 10.1002/glia.10101. [DOI] [PubMed] [Google Scholar]
  54. Kudo I, Murakami M. Phospholipase A2 enzymes. Prostaglandins Other Lipid Mediat. 2002;68–69:3–58. doi: 10.1016/s0090-6980(02)00020-5. [DOI] [PubMed] [Google Scholar]
  55. Kunz T, Oliw EH. The selective cyclooxygenase-2 inhibitor rofecoxib reduces kainate-induced cell death in the rat hippocampus. Eur J Neurosci. 2001;13:569–575. doi: 10.1046/j.1460-9568.2001.01420.x. [DOI] [PubMed] [Google Scholar]
  56. Lafon-Cazal M, Pietri S, Culcasi M, Bockaert J. NMDA-dependent superoxide production and neurotoxicity. Nature. 1993;364:535–537. doi: 10.1038/364535a0. [DOI] [PubMed] [Google Scholar]
  57. Lazarewicz JW, Wroblewski JT, Costa E. N-methyl-D-aspartate-sensitive glutamate receptors induce calcium- mediated arachidonic acid release in primary cultures of cerebellar granule cells. J Neurochem. 1990;55:1875–1881. doi: 10.1111/j.1471-4159.1990.tb05771.x. [DOI] [PubMed] [Google Scholar]
  58. Lazarewicz JW, Salinska E, Wroblewski JT. NMDA receptor-mediated arachidonic acid release in neurons: role in signal transduction and pathological aspects. Adv Exp Med Biol. 1992;318:73–89. doi: 10.1007/978-1-4615-3426-6_7. [DOI] [PubMed] [Google Scholar]
  59. Lazarewicz JW, Wroblewski JT, Palmer ME, Costa E. Activation of N-methyl-D-aspartate-sensitive glutamate receptors stimulates arachidonic acid release in primary cultures of cerebellar granule cells. Neuropharmacology. 1988;27:765–769. doi: 10.1016/0028-3908(88)90088-3. [DOI] [PubMed] [Google Scholar]
  60. Leslie CC. Properties and Regulation of Cytosolic Phospholipase A2. J. Biol. Chem. 1997;272:16709–16712. doi: 10.1074/jbc.272.27.16709. [DOI] [PubMed] [Google Scholar]
  61. Levy BD, Clish CB, Schmidt B, Gronert K, Serhan CN. Lipid mediator class switching during acute inflammation: signals in resolution. Nat Immunol. 2001;2:612. doi: 10.1038/89759. [DOI] [PubMed] [Google Scholar]
  62. Lin LL, Wartmann M, Lin AY, Knopf JL, Seth A, Davis RJ. cPLA2 is phosphorylated and activated by MAP kinase. Cell. 1993;72:269–278. doi: 10.1016/0092-8674(93)90666-e. [DOI] [PubMed] [Google Scholar]
  63. Lio YC, Reynolds LJ, Balsinde J, Dennis EA. Irreversible inhibition of Ca(2+)-independent phospholipase A2 by methyl arachidonyl fluorophosphonate. Biochim Biophys Acta. 1996;1302:55–60. doi: 10.1016/0005-2760(96)00002-1. [DOI] [PubMed] [Google Scholar]
  64. McCullough L, Wu L, Haughey N, Liang X, Hand T, Wang Q, Breyer RM, Andreasson K. Neuroprotective Function of the PGE2 EP2 Receptor in Cerebral Ischemia. J. Neurosci. 2004;24:257–268. doi: 10.1523/JNEUROSCI.4485-03.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Meldrum B, Garthwaite J. Excitatory amino acid neurotoxicity and neurodegenerative disease. Trends Pharmacol Sci. 1990;11:379–387. doi: 10.1016/0165-6147(90)90184-a. [DOI] [PubMed] [Google Scholar]
  66. Meldrum B, Evans M, Griffiths T, Simon R. Ischaemic brain damage: the role of excitatory activity and of calcium entry. Br J Anaesth. 1985;57:44–46. doi: 10.1093/bja/57.1.44. [DOI] [PubMed] [Google Scholar]
  67. Miller B, Sarantis M, Traynelis SF, Attwell D. Potentiation of NMDA receptor currents by arachidonic acid. Nature. 1992;355:722–725. doi: 10.1038/355722a0. [DOI] [PubMed] [Google Scholar]
  68. Molloy GY, Rattray M, Williams RJ. Genes encoding multiple forms of phospholipase A2 are expressed in rat brain. Neurosci Lett. 1998;258:139–142. doi: 10.1016/s0304-3940(98)00838-6. [DOI] [PubMed] [Google Scholar]
  69. Mukherjee AB, Miele L, Pattabiraman N. Phospholipase A2 enzymes: regulation and physiological role. Biochem Pharmacol. 1994;48:1–10. doi: 10.1016/0006-2952(94)90216-x. [DOI] [PubMed] [Google Scholar]
  70. Murakami M, Nakatani Y, Atsumi G, Inoue K, Kudo I. Regulatory functions of phospholipase A2. Crit Rev Immunol. 1997;17:225–283. doi: 10.1615/critrevimmunol.v17.i3-4.10. [DOI] [PubMed] [Google Scholar]
  71. Murakami M, Shimbara S, Kambe T, Kuwata H, Winstead MV, Tischfield JA, Kudo I. The functions of five distinct mammalian phospholipase A2S in regulating arachidonic acid release. Type IIa and type V secretory phospholipase A2S are functionally redundant and act in concert with cytosolic phospholipase A2. J Biol Chem. 1998;273:14411–14423. doi: 10.1074/jbc.273.23.14411. [DOI] [PubMed] [Google Scholar]
  72. Nakagomi T, Sasaki T, Kirino T, Tamura A, Noguchi M, Saito I, Takakura K. Effect of cyclooxygenase and lipoxygenase inhibitors on delayed neuronal death in the gerbil hippocampus. Stroke. 1989;20:925–929. doi: 10.1161/01.str.20.7.925. [DOI] [PubMed] [Google Scholar]
  73. Nakao N, Brundin P. Neurodegeneration and glutamate induced oxidative stress. Prog Brain Res. 1998;116:245–263. doi: 10.1016/s0079-6123(08)60441-0. [DOI] [PubMed] [Google Scholar]
  74. Nakayama M, Uchimura K, Zhu RL, Nagayama T, Rose ME, Stetler RA, Isakson PC, Chen J, Graham SH. Cyclooxygenase-2 inhibition prevents delayed death of CA1 hippocampal neurons following global ischemia. Proc Natl Acad Sci U S A. 1998;95:10954–10959. doi: 10.1073/pnas.95.18.10954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Nogawa S, Zhang F, Ross ME, Iadecola C. Cyclo-oxygenase-2 gene expression in neurons contributes to ischemic brain damage. J Neurosci. 1997;17:2746–2755. doi: 10.1523/JNEUROSCI.17-08-02746.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Ossowska K. The role of excitatory amino acids in experimental models of Parkinson's disease. J Neural Transm Park Dis Dement Sect. 1994;8:39–71. doi: 10.1007/BF02250917. [DOI] [PubMed] [Google Scholar]
  77. Owada Y, Tominaga T, Yoshimoto T, Kondo H. Molecular cloning of rat cDNA for cytosolic phospholipase A2 and the increased gene expression in the dentate gyrus following transient forebrain ischemia. Brain Res Mol Brain Res. 1994;25:364–368. doi: 10.1016/0169-328x(94)90174-0. [DOI] [PubMed] [Google Scholar]
  78. Patel PM, Drummond JC, Sano T, Cole DJ, Kalkman CJ, Yaksh TL. Effect of ibuprofen on regional eicosanoid production and neuronal injury after forebrain ischemia in rats. Brain Res. 1993;614:315–324. doi: 10.1016/0006-8993(93)91050-3. [DOI] [PubMed] [Google Scholar]
  79. Peters-Golden M, Song K, Marshall T, Brock T. Translocation of cytosolic phospholipase A2 to the nuclear envelope elicits topographically localized phospholipids hydrolysis. Biochem J. 1996;318:797–803. doi: 10.1042/bj3180797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  80. Pickard RT, Strifler BA, Kramer RM, Sharp JD. Molecular cloning of two new human paralogs of 85-kDa cytosolic phospholipase A2. J Biol Chem. 1999;274:8823–8831. doi: 10.1074/jbc.274.13.8823. [DOI] [PubMed] [Google Scholar]
  81. Plesniak LA, Boegeman SC, Segelke BW, Dennis EA. Interaction of phospholipase A2 with thioether amide containing phospholipid analogues. Biochemistry. 1993;32:5009–5016. doi: 10.1021/bi00070a006. [DOI] [PubMed] [Google Scholar]
  82. Reynolds IJ, Hastings TG. Glutamate induces the production of reactive oxygen species in cultured forebrain neurons following NMDA receptor activation. J Neurosci. 1995;15:3318–3327. doi: 10.1523/JNEUROSCI.15-05-03318.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  83. Sanfeliu C, Hunt A, Patel AJ. Exposure to N-methyl-D-aspartate increases release of arachidonic acid in primary cultures of rat hippocampal neurons and not in astrocytes. Brain Res. 1990;526:241–248. doi: 10.1016/0006-8993(90)91228-9. [DOI] [PubMed] [Google Scholar]
  84. Sapirstein A, Bonventre JV. Phospholipases A2 in ischemic and toxic brain injury. Neurochemical Research. 2000;25:745–753. doi: 10.1023/a:1007583708713. [DOI] [PubMed] [Google Scholar]
  85. Sasaki T, Nakagomi T, Kirino T, Tamura A, Noguchi M, Saito I, Takakura K. Indomethacin ameliorates ischemic neuronal damage in the gerbil hippocampal CA1 sector. Stroke. 1988;19:1399–1403. doi: 10.1161/01.str.19.11.1399. [DOI] [PubMed] [Google Scholar]
  86. Schaloske RH, Dennis EA. The phospholipase A2 superfamily and its group numbering system. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 2006;1761:1246. doi: 10.1016/j.bbalip.2006.07.011. [DOI] [PubMed] [Google Scholar]
  87. Schevitz RW, Bach NJ, Carlson DG, Chirgadze NY, Clawson DK, Dillard RD, Draheim SE, Hartley LW, Jones ND, Mihelich ED, et al. Structure-based design of the first potent and selective inhibitor of human non-pancreatic secretory phospholipase A2. Nat Struct Biol. 1995;2:458–465. doi: 10.1038/nsb0695-458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  88. Shen Y, Kishimoto K, Linden DJ, Sapirstein A. Cytosolic phospholipase A(2) alpha mediates electrophysiologic responses of hippocampal pyramidal neurons to neurotoxic NMDA treatment. Proc Natl Acad Sci U S A. 2007;104:6078–6083. doi: 10.1073/pnas.0605427104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  89. Silakova JM, Hewett JA, Hewett SJ. Naproxen reduces excitotoxic neurodegeneration in vivo with an extended therapeutic window. J Pharmacol Exp Ther. 2004;309:1060–1066. doi: 10.1124/jpet.103.063867. [DOI] [PubMed] [Google Scholar]
  90. Song C, Chang XJ, Bean KM, Proia MS, Knopf JL, Kriz RW. Molecular characterization of cytosolic phospholipase A2-beta. J Biol Chem. 1999;274:17063–17067. doi: 10.1074/jbc.274.24.17063. [DOI] [PubMed] [Google Scholar]
  91. Stella N, Pellerin L, Magistretti PJ. Modulation of the glutamate-evoked release of arachidonic acid from mouse cortical neurons: involvement of a pH-sensitive membrane phospholipase A2. J Neurosci. 1995;15:3307–3317. doi: 10.1523/JNEUROSCI.15-05-03307.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  92. Stella N, Tence M, Glowinski J, Premont J. Glutamate-evoked release of arachidonic acid from mouse brain astrocytes. J Neurosci. 1994;14:568–575. doi: 10.1523/JNEUROSCI.14-02-00568.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  93. Strauss KI, Marini AM. Cyclooxygenase-2 inhibition protects cultured cerebellar granule neurons from glutamate-mediated cell death. J Neurotrauma. 2002;19:627–638. doi: 10.1089/089771502753754091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  94. Street IP, Lin HK, Laliberte F, Ghomashchi F, Wang Z, Perrier H, Tremblay NM, Huang Z, Weech PK, Gelb MH. Slow- and tight-binding inhibitors of the 85-kDa human phospholipase A2. Biochemistry. 1993;32:5935–5940. doi: 10.1021/bi00074a003. [DOI] [PubMed] [Google Scholar]
  95. Sun GY, Xu J, Jensen MD, Simonyi A. Phospholipase A2 in the central nervous system: implications for neurodegenerative diseases. Journal of Lipid Research. 2004;45:205–213. doi: 10.1194/jlr.R300016-JLR200. [DOI] [PubMed] [Google Scholar]
  96. Sutherland CA, Amin D. Relative activities of rat and dog platelet phospholipase A2 and diglyceride lipase. Selective inhibition of diglyceride lipase by RHC 80267. J Biol Chem. 1982;257:14006–14010. [PubMed] [Google Scholar]
  97. Tapia-Arancibia L, Rage F, Recasens M, Pin JP. NMDA receptor activation stimulates phospholipase A2 and somatostatin release from rat cortical neurons in primary cultures. Eur J Pharmacol. 1992;225:253–262. doi: 10.1016/0922-4106(92)90027-s. [DOI] [PubMed] [Google Scholar]
  98. Taylor AL, Hewett SJ. Potassium-evoked glutamate release liberates arachidonic acid from cortical neurons. J Biol Chem. 2002;277:43881–43887. doi: 10.1074/jbc.M205872200. [DOI] [PubMed] [Google Scholar]
  99. Taylor AL, Hewett SJ. Program No. 951.11. 2003 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, Online; 2003. Regulation of NMDA-stimulated arachidonic acid release from primary mixed cortical cell cultures. [Google Scholar]
  100. Teismann P, Ferger B. Inhibition of the cyclooxygenase isoenzymes COX-1 and COX-2 provide neuroprotection in the MPTP-mouse model of Parkinson's disease. Synapse. 2001;39:167–174. doi: 10.1002/1098-2396(200102)39:2<167::AID-SYN8>3.0.CO;2-U. [DOI] [PubMed] [Google Scholar]
  101. Teismann P, Tieu K, Choi DK, Wu DC, Naini A, Hunot S, Vila M, Jackson-Lewis V, Przedborski S. Cyclooxygenase-2 is instrumental in Parkinson's disease neurodegeneration. Proc Natl Acad Sci U S A. 2003;100:5473–5478. doi: 10.1073/pnas.0837397100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  102. Trackey JL, Uliasz TF, Hewett SJ. SIN-1-induced cytotoxicity in mixed cortical cell culture: peroxynitrite-dependent and -independent induction of excitotoxic cell death. J Neurochem. 2001;79:445–455. doi: 10.1046/j.1471-4159.2001.00584.x. [DOI] [PubMed] [Google Scholar]
  103. Uliasz TF, Hewett SJ. A microtiter trypan blue absorbance assay for the quantitative determination of excitotoxic neuronal injury in cell culture. J Neurosci Methods. 2000;100:157–163. doi: 10.1016/s0165-0270(00)00248-x. [DOI] [PubMed] [Google Scholar]
  104. van der Stelt M, Veldhuis WB, Maccarrone M, Bar PR, Nicolay K, Veldink GA, Di Marzo V, Vliegenthart JF. Acute neuronal injury, excitotoxicity, and the endocannabinoid system. Mol Neurobiol. 2002;26:317–346. doi: 10.1385/MN:26:2-3:317. [DOI] [PubMed] [Google Scholar]
  105. Vazquez E, Herrero I, Miras-Portugal MT, Sanchez-Prieto J. Role of arachidonic acid in the facilitation of glutamate release from rat cerebrocortical synaptosomes independent of metabotropic glutamate receptor responses. Neurosci Lett. 1994;174:9–13. doi: 10.1016/0304-3940(94)90106-6. [DOI] [PubMed] [Google Scholar]
  106. Volterra A. Inhibition of high-affinity glutamate transport in neuronal and glial cells by arachidonic acid and oxygen-free radicals. Molecular mechanisms and neuropathological relevance. Ren Physiol Biochem. 1994;17:165–167. doi: 10.1159/000173809. [DOI] [PubMed] [Google Scholar]
  107. Walton M, Sirimanne E, Williams C, Gluckman PD, Keelan J, Mitchell MD, Dragunow M. Prostaglandin H synthase-2 and cytosolic phospholipase A2 in the hypoxic-ischemic brain: role in neuronal death or survival? Brain Res Mol Brain Res. 1997;50:165–170. doi: 10.1016/s0169-328x(97)00181-2. [DOI] [PubMed] [Google Scholar]
  108. Westerberg E, Deshpande JK, Wieloch T. Regional differences in arachidonic acid release in rat hippocampal CA1 and CA3 regions during cerebral ischemia. J Cereb Blood Flow Metab. 1987;7:189–192. doi: 10.1038/jcbfm.1987.43. [DOI] [PubMed] [Google Scholar]
  109. Wieloch T, Siesjo BK. Ischemic brain injury: the importance of calcium, lipolytic activities, and free fatty acids. Pathol Biol (Paris) 1982;30:269–277. [PubMed] [Google Scholar]
  110. Williams JH, Errington ML, Lynch MA, Bliss TV. Arachidonic acid induces a long-term activity-dependent enhancement of synaptic transmission in the hippocampus. Nature. 1989;341:739–742. doi: 10.1038/341739a0. [DOI] [PubMed] [Google Scholar]
  111. 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. 1999;269:278–288. doi: 10.1006/abio.1999.4053. [DOI] [PubMed] [Google Scholar]
  112. Yedgar S, Cohen Y, Shoseyov D. Control of phospholipase A2 activities for the treatment of inflammatory conditions. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. 2006;1761:1373. doi: 10.1016/j.bbalip.2006.08.003. [DOI] [PubMed] [Google Scholar]
  113. Yoshida S, Inoh S, Asano T, Sano K, Shimasaki H, Ueta N. Brain free fatty acids, edema, and mortality in gerbils subjected to transient, bilateral ischemia, and effect of barbiturate anesthesia. J Neurochem. 1983;40:1278–1286. doi: 10.1111/j.1471-4159.1983.tb13567.x. [DOI] [PubMed] [Google Scholar]
  114. Yu AC, Chan PH, Fishman RA. Effects of arachidonic acid on glutamate and gamma-aminobutyric acid uptake in primary cultures of rat cerebral cortical astrocytes and neurons. J Neurochem. 1986;47:1181–1189. doi: 10.1111/j.1471-4159.1986.tb00738.x. [DOI] [PubMed] [Google Scholar]
  115. Yu M, Ives D, Ramesha CS. Synthesis of prostaglandin E2 ethanolamide from anandamide by cyclooxygenase-2. J Biol Chem. 1997;272:21181–21186. doi: 10.1074/jbc.272.34.21181. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp Fig s1

Supplemental Figure 1. Effect of ionotropic glutamate receptor antagonism on NMDA-stimulated [3H]-AA release. Cortical cell cultures were incubated with [3H]-AA overnight, washed, and then exposed to 100 µM NMDA in the absence or presence of MK-801 (10 µM) or CNQX (30 µM) for 15 min. Thereafter, cell supernatants were collected and assayed for [3H]-AA content. Cultures were then washed and solubilized with warm 0.2% SDS. Cell lysates were collected and assayed for [3H]-AA content. Data are expressed as the % of total incorporated. (*) indicates a value significantly greater than basal [3H]-AA release, whereas (#) represents a significant diminution of NMDA-stimulated [3H]-AA release as determined by one-way ANOVA followed by the Student Newman Keul's t-test for multiple comparisons. Significance was assessed at p < 0.05.

NIHMS56864-supplement-Supp_Fig_s1.tif (85.4KB, tif)
Supp Fig s2

Supplemental Figure 2: Expression cPLA2 paralogs. RNA was isolated from pure astrocyte (Lanes 1, 2) or pure neuronal (Lanes 3,4) cultures. First-strand cDNA was synthesized and cPLA2α̣, cPLA2β, and cPLA2γ expressions were assessed by PCR as described in the text. β-actin mRNA assessed in all RNA samples as an internal control for the amount of RNA in each sample.

NIHMS56864-supplement-Supp_Fig_s2.tif (1.1MB, tif)

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