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The Journal of Neuroscience, August 27, 2003, 23(21):7767-7775

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Palmitoylethanolamide Increases after Focal Cerebral Ischemia and Potentiates Microglial Cell Motility

Allyn Franklin,¹ Sophie Parmentier-Batteur,³ Lisa Walter,¹ David A. Greenberg,³ and Nephi Stella^1,2

Departments of ¹Pharmacology and ²Psychiatry and Behavioral Sciences, University of Washington, Seattle, Washington 98195, and ³Buck Institute for Age Research, Novato, California 94945

	Abstract

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Focal cerebral ischemia (FCI) induces rapid neuronal death in the ischemic core, which gradually expands toward the penumbra, partly as the result of a neuroinflammatory response. It is known that propagation of neuroinflammation involves microglial cells, the resident macrophages of the brain, which are highly motile when activated by specific signals. However, the signals that increase microglial cell motility in response to FCI remain mostly elusive.

Here, we tested the hypothesis that endocannabinoids mediate neuroinflammation propagation by increasing microglial cell motility. We found that, in mouse cerebral cortex, FCI greatly increases palmitoylethanolamide (PEA), only moderately increases anandamide [arachidonylethanolamide (AEA)], and does not affect 2-arachidonoylglycerol levels. We also found that PEA potentiates AEA-induced microglial cell migration, without affecting other steps of microglial activation, such as proliferation, particle engulfment, and nitric oxide production. This potentiation of microglial cell migration by PEA involves reduction in cAMP levels. In line with this, we provide evidence that PEA acts through G_i/o-coupled receptors. Interestingly, these receptors engaged by PEA are pharmacologically distinct from CB₁ and CB₂ cannabinoid receptors, as well as from the WIN and abn-CBD (abnormal-cannabidiol) receptors, two recently identified cannabinoid receptors.

Our results show that PEA and AEA increase after FCI and synergistically enhance microglial cell motility. Because such a response could participate in the propagation of the FCI-induced neuroinflammation within the CNS, and because PEA is likely to act through its own receptor, a better understanding of the receptor engaged by PEA may help guide the search for improved therapies against neuroinflammation.

Key words: stroke; neuroinflammation; marijuana; lipids; nitric oxide; phagocytosis

	Introduction

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In the early stages of stroke, clinical symptoms reflect an impairment of function that precedes permanent structural damage (Dirnagl et al., 1999). With time, however, delayed neuroinflammation, which is likely mediated by microglial cells, contributes to cell death in the periinfarct zone, or penumbra (Dirnagl et al., 1999). Microglial cells are the first immune cells that respond to signals produced by focal cerebral ischemia (FCI) (Stoll et al., 1998). These cells reside within the brain and express membrane receptors that sense pathological changes (Kreutzberg, 1996). Engagement of receptors on microglial cells initiates a rapid, multistep change in their phenotype that is referred to as microglial cell activation (Bruce-Keller, 1999). Specifically, activated microglial cells (1) retract their processes and extend lamellipodia, allowing them to migrate (Stence et al., 2001), (2) proliferate (Hailer et al., 1999), (3) release cytotoxins and proinflammatory cytokines (Becher et al., 2000), and (4) engulf cellular debris (Becher et al., 2000). Pharmacological inhibition of microglial cell activation prevents neuronal damage in the penumbra (Yrjünheikki et al., 1999), indicating that this cell type constitutes a promising target for the treatment of stroke-induced damage. However, the receptors and signals that mediate microglial cell activation after FCI are not well understood.

Cannabinoids—whether plant-derived, endogenous, or synthetic—engage at least four subtypes of cannabinoid receptors: the cloned cannabinoid CB₁ and CB₂ receptors (Matsuda et al., 1990; Munro et al., 1993) and the as-yet-uncloned WIN and abnormal-cannabidiol (abn-CBD) receptors (the latter are also known as anandamide receptors) (Járai et al., 1999; Di Marzo et al., 2000; Breivogel et al., 2001; Hájos et al., 2001). Because cannabinoid compounds have been shown to initiate and modulate microglial cell activation (Stefano et al., 1996; Waksman et al., 1999; Walter et al., 2003), cannabinoid receptors expressed by microglial cells have been regarded as promising pharmaceutical targets (Grundy et al., 2001). CB₁ and CB₂ receptors are coupled to G_i/o-proteins and are normally engaged by endocannabinoids produced by neurons, astrocytes, and microglial cells (Stella and Piomelli, 2001; Walter et al., 2002, 2003). Initial characterizations of the abn-CBD receptors suggest that they also couple to G_i/o-proteins (Mukhopadhyay et al., 2002).

Several endocannabinoids have been identified and are categorized into two main families on the basis of their structure: the acylethanolamides (acyl-EAs), which include arachidonylethanolamide (AEA) (the prototypical endocannabinoid), and the acylesters, which include 2-arachidonoylglycerol (2-AG). AEA acts via CB₁ receptors as a partial agonist and at CB₂ receptors as a weak partial agonist-antagonist. 2-AG acts via both CB₁ and CB₂ receptors as a full agonist (Vogel et al., 1993; Stella et al., 1997; Gonsiorek et al., 2000; Sugiura et al., 2000). Multiple acyl-EAs with cannabinoid-like properties have been identified. For example, homo--linolenoylethanolamide (HEA) and docosatetraenoylethanolamide (DEA) are two endocannabinoid candidates for CB₁ receptors that have never been quantified in brain tissue (Hanus et al., 1993). Palmitoylethanolamide (PEA) has received considerable attention because of its antiinflammatory properties in peripheral tissue (Lambert et al., 2002), yet little is known about the receptor engaged by this lipid and whether it is involved in neuroinflammation. In fact, whether PEA actually belongs to the endocannabinoid lipid family is unclear, primarily because it does not bind to either CB₁ or CB₂ receptors (Felder et al., 1995).

Because endocannabinoid levels are dramatically increased as a result of diverse neuropathological conditions (Mechoulam et al., 2002), we sought to determine whether the amounts of endocannabinoids in mouse cerebral cortex are affected by FCI, and if so, whether these signals act on receptors that initiate and/or modulate microglial cell activation.

	Materials and Methods

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Materials. Acylethanolamides and deuterated acylethanolamides were synthesized in our laboratory, as described previously (Walter et al., 2002). Palmitoylisopropylamide, R-palmitoyl-(1-methyl)ethanolamide, R-palmitoyl-(2-methyl)ethanolamide, and 2-AG were from Cayman Chemical (Ann Arbor, MI). N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A) and N-((1S)-endo-1,3,3-trimethyl-bicycloheptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) (SR144528) were a gift from Sanofi Research (Montpellier, France). abn-CBD and O-1918 were a gift from Dr. G. Kunos (National Institute on Drug Abuse and Alcoholism, Bethesda, MD).

Focal cerebral ischemia and endocannabinoid quantification. FCI was established as described previously (Parmentier-Batteur et al., 2002) after local committee review, and this was conducted according to policies on the use of animals of the Society for Neuroscience. Briefly, 15 adult male CD1 mice (25-30 gm) were separated into three groups of five mice each. In the ischemia group, the left external carotid artery was ligated, and its branches were electrocoagulated; the left common carotid artery was occluded, and a filament was introduced to occlude the left middle cerebral artery and withdrawn after 20 min. The sham surgery group was treated similarly, except that no vessels were occluded, and the control group underwent no surgery. Mice in the ischemia and sham surgery groups were decapitated 24 hr after surgery, and control mice were directly decapitated.

Brains were removed from the skull and placed on ice within 1 min of decapitation. Left cerebral cortices were isolated, and 6 mm coronal slices centered on the middle cerebral artery territory corresponding to the infarct area were dissected out and dropped directly into ice-cold CHCl₃ (20 ml). Samples were kept at -80°C until endocannabinoid analysis was performed as described previously (Walter et al., 2002). Briefly, samples were brought to room temperature, and 200 pmol of deuterated standards were added. Tissue was homogenized, and endocannabinoids were extracted and purified by normal-phase HPLC (Hewlett Packard, Palo Alto, CA) and quantified by chemical ionization-gas chromatography/mass spectrometry (CI-GC/MS) (Varian, Palo Alto, CA).

BV-2 cell culture. The mouse microglial cell line BV-2 [a gift from Dr. E. Blasi (University of Perugia, Perugia, Italy)] (Blasi et al., 1990) was expanded in DMEM supplemented with FBS (3%), HEPES (10 mM), NaHCO₃ (5 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml), and passaged every 3-4 d for 2-12 passages. Approximately 18 hr before experiments, BV-2 cells were recovered at 200,000 cells/ml of MEM-CellGro [MEM supplemented with 10 mM HEPES, 5 mM NaHCO₃, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 10% CellGro (Mediatech, Herndon, VA)]. Cells were harvested for cell migration experiments; plated onto 24-well plates (500 µl/well) for cAMP, nitrite, and 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt (WST-1) quantification; or plated onto 35 mm culture dishes (2 ml/dish) for phagocytosis experiments. For all of the experiments, cannabinoids were dissolved in DMSO (at 1000x) using siliconized glass vials and siliconized pipette tips.

cAMP quantification. BV-2 cells were rinsed once with 500 µl/well HEPES-bicarbonate (HB) buffer (in mM): 145 NaCl, 5.5 KCl, 1.1 CaCl₂, 1.1 MgCl₂, 3.6 NaHCO₃, 5.5 glucose, and 20 HEPES, pH 7.4 at 37°C. Cells were preincubated for 10 min in HB buffer containing IBMX (1 mM) and incubated for 10 min in HB buffer containing IBMX (1 mM) and forskolin (100 µM). All of the cannabinoids were added during both preincubation and incubation. To test for the involvement of G_i/o proteins, BV-2 cells were pretreated with 1 µg/ml pertussis toxin (Calbiochem, La Jolla, CA) for 18 hr. Replacing incubation buffer with ice-cold HEPES (20 mM in water) stopped the incubation. Suspensions were sonicated, boiled for 10 min, and centrifuged for 2 min at 10,000 x g. cAMP levels in supernatants were quantified using a radioimmunoassay Biotrak kit (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK).

Cell migration. BV-2 cells (5 x 10⁴ in 50 µl of MEM-CellGro) were added to the upper chamber and allowed to migrate through polycarbonate filters (pore size, 10 µm; NeuroProbe, Gaithersburg, MD) for 3 hr at 37°C (humidified atmosphere of 95% air and 5% CO₂). Cells that did not migrate and stayed on the upper surface of the filter were wiped off, whereas cells that had migrated to the undersurface were stained with Dif-quick (IMEB, San Marcos, CA) and manually counted at 32x magnification in random fields by three scorers blinded to the experimental conditions.

Proliferation and nitrite release quantification. BV-2 cells were rinsed once with MEM-CellGro at 37°C (500 µl/well) and incubated in MEM-CellGro containing cannabinoids and/or lipopolysaccharide (LPS) plus interferon- (IFN-) (Calbiochem, La Jolla, CA). After 18 hr, nitrites in supernatants were quantified by the Griess reaction [an index of nitric oxide (NO) production] (Granger et al., 1996), and proliferation/metabolic activity was measured with the WST-1 assay (Roche Molecular Biochemicals, Mannheim, Germany) (Tan and Berridge, 2000). Sensitivity of the Griess reaction extended to the low micromolar range of nitrite. In initial experiments, we verified that WST-1 cleavage was linear between 30 and 120 min when incubated with BV-2 cells at 200,000 cells/ml.

Phagocytosis of beads, fluorescence-activated cell sorter analysis, and confocal imaging. BV-2 cells were treated with cannabinoids or LPS/IFN. After 18 hr, medium was replaced with MEM-CellGro containing mouse IgG-opsonized fluorescent latex beads (Fluoresbrite YG carboxylate microspheres; diameter, 1 µm; Polysciences, Warrington, PA) at a final dilution of 100 beads/cell, and cells were maintained for 2 hr at 37°Cin humidified 5% CO₂-95% air. Cells were then washed twice with ice-cold PBS, detached with trypsin-EDTA (0.05%-0.53 mM), harvested, and centrifuged (5 min; 300 x g). Cells were then resuspended in MEM-CellGro and assayed with a Beckman Coulter (Fullerton, CA) flow cytometer such that fluorescence intensity was a measure of bead uptake. We defined the amount of phagocytosing cells as the number of events in region R1 expressed as a percentage of total events (see Fig. 4). The number of beads phagocytosed per cell was set at the geometric mean fluorescence intensity of region R1 (see Fig. 4). Z-section images were acquired with a Leica (Nussloch, Germany) TCS SP/NT confocal microscope (Keck Center, University of Washington).

View larger version (22K): [in this window] [in a new window]   Figure 4. LPS/IFN increases the ability of BV-2 cells to engulf particles. Cells were incubated with mouse IgG-opsonized beads (1 µm) for 2 hr and analyzed by FACS. Unbound beads were too small to be detected. Shown are representative dot plots of forward scatter versus fluorescence intensity for microglial cells incubated with beads at either 4°C (a) or 37°C (b). After analyzing the signal obtained at 4°C, we discounted >95% of the nonspecific signal by gating in R1. BV-2 cells were recovered from R1-FACS and plated onto coverslips, fixed, and labeled with phalloidin (red) to stain actin (c). Engulfed fluorescent latex beads appear yellow-green (scale bar, 50 µm). Inset shows higher magnification of one BV-2 cell that had engulfed eight beads, which was counterstained with 4',6'-diamidino-2-phenylindole (blue) to label the nucleus (scale bar, 20 µm). Fluorescence intensity histograms of BV-2 cells at 37°C pretreated for 18 hr with either vehicle (d) or IFN- (100 U) plus LPS (10 µg/ml) (e) and incubated with beads for 2 hr. Also shown is the cutoff for engulfing versus nonengulfing cells (dotted line).

 

	Results

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Focal cerebral ischemia increases PEA and AEA, but not 2-AG, in cerebral cortex
Several observations suggest that FCI should increase endocannabinoid levels in cerebral cortex: excitotoxicity and ischemia induced by decapitation increase endocannabinoids in whole brain (Schmid et al., 1995; Felder et al., 1996; Kempe et al., 1996; Hansen et al., 2001; Sugiura et al., 2001), and neuronal death induced by FCI is exacerbated when CB₁ receptors are either antagonized or knocked out (Nagayama et al., 1999; Parmentier-Batteur et al., 2002). To determine whether FCI increases endocannabinoid levels in cerebral cortex, we used CI-GC/MS to quantify AEA, 2-AG, HEA, DEA, and PEA in cerebral cortex from control, sham-surgery, and ischemic mouse brain.

In five control animals, the levels of AEA (50 ± 6 pmol/gm), 2-AG (20 ± 2 nmol/gm), and PEA (430 ± 24 pmol/gm) were within the range of previously reported measurements (Sugiura et al., 1996; Cadas et al., 1997; Stella et al., 1997; Di Marzo et al., 2000; Baker et al., 2001; Cravatt et al., 2001). Furthermore, we found that HEA (31 ± 9 pmol/gm) and DEA (48 ± 12 pmol/gm) were as abundant as AEA, which is consistent with these two acyl-EAs being considered endocannabinoids in brain.

In a first set of experiments, we assessed the effect of sham surgery per se on the levels of endocannabinoids in cerebral cortex, because this procedure has been shown to increase the levels of signaling molecules in brain (Barnum et al., 2002). Figure 1 shows that sham surgery induced a significant increase in 2-AG (43 ± 4 nmol/gm) and HEA (68 ± 8 pmol/gm). Whereas amounts of AEA (121 ± 15 pmol/gm) and DEA (89 ± 18 pmol/gm) also tended to increase, amounts of PEA (429 ± 28 pmol/gm) were not affected.

View larger version (23K): [in this window] [in a new window]   Figure 1. Endocannabinoid levels in cerebral cortex after focal cerebral ischemia. Mice underwent either sham surgery (Sham) or 20 min of FCI (Ischemia) or were directly decapitated (Control), as described in Materials and Methods. AEA, 2-AG, PEA, HEA, and DEA in cerebral cortex was quantified by CI-GC/MS. Values shown are the means ± SEMs of quantities measured in n = 5 animals per group. *p < 0.05 and **p < 0.01 compared with sham (ANOVA followed by Dunnett's post-test).

 

To assess the effect of FCI on endocannabinoid levels, left carotid arteries were occluded for 20 min. Twenty-four hours after FCI, the amount of PEA in ischemic cerebral cortex increased 25-fold compared with sham-operated animals, and DEA increased 4-fold. The amount of AEA increased by 3-fold compared with sham-operated animals, but this increase was not significant (Fig. 1). 2-AG levels remained unchanged compared with control (Fig. 1).

These results show that, although FCI does not increase the amount of 2-AG in cerebral cortex, it leads to a large increase in PEA and somewhat smaller increases in AEA and DEA.

Microglial cells express functional CB₁ and CB₂ receptors
Microglial cells express the CB₁ and CB₂ receptor proteins (Walter et al., 2003), suggesting that these cells can sense changes in endocannabinoid levels. CB₁ and CB₂ receptors couple to G_i/o-proteins and inhibit adenylyl cyclase activity (Vogel et al., 1993; Bayewitch et al., 1995; Slipetz et al., 1995). To ascertain whether CB₁ and CB₂ receptors expressed by BV-2 cells inhibit adenylyl cyclase activity, we tested the effects of synthetic cannabinoid agonists on the forskolin-stimulated accumulation of cAMP. CP-55940, a bicyclic cannabinoid agonist that acts with similar potency at both CB₁ and CB₂ receptors (Felder et al., 1995), inhibited the forskolin response by 40% (Fig. 2a). WIN55212-2, an aminoalkylindole cannabinoid agonist that has a higher potency at CB₂ than at CB₁ receptors (Felder et al., 1995), inhibited the forskolin response by 47%, whereas WIN55212-3, its inactive enantiomer, had no significant effect (Table 1). Both the CB₁ receptor antagonist SR141716A (Rinaldi-Carmona et al., 1994) and the CB₂ receptor antagonist SR144528 (Rinaldi-Carmona et al., 1998) antagonized the inhibitory effect of CP-55940 on the forskolin response (Fig. 2a). Together, these results show that, in BV-2 cells, CB₁ and CB₂ receptors are functionally coupled to adenylyl cyclase inhibition.

View larger version (22K): [in this window] [in a new window]   Figure 2. PEA and synthetic cannabinoids inhibit the forskolin-stimulated accumulation of cAMP in BV-2 cells. BV-2 cells were preincubated for 10 min with IBMX (1 mM) and either vehicle (0.1% DMSO), CP-55940 (1 µM), or PEA (0.3 µM). Cells were then incubated for an additional 10 min with the same agents plus forskolin (FSK) (100 µM). During both preincubation and incubation, SR141716A (SR1) (0.3 µM), SR144528 (SR2) (0.3 µM), capsazepine (CPZ) (3 µM), or O-1918 (1 µM) was present. To test for Gi/o-protein involvement, cells were pretreated for 18 hr with 1 µg/ml pertussis toxin (PTX). Results are means ± SEMs of 9-45 independent quantifications from 3 to 15 separate experiments performed in triplicate. *p < 0.05 and **p < 0.01 compared with FSK alone (ANOVA followed by Dunnett's post-test). Basal levels of cAMP were 24.7 ± 3.5 fmol/well, which increased to 87.3 ± 9.8 fmol/well in the presence of FSK (n = 54). Horizontal dotted lines correspond to 100% forskolin response.

 

View this table: [in this window] [in a new window]   Table 1. Effect of synthetic cannabinoids, endocannabinoids, and analogs on forskolin-stimulated accumulation of cAMP in BV-2 cells

 

PEA acts on microglial cells through G_i/o-protein-coupled receptors
Because the level of PEA in cerebral cortex is dramatically increased 24 hr after FCI (Fig. 1), a time corresponding to pronounced neuroinflammation (Dirnagl et al., 1999; Iadecola and Alexander, 2001), we investigated whether microglial cells could sense this lipid. To address this question, we determined whether PEA inhibits the forskolin-stimulated accumulation of cAMP in BV-2. We chose this approach because PEA has been shown to produce CB₂-like responses (Calignano et al., 1998), and CB₂ receptors are G_i/o-protein-coupled receptors that inhibit adenylyl cyclase activity (Bayewitch et al., 1995; Slipetz et al., 1995).

Figure 2 shows that, indeed, PEA inhibited the forskolin-stimulated accumulation of cAMP in BV-2 cells. This response had an IC₅₀ of 6.4 nM and reached a maximal 40% inhibitory effect at 1 µM (Fig. 2b, Table 1). Whereas the PEA response was prevented by pertussis toxin (Fig. 2c), it was not affected by SR141716A or SR144528 (a), suggesting that PEA inhibits adenylyl cyclase activity through a mechanism independent of CB₁ and CB₂ receptors.

Two arguments suggest that PEA does not produce its effect by interacting with WIN receptors. Capsaicin, a vanilloid compound known to activate WIN receptors (Hájos and Freund, 2002), did not reproduce the PEA response (Table 1), and the PEA response was not antagonized by capsazepine (3 µM), which antagonizes both VR1 and WIN receptors (Hájos and Freund, 2002) (Fig. 2c). Two arguments suggest that PEA does not produce its effect by interacting with abn-CBD receptors. Abnormal cannabidiol, a synthetic agonist known to activate abn-CBD receptors (Járai et al., 1999), did not reproduce the PEA response (Table 1), and the PEA response was not antagonized by O-1918 (1 µM), an antagonist at abn-CBD receptors (Offertáler et al., 2003) (Fig. 2c).

Because PEA is effectively taken up by cells (Bisogno et al., 1997) and is a rather good substrate for fatty acid amide hydrolase (Natarajan et al., 1984), we tested whether the effect of PEA could be attributable to a metabolite formed during the 10 min preincubation plus 10 min incubation phase of the assay. However, palmitic acid, a product of PEA hydolysis, did not affect the forskolin-stimulated accumulation of cAMP in BV-2 cells (Table 1), thus ruling out the involvement of a PEA metabolite in this response.

We then assessed the structural requirements of PEA-induced inhibition. PEA contains a saturated, 16 carbon moiety (16:0) linked to ethanolamine through an amide bond. To determine whether the ability of PEA to inhibit adenylyl cyclase depends on its 16 carbon moiety, we treated BV-2 cells with the following acyl-EA analogs: myristoylethanolamide (14:0), pentadecanoylethanolamide (15:0), palmitoleoylethanolamide (16:1), margaroylethanolamide (17:0), and stearoylethanolamide (SEA) (18:0), and measured the forskolin-stimulated cAMP accumulation. Whereas margaroylethanolamide (17:0) had a nonsignificant trend to inhibit the forskolin response, none of these other analogs affected the forskolin response (Table 1). To determine whether the ability of PEA to inhibit adenylyl cyclase depends on its head group, we treated BV-2 cells with palmitoylisopropylamide, R-palmitoyl-(1-methyl)ethanolamide, and R-palmitoyl-(2-methyl)ethanolamide and measured the forskolin-stimulated cAMP accumulation. None of these analogs inhibited the forskolin response (Table 1).

Together, these results show that, in BV-2 cells, PEA inhibits adenylyl cyclase activity with high potency and in a manner dependent on its precise chemical structure. The effect of PEA occurs through G_i/o-protein-coupled receptors that are pharmacologically distinct from CB₁, CB₂, WIN, and abn-CBD receptors.

PEA selectively potentiates AEA-induced microglial cell migration
Increased microglial cell motility (i.e., one of the first steps of microglial cell activation) is essential for neuroinflammation propagation (Dirnagl et al., 1999; Yrjünheikki et al., 1999). Because PEA acts on G_i/o-protein-coupled receptors (Fig. 2), and many G_i/o-protein-coupled receptors, including CB₁ and CB₂ receptors, modulate cell motility (Derocq et al., 2000; Song and Zhong, 2000), we assessed whether PEA affects BV-2 cell motility. To address this possibility, we used the Boyden chamber assay in which BV-2 cells were added to the upper chamber and their migration through a filter toward a lower chamber containing PEA was quantified (Wilkinson, 1998). Over a range of 1 nM to 3 µM, PEA does not affect basal BV-2 cell migration (Walter et al., 2003).

Because AEA induces BV-2 cell migration (Walter et al., 2003), and synergistic interactions between AEA and PEA have been described previously (Calignano et al., 1998), we tested whether PEA modulates AEA-induced BV-2 cell migration. PEA potentiated BV-2 cell migration induced by AEA, whereas it had no effect on BV-2 cell migration induced by 2-AG (Fig. 3). Note that BV-2 cell migration had not reached its ceiling point, because 2-AG at 1 µM is able to increase migration up to 225% of basal (Walter et al., 2003). Myristoylethanolamide, pentadecanoylethanolamide, palmitoleoylethanolamide, margaroylethanolamide, or SEA did not mimic the effect of PEA (data not shown).

View larger version (30K): [in this window] [in a new window]   Figure 3. PEA selectively potentiates the anandamide-induced BV-2 cell migration. Control (0.1% DMSO), PEA (300 nM), db-cAMP (1 mM), or PEA plus db-cAMP were added to the lower compartment of the Boyden chamber in the absence (Basal) or presence of AEA (100 nM) or 2-AG (100 nM). BV-2 cell migration toward these ligands was quantified, and results are expressed as a percentage of basal/control BV-2 cell migration. Results are means ± SEMs of 9-45 independent quantifications from 3 to 15 separate experiments performed in triplicate. *p < 0.05 and **p < 0.01 compared with basal/control migration (ANOVA followed by Dunnett's post-test). The dotted horizontal line corresponds to control basal migration.

 

To determine the molecular mechanism underlying the specific potentiating effect of PEA on AEA-induced migration, we assessed whether BV-2 cell migration induced by AEA and 2-AG could be mechanistically differentiated. Studies using cells transfected with cannabinoid receptors have suggested that activation of MAP kinase—rather than inhibition of adenylyl cyclase—mediates cell migration (Derocq et al., 2000; Song and Zhong, 2000). Differential involvement of MAP kinase activity could be ruled out, because both AEA and 2-AG increased MAP kinase activity to a similar extent (i.e., by approximately threefold) (data not shown) (Walter et al., 2003). Unexpectedly, BV-2 cell migration induced by PEA plus AEA, PEA plus 2-AG, and 2-AG were overridden by the addition of dibutyryl-cAMP (db-cAMP) (Fig. 3). One possibility that arises from this latter result is that inhibition of adenylyl cyclase activity is required to significantly increase BV-2 cell migration. Whereas PEA and 2-AG inhibited the forskolin-stimulated adenylyl cyclase activity to a similar extent, AEA was not effective (Table 1).

These results show that PEA potentiates BV-2 cell migration induced by AEA, without affecting the 2-AG-induced migration. The mechanism underlying this selective effect of PEA on AEA is likely attributable to the fact that increased BV-2 cell migration requires both stimulation of MAP kinase and reduced cAMP levels. Whereas 2-AG both stimulates MAP kinase and reduces cAMP levels, AEA only stimulates MAP kinase. Therefore, PEA is likely to provide the adenylyl cyclase inhibition required to increase the migration of BV-2 cells.

Endocannabinoids do not affect the ability of microglial cells to proliferate, engulf particles, and produce nitric oxide
Although it is clear that microglial cell activation encompasses distinct steps (Bruce-Keller, 1999), it is unclear whether individual activating signals initiate the entire process or only specific steps of microglial cell activation. Thus, we assessed whether endocannabinoids initiate or modulate distinct steps of microglial cell activation, namely, their ability to proliferate, engulf particles, and produce nitric oxide.

To assess whether endocannabinoids or synthetic cannabinoids affect the proliferation rate of BV-2 cells, we preincubated cells with these agents and measured WST-1 conversion (Tan and Berridge, 2000). We used LPS/IFN as positive controls, for these components are known to activate microglial cells (Waksman et al., 1999). Whereas LPS/IFN induced a 37% increase in BV-2 cell proliferation, none of the cannabinoids affected the proliferation rate of BV-2 cells (Table 2).

View this table: [in this window] [in a new window]   Table 2. Cannabinoids do not affect the ability of microglial cells to proliferate, engulf particles, and release nitrites

 

During neuroinflammation, the ability of microglial cells to engulf particles is pivotal, because it promotes cell death (Conradt, 2002) and eliminates ensuing cellular debris (Witting et al., 2000). Because some G_i/o-protein-coupled receptors modulate this function (Lipovsky et al., 1998), we sought to determine whether cannabinoids modulate the ability of BV-2 cells to engulf fluorescent latex beads opsonized with mouse IgG. Using fluorescence-activated cell sorter (FACS) analysis, we saw that, after 2 hr, 58 ± 5% of BV-2 cells had engulfed microspheres (Fig. 4a,b), with each cell engulfing approximately eight microspheres (c). Figure 4, d and e, shows a representative experiment in which LPS/IFN increased the number of BV-2 cells that engulfed particles by 20%. LPS/IFN did not significantly change the number of particles engulfed by each cell (data not shown). Cannabinoids did not affect the number of BV-2 cells that engulfed beads (Table 2), nor did these agents affect the number of beads engulfed by each cell (data not shown).

Activated microglial cells often release a large amount of NO, which leads to the killing of surrounding cells (Chao et al., 1992). Cannabinoids have been reported to increase NO production from microglial cells (Stefano et al., 1996) and to decrease NO production from LPS/IFN-activated microglial cells (Waksman et al., 1999). We revisited this question by treating BV-2 cells with cannabinoids (either alone or in combination with LPS/IFN), and determining nitrite release after 18 hr. Cannabinoids did not

affect the basal release of nitrite (Table 2), nor did they modulate the LPS/IFN-induced production of nitrite (data not shown).

Together, these results show that, although endocannabinoids modulate microglial cell migration (Fig. 3), they do not affect other steps of microglial cell activation, namely, their proliferation rate, particle engulfment, and NO production.

	Discussion

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The signals that follow FCI and mediate the propagation of neuroinflammation within the penumbra are not fully understood. Here, we show that FCI induces a large increase in PEA in cerebral cortex and a somewhat smaller increase in other acyl-EAs. We also show that PEA acts on microglial cells through G_i/o-coupled receptors that are pharmacologically distinct from CB₁, CB₂, WIN, and abn-CBD receptors, and potentiate AEA-induced cell migration. Thus, our results suggest that PEA and AEA signal microglial cells to increase their motility and thus could contribute to the propagation of neuroinflammation in the CNS.

Traumatic brain injury increases 2-AG levels in mouse brain (Panikashvili et al., 2001; Mechoulam et al., 2002). Here, we show that FCI does not affect 2-AG levels in mouse cerebral cortex. In considering that 2-AG levels in cerebral cortex doubled in sham-operated animals, our results suggest that FCI might actually decrease 2-AG production. Thus, changes in endocannabinoid production appear specific to different neuropathological conditions.

Does a common biosynthetic pathway produce all of the acyl-EAs (Piomelli et al., 2000; Schmid et al., 2000)? Several studies show that specific physiological stimuli or pathological conditions increase the production of individual acyl-EAs (Stella et al., 1997; Giuffrida et al., 1999; Berdyshev et al., 2000; Walter et al., 2002, 2003). We found that (1) sham surgery increases HEA production, whereas it does not affect PEA production, and (2) FCI greatly increases PEA production, whereas it does not affect HEA production (Fig. 1). Consequently, our results reinforce the notion that different biosynthetic pathways produce individual acyl-EAs. Along these lines, a recent study showed that cerebral infarction in humans also induces differential increases in acyl-EAs (Schübitz et al., 2002).

To our knowledge, this is the first report describing the ability of PEA to specifically and potently inhibit adenylyl cyclase activity in cells (for negative results, see Felder et al., 1993; Ross et al., 2000). This effect does not involve CB₁ and CB₂ receptors, because SR141716A or SR144528 does not antagonize it. It does not involve WIN and VR1 receptors, because capsaicin does not reproduce it, nor does capsazepine antagonize it. It does not involve abn-CBD receptors, because abn-CBD does not reproduce it, nor does O-1918 antagonize it. It should be emphasized that, whereas micromolar concentrations of SR141716A antagonize WIN and abn-CBD receptors (Járai et al., 1999; Hájos et al., 2001), the PEA-induced inhibition of the forskolin-stimulated cAMP production is not affected by 300 nM SR141716A (Fig. 2). The effect of PEA does not occur through the binding site described for SEA, a higher PEA homolog, found in C6, because SEA did not inhibit adenylyl cyclase activity in the BV-2 cell (Table 1), and PEA does not compete SEA off its described binding site (Maccarrone et al., 2002). Through what receptors, then, does PEA inhibit adenylyl cyclase? We propose that PEA is likely to act through its own receptor. The definite proof for the existence of PEA receptors will come only from their molecular cloning. Note that our results challenge the notion that PEA is a bona fide endocannabinoid. In fact, only experiments demonstrating that PEA receptors are engaged by plant-derived cannabinoids will establish that PEA is indeed an endocannabinoid.

After FCI, is the concentration of PEA sufficient for it to activate its receptors? At this point, this question remains open, primarily because of the issue regarding the extracellular concentration reached by PEA and its compartmentalization. However, it has been shown that PEA increases to 120 nM in microdialysates from humans with stroke, providing supportive evidence that the extracellular concentration of PEA can achieve biologically active levels (Schübitz et al., 2002).

We provide data that suggest a molecular mechanism underlying the specific PEA-induced potentiation of the AEA-induced microglial cell migration. Our results show that increases in BV-2 cell migration also require reduction in cAMP levels. We propose that the potentiating effect of PEA on the AEA-induced BV-2 cell migration is attributable to the ability of PEA to inhibit adenylyl cyclase activity. This is supported by the fact that 2-AG at 100 nM inhibits adenylyl cyclase activity and induces migration, whereas AEA does not. In line with this, G_i/o-protein-coupled receptors increase cell migration by activating the MAP kinase pathway (Klemke et al., 1997). Interestingly, BV-2 cell migration can be further increased by simply increasing 2-AG concentration up to 1 µM (Walter et al., 2003); however, at this concentration, 2-AG does not inhibit adenylyl cyclase, indicating that this endocannabinoid induces a bell-shaped curve inhibition of adenylyl cyclase activity and yet still strongly activates the MAP kinase pathway (data not shown) (Walter et al., 2003). The signal transduction mediating the 1 µM 2-AG-increased BV-2 cell migration is unknown. The effect of PEA cannot be accounted for by the ability of PEA to prevent AEA metabolism, because SEA and myristoylethanolamide inhibit AEA metabolism (Jonsson et al., 2001) but do not potentiate the AEA-induced migration. The synergy described in our study is also different from that seen for the effect of PEA on AEA activation of vanilloid receptors (De Petrocellis et al., 2001), because the latter is also found for SEA and other acylethanolamides (Smart et al., 2002). Furthermore, our results are also different from the study performed in cells transfected with cannabinoid receptors, because in these models, migration is not sensitive to db-cAMP (Bouaboula et al., 1995, 1996; Derocq et al., 2000; Song and Zhong, 2000).

PEA potentiates AEA-induced microglial cell migration without affecting proliferation, particle engulfment, or NO production. This result suggests that PEA receptors couple to signal transduction pathways that specifically modulate cell motility. This modality-specific initiation of microglial cell activation by endocannabinoids contradicts the notion that all of the activating stimuli lead to the same, stereotypical pattern of microglial cell activation.

In summary, we show that PEA and AEA amounts increase after FCI, which possibly signal microglial cells to increase their mobility. Increase in AEA levels may also in turn act on (1) presynaptic CB₁ receptors to reduce excessive release of glutamate and allied excitotoxicity (Shen et al., 1996) and (2) CB₁ receptors expressed by blood vessels to enhance cerebral blood flow (Parmentier-Batteur et al., 2002). We propose that the molecular identification of PEA receptors and a better understanding of their pharmacology should help in developing therapies against the propagation of neuroinflammation, possibly without altering the beneficial effects produced by endocannabinoids that act on CB₁ receptors.

	Footnotes

 
Received May 2, 2003; revised June 26, 2003; accepted July 1, 2003.

We are grateful to the National Institutes of Health (DA14486 to N.S. and NS39912 to D.A.G.) and the National Institute of General Medical Sciences (GM07270 to L.W.) for grant support and to Sanofi Research for providing us with SR141716A and SR144528.

Correspondence should be addressed to Dr. Nephi Stella, Department of Pharmacology, University of Washington, 1959 Northeast Pacific Street, Seattle, WA 98195-7280. E-mail: nstella{at}u.washington.edu.

Copyright © 2003 Society for Neuroscience 0270-6474/03/237767-09$15.00/0

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