P2X receptors-mediated cytosolic phospholipase A 2 activation in primary afferent sensory neurons contributes to neuropathic pain

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1 Journal of Neurochemistry, 2007, 103, doi: /j x P2X receptors-mediated cytosolic phospholipase A 2 activation in primary afferent sensory neurons contributes to neuropathic pain Makoto Tsuda, 1 Shigeo Hasegawa 1 and Kazuhide Inoue Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi, Fukuoka, Japan Abstract Activation of P2X 3 and P2X 2/3 receptors (P2X 3 R/P2X 2/3 R), ionotropic ATP receptor subtypes, in primary sensory neurons is involved in neuropathic pain, a debilitating chronic pain that occurs after peripheral nerve injury. However, the underlying mechanisms remain unknown. We investigated the role of cytosolic phospholipase A 2 (cpla 2 ) as a downstream molecule that mediates the P2X 3 R/P2X 2/3 R-dependent neuropathic pain. We found that applying ATP to cultured dorsal root ganglion (DRG) neurons increased the level of Ser505-phosphorylated cpla 2 and caused translocation of Ser505-phosphorylated cpla 2 to the plasma membrane. The ATP-induced cpla 2 activation was inhibited by a selective antagonist of P2X 3 R/P2X 2/3 R and by a selective inhibitor of cpla 2. In the DRG in vivo, the number of cpla 2 -activated neurons was strikingly increased after peripheral nerve injury but not after peripheral inflammation produced by complete Freund s adjuvant. Pharmacological blockade of P2X 3 R/P2X 2/3 R reversed the nerve injury-induced cpla 2 activation in DRG neurons. Moreover, administering the cpla 2 inhibitor near the DRG suppressed nerve injury-induced tactile allodynia, a hallmark of neuropathic pain. Our results suggest that P2X 3 R/P2X 2/3 R-dependent cpla 2 activity in primary sensory neurons is a key event in neuropathic pain and that cpla 2 might be a potential target for treating neuropathic pain. Keywords: cytosolic phospholipase A 2, neuropathic pain, P2X receptor, primary afferent sensory neurons. J. Neurochem. (2007) 103, A growing body of evidence has indicated that P2X receptors, a family of ligand-gated cation channels activated by extracellular ATP, play an important role in generating and modulating pain signaling (Burnstock 2006). Molecular cloning has so far identified seven genes encoding P2X receptor subunits (P2X 1 R P2X 7 R) (Ralevic and Burnstock 1998). In primary afferent sensory neurons, mrna or protein of almost all P2XRs has been detected (Collo et al. 1996; Kobayashi et al. 2005). However, electrophysiological studies investigating P2XR-mediated ATP responses indicated that homomeric P2X 3 R and heteromeric P2X 2/3 R might be the predominant functional P2X receptors in these cells (Rae et al. 1998; Ueno et al. 1999). Studies using mice lacking P2X 3 R or both P2X 2 R and P2X 3 R clearly corroborated these findings (Cockayne et al. 2000, 2005; Souslova et al. 2000). Various attempts to block P2XRs pharmacologically (Jarvis et al. 2001; McGaraughty et al. 2003) or to suppress their expression molecularly (Honore et al. 2002) and genetically (Cockayne et al. 2000; Souslova et al. 2000) did not affect the basal responses to physiological pain (e.g., noxious heat or mechanical stimuli), but rather preferentially suppressed neuropathic pain in response to peripheral nerve injury. Notably, acute systemic administration of the recently developed selective antagonist for P2X 3 and P2X 2/3 R (P2X 3 R/P2X 2/3 R) A , reversed nerve injury-induced abnormal hypersensitivity to innocuous stimuli (tactile allodynia), which is a hallmark of neuropathic pain syndrome (Jarvis et al. 2002; McGaraughty et al. 2005). As A poorly penetrates into the CNS (Wu et al. 2004; Sharp et al. 2006), these observations suggest a prominent role of peripheral P2X 3 R/P2X 2/3 R in primary sensory neurons for tactile allodynia caused by peripheral nerve injury. However, how P2X 3 R/P2X 2/3 R participates in neuropathic pain has remained unknown. Damaging primary sensory neurons causes ongoing discharges originating at the injured site, which are considered to contribute to neuropathic pain (Chen et al. 1999, 2001; Zhou et al. 2001). Applying ATP excites damaged neurons, however, the spontaneous ongoing discharges in damaged dorsal root ganglion (DRG) neurons are not suppressed by P2XR antagonists (Chen et al. 2001; Zhou et al. 2001), thus the role of the ATP-mediated direct neuronal excitation has remained unclear. As P2X 3 R/ Received April 6, 2007; revised manuscript received June 7, 2007; accepted July 2, Address correspondence and reprint requests to Kazuhide Inoue, Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi, Higashi, Fukuoka , Japan. inoue@phar.kyushu-u.ac.jp 1 These authors contributed equally to this work. Abbreviations used: CFA, complete Freund s adjuvant; cpla 2, cytosolic phosholipase A 2 ; cpla 2 -i, [N-{(2S,4R)-4-(Biphenyl-2-yl- methyl-isobutyl-amino)-1-[2-(2,4-difluorobenzoyl)-benzoyl]-pyrrolidin-2- ylmethyl}-3-[4-(2,4-dioxothiazolidin-5-ylidinemethyl)-phenyl]-acrylamide; DRG, dorsal root ganglion; IgG, immunoglobulin G; IR, immunoreactivity; P2X 3 R/P2X 2/3 R, P2X 3 and P2X 2/3 receptors; p-cpla 2, Ser505-phosphorylated cpla

2 Cytosolic phospholipase A 2 activation via P2X in neuropathic pain 1409 P2X 2/3 R are permeable to Ca 2+ as well as to Na + and K + (Ueno et al. 1998; Khakh et al. 2001; North 2002), we considered the possibility that stimulating P2X 3 R/P2X 2/3 R may lead to activation of Ca 2+ -dependent biochemical cascades in primary sensory neurons. In the present study, we investigated this possibility by focusing on the role of the Ca 2+ -dependent subclass of the PLA 2 family, namely cytosolic phospholipase A 2 (cpla 2 )/ group IVA PLA 2 (Shimizu et al. 2006). cpla 2 is a crucial enzyme for arachidonic acid and lysophospholipids release and the subsequent generation of phospholipid mediators such as prostaglandins and platelet activating factor (Spencer et al. 1998; Hirabayashi and Shimizu 2000). These mediators have been reported to cause sensitization of primary sensory neurons (Park and Vasko 2005) and to produce allodynic behaviors (Minami et al. 1992, 1994; Morita et al. 2004). It is shown here for the first time that nerve injury induces the activation of cpla 2 in primary afferent sensory neurons by P2X 3 R/P2X 2/3 R stimulation. Moreover, cpla 2 activation significantly contributes to nerve injury-induced tactile allodynia. Therefore, the present study suggests neuronal cpla 2 activation as a key intermediary event of P2X 3 R/P2X 2/3 R- dependent neuropathic pain after peripheral nerve injury. Materials and methods Animals Male Wistar rats ( g) were used. Animals were housed at a temperature of 22 ± 1 C with a 12-h light-dark cycle (light on 8:30 to 20:30), and fed food and water ad libitum. All of the animals used in the present study were obtained, housed, cared for and used in accordance with the guidelines of Kyushu University. Culture of rat primary DRG neurons The lumber DRGs (L1 6 segments) were removed from male Wistar rats and were treated in Dulbecco s modified eagle medium with 20 U/mL papain and 2 mg/ml collagenase type II for 1 h at 37 C. At the end of this treatment the enzyme solution was removed and the DRGs were mechanically dissociated by trituration through a Pasteur pipette in Dulbecco s modified eagle medium. They were suspended in F-12 Nutrient Mixture liquid supplemented with 10% horse serum, 2 mmol/l glutamine, 100 units/ml penicillin, 100 lg/ml streptomycin, 100 ng/ml nerve growth factor (Sigma, St Louis, MO, USA) and 100 ng/ml human glial cell-line derived neurotrophic factor (Promega, Madison, WI, USA). They were plated in 35 mm-diameter tissue culture dishes for western blotting or slide glasses coated with 100 lg/ml poly-l-lysine and 10 lg/ml laminin for immunocytochemistry and maintained in an atmosphere of 5% CO 2 /95% ambient air at 37 C for 72 h. Following incubation, the medium was removed, replaced with fresh medium without horse serum, nerve growth factor and human glial cell-line derived neurotrophic factor, and further cultured at 37 C for an additional 24 h. Reagents were added to DRG neuron cultures and incubated for the indicated time. After these treatments, the medium was removed and the cultures were scraped into ice-cold 125 mmol/l Tris-HCl (ph 7.4) containing 20% glycerol, 4% (w/v) sodium dodecyl sulfate, 0.025% (w/v) bromophenol blue and 5% 2-mercaptoethanol. Protein samples were subjected to polyacrylamide gel electrophoresis and transferred electrophoretically to polyvinylidine difluoride membranes. After blocking, the membranes were incubated with anti-phospho-cpla 2 (anti-p-cpla 2 ) antibody (1 : 1000, Cell Signaling, Beverly, MA, USA) or anti-cpla 2 antibody (1 : 1000, Cell Signaling) and then were incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) antibody (1 : 1000, Amersham Biosciences, Buckinghamshire, UK). The blots were detected using a chemiluminescence method (ECL system; Amersham Biosciences). Immunocytochemistry was performed as follows. Immediately after treatment with ATP for 5 min, cells were fixed with 3.7% formaldehyde. After blocking, neurons were incubated with anti-pcpla 2 antibody (1 : 1000) and then were incubated with anti-rabbit IgG-conjugated Alexa Fluor 488 (1 : 1000, Molecular Probes, Eugene, OR, USA), followed by analysis with an LSM510 Imaging System (Zeiss, Oberkochen, Germany). To distinguish among cell size-specific changes, DRG neurons were characterized as small (< 25 lm), medium (25 35 lm), and large (> 35 lm)-sized neurons according to their diameters (Ueno et al. 1999). Neuropathic and inflammatory pain We used the spinal nerve injury model (Kim and Chung 1992) with some modifications (Tsuda et al. 2003): in male Wistar rats a unilateral L5 spinal nerve was tightly ligated and cut just distal to the ligature. For an inflammation pain model, complete Freund s adjuvant (CFA) (0.1 mg/200 ll, Sigma) was injected into the plantar surface of the left hindpaw using a 26-gauge needle. The mechanical allodynia was assessed by using calibrated von Frey filaments ( g) and the paw withdrawal threshold was determined as described previously (Tsuda et al. 2003). Immunohistochemistry Rats were deeply anesthetized by pentobarbital (100 mg/kg, i.p.) and perfused transcardially with 4% paraformaldehyde. DRG sections were removed, post-fixed with the same fixative, and placed in 30% sucrose solution for 24 h at 4 C. The DRG sections (15 lm) were incubated in a blocking solution [3% normal goat serum/0.3% Triton X-100/phosphate-buffered saline(-)] and then with anti-p-cpla 2 antibody (1 : 1000, Cell Signaling). Identification of the type of p-cpla 2 -translocated cells was performed with the following markers: for satellite glia/astrocytes, glial fibrillary acidic protein (1 : 500, Chemicon, Temecula, CA, USA); for neurons, neuronal nuclei (1 : 200, Chemicon) and microtubuleassociated protein 2 (1 : 1000, Chemicon). Neurofilament 200 (1 : 400, Sigma), a marker for myelinated A-fibers, was also used as the primary antibody. Following incubation, the DRG sections were incubated with secondary antibodies (anti-rabbit IgG-conjugated Alexa Fluor 488 or anti-mouse IgG-conjugated Alexa Fluor 546, 1 : 1000, Molecular Probes). The sections were then analyzed by a confocal microscope (LSM510, Zeiss). The number of p-cpla 2 - immunoreactive DRG neurons with translocation was counted in the L5 DRG ipsilateral to the nerve injury. The proportion of the p-cpla 2 -translocated neurons to the total number of DRG neurons was determined in each rat at each time point. For the size-

3 1410 M. Tsuda et al. distribution histogram data, measurement of the cross-sectional areas of p-cpla 2 -translocated neurons was made by using an LSM Image Browser (Zeiss) and only neurons with clearly visible nuclei were used for the quantification. To distinguish among cell sizespecific changes, DRG neurons were characterized as small (< 600 lm 2 ), medium ( lm 2 ), and large (> 1200 lm 2 )- sized neurons according to their cross-sectional areas (Obata et al. 2004). Drug treatment Rats were implanted with catheters for intrathecal injection according to the method described previously (Yaksh et al. 1980). Under isoflurane anesthesia, a sterile 32 gauge intrathecal catheter (ReCathCo) was inserted through the atlanto-occipital membrane and to the L4 or L5 DRG and externalized through the skin (Ji et al. 2002). After the experiments, we confirmed that the tip of the catheter was positioned near the L5 DRG. Rats were injected intrathecally with [N-{(2S,4R)-4-(Biphenyl-2-ylmethyl-isobutyl-amino)-1-[2-(2,4-difluorobenzoyl)-benzoyl]-pyrrolidin-2-ylmethyl}-3-[4-(2,4-dioxothiazolidin-5-ylidinemethyl)-phenyl]-acrylamide (cpla 2 -i, 2.5 nmol/ 10 ll, Calbiochem, San Diego, CA, USA) using a 25 ll Hamilton syringe with a 30-gauge needle once a day from day 0 (just before the nerve injury) to day 13. The paw withdrawal threshold was tested h after the injection of cpla 2 -i at 1, 3, 7 and 14 days post-injury. After the test on day 14, to examine the level of p-cpla 2 in injured DRG neurons in cpla 2 -i- and vehicle-treated groups using immunohistochemistry, the L5 DRG ipsilateral to the nerve injury was removed. For the experiment in which the effect of a single administration of cpla 2 -i on the established allodynia was examined on day 7 after nerve injury, behavioral test was performed immediately before and 45 min after the injection of cpla 2 -i (2.5 nmol/10 ll). A (100 lmol/kg, Sigma) was injected intravenously with a 26-gauge needle 14 days after the nerve injury. To examine the level and translocation of p-cpla 2 in the injured DRG neurons in the A and vehicle-treated groups, the rats were fixed 90 min after injection, and the L5 DRG ipsilateral to the nerve injury was removed and used for immunohistochemistry. Statistical analysis Statistical analyses of the results were made with Student s t test, Student s paired t test or the Mann Whitney U test. Results P2X 3 R/P2X 2/3 R-dependent activation of cpla 2 in cultured primary sensory neurons Full activation of cpla 2 requires two events: phosphorylation of serine residues (including serine505) and translocation of cpla 2 for accessing membrane phospholipids (Hirabayashi and Shimizu 2000). To investigate cpla 2 activation in DRG neurons, we applied ATP to these cells and assessed the level of cpla 2 phosphorylation by western blot analyses using an antibody that recognized Ser505- phosphorylated cpla 2 (p-cpla 2 ) (Su et al. 2004). We found that ATP (10 and 50 lmol/l) caused an increase in the level of p-cpla 2, with maximal effect at 10 lmol/l ATP (Fig. 1a). The bands corresponding to cpla 2 and p-cpla 2 were detected at about 110 kda, which were identical to their molecular weight (Clark et al. 1990; Nemenoff et al. 1993). The level of p-cpla 2 began to increase 3 min after the application of ATP and peaked at 5 min (Fig. 1b). The ATP-induced cpla 2 phosphorylation was prevented by A , a potent and selective antagonist for P2X 3 R/ P2X 2/3 R (Jarvis et al. 2002) (Fig. 1c) as well as by suramin, PPADS and TNP-ATP (data not illustrated). Applying a,bmethylene ATP, an agonist of P2X 1 - and P2X 3 -containing P2XRs (North and Surprenant 2000), also increased p-cpla 2 levels (Fig. 1d). When DRG neurons were pre-treated with the Ca 2+ chelator EGTA (5 and 10 mmol/l) prior to applying ATP, the ATP-induced cpla 2 phosphorylation was significantly inhibited (Fig. 1e). The increase of p-cpla 2 by ATP (Fig. 1g) and by a,b-methylene ATP (data not illustrated) was abolished by cpla 2 -i, a selective inhibitor for cpla 2 (Seno et al. 2000), showing the specificity of the used antibody. Immuocytochemical studies corroborated the western blotting results. Applying ATP caused an increase in p-cpla 2 -immunoreactivity (IR) at the vicinity of the plasma membrane of DRG neurons (Fig. 1f). The cell-size of p-cpla 2 -translocated DRG neurons ranged from small to large (small: 47/90, medium: 86/108 and large: 8/8). These results thus indicate that ATP activates cpla 2 via P2X 3 R/ P2X 2/3 R in cultured DRG neurons. Peripheral nerve injury induces cpla 2 activation in primary sensory neurons in vivo Recent evidence has suggested that P2X 3 R/P2X 2/3 R in DRG neurons play an important role in neuropathic pain (Tsuda et al. 2000; Barclay et al. 2002; Honore et al. 2002; Jarvis et al. 2002), predicting the activation of cpla 2 in DRG neurons under this condition. To determine the possible involvement of cpla 2 in DRG neurons, we first performed immunofluorescent analysis in DRG of rats that had been unilaterally injured in the fifth lumbar (L5) spinal nerve, a model of neuropathic pain (Kim and Chung 1992). Injury to the L5 nerve caused an increase of p-cpla 2 -IR in ipsilateral L5 DRG neurons (Fig. 2a). On the subcellular level, p- cpla 2 -IR did not accumulate around the nuclei (immunostained with the neuronal nuclear marker, neuronal nuclei) (Fig. 2b d), but rather accumulated at the edges of the area immunostained with the neuronal marker microtubule-associated protein 2 (Fig. 2e g). Accumulated p-cpla 2 -IR was never observed in glial fibrillary acidic protein-positive satellite glia/astrocytes (Fig. 2h j) in the contralateral L5 DRG (Fig. 2a) or either side of the non-injured L4 DRG (data not illustrated). In agreement with our in vitro data, cpla 2 -i treatment (see below) markedly decreased p-cpla 2 - IR, indicating again the specificity of p-cpla 2 -IR shown here. The intraplantar injection of CFA is a model of

4 Cytosolic phospholipase A 2 activation via P2X in neuropathic pain 1411 (a) ATP (µmol/l) for 5 min (b) (c) (µmol/l) ATP (µmol/l) for 5 min (µmol/l) (d) (e) (f) (µmol/l) (mmol/l) (g) (µmol/l) (µmol/l) (mmol/l) Fig. 1 Cytosolic phosholipase A 2 (cpla 2 ) is activated via stimulating P2X 3 and P2X 2/3 receptors in rat primary dorsal root ganglion (DRG) neurons. (a g) Western blot (a e, g) and immunocytochemical (f) analyses of Ser505-phosphorylated cpla 2 (p-cpla 2 ) protein after applying ATP or its analog to rat primary DRG neurons. The primary DRG culture was incubated with ATP (1 50 lmol/l) or a,b-methylene ATP (abmeatp; 100 lmol/l) for 1 10 min. A , cpla 2 -i or EGTA was added to the neurons 10 or 30 min before the application of ATP. The total cpla 2 protein loaded on each lane was also detected as loading controls. The bar graphs show the relative values of p-cpla 2 protein induction with respect to control, after normalizing for the cpla 2 protein levels. *p < 0.05, **p < 0.01 compared with the control group. #p < 0.05 compared with the ATP-stimulated group. (f) Increase in the level of p-cpla 2 immunofluorescence and translocation of p-cpla 2 to the plasma membrane in neurons 5 min after the application of 10 lmol/l ATP compared with control. Scale bar, 20 lm. Similar results were observed in each of three experiments. inflammatory pain (Barclay et al. 2002). Although CFA produced a significant decrease in the paw withdrawal threshold (day 0: 14.4 ± 0.6 g, day 7: 2.0 ± 0.3 g, p < 0.001) after 7 days, we did not observe any change in the level and the distribution of p-cpla 2 -IR in both the L5 (Fig. 2k) and L4 DRG (data not illustrated). These results indicate that peripheral nerve injury but not persistent peripheral inflammation causes activation of cpla 2 in DRG neurons. To determine the cell-size distribution of DRG neurons with p-cpla 2 -translocation, we measured the cross-sectional areas of DRG neurons with clearly visible nuclei. Translocated p-cpla 2 was observed in medium- to large-sized neurons as well as in small-sized neurons in the ipsilateral L5 DRG (Fig. 2l). The percentage of p-cpla 2 - translocated DRG neurons for each cell-size, however, was quite different: a much higher proportion was seen in the large-sized neurons than in the small-sized DRG neurons (Fig. 2l). Large-sized cells positive for p-cpla 2 -IR were double-labeled with the marker of myelinated afferent neurons neurofilament 200 (Obata et al. 2004) (Fig. 2m o), suggesting that cpla 2 is predominantly activated myelinated DRG neurons ipsilateral to the injury. P2X 3 R/P2X 2/3 R are involved in nerve injury-induced cpla 2 activation Administration of A (100 lmol/kg, i.v.) markedly suppressed both the level and translocation of p-cpla 2 -IR in injured DRG neurons within 90 min (Fig. 3a) and moreover significantly reduced the number of DRG neurons showing translocated p-cpla 2 in response to nerve injury (p < 0.001, Fig. 3b). In addition, A administration also reduced tactile allodynia in rats as previously demonstrated (Jarvis et al. 2002) (data not illustrated). These data thus strongly indicate that the activation of cpla 2 in damaged DRG neurons following peripheral nerve injury might result from P2X 3 R/P2X 2/3 R stimulation. Inhibition of cpla 2 activation suppresses nerve injury-induced tactile allodynia To determine functional relevance of cpla 2 activation in neuropathic pain, we counted the number of cells with activated cpla 2 in the L5 DRG at various time points subsequent to the nerve injury. The number of cells was increased as early as day 3 (p < 0.001) and the peak was observed on day 14 (p < 0.001) (Fig. 4). The number of

5 1412 M. Tsuda et al. (a) (k) (b) (c) (d) (l) ( ) (e) (f) (g) (h) (i) (j) (m) (n) (o) Fig. 2 Cytosolic phosholipase A 2 (cpla 2 ) is activated in dorsal root ganglion (DRG) neurons after injury to the L5 spinal nerve. (a) Visualization of the Ser505-phosphorylated cpla 2 (p-cpla 2 ) protein detected by p-cpla 2 antibody in the L5 DRG by immunofluorescence analysis with a confocal microscope. Photomicrographs showing the p-cpla 2 immunofluorescence in the L5 DRG 14 days after the nerve injury. (b j) Double immunofluorescence labeling of p-cpla 2 with neuronal nuclei (NeuN) (b d) and microtubule-associated protein 2 (MAP2) (e g), markers of neurons; glial fibrillary acidic protein (GFAP) (h j), a marker of satellite glia/astrocytes in the ipsilateral L5 DRG 14 days after nerve injury. (k) Immunohistochemical analysis of p-cpla 2 protein in the L5 DRG 7 days after injection of complete Freund s adjuvant into the plantar surface of the hindpaw, an inflammatory pain model. (l) Size-distribution histogram of p-cpla 2 - translocated neurons (bar graph) and the percentage of p-cpla 2 - translocated DRG neurons relative to the total number of neurons (line graph) in the ipsilateral L5 DRG 14 days after nerve injury. A crosssectional area of about 650 neurons with clearly visible nuclei was measured. Open bars, total DRG neurons; filled bars, p-cpla 2 - translocated neurons (p-cpla 2 neurons). (m o) Double immunofluorescence labeling of p-cpla 2 with neurofilament 200 (NF200), a marker of myelinated A-fibers, in the ipsilateral L5 DRG 14 days after the nerve injury. Scale bars, 50 lm. neurons in the contralateral L5 DRG was not different from that in naïve rats (data not illustrated). The time-course of the change and the bilateral difference in p-cpla 2 -translocated cells in the DRG were matched closely with the emergence of tactile allodynia (Fig. 4). Furthermore, we injected the selective cpla 2 inhibitor cpla 2 -i through a catheter whose tip was positioned near the L5 DRG (Ji et al. 2002) and examined its effect on the development of tactile allodynia after nerve injury. Rats were treated with cpla 2 -i (2.5 nmol/ 10 ll, n = 5) or vehicle (5% dimethyl sulfoxide 10 ll, n = 5) once a day for 14 days. Vehicle-treated rats displayed a marked decrease in the paw withdrawal threshold following nerve injury (p < 0.001) (Fig. 5a). In contrast, rats treated with cpla 2 -i showed only a slight, but not significant, decrease in the threshold after nerve injury. The significant difference in the threshold between cpla 2 -i and vehicletreated rats was observed on days 3, 7 and 14 after the injury (day 3: p < 0.001, day 7 and 14: p < 0.01) (Fig. 5a). In addition, on day 14 the level of p-cpla 2 -IR in the ipsilateral DRG of cpla 2 -i-treated rats was much lower than in vehicle-treated rats (Fig. 5c). Furthermore, a single administration of cpla 2 -i (2.5 nmol/10 ll, n = 8) near the DRG 7 days after nerve injury also significantly suppressed the expression of tactile allodynia within 45 min (p < 0.001, Fig. 5b). Alteration in motor behavior after cpla 2 -i treatment was not observed (data not shown). Nor did cpla 2 -i affect the paw withdrawal threshold on the side contralateral to the nerve injury: at 14 days the threshold of nerve-injured rats treated with vehicle was 13.1 ± 1.2 g compared with 13.5 ± 1.0 g at 14 days of cpla 2 -i-treated rats (p > 0.1). These results together indicate that inhibiting cpla 2 activation by cpla 2 -i prevents the development and reduces the expression of tactile allodynia caused by nerve injury. Discussion In the present study, we provide the first evidence that cpla 2 is activated in DRG neurons after nerve injury and that the activation of cpla 2 is a key intermediary event of P2X 3 R/ P2X 2/3 R-dependent tactile allodynia, a major behavioral

6 Cytosolic phospholipase A 2 activation via P2X in neuropathic pain 1413 (a) (b) (a) (b) Fig. 3 Cytosolic phosholipase A 2 (cpla 2 ) is activated via stimulating P2X 3 and P2X 2/3 receptors in dorsal root ganglion (DRG) neurons after nerve injury. (a) Photomicrographs showing the Ser505-phosphorylated cpla 2 (p-cpla 2 ) immunofluorescence in the ipsilateral L5 DRG of vehicle- and A treated rats 14 days after the nerve injury. Rats were administered A (100 lmol/kg) or vehicle (PBS) intravenously 90 min before fixation. Scale bar, 50 lm. (b) The number of p-cpla 2 -translocated DRG neurons in the ipsilateral L5 DRG 90 min after administering A About 630 neuron profiles were counted in eight to ten randomly chosen sections from three to four rats in each group. Results are percentages (Mean ± SEM) of p-cpla 2 -translocated DRG neurons (p-cpla 2 neurons) relative to the total number of neurons. ***p < compared with the vehicletreated group. Fig. 4 The time-course of the change in phospho-cytosolic phosholipase A 2 (cpla 2 )-translocated dorsal root ganglion neurons was correlated with the emergence of tactile allodynia. Time course of change in paw withdrawal threshold (Mean ± SEM: bar graph) and the percentages of p-cpla 2 -translocated neurons relative to the total number of neurons in the L5 dorsal root ganglion ipsilateral to the nerve injury (Mean ± SEM: line graph). The paw withdrawal threshold of tactile stimulation to the ipsilateral hindpaw was examined using von Frey filaments neuron profiles were counted in eight to twelve randomly chosen sections from three to four rats at each time point. ***p < 0.001, ###p < compared with pre-injury baseline (Pre). consequence of nerve injury. ATP-dependent phosphorylation of cpla 2 and its translocation in primary cultured DRG neurons was inhibited either by the selective P2X 3 R/P2X 2/3 R antagonist A or by the Ca 2+ chelator EGTA, demonstrating that ATP activates cpla 2 via P2X 3 R/P2X 2/ 3R expressed on DRG neurons in a Ca 2+ -dependent manner. Interestingly, neither the level of the phosphorylated cpla 2 nor its localization was altered in DRG neurons in an animal model of chronic inflammatory pain. It thus seems likely that (c) Fig. 5 Selective cytosolic phosholipase A 2 (cpla 2 ) inhibitor suppresses tactile allodynia and cpla 2 activation caused by injury to the L5 spinal nerve. (a) Effect of a selective cpla 2 inhibitor, [N-{(2S,4R)- 4-(Biphenyl-2-ylmethyl-isobutyl-amino)-1-[2-(2,4-difluorobenzoyl)-benzoyl]-pyrrolidin-2-ylmethyl}-3-[4-(2,4-dioxothiazolidin-5-ylidinemethyl) -phenyl]-acrylamide (cpla 2 -i), on the development of nerve injuryinduced tactile allodynia. cpla 2 -i (2.5 nmol, n = 5) or vehicle (5% DMSO in PBS, n = 5) was administered near the dorsal root ganglion once a day for 14 days. Results are Mean ± SEM of the paw withdrawal threshold. Filled squares, cpla 2 -i; open squares, vehicle. ***p < compared with the threshold on day 0. ##p < 0.01, ###p < compared with the threshold of the vehicle-treated group. (b) Effect of a single administration of cpla 2 -i (2.5 nmol, n = 8) on the decrease in paw withdrawal threshold (Mean ± SEM) 7 days after nerve injury. ***p < compared with pre-injury baseline (Pre). ###p < compared with the threshold on day 7. (c) Photomicrographs showing the p-cpla 2 immunofluorescence in the ipsilateral L5 dorsal root ganglion of vehicle-treated rats and cpla 2 -i-treated rats 14 days after the nerve injury. Scale bar, 50 lm. cpla 2 activation is specifically associated with nerve injury. This notion is consistent with our data showing that cpla 2 was only activated in L5 DRG neurons, but not in L4 DRG neurons, of L5 spinal nerve injured rats. We also found that cpla 2 activation in nerve-injured rats was reversed by acute administration of A , suggesting that the cpla 2 activation depends upon ongoing signaling of ATP-activated P2X 3 R/P2X 2/3 R. Putative sources of ATP could be Schwann cells (Liu and Bennett 2003), satellite glia (Martin 1992), or injured nerves (Franke and Illes 2006). Our present behavioral study reveals the functional relevance of cpla 2 in neuropathic pain: a selective inhibition of cpla 2 activation in DRG neurons significantly suppressed the development and expression of tactile allodynia. Basal sensitivity to mechanical stimuli and motor function were not affected by this treatment. The time-dependent activation of cpla 2 in damaged DRG neurons paralleled the emergence of tactile allodynia, providing substantial support for its contribution

7 1414 M. Tsuda et al. to neuropathic pain. L5 dorsal rhizotomy reduces tactile allodynia following injury of the L5 spinal nerve, indicating an important role of damaged DRG neurons in neuropathic pain (Yoon et al. 1996; Obata et al. 2004). Our data now indicate that P2X 3 R/P2X 2/3 R-dependent activation of cpla 2 in these cells is important for the development and expression of tactile allodynia after peripheral nerve injury. Our present results showing that cpla 2 activation was observed mainly in medium- to large-sized DRG neurons seem to be partly inconsistent with previous observations that P2X 3 R is expressed mainly in small- to medium-sized DRG neurons (Bradbury et al. 1998; Vulchanova et al. 1998; Novakovic et al. 1999; Ramer et al. 2001; Kobayashi et al. 2005). However, several studies confirmed that large-sized DRG neurons also expressed P2X 3 R at both protein and mrna albeit at low levels (Novakovic et al. 1999; Ramer et al. 2001; Kage et al. 2002; Kobayashi et al. 2005). Interestingly, a recent work has demonstrated that nerve damage decreased P2X 3 R protein expression in small-sized DRG neurons, but not in large-sized DRG neurons (Kage et al. 2002). Furthermore, in healthy DRG only 20% of the neurons express P2X 2 R (Kim et al. 2003; Kobayashi et al. 2005), but the number of P2X 2 R-expressing neurons increased to 73% after peripheral nerve injury including large-sized neurons (Kim et al. 2003). These findings and the data presented here suggest that following peripheral nerve injury, functional P2X 3 R and/or P2X 2/3 R may be expressed in medium- to large-sized DRG neurons to activate cpla 2. This is supported by previous data showing that in response to nerve injury ATP-evoked firing is observed ectopically in injured Ab-primary afferent fibers (Chen et al. 1999, 2001; Zhou et al. 2001). Alternatively, it is possible that ATP- P2X 3 R/P2X 2/3 R signaling may indirectly cause cpla 2 activation in large-sized DRG neurons through an unknown neuron-to-neuron communication mechanism in the DRG. The subcellular targeting of cpla 2 is important for accessing membrane lipids to release arachidonic acid (Spencer et al. 1998; Hirabayashi and Shimizu 2000). In the present in vitro and in vivo studies we found the cpla 2 translocation to the plasma membrane in DRG neurons. These results seem to be unique, as almost all the previous in vitro data have shown that cpla 2 translocates to the cytoplasmic membrane of the perinuclear region (Schievella et al. 1995; Evans et al. 2001; Stahelin et al. 2003). The translocation to the cytoplasmic membrane might be because of the binding specificity of its C2 domain to phosphatidylcholine that is abundant at the membranes of the perinuclear region (Stahelin et al. 2003). Considering the fact that the cpla 2 translocation to the perinuclear region was demonstrated in non-neuronal cells (Schievella et al. 1995; Evans et al. 2001; Stahelin et al. 2003), it is possible that the lipid component in damaged neuronal cells could be somewhat different from that in non-neuronal cells. Alternatively, from a recent work showing that physical association of cpla 2 with components of the NADPH oxidase system results in translocation to the plasma membrane (Shmelzer et al. 2003), additional signals and/or proteins may have roles in the P2X 3 R/P2X 2/3 R-mediated cpla 2 translocation to the plasma membrane of DRG neurons after nerve injury. Putative candidate signaling molecules downstream of cpla 2 activation could be prostaglandins (Minami et al. 1992, 1994), platelet activating factor (Morita et al. 2004) or lysophosphatidic acid (Inoue et al. 2004), all of which have been reported to produce tactile allodynia. Phospholipid products mediated by cpla 2 activation may be secreted from DRG neurons and, in turn, increase the excitation of DRG neurons. The expression of many receptors for lipid mediators in DRG neurons (Park and Vasko 2005) supports this notion. Alternatively, cpla 2 -mediated products may also affect the functions of neighboring cells including sympathetic nerve terminals that sprout into the DRG and form abnormal connections to DRG neurons after nerve injury (McLachlan et al. 1993; Chung et al. 1996). Several phospholipids have been implicated as retrograde substances that affect neurotransmission at pre-synapses in the nervous system (Chen and Bazan 2005). Thus, cpla 2 -mediated products might enhance the neuronal connection between sympathetic terminals and DRG neurons and thereby increase the sympathetic nerve-dependent excitation of sensory neurons. Studies in our laboratory determining cpla 2 -mediated products and their roles in neuropathic pain are currently underway. In summary, the present study shows for the first time that stimulating P2X 3 R/P2X 2/3 R activates cpla 2 in primary afferent sensory neurons and identifies cpla 2 activity as a key event in the pathogenesis of neuropathic pain. In our behavioral study, cpla 2 -i suppressed neuropathic allodynia without any substantial deficit in the basal sensitivity. Thus, interfering with the cpla 2 activity may also be a potential therapeutic target for treating neuropathic pain. Acknowledgements We thank Dr Knut Biber for critical reading and suggestions. 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