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Carrageenan-induced Paw Edema in Rat Elicits a Predominant Prostaglandin E2 (PGE2) Response in the Central Nervous System Associated with the Induction of Microsomal PGE2 Synthase-1*

Open AccessPublished:March 24, 2004DOI:https://doi.org/10.1074/jbc.M403106200
      Peripheral inflammation involves an increase in cyclooxygenase-2 (COX-2)-mediated prostaglandin (PG) synthesis in the central nervous system (CNS), which contributes to allodynia and hyperalgesia. In the present study we have determined the changes in prostanoid tissue levels and in expression of terminal prostanoid synthases in both the CNS and inflamed peripheral tissue during carrageenan-induced paw inflammation in the rat. Prostanoid levels were measured by liquid chromatography-mass spectrometry and enzyme expression at the RNA level by quantitative PCR analysis during both the early (1-6 h) and late (12 and 24 h) phases of the inflammatory response. In the paw, the early phase was associated with increases in PGE2 and thromboxane (TX)B2 levels and with a peak of COX-2 expression that preceded that of microsomal prostaglandin-E2 synthase-1 (mPGES-1). COX-2 and mPGES-1 remained elevated during the late phase, and PGE2 continued to further increase through 24 h. The cytosolic PGE2 synthase (cPGES) showed a small transient increase during the early phase, whereas mPGES-2 expression was not affected by inflammation. In the cerebrospinal fluid, elevated levels of PGE2, 6-keto-PGF, PGD2, and TXB2 were detected during the early phase. PGE2 levels also increased in the spinal cord and, to a lesser extent, in the brain and remained elevated in both the cerebrospinal fluid and the spinal cord during the late phase. The expression of mPGES-1 was strongly up-regulated in the brain and spinal cord during inflammation, whereas no change was detected for the expression of cPGES, mPGES-2, COX-1, and terminal PGD, TX, or PGI synthases. The results show that the carrageenan-induced edema in the paw elicits an early phase of COX-2 induction in the CNS leading to an increase synthesis in PGD2, 6-keto-PGF, and TXB2 in addition to the major PGE2 response. The data also indicate that the up-regulation of mPGES-1 contributes to COX-2-mediated PGE2 production in the CNS during peripheral inflammation.

      Introduction

      Carrageenan-induced inflammation in the rat paw represents a classical model of edema formation and hyperalgesia, which has been extensively used in the development of nonsteroidal anti-inflammatory drugs and selective COX
      The abbreviations used are: COX, cyclooxygenase; CNS, central nervous system; CSF, cerebrospinal fluid; cPGES, cytosolic prostaglandin-E2 synthase; mPGES, microsomal prostaglandin-E2 synthase; PG, prostaglandin; PGDS, lipocalin-type prostaglandin D synthase; PGIS, prostacyclin synthase; TX, thromboxane; TXS, thromboxane synthase; IL, interleukin; LCMS, liquid chromatography-mass spectrometry; IP, prostacyclin receptor.
      -2 inhibitors. Several lines of evidence indicate that the COX-2-mediated increase in prostaglandin (PG) E2 production in the central nervous system (CNS) contributes to the severity of the inflammatory and pain responses in this model. COX-2 is rapidly induced in the spinal cord and other regions of the CNS following carrageenan injection in the paw (
      • Ichitani Y.
      • Shi T.
      • Haeggstrom J.Z.
      • Samuelsson B.
      • Hokfelt T.
      ). The administration of selective COX-2 inhibitors, but not COX-1 inhibitors, reduces the levels of PGE2 in the cerebrospinal fluid (CSF) and hyperalgesia (
      • Smith C.J.
      • Zhang Y.
      • Koboldt C.M.
      • Muhammad J.
      • Zweifel B.S.
      • Shaffer A.
      • Talley J.J.
      • Masferrer J.L.
      • Seibert K.
      • Isakson P.C.
      ,
      • Dirig D.M.
      • Isakson P.C.
      • Yaksh T.L.
      ,
      • Zhang Y.
      • Shaffer A.
      • Portanova J.
      • Seibert K.
      • Isakson P.C.
      ,
      • Riendeau D.
      • Percival M.D.
      • Boyce S.
      • Brideau C.
      • Charleson S.
      • Cromlish W.
      • Ethier D.
      • Evans J.
      • Falgueyret J.P.
      • Ford-Hutchinson A.W.
      • Gordon R.
      • Greig G.
      • Gresser M.
      • Guay J.
      • Kargman S.
      • Leger S.
      • Mancini J.A.
      • O'Neill G.
      • Ouellet M.
      • Rodger I.W.
      • Therien M.
      • Wang Z.
      • Webb J.K.
      • Wong E.
      • Chan C.C.
      ). In addition, it has been shown that the intrathecal administration of PGE2 potentiates carrageenan-induced inflammation (
      • Daher J.B.
      • Tonussi C.R.
      ) and that the direct microinjection of PGE2 in the brain causes hyperalgesia (
      • Hosoi M.
      • Oka T.
      • Hori T.
      ). Selective COX-2 inhibitors can also inhibit peripheral pain responses when given intrathecally (
      • Dirig D.M.
      • Isakson P.C.
      • Yaksh T.L.
      ,
      • Samad T.A.
      • Moore K.A.
      • Sapirstein A.
      • Billet S.
      • Allchorne A.
      • Poole S.
      • Bonventre J.V.
      • Woolf C.J.
      ,
      • Yamamoto T.
      • Nozaki-Taguchi N.
      ), whereas a selective COX-1 inhibitor has no effect (
      • Yaksh T.L.
      • Dirig D.M.
      • Conway C.M.
      • Svensson C.
      • Luo Z.D.
      • Isakson P.C.
      ). The central effects of PGE2 appear to be mediated via the EP3 receptor based on observations that the microinjection of an agonist of the EP3 receptor in the brain directly causes hyperalgesia (
      • Hosoi M.
      • Oka T.
      • Hori T.
      ), and the inflammatory responses are strongly reduced in the mice deficient in the EP3 receptor (
      • Minami T.
      • Nakano H.
      • Kobayashi T.
      • Sugimoto Y.
      • Ushikubi F.
      • Ichikawa A.
      • Narumiya S.
      • Ito S.
      )
      Altogether these studies indicate that the central production of PGE2 mediated by COX-2 during inflammation contributes to nociception and hyperalgesia at the site of peripheral inflammation. However, the role of the different prostanoid synthases in inflammation and the identity of the critical prostanoids involved in the inflammatory processes have not been well defined. In addition to PGE2, PGI2 also has been proposed to represent an important mediator of inflammation based on the reduced edema and pain response in the prostacyclin receptor (IP) knock-out mice (
      • Murata T.
      • Ushikubi F.
      • Matsuoka T.
      • Hirata M.
      • Yamasaki A.
      • Sugimoto Y.
      • Ichikawa A.
      • Aze Y.
      • Tanaka T.
      • Yoshida N.
      • Ueno A.
      • Oh-Ishi S.
      • Narumiya S.
      ,
      • Ueno A.
      • Naraba H.
      • Ikeda Y.
      • Ushikubi F.
      • Murata T.
      • Narumiya S.
      • Oh-Ishi S.
      ). Clinical studies have indicated that selective COX-2 inhibitors inhibit the production of urinary PGI2 metabolites in addition to those of the PGE2 pathway (
      • Catella-Lawson F.
      • McAdam B.
      • Morrison B.W.
      • Kapoor S.
      • Kujubu D.
      • Antes L.
      • Lasseter K.C.
      • Quan H.
      • Gertz B.J.
      • FitzGerald G.A.
      ) indicating that COX-2 plays a role in the synthesis of both prostanoid mediators in humans.
      PGE2 is synthesized by the sequential reactions catalyzed by COX, which converts arachidonic acid to PGH2, and by PGE synthase, which converts PGH2 to PGE2. Several proteins that possess variable levels of PGE synthase activity have been identified, microsomal PGES-1 (mPGES-1) (
      • Jakobsson P.J.
      • Thoren S.
      • Morgenstern R.
      • Samuelsson B.
      ), glutathione transferases (
      • Ujihara M.
      • Tsuchida S.
      • Satoh K.
      • Sato K.
      • Urade Y.
      ), cytosolic PGES (cPGES)/p23 (
      • Tanioka T.
      • Nakatani Y.
      • Semmyo N.
      • Murakami M.
      • Kudo I.
      ) and mPGES-2 (
      • Tanikawa N.
      • Ohmiya Y.
      • Ohkubo H.
      • Hashimoto K.
      • Kangawa K.
      • Kojima M.
      • Ito S.
      • Watanabe K.
      ,
      • Murakami M.
      • Kudo I.
      ). The mPGES-1 is considered to be the major form implicated in COX-2-mediated PGE2 production based on co-transfection experiments in cultured cells indicating a better coupling with COX-2 than with COX-1 for the production of PGE2 (
      • Murakami M.
      • Naraba H.
      • Tanioka T.
      • Semmyo N.
      • Nakatani Y.
      • Kojima F.
      • Ikeda T.
      • Fueki M.
      • Ueno A.
      • Oh S.
      • Kudo I.
      ) and based on the inducibility of the enzyme in response to pro-inflammatory mediators (
      • Jakobsson P.J.
      • Thoren S.
      • Morgenstern R.
      • Samuelsson B.
      ,
      • Stichtenoth D.O.
      • Thoren S.
      • Bian H.
      • Peters-Golden M.
      • Jakobsson P.J.
      • Crofford L.J.
      ,
      • Han R.
      • Tsui S.
      • Smith T.J.
      ), and in various models of inflammation and fever in vivo (
      • Yamagata K.
      • Matsumura K.
      • Inoue W.
      • Shiraki T.
      • Suzuki K.
      • Yasuda S.
      • Sugiura H.
      • Cao C.
      • Watanabe Y.
      • Kobayashi S.
      ,
      • Inoue W.
      • Matsumura K.
      • Yamagata K.
      • Takemiya T.
      • Shiraki T.
      • Kobayashi S.
      ,
      • Mancini J.A.
      • Blood K.
      • Guay J.
      • Gordon R.
      • Claveau D.
      • Chan C.C.
      • Riendeau D.
      ,
      • Claveau D.
      • Sirinyan M.
      • Guay J.
      • Gordon R.
      • Chan C.C.
      • Bureau Y.
      • Riendeau D.
      • Mancini J.A.
      ). Furthermore, the mPGES-1 has been shown to co-localize with COX-2 in endothelial cells of the brain vasculature after induction with interleukin (IL)-1β (
      • Yamagata K.
      • Matsumura K.
      • Inoue W.
      • Shiraki T.
      • Suzuki K.
      • Yasuda S.
      • Sugiura H.
      • Cao C.
      • Watanabe Y.
      • Kobayashi S.
      ,
      • Ek M.
      • Engblom D.
      • Saha S.
      • Blomqvist A.
      • Jakobsson P.J.
      • Ericsson-Dahlstrand A.
      ). The profile of the mPGES-1 knock-out mice is strongly supportive for a role of this enzyme in the production of inflammatory PGE2 production with a marked reduction of lipopolysaccharide-stimulated PGE2 synthesis in peritoneal macrophages (
      • Uematsu S.
      • Matsumoto M.
      • Takeda K.
      • Akira S.
      ) and a reduction in inflammation in the collagen-induced arthritis model (
      • Trebino C.E.
      • Stock J.L.
      • Gibbons C.P.
      • Naiman B.M.
      • Wachtmann T.S.
      • Umland J.P.
      • Pandher K.
      • Lapointe J.M.
      • Saha S.
      • Roach M.L.
      • Carter D.
      • Thomas N.A.
      • Durtschi B.A.
      • McNeish J.D.
      • Hambor J.E.
      • Jakobsson P.J.
      • Carty T.J.
      • Perez J.R.
      • Audoly L.P.
      ).
      Most of the studies on the regulation of mPGES-1 in vivo have been performed using models of IL-1-or lipopolysaccharide-induced pyresis in which the expression of the enzyme has been shown to increase in the CNS and in certain peripheral tissues (
      • Yamagata K.
      • Matsumura K.
      • Inoue W.
      • Shiraki T.
      • Suzuki K.
      • Yasuda S.
      • Sugiura H.
      • Cao C.
      • Watanabe Y.
      • Kobayashi S.
      ,
      • Inoue W.
      • Matsumura K.
      • Yamagata K.
      • Takemiya T.
      • Shiraki T.
      • Kobayashi S.
      ,
      • Mancini J.A.
      • Blood K.
      • Guay J.
      • Gordon R.
      • Claveau D.
      • Chan C.C.
      • Riendeau D.
      ,
      • Ek M.
      • Engblom D.
      • Saha S.
      • Blomqvist A.
      • Jakobsson P.J.
      • Ericsson-Dahlstrand A.
      ,
      • Uematsu S.
      • Matsumoto M.
      • Takeda K.
      • Akira S.
      ,
      • Engblom D.
      • Ek M.
      • Andersson I.M.
      • Saha S.
      • Dahlstrom M.
      • Jakobsson P.J.
      • Ericsson-Dahlstrand A.
      • Blomqvist A.
      ,
      • Chan C.C.
      • Boyce S.
      • Brideau C.
      • Ford-Hutchinson A.W.
      • Gordon R.
      • Guay D.
      • Hill R.G.
      • Li C.S.
      • Mancini J.
      • Penneton M.
      • Prasit P.
      • Rasori R.
      • Riendeau D.
      • Roy P.
      • Tagari P.
      • Vickers P.
      • Wong E.
      • Roger I.W.
      ). Recently, mPGES-1 has also been shown to be inducible during a chronic model of adjuvant-induced arthritis (
      • Claveau D.
      • Sirinyan M.
      • Guay J.
      • Gordon R.
      • Chan C.C.
      • Bureau Y.
      • Riendeau D.
      • Mancini J.A.
      ,
      • Engblom D.
      • Ek M.
      • Andersson I.M.
      • Saha S.
      • Dahlstrom M.
      • Jakobsson P.J.
      • Ericsson-Dahlstrand A.
      • Blomqvist A.
      ). In the present study we report on the up-regulation of mPGES-1 in the CNS during the acute model of carrageenan-induced inflammation of the paw in the rat, providing further evidence for the role of mPGES-1 in the increased synthesis of PGE2 associated with inflammatory responses.

      MATERIALS AND METHODS

      Model of Carrageenan-induced Edema in Rat—All procedures for in vivo experiments were approved by the Animal Care Committee at the Merck Frosst Centre for Therapeutic Research (Kirkland, Quebec, Canada) and were performed according to guidelines established by the Canadian Council on Animal Care. Paw edema was induced in Sprague-Dawley rats (150-200 g) (Charles River Laboratories, St-Constant, Quebec, Canada) by injection of 100 μl of 1% carrageenan (λ-carrageenan, type IV, Sigma) diluted in saline in the left hind foot pad (
      • Chan C.C.
      • Boyce S.
      • Brideau C.
      • Ford-Hutchinson A.W.
      • Gordon R.
      • Guay D.
      • Hill R.G.
      • Li C.S.
      • Mancini J.
      • Penneton M.
      • Prasit P.
      • Rasori R.
      • Riendeau D.
      • Roy P.
      • Tagari P.
      • Vickers P.
      • Wong E.
      • Roger I.W.
      ). Paw volumes were determined using a water plethysmometer (Ugo Basil, Italy). At times 0, 1, 3, 5, 6, 12, and 24 h, rats were euthanized by carbon dioxide inhalation. The tissues (brain, spinal cord, and soft paw) were flash frozen in liquid nitrogen and kept at -80°C until processing. The CSF was collected and kept at -80°C for prostanoid analyses.
      Preparation of Tissue Extracts—Frozen tissues were pulverized in liquid nitrogen using a mortar and pestle, homogenized in 5 volumes of ice-cold phosphate-buffered saline, pH 7.4 (2.5 mm glutathione, 2 mm dithiothreitol and 1× Complete Protease Inhibitor mixture (Roche Diagnostics GmbH, Manheim, Germany)), with a tissue tearer (polytron PRO 200) and sonicated on an ice bath for 12 s (Kontes, micro-ultrasonic cell disrupter, output 70%). The tissue homogenates were centrifuged at 1000 × g at 4°C for 10 min, and the resulting supernatant was further centrifuged at 100,000 × g at 4°C for 90 min. The pellet (microsomal fraction) was resuspended in the cold homogenization buffer, and the supernatant was kept as the soluble fraction. Protein levels were determined using a Bradford protein assay (Bio-Rad). Fractions were kept at -80°C until further analysis.
      Measurements of Prostanoids by Liquid Chromatography-Mass Spectrometry (LC-MS)—PGE2, PGF, PGD2, 6-keto-PGF and thromboxane (TX) B2 in the soluble fractions were analyzed by LC-MS. PG standards and deuterated PGs were purchased from Cayman Chemical (Ann Arbor, MI); the non-deuterated PGs were purchased as solids.
      Samples (100 μl) for LC-MS analysis were protein-precipitated by the addition of acetonitrile (150 μl) containing deuterated prostaglandins (2 ng/ml) as internal standards (d4-PGE2 served as an internal standard for both PGE2 and PGD2). Samples were vortexed and centrifuged (3000 × g, 10 min) and the supernatant transferred to a new 96-well plate. The plates were sealed using a TopSeal™ microplate heat sealing film (Packard Instrument B.V., Meriden, CT). Samples (50 μl) were injected onto a 4.6 × 150-mm YMC ODS-A column using a Shimadzu SIL-HTc autosampler and LC-10ADVP pumps. Prostaglandins were eluted at 1 ml/min using a linear gradient from 10 to 90% acetonitrile versus 0.1% formic acid over 10 min.
      Detection of prostaglandins was achieved using a Sciex API-4000 triple quadrupole mass spectrometer. Analysis was carried out using negative ion electrospray with 1 ml/min entering the source. The source was operated at 500 °C, electrospray voltage was-4500 V, and gas 1 and gas 2 were 60 and 50, respectively. Each prostaglandin was optimized individually for parent mass and fragment mass sensitivity as shown in Table I. The limit of detection ranged from 16 to 200 pg/ml depending on the prostaglandin.
      Table IMS conditions for the analyses of prostaglandins and TXB2
      ProstaglandinQ1
      Q, quadrupole.
      mass
      Q3 massDP
      DP, declustering potential.
      voltage
      Collision energy
      PGE2 and PGD2351.3315.1−70−16
      d4-PGE2355.3319.1−70−16
      PGF353.3309.0−85−26
      d4-PGF357.3313.0−85−26
      6-keto-PGF369.1163.2−75−36
      d4-6-keto-PGF373.1167.2−75−36
      TXB2369.1168.8−65−28
      d4-TXB2373.1168.8−65−28
      a Q, quadrupole.
      b DP, declustering potential.
      Western Blot Analyses—Aliquots containing 25 or 50 μg of protein were loaded on SDS-PAGE using 4-20% gradient gels (Novex, Invitrogen) and transferred electrophoretically to polyvinylidene difluoride membranes using a Novex immunoblot apparatus according to the manufacturer's instructions. Immunodetection was performed using COX-2 (1/300 dilution) and mPGES-1 (1/300) purified antibodies (Cayman Chemical). Chemiluminescence detection was performed using Fuji Film LAS-1000 charge-couple device and Image Gauge software for quantitative analysis or by film exposure (Kodak BioMax MR, Eastman Kodak Co.) using a Bio-Rad GS-800 densitometer and Quantity One software for analysis.
      Analyses of mRNA by Quantitative PCR—The mRNA isolation and quantification were performed as described previously (
      • Claveau D.
      • Sirinyan M.
      • Guay J.
      • Gordon R.
      • Chan C.C.
      • Bureau Y.
      • Riendeau D.
      • Mancini J.A.
      ) with some modifications. Briefly, frozen tissues were pulverized in liquid nitrogen using mortar and pestle and then homogenized in TRIzol reagent (Canadian Life Technologies, Ontario, Canada), and total RNA was isolated according to manufacturer's instructions. A clean up of the RNA was performed using the RNeasy mini kit (Qiagen, Ontario, Canada) as described in the manufacturer's instruction and including a DNase 1 treatment (RNase-free DNase set from Qiagen). The quality of mRNA was evaluated using RNA 6000 Nano assay and Agilent 2100 Bioanalyser (Agilent Technologies, Waldbron, Germany).
      Reverse transcription of RNA (50 ng) was performed using TaqMan transcription reagents (PE Biosystems, Branchburg, NJ) using 1× Taq-Man reverse transcription buffer, 5.5 mm magnesium chloride, 500 μm dNTP, 2.5 μm random hexamers, 0.4 units/μl RNase inhibitors, and 1.25 units/μl Multiscribe reverse transcriptase. Real time quantitative PCR for mPGES-1, COX-2, COX-1, cPGES, mPGES-2, and prostacyclin synthase (PGIS) was performed using probes and primers as fully described by Claveau et al. (
      • Claveau D.
      • Sirinyan M.
      • Guay J.
      • Gordon R.
      • Chan C.C.
      • Bureau Y.
      • Riendeau D.
      • Mancini J.A.
      ). The oligonucleotide sequences used for lipocalin-type prostaglandin-D synthase (PGDS) and thromboxane synthase (TXS) probes and primers were as follows: PGDS probe, 5′-ACGCGTACTCATCGTAGTCGGTTTCTACCA-3′; PGDS forward primer, 5′-CCCGGACAGTACACCTACAACAG-3′; PGDS reverse primer, 5′-TGGTGCCCTTGCTGAACAG-3′; TXS probe, 5′-TCGATGCCAAAGGCCACACTGG-3′; TXS forward primer, 5′-CCAGAGGTGTTACTGCTGTTTTACC-3′; TXS reverse primer, 5′-GGAGCATCCTGGGAGTTCAC-3′.
      Statistical Analysis—Each n value corresponds to a different animal and is indicated in the figure legend. Standard one-way analysis of variance was used to compare the multiple groups. The data were log-scaled so that underlying assumptions of equal variance and normality were better satisfied, and expressions of effects could be made in terms of the percent change. The means and standard errors for each group were estimated from the analysis of variance, which pools the data variability from the 14 groups under the common assumption that the population variances are equal. All follow-up comparisons were deemed statistically significant at the p < 0.05 level unless noted otherwise.

      RESULTS

      Time Course of Edema Formation—Carrageenan-induced edema in the hind foot pad was used as a model to determine the temporal relationships between edema formation, prostanoid synthesis, and expression of selected terminal prostanoid synthases both at the site of peripheral inflammation and in the CNS. Under the conditions used for carrageenan-induced inflammation, swelling of the paw occurred rapidly after the injection of carrageenan with an increase in volume of 1.5-fold at 1 h, which reached a maximum at 6 h (2.7-fold) and remained elevated until the last measurement at 24 h (Fig. 1).
      Figure thumbnail gr1
      Fig. 1Time course of carrageenan-induced edema in the rat paw. Results are expressed as paw volumes (means ± S.E., n = 4 animals) determined at different times after injection of carrageenan (or saline) in the left hind foot pad.
      Increased PGE2 and TXB2 Levels in Inflamed Paw—The soft tissues of the paws were collected at different times after carrageenan or saline injection and were profiled for their content in PGE2, PGD2, PGF, 6-keto-PGF, and TXB2 by LC-MS analysis. PGE2 levels in the paw were found to increase in two phases (Fig. 2A). An initial 2-4-fold increase over the measurements at time 0 and saline controls was observed from 1 to 6 h after the carrageenan injection. PGE2 levels continued to increase with time, reaching 6-and 8-fold increases over their respective saline controls at 12 and 24 h.
      Figure thumbnail gr2
      Fig. 2Increase in the levels of prostanoids in paw tissues during carrageenan-induced edema. The contents in PGE2 (A) and TXB2 (B) were determined in soluble extracts prepared from the soft tissues of the paw collected at different times after carrageenan administration. Analyses were performed by LC-MS. Data are reported as means ± S.E. (n = 5 or 6 for carrageenan samples, n = 2 for saline controls). *, different from both carrageenan at time 0 and corresponding saline-injected control at p < 0.05.
      Among the other prostanoids, the most noticeable changes were observed for TXB2 with a modest but significant increase of ∼2-4-fold at 3-5 h similar to that of PGE2 at these time points (Fig. 2B). In contrast to PGE2, however, the increase was transient and levels of TXB2 returned to near base line at the 12 and 24 h time points. Basal levels of PGD2 (0.05-0.08 ng/mg protein) and 6-keto-PGF (0.1-0.4 ng/mg protein) were detected but did not show any significant increase during inflammation as compared with controls (data not shown). The content in PGF was low throughout the time course (a small peak of 0.04 ng/ml at 3 h after carrageenan, data not shown).
      Expression of Terminal Synthases in the Inflamed Paw—The expression of the various prostanoid synthases was measured by real time quantitative PCR. The data are expressed as the relative expression of mRNA as compared with saline control paw at time 0 (and using 18 S rRNA to normalize). Both COX-2 and mPGES-1 mRNAs were elevated in inflamed paws during the whole time course, with the peak of COX-2 mRNA (5-fold at 1 h versus time 0) preceding that of mPGES-1 (60-fold at 5 h) (Fig. 3A). At 24 h the mPGES-1 mRNA was still 4-fold higher and statistically different from the saline-injected control.
      Figure thumbnail gr3
      Fig. 3Real time quantitative PCR analysis of the expression of the mRNAs of mPGES-1, COX-2, COX-1, cPGES, and mPGES-2 in paw tissues during carrageenan-induced edema. At the different time points total RNA was isolated from paw soft tissues, reverse-transcribed, and analyzed by real time quantitative PCR analysis. The levels of mRNA are expressed relative to that of the saline control at time 0, after normalization to 18 S rRNA. Data are reported as means ± S.E. (n = 5 or 6 for carrageenan samples, n = 2 for saline). *, significantly different from both the value at time 0 and the corresponding saline-injected control (data not shown) at p < 0.05.
      In contrast to COX-2 and mPGES-1, the mRNAs of COX-1 and mPGES-2 did not increase during edema (Fig. 3B). The cPGES showed only very modest changes with a small peak of induction detected at 5 h (∼2-fold versus time 0) (Fig. 3B).
      The induction of COX-2 and mPGES-1 was also determined at the protein level using Western blot analysis (Fig. 4). No signal could be detected for COX-2 in the tissues from the saline controls at any time point, nor at time 0, or 1 h after carrageenan injection. An increase in COX-2 protein expression was detected at 3 h and a large accumulation in protein at 24 h. The induction of the mPGES-1 in the carrageenan-injected paw was evident at 5 h (5-fold) with a larger accumulation in protein at 12 and 24 h (Fig. 4). A low level of basal mPGES-1 protein was also detected at time 0 and was not altered with time in saline-injected controls. The cPGES and mPGES-2 proteins were detected at all time points but were not affected significantly by the inflammatory response (data not shown).
      Figure thumbnail gr4
      Fig. 4Increases in the expression of COX-2 and mPGES-1 proteins in the inflamed paw following the administration of carrageenan. 50 μg of protein of the microsomal fraction from extracts of paw tissues collected at the indicated times after the injection of saline (s) or carrageenan (c) were analyzed by immunoblot for the mPGES-1 or COX-2 protein. Std, standard.
      Characteristics of the PGE2 Response in the CNS during Inflammation of the Paw—To better define the CNS response during peripheral inflammation, we also investigated the effect of carrageenan-induced paw inflammation changes on the CNS prostanoid profile and on the expression of terminal prostanoid synthases in the brain and spinal cord. PGE2 was detectable barely in the CSF from naïve or saline-injected control animals (<0.02 ng/ml). A significant augmentation in PGE2 was measured at 3 h after carrageenan injection and reached a peak at 6 h representing at least a 50-fold increase (Fig. 5A). In contrast to the paw tissues wherein PGE2 levels continued to increase until 24 h, PGE2 levels in the CSF declined at 12 and 24 h but still remained significantly elevated as compared with corresponding saline controls (Fig. 5A).
      Figure thumbnail gr5
      Fig. 5Increase in the level of PGE2 in CSF, spinal cord, and brain extracts during carrageenan-induced paw edema. PGE2 levels in the CSF are expressed in ng/ml as mean ± S.E. (n = 4) (A). PGE2 levels in brain (n = 5 for carrageenan samples, n = 2 for saline) and spinal cord (n = 3 for carrageenan, n = 1 for saline) are expressed in ng/mg of protein of soluble extracts (B). *, different from both time 0 and saline controls at p < 0.05.
      Low basal levels of PGE2 were detected in the extracts from spinal cord and brain tissues that were not altered with time in control animals injected with saline. Carrageenan injection in the paw caused a marked increase of PGE2 in the spinal cord with a maximum at 6 h (6-fold), and a time course (Fig. 5B) that was similar to that observed for the CSF. In the brain, the increase in PGE2 levels (2-fold increase at 3-6 h) was less pronounced than in the spinal cord (Fig. 5B).
      Elevation in Prostaglandin and TXB2 Levels in the CNS during the Early Phase of Inflammation—The tissues from the CNS were also analyzed for their contents of the other prostanoids. In the CSF, 6-keto-PGF, PGD2, and TXB2 levels were all at the limit of detection of the LC-MS analysis for samples from untreated animals or from the control animals collected at different times after saline injection. After carrageenan injection, 6-keto-PGF was detected at elevated levels in the CSF at 3, 5, and 6 h, while PGD2 and TXB2 showed increases at 6 h (Fig. 6A). PGF could not be detected at any time point in the CSF. Only small changes for these prostanoids were detected in the brain and spinal cord extracts. In the spinal cord, small transient increases (∼2-fold) were observed at 6 h for 6-keto-PGF, PGD2, TXB2, and PGF (data not shown). In the brain, PGD2, PGF, TXB2, and 6-keto-PGF all showed minor increases at 3-6 h (1.5-2-fold), which reached statistical significance over time 0 and saline only for PGD2 and PGF (Fig. 6B).
      Figure thumbnail gr6
      Fig. 6Variation in prostanoid levels of the CSF and brain during carrageenan-induced paw edema. The tissue content in 6-keto-PGF, PGD2, TXB2, and PGF, was determined by LC-MS. Data are reported as means ± S.E. for CSF (n = 4) (A) and for brain soluble extracts (n = 5) (B). *, significantly different from both time 0 and saline controls (data not shown) at p < 0.05.
      A Marked Up-regulation of mPGES-1 but Not of PGIS, TXS, and PGDS Expression in the CNS during Inflammation of the Paw—Total brain and spinal cord extracts were prepared from animals treated with carrageenan for different periods of time and were analyzed by quantitative PCR for expression of COX and PGE terminal synthases. As shown in Fig. 7A, mPGES-1 and COX-2 are both strongly induced in the spinal cord with maximal increases between 3 and 6 h. In the brain, we observed an 8-10-fold induction of mPGES-1 at 3-6 h, whereas the COX-2 showed only a small but significant 2-fold induction at 1-5 h (Fig. 7B). In contrast, there was no induction at the mRNA level for COX-1, cPGES, or mPGES-2 in the spinal cord or in the brain (Fig. 7, A and B). Microsomal fractions were prepared from spinal cord homogenates to enrich mPGES-1 protein for Western blot analysis. A protein band corresponding to mPGES-1 was detected at 6 and 24 h after the initiation of inflammation at about 3× higher levels than in the control, indicating that the induction of mPGES-1 also occurs at the protein level (Fig. 7C). Tissues were also analyzed for PGIS mRNAs because deletion of the IP receptor has suggested a role for prostacyclin in edema formation and pain (
      • Murata T.
      • Ushikubi F.
      • Matsuoka T.
      • Hirata M.
      • Yamasaki A.
      • Sugimoto Y.
      • Ichikawa A.
      • Aze Y.
      • Tanaka T.
      • Yoshida N.
      • Ueno A.
      • Oh-Ishi S.
      • Narumiya S.
      ,
      • Ueno A.
      • Matsumoto H.
      • Naraba H.
      • Ikeda Y.
      • Ushikubi F.
      • Matsuoka T.
      • Narumiya S.
      • Sugimoto Y.
      • Ichikawa A.
      • Oh-Ishi S.
      ). As shown in Fig. 8A, no significant induction of PGIS could be observed during the development of paw edema in brain, spinal cord or inflamed tissue from the paw. Similarly, PGDS and TXS expression in the brain and spinal cord did not show any significant changes during the inflammation response (Fig. 8, B and C). These results confirm the involvement of COX-2 in the production of PGs in the CNS during inflammation and show that the selective induction of mPGES-1 is associated with the pronounced increase in PGE2 levels.
      Figure thumbnail gr7
      Fig. 7Real time quantitative PCR analysis of the expression of the mRNA of mPGES-1, COX-2, COX-1, cPGES, and mPGES-2 in the CNS during carrageenan-induced paw edema. Data are reported as ratios (means ± S.E.) to the corresponding saline controls at time 0 for the spinal cord (n = 3) (A) and brain tissues (n = 5) (B). *, significantly different from both time 0 and saline controls (data not shown) at p < 0.05. Immunoblot analysis of mPGES-1 in microsomal fractions from spinal cords of carrageenan-treated animals collected at time 0, 6, and 24 h (C). Purified rat mPGES-1 was used as standard (Std).
      Figure thumbnail gr8
      Fig. 8Expression of PGIS, PGDS, and TXS during carrageenan-induced paw edema. The expression of mRNA was monitored by real time quantitative PCR analysis in selected tissues collected at different time points after carrageenan injection. Data are reported as means ± S.E. for the spinal cord (n = 3), brain (n = 5), or paw soft tissues (n = 5 or 6). No significant difference was detected when compared with both time 0 and the corresponding saline controls (data not shown).

      DISCUSSION

      Both PGE2 and PGI2 have been implicated as mediators of the inflammatory and pain responses. In the present study, we seek to obtain more information on the role of these and other prostanoids by examining the changes in tissue levels of the major prostanoids and the expression of terminal prostanoid synthases in the CNS as compared with the site of peripheral footpad edema.
      Induction of COX-2 and mPGES-1 during Sustained PGE2 Production in Inflamed Paws—Previous studies (
      • Ibuki T.
      • Matsumura K.
      • Yamazaki Y.
      • Nozaki T.
      • Tanaka Y.
      • Kobayashi S.
      ,
      • Seibert K.
      • Zhang Y.
      • Leahy K.
      • Hauser S.
      • Masferrer J.
      • Perkins W.
      • Lee L.
      • Isakson P.
      ,
      • Nantel F.
      • Denis D.
      • Gordon R.
      • Northey A.
      • Cirino M.
      • Metters K.M.
      • Chan C.C.
      ) have shown that COX-2 is detected at elevated levels in paw tissues and in the CNS following carrageenan-induced inflammation. In the current model, swelling of the paw progressively increased over the first 6 h, and the edema persisted for at least 24 h. A very early induction of COX-2 occurred in the paw, with maximal mRNA expression at 1 h, together with an increased expression of mPGES-1 that peaked at 6 h and remained elevated over controls until the last 24-h time point. The sequential induction of COX-2 and mPGES-1 observed in vivo paralleled the in vitro data obtained with cell lines during stimulation of PGE2 production (
      • Stichtenoth D.O.
      • Thoren S.
      • Bian H.
      • Peters-Golden M.
      • Jakobsson P.J.
      • Crofford L.J.
      ,
      • Kojima F.
      • Naraba H.
      • Sasaki Y.
      • Okamoto R.
      • Koshino T.
      • Kawai S.
      ). Both enzymes accumulate at the protein level in paws at later time points during the maintenance of inflammation as PGE2 levels continued to increase. These data indicate the early phase of the modest increase in PGE2 might be primarily COX-2-dependent and that both elevated COX-2 and mPGES-1 contribute to a more sustained production and accumulation of PGE2 at the inflammation site. Of the three PGE2 synthases evaluated (cPGES, mPGES-1, and mPGES-2), only mPGES-1 mRNA was strongly up-regulated (a transient doubling of expression was detected for cPGES) as observed in the adjuvant-induced arthritis model in rat (
      • Claveau D.
      • Sirinyan M.
      • Guay J.
      • Gordon R.
      • Chan C.C.
      • Bureau Y.
      • Riendeau D.
      • Mancini J.A.
      ) and after IL-1β-induced inflammation in mouse brain (
      • Moore A.H.
      • Olschowka J.A.
      • O'Banion M.K.
      ), although a small increase in cPGES protein could also be detected in these models. Although no induction was seen in the current model, mPGES-2 (designated as GBF-1) has been reported recently to be inducible in response to interferon-γ (
      • Hu J.
      • Meng Q.
      • Roy S.K.
      • Raha A.
      • Hu J.
      • Zhang J.
      • Hashimoto K.
      • Kalvakolanu D.V.
      ). It should be noted that the mPGES-1 protein was detected in extracts of normal paws and thus the constitutive expression of the enzyme might contribute to basal PGE2 levels as well as to the early phase of PGE2 production. Constitutive expression of mPGES-1 has also been reported previously in the mouse hypothalamus (
      • Matsuoka Y.
      • Furuyashiki T.
      • Bito H.
      • Ushikubi F.
      • Tanaka Y.
      • Kobayashi T.
      • Muro S.
      • Satoh N.
      • Kayahara T.
      • Higashi M.
      • Mizoguchi A.
      • Shichi H.
      • Fukuda Y.
      • Nakao K.
      • Narumiya S.
      ).
      Carrageenan-induced Edema of the Paw Causes an Up-regulation of COX-2 and a General Increase in Prostanoids in the CNS during the Early Phase of Inflammation—The up-regulation of expression of COX-2 mRNA in the CNS was rapid, detectable at the first 1-h time point, and was more pronounced in the spinal cord than in whole brain tissue extracts. In both tissues the elevation of COX-2 was accompanied by an increase in PGE2 (2-5-fold) and a more modest increase in PGD2, PGF, TXB2, and 6-keto-PGF during the early phase. PGF and PGD2 have also been implicated in centrally mediated pain responses. It has been shown that the intrathecal administration of PGE2 and PGF at low doses produced hyperalgesia to mechanical stimuli in rats (
      • Turnbach M.E.
      • Spraggins D.S.
      • Randich A.
      ). These two prostanoids have also been reported to cause touch-evoked allodynia in mice (
      • Minami T.
      • Uda R.
      • Horiguchi S.
      • Ito S.
      • Hyodo M.
      • Hayaishi O.
      ,
      • Minami T.
      • Uda R.
      • Horiguchi S.
      • Ito S.
      • Hyodo M.
      • Hayaishi O.
      ) but have much weaker effects in rats (
      • Turnbach M.E.
      • Spraggins D.S.
      • Randich A.
      ), which may reflect species differences in prostanoid responses. In mice, there is strong evidence that PGD2 plays a role in nociception (
      • Horiguchi S.
      • Ueno R.
      • Hyodo M.
      • Hayaishi O.
      ) and is essential for PGE2-induced allodynia (
      • Eguchi N.
      • Minami T.
      • Shirafuji N.
      • Kanaoka Y.
      • Tanaka T.
      • Nagata A.
      • Yoshida N.
      • Urade Y.
      • Ito S.
      • Hayaishi O.
      ). In the present study with rats, PGD2 was the most abundant prostanoid in brain extracts, consistent with previous reports (
      • Abdel-Halim M.S.
      • Lunden I.
      • Cseh G.
      • Anggard E.
      ), and the increase in PGD2 in the brain during the early phase of inflammation was statistically significant compared with both time 0 and saline controls. Although the level of stimulation was very low, the maximal levels reached in the spinal cord during the early phase were 1.3 ± 0.3 ng/mg protein for PGD2 and 0.28 ± 0.04 ng/mg protein for PGF as compared with 0.6 ± 0.1 ng/mg protein for PGE2. Thus it is possible that both PGD2 and PGF, in addition to PGE2, contribute to pain responses in carrageenan-induced inflammation. In the CSF where the amounts of prostanoids are extremely low in untreated animals, marked increases in PGD2, TXB2, and 6-keto-PGF were detected during the early phase. These prostanoids were also found to increase in the CNS after kainate-induced COX-2 expression (
      • Ciceri P.
      • Zhang Y.
      • Shaffer A.F.
      • Leahy K.M.
      • Woerner M.B.
      • Smith W.G.
      • Seibert K.
      • Isakson P.C.
      ) and neuronal COX-2 overexpression (
      • Vidensky S.
      • Zhang Y.
      • hand T.
      • Goellner J.
      • Shaffer A.
      • Isakson P.
      • Andreasson K.
      ). All these observations are consistent with the present data suggesting that COX-2 plays a role in the activation of prostanoid synthetic pathways other than the PGE2 pathway in the CNS. Although 6-keto-PGF, TXB2, and PGD2 were all found to increase in the CSF, no increase in the corresponding terminal synthases could be detected. These data are consistent with the up-regulation of COX-2 resulting in an increased synthesis of the PGH2 substrate available for each of the prostacyclin, TX, and PGD synthetic pathways during the acute phase of inflammation.
      Peripheral Inflammation Causes a Pronounced Elevation of PGE2 and the Selective Induction of mPGES-1 in the CNS—As compared with the other prostanoids measured, PGE2 showed the largest induction in the spinal cord and reached the highest levels in the CSF among the prostanoids tested (by ∼3-fold) during the early phase. In contrast to the large accumulation of PGE2 observed for the paw, PGE2 levels in the CNS decreased after the peak at 6 h (but were still slightly higher in the spinal cord and in the CSF than in the corresponding saline controls at 24 h). The present data indicate that this elevation in PGE2 correlated with the marked and selective induction of mPGES-1 in the spinal cord. In whole brain extracts, a large increase of mPGES-1 also occurred, but the increases in PGE2 and COX-2 were less pronounced.
      COX-2 induction in the CNS has been proposed to result from the afferent neuronal input and from an increase in circulating of pro-inflammatory cytokines. Strong evidence that IL-1β, whose levels are highly up-regulated in the paw and in the CSF following injection of the paw with Freund adjuvant, represents a major mediator of COX-2 induction in the CNS (
      • Samad T.A.
      • Moore K.A.
      • Sapirstein A.
      • Billet S.
      • Allchorne A.
      • Poole S.
      • Bonventre J.V.
      • Woolf C.J.
      ). The inducibility of mPGES-1 has been extensively characterized in rat brain following treatment with IL-1β or lipopolysaccharide (
      • Yamagata K.
      • Matsumura K.
      • Inoue W.
      • Shiraki T.
      • Suzuki K.
      • Yasuda S.
      • Sugiura H.
      • Cao C.
      • Watanabe Y.
      • Kobayashi S.
      ,
      • Inoue W.
      • Matsumura K.
      • Yamagata K.
      • Takemiya T.
      • Shiraki T.
      • Kobayashi S.
      ,
      • Ek M.
      • Engblom D.
      • Saha S.
      • Blomqvist A.
      • Jakobsson P.J.
      • Ericsson-Dahlstrand A.
      ,
      • Schuligoi R.
      • Ulcar R.
      • Peskar B.A.
      • Amann R.
      ,
      • Ivanov A.I.
      • Pero R.S.
      • Scheck A.C.
      • Romanovsky A.A.
      ) and during adjuvant-induced arthritis (
      • Engblom D.
      • Ek M.
      • Andersson I.M.
      • Saha S.
      • Dahlstrom M.
      • Jakobsson P.J.
      • Ericsson-Dahlstrand A.
      • Blomqvist A.
      ). In these studies, the peak of expression of COX-2 preceded that of mPGES-1 following the induction of fever with lipopolysaccharide or the intravenous administration of IL-1β (
      • Samad T.A.
      • Moore K.A.
      • Sapirstein A.
      • Billet S.
      • Allchorne A.
      • Poole S.
      • Bonventre J.V.
      • Woolf C.J.
      ,
      • Inoue W.
      • Matsumura K.
      • Yamagata K.
      • Takemiya T.
      • Shiraki T.
      • Kobayashi S.
      ,
      • Ek M.
      • Engblom D.
      • Saha S.
      • Blomqvist A.
      • Jakobsson P.J.
      • Ericsson-Dahlstrand A.
      ). The characteristics of the present model are thus consistent with an IL-1 mediated process where both COX-2 and mPGES-1 are rapidly induced in sequence to contribute the maximal PGE2 production (at 6 h) and elevated levels throughout the inflammation response. PGE2 in the spinal cord can contribute to potentiation of peripheral edema (
      • Daher J.B.
      • Tonussi C.R.
      ), to the enhanced neuron hyperexcitability (
      • Vasquez E.
      • Bar K.J.
      • Ebersberger A.
      • Klein B.
      • Vanegas H.
      • Schaible H.G.
      ), and to hyperalgesia (
      • Hosoi M.
      • Oka T.
      • Hori T.
      ,
      • Ibuki T.
      • Matsumura K.
      • Yamazaki Y.
      • Nozaki T.
      • Tanaka Y.
      • Kobayashi S.
      ).
      Prostacyclin Levels Are Elevated in the CNS during the Early Phase Carrageenan-induced Paw Edema without Significant Up-regulation of PGIS—Studies with the IP receptor knock-out mice have suggested a role for prostacyclin in this model (
      • Murata T.
      • Ushikubi F.
      • Matsuoka T.
      • Hirata M.
      • Yamasaki A.
      • Sugimoto Y.
      • Ichikawa A.
      • Aze Y.
      • Tanaka T.
      • Yoshida N.
      • Ueno A.
      • Oh-Ishi S.
      • Narumiya S.
      ). We have not detected any significant increase in the stable prostacyclin metabolite 6-keto-PGF in inflamed paws. In another model of inflammation in the rat (adjuvant-induced arthritis), 6-keto-PGF was found to show a transient increase in the paw (
      • Claveau D.
      • Sirinyan M.
      • Guay J.
      • Gordon R.
      • Chan C.C.
      • Bureau Y.
      • Riendeau D.
      • Mancini J.A.
      ) and to be elevated in the spinal cord during chronic inflammation (
      • Hay C.H.
      • Trevethick M.A.
      • Wheeldon A.
      • Bowers J.S.
      • de Belleroche J.S.
      ). In the early phase of carrageenan-induced edema we observed small increases in 6-keto-PGF in the brain and the spinal cord comparable with those observed for the other prostanoids. Interestingly, the increase in 6-keto-PGF in the CSF appears to occur earlier than that of PGD2 and TXB2. In whole extracts of brain, spinal cord, or paw tissues, no major change of PGIS expression (<2-fold) was detected in contrast to mPGES-1. The increase in 6-keto-PGF detected in the CNS thus appears to reflect the increase in COX-2 activity leading to an increase in PGH2 substrate availability for the different prostaglandins, prostacyclin, and TX pathways. It has been shown recently (
      • Doi Y.
      • Minami T.
      • Nishizawa M.
      • Mabuchi T.
      • Mori H.
      • Ito S.
      ) that the expression of the IP receptor increases in the spinal cord and that the IP receptor agonist cicaprost induces mechanical hyperalgesia in the inflamed paw, suggesting of a role for PGI2 in acute inflammation.
      In summary, we have found that the induction of COX-2 that occurs in the CNS during carrageenan-induced paw inflammation leads to an increase in PGs, prostacyclin, and TX in the early phase and to a large increase in PGE2 production associated with selective up-regulation of mPGES-1. The results of this study provide further evidence for the implication of mPGES-1 as an important terminal enzyme for COX-2-mediated synthesis of inflammatory PGE2 not only at the site of inflammation but also in the CNS. The data from this study, along with other observations on the induction of the mPGES-1 during fever and adjuvant-induced arthritis, and on the profile of the mPGES-1 knock-out mice showing reduced sensitivity to collagen-induced arthritis are all consistent with the proposals that mPGES-1 plays a role in inflammatory responses and represents a potential therapeutic target for novel anti-inflammatory agents.

      Acknowledgments

      We thank David Claveau (Merck Frosst) for the supply of primers for quantitative PCR analysis and technical support in RNA analysis, Chi-Chung Chan and the staff of the Department of Comparative Medicine (Merck Frosst) for their support for the in vivo studies, and Bill Pikounis from the Department of Biometrics Research (Merck) for his help on the statistical analysis of the data.

      REFERENCES

        • Ichitani Y.
        • Shi T.
        • Haeggstrom J.Z.
        • Samuelsson B.
        • Hokfelt T.
        Neuroreport. 1997; 8: 2949-2952
        • Smith C.J.
        • Zhang Y.
        • Koboldt C.M.
        • Muhammad J.
        • Zweifel B.S.
        • Shaffer A.
        • Talley J.J.
        • Masferrer J.L.
        • Seibert K.
        • Isakson P.C.
        Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13313-13318
        • Dirig D.M.
        • Isakson P.C.
        • Yaksh T.L.
        J. Pharmacol. Exp. Ther. 1998; 285: 1031-1038
        • Zhang Y.
        • Shaffer A.
        • Portanova J.
        • Seibert K.
        • Isakson P.C.
        J. Pharmacol. Exp. Ther. 1997; 283: 1069-1075
        • Riendeau D.
        • Percival M.D.
        • Boyce S.
        • Brideau C.
        • Charleson S.
        • Cromlish W.
        • Ethier D.
        • Evans J.
        • Falgueyret J.P.
        • Ford-Hutchinson A.W.
        • Gordon R.
        • Greig G.
        • Gresser M.
        • Guay J.
        • Kargman S.
        • Leger S.
        • Mancini J.A.
        • O'Neill G.
        • Ouellet M.
        • Rodger I.W.
        • Therien M.
        • Wang Z.
        • Webb J.K.
        • Wong E.
        • Chan C.C.
        Br. J. Pharmacol. 1997; 121: 105-117
        • Daher J.B.
        • Tonussi C.R.
        Brain Res. 2003; 962: 207-212
        • Hosoi M.
        • Oka T.
        • Hori T.
        Pain. 1997; 71: 303-311
        • Samad T.A.
        • Moore K.A.
        • Sapirstein A.
        • Billet S.
        • Allchorne A.
        • Poole S.
        • Bonventre J.V.
        • Woolf C.J.
        Nature. 2001; 410: 471-475
        • Yamamoto T.
        • Nozaki-Taguchi N.
        Neuroreport. 1997; 8: 2179-2182
        • Yaksh T.L.
        • Dirig D.M.
        • Conway C.M.
        • Svensson C.
        • Luo Z.D.
        • Isakson P.C.
        J. Neurosci. 2001; 21: 5847-5853
        • Minami T.
        • Nakano H.
        • Kobayashi T.
        • Sugimoto Y.
        • Ushikubi F.
        • Ichikawa A.
        • Narumiya S.
        • Ito S.
        Br. J. Pharmacol. 2001; 133: 438-444
        • Murata T.
        • Ushikubi F.
        • Matsuoka T.
        • Hirata M.
        • Yamasaki A.
        • Sugimoto Y.
        • Ichikawa A.
        • Aze Y.
        • Tanaka T.
        • Yoshida N.
        • Ueno A.
        • Oh-Ishi S.
        • Narumiya S.
        Nature. 1997; 388: 678-682
        • Ueno A.
        • Naraba H.
        • Ikeda Y.
        • Ushikubi F.
        • Murata T.
        • Narumiya S.
        • Oh-Ishi S.
        Life Sci. 2000; 66: L155-L160
        • Catella-Lawson F.
        • McAdam B.
        • Morrison B.W.
        • Kapoor S.
        • Kujubu D.
        • Antes L.
        • Lasseter K.C.
        • Quan H.
        • Gertz B.J.
        • FitzGerald G.A.
        J. Pharmacol. Exp. Ther. 1999; 289: 735-741
        • Jakobsson P.J.
        • Thoren S.
        • Morgenstern R.
        • Samuelsson B.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7220-7225
        • Ujihara M.
        • Tsuchida S.
        • Satoh K.
        • Sato K.
        • Urade Y.
        Arch. Biochem. Biophys. 1988; 264: 428-437
        • Tanioka T.
        • Nakatani Y.
        • Semmyo N.
        • Murakami M.
        • Kudo I.
        J. Biol. Chem. 2000; 275: 32775-32782
        • Tanikawa N.
        • Ohmiya Y.
        • Ohkubo H.
        • Hashimoto K.
        • Kangawa K.
        • Kojima M.
        • Ito S.
        • Watanabe K.
        Biochem. Biophys. Res. Commun. 2002; 291: 884-889
        • Murakami M.
        • Kudo I.
        Prog. Lipid Res. 2004; 43: 3-35
        • Murakami M.
        • Naraba H.
        • Tanioka T.
        • Semmyo N.
        • Nakatani Y.
        • Kojima F.
        • Ikeda T.
        • Fueki M.
        • Ueno A.
        • Oh S.
        • Kudo I.
        J. Biol. Chem. 2000; 275: 32783-32792
        • Stichtenoth D.O.
        • Thoren S.
        • Bian H.
        • Peters-Golden M.
        • Jakobsson P.J.
        • Crofford L.J.
        J. Immunol. 2001; 167: 469-474
        • Han R.
        • Tsui S.
        • Smith T.J.
        J. Biol. Chem. 2002; 277: 16355-16364
        • Yamagata K.
        • Matsumura K.
        • Inoue W.
        • Shiraki T.
        • Suzuki K.
        • Yasuda S.
        • Sugiura H.
        • Cao C.
        • Watanabe Y.
        • Kobayashi S.
        J. Neurosci. 2001; 21: 2669-2677
        • Inoue W.
        • Matsumura K.
        • Yamagata K.
        • Takemiya T.
        • Shiraki T.
        • Kobayashi S.
        Neurosci. Res. 2002; 44: 51-61
        • Mancini J.A.
        • Blood K.
        • Guay J.
        • Gordon R.
        • Claveau D.
        • Chan C.C.
        • Riendeau D.
        J. Biol. Chem. 2001; 276: 4469-4475
        • Claveau D.
        • Sirinyan M.
        • Guay J.
        • Gordon R.
        • Chan C.C.
        • Bureau Y.
        • Riendeau D.
        • Mancini J.A.
        J. Immunol. 2003; 170: 4738-4744
        • Ek M.
        • Engblom D.
        • Saha S.
        • Blomqvist A.
        • Jakobsson P.J.
        • Ericsson-Dahlstrand A.
        Nature. 2001; 410: 430-431
        • Uematsu S.
        • Matsumoto M.
        • Takeda K.
        • Akira S.
        J. Immunol. 2002; 168: 5811-5816
        • Trebino C.E.
        • Stock J.L.
        • Gibbons C.P.
        • Naiman B.M.
        • Wachtmann T.S.
        • Umland J.P.
        • Pandher K.
        • Lapointe J.M.
        • Saha S.
        • Roach M.L.
        • Carter D.
        • Thomas N.A.
        • Durtschi B.A.
        • McNeish J.D.
        • Hambor J.E.
        • Jakobsson P.J.
        • Carty T.J.
        • Perez J.R.
        • Audoly L.P.
        Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 9044-9049
        • Engblom D.
        • Ek M.
        • Andersson I.M.
        • Saha S.
        • Dahlstrom M.
        • Jakobsson P.J.
        • Ericsson-Dahlstrand A.
        • Blomqvist A.
        J. Comp. Neurol. 2002; 452: 205-214
        • Chan C.C.
        • Boyce S.
        • Brideau C.
        • Ford-Hutchinson A.W.
        • Gordon R.
        • Guay D.
        • Hill R.G.
        • Li C.S.
        • Mancini J.
        • Penneton M.
        • Prasit P.
        • Rasori R.
        • Riendeau D.
        • Roy P.
        • Tagari P.
        • Vickers P.
        • Wong E.
        • Roger I.W.
        J. Pharmacol. Exp. Ther. 1995; 274: 1531-1537
        • Ueno A.
        • Matsumoto H.
        • Naraba H.
        • Ikeda Y.
        • Ushikubi F.
        • Matsuoka T.
        • Narumiya S.
        • Sugimoto Y.
        • Ichikawa A.
        • Oh-Ishi S.
        Biochem. Pharmacol. 2001; 62: 157-160
        • Ibuki T.
        • Matsumura K.
        • Yamazaki Y.
        • Nozaki T.
        • Tanaka Y.
        • Kobayashi S.
        J. Neurochem. 2003; 86: 318-328
        • Seibert K.
        • Zhang Y.
        • Leahy K.
        • Hauser S.
        • Masferrer J.
        • Perkins W.
        • Lee L.
        • Isakson P.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12013-12017
        • Nantel F.
        • Denis D.
        • Gordon R.
        • Northey A.
        • Cirino M.
        • Metters K.M.
        • Chan C.C.
        Br. J. Pharmacol. 1999; 128: 853-859
        • Kojima F.
        • Naraba H.
        • Sasaki Y.
        • Okamoto R.
        • Koshino T.
        • Kawai S.
        J. Rheumatol. 2002; 29: 1836-1842
        • Moore A.H.
        • Olschowka J.A.
        • O'Banion M.K.
        J. Neuroimmunol. 2004; 148: 32-40
        • Hu J.
        • Meng Q.
        • Roy S.K.
        • Raha A.
        • Hu J.
        • Zhang J.
        • Hashimoto K.
        • Kalvakolanu D.V.
        J. Biol. Chem. 2002; 277: 30253-30263
        • Matsuoka Y.
        • Furuyashiki T.
        • Bito H.
        • Ushikubi F.
        • Tanaka Y.
        • Kobayashi T.
        • Muro S.
        • Satoh N.
        • Kayahara T.
        • Higashi M.
        • Mizoguchi A.
        • Shichi H.
        • Fukuda Y.
        • Nakao K.
        • Narumiya S.
        Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4132-4137
        • Turnbach M.E.
        • Spraggins D.S.
        • Randich A.
        Pain. 2002; 97: 33-45
        • Minami T.
        • Uda R.
        • Horiguchi S.
        • Ito S.
        • Hyodo M.
        • Hayaishi O.
        Pain. 1992; 50: 223-229
        • Minami T.
        • Uda R.
        • Horiguchi S.
        • Ito S.
        • Hyodo M.
        • Hayaishi O.
        Pain. 1994; 57: 217-223
        • Horiguchi S.
        • Ueno R.
        • Hyodo M.
        • Hayaishi O.
        Eur. J. Pharmacol. 1986; 122: 173-179
        • Eguchi N.
        • Minami T.
        • Shirafuji N.
        • Kanaoka Y.
        • Tanaka T.
        • Nagata A.
        • Yoshida N.
        • Urade Y.
        • Ito S.
        • Hayaishi O.
        Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 726-730
        • Abdel-Halim M.S.
        • Lunden I.
        • Cseh G.
        • Anggard E.
        Prostaglandins. 1980; 19: 249-258
        • Ciceri P.
        • Zhang Y.
        • Shaffer A.F.
        • Leahy K.M.
        • Woerner M.B.
        • Smith W.G.
        • Seibert K.
        • Isakson P.C.
        J. Pharmacol. Exp. Ther. 2002; 302: 846-852
        • Vidensky S.
        • Zhang Y.
        • hand T.
        • Goellner J.
        • Shaffer A.
        • Isakson P.
        • Andreasson K.
        Neuromolecular Med. 2003; 3: 15-28
        • Schuligoi R.
        • Ulcar R.
        • Peskar B.A.
        • Amann R.
        Neuroscience. 2003; 116: 1043-1052
        • Ivanov A.I.
        • Pero R.S.
        • Scheck A.C.
        • Romanovsky A.A.
        Am. J. Physiol. 2002; 283: R1104-R1117
        • Vasquez E.
        • Bar K.J.
        • Ebersberger A.
        • Klein B.
        • Vanegas H.
        • Schaible H.G.
        J. Neurosci. 2001; 21: 9001-9008
        • Hay C.H.
        • Trevethick M.A.
        • Wheeldon A.
        • Bowers J.S.
        • de Belleroche J.S.
        Neuroscience. 1997; 78: 843-850
        • Doi Y.
        • Minami T.
        • Nishizawa M.
        • Mabuchi T.
        • Mori H.
        • Ito S.
        Neuroreport. 2002; 13: 93-96