The p21-activated kinase PAK 5 is involved in formalin-induced nociception through regulation of MAP-kinase signaling and formalin-specific receptors.

p21-activated kinases (PAKs) are involved in signal cascades relevant for nociceptive processing and neuropathic pain. Particularly, the recently described group B PAKs 4, 5 and 6 regulate MAP-kinases and the rearrangement of the actin cytoskeleton, both of which have been linked to pain processing. However, a specific role of these PAKs in nociception has not yet been demonstrated. We found PAK 4, 5 and 6 expression in pain-relevant tissues in peripheral and CNS. Since viable knock-out mice only exist for the PAK isoform 5, we further assessed the impact of this PAK on acute and chronic pain using different behavioral models in mice. PAK 5 knock-out mice showed normal acute nociception and did not differ from wild type mice in their neuropathic pain behavior. However, the nociceptive response in formalin-induced paw inflammation was significantly reduced in knock-out mice associated with inhibition of MAP-kinase activation and a decreased number of formalin-induced c-Fos positive neurons in the spinal cord. Furthermore, in isolated neurons, we found a significantly reduced calcium response after stimulation of TRPA1-channels in PAK 5(-/-)- compared to PAK 5(+/+)-cells. Our results indicate that PAK 5 is involved in formalin-induced inflammatory nociception through regulation of MAPK-induced c-Fos-activation and formalin-specific TRP-channels.

Analysis of sphingolipid and prostaglandin synthesis during zymosan-induced inflammation.

Sphingosine-1-phosphate (S1P) is generated through phosphorylation of sphingosine by two sphingosine kinases (SPHK-1 and -2). As extra- and intracellular messenger S1P fulfils multiple roles in inflammation such as mediating proinflammatory inputs or acting as chemoattractant. In addition, S1P induces cyclooxygenase-2 (COX-2) expression and the synthesis of proinflammatory prostanoids in several cell types. Here, we analysed in vivo the regulation of S1P level as well as potential interactions between S1P and COX-dependent prostaglandin synthesis during zymosan-induced inflammation. S1P and prostanoid levels were determined in the blood and at the site of inflammation under basal conditions and during zymosan-induced inflammation using wild type and SPHK-1 and -2 knockout mice. We found that alterations in S1P levels did not correlate with changes in plasma- or tissue-concentrations of the prostanoids as well as COX-2 expression. In the inflamed tissue S1P and prostanoid concentrations were reciprocally regulated. Prostaglandin levels increased over 6h, while S1P and sphingosine level decreased during the same time, which makes an induction of prostanoid synthesis by S1P in zymosan-induced inflammation unlikely. Additionally, despite altered S1P levels wild type and SPHK knockout mice showed similar behavioural nociceptive responses and oedema sizes suggesting minor functions of S1P in this inflammatory model.

Soluble epoxide hydrolase limits mechanical hyperalgesia during inflammation.

BACKGROUND: Cytochrome-P450 (CYP450) epoxygenases metabolise arachidonic acid (AA) into four different biologically active epoxyeicosatrienoic acid (EET) regioisomers. Three of the EETs (i.e., 8,9-, 11,12- and 14,15-EET) are rapidly hydrolysed by the enzyme soluble epoxide hydrolase (sEH). Here, we investigated the role of sEH in nociceptive processing during peripheral inflammation. RESULTS: In dorsal root ganglia (DRG), we found that sEH is expressed in medium and large diameter neurofilament 200-positive neurons. Isolated DRG-neurons from sEH(-/-) mice showed higher EET and lower DHET levels. Upon AA stimulation, the largest changes in EET levels occurred in culture media, indicating both that cell associated EET concentrations quickly reach saturation and EET-hydrolyzing activity mostly effects extracellular EET signaling. In vivo, DRGs from sEH-deficient mice exhibited elevated 8,9-, 11,12- and 14,15-EET-levels. Interestingly, EET levels did not increase at the site of zymosan-induced inflammation. Cellular imaging experiments revealed direct calcium flux responses to 8,9-EET in a subpopulation of nociceptors. In addition, 8,9-EET sensitized AITC-induced calcium increases in DRG neurons and AITC-induced calcitonin gene related peptide (CGRP) release from sciatic nerve axons, indicating that 8,9-EET sensitizes TRPA1-expressing neurons, which are known to contribute to mechanical hyperalgesia. Supporting this, sEH(-/-) mice showed increased nociceptive responses to mechanical stimulation during zymosan-induced inflammation and 8,9-EET injection reduced mechanical thresholds in naive mice. CONCLUSION: Our results show that the sEH can regulate mechanical hyperalgesia during inflammation by inactivating 8,9-EET, which sensitizes TRPA1-expressing nociceptors. Therefore we suggest that influencing the CYP450 pathway, which is actually highly considered to treat cardiovascular diseases, may cause pain side effects.

Anti-inflammatory role of microsomal prostaglandin E synthase-1 in a model of neuroinflammation.

A major immunological response during neuroinflammation is the activation of microglia, which subsequently release proinflammatory mediators such as prostaglandin E(2) (PGE(2)). Besides its proinflammatory properties, cyclooxygenase-2 (COX-2)-derived PGE(2) has been shown to exhibit anti-inflammatory effects on innate immune responses. Here, we investigated the role of microsomal PGE(2) synthase-1 (mPGES-1), which is functionally coupled to COX-2, in immune responses using a model of lipopolysaccharide (LPS)-induced spinal neuroinflammation. Interestingly, we found that activation of E-prostanoid (EP)2 and EP4 receptors, but not EP1, EP3, PGI(2) receptor (IP), thromboxane A(2) receptor (TP), PGD(2) receptor (DP), and PGF(2) receptor (FP), efficiently blocked LPS-induced tumor necrosis factor α (TNFα) synthesis and COX-2 and mPGES-1 induction as well as prostaglandin synthesis in spinal cultures. In vivo, spinal EP2 receptors were up-regulated in microglia in response to intrathecally injected LPS. Accordingly, LPS priming reduced spinal synthesis of TNFα, interleukin 1ß (IL-1ß), and prostaglandins in response to a second intrathecal LPS injection. Importantly, this reduction was only seen in wild-type but not in mPGES-1-deficient mice. Furthermore, intrathecal application of EP2 and EP4 agonists as well as genetic deletion of EP2 significantly reduced spinal TNFα and IL-1ß synthesis in mPGES-1 knock-out mice after LPS priming. These data suggest that initial inflammation prepares the spinal cord for a negative feedback regulation by mPGES-1-derived PGE(2) followed by EP2 activation, which limits the synthesis of inflammatory mediators during chronic inflammation. Thus, our data suggest a role of mPGES-1-derived PGE(2) in resolution of neuroinflammation.

The ubiquitin ligase MYCBP2 regulates transient receptor potential vanilloid receptor 1 (TRPV1) internalization through inhibition of p38 MAPK signaling.

The E3 ubiquitin ligase MYCBP2 negatively regulates neuronal growth, synaptogenesis, and synaptic strength. More recently it was shown that MYCBP2 is also involved in receptor and ion channel internalization. We found that mice with a MYCBP2-deficiency in peripheral sensory neurons show prolonged thermal hyperalgesia. Loss of MYCBP2 constitutively activated p38 MAPK and increased expression of several proteins involved in receptor trafficking. Surprisingly, loss of MYCBP2 inhibited internalization of transient receptor potential vanilloid receptor 1 (TRPV1) and prevented desensitization of capsaicin-induced calcium increases. Lack of desensitization, TRPV internalization and prolonged hyperalgesia were reversed by inhibition of p38 MAPK. The effects were TRPV-specific, since neither mustard oil-induced desensitization nor behavioral responses to mechanical stimuli were affected. In summary, we show here for the first time that p38 MAPK activation can inhibit activity-induced ion channel internalization and that MYCBP2 regulates internalization of TRPV1 in peripheral sensory neurons as well as duration of thermal hyperalgesia through p38 MAPK.

Cofilin phosphorylation is involved in nitric oxide/cGMP-mediated nociception.

There is convincing evidence that nitric oxide (NO), cGMP and cGMP-dependent protein kinase I (PKG-I) are involved in the development of hyperalgesia in response to noxious stimuli. However, downstream target proteins contributing to nociception have not been completely identified so far. Several reports indicate a role of the NO/cGMP/PKG cascade in the regulation of neurite outgrowth which is suggested to be involved in specific mechanisms of nociception. Since neurite outgrowth is strongly dependent on modulation of cytoskeleton proteins we were interested in the impact of PKG-I activation on the actin cytoskeleton and its role in inflammatory hyperalgesia. Therefore we investigated the actin-destabilising protein cofilin and its NO-dependent effects in vitro in primary neuronal cultures as well as in vivo in the zymosan-induced paw inflammation model in rats. In primary neurons from rats, treatment with the PKG-I activator 8-Br-cGMP induced a time-dependent phosphorylation of cofilin and significantly increased neurite outgrowth. Further functional analysis revealed that the underlying signal transduction pathways involve activation of the Rho-GTPases RhoA, Rac1 and Cdc42 and their corresponding downstream targets Rho-kinase (ROCK) and p21-activated kinase (PAK). In vivo, treatment of rats with the NO-synthase inhibitor l-NAME and the ROCK-inhibitor Y-27632, respectively, led to a significant decrease of cofilin phosphorylation in the spinal cord and resulted in antinociceptive effects in a model of inflammatory hyperalgesia. Our results suggest that cofilin represents a downstream target of NO/cGMP/PKG signal transduction in neurons thus indicating that it is involved in NO-mediated nociception.

Toponomics analysis of drug-induced changes in arachidonic acid-dependent signaling pathways during spinal nociceptive processing.

Multi-Epitope-Ligand-Carthography (MELC) allows consecutive immunohistochemical visualization of up to 100 proteins on the same tissue sample. Subsequent biomathematical analysis of these images allows a quantitative description of changes in protein networks. We used the MELC technology to study the effect of the nonopioid analgesic drug dipyrone on protein network profiles associated with arachidonic acid-dependent signaling pathways. MELC analysis with 31 different fluorescence-labeled tags was used to compare the effect of dipyrone on protein networks in spinal cords of mice with zymosan-induced hyperalgesia, a common model for inflammatory pain. We found that the number of motifs which describe the colocalization of 5-lipoxygenase (5-LO) or 12-LO with other proteins increased disproportionally after dipyrone treatment. Activation of 5-LO and 12-LO induces their translocation to membrane compartments which was also reflected by MELC results. Although no changes in 5-LO or 12-LO expression were seen by Western blot analysis or by immunohistochemistry in spinal cords of dipyrone-treated mice, the activation of both enzymes was verified by determining LO-products. Spinal amounts of 5(S)-hydroxyeicosatetraenoic acid (HETE) and 12(S)-HETE, which are generated by 5-LO and 12-LO, respectively, were significantly increased in spinal cords of dipyrone-treated animals. In primary spinal cord neurons, dipyrone selectively and dose-dependently increased 5(S)-(HETE) and 12(S)-HETE synthesis. Thus, we show for the first time that monitoring protein network profiles by topological proteomic analysis is a useful tool to identify mechanisms of drug actions.

New analgesics synthetically derived from the paracetamol metabolite N-(4-hydroxyphenyl)-(5Z,8Z,11Z,14Z)-icosatetra-5,8,11,14-enamide.

N-(4-hydroxyphenyl)-(5Z,8Z,11Z,14Z)-icosatetra-5,8,11,14-enamide (AM404) is a metabolite of the well-known analgesic paracetamol. AM404 inhibits endocannabinoid cellular uptake, binds weakly to CB1 and CB2 cannabinoid receptors, and is formed by fatty acid amide hydrolase (FAAH) in vivo. We prepared three derivatives of this new (endo)cannabinoid using bioisosteric replacement (1), homology (2), and derivatization (3) of the 4-aminophenol moiety in AM404 and tested them against CB1, CB2, and FAAH. We found affinities toward both cannabinoid receptors equal to or greater than that of AM404. Shortening the acyl chain from C20 to C2 led to three new paracetamol analogues: N-(1H-indazol-5-yl)acetamide (5), N-(4-hydroxybenzyl)acetamide (6), and N-(4-hydroxy-3-methoxyphenyl)acetamide (7). Again, 5, 6, and 7 were tested against CB1, CB2, and FAAH without significant activity. However, 5 and 7 behaved like inhibitors of cyclooxygenases in whole blood assays. Finally, 5 (50 mg/kg) and 6 (275 mg/kg) displayed analgesic activities comparable to paracetamol (200 mg/kg) in the mouse formalin test.

Sphingosine 1-phosphate modulates spinal nociceptive processing.

Sphingosine 1-Phosphate (S1P) modulates various cellular functions such as apoptosis, cell differentiation, and migration. Although S1P is an abundant signaling molecule in the central nervous system, very little is known about its influence on neuronal functions. We found that S1P concentrations were selectively decreased in the cerebrospinal fluid of adult rats in an acute and an inflammatory pain model. Pharmacological inhibition of sphingosine kinases (SPHK) decreased basal pain thresholds and SphK2 knock-out mice, but not SphK1 knock-out mice, had a significant decrease in withdrawal latency. Intrathecal application of S1P or sphinganine 1-phosphate (dihydro-S1P) reduced the pain-related (nociceptive) behavior in the formalin assay. S1P and dihydro-S1P inhibited cyclic AMP (cAMP) synthesis, a key second messenger of spinal nociceptive processing, in spinal cord neurons. By combining fluorescence resonance energy transfer (FRET)-based cAMP measurements with Multi Epitope Ligand Cartography (MELC), we showed that S1P decreased cAMP synthesis in excitatory dorsal horn neurons. Accordingly, intrathecal application of dihydro-S1P abolished the cAMP-dependent phosphorylation of NMDA receptors in the outer laminae of the spinal cord. Taken together, the data show that S1P modulates spinal nociceptive processing through inhibition of neuronal cAMP synthesis.

Toponomics analysis of functional interactions of the ubiquitin ligase PAM (Protein Associated with Myc) during spinal nociceptive processing.

Protein associated with Myc (PAM) is a giant E3 ubiquitin ligase of 510 kDa. Although the role of PAM during neuronal development is well established, very little is known about its function in the regulation of synaptic strength. Here we used multiepitope ligand cartography (MELC) to study protein network profiles associated with PAM during the modulation of synaptic strength. MELC is a novel imaging technology that utilizes biomathematical tools to describe protein networks after consecutive immunohistochemical visualization of up to 100 proteins on the same sample. As an in vivo model to modulate synaptic strength we used the formalin test, a common model for acute and inflammatory pain. MELC analysis was performed with 37 different antibodies or fluorescence tags on spinal cord slices and led to the identification of 1390 PAM-related motifs that distinguish untreated and formalin-treated spinal cords. The majority of these motifs related to ubiquitin-dependent processes and/or the actin cytoskeleton. We detected an intermittent colocalization of PAM and ubiquitin with TSC2, a known substrate of PAM, and the glutamate receptors mGluR5 and GLUR1. Importantly these complexes were detected exclusively in the presence of F-actin. A direct PAM/F-actin interaction was confirmed by colocalization and cosedimentation. The binding of PAM toward F-actin varied strongly between the PAM splice forms found in rat spinal cords. PAM did not ubiquitylate actin or alter actin polymerization and depolymerization. However, F-actin decreased the ubiquitin ligase activity of purified PAM. Because PAM activation is known to involve its translocation, the binding of PAM to F-actin may serve to control its subcellular localization as well as its activity. Taken together we show that defining protein network profiles by topological proteomics analysis is a useful tool to identify previously unknown protein/protein interactions that underlie synaptic processes.

Celecoxib inhibits 5-lipoxygenase.

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor used in the therapy of inflammatory and painful conditions. Various COX-2-independent pharmacological effects, such as a chemo-preventive and tumor-regressive activity have been suggested, but the respective non-COX-2 targets of celecoxib are still a matter of research. We now demonstrate that celecoxib inhibits 5-lipoxygenase (5-LO), a key enzyme in leukotriene (LT) biosynthesis. Celecoxib suppressed 5-LO product formation in ionophore A23187-activated human polymorphonuclear leukocytes (IC(50) approximately 8 microM). Similarly, celecoxib inhibited LTB(4) formation in human whole blood (IC(50) approximately 27.3 microM). Direct interference of 5-LO with celecoxib was visualized by inhibition of enzyme catalysis both in cell homogenates and with purified 5-LO (IC(50) approximately 23.4 and 24.9 microM, respectively). Related lipoxygenases (12-LO and 15-LO) were not affected by celecoxib. Other COX-2 inhibitors (etoricoxib and rofecoxib) or unselective NSAIDs (non-steroidal anti-inflammatory drugs, diclofenac) failed to inhibit 5-LO. In rats which received celecoxib (i.p.), the blood LTB(4) levels were dose-dependently reduced with an ED(50) value approximately 35.2 mg/kg. Together, celecoxib is a direct inhibitor of 5-LO in vitro and in vivo. These findings provide a potential molecular basis for some of the described COX-2-independent pharmacological effects of celecoxib.

Antinociceptive activity of the S1P-receptor agonist FTY720.

FTY720 is a novel immunosuppressive drug that inhibits the egress of lymphocytes from secondary lymphoid tissues and thymus. In its phosphorylated form FTY720 is a potent S1P receptor agonist. Recently it was also shown that FTY720 can reduce prostaglandin synthesis through the direct inhibition of the cytosolic phospholipase A2 (cPLA2). Since prostaglandins are important mediators of nociception, we studied the effects of FTY720 in different models of nociception. We found that intraperitoneal administration of FTY720 reduced dose-dependently the nociceptive behaviour of rats in the formalin assay. Although the antinociceptive doses of FTY720 were too low to alter the lymphocyte count, prostanoid concentrations in the plasma were dramatically reduced. Surprisingly, intrathecally administered FTY720 reduced the nociceptive behaviour in the formalin assay without altering spinal prostaglandin synthesis, indicating that additional antinociceptive mechanisms beside the inhibition of prostaglandin synthesis are involved. Accordingly, FTY720 reduced also the nociceptive behaviour in the spared nerve injury model for neuropathic pain which does not depend on prostaglandin synthesis. In this model the antinociceptive effect of FTY720 was similar to gabapentin, a commonly used drug to treat neuropathic pain. Taken together we show for the first time that FTY720 possesses antinociceptive properties and that FTY720 reduces nociceptive behaviour during neuropathic pain.

Consequences of altered eicosanoid patterns for nociceptive processing in mPGES-1-deficient mice.

Cyclooxygenase-2 (COX-2)-dependent prostaglandin (PG) E(2) synthesis in the spinal cord plays a major role in the development of inflammatory hyperalgesia and allodynia. Microsomal PGE(2) synthase-1 (mPGES-1) isomerizes COX-2-derived PGH(2) to PGE(2). Here, we evaluated the effect of mPGES-1-deficiency on the nociceptive behavior in various models of nociception that depend on PGE(2) synthesis. Surprisingly, in the COX-2-dependent zymosan-evoked hyperalgesia model, the nociceptive behavior was not reduced in mPGES-1-deficient mice despite a marked decrease of the spinal PGE(2) synthesis. Similarly, the nociceptive behavior was unaltered in mPGES-1-deficient mice in the formalin test. Importantly, spinal cords and primary spinal cord cells derived from mPGES-1-deficient mice showed a redirection of the PGE(2) synthesis to PGD(2), PGF(2alpha) and 6-keto-PGF(1alpha) (stable metabolite of PGI(2)). Since the latter prostaglandins serve also as mediators of nociception they may compensate the loss of PGE(2) synthesis in mPGES-1-deficient mice.

The impact of CREB and its phosphorylation at Ser142 on inflammatory nociception.

Peripheral noxious stimulation leads to phosphorylation and thereby activation of the transcription factor CREB in the spinal cord. CREB phosphorylation occurs mainly at serine 133, but the phosphorylation site at serine 142 may also be important. We investigated the impact of spinal CREB protein levels and phosphorylation at Ser142 on the nociceptive behaviour in rat and mouse models of inflammatory nociception. Downregulation of total CREB protein in the rat spinal cord by antisense-oligonucleotides resulted in antinociceptive effects. After peripheral noxious stimulation CREB was phosphorylated in the spinal cord at serine 133 and 142 indicating a potential role of both residues in nociceptive processing. However, Ser142 mutant mice developed equal behavioural correlates of hyperalgesia as wild-type mice in different inflammatory models. Thus, our data confirm that CREB is essential for spinal nociceptive processing. However, prevention of phosphorylation only at serine 142 is not sufficient to modulate the nociceptive response.

The glutamate transporter GLAST is involved in spinal nociceptive processing.

GLAST and GLT-1 are the most abundant glutamate transporters in the CNS and protect neurons from glutamate neurotoxicity. Here, we investigated the role of GLAST in spinal nociceptive processing. GLAST protein expression was not altered after treatment of rats with either formalin or zymosan. Surprisingly, knock-down of GLAST in the spinal cord using antisense-oligonucleotides decreased glutamate concentrations in cerebrospinal fluid (CSF) and reduced the nociceptive behaviour in the rat formalin assay. However, it did not influence thermal hyperalgesia in the zymosan-induced paw inflammation model indicating that GLAST is associated with spontaneous rather than inflammatory nociception. Mechanisms that might explain the decreased response in the formalin assay may include compensatory activation of other glutamate transporters, inhibition of glutamate release or disturbance of glutamate recycling. In conclusion, these data suggest that inhibition of GLAST expression in the spinal cord reduces excitatory synaptic activity and thereby spontaneous responses after nociceptive stimulation of the paw.

Evidence of COX-2 independent induction of apoptosis and cell cycle block in human colon carcinoma cells after S- or R-ibuprofen treatment.

Ibuprofen belongs to the 2-aryl propionic-acid derivatives and consists of two enantiomers, of which S-ibuprofen is a potent cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibitor whereas the R-enantiomer is two to three orders of magnitude less potent to inhibit cyclooxygenases. Beside its positive effects on inflammation and pain several animal studies have shown that ibuprofen also inhibits tumor initiation and proliferation but the molecular mechanisms are not fully understood. To investigate to which extent the antiproliferative effect of ibuprofen depends on COX-inhibition we tested both enantiomers in different human colon carcinoma cell lines (HCA-7 express COX-1, COX-2 and produce high prostaglandin E2 level; HCT-15 express only COX-1 and produce nearly no prostaglandin E2). S- and R-ibuprofen reduced concentration dependently cell survival in both cell lines to a similar extent and caused a G0/G1 phase block as well as apoptosis. The cell cycle block was accompanied by a down regulation of cyclin A and B and an increase of the cell cycle inhibitory protein p27Kip-1. HCA-7 cells were less sensitive against the antiproliferative effects of ibuprofen enantiomers which was probably due to lower ibuprofen concentrations in this cell type. Also in the nude mice model both enantiomers inhibited tumor growth of HCA-7 and HCT-15 xenografts to a similar extent. However, in mice about 54% of R-ibuprofen was unidirectionally inverted to S-ibuprofen, thus the observed antitumorigenic effect of R-ibuprofen in vivo cannot solely be assigned to this enantiomer. In conclusion our data indicate that S- and R-ibuprofen show similar antiproliferative effects in human colon carcinoma cell lines irrespective of its COX-inhibiting potencies.

LC-MS/MS-analysis of prostaglandin E2 and D2 in microdialysis samples of rats.

For the determination of prostaglandins in microdialysis samples, usually immunoassays are used. However, these assays may show cross-reactivity among various prostaglandins. To overcome this problem a specific method for the determination of prostaglandin E2 and D2 in rat microdialysis samples by using liquid chromatography-electrospay ionization-tandem mass spectrometry (LC-ESI-MS/MS) is described. Prostaglandin E2 and D2 were extracted from microdialysis samples with liquid-liquid extraction using deuterated prostaglandin D2, [2H4]-PGD2, as internal standard. Subsequently, prostaglandins were separated with a phenomenex Synergi Hydro-RP column and determined with a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface operating in negative ionization mode. The method showed a LLOQ of 25 pg/ml for prostaglandin E2 and 50 pg/ml for prostaglandin D2. The applicability of the method is shown in rat spinal cord microdialysis samples following peripheral nociceptive stimulation.

Downregulation of cytosolic prostaglandin E2 synthase results in decreased nociceptive behavior in rats.

Nociception-evoked prostaglandin E2 (PGE2) release in the spinal cord contributes considerably to the development of hyperalgesia and allodynia. Biosynthesis of PGE2 involves the conversion of arachidonic acid to PGH2 by cyclooxygenases (COXs), followed by an isomerization of PGH2 to PGE2 by PGE2 synthases (PGESs). The roles of COX-1, COX-2, and the inducible microsomal PGES-1 have been studied in models of pain and inflammation. In contrast, in nociceptive processes, very little is known about the role of cytosolic PGES (cPGES), which has been described as being functionally coupled to COX-1. Here we show by in situ hybridization and immunohistological analysis that COX-1 and cPGES are constitutively expressed in neuronal and non-neuronal cells of the dorsal and ventral horns in the spinal cord of adult rats. The protein levels of both enzymes were not regulated by nociceptive stimuli; however, reduction of cPGES in rat spinal cord with intrathecal application of cPGES antisense oligonucleotides reduced the nociceptive behavior in zymosan-evoked thermal hyperalgesia and in the formalin assay. The data indicate that cPGES plays an important role in mediating early responses during spinal nociceptive processing.

Comparative proteomic analysis of the rat spinal cord in inflammatory and neuropathic pain models.

Pathological pain associated either with peripheral tissue damage and inflammation (inflammatory pain) or peripheral nerve injury (neuropathic pain) is characterized by persistent pain hypersensitivity. This hypersensitivity is believed to be mediated by sensitization of nociceptors and spinal dorsal horn neurons leading to hyperalgesia and allodynia. Changes of protein expression and/or phosphorylation are known to contribute to the development of this hyperexcitability of the nociceptive system. In the present study we analyzed protein patterns in the spinal cord following paw inflammation or sciatic nerve injury using two-dimensional (2D) gel electrophoresis combined with MALDI-TOF mass spectrometry. 2D-PAGE revealed nine and five regulated proteins following paw inflammation and sciatic nerve damage, respectively. These regulated proteins had not been identified previously with other methods. There was no overlap of regulated proteins between models except for the small heat shock protein alpha-crystallin, which was decreased in both models. In conclusion, this study illustrates that employment of the proteomic 2D-PAGE approach allows for identification of novel regulated proteins that may be involved in the central sensitization and possibly manifestation of chronic pain.

Essential role of the synaptic vesicle protein synapsin II in formalin-induced hyperalgesia and glutamate release in the spinal cord.

The synaptic vesicle protein synapsin II plays an important role in the regulation of neurotransmitter release and synaptic plasticity. Here, we investigated its involvement in the synaptic transmission of nociceptive signals in the spinal cord and the development of pain hypersensitivity. We show that synapsin II is predominantly expressed in terminals and neuronal fibers in superficial laminae of the dorsal horn (laminae I-II). Formalin injection into a mouse hindpaw normally causes an immediate and strong release of glutamate in the dorsal horn. In synapsin II deficient mice this glutamate release is almost completely missing. This is associated with reduced nociceptive behavior in the formalin test and in the zymosan-induced paw inflammation model. In addition, the formalin evoked increase in the number of c-Fos IR neurons is significantly reduced in synapsin II knockout mice. Touch perception and motor coordination, however, are normal indicating that synapsin II deficiency does not generally disrupt sensory and/or motor functions. Antisense-mediated transient knockdown of synapsin II in the spinal cord of adult animals also reduced the nociceptive behavior. As the antisense effect is independent of a potential role of synapsin II during development we suggest that the hypoalgesia in synapsin II deficient mice does involve a direct 'pain-facilitating' effect of synapsin II and is not essentially dependent on potentially occurring developmental alterations. The distinctive role of synapsin II for pain signaling probably results from its specific localization and possibly from a specific control of glutamate release.