It is widely accepted that peripheral injury increases spinal inducible cyclooxygenase (COX-2) expression and prostaglandin E 2 (PGE 2 ) formation as key mediators of nociceptive sensitization. Here, we used inducible nitric oxide synthase (iNOS) genedeficient (iNOSϪ/Ϫ) mice to determine the contribution of iNOSderived nitric oxide (NO) to this process. iNOSϪ/Ϫ mice exhibited reduced thermal hyperalgesia after zymosan injection. Spinal NO and PGE 2 formation both remained at baseline levels, in contrast to wild-type (wt) mice. In wt mice reduced hyperalgesia similar to that seen in iNOSϪ/Ϫ mice was induced by local spinal, but not by systemic treatment with the iNOS inhibitor L-NIL, suggesting that the reduced heat sensitization in iNOSϪ/Ϫ mice was attributable to the lack of spinal rather than peripheral iNOS. Two additional observations indicate that the antinociceptive effects of iNOS inhibition are dependent on a loss of stimulation of PG synthesis. First, intrathecal injection of the COX inhibitor indomethacin, which exerted pronounced antinociceptive effects in wt mice, was completely ineffective in iNOSϪ/Ϫ mice. Second, treatment with the NO donor RE-2047 not only completely restored spinal PG production and thermal sensitization in iNOSϪ/Ϫ mice but also its sensitivity to indomethacin. In both types of mice induction of thermal hyperalgesia was accompanied by similar increases in COX-1 and COX-2 mRNA expression. The stimulation of PG production by NO therefore involves an increase in enzymatic activity, rather than an alteration of COX gene expression. These results indicate that NO derived from spinal iNOS acts as a fast inductor of spinal thermal hyperalgesia. Key words: nitric oxide; inducible nitric oxide synthase; zymosan; thermal hyperalgesia; paw edema; spinal microdialysis; L-NIL; RE-2047; prostaglandins; cyclooxygenaseAcute tissue damage is often accompanied by the fast development of hyperalgesia and allodynia (Andrew and Greenspan, 1999). Both peripheral mechanisms at the site of injury and central processes particularly in the spinal cord contribute to this phenomenon. Prostaglandins (PGs) Brune, 1994) as well as nitric oxide (NO) (Lawand et al., 1997) are produced in response to tissue damage peripherally and centrally. Whereas PGs are generally accepted to play a dominant role in nociceptive sensitization (Bley et al., 1998), the role of NO is less clear. Also some authors claim of an anti-nociceptive action of NO (Goettl and Larson, 1996;Hamalainen and Lovick, 1997), most favor a pronociceptive activity Kawabata et al., 1994;Chen and Levine, 1999). Part of this controversy may arise from the existence of three different isoenzymes of NO synthase (NOS) (Gonzalez-Hernandez and Rustioni, 1999), which may have distinct effects on nociception, and from the lack of specific inhibitors for these different isoforms. In the CNS including the spinal cord, NO is thought to be primarily produced by the neuronal isoform of NOS (nNOS) (Downen et al., 1999). However, endothelial NOS is also found in neur...

Since long-term hyperexcitability of nociceptive neurons in the spinal cord has been suggested to be caused and maintained by changes of protein expression we assessed protein patterns in lumbar spinal cord during a zymosan induced paw inflammation employing two-dimensional (2D) gel electrophoresis. 2D PAGE revealed a time-dependent breakdown of scaffolding proteins one of which was neurofilament light chain (NFL) protein, which has been previously found to be important for axonal architecture and transport. Nociception induced breakdown of NFL in the spinal cord and dorsal root ganglias was prevented by pretreatment of the animals with a single dose of the specific inhibitor of the protease calpain (MDL-28170) which has been shown to be the primary protease involved in neurofilament degradation in neurodegenerative diseases. Treatment with the calpain inhibitor also provided anti-inflammatory and anti-hyperalgesic effects in the zymosan-induced paw inflammation model irrespective of whether the drug was administered systemically (i.p.) or delivered onto the lumbar spinal cord. This suggests that the activation of calpain is involved in the sensitization of nociceptive neurons what is partly due to neurofilament breakdown but cleavage of other calpain substrates may also be involved. Our results indicate that inhibition of pathological calpain activity may present an interesting novel drug target in the treatment of pain and inflammation.

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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. Molecular & Cellular Proteomics 7:2475-2485, 2008.Synapses are dynamic structures that expand, retract, and remodel to accomplish activity-dependent modification of neuronal circuits. During peripheral inflammation, the synaptic contacts between primary sensory neurons and dorsal horn neurons are modified in a way that the responsiveness of the system to subsequent stimuli is increased, resulting in hypersensitivity to noxious stimuli (1, 2). These activity-dependent changes in synaptic morphology and strength are based on many different mechanisms including alterations in ion channel and receptor activities due to phosphorylation by protein kinases, the translocation of ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) 1 receptors to the postsynaptic membrane (3, 4), transcription-and translation-dependent changes in protein expression (2, 5), and ubiquitylation-mediated protein degradation (6). Often these changes occur only at a few synapses, making the analysis by common proteomics techniques extremely difficult. Multiepitope ligand cartography (MELC) is a novel i...

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.