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R-flurbiprofen is the non-COX-inhibiting enantiomer of flurbiprofen and is not converted to S-flurbiprofen in human cells. Nevertheless, it reduces extracellular prostaglandin E2 (PGE2) in cancer or immune cell cultures and human extracellular fluid. Here, we show that R-flurbiprofen acts through a dual mechanism: (i) it inhibits the translocation of cPLA2α to the plasma membrane and thereby curtails the availability of arachidonic acid and (ii) R-flurbiprofen traps PGE2 inside of the cells by inhibiting multidrug resistance–associated protein 4 (MRP4, ABCC4), which acts as an outward transporter for prostaglandins. Consequently, the effects of R-flurbiprofen were mimicked by RNAi-mediated knockdown of MRP4. Our data show a novel mechanism by which R-flurbiprofen reduces extracellular PGs at physiological concentrations, particularly in cancers with high levels of MRP4, but the mechanism may also contribute to its anti-inflammatory and immune-modulating properties and suggests that it reduces PGs in a site- and context-dependent manner.
Background Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter which mainly mediates its effects on neurons via ionotropic (GABAA) and metabotropic (GABAB) receptors. GABAB receptors are widely expressed in the central and the peripheral nervous system. Although there is evidence for a key function of GABAB receptors in the modulation of pain, the relative contribution of peripherally- versus centrally-expressed GABAB receptors is unclear. Results In order to elucidate the functional relevance of GABAB receptors expressed in peripheral nociceptive neurons in pain modulation we generated and analyzed conditional mouse mutants lacking functional GABAB(1) subunit specifically in nociceptors, preserving expression in the spinal cord and brain (SNS-GABAB(1)-/- mice). Lack of the GABAB(1) subunit precludes the assembly of functional GABAB receptor. We analyzed SNS-GABAB(1)-/- mice and their control littermates in several models of acute and neuropathic pain. Electrophysiological studies on peripheral afferents revealed higher firing frequencies in SNS-GABAB(1)-/- mice compared to corresponding control littermates. However no differences were seen in basal nociceptive sensitivity between these groups. The development of neuropathic and chronic inflammatory pain was similar across the two genotypes. The duration of nocifensive responses evoked by intraplantar formalin injection was prolonged in the SNS-GABAB(1)-/- animals as compared to their control littermates. Pharmacological experiments revealed that systemic baclofen-induced inhibition of formalin-induced nociceptive behaviors was not dependent upon GABAB(1) expression in nociceptors. Conclusion This study addressed contribution of GABAB receptors expressed on primary afferent nociceptive fibers to the modulation of pain. We observed that neither the development of acute and chronic pain nor the analgesic effects of a systematically-delivered GABAB agonist was significantly changed upon a specific deletion of GABAB receptors from peripheral nociceptive neurons in vivo. This lets us conclude that GABAB receptors in the peripheral nervous system play a less important role than those in the central nervous system in the regulation of pain.
Background: Descending inhibitory pain control contributes to the endogenous defense against chronic pain and involves noradrenergic and serotonergic systems. The clinical efficacy of antidepressants suggests that serotonin may be particularly relevant for neuropathic pain conditions. Serotonergic signaling is regulated by synthesis, metabolisms, reuptake and receptors. To address the complexity, we used inbred mouse strains, C57BL/6J, 129 Sv, DBA/2J and Balb/c, which differ in brain serotonin levels. Results: Serotonin analysis after nerve injury revealed inter-strain differences in the adaptation of descending serotonergic fibers. Upregulation of spinal cord and midbrain serotonin was apparent only in 129 Sv mice and was associated with attenuated nerve injury evoked hyperalgesia and allodynia in this strain. The increase of dorsal horn serotonin was blocked by hemisectioning of descending fibers but not by rhizotomy of primary afferents indicating a midbrain source. Para-chlorophenylalanine-mediated serotonin depletion in spinal cord and midbrain intensified pain hypersensitivity in the nerve injury model. In contrast, chronic inflammation of the hindpaw did not evoke equivalent changes in serotonin levels in the spinal cord and midbrain and nociceptive thresholds dropped in a parallel manner in all strains. Conclusion: The results suggest that chronic nerve injury evoked hypernociception may be contributed by genetic differences of descending serotonergic inhibitory control.
Background: R-flurbiprofen, one of the enantiomers of flurbiprofen racemate, is inactive with respect to cyclooxygenase inhibition, but shows analgesic properties without relevant toxicity. Its mode of action is still unclear. Methodology/Principal Findings: We show that R-flurbiprofen reduces glutamate release in the dorsal horn of the spinal cord evoked by sciatic nerve injury and thereby alleviates pain in sciatic nerve injury models of neuropathic pain in rats and mice. This is mediated by restoring the balance of endocannabinoids (eCB), which is disturbed following peripheral nerve injury in the DRGs, spinal cord and forebrain. The imbalance results from transcriptional adaptations of fatty acid amide hydrolase (FAAH) and NAPE-phospholipase D, i.e. the major enzymes involved in anandamide metabolism and synthesis, respectively. R-flurbiprofen inhibits FAAH activity and normalizes NAPE-PLD expression. As a consequence, R-Flurbiprofen improves endogenous cannabinoid mediated effects, indicated by the reduction of glutamate release, increased activity of the anti-inflammatory transcription factor PPAR gamma and attenuation of microglia activation. Antinociceptive effects are lost by combined inhibition of CB1 and CB2 receptors and partially abolished in CB1 receptor deficient mice. R-flurbiprofen does however not cause changes of core body temperature which is a typical indicator of central effects of cannabinoid-1 receptor agonists. Conclusion: Our results suggest that R-flurbiprofen improves the endogenous mechanisms to regain stability after axonal injury and to fend off chronic neuropathic pain by modulating the endocannabinoid system and thus constitutes an attractive, novel therapeutic agent in the treatment of chronic, intractable pain.
Progranulin haploinsufficiency is associated with frontotemporal dementia in humans. Deficiency of progranulin led to exaggerated inflammation and premature aging in mice. The role of progranulin in adaptations to nerve injury and neuropathic pain are still unknown. Here we found that progranulin is up-regulated after injury of the sciatic nerve in the mouse ipsilateral dorsal root ganglia and spinal cord, most prominently in the microglia surrounding injured motor neurons. Progranulin knockdown by continuous intrathecal spinal delivery of small interfering RNA after sciatic nerve injury intensified neuropathic pain-like behaviour and delayed the recovery of motor functions. Compared to wild-type mice, progranulin-deficient mice developed more intense nociceptive hypersensitivity after nerve injury. The differences escalated with aging. Knockdown of progranulin reduced the survival of dissociated primary neurons and neurite outgrowth, whereas addition of recombinant progranulin rescued primary dorsal root ganglia neurons from cell death induced by nerve growth factor withdrawal. Thus, up-regulation of progranulin after neuronal injury may reduce neuropathic pain and help motor function recovery, at least in part, by promoting survival of injured neurons and supporting regrowth. A deficiency in this mechanism may increase the risk for injury-associated chronic pain.
Oral presentation from 4th International Conference of cGMP Generators, Effectors and Therapeutic Implications ; Regensburg, Germany. 19–21 June 2009 Background: An exaggerated pain sensitivity is the dominant feature of inflammatory and neuropathic pain both in the clinical setting and in experimental animal models. It manifests as pain in response to normally innocuous stimuli (allodynia), increased response to noxious stimuli (hyperalgesia) or spontaneous pain, and can persist long after the initial injury is resolved. Research over the last decades has revealed that several signaling pathways in the spinal cord essentially contribute to the pain sensitization. To test the contribution of cGMP produced by NO-sensitive guanylyl cyclase (NO-GC) to pain sensitization, we investigated the localization of NO-GC in the spinal cord and in dorsal root ganglia, and we characterized the nociceptive behavior of mice deficient in NO-GC (GC-KO mice). Results: We show that NO-GC (β1 subunit) is distinctly expressed in neurons of the mouse spinal cord, while its distribution in dorsal root ganglia is restricted to non-neuronal cells. GC-KO mice exhibited a considerably reduced nociceptive behavior in models of inflammatory or neuropathic pain, but their responses to acute pain were not impaired. Moreover, GC-KO mice failed to develop pain sensitization induced by spinal administration of drugs releasing NO. Surprisingly, during spinal nociceptive processing cGMP produced by NO-GC may activate signaling pathways different from cGMP-dependent protein kinase I (cGKI), while cGKI can be activated by natriuretic peptide receptor-B (NPR-B) dependent cGMP production. Conclusion: Taken together, our results provide evidence that NO-GC has a dominant role in the development of exaggerated pain sensitivity during inflammatory and neuropathic pain. Furthermore, beside the NO-mediated cGMP synthesis, cGMP produced by NPR-B contributes to pain sensitization by activation of cGKI.
Nerve injury leads to sensitization mechanisms in the peripheral and central nervous system which involve transcriptional and post-transcriptional modifications in sensory nerves. To assess protein regulations in the spinal cord after injury of the sciatic nerve in the Spared Nerve Injury model (SNI) we performed a proteomic analysis using 2D-difference gel electrophoresis (DIGE) technology. Among approximately 2300 protein spots separated on each gel we detected 55 significantly regulated proteins after SNI whereof 41 were successfully identified by MALDI-TOF MS. Out of the proteins which were regulated in the DIGE analyses after SNI we focused on the carboxypeptidase A inhibitor latexin because protease dysfunctions contribute to the development of neuropathic pain. Latexin protein expression was reduced after SNI which could be confirmed by Western Blot analysis, quantitative RT-PCR and in-situ hybridisation. The decrease of latexin was associated with an increase of the activity of carboxypeptidase A indicating that the balance between latexin and carboxypeptidase A was impaired in the spinal cord after peripheral nerve injury due to a loss of latexin expression in spinal cord neurons. This may contribute to the development of cold allodynia because normalization of neuronal latexin expression in the spinal cord by AAV-mediated latexin transduction or administration of a small molecule carboxypeptidase A inhibitor significantly reduced acetone-evoked nociceptive behavior after SNI. Our results show the usefulness of proteomics as a screening tool to identify novel mechanisms of nerve injury evoked hypernociception and suggest that carboxypeptidase A inhibition might be useful to reduce cold allodynia.
The manifestation of chronic back pain depends on structural, psychosocial, occupational and genetic influences. Heritability estimates for back pain range from 30% to 45%. Genetic influences are caused by genes affecting intervertebral disc degeneration or the immune response and genes involved in pain perception, signalling and psychological processing. This inter-individual variability which is partly due to genetic differences would require an individualized pain management to prevent the transition from acute to chronic back pain or improve the outcome. The genetic profile may help to define patients at high risk for chronic pain. We summarize genetic factors that (i) impact on intervertebral disc stability, namely Collagen IX, COL9A3, COL11A1, COL11A2, COL1A1, aggrecan (AGAN), cartilage intermediate layer protein, vitamin D receptor, metalloproteinsase-3 (MMP3), MMP9, and thrombospondin-2, (ii) modify inflammation, namely interleukin-1 (IL-1) locus genes and IL-6 and (iii) and pain signalling namely guanine triphosphate (GTP) cyclohydrolase 1, catechol-O-methyltransferase, μ opioid receptor (OPMR1), melanocortin 1 receptor (MC1R), transient receptor potential channel A1 and fatty acid amide hydrolase and analgesic drug metabolism (cytochrome P450 [CYP]2D6, CYP2C9).
Nucleoredoxin is a thioredoxin-like redoxin that has been recognized as redox modulator of WNT signaling. Using a Yeast-2-Hybrid screen, we identified calcium calmodulin kinase 2a, Camk2a, as a prominent prey in a brain library. Camk2a is crucial for nitric oxide dependent processes of neuronal plasticity of learning and memory. Therefore, the present study assessed functions of NXN in neuronal Nestin-NXN-/- deficient mice. The NXN-Camk2a interaction was confirmed by coimmunoprecipitation, and by colocalization in neuropil and dendritic spines. Functionally, Camk2a activity was reduced in NXN deficient neurons and restored with recombinant NXN. Proteomics revealed reduced oxidation in the hippocampus of Nestin-NXN-/- deficient mice, including Camk2a, further synaptic and mitochondrial proteins, and was associated with a reduction of mitochondrial respiration. Nestin-NXN-/- mice were healthy and behaved normally in behavioral tests of anxiety, activity and sociability. They had no cognitive deficits in touchscreen based learning & memory tasks, but omitted more trials showing a lower interest in the reward. They also engaged less in rewarding voluntary wheel running, and in exploratory behavior in IntelliCages. Accuracy was enhanced owing to the loss of exploration. The data suggested that NXN maintained the oxidative state of Camk2a and thereby its activity. In addition, it supported oxidation of other synaptic and mitochondrial proteins, and mitochondrial respiration. The loss of NXN-dependent pro-oxidative functions manifested in a loss of exploratory drive and reduced interest in reward in behaving mice.
Genes encoding endocannabinoid and sphingolipid metabolism pathways were suggested to contribute to the genetic risk towards attention deficit hyperactivity disorder (ADHD). The present pilot study assessed plasma concentrations of candidate endocannabinoids, sphingolipids and ceramides in individuals with adult ADHD in comparison with healthy controls and patients with affective disorders. Targeted lipid analyses of 23 different lipid species were performed in 71 mental disorder patients and 98 healthy controls (HC). The patients were diagnosed with adult ADHD (n = 12), affective disorder (major depression, MD n = 16 or bipolar disorder, BD n = 6) or adult ADHD with comorbid affective disorders (n = 37). Canonical discriminant analysis and CHAID analyses were used to identify major components that predicted the diagnostic group. ADHD patients had increased plasma concentrations of sphingosine-1-phosphate (S1P d18:1) and sphinganine-1-phosphate (S1P d18:0). In addition, the endocannabinoids, anandamide (AEA) and arachidonoylglycerol were increased. MD/BD patients had increased long chain ceramides, most prominently Cer22:0, but low endocannabinoids in contrast to ADHD patients. Patients with ADHD and comorbid affective disorders displayed increased S1P d18:1 and increased Cer22:0, but the individual lipid levels were lower than in the non-comorbid disorders. Sphingolipid profiles differ between patients suffering from ADHD and affective disorders, with overlapping patterns in comorbid patients. The S1P d18:1 to Cer22:0 ratio may constitute a diagnostic or prognostic tool.
