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Abstract Background The role of the diffusible messenger nitric oxide (NO) in the regulation of pain transmission is still a debate of matter, pro-nociceptive and/or anti-nociceptive. S-Nitrosylation, the reversible post-translational modification of selective cysteine residues in proteins, has emerged as an important mechanism by which NO acts as a signaling molecule. The occurrence of S-nitrosylation in the spinal cord and its targets that may modulate pain transmission remain unclarified. The "biotin-switch" method and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were employed for identifying S-nitrosylated proteins. Results Here we show that actin was a major protein S-nitrosylated in the spinal cord by the NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP). Interestingly, actin was S-nitrosylated, more in the S2 fraction than in the P2 fraction of the spinal homogenate. Treatment of PC12 cells with SNAP caused rapid S-nitrosylation of actin and inhibited dopamine release from the cells. Just like cytochalasin B, which depolymerizes actin, SNAP decreased the amount of filamentous actin cytoskeleton just beneath the membrane. The inhibition of dopamine release was not attenuated by inhibitors of soluble guanylyl cyclase and cGMP-dependent protein kinase. Conclusion The present study demonstrates that actin is a major S-nitrosylated protein in the spinal cord and suggests that NO directly regulates neurotransmitter release by S-nitrosylation in addition to the well-known phosphorylation by cGMP-dependent protein kinase.

We demonstrated previously that sorting nexin 25 (SNX25) in nerve-associated macrophages plays critical roles in pain sensation by regulating tissue NGF content under both physiological and neuropathic conditions. In the present study, we apply the SNX25-NGF paradigm to tactile perception by showing that Snx25 +/- mice or macrophage-specific Snx25 conditional knock-out (mcKO) mice had weaker responses to tactile stimuli in normal conditions. Snx25 mcKO mice responded poorly to transcutaneous electrical stimuli at a frequency of 5 Hz (C fiber responses), but normally to stimuli at a frequency of 250 Hz (Aδ fiber responses) or of 2000 Hz (Aβ fiber responses). CX3CR1-positive dermal macrophages were frequently found near calcitonin gene-related peptide (CGRP)- positive nerves and, less frequently, tyrosine hydroxylase (TH)-positive nerves. We confirmed that the tissue content of NGF was lower in Snx25 mcKO mice than in wild-type mice, and in turn, dermal NGF injection restored tactile sensitivity in Snx25 +/- mice and Snx25 mcKO mice to normal levels. These results indicate that CGRP-positive C-nociceptors (possibly also TH-positive C-LTMRs) associated dermal macrophages control tactile perception by producing NGF and secreting it into the dermis., (© 2024. The Author(s).)

Increased levels of lactate, an end-product of glycolysis, have been proposed as a potential surrogate marker for metabolic changes during neuronal excitation. These changes in lactate levels can result in decreased brain pH, which has been implicated in patients with various neuropsychiatric disorders. We previously demonstrated that such alterations are commonly observed in five mouse models of schizophrenia, bipolar disorder, and autism, suggesting a shared endophenotype among these disorders rather than mere artifacts due to medications or agonal state. However, there is still limited research on this phenomenon in animal models, leaving its generality across other disease animal models uncertain. Moreover, the association between changes in brain lactate levels and specific behavioral abnormalities remains unclear. To address these gaps, the International Brain pH Project Consortium investigated brain pH and lactate levels in 109 strains/conditions of 2294 animals with genetic and other experimental manipulations relevant to neuropsychiatric disorders. Systematic analysis revealed that decreased brain pH and increased lactate levels were common features observed in multiple models of depression, epilepsy, Alzheimer's disease, and some additional schizophrenia models. While certain autism models also exhibited decreased pH and increased lactate levels, others showed the opposite pattern, potentially reflecting subpopulations within the autism spectrum. Furthermore, utilizing large-scale behavioral test battery, a multivariate cross-validated prediction analysis demonstrated that poor working memory performance was predominantly associated with increased brain lactate levels. Importantly, this association was confirmed in an independent cohort of animal models. Collectively, these findings suggest that altered brain pH and lactate levels, which could be attributed to dysregulated excitation/inhibition balance, may serve as transdiagnostic endophenotypes of debilitating neuropsychiatric disorders characterized by cognitive impairment, irrespective of their beneficial or detrimental nature., Competing Interests: HH, HS, SH, GS, YT, MT, MI, MS, IH, KH, MH, AN, YM, SO, MM, AU, YK, AM, KN, SK, TS, TI, HN, YG, MR, TT, KY, NT, HK, JI, IN, TK, TT, MD, HN, KT, KS, KN, TT, HF, YS, KY, LR, NH, HM, TY, SW, TA, KT, TN, HK, FH, AH, HH, KT, TT, TK, TK, IG, GC, NA, HH, SK, TK, MH, YH, RM, KO, AH, LB, IY, YH, HK, YM, KI, HK, BK, HO, YY, AT, NK, SG, MI, MN, KM, EO, IO, TS, KY, HA, KT, KI, TS, AK, MF, NU, TK, TT, YY, MI, TH, KI, KN, SH, KE, KH, YT, SN, NS, TY, NU, TY, YK, RO, NS, KS, SH, KO, HM, TF, MT, HS, DS, YI, TI, YK, TY, KS, TM No competing interests declared, SY, NN Employee of Takeda Pharmaceutical Company, Ltd

Deficiency of an extracellular matrix glycoprotein tenascin-X (TNX) leads to a human heritable disorder Ehlers-Danlos syndrome, and TNX-deficient patients complain of chronic joint pain, myalgia, paresthesia, and axonal polyneuropathy. We previously reported that TNX-deficient (Tnxb -/- ) mice exhibit mechanical allodynia and hypersensitivity to myelinated A-fibers. Here, we investigated the pain response of Tnxb -/- mice using pharmacological silencing of A-fibers with co-injection of N-(2,6-Dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314), a membrane-impermeable lidocaine analog, plus flagellin, a toll-like receptor 5 (TLR5) ligand. Intraplantar co-injection of QX-314 and flagellin significantly increased the paw withdrawal threshold to transcutaneous sine wave stimuli at frequencies of 250 Hz (Aδ fiber responses) and 2000 Hz (Aβ fiber responses), but not 5 Hz (C fiber responses) in wild-type mice. The QX-314 plus flagellin-induced silencing of Aδ- and Aβ-fibers was also observed in Tnxb -/- mice. Co-injection of QX-314 and flagellin significantly inhibited the mechanical allodynia and neuronal activation of the spinal dorsal horn in Tnxb -/- mice. Interestingly, QX-314 alone inhibited the mechanical allodynia in Tnxb -/- mice, and it increased the paw withdrawal threshold to stimuli at frequencies of 250 Hz and 2000 Hz in Tnxb -/- mice, but not in wild-type mice. The inhibition of mechanical allodynia induced by QX-314 alone was blocked by intraplantar injection of a TLR5 antagonist TH1020 in Tnxb -/- mice. These results suggest that mechanical allodynia due to TNX deficiency is caused by the hypersensitivity of Aδ- and Aβ-fibers, and it is induced by constitutive activation of TLR5., (© 2023. The Author(s).)

Tenascin-X (TNX) is an extracellular matrix glycoprotein for which a deficiency results in a recessive form of classical-like Ehlers-Danlos syndrome (clEDS), a heritable connective tissue disorder with hyperextensible skin without atrophic scarring, joint hypermobility, and easy bruising. Notably, patients with clEDS also suffer from not only chronic joint pain and chronic myalgia but also neurological abnormalities such as peripheral paresthesia and axonal polyneuropathy with high frequency. By using TNX-deficient ( Tnxb -/- ) mice, well-known as a model animal of clEDS, we recently showed that Tnxb -/- mice exhibit hypersensitivity to chemical stimuli and the development of mechanical allodynia due to the hypersensitization of myelinated A-fibers and activation of the spinal dorsal horn. Pain also occurs in other types of EDS. First, we review the underlying molecular mechanisms of pain in EDS, especially that in clEDS. In addition, the roles of TNX as a tumor suppressor protein in cancer progression have been reported. Recent in silico large-scale database analyses have shown that TNX is downregulated in various tumor tissues and that high expression of TNX in tumor cells has a good prognosis. We describe what is so far known about TNX as a tumor suppressor protein. Furthermore, some patients with clEDS show delayed wound healing. Tnxb -/- mice also exhibit impairment of epithelial wound healing in corneas. TNX is also involved in liver fibrosis. We address the molecular mechanism for the induction of COL1A1 by the expression of both a peptide derived from the fibrinogen-related domain of TNX and integrin α11., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Okuda-Ashitaka and Matsumoto.)

Nucleophosmin 1 (NPM1) primarily localizes to the nucleus and is passively released into the extracellular milieu by necrotic or damaged cells, or is secreted by monocytes and macrophages. Extracellular NPM1 acts as a potent inflammatory stimulator by promoting cytokine production [e.g., tumor necrosis factor-α (TNF-α)], which suggests that NPM1 acts as a damage-associated molecular pattern. However, the receptor of NPM1 is unknown. Evidence indicates that DAMPs, which include high mobility group box 1 and histones, may bind Toll-like receptors (TLRs). In the present study, it was shown that NPM1 signaling was mediated via the TLR4 pathway, which suggests that TLR4 is an NPM1 receptor. TLR4 binds myeloid differentiation protein-2 (MD-2), which is essential for intracellular signaling. Furthermore, the TLR4 antagonist, LPS- Rhodobacter sphaeroides (an MD-2 antagonist) and TAK-242 (a TLR4 signaling inhibitor) significantly inhibited NPM1-induced TNF-α production by differentiated THP-1 cells as well as reducing ERK1/2 activation. Far-western blot analysis revealed that NPM1 directly bound MD-2. Thus, the results of the present study provide compelling evidence that TLR4 binds NPM1, and it is hypothesized that inhibiting NPM1 activity may serve as a novel strategy for treating TLR4-related diseases., (Copyright: © Nakatomi et al.)

Tenascin-X (TNX) is a member of the extracellular matrix glycoprotein tenascin family, and TNX deficiency leads to Ehlers-Danlos syndrome, a heritable human disorder characterized mostly by skin hyperextensibility, joint hypermobility, and easy bruising. TNX-deficient patients complain of chronic joint pain, myalgia, paresthesia, and axonal polyneuropathy. However, the molecular mechanisms by which TNX deficiency complicates pain are unknown. Here, we examined the nociceptive behavioral responses of TNX-deficient mice. Compared with wild-type mice, TNX-deficient mice exhibited mechanical allodynia but not thermal hyperalgesia. TNX deficiency also increased pain sensitivity to chemical stimuli and aggravated early inflammatory pain elicited by formalin. TNX-deficient mice were significantly hypersensitive to transcutaneous sine wave stimuli at frequencies of 250 Hz (Aδ fiber responses) and 2000 Hz (Aβ fiber responses), but not to stimuli at frequency of 5 Hz (C fiber responses). In addition, the phosphorylation levels of extracellular signal-related kinase, an active neuronal marker, and the activity of NADPH-diaphorase, a neuronal nitric oxide activation marker, were enhanced in the spinal dorsal horns of TNX-deficient mice. These results suggest that TNX deficiency contributes to the development of mechanical allodynia and hypersensitivity to chemical stimuli, and it induces hypersensitization of myelinated A fibers and activation of the spinal dorsal horn.

Nocistatin (NST) is a neuropeptide produced from the same precursor protein of opioid peptide nociceptin/orphanin FQ, and it is involved in a broad range of central functions including pain transmission in the nervous system. However, the composition and structure of the receptor(s) for NST remain unclear. Here, we developed NST photoaffinity probe to identify NST receptor. The NST photoaffinity probe contains an azide moiety for the tagging of the binding protein as well as biotin for protein detection. Intrathecal administration of a NST photoaffinity probe, biotin-(AC 5 ) 2 -[Y 6 ,azF 14 ]bNST, inhibited the nociceptin/orphanin FQ-evoked tactile pain allodynia in a manner similar to that of NST. The biotin-(AC 5 ) 2 -[Y 6 ,azF 14 ]bNST-binding proteins were primarily localized in the gray matter of the spinal cord. After photo-crosslinking of the protein complex with biotin-(AC 5 ) 2 -[Y 6 ,azF 14 ]bNST, two dominant binding protein bands were observed at 58 and 64 kDa. Thus, biotin-(AC 5 ) 2 -[Y 6 ,azF 14 ]bNST has pharmacological activity and is useful for characterizing the NST receptor., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Pathological chronic pain, which is frequently associated with prolonged tissue damage, inflammation, tumour invasion, and neurodegenerative diseases, gives rise to hyperalgesia and allodynia. We previously reported that intrathecal administration of nociceptin/orphanin FQ (N/OFQ), an endogenous ligand for the orphan opioid receptor-like receptor, in the femtomole range induces touch-evoked allodynia. N/OFQ has been implicated in multiple signalling pathways, such as inhibition of cAMP production and Ca(2+) channels, or activation of K(+) channels and mitogen-activated protein kinase, although the signalling pathways of N/OFQ-induced allodynia remain unclear. To address these issues, we developed an ex vivo mitogen-activated protein kinase assay by using intact slices of mouse spinal cord. N/OFQ markedly increased the phosphorylation of c-Jun N-terminal kinase (JNK) in the superficial dorsal horn of the spinal cord. The N/OFQ-stimulated JNK phosphorylation was significantly inhibited by pertussis toxin, the phospholipase C inhibitor U73122, and the inositol trisphosphate receptor antagonist Xestospongin C. Intrathecal administration of the JNK inhibitor SP600125 inhibited N/OFQ-evoked allodynia. The N/OFQ-induced increase in JNK phosphorylation was observed in astrocytes that expressed glial fibrillary acidic protein. N/OFQ also induced monocyte chemoattractant protein-1 (MCP-1) release via the JNK pathway, and N/OFQ-induced JNK phosphorylation was observed in MCP-1-immunoreactive astrocytes. Intrathecal administration of the MCP-1 receptor antagonist RS504393 inhibited N/OFQ-evoked allodynia. These results suggest that, in the spinal dorsal horn, N/OFQ induces allodynia through activation of JNK via the phospholipase C-inositol trisphosphate pathway, which is coupled to pertussis toxin-sensitive G-protein, and following the release of MCP-1 from astrocytes., (© 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Background: Chronic pain associated with inflammation is an important clinical problem, and the underlying mechanisms remain poorly understood. 4-Nitrophenylphosphatase domain and nonneuronal SNAP25-like protein homolog (NIPSNAP) 1, an interacting protein with neuropeptide nocistatin, is implicated in the inhibition of tactile pain allodynia. Although nocistatin inhibits some inflammatory pain responses, whether NIPSNAP1 affects inflammatory pain appears to be unclear. Here, we examined the nociceptive behavioral response of NIPSNAP1-deficient mice and the expression of NIPSNAP1 following peripheral inflammation to determine the contribution of NIPSNAP1 to inflammatory pain., Results: Nociceptive behavioral response increased in phase II of the formalin test, particularly during the later stage (26-50 min) in NIPSNAP1-deficient mice, although the response during phase I (0-15 min) was not significantly different between the deficient and wild-type mice. Moreover, phosphorylation of extracellular signal-related kinase was enhanced in the spinal dorsal horn of the deficient mice. The prolonged inflammatory pain induced by carrageenan and complete Freund's adjuvant was exacerbated in NIPSNAP1-deficient mice. NIPSNAP1 mRNA was expressed in small- and medium-sized neurons of the dorsal root ganglion and motor neurons of the spinal cord. In the formalin test, NIPSNAP1 mRNA was slightly increased in dorsal root ganglion but not in the spinal cord. In contrast, NIPSNAP1 mRNA levels in dorsal root ganglion were significantly decreased during 24-48 h after carrageenan injection. Prostaglandin E2, a major mediator of inflammation, stimulated NIPSNAP1 mRNA expression via the cAMP-protein kinase A signaling pathway in isolated dorsal root ganglion cells., Conclusions: These results suggest that changes in NIPSNAP1 expression may contribute to the pathogenesis of inflammatory pain., (© The Author(s) 2016.)

Nociceptin/orphanin FQ (N/OFQ) and nocistatin (NST) are neuropeptides produced from the same precursor protein. N/OFQ is involved in a broad range of central functions including pain, learning, memory, anxiety, and feeding. However, NST has opposite effects on various central functions evoked by N/OFQ. The regulation of their receptors may be important for these opposite functions of NST and N/OFQ. Although N/OFQ binds to a specific N/OFQ receptor, the target molecule of NST remains unclear. Some biological effects of NST are mediated by a G protein-coupled receptor. Furthermore, using high-performance affinity nanobeads, we recently identified a 4-nitrophenylphosphatase domain and nonneuronal SNAP25-like protein homolog 1 (NIPSNAP1) as a protein that interacts with NST in the mouse spinal cord. The inhibition of N/OFQ-evoked tactile pain allodynia by NST is mediated by NIPSNAP1. This review focuses on the molecular mechanisms of pain regulation by the target molecules of NST including a G protein-coupled receptor and NIPSNAP1., (© 2015 Elsevier Inc. All rights reserved.)

A neuropeptide nociceptin or orphanin FQ (N/OFQ) is an endogenous ligand for the orphan opioid receptor-like receptor. During studies on the analysis of the precursor of N/OFQ, we identified a novel neuropeptide produced from the same precursor and named it "nocistatin (NST)". Intrathecal (i.t.) administration of N/OFQ induces pain responses including touch-evoked allodynia and thermal hyperalgesia, and simultaneous administration of NST blocks the allodynia and hyperalgesia induced by N/OFQ. In the years since these discoveries, N/OFQ has been shown to be involved in a wide range of pharmacological activities, such as relaying pain perception in peripheral tissues, to the central nervous system, and NST was shown to have opposite effects on various central functions evoked by N/OFQ. Pharmacological characterization using various neurotransmitter agents, agonists, antagonists and knockout mice in vivo; electrophysiological and immunohistological analysis ex vivo; and molecular cloning using affinity chromatography of high-performance affinity nanobeads; and protein processing measurement using bioluminescence resonance energy transfer (BRET) in vitro have generated new insights into pain transmission regulated by NST and N/OFQ. This review focuses on the molecular and cellular mechanisms of pain transmission regulated by NST.

4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) is a molecule of physiologically unknown function, although it is predominantly expressed in the brain, spinal cord, liver, and kidney. We identified NIPSNAP1 as a protein that interacts with the neuropeptide nocistatin (NST) from synaptosomal membranes of mouse spinal cord using high-performance affinity latex beads. NST, which is produced from the same precursor protein as an opioid-like neuropeptide nociceptin/orphanin FQ (N/OFQ), has opposite effects on pain transmission evoked by N/OFQ. The calculated full-length pre-protein of NIPSNAP1 was 33 kDa, whereas the N-terminal truncated form of NIPSNAP1 (29 kDa) was ubiquitously expressed in the neuronal tissues, especially in synaptic membrane and mitochondria of brain. The 29-kDa NIPSNAP1 was distributed on the cell surface, and NST interacted with the 29-kDa but not the 33-kDa NIPSNAP1. Although intrathecal injection of N/OFQ induced tactile allodynia in both wild-type and NIPSNAP1-deficient mice, the inhibition of N/OFQ-evoked tactile allodynia by NST seen in wild-type mice was completely lacking in the deficient mice. These results suggest that NIPSNAP1 is an interacting molecule of NST and plays a crucial role in pain transmission.

Purpose: The authors previously reported ornithine cytotoxicity in ornithine-δ-aminotransferase (OAT)-deficient human retinal pigment epithelial (RPE) cells as an in vitro model of gyrate atrophy of the choroid and retina (GA). Given that RPE cells are severely damaged by arginine combined with ornithine, they investigated the role of arginine metabolism using that in vitro model., Methods: Human telomerase reverse transcriptase (hTERT)-RPE cells were incubated with ornithine or other agents in the presence of 5-fluoromethylornithine (5-FMO), an OAT-specific inhibitor. mRNA expression was determined by quantitative real-time polymerase chain reaction, and the concentration of nitric oxide (NO) was quantified using a Griess assay. Furthermore, cytotoxicity was examined by morphologic observations and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assays, with the effect of arginase II examined using short interfering (si) RNA for arginase II and S-(2-boronoethyl)-L-cysteine (BEC), an arginase inhibitor., Results: NO production in 5-FMO-treated hTERT-RPE cells was increased by ornithine, and the NO donors S-nitroso-N-acetyl-DL-penicillamine (SNAP) and S-nitrosoglutathione induced cytotoxicity. Ornithine increased the expression of arginase II mRNA in 5-FMO-treated cells. Arginase II upregulation was partially inhibited by an NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester, which was mimicked by SNAP. Arginase II siRNA and BEC enhanced ornithine cytotoxicity, and arginase II silencing resulted in a further increase in NO production., Conclusions: These results demonstrate that NO is produced in our in vitro GA model, which induced cytotoxicity of RPE cells and upregulation of arginase II. NO may be involved in RPE degeneration in GA through the regulation of arginase II mRNA expression.

Neuropathic pain produced by damage to or dysfunction of the nervous system is a common and severely disabling state that affects millions of people worldwide. Recent evidence indicates that activated microglia are key cellular intermediaries in the pathogenesis of neuropathic pain and that ATP serves as the mediator. However, the in vivo mechanism underlying the retention of activated microglia in the injured region has not yet been completely elucidated. Prostaglandin E(2) (PGE(2)) is the principal proinflammatory prostanoid and plays versatile roles by acting via four PGE receptor subtypes, EP1-EP4. In the present study, we investigated the role of PGE(2) in spinal microglial activation in relation to neuropathic pain by using genetic and pharmacological methods. Mice deficient in microsomal prostaglandin E synthase-1 impaired the activation of microglia and the NMDA-nitric oxide (NO) cascade in spinal neurons in the dorsal horn and did not exhibit mechanical allodynia after peripheral nerve injury. The intrathecal injection of indomethacin, a nonsteroidal anti-inflammatory drug, ONO-8713, a selective EP1 antagonist, or 7-nitroindole, a neuronal NO synthase inhibitor, attenuated mechanical allodynia and the increase in activated microglia observed in the established neuropathic-pain state. We further demonstrated that ATP-induced microglial migration was blocked in vitro by PGE(2) via EP2 and by S-nitrosoglutathione, an NO donor. Taken together, the present study suggests that PGE(2) participated in the maintenance of neuropathic pain in vivo not only by activating spinal neurons, but also by retaining microglia in the central terminals of primary afferent fibers via EP2 subtype and via EP1-mediated NO production., (©2010 Wiley-Liss, Inc.)

Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) is a key mediator of long-term potentiation (LTP), which can be triggered by N-methyl-d-aspartate (NMDA) receptor-mediated Ca(2+) influx. We previously demonstrated that Fyn kinase-mediated phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 in the dorsal horn was involved in a neuropathic pain state even 1 week after nerve injury. Here we show that Y1472F-KI mice with a knock-in mutation of the Tyr1472 site to phenylalanine did not exhibit neuropathic pain induced by L5 spinal nerve transection, whereas they did retain normal nociceptive responses and induction of inflammatory pain. Phosphorylation of NR2B at Tyr1472 was only impaired in the spinal cord of Y1472F-KI mice among the major phosphorylation sites. There was no difference in the Ca(2+) response to glutamate and sensitivity to NMDA receptor antagonists between naive wild-type and Y1472F-KI mice, and the Ca(2+) response to glutamate was attenuated in the Y1472F-KI mice after nerve injury. Autophosphorylation of CaMKII at Thr286 was markedly impaired in Y1472F-KI mice after nerve injury, but there was no difference in phosphorylation of CaMKII at Thr305 or protein kinase Cγ at Thr674, and activation of neuronal nitric oxide synthase and microglia in the superficial layer of spinal cord between wild-type and Y1472F-KI mice after the operation. These results demonstrate that the attenuation of neuropathic pain is caused by the impaired NMDA receptor-mediated CaMKII signaling in Y1472F-KI mice, and suggest that autophosphorylation of CaMKII at Thr286 plays a central part not only in LTP, but also in persistent neuropathic pain., (© 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Background: The role of the diffusible messenger nitric oxide (NO) in the regulation of pain transmission is still a debate of matter, pro-nociceptive and/or anti-nociceptive. S-Nitrosylation, the reversible post-translational modification of selective cysteine residues in proteins, has emerged as an important mechanism by which NO acts as a signaling molecule. The occurrence of S-nitrosylation in the spinal cord and its targets that may modulate pain transmission remain unclarified. The "biotin-switch" method and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were employed for identifying S-nitrosylated proteins., Results: Here we show that actin was a major protein S-nitrosylated in the spinal cord by the NO donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP). Interestingly, actin was S-nitrosylated, more in the S2 fraction than in the P2 fraction of the spinal homogenate. Treatment of PC12 cells with SNAP caused rapid S-nitrosylation of actin and inhibited dopamine release from the cells. Just like cytochalasin B, which depolymerizes actin, SNAP decreased the amount of filamentous actin cytoskeleton just beneath the membrane. The inhibition of dopamine release was not attenuated by inhibitors of soluble guanylyl cyclase and cGMP-dependent protein kinase., Conclusion: The present study demonstrates that actin is a major S-nitrosylated protein in the spinal cord and suggests that NO directly regulates neurotransmitter release by S-nitrosylation in addition to the well-known phosphorylation by cGMP-dependent protein kinase.

In the central nervous system, the activation of neuronal nitric oxide synthase (nNOS) is closely associated with activation of NMDA receptor, and trafficking of nNOS may be a prerequisite for efficient NO production at synapses. We recently demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) and NMDA synergistically caused the translocation of nNOS to the membrane and stimulated NO production in PC12 (pheochromocytoma) cells. However, the mechanisms responsible for trafficking and activation of nNOS are largely unknown. To address these issues, here we constructed a yellow fluorescent protein (YFP)-tagged nNOS N-terminal (1-299 a.a.) mutant, nNOSNT-YFP, and visualized its translocation in PC12 cells stably expressing it. PACAP enhanced the translocation synergistically with NMDA in a time- and concentration-dependent manner. The translocation was blocked by inhibitors of protein kinase A (PKA), protein kinase C (PKC), and Src kinase; and the effect of PACAP could be replaced with PKA and PKC activators. The beta-finger region in the PSD-95/disc large/zonula occludens-1 domain of nNOS was required for the translocation of nNOS and its interaction with post-synaptic density-95 (PSD-95), and NO formation was attenuated by dominant negative nNOSNT-YFP. These results demonstrate that PACAP stimulated nNOS translocation mediated by PKA and PKC via PAC(1)-receptor (a PACAP receptor) and suggest cross-talk between PACAP and NMDA for nNOS activation by Src-dependent phosphorylation of NMDA receptors.

Central sensitization, similar to long-term potentiation in the hippocampus, refers to the increased synaptic efficacy established in somatosensory neurons in the dorsal horn of the spinal cord following tissue injury or nerve damage. In the course of inflammation, many proteins including glutamate receptors are assumed to be dynamically reorganized in the postsynaptic density (PSD) and involved in persistent pain. Mechanical hyperalgesia induced by intraplantar injection of complete Freund's adjuvant (CFA) was inhibited at 4 h, but not at 24 h, by indomethacin, an inhibitor of prostanoid synthesis. To elucidate the nature of the molecule(s) involved in the late phase of inflammatory pain, we analysed the PSD fraction prepared from the lumbar spinal cord of rats before and 24 h after CFA injection by conducting two-dimensional differential gel electrophoresis. N-ethylmaleimide-sensitive fusion protein (NSF) was identified as a downregulated protein in the PSD by MALDI-TOF MS and immunoblotting. Concomitant with the decrease in NSF, GluR2 and GluR3 were decreased and GluR1 was conversely increased in the PSD fraction 24 h after CFA injection. In vivo patch-clamp recordings of rats 24 h after CFA injection showed that excitatory postsynaptic currents of dorsal horn neurons evoked by pinch stimuli to inflamed skin were inwardly rectified and inhibited by 60% by philanthotoxin-433, a selective inhibitor of the Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. These results suggest that peripheral inflammation gives rise to central sensitization in the spinal cord through subunit composition switch of AMPA receptors in the late phase.

Prostaglandin F (PGF) ethanolamide (prostamide F) synthase, which catalyzed the reduction of prostamide H(2) to prostamide F(2alpha), was found in mouse and swine brain. The enzyme was purified from swine brain, and its amino acid sequence was defined. The mouse enzyme consisted of a 603-bp open reading frame coding for a 201-amino acid polypeptide with a molecular weight of 21,669. The amino acid sequence placed the enzyme in the thioredoxin-like superfamily with Cys(44) being the active site. The enzyme expressed in Escherichia coli as well as the native enzyme catalyzed not only the reduction of prostamide H(2) to prostamide F(2alpha) but also that of PGH(2) to PGF(2alpha). The V(max) and K(m) values for prostamide H(2) were about 0.25 micromol/min.mg of protein and 7.6 microm, respectively, and those for PGH(2) were about 0.69 micromol/min.mg of protein and 6.9 microm, respectively. Neither PGE(2) nor PGD(2) served as a substrate for this synthase. Based on these data, we named the enzyme prostamide/PGF synthase. Although the enzyme showed a broad specificity for reductants, reduced thioredoxin preferentially served as a reducing equivalent donor for this enzyme. Moreover, Northern and Western blot analyses in addition to the prostamide F synthase activity showed that the enzyme was mainly distributed in the brain and spinal cord, and the immunohistochemical study in the spinal cord showed that the enzyme was found mainly in the cytosol. These results suggest that prostamide/PGF synthase may play an important functional role in the central nervous system.

We have recently demonstrated that nitric oxide (NO) produced by neuronal NO synthase (nNOS) in the spinal cord is involved in the maintenance of neuropathic pain. To clarify whether NO itself affected nNOS activity in the spinal cord as a retrograde messenger, we examined the involvement of the NO/cGMP signaling pathway in the regulation of nNOS activity by NADPH-diaphorase histochemistry. NO-generating agents NOR3 (t(1/2)=30min) and SNAP (t(1/2)=5h), but not NOR1 (t(1/2)=1.8min), significantly enhanced NADPH-diaphorase staining in the spinal cord. 8-Br-cGMP also enhanced it similar to that by NOR3, and 8-Br-cAMP and forskolin, an activator of adenylate cyclase, enhanced it moderately. NOR1 and NOR3 markedly increased the cGMP level in the spinal cord. The enhancement of NADPH-diaphorase staining by NOR3 was significantly inhibited by CPTIO, an NO scavenger, ODQ, a soluble guanylate cyclase inhibitor, and KT5823, an inhibitor of cGMP-dependent protein kinase. Additionally, the NOR3-enhanced nNOS activity was completely inhibited by NMDA antagonists MK-801 and d-AP5, partially by the GluRepsilon2-selective antagonist CP-101,606, and was attenuated in GluRepsilon1(-/-) and GluRepsilon1(-/-)/epsilon4(-/-) mice. These results suggest that NO may regulate nNOS activity as a retrograde messenger in the spinal cord via activation of NMDA receptor containing GluRepsilon1 and GluRepsilon2 subunits.

We previously showed that ornithine was mainly transported via cationic amino acid transporter (CAT)-1 in human retinal pigment epithelial (RPE) cell line, human telomerase RT (hTERT)-RPE, and that CAT-1 was involved in ornithine cytotoxicity in ornithine-delta-aminotransferase (OAT)-deficient cell produced by a OAT specific inhibitor, 5-fluoromethylornithine (5-FMO). We showed here that CAT-1 mRNA expression was increased by ornithne in OAT-deficient RPE cells, which was reversed by an inhibitor of ornithine decarboxylase (ODC), alpha-difluoromethylornithine (DFMO). Polyamines, especially spermine, one of the metabolites of ODC, also enhanced the expression of CAT-1 mRNA. ODC mRNA expression was also increased by ornithine and polyamines, and gene silencing of ODC by siRNA decreased ornithine transport activity and its cytotoxicity. In addition, the mRNA of nuclear protein c-myc was also increased in 5-FMO- and ornithine-treated hTERT-RPE cells, and gene silencing of c-myc prevented the induction of CAT-1 and ODC. Increases in expression of CAT-1, ODC, and c-myc, and the inhibition of these stimulated expression by DFMO were also observed in primary porcine RPE cells. These results suggest that spermine plays an important role in stimulation of mRNA expression of CAT-1, which is a crucial role in ornithine cytotoxicity in OAT-deficient hTERT-RPE cells.

Interleukin (IL)-1 and tumor necrotic factor alpha (TNFalpha) are pivotal in the pathogenesis of endotoxemia. In spite of the in vitro finding that IL-1beta, but not TNFalpha, can induce iNOS mRNA and NO production as a single stimulus in hepatocytes in primary culture, the involvement of IL-1 in iNOS induction in the liver has been less clear in vivo. To address this, we challenged IL-1alpha/beta double-knockout (IL-1alpha/beta(-/-)) and TNFalpha(-/-) mice with lipopolysaccharide (LPS). As compared with wild-type mice, the increases in the plasma NO level measured as nitrite and nitrate and hepatic iNOS were significantly reduced in IL-1alpha/beta(-/-) and TNFalpha(-/-) mice 8 and 12h after the LPS challenge. In the wild-type mice, iNOS protein was first detected in Kupffer cells around the portal vein 2h after LPS challenge; and then it spread to hepatocytes throughout the intralobular region of the liver by 8h. Although the expression of iNOS protein was detected in Kupffer cells of both IL-1alpha/beta(-/-) and TNFalpha(-/-) mice, its level was moderate in hepatocytes of IL-1alpha/beta(-/-) mice, but negligible in those of TNFalpha(-/-) mice, 8h after LPS challenge. Concomitant with the expression of iNOS protein in the liver, Toll-like receptor 4, the signaling receptor for LPS, was expressed in hepatocytes of wild-type and IL-1alpha/beta(-/-) mice, but not of TNFalpha(-/-) mice. These results demonstrate that the expression of Toll-like receptor 4 is well correlated with that of iNOS protein in hepatocytes in vivo after LPS challenge and that IL-1 is not essential for the induction of iNOS in hepatocytes in vivo.

Nociceptin/orphanin FQ (N/OFQ) was earlier shown to be involved in the maintenance of neuropathic pain by activating neuronal nitric oxide synthase (nNOS). We recently established an ex vivo system to elucidate biochemical and molecular mechanisms for nNOS activation by the use of a combination of isolated intact spinal cord preparations and NADPH-diaphorase histochemistry. Here we examined the N/OFQ signal pathways coupled to nNOS activation in the spinal cord by using this ex vivo system. N/OFQ enhanced nNOS activity in the superficial layer of the spinal cord, as assessed by NADPH-diaphorase histochemistry, in a time- and dose-dependent manner. The maximum effect was observed at 3-10 nM. The N/OFQ-stimulated nNOS activity was inhibited by NMDA receptor antagonists MK-801 and D-AP5, but not by the NR2B-selective antagonist CP-101,606; and the stimulated activity was observed in NR2D(-/-) mice, but not in NR2A(-/-) or NR2A(-/-)/NR2D(-/-) mice. N/OFQ receptor antagonists attenuated the nNOS activity stimulated by N/OFQ, but not that by NMDA. Furthermore, the potentiation of nNOS by N/OFQ was inhibited by calphostin C and Ro 31-8220, PP2, and KN-62, but not by H-89. These results suggest that N/OFQ stimulated nNOS activity by a biochemical cascade initiated by activation of NMDA receptors containing NR2A.

Purpose: A prior report showed ornithine cytotoxicity in ornithine-delta-aminotransferase (OAT)-deficient human retinal pigment epithelial (RPE) cells in an in vitro model of gyrate atrophy of the choroid and retina. This study was intended to clarify the mechanism of ornithine cytotoxicity and to determine the responsible amino acid transporters., Methods: The mRNA expression of amino acid transporters in human telomerase reverse transcriptase (hTERT)-RPE cells was examined by reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis. Carrier-mediated ornithine transport via the L-type amino acid transporter (LAT)1, LAT2, cationic amino acid transporter (CAT)-1, and y(+)LAT2 systems was evaluated by short interfering (si)RNA-mediated gene silencing. The cytoprotective effect of CAT-1-specific siRNA on ornithine cytotoxicity was measured using quantitative analysis of cellular adenosine triphosphate (ATP) at 24 hours after treatment with ornithine in OAT-deficient RPE cells., Results: LAT1, LAT2, CAT-1, and y(+)LAT2 mRNA expression was detected by Northern blot analysis, whereas RT-PCR revealed that LAT1, LAT2, y(+)LAT1, y(+)LAT2, CAT-1, and b(0,+)AT mRNAs were expressed together with the heterodimeric glycoproteins 4F2hc and rBAT in hTERT-RPE cells. l-[(14)C]ornithine uptake in hTERT-RPE cells was decreased by 46.6% and 22.0% by CAT-1 and y(+)LAT2 siRNA, respectively, whereas LAT1 and LAT2 siRNA had no significant effect. Further, CAT-1 silencing by siRNA reduced ornithine cytotoxicity in OAT-deficient RPE cells., Conclusions: The results suggest that ornithine transport via CAT-1 may play a crucial role in ornithine cytotoxicity in hTERT-RPE cells. Reduction of the ornithine transport via CAT-1 may be a new target for treatment of gyrate atrophy.

Purpose: A prior study showed inactivation of ornithine-delta-aminotransferase (OAT)-deficient human retinal pigment epithelial (RPE) cells by a specific irreversible inhibitor (5-fluoromethylornithine; 5-FMO) leading to cell death, in an in vitro model of gyrate atrophy (GA) of the choroid and retina. In the present study, the cytotoxicity of metabolites of ornithine, especially spermine, in RPE cells was investigated, to clarify the mechanism of ornithine cytotoxicity in RPE cells., Methods: RPE cells were incubated with ornithine or compounds involved in ornithine metabolic pathways. The effects on RPE cell viability and proliferative activity were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric and [(3)H]thymidine incorporation assays. Incorporation of spermine into RPE cells was examined by using [(14)C]spermine and dansyl-spermine. To assess spermine-induced RPE cell death, cells were double stained with annexin V and propidium iodide and subjected to flow cytometry., Results: Ornithine, arginine, glutamate, proline, creatine, glycine, and putrescine exhibited no effects on the viability and proliferative activities of RPE cells, whereas spermidine and spermine (10 mM) inhibited [(3)H]thymidine incorporation by 13% and 89%, respectively. The inhibition of [(3)H]thymidine incorporation by spermine was dose dependent and was observed as early as 4 hours after addition. Further, spermine was incorporated and accumulated in the perinuclear region of RPE cells. Apoptotic RPE cell death was induced by spermine in a dose-dependent manner., Conclusions: The present results demonstrated that excessive spermine is cytotoxic to RPE cells and suggest that metabolites of ornithine, especially spermine, may be involved in the mechanism of RPE degeneration in GA.

Primary afferent fibers are originated from pseudounipolar sensory cells in dorsal root ganglia (DRG) and transmit external stimuli received in the skin to the spinal cord. Here we undertook a proteomic approach to uncover the polarity of primary afferent fibers. Lumbar spinal nerve segments, peripheral and central to DRG, were dissected from 5-wk-old Wistar rats and the lysates were subjected to large-sized 2-DE at pH 5-6. Among approximately 800 protein spots detected in the central and peripheral fractions, one of the unique spots in the peripheral fraction with MW of 60 kDa and pI of 5.6 was identified as an isoform of collapsin response mediator protein-2 (CRMP-2) by MALDI-TOF MS and Western blots with anti-CRMP-2 antibodies that recognize 1-17 and 486-528 residues. Since this novel spot was detected only in the peripheral fraction, but not in the central fraction, DRG, and other regions of the brain, it was named periCRMP-2. The C-terminal fragment of CRMP-2 was not detected in periCRMP-2 by MS analyses. Expression of periCRMP-2 decreased following sciatic nerve injury. These results suggest that periCRMP-2 is a C-terminal truncated isoform polarized in the peripheral side of spinal nerves and may be involved in nerve degeneration and regeneration.

Pain often outlasts its usefulness as warning and aid in wound healing, and becomes chronic and intractable after tissue damage and nerve injury. Many molecules have been implicated as mediators and modulators in persistent pain such as hyperalgesia and tactile pain (allodynia). We previously showed that prostaglandin (PG) E(2), PGF(2alpha) or the neuropeptide nociceptin, also called orphanin FQ (N/OFQ) administered intrathecally (i.t.) produced allodynia in conscious mice. In the present study, we examined the relationship of pain responses between PGs and N/OFQ using the N/OFQ receptor (NOP) antagonist, N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxy-methyl)benzamide monohydrochloride (JTC-801), and in mice lacking the N/OFQ prepropeptide (ppN/OFQ(-/-)) and the NOP receptor (NOP(-/-)). JTC-801 dose-dependently blocked the N/OFQ- and PGE(2)-induced allodynia, but not the PGF(2alpha)-induced one. Neither N/OFQ nor PGE(2) induced allodynia in NOP(-/-) mice. By contrast, the N/OFQ-induced allodynia was not affected by inhibition of PG production by a 60-min pretreatment with the non-steroidal anti-inflammatory drug, indomethacin. Among PGE receptor (EP) subtype-selective agonists, the EP4 agonist, AE1-329, markedly stimulated the release of N/OFQ from spinal slices and induced allodynia. AE1-329 also increased nitric oxide production in spinal slices using fluorescent nitric oxide detection, which was blocked by pretreatment with JTC-801. Conversely, PGE(2)-induced allodynia was not observed in ppN/OFQ(-/-) mice. N/OFQ immunoreactive puncta were colocalized with EP4. Taken together, these results demonstrate that PGE(2) induced allodynia by stimulation of N/OFQ release in the spinal cord via EP4 receptor subtypes.

Prostaglandin E2 (PGE2), the principal pro-inflammatory prostanoid, is known to play versatile roles in pain transmission via four PGE receptor subtypes, EP1-EP4. We recently demonstrated that continuous production of nitric oxide (NO) by neuronal NO synthase (nNOS) following phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) and NMDA receptor NR2B subunits is essential for neuropathic pain. These phosphorylation and nNOS activity visualized by NADPH-diaphorase histochemistry were blocked by indomethacin, a PG synthesis inhibitor. To clarify the interaction between cyclooxygenase and nNOS pathways in the spinal cord, we examined the effect of EP subtype-selective agonists on NO production. NO formation was stimulated in the spinal superficial layer by EP1, EP3, and EP4 agonists. While the EP1- and the EP4-stimulated NO formation was markedly blocked by MK-801, an NMDA receptor antagonist, the EP3-stimulated one was completely inhibited by H-1152, a Rho-kinase inhibitor. Phosphorylation of MARCKS and NADPH-diaphorase activity stimulated by the EP3 agonist were also blocked by H-1152. These results suggest that PGE2 stimulates NO formation by Rho-kinase via EP3, a mechanism(s) different from EP1 and EP4.

It has been reported that nipradilol, a nonselective beta- and selective alpha1-receptor antagonist, has cytoprotective effects. We attempted to clarify the effects of nipradilol on the expression of apoptosis associated genes and the activity of nuclear factor-kappaB, a transcription factor, in PC12 cells during serum deprivation induced apoptosis. PC12 cells were cultured in serum free RPMI1640 medium with or without 0.01, 0.1, 1, or 10 microM of nipradilol, or in serum-added medium as a control. The gene expressions of Bax, Bcl-2, Fas, FasL, Caspase-1, 2, 3, and 9, p53, and Smac/DIABLO were examined using a quantitative real time polymerase chain reaction method, while nuclear factor-kappaB activity was examined using an electrophoresis mobility shift assay with a nuclear factor-kappaB consensus sequenced DNA probe. The effects of denitronipradilol were also examined to clarify the effect of nitric oxide donative action. Nipradilol down-regulated Bax gene expression 12 hr after serum deprivation, and that of the capase-9 and Smac/DIABLO genes at 24 hr, compared to the serum-free sample, while it also increased cell viability and decreased DNA ladder formation at 48 hr. However, the expressions of other examined genes were not affected by the agent. In addition, nuclear factor-kappaB activity was increased 2 hr after the addition of 0.1 or 1 microM of nipradilol. In contrast, denitronipradilol did not show any effects toward PC12 cells. Our results suggest that nipradilol may have an effect on apoptosis associated gene expression and nuclear factor-kappaB activity during the prevention of apoptosis via nitric oxide donative action.