Truncated GPNMB, a microglial transmembrane protein, serves as a scavenger receptor for oligomeric ß-amyloid peptide1-42 in primary type 1 microglia.

Glycoprotein non-metastatic melanoma protein B (GPNMB) is up-regulated in one subtype of microglia (MG) surrounding senile plaque depositions of amyloid-beta (Aß) peptides. However, whether the microglial GPNMB can recognize the fibrous Aß peptides as ligands remains unknown. In this study, we report that the truncated form of GPNMB, the antigen for 9F5, serves as a scavenger receptor for oligomeric Aß1-42 (o-Aß1-42 ) in rat primary type 1 MG. 125 I-labeled o-Aß1-42 exhibited specific and saturable endosomal/lysosomal degradation in primary-cultured type 1 MG from GPNMB-expressing wild-type mice, whereas the degradation activity was markedly reduced in cells from Gpnmb-knockout mice. The Gpnmb-siRNA significantly inhibits the degradation of 125 I-o-Aß1-42 by murine microglial MG5 cells. Therefore, GPNMB contributes to mouse MG's o-Aß1-42 clearance. In rat primary type 1 MG, the cell surface expression of truncated GPNMB was confirmed by a flow cytometric analysis using a previously established 9F5 antibody. 125 I-labeled o-Aß1-42 underwent endosomal/lysosomal degradation by rat primary type 1 MG in a dose-dependent fashion, while the 9F5 antibody inhibited the degradation. The binding of 125 I-o-Aß1-42 to the rat primary type 1 MG was inhibited by 42% by excess unlabeled o-Aß1-42 , and by 52% by the 9F5 antibody. Interestingly, the 125 I-o-Aß1-42 degradations by MG-like cells from human-induced pluripotent stem cells was inhibited by the 9F5 antibody, suggesting that truncated GPNMB also serve as a scavenger receptor for o-Aß1-42 in human MG. Our study demonstrates that the truncated GPNMB (the antigen for 9F5) binds to oligomeric form of Aß1-42 and functions as a scavenger receptor on MG, and 9F5 antibody can act as a blocking antibody for the truncated GPNMB.

Synthesis and evaluation of radioiodinated 1-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}piperidin-4-amine derivatives for platelet-derived growth factor receptor ß (PDGFRß) imaging.

Platelet-derived growth factor receptor ß (PDGFRß) is a transmembrane tyrosine kinase receptor and it is upregulated in various malignant tumors. Radiolabeled PDGFRß inhibitors can be a convenient tool for the imaging of tumors overexpressing PDGFRß. In this study, [125I]-1-{5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinoline-8-yl}piperidin-4-amine ([125I]IIQP) and [125I]-N-3-iodobenzoyl-1-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}-piperidin-4-amine ([125I]IB-IQP) were designed and synthesized, and their potential as PDGFRß imaging agents was evaluated. In cellular uptake experiments, [125I]IIQP and [125I]IB-IQP showed higher uptake by PDGFRß-positive cells than by PDGFRß-negative cells, and the uptake in PDGFRß-positive cells was inhibited by co-culture with PDGFRß ligands. The biodistribution of both radiotracers in normal mice exhibited hepatobiliary excretion as the main route. In mice inoculated with BxPC3-luc (PDGFRß-positive), the tumor uptake of radioactivity at 1h after the injection of [125I]IIQP was significantly higher than that after the injection of [125I]IB-IQP. These results indicated that [125I]IIQP can be a suitable PDGFRß imaging agent. However, further modification of its structure will be required to obtain a more appropriate PDGFRß-targeted imaging agent with a higher signal/noise ratio.

Autocrine Semaphorin3A signaling is essential for the maintenance of stem-like cells in lung cancer.

Cancer stem-like cells (CSCs) exist in tumor tissues composed of heterogeneous cell population and are characterized by their self-renewal capacity and tumorigenicity. Many studies demonstrate that eradication of CSCs prevents development and recurrences of tumor; yet, molecules critical for the maintenance of CSCs have not been completely understood. We previously reported that Semaphorin3A (Sema3a) knockdown suppressed the tumorigenicity and proliferative capacity of Lewis lung carcinoma (LLC) cells. Therefore, we identified Sema3a as an essential factor for the establishment or maintenance of CSCs derived from LLC (LLC-stem cell). shRNA against Sema3a was introduced into LLC cells to establish a LLC-stem cell line and its effects on tumorigenesis, sphere formation, and mTORC1 activity were tested. Sema3a knockdown completely abolished tumorigenicity and the sphere-formation and self-renewal ability of LLC-stem cells. The Sema3a knockdown was also associated with decreased expression of mRNA for stem cell markers. The self-renewal ability abolished by Sema3a knockdown could not be recovered by exogenous addition of recombinant SEMA3A. In addition, the activity of mammalian target of rapamycin complex 1 (mTORC1) and the expression of its substrate p70S6K1 were also decreased. These results demonstrate that Sema3a is a potential therapeutic target in eradication of CSCs.

Plexin A1 signaling confers malignant phenotypes in lung cancer cells.

Aberrant changes to several signaling pathways because of genetic mutations or increased cytokine production are critical for tumor cells to become malignant. Semaphorin 3A (SEMA3A) acts as a bivalent factor that suppresses or promotes tumor development in different pathological backgrounds. Previously, we showed that SEMA3A positively regulated the proliferative and glycolytic activities of mouse-derived Lewis lung carcinoma (LLC) cells. Plexins A1-A4 (PLXNA1-PLXNA4) are SEMA3A receptors; however, it is not known which subtype is critical for oncogenic SEMA3A signaling. We used LLC cells to investigate the role of PLXNA1 in oncogenic SEMA3A signaling. Using short hairpin RNA-mediated knockdown, we investigated the effects of constitutive inhibition of Plxna1 on cell proliferation, metabolic dependency, and epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) sensitivity. We found that Plxna1 knockdown did not affect apoptosis but resulted in decreased cell proliferation and reductions in mRNA expression levels of proliferation-marker genes, such as Ccnd1, Pcna, and Myc. In addition, we found decreased mRNA expression levels of glycolysis-associated genes, such as Pkm2 and Ldha, and decreased lactate production. In contrast, we found no changes in the mRNA expression levels of oxidative phosphorylation-associated genes, such as Cycs, Cox5a, and Atp5g1. We found that Plxna1 knockdown conferred resistance to glucose starvation but increased cytotoxicity to oligomycin. Plxna1 or Sema3a knockdown caused an increased sensitivity to the EGFR-TKIs gefitinib and erlotinib, in Lewis lung carcinoma (LLC) cells. These findings demonstrate that PLXNA1 mediates the acquisition of malignant phenotypes induced by autocrine SEMA3A signaling.

Cancer pain relief achieved by disrupting tumor-driven semaphorin 3A signaling in mice.

Cancer-induced bone pain (CIBP) is the most common pain arising from cancer and is inadequately managed with current standard therapeutics. While the etiology of CIBP remains to be fully elucidated, increasing evidence suggests that CIBP is uniquely complex. We tested whether semaphorin 3A (Sema3A) signals were involved in the development of CIBP in mice. The mouse model employed for CIBP - mice inoculated with Lewis lung carcinoma (LLC) cells injected into the femur intramedullary space - showed progressive decline in the weight bearing of the ipsilateral hind limb. The LLC cell inoculation resulted in a progressive increase in Sema3A mRNA expression over time and an increase in the number of Sema3A-immunoreactive cells in the ipsilateral femur. To define the role of Sema3A in development of CIBP, we employed a lentiviral expression system to establish a stable LLC cell line expressing scrambled shRNA for the control group (LLC/scramble) and shRNAs directed against Sema3A mRNA for the loss-of-function group (LLC/shSema3A). Inoculation of LLC/shSema3A did not cause upregulation of Sema3A mRNA expression and proliferation of the inoculated cells in the femur compared to that in mice inoculated with LLC/scramble. Mice inoculated with LLC/shSema3A, but not LLC/scramble, showed an attenuation of the significant decline in the weight bearing of the ipsilateral hind paw. Our findings indicate that Sema3A serves as a potential therapeutic target for CIBP.

The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin-like protease(s) in a subpopulation of microglia in neonatal rat brain.

To differentiate subtypes of microglia (MG), we developed a novel monoclonal antibody, 9F5, against one subtype (type 1) of rat primary MG. The 9F5 showed high selectivity for this cell type in Western blot and immunocytochemical analyses and no cross-reaction with rat peritoneal macrophages (Mφ). We identified the antigen molecule for 9F5: the 50- to 70-kDa fragments of rat glycoprotein nonmetastatic melanoma protein B (GPNMB)/osteoactivin, which started at Lys(170) . In addition, 9F5 immunoreactivity with GPNMB depended on the activity of furin-like protease(s). More important, rat type 1 MG expressed the GPNMB fragments, but type 2 MG and Mφ did not, although all these cells expressed mRNA and the full-length protein for GPNMB. These results suggest that 9F5 reactivity with MG depends greatly on cleavage of GPNMB and that type 1 MG, in contrast to type 2 MG and Mφ, may have furin-like protease(s) for GPNMB cleavage. In neonatal rat brain, amoeboid 9F5+ MG were observed in specific brain areas including forebrain subventricular zone, corpus callosum, and retina. Double-immunοstaining with 9F5 antibody and anti-Iba1 antibody, which reacts with MG throughout the CNS, revealed that 9F5+ MG were a portion of Iba1+ MG, suggesting that MG subtype(s) exist in vivo. We propose that 9F5 is a useful tool to discriminate between rat type 1 MG and other subtypes of MG/Mφ and to reveal the role of the GPNMB fragments during developing brain. GLIA 2016;64:1938-1961.

mTORC1 is a critical mediator of oncogenic Semaphorin3A signaling.

Aberration of signaling pathways by genetic mutations or alterations in the surrounding tissue environments can result in tumor development or metastasis. However, signaling molecules responsible for these processes have not been completely elucidated. Here, we used mouse Lewis lung carcinoma cells (LLC) to explore the mechanism by which the oncogenic activity of Semaphorin3A (Sema3A) signaling is regulated. Sema3A knockdown by shRNA did not affect apoptosis, but decreased cell proliferation in LLCs; both the mammalian target of rapamycin complex 1 (mTORC1) level and glycolytic activity were also decreased. In addition, Sema3A knockdown sensitized cells to inhibition of oxidative phosphorylation by oligomycin, but conferred resistance to decreased cell viability induced by glucose starvation. Furthermore, recombinant SEMA3A rescued the attenuation of cell proliferation and glycolytic activity in LLCs after Sema3A knockdown, whereas mTORC1 inhibition by rapamycin completely counteracted this effect. These results demonstrate that Sema3A signaling exerts its oncogenic effect by promoting an mTORC1-mediated metabolic shift from oxidative phosphorylation to aerobic glycolysis.

Tumor cell-derived secretory factor downregulates Semaphorin-3a in osteoblasts by activating mammalian target of rapamycin pathway.

We found that conditioned medium derived from Lewis Lung Carcinoma cells down-regulated Semaphorin3a (Sema3a) mRNA expression and increased the activity of mammalian target of rapamycin complex 1 (mTORC1) in osteoblast-like MC3T3-E1 cells. Furthermore, mTORC1 inhibition with rapamycin counteracted the effect of conditioned media on Sema3a mRNA expression. These results suggest that tumor cells decrease Sema3a mRNA expression in osteoblast in an mTORC1-dependent manner.

Macrophage-T cell interactions mediate neuropathic pain through the glucocorticoid-induced tumor necrosis factor ligand system.

Peripheral neuroinflammation caused by activated immune cells can provoke neuropathic pain. Herein, we investigate the actions of macrophages and T cells through glucocorticoid-induced tumor neurosis factor receptor ligand (GITRL) and its receptor (GITR) in neuropathic pain. After partial sciatic nerve ligation (PSL) in enhanced green fluorescent protein (eGFP) chimeric mice generated by the transplantation of eGFP(+) bone marrow cells, eGFP(+) macrophages, and T cells markedly migrated to the injured site after PSL. Administration of agents to deplete macrophages (liposome-clodronate and Clophosome-A(TM)) or T cells (anti-CD4 antibody and FTY720) could suppress PSL-induced thermal hyperalgesia and tactile allodynia. The expression levels of co-stimulatory molecules GITRL and GITR were increased on infiltrating macrophages and T cells, respectively. The perineural injection of a GITRL neutralizing antibody that could inhibit the function of the GITRL-GITR pathway attenuated PSL-induced neuropathic pain. Additionally, the induction of inflammatory cytokines and the accumulation of GITR(+) T cells in the injured SCN were abrogated after macrophage depletion by Clophosome-A(TM). In conclusion, GITRL expressed on macrophages drives cytokine release and T cell activation, resulting in neuropathic pain via GITR-dependent actions. The GITRL-GITR pathway might represent a novel target for the treatment of neuropathic pain.

Peripheral interleukin-4 ameliorates inflammatory macrophage-dependent neuropathic pain.

There is increasing evidence that inflammatory (M1-polarized) macrophages drive the nonresolving neuroinflammation that causes neuropathic pain after nerve injury. As interleukin-4 (IL-4) promotes the suppressive (M2-polarized) state in macrophages, we examined whether exploiting an IL-4-mediated pathway could ameliorate M1 macrophage-dependent neuropathic pain. The mRNA and protein expression of IL-4 receptor α chain (IL-4Rα) were upregulated in accumulating F4/80 macrophages in injured sciatic nerve (SCN). In mouse macrophage cell line J774A.1, IL-4 downregulated the mRNA expression of M1 macrophage-specific molecules (IL-1ß, CC chemokine ligand 3, and CD86) normally provoked by lipopolysaccharide, while increasing the mRNA expression of M2 macrophage-specific molecules (arginase-1, IL-10, and CD206) through a STAT6-mediated pathway. In ex vivo SCN culture, M1 molecules were highly expressed in the injured SCN on day 7 after partial SCN ligation (PSL) but were decreased by IL-4 treatment. In contrast, M2 molecules were upregulated by IL-4. IL-4 also increased phosphorylated STAT6 (pSTAT6) expression and shifted IL-1ß M1 macrophages toward a CD206 M2 phenotype. Perineural administration of IL-4 in mice subject to PSL ameliorated development and maintenance of tactile allodynia and thermal hyperalgesia. These effects of IL-4 were based on that IL-4 treatment increased the proportions of pSTAT6 and CD206 macrophages in injured SCN on day 14 after PSL. We found that neuropathic pain can be ameliorated by IL-4 treatment, which exerts its therapeutic effect on accumulating macrophages through a STAT6-dependent pathway. A shift in macrophage phenotype from the inflammatory to the suppressive phenotype, driven by IL-4R signaling, may have benefits in the treatment of neuropathic pain.

[Study on novel mechanism underlying analgesia targeting TRPV1].

Transient receptor potential protein (TRP) channels are distributed in pain pathways including primary afferent neurons and function as transduction of various noxious stimuli to innocuous stimuli. TRP channels are considered as molecular basis of chronic pain. Targeting TRPs may lead to novel class of analgesics, and so drug-discovery efforts are focused on TRP agonists and its antagonists. Few products have, however, been placed on the market, because most of candidates have adverse effects. A lesion or disease of the somatosensory nervous system causes neuropathic pain, a type of chronic pain. Neuropathic pain is intolerable and obstinate and therefore, debilitates the affected patients. A great deal of effort has been made to develop medicine targeting molecules involved in neuropathic pain, whereby the promising therapeutically targeted molecules have been identified. Neuroinflammation, based on pathological alteration in crosstalk between nervous system and immune system, has been a focus of attention as pathological mechanism involved in development of neuropathic pain. Recently, we used an animal model for neuropathic pain to find the possibility that neuropathic pain was exacerbated by adipokines derived from perineural adipocytes distributed in injured peripheral neurons. A working hypothesis is therefore proposed that the perineural adipocytes interacts with the immune cells, which also have TRPV1, in injured peripheral nerve, followed by a paracrine loop involving proinflammatory cytokines, chemokines and adipokines derived from them which aggravates and prolongs pain. Here, we overview the developmental status in TRPV1-targetting analgesics and illustrate our recent findings in terms of neuroinflammation.

Possible involvement of endogenous opioid system located downstream of α7 nicotinic acetylcholine receptor in mice with physical dependence on nicotine.

We previously reported that nicotine (NIC)-induced analgesia was elicited in part by activation of the endogenous opioid system. Moreover, it is well known that NIC has physical-dependence liability, but its mechanism is unclear. Therefore, we examined whether physical dependence on NIC was mediated by activation of the endogenous opioid system in ICR mice. We evaluated increased serum corticosterone (SCS) as an indicator of NIC withdrawal, as it is a quantitative indicator of naloxone (opioid receptor antagonist, NLX)-precipitated morphine withdrawal in mice. In this study, NLX precipitated an SCS increase in mice receiving repeated NIC, by a dose-dependent mechanism, and correlated with the dose and number of days of repeated NIC administration. When an opioid receptor antagonist (naltrexone) was concomitantly administered with repeated NIC, the NLX-precipitated SCS increase was not elicited. Concomitant administration of the α7 nicotinic acetylcholine receptor (nAChR) antagonist (methyllycaconitine) with repeated NIC, but not the α4ß2 nAChR antagonist (dihydro-ß-erythroidine), did not elicit an SCS increase by NLX. Thus, a physical dependence on NIC was in part mediated by the activation of the endogenous opioid system, located downstream of α7 nAChR.

HMGB1 as a potential therapeutic target for neuropathic pain.

Neuropathic pain, which is intolerable and persistent, arises as a direct consequence of a lesion or disease affecting the somatosensory system and can be debilitating for the affected patients. Accumulating evidence from animal studies has revealed the potential molecular basis for neuropathic pain, resulting in many promising therapeutic targets. While efforts at drug discovery have been made, conventional pharmacotherapy, including the use of opioid analgesics, is still insufficient for the relief of neuropathic pain. Therefore, novel target molecules that may lead to the development of promising analgesics are eagerly anticipated for improved treatment of neuropathic pain. In various insults such as sepsis and ischemia, high-mobility group box 1 (HMGB1) is released extracellularly to induce inflammation. HMGB1 was originally identified as a ubiquitous nuclear protein, but emerging evidence has suggested that HMGB1 also plays a role in neuroinflammation as a pro-inflammatory mediator. These findings suggest that HMGB1 may be involved in the pathology of neuropathic pain. In fact, some reports demonstrate an involvement of HMGB1 in the development and maintenance of neuropathic pain in experimental animals. Here, we overview the characteristics of HMGB1 as a pro-inflammatory mediator and show the promise of HMGB1 as a therapeutic target for neuropathic pain.

Activation of nicotinic acetylcholine receptors on bone marrow-derived cells relieves neuropathic pain accompanied by peripheral neuroinflammation.

Emerging evidence indicates that chronic neuroinflammation plays a pivotal role in neuropathic pain. We explored whether activation of the nicotinic acetylcholine receptor (nAChRs) pathway on peripheral immune cells improves neuropathic pain. Mice were subjected to partial sciatic nerve ligation (PSL). Enhanced green fluorescent protein (EGFP)-chimeric mice were generated by transplantation of EGFP(+) bone marrow (BM) cells from EGFP-transgenic mice into wild-type mice. EGFP(+) BM-derived cells infiltrated the injured sciatic nerve (SCN) of EGFP-chimeric mice, and these cells were found to be F4/80(+) macrophages and Ly6G(+) neutrophils. The protein expression of nAChR subunit α4 and α7 were up-regulated in the injured SCN. Increased α4 and α7 subunits were localized on both BM-derived macrophages and neutrophils. When nicotine (20nmol) was perineurally administered once a day for 4days (days 0-3), PSL-induced tactile allodynia and thermal hyperalgesia were significantly prevented. Relieving effects of nicotine on neuropathic pain were reversed by co-administration of mecamylamine (20nmol), a non-selective antagonist for nAChRs. PSL-induced up-regulation of inflammatory cytokines and chemokines was suppressed by perineural administration of nicotine. Taken together, the expression of α4ß2 and α7 subtypes of nAChRs may be increased on circulating macrophages and neutrophils in injured peripheral nerves. Activation of nAChRs on immune cells may relieve neuropathic pain accompanied by the suppression of neuroinflammation.

The critical role of spinal ceramide in the development of partial sciatic nerve ligation-induced neuropathic pain in mice.

Recent observations indicate that peripheral nerve injury induces central sensitization through microglial activation and the release of inflammatory cytokines, resulting in the development of neuropathic pain. However, the underlying mechanisms of this phenomenon remain to be fully elucidated. In this study, we examined the involvement of spinal ceramide, a bioactive lipid, in the development of neuropathic pain induced by partial sciatic nerve ligation (PSL). We found that the mRNA expression levels for ceramide synthase and neutral sphingomyelinase, which are enzymes of ceramide biosynthesis, were up-regulated in the spinal cord from 3h to 1 day after PSL. The mRNA expressions of cytokines (interleukin-1ß and tumor necrosis factor-α) and the microglial specific molecules (Iba-1 and CD11b) were also increased in the spinal cord after PSL. In the von Frey test, intrathecal injection of the ceramide biosynthesis inhibitors Fumonisin B1 and GW4869 at 3h and day 3 after PSL significantly attenuated PSL-induced tactile allodynia. By immunohistochemistry, microglial activation in the dorsal horn was suppressed by Fumonisin B1 and GW4869. Therefore, we conclude that spinal ceramide may play a crucial role in PSL-induced neuropathic pain through the activation of microglia.

Epigenetic augmentation of the macrophage inflammatory protein 2/C-X-C chemokine receptor type 2 axis through histone H3 acetylation in injured peripheral nerves elicits neuropathic pain.

Although there is growing evidence showing that the involvement of chemokines in the pathogenesis of neuropathic pain is associated with neuroinflammation, the details are unclear. We investigated the C-X-C chemokine ligand type 2 [macrophage inflammatory protein 2 (MIP-2)]/C-X-C chemokine receptor type 2 (CXCR2) axis and epigenetic regulation of these molecules in neuropathic pain after peripheral nerve injury. Expression of MIP-2 and CXCR2 were up-regulated and localized on accumulated neutrophils and macrophages in the injured sciatic nerve (SCN) after partial sciatic nerve ligation (PSL). Perineural injection of MIP-2-neutralizing antibody (anti-MIP-2) or the CXCR2 antagonist N-(2-bromophenyl)-N'-(2-hydroxy-4-nitrophenyl)urea (SB225002) prevented PSL-induced tactile allodynia and thermal hyperalgesia. Perineural injection of recombinant MIP-2 elicited neuropathic pain-like behaviors. Anti-MIP-2 suppressed neutrophil accumulation in the SCN after PSL. Neutrophil depletion by intraperitoneal injection of Ly6G antibody attenuated PSL-induced neuropathic pain. Both anti-MIP-2 and SB225002 suppressed up-regulation of inflammatory cytokines and chemokines in the injured SCN. In addition, acetylation of histone H3 [lysine (Lys9)-acetylated histone H3 (AcK9-H3)] on the promoter region of MIP-2 and CXCR2 was increased in the injured SCN after PSL. Expression of AcK9-H3 was observed in the nuclei of neutrophils and macrophages surrounding the epineurium. Administration of the histone acetyltransferase inhibitor anacardic acid suppressed the up-regulation of MIP-2 and CXCR2 in the SCN after PSL and resulted in the prevention of PSL-induced neuropathic pain. Taken together, these results show that augmentation of the MIP-2/CXCR2 axis by hyperacetylation of histone H3 on the promoter region of MIP-2 and CXCR2 located in the injured peripheral nerve elicits chronic neuroinflammation through neutrophil accumulation, leading to neuropathic pain.

Pharmacological relationship between nicotinic and opioid systems in analgesia and corticosterone elevation.

AIMS: Although a pharmacological relationship is known to exist between nicotine and morphine, the exact mechanisms are unclear. Here, we investigated crosstalk between the endogenous opioid system and nicotinic acetylcholine receptors (nAChRs), specifically in nicotine-induced analgesia and activation of the hypothalamic-pituitary-adrenal (HPA) axis. MAIN METHODS: Nicotine and morphine were administered subcutaneously to mice and the effects of these drugs on analgesia and serum corticosterone (SCS) levels were evaluated by the tail-pinch method and fluorometric assay, respectively. KEY FINDINGS: Both nicotine and morphine produced analgesia and SCS increase after a single injection. Nicotine-induced analgesia was prevented by both mecamylamine (MEC; 1mg/kg) and naloxone (NLX; 1mg/kg), and also by repeated administration of morphine or nicotine. Morphine-induced analgesia was prevented by NLX, but not MEC, and by repeated administration of morphine, but not nicotine. Conversely, the nicotine-induced increase in SCS level was prevented by MEC, but not NLX. Morphine-induced SCS increase was prevented by NLX, but not MEC. Moreover, nicotine-induced analgesia was suppressed by dihydro-ß-erythroidine (DHßE; an antagonist for the α4ß2 nAChR) or methyllycaconitine (MLA; an antagonist for the α7 nAChR). The nicotine-induced increase in SCS level was suppressed by DHßE, but not MLA. SIGNIFICANCE: Nicotine-induced analgesia may involve the endogenous opioid system through crosstalk with nicotinic pathways. However, the relationship between these systems does not extend to cooperative actions in nicotine-induced HPA-axis activation.

CC-chemokine MIP-1α in the spinal cord contributes to nerve injury-induced neuropathic pain.

We investigated the involvement of spinal macrophage inflammatory protein-1α (MIP-1α), an inflammatory chemokine, in partial sciatic nerve ligation (PSL)-induced neuropathic pain in mice. PSL increased MIP-1α mRNA levels as well as levels of the MIP-1α receptor, CCR1, but not CCR5 in the spinal dorsal horn. PSL-induced tactile allodynia and thermal hyperalgesia were prevented by intrathecal (i.t.) injection of a neutralizing antibody of MIP-1α (2ng). Recombinant MIP-1α (10pmol, i.t.) elicited long-lasting tactile allodynia and thermal hyperalgesia in naïve mice. These results suggest that peripheral nerve injury elicits the up-regulation of spinal MIP-1α and CCR1 to participate in neuropathic pain.

Increment of activated serine/threonine protein phosphatase in brain membrane fraction synchronized with antinociceptive effect of morphine in mice.

We have examined the involvement of serine/threonine protein phosphatase (PP) sensitive to okadaic acid (OA) in the antinociceptive effect of morphine in mice. The present study was performed to elucidate subcellular distribution and activity of OA-sensitive PPs in the brain when mice exposed to morphine. Subcutaneous administration of morphine (5 mg/kg) produced the antinociceptive effect with the maximum 30 min after its administration, evaluated by tail-pinch test. The antinociception was accompanied by an increment of activity in OA-sensitive PPs in the membrane fraction prepared from the whole brain of mice treated with morphine: The temporal profile of the morphine-induced increment of OA-sensitive PP activity was consistent with that of antinociceptive effects of morphine. The morphine-induced increase in OA-sensitive PP activity was dependent on the dose and attenuated by the concurrent administration of naloxone (1 mg/kg). To identify the subtype of OA-sensitive PPs in morphine-enhanced activity, we examined the level of PP2A and PP5, OA-sensitive PPs, in the subcellular fraction prepared from the whole brain of mice receiving morphine. Western blot revealed that morphine elicited the significant increase in the level of PP5, but not PP2A, in the membrane fraction, with the same peak time for the increment of PP5 as the antinociception. No significant change was observed in the level of OA-sensitive PPs in the cytosolic fraction at any examined time after morphine. These results suggest that the translocation of PP5 to the membrane fraction is, at least in part, involved in the antinociceptive effect of morphine in mice.