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Spinal Anti-Allodynia Action of Glycine Transporter Inhibitors In JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 JPET ThisFast article Forward. has not been Published copyedited andon formatted.April 30, The 2008 final versionas DOI:10.1124/jpet.108.136267 may differ from this version. JPET #136267 Spinal Anti-Allodynia Action of Glycine Transporter Inhibitors in Neuropathic Pain Models in Mice Downloaded from Katsuya Morita, Naoyo Motoyama, Tomoya Kitayama, Norimitsu Morioka, Koki jpet.aspetjournals.org Kifune, and Toshihiro Dohi at ASPET Journals on September 26, 2021 Department of Pharmacology, Division of Integrated Medical Science, Hiroshima University Graduate School of Biomedical Sciences, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553, Japan (KM, NM, TK, NM, KK, TD) 1 Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 This article has not been copyedited and formatted. The final version may differ from this version. JPET #136267 Running title: Anti-Allodynia and Glycine Transporter Inhibitors Address correspondence to: Professor Toshihiro Dohi, Ph.D., Department of Pharmacology, Division of Integrated Medical Science, Hiroshima University Graduate School of Biomedical Sciences, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553, Japan. E-mail Downloaded from address: [email protected] Tel.: +81-82-257-5640; fax: +81-82-257-5644. jpet.aspetjournals.org ABBREVIATIONS: ACSF, artificial cerebrospinal fluid; ALX 1393, at ASPET Journals on September 26, 2021 (O-[(2-benzyloxyphenyl-3-fluorophenyl)methyl]-L-serine); BDNF, brain-derived neurotrophic factor; CFA, complete Freund's adjuvant; DCK, 5,7-dichlorokynurenic acid (5,7-dichloro-4-hydroxyquinoline-2-carboxylic acid); GlyR, glycine receptor; GlyT, glycine transporter; HVJ, hemagglutinating virus of the Japan; i.t., intrathecal; i.v., intravenous; L-701,324, 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(1H)-quinolinone; NMDA, N-methyl-D-aspartate; ORG25543, 4-benzyloxy-3,5-dimethoxy-N-[1-(dimethylaminocyclopently)-methyl]benzamide; ORG25935, cis-N-methyl-N-(6-methoxy-1-phenyl-1,2,3,4-tetrahydronaphthalen-2-yl methyl)amino methylcarboxylic acid; SDS, sodium dodecylsulfate; siRNA, small 2 JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 This article has not been copyedited and formatted. The final version may differ from this version. JPET #136267 interfering RNA; STZ, streptozotocin. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 3 JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 This article has not been copyedited and formatted. The final version may differ from this version. JPET #136267 The number of text pages is 55 (figures not included). The number of tables is 2. The number of figures is 6. The number of words Abstract: 242 words Downloaded from Introduction: 755 words Discussion: 1708 words jpet.aspetjournals.org The number of references: 50 Section: Behavioral Pharmacology at ASPET Journals on September 26, 2021 4 JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 This article has not been copyedited and formatted. The final version may differ from this version. JPET #136267 ABSTRACT Neuropathic pain is refractory against conventional analgesics and thus novel medicaments are desired for the treatment. Glycinergic neurons are localized in specific brain regions, including the spinal cord, where they play an important role in the regulation of pain signal transduction. Glycine transporter (GlyT) 1, present in glial Downloaded from cells, and GlyT2, located in neurons, play roles in modulating glycinergic neurotransmission by clearing synaptically released glycine or supplying glycine to the jpet.aspetjournals.org neurons and thus could modify pain signal transmission in the spinal cord. Here we demonstrated that intravenous or intrathecal administration of GlyT1 inhibitors, ORG at ASPET Journals on September 26, 2021 25935 or sarcosine, and GlyT2 inhibitors, ORG 25543 and ALX 1393, or knockdown of spinal GlyTs by siRNA of GlyTs mRNA produced a profound anti-allodynia effect in a partial peripheral nerve ligation model and other neuropathic pain models in mice. The anti-allodynia effect is mediated through spinal glycine receptor α3. These results established GlyTs as the target molecules for the development of medicaments for neuropathic pain. These manipulations to stimulate glycinergic neuronal activity, however, were without effect during the 4 days post-nerve injury, while manipulations to inhibit glycinergic neuronal activity protected against the development of allodynia in this phase. The results implied that the timing of medication with their inhibitors should 5 JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 This article has not been copyedited and formatted. The final version may differ from this version. JPET #136267 be considered, because glycinergic control of pain was reversed in the critical period of 3 to 4 days post-surgery. This may also provide important information for understanding the underlying molecular mechanisms of the development of neuropathic pain. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021 6 JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 This article has not been copyedited and formatted. The final version may differ from this version. JPET #136267 Introduction Neuropathic pain arising from peripheral or spinal nerve injury, diabetes, or infection with herpes virus is a result of the final product of an altered peripheral, spinal, and supraspinal process for which the usual analgesics are not effective and novel analgesics are desired. Recent progress of research on the underlying mechanism of the Downloaded from pathology revealed more complexity depending on the cause and stage of ongoing neuropathy. Among various mechanisms involved in the pathology, alterations of jpet.aspetjournals.org synaptic transmission within the spinal cord dorsal horn as well as peripheral nerves following peripheral nerve injury play key roles. In addition to the activation of at ASPET Journals on September 26, 2021 stimulatory spinal neurotransmission, disinhibition of inhibitory neurotransmission has been implicated in the generation of inflammatory and neuropathic pain (Woolf and Mannion, 1999). Glycine as well as GABA serves as a major inhibitory neurotransmitter in the spinal cord of vertebrates. Actually, relief from glycinergic inhibition by an inhibitor of glycine release (Ito et al., 2001) or blockers of glycine receptors (Sivilotti and Woolf, 1994; Sherman et al., 1997; Loomis et al., 2001; Gomeza et al., 2003a) in the dorsal horn can elicit tactile allodynia, a major symptom of neuropathic pain. Recent studies suggested that reduction of the chloride gradient across the neuronal membrane, which in turn leads to reduction of anion reversal potential, 7 JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 This article has not been copyedited and formatted. The final version may differ from this version. JPET #136267 occurred in neurons of lamina I of the superficial dorsal horn following peripheral nerve injury (Coull et al., 2003). The change in the driving force means that both glycine and GABAA receptor-mediated inputs produce less hyperpolarization and could even paradoxically depolarize the neuron (Coull et al., 2003, 2005; Prescott et al., 2006). This may be one of likely mechanism which contributes to the production of allodynia and Downloaded from hyperalgesia associated with neuropathic pain. In contrast, the enhancement of the activity of glycinergic neurons exerts an anti-allodynia in a nerve injury model (Abdin jpet.aspetjournals.org et al., 2006). The evidence suggests the importance of glycinergic control of spinal pain processing. at ASPET Journals on September 26, 2021 Extracellular concentrations of glycine at glycinergic nerve terminals are regulated by Na+/Cl--dependent glycine transporters (GlyTs). GlyT1 is widely expressed in glial cells. The evidence suggests an essential role of GlyT1 in lowering extracellular glycine concentration at glycinergic synaptic cleft (Jursky and Nelson, 1996; Roux and Supplisson, 2000). The inhibition of GlyT1 and enhancement of strychnine-sensitive glycine receptors would compromise the ability of GlyT1 inhibitors to be beneficial for pain control (Armer, 2000; Aragón and López-Corcuera, 2003). On the other hand, GlyT1 prevents saturation of the glycine binding site on N-methyl-D-aspartate (NMDA) receptors and reduces the activity of excitatory synapses. The inhibition of this role of 8 JPET Fast Forward. Published on April 30, 2008 as DOI: 10.1124/jpet.108.136267 This article has not been copyedited and formatted. The final version may differ from this version. JPET #136267 GlyT1 would counteract the ability of GlyT1 inhibitor to be beneficial for pain control. GlyT2 is largely localized to the presynaptic terminals of glycinergic neurons in the spinal cord as well as in the brain stem and cerebellum (Zafra et al., 1995a, 1995b). GlyT2 is present only in glycinergic neurons in association with postsynaptic glycine receptors; therefore, there is a concept that GlyT2 mediates the clearance of Downloaded from postsynaptically released glycine at inhibitory synaptic cleft (Brasnjo and Otis, 2003). It is expected that GlyT2 inhibitors would be useful in the treatment of pain by
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