title: Electrophysiological evidence of increased glycine receptor-mediated phasic and tonic inhibition by blockade of glycine transporters in spinal superficial dorsal horn neurons of adult mice.
abstract: To understand the synaptic and/or extrasynaptic mechanisms underlying pain relief by blockade of glycine transporter subtypes GlyT1 and GlyT2, whole-cell recordings were made from dorsal horn neurons in spinal slices from adult mice, and the effects of NFPS and ALX-1393, selective GlyT1 and GlyT2 inhibitors, respectively, on phasic evoked or miniature glycinergic inhibitory postsynaptic currents (eIPSCs or mIPSCs) were examined. NFPS and ALX-1393 prolonged the decay phase of eIPSCs without affecting their amplitude. In the presence of tetrodotoxin to record mIPSCs, NFPS and ALX-1393 induced a tonic inward current that was reversed by strychnine. Although NFPS had no statistically significant influences on mIPSCs, ALX-1393 significantly increased their frequency. We then further explored the role of GlyTs in the maintenance of glycinergic IPSCs. To facilitate vesicular release of glycine, repetitive high-frequency stimulation (HFS) was applied at 10 Hz for 3 min during continuous recordings of eIPSCs at 0.1 Hz. Prominent suppression of eIPSCs was evident after HFS in the presence of ALX-1393, but not NFPS. Thus, it appears that phasic and tonic inhibition may contribute to the analgesic effects of GlyT inhibitors. However, reduced glycinergic inhibition due to impaired vesicular refilling could hamper the analgesic efficacy of GlyT2 inhibitors.
authors.affiliation: Laboratory of Pharmacology, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan; Medicinal Research Laboratories, School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Misa
authors.initials: M
authors.lastname: Oyama
authors.affiliation: Pain & Neuroscience Laboratories, Daiichi Sankyo Co. Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan.
authors.forename: Seiya
authors.initials: S
authors.lastname: Kuraoka
authors.affiliation: Laboratory of Pharmacology, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan; Medicinal Research Laboratories, School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Shun
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Laboratory of Pharmacology, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan; Medicinal Research Laboratories, School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Iwai
authors.affiliation: Laboratory of Pharmacology, School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan; Medicinal Research Laboratories, School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Electronic address: tanabemi@pharm.kitasato-u.ac.jp.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Tanabe
mesh_terms: D000818:Animals; D050487:Glycine Plasma Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D053444:Inhibitory Postsynaptic Potentials / Q000187:drug effects; D008297:Male; D051379:Mice; D020671:Posterior Horn Cells / Q000187:drug effects / Q000502:physiology*; D012521:Sarcosine / Q000031:analogs & derivatives / Q000494:pharmacology; D012694:Serine / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: C539864:ALX 1393; D050487:Glycine Plasma Membrane Transport Proteins; C435025:N-(3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)-3-(4'-phenylphenoxy)propyl)sarcosine; C493270:Slc6a5 protein, mouse; C493267:Slc6a9 protein, mouse; D012694:Serine; D012521:Sarcosine
keywords: Glycine transporter; Inhibitory postsynaptic synaptic currents; Patch clamp; Spinal cord; Strychnine-sensitive glycine receptor