title: Positive N-methyl-D-aspartate receptor modulation by selective glycine transporter-1 inhibition in the rat dorsal spinal cord in vivo.
abstract: In this study we have employed the selective glycine transporter-1 (GlyT-1) and GlyT-2 transporter inhibitors R-(-)-N-methyl-N-[3-[(4-trifluoromethyl)phenoxy]-3-phenyl-propyl]glycine (1:1) lithium salt (Org 24598) and 4-benzyloxy-3,5-dimethoxy-N-[1-(dimethylaminocyclopently)methyl]benzamide (Org 25543), respectively, and microdialysis perfusion to determine the effect of GlyT transporter inhibition on extracellular amino acid concentrations in the lumbar dorsal spinal cord of the halothane-anaesthetised rat. Reverse dialysis of Org 24598 (0.1-10 microM) induced a concentration-related increase in extracellular glycine accompanied by a progressive increase in citrulline, but not aspartate, glutamate or GABA, efflux. Org 25543 (10 microM) by the same route induced a similar increase in glycine levels without affecting the efflux of other amino acids quantified. To test the hypothesis that the increase in citrulline efflux resulted from activation of the N-methyl-D-aspartate receptor (NMDA-R)/nitric oxide synthase (NOS) signalling cascade, the sensitivity was determined of GlyT-1 inhibition-induced effects to NMDA-R antagonism or NOS inhibition. Co-administration by reverse dialysis of the selective NMDA-R channel blocker MK-801 (0.5 mM) or the selective antagonist of the strychnine-insensitive glycine site, 7-chlorokynurenic acid (1 mM), with Org 24598 (10 microM) did not affect the uptake inhibition-induced increase in glycine efflux, but did significantly attenuate the increase in extracellular citrulline. Similarly, co-administration with Org 24598 of the isoform non-selective and selective neuronal NOS inhibitors Nomega-nitro-L-arginine methyl ester (1 mM) or 1-(2-trifluoromethylphenyl)imidazole (0.2 mM), respectively, prevented Org 24598-induced citrulline efflux with no effect on increased glycine efflux. These data provide evidence that the observed increased in extracellular citrulline is a consequence of positive modulation of NMDA-R, secondary to increased extracellular glycine and support a protective role for GlyT-1 against fluctuations in extracellular glycine uptake at glutamatergic synapses in the dorsal spinal cord. Such a mechanism could be important to NMDA-R-mediated synaptic plasticity in the spinal cord and be of relevance to the clinical usage of GlyT-1 inhibitors.
authors.affiliation: Department of Pharmacology, Division of Neuroscience, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. k.j.whitehead@bham.ac.uk
authors.forename: K J
authors.initials: KJ
authors.lastname: Whitehead
authors.forename: S M
authors.initials: SM
authors.lastname: Pearce
authors.forename: G
authors.initials: G
authors.lastname: Walker
authors.forename: H
authors.initials: H
authors.lastname: Sundaram
authors.forename: D
authors.initials: D
authors.lastname: Hill
authors.forename: N G
authors.initials: NG
authors.lastname: Bowery
mesh_terms: D026921:Amino Acid Transport Systems, Neutral / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000818:Animals; D005998:Glycine / Q000031:analogs & derivatives* / Q000494:pharmacology; D050487:Glycine Plasma Membrane Transport Proteins; D008297:Male; D020671:Posterior Horn Cells / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*
chemical_list: D026921:Amino Acid Transport Systems, Neutral; D050487:Glycine Plasma Membrane Transport Proteins; D016194:Receptors, N-Methyl-D-Aspartate; C493269:Slc6a5 protein, rat; C493266:Slc6a9 protein, rat; C435781:org 24598; D005998:Glycine