Molecular cloning and functional expression of the human glycine transporter GlyT2 and chromosomal localisation of the gene in the human genome<sup>1</sup>

Morrow, John A.; Collie, Iain T.; Dunbar, Donald R.; Walker, Glenn; Shahid, Mansoor; Hill, David R.

doi:10.1016/s0014-5793(98)01390-8

Cited by 72 publications

(54 citation statements)

References 54 publications

(74 reference statements)

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“…Tricyclic antidepressants are one of the major clinical medicaments for neuropathic pain, and some can inhibit GlyTs. Sarcosine blocks GlyT1 (Morrow et al, 1998), and amoxapine inhibits GlyT2 more selectively (Nú ñ ez et al, 2000). Doxepin, amitriptyline, and nortriptyline block both GlyT1 and GlyT2 (Nú ñ ez et al, 2000).…”

Section: Discussionmentioning

confidence: 99%

Spinal Antiallodynia Action of Glycine Transporter Inhibitors in Neuropathic Pain Models in Mice

Morita¹,

Motoyama²,

Kitayama³

et al. 2008

J Pharmacol Exp Ther

115

143

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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 cells, and GlyT2, located in neurons, play roles in modulating glycinergic neurotransmission by clearing synaptically released glycine or supplying glycine to the neurons and thus could modify pain signal transmission in the spinal cord. In this study, we demonstrated that i.v. or intrathecal administration of GlyT1 inhibitors, cis-N-methyl-N-(6-methoxy-1-phenyl-1,2,3,4-tetrahydronaphthalen-2-yl methyl)amino methylcarboxylic acid (ORG25935) or sarcosine, and GlyT2 inhibitors, 4-benzyloxy-3,or knockdown of spinal GlyTs by small interfering RNA of GlyTs mRNA produced a profound antiallodynia effect in a partial peripheral nerve ligation model and other neuropathic pain models in mice. The antiallodynia effect is mediated through spinal glycine receptor ␣3. These results established GlyTs as the target molecules for the development of medicaments for neuropathic pain. However, these manipulations to stimulate glycinergic neuronal activity were without effect during the 4 days after nerve injury, whereas 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 be considered, because glycinergic control of pain was reversed in the critical period of 3 to 4 days after surgery. This may also provide important information for understanding the underlying molecular mechanisms of the development of neuropathic pain.Neuropathic pain arising from peripheral or spinal nerve injury and 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 pathology revealed more complexity, depending on the cause and stage of ongoing neuropathy. Among various mechanisms involved in the pathology, alterations of synaptic transmission within the spinal cord dorsal horn as well as peripheral nerves after peripheral nerve injury play key roles. In addition to the activation of 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 serve as major inhibitory neurotransmitters in the spinal cord of vertebrates. In fact, relief from glycinergic inhibition by an inhib-

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Section: Discussionmentioning

confidence: 99%

Spinal Antiallodynia Action of Glycine Transporter Inhibitors in Neuropathic Pain Models in Mice

Morita¹,

Motoyama²,

Kitayama³

et al. 2008

J Pharmacol Exp Ther

115

143

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“…The GLYT1 transport inhibitor ORG23798 [bis(4-fluorophenyl)methylenepiperidineacetic acid, lithium salt] was synthesized at Organon Laboratories. Inhibition of glycine transporters was determined from uptake of [ 3 H]glycine into Chinese hamster ovary (CHO) cells stably expressing either hGLYT1b or hGLYT2 by methods previously described (28). Interaction with other NTT systems was assessed by using rat brain synaptosomal preparations and, for a range of receptors, by using membranes derived from NIH 3T3 (mouse) or CHO cells stably transfected with human cloned receptors (29).…”

Section: Methodsmentioning

confidence: 99%

The glycine neurotransmitter transporter GLYT1 is an organic osmolyte transporter regulating cell volume in cleavage-stage embryos

Steeves

Hammer

Walker

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

145

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Cells subjected to sustained high osmolarity almost universally respond by accumulating compatible organic osmolytes that, in contrast to inorganic ions, are not deleterious even at high intracellular concentrations. Their accumulation from the external environment by known organic osmolyte transporters, such as the four identified in mammals, occurs only slowly in response to sustained high osmolarity, by synthesis of new transporter proteins. Most cells, however, are not subject to high or varying osmolarity, and it is not clear whether organic osmolytes are generally required at normal osmolarities or how they are regulated. The fertilized egg of the mouse is protected in the oviduct from perturbations in osmolarity. However, deleterious effects of osmotic stress were evident in vitro even at normal oviductal osmolarity. Glycine was found to protect development, indicating that early mouse embryos may use glycine as an organic osmolyte at physiological osmolarity. We have now found that GLYT1, a glycine transporter of the neurotransmitter transporter gene family, functions as the organic osmolyte transporter that mediates the osmotically regulated accumulation of glycine and regulates cell volume in early embryos. Furthermore, osmotic stimulation of GLYT1 transport was immediate, without a requirement for protein synthesis, implying regulation different from known organic osmolyte transporters. Thus, GLYT1 appears to have a previously unidentified role as an organic osmolyte transporter that functions in acute organic osmolyte and volume homeostasis near normal osmolarity.

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“…6,17,21 Thus, we tested the effect of varying buffer compositions on V m fluorescence responses to glycine. The ionic compositions of the 3 buffers tested (NaCl control, LiCl, and NaI) are described in Table 1.…”

Section: Resultsmentioning

confidence: 99%

Validation of a Fluorescent Imaging Plate Reader Membrane Potential Assay for High-Throughput Screening of Glycine Transporter Modulators

Benjamin

Skelton

Hanway³

et al. 2005

SLAS Discovery

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A fluorescent imaging plate reader (FLIPR) membrane potential (V m ) assay was evaluated for pharmacological characterization and high-throughput screening (HTS) of rat glycine transporter type 2 (rGlyT 2 ) in a stable rGlyT 2 -HEK cell line. Data show that glycine activation of rGlyT 2 consistently results in a concentration-dependent V m response on the FLIPR that is blocked by the potent and selective GlyT 2 antagonist 4-benzyloxy-3,5-dimethoxy-N-[1-dimethylaminocyclopentyl)methyl]-benz-amide (Org-25543). Agonist and antagonist pharmacologies match those reported using conventional [ 3 H]glycine uptake assays and electrophysiology. The glycine response is dependent on buffer ionic composition consistent with GlyT 2 physiology. Assay signal-to-background and coefficient of variation meets sufficient statistical criteria to conduct HTS. The results of a screen of the chemical inventory demonstrate that the assay is able to successfully identify and confirm GlyT 2 inhibitors. The advantages of this assay are its homogeneity, compatibility with both 96-and 384-well formats, and lack of radioactivity usage. Thus, the authors conclude that a fluorescence-based V m assay on FLIPR is a viable approach for identification and pharmacological profiling of small molecule modulators of the electrogenic transporter rGlyT 2 . (Journal of Biomolecular Screening 2005:365-373)

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Molecular cloning and functional expression of the human glycine transporter GlyT2 and chromosomal localisation of the gene in the human genome¹

Cited by 72 publications

References 54 publications

Spinal Antiallodynia Action of Glycine Transporter Inhibitors in Neuropathic Pain Models in Mice

Spinal Antiallodynia Action of Glycine Transporter Inhibitors in Neuropathic Pain Models in Mice

The glycine neurotransmitter transporter GLYT1 is an organic osmolyte transporter regulating cell volume in cleavage-stage embryos

Validation of a Fluorescent Imaging Plate Reader Membrane Potential Assay for High-Throughput Screening of Glycine Transporter Modulators

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Molecular cloning and functional expression of the human glycine transporter GlyT2 and chromosomal localisation of the gene in the human genome1

Cited by 72 publications

References 54 publications

Spinal Antiallodynia Action of Glycine Transporter Inhibitors in Neuropathic Pain Models in Mice

Spinal Antiallodynia Action of Glycine Transporter Inhibitors in Neuropathic Pain Models in Mice

The glycine neurotransmitter transporter GLYT1 is an organic osmolyte transporter regulating cell volume in cleavage-stage embryos

Validation of a Fluorescent Imaging Plate Reader Membrane Potential Assay for High-Throughput Screening of Glycine Transporter Modulators

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Molecular cloning and functional expression of the human glycine transporter GlyT2 and chromosomal localisation of the gene in the human genome¹