Specific GlyT1 Research Articles

Reduction of Rapid Proliferating Tumour Cell Lines by Inhibition of the Specific Glycine Transporter GLYT1.

Studies have highlighted the relevance of extracellular glycine and serine in supporting high growth rates of rapidly proliferating tumours. The present study analysed the role of the specific glycine transporter GLYT1 in supplying glycine to cancer cells and maintaining cell proliferation. GLYT1 knockdown in the rapidly proliferating tumour cell lines A549 and HT29 reduced the number of viable cells by approximately 30% and the replication rate presented a decrease of about 50% when compared to cells transfected with control siRNA. In contrast, when compared to control, GLYT1 siRNA had only a minimal effect on cell number of the slowly proliferating tumour cell line A498, reducing the number of viable cells by 7% and no significant difference was observed when analysing the replication rate between GLYT1 knockdown and control group. When utilising a specific GLYT1 inhibitor, ALX-5407, the doubling time of rapidly proliferating cells increased by about 8 h presenting a significant reduction in the number of viable cells after 96 h treatment when compared to untreated cells. Therefore, these results suggest that GLYT1 is required to maintain high proliferation rates in rapidly proliferating cancer cells and encourage further investigation of GLYT1 as a possible target in a novel therapeutic approach.

The GlyT1 Inhibitor Bitopertin Ameliorates Allodynia and Hyperalgesia in Animal Models of Neuropathic and Inflammatory Pain.

Background: Chronic pain conditions are difficult to treat and the therapeutic outcome is frequently unsatisfactory. Changes in excitation/inhibition balance within the dorsal horn contribute to the establishment and persistence of chronic pain. Thus, facilitation of inhibitory neurotransmission is a promising approach to treat chronic pain pharmacologically. Glycine transporter 1 (GlyT1) plays an important role in regulating extracellular glycine concentrations. Aim of the present study therefore was to investigate whether the specific GlyT1 inhibitor bitopertin (RG1678; RO4917838) might constitute a novel treatment for chronic pain by facilitating glycinergic inhibition.Methods: Mechanical allodynia and thermal hyperalgesia were induced by chronic constriction injury of the sciatic nerve or carrageenan injections into the plantar surface of the hind paw in rodents. The effect of acute and long-term bitopertin application on the reaction threshold to mechanical and thermal stimuli was determined. General activity was determined in open field experiments. The glycine concentration in cerebrospinal fluid and blood was measured by HPLC.Results: Systemic application of bitopertin in chronic pain conditions lead to a significant increase of the reaction thresholds to mechanical and thermal stimuli in a time and dose-dependent manner. Long-term application of bitopertin effectuated stable beneficial effects over 4 weeks. Bitopertin did not alter reaction thresholds to stimuli in control animals and had no effect on general locomotor activity and anxiety but lead to an increased glycine concentration in cerebrospinal fluid.Conclusion: These findings suggest that inhibition of the GlyT1 by bitopertin represents a promising new approach for the treatment of chronic pain.

Long-term Application of Glycine Transporter Inhibitors Acts Antineuropathic and Modulates SpinalN-methyl-d-aspartate Receptor Subunit NR-1 Expression in Rats

Dysfunction of spinal glycinergic neurotransmission is a major pathogenetic factor in neuropathic pain. The synaptic glycine concentration is controlled by the two glycine transporters (GlyT) 1 and 2. GlyT inhibitors act antinociceptive in various animal pain models when applied as bolus. Yet, in some studies, severe neuromotor side effects were reported. The aim of the current study was to elucidate whether continuous inhibition of GlyT ameliorates neuropathic pain without side effects and whether protein expression of GlyT1, GlyT2, or N-methyl-D-aspartate receptor subunit NR-1 in the spinal cord is affected. In the chronic constriction injury model of neuropathic pain, male Wistar rats received specific GlyT1 and GlyT2 inhibitors (ALX5407 and ALX1393; Sigma-Aldrich, St. Louis, MO) or vehicle for 14 days via subcutaneous osmotic infusion pumps (n = 6). Mechanical allodynia and thermal hyperalgesia were assessed before, after chronic constriction injury, and every 2 days during substance application. At the end of behavioral assessment, the expression of GlyT1, GlyT2, and NR-1 in the spinal cord was determined by Western blot analysis. Both ALX5407 and ALX1393 ameliorated thermal hyperalgesia and mechanical allodynia in a time- and dose-dependent manner. Respiratory or neuromotor side effects were not observed. NR-1 expression in the ipsilateral spinal cord was significantly reduced by ALX5407, but not by ALX1393. The expression of GlyT1 and GlyT2 remained unchanged. Continuous systemic inhibition of GlyT significantly ameliorates neuropathic pain in rats. Thus, GlyT represent promising targets in pain research. Modulation of N-methyl-D-aspartate receptor expression might represent a novel mechanism for the antinociceptive action of GyT1 inhibitors.

The epithelial glycine transporter GLYT1: protecting the gut from inflammation

The epithelial glycine transporter GLYT1: protecting the gut from inflammation

Glycine transporter GLYT1 is essential for glycine-mediated protection of human intestinal epithelial cells against oxidative damage.

Glycine protects mammalian intestine against oxidative damage caused by ischaemia-reperfusion (IR) injury and prevents or reverses experimentally-induced colitis. However the mechanism of protection remains largely unknown. The objectives of the current study were to demonstrate directly glycine-mediated protection of human intestinal epithelial cells and to determine the requirement for glycine uptake by the specific transporter GLYT1. Exogenous glycine protected human intestinal Caco-2 and HCT-8 cells against the oxidative agent tert-butylhydroperoxide and reduced the intracellular concentration of reactive oxygen species, when applied prior to but not concomitant with the oxidative challenge. Glycine given prior to oxidative challenge preserved intracellular glutathione concentration but had no effect on the rate of glycine uptake. Protection was dependent on GLYT1 activity, being blocked by a specific GLYT1 inhibitor, supporting a requirement for intracellular glycine accumulation. Maintained intracellular glutathione content is indicated as a mechanism through which the protective effect may in part be mediated. However expression of the genes encoding GLYT1 and the glutathione synthesising enzymes glutamate-cysteine ligase, both catalytic and modifier subunits, and glutathione synthetase was not altered by glycine or tert-butylhydroperoxide, suggesting transcriptional regulation is not involved. This work has demonstrated a novel role of GLYT1 in intestine and shown that intestinal epithelial cells respond directly to oxidative challenge without reliance on extra-epithelial tissues or functions such as neurone, blood-flow or immune responses for antioxidant defence. The protective actions of glycine and maintenance of epithelial antioxidant defences suggest it may be beneficial in treatment of inflammatory bowel disease.

Glycine tranporter-1 blockade potentiates NMDA-mediated responses in rat prefrontal cortical neurons in vitro and in vivo.

The N-methyl-D-aspartate (NMDA) receptor (NMDA-R) has pivotal roles in neural development, learning, memory, and synaptic plasticity. Functional impairment of NMDA-R has been implicated in schizophrenia. NMDA-R activation requires glycine to act on the glycine-B (GlyB) site of the NMDA-R as an obligatory co-agonist with glutamate. Extracellular glycine near NMDA-R is regulated effectively by a glial glycine transporter (GlyT1). Using whole-cell voltage-clamp recordings in prefrontal cortex (PFC) slices, we have shown that exogenous GlyB site agonists glycine and D-serine, or a specific GlyT1 inhibitor N[3-(4'-fluorophenyl)-3-(4'-phenylphenoxy)propyl]sarcosine (NFPS) in the presence of exogenous glycine (10 microM), potentiated synaptically evoked NMDA excitatory postsynaptic currents (EPSCs) in vitro. Furthermore, in urethan-anesthetized rats, microiontophoretic NMDA pulses excite single PFC neurons. When these responses were blocked by approximately 50% to approximately 90% on continuous iontophoretic application of the GlyB site, antagonist (+)HA-966, intravenous NFPS (5 mg/kg), or a GlyB site agonist D-serine (50 mg/kg iv) reversed this (+)HA-966 block. NFPS may elevate endogenous glycine levels sufficiently to displace (+)HA-966 from the GlyB sites of the NMDA-R, thus enabling reactivation of the NMDA-Rs by iontophoretic NMDA applications. D-Serine (50-100 mg/kg iv) or NFPS (1-2 mg/kg iv) alone also augmented NMDA-evoked excitatory responses. These data suggest that direct GlyB site stimulation by D-serine, or blockade of GLYT1 to elevate endogenous glycine to act on unsaturated GlyB sites on NMDA-Rs, potentiated NMDA-R-mediated firing responses in rat PFC. Hence, blockade of GlyT1 to elevate glycine near the NMDA-R may activate hypofunctional NMDA-R, which has been implicated to play a critical role in the pathophysiology of schizophrenia.