Glycine transporter type 2 (GlyT2) inhibitor ameliorates bladder overactivity and nociceptive behavior in rats.

BACKGROUND: Glycine is a major inhibitory neurotransmitter in the spinal cord, the concentration of which is regulated by two types of glycine transporters (GlyTs): GlyT1 and GlyT2. We hypothesized that the inhibition of GlyTs could ameliorate bladder overactivity and/or pain sensation in the lower urinary tract. OBJECTIVE: Investigate the effects of GlyT inhibitors on bladder overactivity and pain behavior in rats. DESIGN, SETTING, AND PARTICIPANTS: Cystometry was performed under urethane anesthesia in cyclophosphamide (CYP)-treated rats. In behavioral studies using conscious rats, nociceptive responses were induced by intravesical administration of resiniferatoxin (3µM). Selective GlyT1 or GlyT2 inhibitors were administered intrathecally to evaluate their effects. MEASUREMENTS: Cystometric parameters, nociceptive behaviors (licking and freezing), and messenger RNA (mRNA) levels of GlyTs and glycine receptor (GlyR) subunits in the dorsal spinal cord (L6-S1) were measured. RESULTS AND LIMITATIONS: During cystometry in CYP-treated rats, significant increases in intercontraction interval and micturition pressure threshold were elicited by ALX-1393, a selective GlyT2 inhibitor, but not by sarcosine, a GlyT1 inhibitor. These effects were completely reversed by strychnine, a GlyR antagonist. ALX-1393 also significantly suppressed nociceptive behaviors in a dose-dependent manner. In sham rats, GlyT2 mRNA was expressed at a much higher level (23-fold) in the dorsal spinal cord than GlyT1 mRNA. In CYP-treated rats, mRNA levels of GlyT2 and the GlyR α1 and ß subunits were significantly reduced. CONCLUSIONS: These results indicate that GlyT2 plays a major role in the clearance of extracellular glycine in the spinal cord and that GlyT2 inhibition leads to amelioration of CYP-induced bladder overactivity and pain behavior. GlyT2 may be a novel therapeutic target for the treatment of overactive bladder and/or bladder hypersensitive disorders such as bladder pain syndrome/interstitial cystitis.

Quantification of the glycine transporter 1 in rhesus monkey brain using [18F]MK-6577 and a model-based input function.

BACKGROUND: Glycine transporter 1 (GlyT1) inhibitors have emerged as potential treatments for schizophrenia due to their potentiation of NMDA receptor activity by modulating the local concentrations of the NMDA co-agonist glycine. [18F]MK-6577 is a potent and selective GlyT1 inhibitor PET tracer. Although differences in ligand kinetics can be expected between non-human primates and humans, the tracer pre-clinical evaluation can provide valuable information supporting protocol design and quantification in the clinical space. The main objective of this work was to evaluate the in vivo kinetics of [18F]MK-6577 in rhesus monkey brain. Additionally, a method for estimating the tracer input function from the tracer brain tissue kinetics and venous sampling was validated. This technique was applied for determination of the dose-occupancy relationship of a GlyT1 inhibitor in monkey brain. METHODS: Compartmental and Logan graphical analysis were utilized for quantification of the [18F]MK-6577 binding using the measured tracer arterial input function. The stability of the tracer volume of distribution relative to scan length was assessed. The proposed model-based input function method takes advantage of the agreement between the tracer concentration in arterial and venous plasma from ~5 min. The approach estimates the initial peak of the input curve by adding a gamma like function term to the measured venous curve. The parameters of the model function were estimated by simultaneously fitting several brain time activity curves to a compartmental model. RESULTS: Good agreement was found between the model-based and the measured arterial plasma curve and the corresponding distribution volumes. The Logan analysis was the preferred method of analysis providing reliable and stable volume of distribution and occupancy results using a 90 and possibly 60 min scan length. CONCLUSION: The model-based input function method and Logan analysis are well suited for quantification of [18F]MK-6577 binding and GlyT1 occupancy in monkey brain.

Synthesis and characterization of [¹²5I]2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoromethyl-benzamide as novel imaging probe for glycine transporter 1.

In this study, 2-iodo substituted 1-methylpiperidin-2-yl benzamide derivatives were synthesized and evaluated as candidate SPECT imaging agents for glycine transporter 1 (GlyT1). In JAR cells, which predominantly express GlyT1, 2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoromethyl-benzamide (5) showed excellent inhibitory activity of [(3)H]glycine uptake (IC(50)=2.4 nM). Saturation assay in rat cortical membranes revealed that [(125)I]5 had a single high affinity binding site with a K(d) of 1.54 nM and a B(max) of 3.40 pmol/mg protein. In vitro autoradiography demonstrated that [(125)I]5 showed consistent accumulation with GlyT1 expression. The in vitro binding was greatly inhibited by GlyT1 inhibitors but not by other site ligands, which suggested the high specific binding of [(125)I]5 with GlyT1. In the biodistribution and ex vivo autoradiography studies using mice, [(125)I]5 showed high blood-brain barrier permeability (1.68-2.17% dose/g at 15-60 min) and similar regional brain distribution pattern with in vitro results. In addition, pre-treatment of GlyT1 ligands resulted in significant decrease of [(125)I]5 binding in the GlyT1-rich regions. This preliminary study demonstrated that radio-iodinated 5 is a promising SPECT imaging probe for GlyT1.

Discovery of benzoylisoindolines as a novel class of potent, selective and orally active GlyT1 inhibitors.

Benzoylisoindolines were discovered as a novel structural class of GlyT1 inhibitors. SAR studies and subsequent lead optimization efforts focused primarily on addressing hERG liability and on improving in vivo efficacy resulted in the identification of potent GlyT1 inhibitors displaying excellent selectivity and in vivo PD and PK profiles.

Molecular identification and characterisation of the glycine transporter (GLYT1) and the glutamine/glutamate transporter (ASCT2) in the rat lens.

Glutathione (GSH) is an essential antioxidant required for the maintenance of lens transparency. In the lens, GSH is maintained at unusually high concentrations as a result of direct GSH uptake and/or intracellular de novo synthesis from its precursor amino acids; cysteine, glycine and glutamine/glutamate. With increasing age, the levels of GSH, particularly in the core of the lens, are significantly reduced. It has been proposed that alterations in the transport of GSH and/or its precursor amino acids may contribute to the changes in GSH levels in older lenses. As considerable uncertainty exists about the molecular identity of GSH transporters in the lens, we have focused on identifying transporters involved in the uptake of the precursor amino acids required for GSH synthesis. Previously, we identified an uptake system for cyst(e)ine mediated by the Xc(-) exchanger and the Excitatory Amino Acid Transporters (EAATs) in the rat lens. In this current study, we have identified and localised additional uptake systems that contribute to GSH synthesis. Transcripts for GLYT1 (glycine transporter) and ASCT2 (glutamine/glutamate transporter) were detected in rat lens fiber cells using the reverse transcription-polymerase chain reaction (RT-PCR). Western blot analysis confirmed the expression of both GLYT1 and ASCT2 at the protein level. Immunocytochemistry revealed GLYT1 expression to be restricted to cortical regions of the lens. Labelling was predominantly cytoplasmic with some labelling of the membrane. In contrast, ASCT2 was expressed throughout the lens extending from the outer cortex through to the core. In the outer cortex, ASCT2 expression was predominantly cytoplasmic. However, with deeper distance into the lens, labelling became more membraneous indicating insertion of ASCT2 into the membranes of mature fiber cells of the lens core. The molecular identification and localisation of GLYT1 and ASCT2 in the lens suggests that these transporters may be responsible for the uptake of the precursor amino acids, glycine and glutamine, which are involved in GSH synthesis. Moreover, the presence of ASCT2 in the centre of the lens raises the possibility that ASCT2 may work with the Xc(-) exchanger to accumulate cysteine where it can potentially act as a low molecular mass antioxidant.

Differential projections of excitatory and inhibitory dorsal horn interneurons relaying information from group II muscle afferents in the cat spinal cord.

Dorsal horn interneurons with input from group II muscle spindle afferents are components of networks involved in motor control. Thirteen dorsal horn interneurons with monosynaptic group II input were characterized electrophysiologically and labeled intracellularly with Neurobiotin. Their axonal projections were traced, and neurotransmitter content was established by using immunocytochemistry. Two subpopulations were identified: five interneurons had axons that contained vesicular glutamate transporter 2 and hence were glutamatergic and excitatory. Terminals of the remaining eight interneurons were immunoreactive for the glycine transporter 2 or were apposed to gephyrin but did not contain the GABA-synthesizing enzyme glutamic acid decarboxylase and were therefore glycinergic and inhibitory. Excitatory cells were located mainly in the central region of lamina IV and had relatively small somata and restricted dendritic trees. In contrast, inhibitory interneurons were located more ventrally, in lamina V and had relatively larger somata and more extensive dendritic trees. Axonal projections of the two subpopulations differed considerably. Excitatory interneurons predominantly projected ipsilaterally, whereas most inhibitory interneurons projected both ipsilaterally and contralaterally. Three inhibitory axons formed contacts with large cholinergic cells in motor nuclei, thus revealing a novel direct coupling between inhibitory dorsal horn interneurons and motoneurons. The organization of the excitatory interneurons is consistent with current knowledge of reflex pathways to motoneurons, but the existence and connections of the inhibitory subpopulation could not be predicted from previous data. Our results indicate that these latter interneurons exercise widespread inhibitory control over a variety of cell types located on both sides of the spinal cord.

Glycine-gated chloride channels depress synaptic transmission in rat hippocampus.

An inhibitory role for strychnine-sensitive glycine-gated chloride channels (GlyRs) in mature hippocampus is beginning to be appreciated. We have reported previously that CA1 pyramidal cells and GABAergic interneurons recorded in 3- to 4-wk-old rat hippocampal slices express functional GlyRs, dispelling previous misconceptions that GlyR expression ceases in early development. However, the effect of GlyR activation on cell excitability and synaptic circuits in hippocampus has not been fully explored. Using whole cell current-clamp recordings, we show that activation of strychnine-sensitive GlyRs through exogenous glycine application causes a significant decrease in input resistance and prevents somatically generated action potentials in both CA1 pyramidal cells and interneurons. Furthermore, GlyR activation depresses the synaptic network by reducing suprathreshold excitatory postsynaptic potentials (EPSPs) to subthreshold events in both cell types. Blockade of postsynaptic GlyRs with the chloride channel blocker 4, 4'-diisothiocyanatostilbene-2-2'-disulfonic acid (DIDS) or altering the chloride ion driving force in recorded cells attenuates the synaptic depression, strongly indicating that a postsynaptic mechanism is responsible. Increasing the local glycine concentration by blocking reuptake causes a strychnine-sensitive synaptic depression in interneuron recordings, suggesting that alterations in extracellular glycine will impact excitability in hippocampal circuits. Finally, using immunohistochemical methods, we show that glycine and the glycine transporter GlyT2 are co-localized selectively in GABAergic interneurons, indicating that interneurons contain both inhibitory neurotransmitters. Thus we report a novel mechanism whereby activation of postsynaptic GlyRs can function to depress activity in the synaptic network in hippocampus. Moreover, the co-localization of glycine and GABA in hippocampal interneurons, similar to spinal cord, brain stem, and cerebellum, suggests that this property is likely to be a general characteristic of inhibitory interneurons throughout the CNS.