ABSTRACT
The neurotransmitter glycine is an agonist at the strychnine-sensitive glycine receptors. In addition, it has recently been discovered to act at two new receptors, the excitatory glycine receptor and metabotropic glycine receptor. Glycine's neurotransmitter roles have been most extensively investigated in the spinal cord, where it is known to play essential roles in pain, itch, and motor function. In contrast, less is known about supraspinal glycinergic functions, and their contributions to pain circuits are largely unrecognized. As glycinergic neurons are absent from cortical regions, a clearer understanding of how supraspinal glycine modulates pain could reveal new pharmacological targets. This review aims to synthesize the published research on glycine's role in the adult brain, highlighting regions where glycine signaling may modulate pain responses. This was achieved through a scoping review methodology identifying several key regions of supraspinal pain circuitry where glycine signaling is involved. Therefore, this review unveils critical research gaps for supraspinal glycine's potential roles in pain and pain-associated responses, encouraging researchers to consider glycinergic neurotransmission more widely when investigating neural mechanisms of pain.
ABSTRACT
Chronic neuropathic pain is a debilitating pain syndrome caused by damage to the nervous system that is poorly served by current medications. Given these problems, clinical studies have pursued extracts of the plant Cannabis sativa as alternative treatments for this condition. The vast majority of these studies have examined cannabinoids which contain the psychoactive constituent delta-9-tetrahydrocannabinol (THC). While there have been some positive findings, meta-analyses of this clinical work indicates that this effectiveness is limited and hampered by side-effects. This review focuses on how recent preclinical studies have predicted the clinical limitations of THC-containing cannabis extracts, and importantly, point to how they might be improved. This work highlights the importance of targeting channels and receptors other than cannabinoid CB1 receptors which mediate many of the side-effects of cannabis.
ABSTRACT
The midbrain periaqueductal gray (PAG), particularly its ventrolateral column (vlPAG), is part of a key descending pathway that modulates nociception, fear and anxiety behaviors in both humans and rodents. It has been previously demonstrated that inhibitory GABAergic neurons within the vlPAG have a major role in this nociceptive modulation. However, the PAG contains a diverse range of neuronal subtypes and the contribution of different subtypes of inhibitory neurons to nociceptive control has not been investigated. Here, we employed a chemogenetic strategy in mice that express Cre recombinase under the promotor for the glycine transporter 2 (GlyT2::cre) to modulate a novel group of glycinergic neurons within the vlPAG and then investigate their role in nociceptive control. We show that activation of GlyT2-PAG neurons enhances cold and noxious heat responses and increases locomotor activity (LMA) in both male and female mice. In contrast, inhibition of GlyT2-PAG neurons reduced nociceptive responses, while locomotor behaviors were unaffected. Our findings demonstrate that GlyT2+ neurons in the vlPAG modulate nociception and suggest that strategies targeting GlyT2-PAG neurons could be used to design novel analgesic therapies.
Subject(s)
Nociception , Periaqueductal Gray , Humans , Male , Female , Mice , Animals , Periaqueductal Gray/metabolism , Nociception/physiology , Neurons/physiology , Fear , AnxietyABSTRACT
Animal models have consistently indicated that central sensitization and the development of chronic neuropathic pain are linked to changes to inhibitory signaling in the dorsal horn of the spinal cord. However, replication of data investigating the cellular mechanisms that underlie these changes remains a challenge and there is still a lack of understanding about what aspects of spinal inhibitory transmission most strongly contribute to the disease. Here, we compared the effect of two different sciatic nerve injuries commonly used to generate rodent models of neuropathic pain on spinal glycinergic signaling. Using whole cell patch-clamp electrophysiology in spinal slices, we recorded from neurons in the lamina II of the dorsal horn and evoked inhibitory postsynaptic currents with a stimulator in lamina III, where glycinergic cell bodies are concentrated. We found that glycine inputs onto radial neurons were reduced following partial nerve ligation (PNL) of the sciatic nerve, consistent with a previous report. However, this finding was not replicated in animals that underwent chronic constriction injury (CCI) to the same nerve region. To limit the between-experiment variability, we kept the rat species, sex, and age consistent and had a single investigator carry out the surgeries. These data show that PNL and CCI cause divergent spinal signaling outcomes in the cord and add to the body of evidence suggesting that treatments for neuropathic pain should be triaged according to nerve injury or cellular dysfunction rather than the symptoms of the disease.NEW & NOTEWORTHY Neuropathic pain models are used in preclinical research to investigate the mechanisms underlying allodynia, a common symptom of neuropathic pain, and to test, develop, and validate therapies for persistent pain. We demonstrate that a glycinergic dysfunction is consistently associated with partial nerve ligation but not the chronic constriction injury model. This suggests that the cellular effects produced by each injury are distinct and that data from different neuropathic pain models should be considered separately.
Subject(s)
Neuralgia , Substantia Gelatinosa , Rats , Animals , Rats, Sprague-Dawley , Constriction , Neurons , Spinal CordABSTRACT
Descending projections from neurons in the rostral ventromedial medulla (RVM) make synapses within the superficial dorsal horn (SDH) of the spinal cord that are involved in the modulation of nociception, the development of chronic pain and itch, and an important analgesic target for opioids. This projection is primarily inhibitory, but the relative contribution of GABAergic and glycinergic transmission is unknown and there is limited knowledge about the SDH neurons targeted. Additionally, the details of how spinal opioids mediate analgesia remain unclear, and no study has investigated the opioid modulation of this synapse. We address this using ex vivo optogenetic stimulation of RVM fibres in conjunction with whole-cell patch-clamp recordings from the SDH in spinal cord slices. We demonstrate that both GABAergic and glycinergic neurotransmission is employed and show that SDH target neurons have diverse morphological and electrical properties, consistent with both inhibitory and excitatory interneurons. Then, we describe a subtype of SDH neurons that has a glycine-dominant input, indicating that the quality of descending inhibition across cells is not uniform. Finally, we discovered that the kappa-opioid receptor agonist U69593 presynaptically suppressed most RVM-SDH synapses. By contrast, the mu-opioid receptor agonist DAMGO acted both pre- and postsynaptically at a subset of synapses, and the delta-opioid receptor agonist deltorphin II had little effect. These data provide important mechanistic information about a descending control pathway that regulates spinal circuits. This information is necessary to understand how sensory inputs are shaped and develop more reliable and effective alternatives to current opioid analgesics.
Subject(s)
Analgesics, Opioid , Glycine , Analgesics, Opioid/pharmacology , Glycine/pharmacology , Receptors, Opioid, kappa , Spinal Cord/physiology , Spinal Cord Dorsal Horn , gamma-Aminobutyric AcidABSTRACT
Replication studies play an essential role in corroborating research findings and ensuring that subsequent experimental works are interpreted correctly. A previously published paper indicated that the neurotransmitter glutamate, along with the compounds N-methyl-d-aspartate (NMDA) and d-(-)-2-amino-5-phosphonopentanoic acid (AP5), acts as positive allosteric modulators of inhibitory glycine receptors. The paper further suggested that this form of modulation would play a role in setting the spinal inhibitory tone and influencing sensory signaling, as spillover of glutamate onto nearby glycinergic synapses would permit rapid crosstalk between excitatory and inhibitory synapses. Here, we attempted to replicate this finding in primary cultured spinal cord neurons, spinal cord slice, and Xenopus laevis oocytes expressing recombinant human glycine receptors. Despite extensive efforts, we were unable to reproduce the finding that glutamate, AP5, and NMDA positively modulate glycine receptor currents. We paid careful attention to critical aspects of the original study design and took into account receptor saturation and protocol deviations such as animal species. Finally, we explored possible explanations for the experimental discrepancy. We found that solution contamination with a high-affinity modulator such as zinc is most likely to account for the error, and we suggest methods for preventing this kind of misinterpretation in future studies aimed at characterizing high-affinity modulators of the glycine receptor. SIGNIFICANCE STATEMENT: A previous study indicates that glutamate spillover onto inhibitory synapses can directly interact with glycine receptors to enhance inhibitory signalling. This finding has important implications for baseline spinal transmission and may play a role when chronic pain develops. However, we failed to replicate the results and did not observe glutamate, d-(-)-2-amino-5-phosphonopentanoic acid, or N-methyl-d-aspartate modulation of native or recombinant glycine receptors. We ruled out various sources for the discrepancy and found that the most likely cause is solution contamination.
Subject(s)
Receptors, Glycine/metabolism , 2-Amino-5-phosphonovalerate/metabolism , Animals , Buffers , Cells, Cultured , Chronic Pain/pathology , Glutamic Acid/metabolism , Humans , Mice , N-Methylaspartate/metabolism , Neurons/metabolism , Oocytes , Patch-Clamp Techniques , Primary Cell Culture , Rats , Recombinant Proteins/metabolism , Reproducibility of Results , Spinal Cord/cytology , Spinal Cord/metabolism , Synapses/metabolism , Synaptic Transmission/physiology , Xenopus laevis , Zinc/pharmacologyABSTRACT
Over the past decade there has been an increasing reliance on strong opioids to treat acute and chronic pain, which has been associated with a rising epidemic of prescription opioid misuse, abuse, and overdose-related deaths. Deaths from prescription opioids have more than quadrupled in the USA since 1999, and this pattern is now occurring globally. Inappropriate opioid prescribing after surgery, particularly after discharge, is a major cause of this problem. Chronic postsurgical pain, occurring in approximately 10% of patients who have surgery, typically begins as acute postoperative pain that is difficult to control, but soon transitions into a persistent pain condition with neuropathic features that are unresponsive to opioids. Research into how and why this transition occurs has led to a stronger appreciation of opioid-induced hyperalgesia, use of more effective and safer opioid-sparing analgesic regimens, and non-pharmacological interventions for pain management. This Series provides an overview of the epidemiology and societal effect, basic science, and current recommendations for managing persistent postsurgical pain. We discuss the advances in the prevention of this transitional pain state, with the aim to promote safer analgesic regimens to better manage patients with acute and chronic pain.
Subject(s)
Acute Pain/therapy , Chronic Pain/therapy , Pain, Postoperative/therapy , Acute Pain/diagnosis , Acute Pain/etiology , Analgesics, Opioid/therapeutic use , Chronic Pain/diagnosis , Chronic Pain/etiology , Humans , Pain, Postoperative/diagnosis , Pain, Postoperative/etiologyABSTRACT
The corelease of several neurotransmitters from a single synaptic vesicle has been observed at many central synapses. Nevertheless, the signaling synergy offered by cotransmission and the mechanisms that maintain the optimal release and detection of neurotransmitters at mixed synapses remain poorly understood, thus limiting our ability to interpret changes in synaptic signaling and identify molecules important for plasticity. In the brainstem and spinal cord, GABA and glycine cotransmission is facilitated by a shared vesicular transporter VIAAT (also named VGAT), and occurs at many immature inhibitory synapses. As sensory and motor networks mature, GABA/glycine cotransmission is generally replaced by either pure glycinergic or GABAergic transmission, and the functional role for the continued corelease of GABA and glycine is unclear. Whether or not, and how, the GABA/glycine content is balanced in VIAAT-expressing vesicles from the same terminal, and how loading variability effects the strength of inhibitory transmission is not known. Here, we use a combination of loose-patch (LP) and whole-cell (WC) electrophysiology in cultured spinal neurons of GlyT2:eGFP mice to sample miniature inhibitory post synaptic currents (mIPSCs) that originate from individual GABA/glycine co-releasing synapses and develop a modeling approach to illustrate the gradual change in mIPSC phenotypes as glycine replaces GABA in vesicles. As a consistent GABA/glycine balance is predicted if VIAAT has access to both amino-acids, we test whether vesicle exocytosis from a single terminal evokes a homogeneous population of mixed mIPSCs. We recorded mIPSCs from 18 individual synapses and detected glycine-only mIPSCs in 4/18 synapses sampled. The rest (14/18) were co-releasing synapses that had a significant proportion of mixed GABA/glycine mIPSCs with a characteristic biphasic decay. The majority (9/14) of co-releasing synapses did not have a homogenous phenotype, but instead signaled with a combination of mixed and pure mIPSCs, suggesting that there is variability in the loading and/or storage of GABA and glycine at the level of individual vesicles. Our modeling predicts that when glycine replaces GABA in synaptic vesicles, the redistribution between the peak amplitude and charge transfer of mIPSCs acts to maintain the strength of inhibition while increasing the temporal precision of signaling.
ABSTRACT
KEY POINTS: The midbrain periaqueductal grey (PAG) forms part of an endogenous analgesic system which is tightly regulated by the neurotransmitter GABA. The role of endocannabinoids in regulating GABAergic control of this system was examined in rat PAG slices. Under basal conditions GABAergic neurotransmission onto PAG output neurons was multivesicular. Activation of the endocannabinoid system reduced GABAergic inhibition by reducing the probability of release and by shifting release to a univesicular mode. Blockade of endocannabinoid system unmasked a tonic control over the probability and mode of GABA release. These findings provides a mechanistic foundation for the control of the PAG analgesic system by disinhibition. ABSTRACT: The midbrain periaqueductal grey (PAG) has a crucial role in coordinating endogenous analgesic responses to physiological and psychological stressors. Endocannabinoids are thought to mediate a form of stress-induced analgesia within the PAG by relieving GABAergic inhibition of output neurons, a process known as disinhibition. This disinhibition is thought to be achieved by a presynaptic reduction in GABA release probability. We examined whether other mechanisms have a role in endocannabinoid modulation of GABAergic synaptic transmission within the rat PAG. The group I mGluR agonist DHPG ((R,S)-3,5-dihydroxyphenylglycine) inhibited evoked IPSCs and increased their paired pulse ratio in normal external Ca2+ , and when release probability was reduced by lowering Ca2+ . However, the effect of DHPG on the coefficient of variation and kinetics of evoked IPSCs differed between normal and low Ca2+ . Lowering external Ca2+ had a similar effect on evoked IPSCs to that observed for DHPG in normal external Ca2+ . The low affinity GABAA receptor antagonist TPMPA ((1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid) inhibited evoked IPSCs to a greater extent in low than in normal Ca2+ . Together these findings indicate that the normal mode of GABA release is multivesicular within the PAG, and that DHPG and lowering external Ca2+ switch this to a univesicular mode. The effects of DHPG were mediated by mGlu5 receptor engagement of the retrograde endocannabinoid system. Blockade of endocannabinoid breakdown produced a similar shift in the mode of release. We conclude that endocannabinoids control both the mode and the probability of GABA release within the PAG.
Subject(s)
Endocannabinoids/physiology , Periaqueductal Gray/physiology , Synapses/physiology , gamma-Aminobutyric Acid/physiology , Animals , Calcium/physiology , Female , GABA Antagonists/pharmacology , In Vitro Techniques , Inhibitory Postsynaptic Potentials/drug effects , Male , Methoxyhydroxyphenylglycol/analogs & derivatives , Methoxyhydroxyphenylglycol/pharmacology , Periaqueductal Gray/drug effects , Phosphinic Acids/pharmacology , Piperidines/pharmacology , Pyrazoles/pharmacology , Pyridines/pharmacology , Rats, Sprague-Dawley , Receptors, Metabotropic Glutamate/agonists , Receptors, Metabotropic Glutamate/physiologyABSTRACT
In mammals, fast inhibitory neurotransmission is carried out by two amino acid transmitters, γ-aminobutyric acid (GABA) and glycine. The higher brain uses only GABA, but in the spinal cord and brain stem both GABA and glycine act as inhibitory signals. In some cases GABA and glycine are co-released from the same neuron where they are co-packaged into synaptic vesicles by a shared vesicular inhibitory amino acid transporter, VIAAT (also called vGAT). The vesicular content of all other classical neurotransmitters (eg. glutamate, monoamines, acetylcholine) is determined by the presence of a specialized vesicular transporter. Because VIAAT is non-specific, the phenotype of inhibitory synaptic vesicles is instead predicted to be dependent on the relative concentration of GABA and glycine in the cytosol of the presynaptic terminal. This predicts that changes in GABA or glycine supply should be reflected in vesicle transmitter content but as yet, the mechanisms that control GABA versus glycine uptake into synaptic vesicles and their potential for modulation are not clearly understood. This review summarizes the most relevant experimental data that examines the link between GABA and glycine accumulation in the presynaptic cytosol and the inhibitory vesicle phenotype. The accumulated evidence challenges the hypothesis that vesicular phenotype is determined simply by the competition of inhibitory transmitter for VIAAT and instead suggest that the GABA/glycine balance in vesicles is dynamically regulated.
Subject(s)
Neurotransmitter Agents/metabolism , Presynaptic Terminals/physiology , Synaptic Vesicles/metabolism , Synaptic Vesicles/physiology , Vesicular Inhibitory Amino Acid Transport Proteins/metabolism , Animals , Glycine/metabolism , Humans , Synaptic Transmission/physiology , gamma-Aminobutyric Acid/metabolismABSTRACT
Sustained synaptic transmission requires vesicle recycling and refilling with transmitter, two processes considered to proceed independently. Contrary to this assumption, we show here that depletion of cytosolic transmitter at GABAergic synapses reversibly reduces the number of recycling vesicles. Using paired recordings in hippocampal cultures, we show that repetitive activity causes two phases of reduction of the postsynaptic response. The first involves the classical depletion of the readily releasable and recycling pools, while the second reflects impairment of vesicle filling as GABA is consumed, since it can only be reversed by uptake of GABA or its precursors, glutamate or glutamine. Surprisingly, this second phase is associated with reduced quantal release, a faster depression rate and lower FM5-95 labeling, suggesting that the size of the cycling vesicular pool is regulated by cytosolic transmitter availability. Regulation of vesicular cycling may represent a general mechanism of presynaptic plasticity, matching synaptic release to transmitter supply.
Subject(s)
Cytosol/metabolism , Hippocampus/metabolism , Synaptic Transmission/physiology , Synaptic Vesicles/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Exocytosis/physiology , Glutamic Acid/metabolism , Mice , Mice, Inbred C57BL , Neuronal Plasticity/physiology , Presynaptic Terminals/metabolism , Synaptic Vesicles/physiologyABSTRACT
At inhibitory synapses, glycine and GABA are accumulated into synaptic vesicles by the same vesicular transporter VGAT/VIAAT (vesicular GABA transporter/vesicular inhibitory amino acid transporter), enabling a continuum of glycine, GABA, and mixed phenotypes. Many fundamental aspects of the presynaptic contribution to the inhibitory phenotypes remain unclear. The neuronal transporter GlyT2 is one of the critical presynaptic factors, because glycinergic transmission is impaired in knock-out GlyT2(-/-) mice and mutations in the human GlyT2 gene slc6a5 are sufficient to cause hyperekplexia. Here, we establish that GlyT2-mediated uptake is directly coupled to the accumulation of glycine into recycling synaptic vesicles using cultured spinal cord neurons derived from GlyT2-enhanced green fluorescent protein transgenic mice. Membrane expression of GlyT2 was confirmed by recording glycine-evoked transporter current. We show that GlyT2 inhibition induces a switch from a predominantly glycine to a predominantly GABA phenotype. This effect was mediated by a reduction of glycinergic quantal size after cytosolic depletion of glycine and was entirely reversed by glycine resupply, illustrating that the filling of empty synaptic vesicles is tightly coupled to GlyT2-mediated uptake. Interestingly, high-frequency trains of stimuli elicit two phases of vesicle release with distinct kinetic requirements for glycine refilling. Thus, our results demonstrate the central role played by GlyT2 in determining inhibitory phenotype and therefore in the physiology and pathology of inhibitory circuits.
Subject(s)
Glycine Plasma Membrane Transport Proteins/physiology , Neural Inhibition/physiology , Neurons/physiology , Spinal Cord/cytology , Synaptic Vesicles/metabolism , Animals , Benzamides/pharmacology , Cell Culture Techniques , Dose-Response Relationship, Radiation , Drug Interactions , Electric Stimulation/methods , Excitatory Amino Acid Antagonists/pharmacology , GABA Antagonists/pharmacology , Glycine/metabolism , Glycine/pharmacology , Glycine Agents/pharmacology , Glycine Plasma Membrane Transport Proteins/genetics , Green Fluorescent Proteins/genetics , Inhibitory Postsynaptic Potentials/drug effects , Inhibitory Postsynaptic Potentials/physiology , Inhibitory Postsynaptic Potentials/radiation effects , Membrane Potentials/drug effects , Membrane Potentials/genetics , Membrane Potentials/radiation effects , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nonlinear Dynamics , Patch-Clamp Techniques/methods , Pyridazines/pharmacology , Strychnine/pharmacology , Time FactorsABSTRACT
Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord motor and pain sensory neurons. Recent studies demonstrated apparently contradictory (potentiating versus inhibitory) effects of the endocannabinoid anandamide on these receptors. The present study characterised the effects of cannabinoid agonists on alpha1, alpha1beta, alpha2 and alpha3 GlyRs recombinantly expressed in HEK293 cells with the aims of reconciling effects of cannabinoids on these receptor subtypes and to establish the potential of different GlyR isoforms as novel physiological or analgesic targets for cannabinoids. The compounds investigated were anandamide, HU-210, HU-308, WIN55,212-2 and the endogenous non-cannabinoid, N-arachidonyl-glycine. The latter compound was chosen due to the structural similarity with anandamide and known analgesic actions in the spinal cord. Recombinant alpha1 and alpha1beta GlyRs were potentiated by anandamide and HU-210 at submicromolar concentrations, whereas WIN55,212-2 had no effect and HU-308 produced only weak inhibition. By contrast, N-arachidonyl-glycine exerted complex effects including both potentiation and inhibition. Anandamide had no effect at alpha2 or alpha3 GlyRs although the other cannabinoids produced potent inhibition. On alpha2 GlyRs, the inhibitory potency sequence was HU-210=WIN55,212-2>HU-308>N-arachidonyl-glycine but on alpha3 GlyRs it was HU-210=WIN55212=HU-308>N-arachidonyl-glycine. These results suggest that alpha1, alpha2 and alpha3 containing GlyRs exhibit distinct pharmacological profiles for cannabinoids. We conclude that cannabinoid agonists may be useful as pharmacological tools for selectively inhibiting alpha2 and alpha3 GlyRs. Our results also establish GlyRs as potential novel targets for endogenous and exogenous cannabinoids.
Subject(s)
Arachidonic Acids/pharmacology , Cannabinoids/pharmacology , Glycine/analogs & derivatives , Receptors, Glycine/physiology , Cloning, Molecular , DNA, Complementary/genetics , Dronabinol/analogs & derivatives , Dronabinol/pharmacology , Glycine/pharmacology , Glycine/physiology , Humans , Mutagenesis , Protein Subunits/drug effects , Protein Subunits/physiology , Receptors, Glycine/chemistry , Receptors, Glycine/drug effectsABSTRACT
The mechanisms that specify the vesicular phenotype of inhibitory interneurons in vertebrates are poorly understood because the two main inhibitory transmitters, glycine and GABA, share the same vesicular inhibitory amino acid transporter (VIAAT) and are both present in neurons during postnatal development. We have expressed VIAAT and the plasmalemmal transporters for glycine and GABA in a neuroendocrine cell line and measured the quantal release of glycine and GABA using a novel double-sniffer patch-clamp technique. We found that glycine is released from vesicles when VIAAT is coexpressed with either the neuronal transporter GlyT2 or the glial transporter GlyT1. However, GlyT2 was more effective than GlyT1, probably because GlyT2 is unable to operate in the reverse mode, which gives it an advantage in maintaining the high cytosolic glycine concentration required for efficient vesicular loading by VIAAT. The vesicular inhibitory phenotype was gradually altered from glycinergic to GABAergic through mixed events when GABA is introduced into the secretory cell and competes for uptake by VIAAT. Interestingly, the VIAAT ortholog from Caenorhabditis elegans (UNC-47), a species lacking glycine transmission, also supports glycine exocytosis in the presence of GlyT2, and a point mutation of UNC-47 that abolishes GABA transmission in the worm confers glycine specificity. Together, these results suggest that an increased cytosolic availability of glycine in VIAAT-containing terminals was crucial for the emergence of glycinergic transmission in vertebrates.
Subject(s)
Glycine Plasma Membrane Transport Proteins/biosynthesis , Glycine/metabolism , Phenotype , Synaptic Vesicles/metabolism , Vesicular Inhibitory Amino Acid Transport Proteins/biosynthesis , Animals , Caenorhabditis elegans , Cell Line , Glycine/genetics , Glycine Plasma Membrane Transport Proteins/genetics , Humans , Rats , Synaptic Vesicles/genetics , Vesicular Inhibitory Amino Acid Transport Proteins/geneticsABSTRACT
N-arachidonyl-glycine is one of a series of N-arachidonyl-amino acids that are derived from arachidonic acid. N-arachidonyl-glycine is produced in a wide range of tissues with greatest abundance in the spinal cord. Here we report that N-arachidonyl-glycine is a reversible and non-competitive inhibitor of glycine transport by GLYT2a, but has little effect on glycine transport by GLYT1b or gamma-amino butyric acid transport by GAT1. It has previously been reported that the activity of GLYT2a is down-regulated by protein kinase C and therefore we investigated whether the actions of N-arachidonyl-glycine on GLYT2a are mediated by second messenger systems that lead to the activation of protein kinase C. However, the protein kinase C inhibitor, staurosporine, had no effect on the actions of N-arachidonyl-glycine on GLYT2a. Thus, the actions of N-arachidonyl-glycine are likely to be mediated by a direct interaction with the transporter. We have further defined the pharmacophore by investigating the actions of other N-arachidonyl amino acids as well as the closely related compounds arachidonic acid, anandamide and R1-methanandamide. Arachidonic acid, anandamide and R1-methanandamide have no effect on glycine transport, but N-arachidonyl-l-alanine has similar efficacy at GLYT2a to N-arachidonyl-glycine, and N-arachidonyl-gamma-amino butyric acid is less efficacious. These observations define a novel recognition site for the N-arachidonyl amino acids.
Subject(s)
Arachidonic Acids/pharmacology , Glycine Plasma Membrane Transport Proteins/antagonists & inhibitors , Glycine/analogs & derivatives , Animals , DNA/genetics , Dose-Response Relationship, Drug , Electrophysiology , Endocannabinoids , Enzyme Inhibitors/pharmacology , Female , GABA Plasma Membrane Transport Proteins/metabolism , GABA Uptake Inhibitors , Glycine/metabolism , Glycine/pharmacology , Glycine Plasma Membrane Transport Proteins/metabolism , Humans , Kinetics , Oocytes/metabolism , Patch-Clamp Techniques , Polyunsaturated Alkamides/pharmacology , Staurosporine/pharmacology , Xenopus laevisABSTRACT
Glycine is a coagonist at the N-methyl-D-aspartate receptor. Changes in extracellular glycine concentration may modulate N-methyl-D-aspartate receptor function and excitatory synaptic transmission. The GLYT1 glycine transporter is present in glia surrounding excitatory synapses, and plays a key role in regulating extracellular glycine concentration. We investigated the kinetic and other biophysical properties of GLYT1b, stably expressed in CHO cells, using whole-cell patch-clamp techniques. Application of glycine produced an inward current, which decayed within a few seconds to a steady-state level. When glycine was removed, a transient outward current was observed, consistent with reverse transport of accumulated glycine. The outward current was enhanced by elevating intracellular or lowering extracellular [Na(+)], and was modulated by changes in extracellular [glycine] and time of glycine application. We developed a model of GLYT1b function, which accurately describes the time course of the transporter current under a range of experimental conditions. The model predicts that glial uptake of glycine will decay toward zero during a sustained period of elevated glycine concentration. This property of GLYT1b may permit spillover from glycinergic terminals to nearby excitatory terminals during a prolonged burst of inhibitory activity, and reverse transport may extend the period of elevated glycine concentration beyond the end of the inhibitory burst.
Subject(s)
Biophysics/methods , Glycine Plasma Membrane Transport Proteins/chemistry , Glycine/chemistry , Animals , Biological Transport , CHO Cells , Cricetinae , Dose-Response Relationship, Drug , GABA Plasma Membrane Transport Proteins/metabolism , Glycine Plasma Membrane Transport Proteins/physiology , Kinetics , Models, Biological , Patch-Clamp Techniques , Receptors, N-Methyl-D-Aspartate/chemistry , Sodium/chemistry , Synapses/physiology , Time FactorsABSTRACT
The regulation of glutamate and glycine concentrations within excitatory synapses plays an important role in maintaining a dynamic signalling process between neurones, but the failure to regulate the concentrations of these neurotransmitters has been implicated in the pathogenesis of various neurological disorders. In this review we shall discuss how glutamate and glycine transporters regulate synaptic concentrations of these neurotransmitters and how endogenous allosteric modulators influence transporter function. Whilst glutamate transport inhibitors are unlikely to be of therapeutic value because their potential to cause excitoxicity and cell death, a greater understanding of how endogenous compounds allosterically modulate glutamate transporters may provide alternate drug targets. On the other hand, there are some promising drugs that inhibit glycine transporters, which are being trialled as an alternate treatment for schizophrenia. We shall discuss how the activity of one such compound may be expected to influence excitatory neurotransmission.
Subject(s)
Carrier Proteins/metabolism , Neurotransmitter Agents/metabolism , Receptors, Glutamate/metabolism , Synapses/metabolism , Amino Acid Transport System X-AG/metabolism , Amino Acid Transport Systems, Neutral/metabolism , Animals , Carrier Proteins/chemistry , Glycine Plasma Membrane Transport Proteins , Humans , Protein Conformation , Receptors, Glutamate/chemistryABSTRACT
In the central nervous system, glycine is a co-agonist with glutamate at the N-methyl-D-aspartate subtype of glutamate receptors and also an agonist at inhibitory, strychnine-sensitive glycine receptors. The GLYT1 subtypes of glycine transporters (GLYTs) are responsible for regulation of glycine at excitatory synapses, whereas a combination of GLYT1 and GLYT2 subtypes of glycine transporters are used at inhibitory glycinergic synapses. Zn2+ is stored in synaptic vesicles with glutamate in a number of regions of the brain and is believed to play a role in modulation of excitatory neurotransmission. In this study we have investigated the actions of Zn2+ on the glycine transporters, GLYT1b and GLYT2a, expressed in Xenopus laevis oocytes and we demonstrate that Zn2+ is a noncompetitive inhibitor of GLYT1 but has no effect on GLYT2. We have also investigated the molecular basis for these differences and the relationship between the Zn2+ and proton binding sites on GLYT1. Using site-directed mutagenesis, we identified 2 histidine residues, His-243 in the large second extracellular loop (ECL2) and His-410 in the fourth extracellular loop (ECL4), as two coordinates in the Zn2+ binding site of GLYT1b. In addition, our study suggests that the molecular determinants of proton regulation of GLYT1b are localized to the 2 histidine residues (His-410 and His-421) of ECL4. The ability of Zn2+ and protons to regulate the rate of glycine transport by interacting with residues situated in ECL4 of GLYT1b suggests that this region may influence the substrate translocation mechanism.
Subject(s)
Amino Acid Transport Systems, Neutral/metabolism , Zinc/metabolism , Amino Acid Sequence , Amino Acid Transport Systems, Neutral/genetics , Animals , Biological Transport , Brain/metabolism , Glycine Plasma Membrane Transport Proteins , Humans , Molecular Sequence Data , Protons , Receptors, N-Methyl-D-Aspartate/metabolism , Sequence Alignment , XenopusABSTRACT
The GLYT1 subtypes of glycine transporter are expressed in glia surrounding excitatory synapses in the mammalian CNS and may regulate synaptic glycine concentrations required for activation of the NMDA subtypes of glutamate receptor. In this report we demonstrate that the rate of glycine transport by GLYT1 is inhibited by arachidonic acid. The cyclo-oxygenase and lipoxygenase inhibitors indomethacin and nordihydroguaiaretic acid, and the protein kinase C inhibitor staurosporine, had no effect on the extent of arachidonic acid inhibition of transport, which suggests that the inhibitory effects of arachidonic acid result from a direct interaction with the transporter. In contrast to arachidonic acid, its amide derivative, anandamide, and the more stable analogue R1-methanandamide stimulate glycine transport. This stimulation is unlikely to be a secondary effect of cannabinoid receptor stimulation because the cannabinoid receptor agonist WIN 55 212-2 had no effect on transport. We suggest that the stimulatory effects of anandamide on GLYT1 are due to a direct interaction with the transporter.
Subject(s)
Amino Acid Transport Systems, Neutral/drug effects , Amino Acid Transport Systems, Neutral/metabolism , Arachidonic Acid/pharmacology , Arachidonic Acids/pharmacology , Amino Acid Transport Systems, Neutral/genetics , Animals , Biological Transport/drug effects , Endocannabinoids , Glioma/metabolism , Glycine/metabolism , Glycine Plasma Membrane Transport Proteins , Oocytes/drug effects , Oocytes/metabolism , Patch-Clamp Techniques , Polyunsaturated Alkamides , Protein Isoforms/drug effects , Protein Isoforms/genetics , Protein Isoforms/metabolism , Rats , Transfection , Xenopus laevisABSTRACT
Current antipsychotic drugs are only partially effective in treating schizophrenia and there is a clear need to develop better therapies. An alternative approach to develop new antipsychotics has come from the NMDA receptor hypofunction model for schizophrenia. It has been hypothesised that stimulation of NMDA receptors with glycine site agonists may be therapeutic, and a number of clinical trials of glycine together with standard antipsychotic drugs have been recently been conducted. Modest improvements in negative symptoms have been reported in some studies but a potentially more effective treatment is to use inhibitors of the GLYT1 subtype of glycine transporters. Expression of GLYT1 within the brain correlates with NMDA receptor expression patterns and it has been suggested that GLYT1 may regulate synaptic glycine concentrations. With the development of selective and potent non-transported inhibitors of GLYT1 it should be possible to elevate synaptic glycine concentrations more effectively and thereby to increase NMDA receptor activity. Recent in vitro studies demonstrate that the glycine transport inhibitor, N[3-(4-fluorophenyl)-3-(4'-phenylphenoxy)] propylsarcosine, enhances NMDA receptor activity and the use of this class of compounds in clinical studies is eagerly awaited.