AMPA and Kainate Receptors in Turtle Retina: An Immunocytochemical Study

Summary 1. Glutamate is one of the main neurotransmitters in the retina. Its effects are mediated by a large number of ionotropic and metabotropic receptors. 2. The distribution of ionotropic AMPA receptor subunits GluR1–4, kainate receptor subunits GluR5–7 and KA2, as well as delta receptors 1–2 was studied in turtle retina. Indirect immunofluorescence was used to localize the different receptor subunits viewed using light microscopy. 3. Results show that all subunits, with excerption of GluR1 and GluR5, are widely distributed in the turtle retina. 4. They are mainly located in the both plexiform layers of the retina where punctate staining, a sign for synaptic localization, is observed. 5. The vast majority of the subunits possess specific pattern of staining that allow to suppose that they are involved in different retinal circuits. 6. It can be assumed that the GluR2/3 and GluR6/7 subunits are expressed on the dendrites of a subpopulation of bipolar cells that are immunopositive for α-isoform of protein kinase C (PKCα). The GluR2/3 and GluR6/7 subunits are most probably used by the same PKCα immunopositive bipolar cells in their synaptic contacts with the third-order retinal neurons, the amacrine and ganglion cells.     

In vitro and in vivo characterization of MPEP,an allosteric modulator of the metabotropic glutamate receptor subtype 5: review article

Summary.  There is a need to identify subtype-specific ligands for mGlu receptors to elucidate the potential of these receptors for the treatment of nervous system disorders. To date, most mGlu receptor antagonists are amino acid-like compounds acting as competitive antagonists at the glutamate binding site located in the large extracellular N-terminal domain. We have characterized novel subtype-selective mGlu₅ receptor antagonists which are structurally unrelated to competitive mGlu receptor ligands. Using a series of chimeric receptors and point mutations we demonstrate that these antagonists act as inverse agonists with a novel allosteric binding site in the seven-transmembrane domain. Recent studies in animal models implicate mGlu₅ receptors as a potentially important therapeutic target particularly for the treatment of pain and anxiety. Received July 2, 2001 Accepted August 6, 2001 Published online September 10, 2002     

Variability of calcium responses to agonists of glutamate receptors in hippocampal neurons

The subject of this work was to study the reasons of the variability of the calcium response amplitudes in individual neurons of the hippocampal cell culture to agonists of ionotropic glutamate receptors and the regularities of the calcium response amplitude distribution. Changes of [Ca²⁺] _i in the neurons in response to the NMDA-, AMPA-, and KA-receptor agonists were recorded using fluorescence probe Fura-2. The calcium response amplitudes (expressed as the ratio of fluorescence intensities of Fura-2 upon excitation at wave-lengths 340 and 380 nm) to short-term application of glutamate receptor agonists N-methyl-D-aspartate (NMDA), domoic acid (DA), α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), and (S)-(−)-5-fluorowillardiine (FW) were measured. Calcium responses of individual cells differed in shape and amplitude but always reproduced upon the second application of the agonist. To elucidate the nature of calcium response variability, we compared distributions of calcium response amplitudes to the NMDA-, KA-, and AMPA-receptor agonists in cultures of various ages in the presence of receptor desensitization inhibitors and different agonist concentrations. An even increase from 0.05 to 1.6 was characteristic for distributions of calcium response amplitudes. Nevertheless, in 1–3% neurons of the cell culture, calcium response amplitudes reached much higher values. The efficiency of the ligands usually increased in the following order: FW ≈ NMDA > DA. However, this regularity varied with age and depended on the presence of the receptor desensitization inhibitor. In the process of growth and differentiation of neurons in culture from 1 to 14 day in vitro, calcium response amplitude to AMPA- and KA-receptor agonists increased. Desensitization inhibitors transformed the response from pulse-like with a sharp peak into stepwise and increased the amplitude of calcium responses but did not abolish the character of even amplitude distribution. The effect of AMPA- and KA-receptor desensitization inhibitor decreased with calcium response amplitude growth in the control and approached zero in neurons with initially maximal amplitude. KA- and AMPA-receptor agonists at high concentrations possessed a property of desensitization inhibitors and transformed a transient response into a continuous one that lasted throughout the application time. Thus, the amplitude and shape of the calcium response to glutamate receptor agonists is a characteristic parameter of an individual cell.     

Novel approaches to targeting glutamate receptors for the treatment of chronic pain: review article

Summary.  Glutamatergic mechanisms are implicated in acute and chronic pain, and there is a great diversity of glutamate receptors that can be used as targets for novel analgesics. Some approaches, e.g. NMDA receptor antagonism, have been validated clinically, however, the central side-effects have remained the main problem with most compounds. Recently, some novel approaches have been explored as new compounds targeting some modulatory sites at the NMDA receptor (glycine_B and NR2B-subtype selective antagonists), as well as kainate and metabotropic glutamate receptors, have been discovered. Many of these compounds have demonstrated efficacy in animal models of chronic pain, and some of them appear to have a reduced side-effect liability compared to clinically tested NMDA antagonists. These recent advances are reviewed in the present work. Received July 6, 2001 Accepted August 6, 2001 Published online June 26, 2002     

Localization and physiological roles of metabotropic glutamate receptors in the direct and indirect pathways of the basal ganglia

Summary.  Our current understanding of the circuitry of the basal ganglia, and the pathophysiology of Parkinson's disease has led to major breakthroughs in the treatment of this debilitating movement disorder. Unfortunately, there are significant problems with the currently available pharmacological therapies that focus on dopamine replacement or dopaminergic agonists. Because of this, much effort has been focused on developing novel targets for the treatment of Parkinson's disease. The metabotropic glutamate receptors are a family of G-protein coupled receptors activated by glutamate. These receptors are differentially distributed throughout the basal ganglia in a manner suggesting that they may provide novel targets for the treatment of movement disorders. In this review we summarize anatomical and physiological data from our work and the work of other laboratories describing the distribution and physiological roles of metabotropic glutamate receptors in the basal ganglia with emphasis on possible therapeutic targets. Received July 2, 2001 Accepted August, 6, 2001 Published online June 26, 2002     

大鼠新鲜分离DRG神经元胞体膜谷氨酸受体亚型及其分布

本文目的是研究ＤＲＧ神经元膜谷氨酸受体亚型的分布及其共存情况。实验在ＤＲＧ分离细胞上应用膜片带技术记录ＮＭＤＡ－,ＫＡ－和ＱＡ／ＡＭＰＡ－激活电流。在受检的３７个细胞中７０．３％的细胞对ＮＭＤＡ敏感;１８．９％的细胞对ＫＡ敏感;５６．８％的细胞对ＱＡ敏感。其分布的情况是：单独存在一种受体的细胞为１５个;二种受体共存的细胞为１３个;三种受体共存的细胞为４个．另外有４个细胞三种受体均无。     

Group III Metabotropic Glutamate Receptors: Pharmacology,Physiology and Therapeutic Potential

Glutamate, the primary excitatory neurotransmitter in the central nervous system (CNS), exerts neuromodulatory actions via the activation of metabotropic glutamate (mGlu) receptors. There are eight known mGlu receptor subtypes (mGlu1-8), which are widely expressed throughout the brain, and are divided into three groups (I–III), based on signalling pathways and pharmacological profiles. Group III mGlu receptors (mGlu4/6/7/8) are primarily, although not exclusively, localised on presynaptic terminals, where they act as both auto- and hetero-receptors, inhibiting the release of neurotransmitter. Until recently, our understanding of the role of individual group III mGlu receptor subtypes was hindered by a lack of subtype-selective pharmacological tools. Recent advances in the development of both orthosteric and allosteric group III-targeting compounds, however, have prompted detailed investigations into the possible functional role of these receptors within the CNS, and revealed their involvement in a number of pathological conditions, such as epilepsy, anxiety and Parkinson’s disease. The heterogeneous expression of group III mGlu receptor subtypes throughout the brain, as well as their distinct distribution at glutamatergic and GABAergic synapses, makes them ideal targets for therapeutic intervention. This review summarises the advances in subtype-selective pharmacology, and discusses the individual roles of group III mGlu receptors in physiology, and their potential involvement in disease.     

Metabotropic glutamate receptors in mechanisms of plasticity of the central nervous system in the honeybee <Emphasis Type="Italic">Apis mellifera</Emphasis>

Localization of metabotropic glutamate receptors (MGR) in head ganglion of honeybee Apis mellifera, and mechanisms of participation of activated MGR in CNS plasticity are investigated by means of complex approach using immunochemical, electrophysiological and behavioral methods. Influence of MGR activation on GABAergic system and inotropic glutamate receptors (IGR) of AMPA- and NMDA-subtypes is studied. MGR are revealed in lateral and medial calices of mushroom bodies. The inhibiting influence of MGR on AMPA- and NMDA receptors is shown using method of conditioned reflex. Previous activation of MGR neutralizes the inhibiting effect of GABA. Modulating role of heterogeneous MGR population in mechanisms of CNS plasticity on the level of glutamatergic synapse, and at interaction with GABAergic system is discussed.     

Unstructured loop is essential for the activation of mGluR2

Metabotropic Glutamate Receptors (mGluRs) are Class C G-protein coupled receptors (GPCRs) that are expressed throughout the central nervous system and are involved in several neurological and psychiatric disorders. Although, many studies focused on Glutamate induced activation of mGluR2, however, the role of unstructured loop (or “BC loop”) in activation of metabotropic Glutamate receptors is currently unknown. Here, using Förster Resonance Energy Transfer (FRET) based assay in live cells we show that unstructured loop is required for Glutamate induced conformation and hence the activation of the receptor.     

Glutamate Signaling in Benign and Malignant Disorders: Current Status,Future Perspectives,and Therapeutic Implications

Glutamate, a nonessential amino acid, is the major excitatory neurotransmitter in the central nervous system. As such, glutamate has been shown to play a role in not only neural processes, such as learning and memory, but also in bioenergetics, biosynthetic and metabolic oncogenic pathways. Glutamate has been the target of intense investigation for its involvement not only in the pathogenesis of benign neurodegenerative diseases (NDDs) such as Parkinson''s disease, Alzheimer''s disease, schizophrenia, multiple sclerosis, and amyotropic lateral sclerosis (ALS), but also in carcinogenesis and progression of malignant diseases. In addition to its intracellular activities, glutamate in secreted form is a phylogenetically conserved cell signaling molecule. Glutamate binding activates multiple major receptor families including the metabotropic glutamate receptors (mGluRs) and ionotropic glutamate receptors (iGluRs), both of which have been implicated in various signaling pathways in cancer. Inhibition of extracellular glutamate release or glutamate receptor activation via competitive or non-competitive antagonists decreases growth, migration and invasion and induces apoptosis in breast cancer, melanoma, glioma and prostate cancer cells. In this review, we discuss the current state of glutamate signaling research as it relates to benign and malignant diseases. In addition, we provide a synopsis of clinical trials using glutamate antagonists for the treatment of NDD and malignant diseases. We conclude that in addition to its potential role as a metabolic biomarker, glutamate receptors and glutamate-initiated signaling pathways may provide novel therapeutic opportunities for cancer.     

NMDA受体与Ⅰ组代谢型谷氨酸受体的相互作用

谷氨酸是哺乳动物中枢神经系统重要兴奋性神经递质,参与学习、记忆、药物依赖成瘾及神经系统退行性疾病等多种病理生理过程。谷氨酸通过激活离子型（iGluRs）和代谢型谷氨酸受体（mGluRs）发挥作用。业已有研究提示iGluRs和mGluRs之间存在相互作用,但具体机制尚待阐明。本文从蛋白分子结构、突触可塑性、相互作用可能涉及的信号分子和通路等方面综述了NMDAR与Ⅰ组mGluRs之间的相互作用,旨在为深入研究谷氨酸受体之间的相互作用提供线索。     

GABAC receptors in the vertebrate retina

In the central nervous system (CNS), the inhibitory transmitter GABA interacts with three subtypes of GABA receptors, type A, type B, and type C. Historically, GABA receptors have been classified as either the inotropic GABA_A receptors or the metabotropic GABA_B receptors. Over the past 10 yr, studies have shown that a third class, called the GABA_C receptor, also exists. GABA_C receptors are found primarily in the vertebrate retina and to some extent in other parts of the CNS. Although GABA_A and GABA_C receptors both gate chloride channels, they are pharmacologically, molecularly, and functionally distinct. The ρ subunit of the GABA_C receptor, which has about 35% amino acid homology to GABA_A receptor subunits, was cloned from the retina and, when expressed inXenopus oocytes, has properties similar to retinal GABA_C receptors. There are probably distinct roles for GABA_C receptors in the retina, because they are found on only a subset of neurons, whereas GABA_A receptors are ubiquitous. This article reviews recent electrophysiological and molecular studies that have characterized the unique properties of GABA_C receptors and describes the roles that these receptors may play in visual information processing in the retina.     

Phagocytic functions of microglial cells in the central nervous system and their importance in two neurodegenerative diseases: multiple sclerosis and Alzheimer’s disease

Microglial cells are the resident phagocytic cells of the central nervous system (CNS). They possess a wide range of receptors allowing them to identify and internalize numerous pathogens. We will discuss here the role of the most important receptors of microglia involved in non-opsonin-dependent phagocytosis (mannose receptor, β-glucan receptor, scavenger receptor) and that of receptors involved in the opsonin-dependent phagocytosis, namely the complement 3 (CR3) and the Fcγ receptors (FcγR). First, the molecular and cellular mechanisms induced when these receptors are conducting a phagocytic event are presented. In the second part, we will discuss the role these receptors may play in multiple sclerosis and Alzheimer’s disease, in the elimination by phagocytosis of myelin and beta amyloid peptide respectively. The first two authors contributed equally to this work.     

The Structure of (?)-Kaitocephalin Bound to the Ligand Binding Domain of the (S)-α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA)/Glutamate Receptor,GluA2

Glutamate receptors mediate the majority of excitatory synaptic transmission in the central nervous system, and excessive stimulation of these receptors is involved in a variety of neurological disorders and neuronal damage from stroke. The development of new subtype-specific antagonists would be of considerable therapeutic interest. Natural products can provide important new lead compounds for drug discovery. The only natural product known to inhibit glutamate receptors competitively is (−)-kaitocephalin, which was isolated from the fungus Eupenicillium shearii and found to protect CNS neurons from excitotoxicity. Previous work has shown that it is a potent antagonist of some subtypes of glutamate receptors (AMPA and NMDA, but not kainate). The structure of kaitocephalin bound to the ligand binding domain of the AMPA receptor subtype, GluA2, is reported here. The structure suggests how kaitocephalin can be used as a scaffold to develop more selective and high affinity antagonists for glutamate receptors.     

Application of photoaffinity crosslinking in determining the interaction between calcitonin and its receptor

Summary Understanding the molecular mechanism underlying how the peptide ligands bind to their receptors with subsequent receptor activation and cellular response is of great long-term value in designing receptor-targeted drugs. This is more difficult for class-II G protein-coupled receptors as only minimal structural data is available and their natural peptide ligands contain a large and diffuse pharmacophore. To address this problem, photoaffinity labeling studies have been developed to identify the spatial proximity between the photophore-modified ligand and its receptor. This minireview looks at the application of this approach in determining the proximal sites between class-II G protein-coupled receptor peptide ligands and their corresponding receptors, including parathyroid hormone, secretin and vasoactive intestinal polypeptide. More specifically, we will highlight interaction sites between positions 19, 16 and 26 of calcitonin with C¹³⁴−K¹⁴¹, and F¹³⁷ and T³⁰ of the receptor, respectively.     

Muscarinic toxins: tools for the study of the pharmacological and functional properties of muscarinic receptors

Muscarinic receptors mediate metabotropic actions of acetylcholine in the CNS and PNS and autocrine functions of acetylcholine in non-neuronal systems. Because of the lack of highly selective muscarinic ligands, the precise location, functional role, and roles in various diseases of the five muscarinic receptor subtypes remain unclear. Muscarinic toxins isolated from the venom of Dendroaspis snakes have a natural high affinity and selectivity, associated with roles as competitive antagonists, allosteric modulators, and potential agonists. These toxins may therefore be invaluable tools for studying muscarinic receptors. We review data on the structural and pharmacological characterization of the muscarinic toxins, focusing on recent structure–function studies on toxin–receptor interactions. We discuss the potential benefits of using these toxins for investigating muscarinic function in vivo .     

Advances in understanding neuronal somatostatin receptors

It has long been considered that somatostatin acts as a neuromodulator in the mammalian central nervous system but its precise physiological roles remain elusive. Early studies to identify somatostatin-binding sites revealed a widespread heterogeneous pattern, especially in the CNS. More recently, a family of somatostatin receptors have been identified, of which five genes (sst_1–5) have been cloned. In this review, we discuss current data describing the localisation of the five receptor types. Recent progress in understanding their function has been made using high-affinity, selective receptor ligands and transgenic animal technology. Finally, the therapeutic potential for somatostatin receptor-selective compounds as analgesics is considered.     

Glutamate Stimulates Local Protein Synthesis in the Axons of Rat Cortical Neurons by Activating α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors and Metabotropic Glutamate Receptors

Glutamate is the principal excitatory neurotransmitter in the mammalian CNS. By analyzing the metabolic incorporation of azidohomoalanine, a methionine analogue, in newly synthesized proteins, we find that glutamate treatments up-regulate protein translation not only in intact rat cortical neurons in culture but also in the axons emitting from cortical neurons before making synapses with target cells. The process by which glutamate stimulates local translation in axons begins with the binding of glutamate to the ionotropic AMPA receptors and metabotropic glutamate receptor 1 and members of group 2 metabotropic glutamate receptors on the plasma membrane. Subsequently, the activated mammalian target of rapamycin (mTOR) signaling pathway and the rise in Ca²⁺, resulting from Ca²⁺ influxes through calcium-permeable AMPA receptors, voltage-gated Ca²⁺ channels, and transient receptor potential canonical channels, in axons stimulate the local translation machinery. For comparison, the enhancement effects of brain-derived neurotrophic factor (BDNF) on the local protein synthesis in cortical axons were also studied. The results indicate that Ca²⁺ influxes via transient receptor potential canonical channels and activated the mTOR pathway in axons also mediate BDNF stimulation to local protein synthesis. However, glutamate- and BDNF-induced enhancements of translation in axons exhibit different kinetics. Moreover, Ca²⁺ and mTOR signaling appear to play roles carrying different weights, respectively, in transducing glutamate- and BDNF-induced enhancements of axonal translation. Thus, our results indicate that exposure to transient increases of glutamate and more lasting increases of BDNF would stimulate local protein synthesis in migrating axons en route to their targets in the developing brain.     

P2 receptors in atherosclerosis and postangioplasty restenosis

Atherosclerosis is an immunoinflammatory process that involves complex interactions between the vessel wall and blood components and is thought to be initiated by endothelial dysfunction [Ross (Nature 362:801–809, 1993); Fuster et al. (N Engl J Med 326:242–250, 1992); Davies and Woolf (Br Heart J 69:S3–S11, 1993)]. Extracellular nucleotides that are released from a variety of arterial and blood cells [Di Virgilio and Solini (Br J Pharmacol 135:831–842, 2002)] can bind to P2 receptors and modulate proliferation and migration of smooth muscle cells (SMC), which are known to be involved in intimal hyperplasia that accompanies atherosclerosis and postangioplasty restenosis [Lafont et al. (Circ Res 76:996–1002, 1995)]. In addition, P2 receptors mediate many other functions including platelet aggregation, leukocyte adherence, and arterial vasomotricity. A direct pathological role of P2 receptors is reinforced by recent evidence showing that upregulation and activation of P2Y₂ receptors in rabbit arteries mediates intimal hyperplasia [Seye et al. (Circulation 106:2720–2726, 2002)]. In addition, upregulation of functional P2Y receptors also has been demonstrated in the basilar artery of the rat double-hemorrhage model [Carpenter et al. (Stroke 32:516–522, 2001)] and in coronary artery of diabetic dyslipidemic pigs [Hill et al. (J Vasc Res 38:432–443, 2001)]. It has been proposed that upregulation of P2Y receptors may be a potential diagnostic indicator for the early stages of atherosclerosis [Elmaleh et al. (Proc Natl Acad Sci U S A 95:691–695, 1998)]. Therefore, particular effort must be made to understand the consequences of nucleotide release from cells in the cardiovascular system and the subsequent effects of P2 nucleotide receptor activation in blood vessels, which may reveal novel therapeutic strategies for atherosclerosis and restenosis after angioplasty.     

Development of a homogeneous binding assay for histamine receptors

Histamine is critically involved in a wide range of physiological and pathological processes through its actions at different receptors. Thus, histamine receptors have been actively pursued as therapeutic targets in the pharmaceutical industry for the treatment of a variety of diseases. There are currently four histamine receptors that have been cloned, all of which are G protein-coupled receptors. Studies from both academia and pharmaceutical companies have identified compounds that modulate the function of specific histamine receptors. These efforts led to the successful introduction of histamine H(1) and H(2) receptor antagonists for the treatment of allergy and excess gastric acid secretion, respectively. Histamine H(3) receptor ligands are currently under investigation for the treatment of obesity and neurological disorders. The recently identified histamine H(4) receptor is preferentially expressed in the immune tissues, suggesting a potential role in normal immune functions and possibly in the pathogenesis of inflammatory diseases. Even with the long history of histamine research and the important applications of histamine receptor ligands, assays to measure the affinity of compounds binding to histamine receptors are still routinely analyzed using a filtration assay, a very low-throughput assay involving washing and filtration steps. This article describes a simple, robust, and homogeneous binding assay based on the scintillation proximity assay (SPA) technology that provides results equivalent to those obtained using the more complex filtration assay. The SPA format is easily adapted to high-throughput screening because it is amenable to automation. In summary, this technique allows high-throughput screening of compounds against multiple histamine receptors and, thus, facilitates drug discovery efforts.