Pharmacological topics of bone metabolism: glutamate as a signal mediator in bone

The view that L-glutamate (Glu) is an excitatory amino acid neurotransmitter in the mammalian central nervous system is prevailing on the basis of successful cloning of a number of genes encoding different signaling molecules, such as Glu receptors for the signal input, Glu transporters for the signal termination and vesicular Glu transporters for the signal output through exocytotic release. Little attention has been paid to an extracellular transmitter role of Glu in peripheral neuronal and non-neuronal tissues, by contrast, whereas recent molecular biological and pharmacological analyses including ours give rise to a novel function for Glu as an autocrine and/or paracrine signal mediator in bone comprised of osteoblasts, osteoclasts and osteocytes, in addition to other peripheral tissues including pancreas, adrenal and pituitary glands. Emerging evidence suggests that Glu could play a dual role in mechanisms underlying the maintenance of cellular homeostasis as an excitatory neurotransmitter in the central nervous system and as an extracellular signal mediator in peripheral autocrine and/or paracrine tissues. In this review, therefore, we would outline the possible signaling system for Glu to play a role as an extracellular signal mediator in mechanisms underlying maintenance of the cellular homeostasis in bone.     

Possible involvement of glutamatergic signaling machineries in pathophysiology of rheumatoid arthritis

The prevailing view is that L-glutamate (Glu) functions as an excitatory amino acid neurotransmitter through a number of molecular machineries required for the neurocrine signaling at synapses in the brain. These include Glu receptors for signal input, Glu transporters for signal termination, and vesicular Glu transporters for signal output through exocytotic release. Although relatively little attention has been paid to the functional expression of these molecules required for glutamatergic signaling in peripheral tissues, recent molecular biological analyses including ours give rise to a novel function for Glu as an extracellular signal mediator in the autocrine and/or paracrine system in several peripheral and non-neuronal tissues, including bone and cartilage. In particular, a drastic increase is demonstrated in the endogenous levels of both Glu and aspartate in the synovial fluid with intimate relevance to increased edema and sensitization to thermal hyperalgesia in experimental arthritis models. However, to date, there is only limited information about the physiological and pathological significance of glutamatergic signaling machineries expressed by articular synovial tissues. In this review, we have outlined the role of Glu in synovial fibroblasts in addition to the possible involvement of glutamatergic signaling machineries in the pathogenesis of joint diseases such as rheumatoid arthritis.     

Roles and regulation of glutamate transporters in the central nervous system

1. Glutamate transporters (also known as excitatory amino acid transporters or EAAT) are solely responsible for the removal of the excitatory neurotransmitter l-glutamate (Glu) from the extracellular space and, thus, permit normal transmission, as well as preventing cell death due to the excessive activation of Glu receptors. 2. Five subtypes of glutamate transporter (EAAT1-5) exist, possessing distinct pharmacology, cellular localization and modulatory mechanisms. 3. Experimental inhibition of EAAT activity in vitro and in vivo results in increased extracellular concentrations of Glu and in neuronal death via excitotoxicity, highlighting the importance of EAAT in normal excitatory neurotransmission. 4. Dysfunction of EAAT may contribute to the pathology of both acute neuronal injury and chronic neurodegenerative conditions, so correction of EAAT function under these conditions may provide a valuable therapeutic strategy. 5. The present review describes basic pharmacological studies that allow new insights into EAAT function and suggest possible strategies for the therapeutic modulation of EAAT.     

Glutamate transporters as drug targets

The L-glutamate (Glu) has been hypothesized as an excitatory amino acid neurotransmitter in the mammalian central nervous system after successful cloning and identification of a number of genes encoding signaling machineries required for the neurocrine at synapses in the brain. These include excitatory amino acid transporters (EAATs) for signal termination and vesicular Glu transporters (VGLUTs) for signal output through exocytotic release, in addition to Glu receptors (GluRs) for signal input. These Glu signaling molecules not only play key roles in mechanisms associated with synaptic plasticity such as learning and memory, but also participate in the etiology and pathology of different neuropsychiatric disorders and neuronal cell death seen in various neurodegenerative diseases. Of the aforementioned Glu signaling molecules, EAATs are essential for the termination of signal transmission mediated by Glu as well as for the prevention of neurotoxicity mediated by this endogenous excitotoxin, while VGLUTs are crucial for the storage of Glu in synaptic vesicles to suffice for the definition of a glutamatergic phenotype. Many early desperate efforts were devoted to the search and development of novel compounds with a therapeutic window toward GluRs, while relatively little attention was paid to either EAATs or VGLUTs in this aspect. In this review, therefore, we will summarize the classification and functionality of EAATs and VGLUTs with a focus on their possibilities as potential therapeutic targets for different neurodegenerative and neuropsychiatric disorders related to malfunction of Glu signaling in human beings.     

Glutamate as a modulator of embryonic and adult neurogenesis

It has been widely accepted that neurogenesis continues throughout life. Neural stem cells can be found in the ventricular zone of the embryonic and in restricted regions of the adult central nervous system, including subventricular and subgranular zones of the hippocampal dentate gyrus. The network of signaling mechanisms determining whether neural stem cells remain in a proliferative state or differentiate is only partly discovered. Recent advances indicate that glutamate (Glu), the predominant excitatory neurotransmitter in mature neurons, can influence immature neural cell proliferation and differentiation, as well. Despite many similarities, Glu actions on neurogenesis in the developing and adult brain show distinct differences and are far from being clear. Due to alterations of Glu transport mechanisms, extracellular Glu level is high in the embryonic CNS. Glu acts non-synaptically on dividing progenitors either by directly activating ionotropic and/or metabotropic Glu receptors or can influence other cells which are located in the vicinity of proliferating cells and produce molecules regulating neural precursor cell proliferation by other mechanisms. Due to the complexity of signaling pathways and to regional differences in neural precursors, Glu can influence proliferation and neuronal commitment as well, and acts as a positive regulator of neurogenesis. Brain injuries like ischemia, epilepsy or stress lead to severe neuronal death and additionally, influence neurogenesis, as well. Glu homeostasis is altered under these pathological circumstances, implying that therapeutic treatments mediating Glu signaling might be useful to increase neuronal replacement after cell loss in the brain.     

Glutamate transporters in brain ischemia： to modulate or not？

In this review, we briefly describe glutamate （Glu） metabolism and its specific transports and receptors in the central nervous system （CNS）. Thereafter, we focus on excitatory amino acid transporters, cystine/glutamate antiporters （system xc-） and vesicular glutamate transporters, specifically addressing their location and roles in CNS and the molecular mechanisms underlying the regulation of Glu transporters. We provide evidence from in vitro or in vivo studies concerning alterations in Glu transporter expression in response to hypoxia or ischemia, including limited human data that supports the role of Glu transporters in stroke patients. Moreover, the potential to induce brain tolerance to ischemia through modulation of the expression and/or activities of Glu transporters is also discussed. Finally we present strategies involving the application of ischemic preconditioning and pharmacological agents, eg β-lactam antibiotics, amitriptyline, riluzole and N-acetylcysteine, which result in the significant protection of nervous tissues against ischemia.     

Pharmacological manipulation of glutamate transport

L-Glutamic acid acts as the major excitatory neurotransmitter and, at the same time, represents a potential neurotoxin for the mammalian central nervous system (CNS). The termination of excitatory transmission and the maintenance of physiologic levels of extracellular glutamate, which is necessary to prevent excitotoxicity, are prominently mediated by a family of high-affinity sodium-dependent excitatory amino acid transporters (EAATs). Five subtypes of EAATs have been cloned, possessing distinct pharmacology, localization, sensitivity to transport inhibitors and modulatory mechanisms. Expression and activity of EAATs have been shown to be amenable to fine endogenous and, potentially, pharmacological regulation by substrate itself, growth factors, second messengers, hormones, biological oxidants, inflammatory mediators and pathological conditions. The present review describes basic pharmacological studies, mostly performed on animal models or cell preparations, in order to obtain an updated picture of the known regulatory mechanisms of single EAAT expression and activity. New insight into molecular pathways involved in EAAT regulation will allow pharmacological manipulation of excitatory CNS activity, possibly avoiding adverse effects of glutamate receptor blockade.     

The glutamatergic system outside the CNS and in cancer biology

Glutamate is a major excitatory neurotransmitter in the CNS. The signalling machinery consists of: glutamate receptors, which are responsible for signal input; plasma glutamate transporters, which are responsible for signal termination; and vesicular glutamate transporters for signal output through exocytic release. Recently, data have suggested that the glutamatergic system plays an important role in non-neuronal tissues. In addition, the expression of glutamatergic system has been implicated in tumour biology. This review outlines the evidence, which suggests that the glutamatergic system may have an important role in cancer biology.     

The glutamatergic system outside the CNS and in cancer biology

Glutamate is a major excitatory neurotransmitter in the CNS. The signalling machinery consists of: glutamate receptors, which are responsible for signal input; plasma glutamate transporters, which are responsible for signal termination; and vesicular glutamate transporters for signal output through exocytic release. Recently, data have suggested that the glutamatergic system plays an important role in non-neuronal tissues. In addition, the expression of glutamatergic system has been implicated in tumour biology. This review outlines the evidence, which suggests that the glutamatergic system may have an important role in cancer biology.     

Role for GABA and Glu plasma membrane transporters in the interplay of inhibitory and excitatory neurotransmission

Neurotransmitter plasma membrane transporters do have much more to perform than simply terminating synaptic transmission and replenishing neurotransmitter pools. Findings in the past decade have evidenced their function in maintaining physiological synaptic excitability, and their actions in critical or pathological conditions, also. Conclusively these findings indicated a previously unrecognized role for neurotransmitter plasma membrane transporters in both, synaptic and nonsynaptic signaling. Major inhibitory and excitatory neurotransmitters within the brain, GABA and Glu, have long been considered to operate through independent systems (GABAergic or Gluergic), each of them characterized by its own localization, function and dedicated GABAergic or Gluergic cell phenotypes. Recent advances, however, have challenged this long-standing paradigm. Localization of GABA in Gluergic terminals and Glu in GABAergic cells were reported. Specific plasma membrane transporters for GABA and Glu are also co-localized in different brain areas. Although, their role in regulating each other's signal is still far from being understood, emerging lines of evidence on interplaying GABAergic and Gluergic processes through plasma membrane transporters opens up a new avenue in the field of more specific therapeutic intervention.     

Neuropsychiatric disorders and GABA]

In the mammalian central nervous system (CNS), the inhibitory GABAergic system is composed of different signaling molecules such as glutamate decaroxylase, vesicular GABA transporters, GABA receptors, GABA transporters and GABA transaminase. A prevailing view is that the balance between excitatory signaling mediated by glutamate and inhibitory signaling mediated by GABA plays a pivotal role in mechanisms underlying the modulation and maintenance of a variety of neural functions. Therefore, abnormalities in a GABAergic signaling molecule would lead to a crisis of severe symptoms relevant to a number of neuropsychiatric disorders. These include epilepsy, depression, schizophrenia, stiff-person syndrome, drug addiction and so on. In this review article, we will summarize recent studies on the relationship between the malfunction of GABAergic signaling molecules and the etiology of these neuropsychiatric disorders. We will also refer to novel strategies on GABAergic signaling molecules other than GABA receptors for therapeutic usefulness in the future.     

Neuronal high-affinity sodium-dependent glutamate transporters (EAATs): targets for the development of novel therapeutics against neurodegenerative diseases

L-Glutamate is the major excitatory neurotransmitter in mammalian central nervous system, and excitatory amino acid transporters (EAATs) are essential for terminating synaptic excitation and for maintaining extracellular glutamate concentration below toxic levels. Although the structure of these channel-like proteins has not been yet reported, their membrane topology has been hypothesised based on biochemical and protein sequence analyses. In the case of an inadequate clearance from synaptic cleft and from the extrasynaptic space, glutamate behaves as a potent neurotoxin, and it may be related to several neurodegenerative pathologies including epilepsy, ischemia, amyotrophic lateral sclerosis, and Alzheimer disease. The recent boom of glutamate is demonstrated by the enormous amount of publications dealing with the function of glutamate, with its role on modulation of synaptic transmission throughout the brain, mainly focusing: i). on the structure of its receptors, ii). on molecular biology and pharmacology of Glu transporters, and iii). on the role of glutamate uptake and reversal uptake in several neuropathologies. This review will deal with the recent and most interesting published results on Glu transporters membrane topology, Glu transporters physiopathological role and Glu transporters medicinal chemistry, highlighting the guidelines for the development of potential neuroprotective agents targeting neuronal high-affinity sodium-dependent glutamate transporters.     

Regulation by astrocytic ATP of synaptic transmission

Originally ascribed to having only passive roles in the CNS, astrocytes are now known to have an active role in the regulation of synaptic transmission. Neuronal activity can evoke Ca(2+) transients in astrocytes and Ca(2+) transients in astrocytes can evoke changes in neuronal activity. The excitatory neurotransmitter glutamate has been shown to mediate such bi-directional communication between astrocytes and neurons. We demonstrate here that ATP, a primary mediator of intercellular Ca(2+) signaling among astrocytes, also mediates intercellular signaling between astrocytes and neurons in hippocampal cultures. Mechanical stimulation of astrocytes evoked Ca(2+) waves mediated by the release of ATP and activation of P2 receptors. Mechanically evoked Ca(2+) waves led to decreased excitatory glutamatergic synaptic transmission in an ATP-dependent manner. Exogenous application of ATP does not affect post-synaptic glutamatergic responses but decreased pre-synaptic exocytotic events. Finally, we show that astrocytes exhibit spontaneous Ca(2+) oscillations mediated by extracellular ATP and that inhibition of these Ca(2+) responses enhanced excitatory glutamatergic transmission. We therefore conclude that ATP released from astrocytes exerts tonic and activity-dependent down-regulation of synaptic transmission via pre-synaptic mechanisms.     

Insulin and the insulin receptor in experimental models of learning and memory

Insulin is best known for its action on peripheral insulin target tissues such as the adipocyte, muscle and liver to regulate glucose homeostasis. In the central nervous system (CNS), insulin and the insulin receptor are found in specific brain regions where they show evidence of participation in a variety of region-specific functions through mechanisms that are different from its direct glucose regulation in the periphery. While the insulin/insulin receptor associated with the hypothalamus plays important roles in regulation of the body energy homeostasis, the hippocampus- and cerebral cortex-distributed insulin/insulin receptor has been shown to be involved in brain cognitive functions. Emerging evidence has suggested that insulin signaling plays a role in synaptic plasticity by modulating activities of excitatory and inhibitory receptors such as glutamate and GABA receptors, and by triggering signal transduction cascades leading to alteration of gene expression that is required for long-term memory consolidation. Furthermore, deterioration of insulin receptor signaling appears to be associated with aging-related brain degeneration such as the Alzheimer's dementia and cognitive impairment in aged subjects suffering type 2 diabetes mellitus.     

Transporters for L-glutamate: an update on their molecular pharmacology and pathological involvement

L-Glutamate (Glu) is the major excitatory neurotransmitter in the mammalian CNS and five types of high-affinity Glu transporters (EAAT1-5) have been identified. The transporters EAAT1 and EAAT2 in glial cells are responsible for the majority of Glu uptake while neuronal EAATs appear to have specialized roles at particular types of synapses. Dysfunction of EAATs is specifically implicated in the pathology of neurodegenerative conditions such as amyotrophic lateral sclerosis, epilepsy, Huntington's disease, Alzheimer's disease and ischemic stroke injury, and thus treatments that can modulate EAAT function may prove beneficial in these conditions. Recent advances have been made in our understanding of the regulation of EAATs, including their trafficking, splicing and post-translational modification. This article summarises some recent developments that improve our understanding of the roles and regulation of EAATs.     

Regulation and dysregulation of glutamate transporters

Glutamate is the primary excitatory neurotransmitter in the central nervous system. During synaptic activity, glutamate is released into the synaptic cleft and binds to glutamate receptors on the pre- and postsynaptic membrane as well as on neighboring astrocytes in order to start a number of intracellular signaling cascades. To allow for an efficient signaling to occur, glutamate levels in the synaptic cleft have to be maintained at very low levels. This process is regulated by glutamate transporters, which remove excess extracellular glutamate via a sodium-potassium coupled uptake mechanism. When extracellular glutamate levels rise to about normal, glutamate overactivates glutamate receptors, triggering a multitude of intracellular events in the postsynaptic neuron, which ultimately results in neuronal cell death. This phenomenon is known as excitotoxicity and is the underlying mechanisms of a number of neurodegenerative diseases. A dysfunction of the glutamate transporter is thought to contribute to cell death during excitotoxicity. Therefore, efforts have been made to understand the regulation of glutamate transporter function. Transporter activity can be regulated in different ways, including through gene expression, transporter protein targeting and trafficking and through posttranslational modifications of the transporter protein. The identification of these mechanisms has helped to understand the role of glutamate transporters during pathology and will aid in the development of therapeutic strategies with the transporter as a desirable target.     

SLC6 neurotransmitter transporters: structure, function, and regulation

The neurotransmitter transporters (NTTs) belonging to the solute carrier 6 (SLC6) gene family (also referred to as the neurotransmitter-sodium-symporter family or Na(+)/Cl(-)-dependent transporters) comprise a group of nine sodium- and chloride-dependent plasma membrane transporters for the monoamine neurotransmitters serotonin (5-hydroxytryptamine), dopamine, and norepinephrine, and the amino acid neurotransmitters GABA and glycine. The SLC6 NTTs are widely expressed in the mammalian brain and play an essential role in regulating neurotransmitter signaling and homeostasis by mediating uptake of released neurotransmitters from the extracellular space into neurons and glial cells. The transporters are targets for a wide range of therapeutic drugs used in treatment of psychiatric diseases, including major depression, anxiety disorders, attention deficit hyperactivity disorder and epilepsy. Furthermore, psychostimulants such as cocaine and amphetamines have the SLC6 NTTs as primary targets. Beginning with the determination of a high-resolution structure of a prokaryotic homolog of the mammalian SLC6 transporters in 2005, the understanding of the molecular structure, function, and pharmacology of these proteins has advanced rapidly. Furthermore, intensive efforts have been directed toward understanding the molecular and cellular mechanisms involved in regulation of the activity of this important class of transporters, leading to new methodological developments and important insights. This review provides an update of these advances and their implications for the current understanding of the SLC6 NTTs.     

Can selective ligands for glutamate binding proteins be rationally designed?

A major neurotransmitter, L-Glutamate must be stored, transported and received, and these processes are mediated by proteins that bind this simple yet essential amino acid. Detailed evidence continues to emerge on the structure of Glu binding proteins, which includes both receptors and transporters. It appears that receptors and transporters bind to Glu in different conformations, which may present a pharmacological opportunity. This review will compare and contrast information available on Glu Receptors (AMPA, NMDA, KA and mGlu), excitatory amino acid transporters (EAATs), the system Xc- transporter (XCT) and the vesicular Glutamate transporter (GVT). The cross-reactivity of ligands which have been previously used to characterize the glutamate binding proteins with system Xc- raises some fundamental interpretational issues regarding the mechanisms through which these analogues produce CNS damage. Although at one time it was thought that unraveling selectivity among glutamate binding proteins was an intractable problem, recently the NMDA antagonist (memantine) has been approved for general medical practice for treatment of Alzheimer's disease. Two other agents are in advanced clinical trials: an Ampakine for potential improvement of cognitive disorders, and a selective mGlu agonist for treatment of anxiety. The prospects for unraveling cross-reactivity will be weighed in light of a critical comparison of the glutamate binding protein targets.     

A pharmacological review of competitive inhibitors and substrates of high-affinity, sodium-dependent glutamate transport in the central nervous system

The acidic amino acid L-glutamate acts as both a primary excitatory neurotransmitter and a potential neurotoxin within the mammalian central nervous system. Functionally juxtaposed between these neurophysiological and pathological actions are an assorted group of integral membrane transporter proteins that rapidly and efficiently sequester glutamate into cellular and subcellular compartments. While multiple systems exist that are capable of mediating the uptake of L-glutamate, the high-affinity, sodium-dependent transporters have emerged as the most prominent players in the CNS with respect to terminating the excitatory signal, recycling the transmitter, and regulating extracellular levels of glutamate below those which could induce excitotoxic pathology. The focus of the present review is on the pharmacological specificity of these sodium-dependent transporters and, more specifically, on the competitive inhibitors that have been used to delineate the chemical requirements for binding and translocation. Analogues of glutamate that are conformationally constrained as a consequence of either the addition of substituents to the carbon backbone of glutamate or aspartate (e.g., beta-hydroxyaspartate or methylglutamate derivatives) or the incorporation of ring systems (e.g., (carboxycyclopropyl)glycines, aminocyclobutane dicarboxylates, or pyrrolidine dicarboxylates), have been especially valuable in these efforts. In this review, a particular emphasis is placed on the identification of analogues that exhibit preferential activity among the recently cloned transporter subtypes and on the differentiation of substrates from non-transportable inhibitors.