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1.
PLoS Comput Biol ; 17(6): e1009019, 2021 06.
Article in English | MEDLINE | ID: mdl-34143772

ABSTRACT

The anatomical and functional organization of neurons and astrocytes at 'tripartite synapses' is essential for reliable neurotransmission, which critically depends on ATP. In low energy conditions, synaptic transmission fails, accompanied by a breakdown of ion gradients, changes in membrane potentials and cell swelling. The resulting cellular damage and cell death are causal to the often devastating consequences of an ischemic stroke. The severity of ischemic damage depends on the age and the brain region in which a stroke occurs, but the reasons for this differential vulnerability are far from understood. In the present study, we address this question by developing a comprehensive biophysical model of a glutamatergic synapse to identify key determinants of synaptic failure during energy deprivation. Our model is based on fundamental biophysical principles, includes dynamics of the most relevant ions, i.e., Na+, K+, Ca2+, Cl- and glutamate, and is calibrated with experimental data. It confirms the critical role of the Na+/K+-ATPase in maintaining ion gradients, membrane potentials and cell volumes. Our simulations demonstrate that the system exhibits two stable states, one physiological and one pathological. During energy deprivation, the physiological state may disappear, forcing a transit to the pathological state, which can be reverted when blocking voltage-gated Na+ and K+ channels. Our model predicts that the transition to the pathological state is favoured if the extracellular space fraction is small. A reduction in the extracellular space volume fraction, as, e.g. observed with ageing, will thus promote the brain's susceptibility to ischemic damage. Our work provides new insights into the brain's ability to recover from energy deprivation, with translational relevance for diagnosis and treatment of ischemic strokes.


Subject(s)
Ions/metabolism , Synapses/metabolism , Action Potentials/physiology , Adenosine Triphosphate/metabolism , Animals , Brain/blood supply , Brain/metabolism , Brain/physiology , Energy Metabolism , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , Homeostasis , Ischemia/physiopathology , Mice , Models, Neurological , Neurons/drug effects , Neurons/physiology , Synaptic Transmission
2.
J Neurophysiol ; 123(6): 2122-2135, 2020 06 01.
Article in English | MEDLINE | ID: mdl-32347148

ABSTRACT

Peripheral viscerosensory afferent signals are transmitted to the nucleus tractus solitarii (nTS) via release of glutamate. Following release, glutamate is removed from the extrasynaptic and synaptic cleft via excitatory amino acid transporters (EAATs), thus limiting glutamate receptor activation or over activation, and maintaining its working range. We have shown that EAAT block with the antagonist threo-ß-benzyloxyaspartic acid (TBOA) depolarized nTS neurons and increased spontaneous excitatory postsynaptic current (sEPSC) frequency yet reduced the amplitude of afferent (TS)-evoked EPSCs (TS-EPSCs). Interestingly, chronic intermittent hypoxia (CIH), a model of obstructive sleep apnea (OSA), produces similar synaptic responses as EAAT block. We hypothesized EAAT expression or function are downregulated after CIH, and this reduction in glutamate removal contributes to the observed neurophysiological responses. To test this hypothesis, we used brain slice electrophysiology and imaging of glutamate release and TS-afferent Ca2+ to compare nTS properties of rats exposed to 10 days of normoxia (Norm; 21%O2) or CIH. Results show that EAAT blockade with (3S)-3-[[3-[[4-(trifluoromethyl)benzoyl]-amino]phenyl]methoxy]-l-aspartic acid (TFB-TBOA) in Norm caused neuronal depolarization, generation of an inward current, and increased spontaneous synaptic activity. The latter augmentation was eliminated by inclusion of tetrodotoxin in the perfusate. TS stimulation during TFB-TBOA also elevated extracellular glutamate and decreased presynaptic Ca2+ and TS-EPSC amplitude. In CIH, the effects of EAAT block are eliminated or attenuated. CIH reduced EAAT expression in nTS, which may contribute to the attenuated function seen in this condition. Therefore, CIH reduces EAAT influence on synaptic and neuronal activity, which may lead to the physiological consequences seen in OSA and CIH.NEW & NOTEWORTHY Removal of excitatory amino acid transporter (EAAT) restraint increases spontaneous synaptic activity yet decreases afferent [tractus solitarius (TS)]-driven excitatory postsynaptic current (EPSC) amplitude. In the chronic intermittent hypoxia model of obstructive sleep apnea, this restraint is lost due to reduction in EAAT expression and function. Thus EAATs are important in controlling elevated glutamatergic signaling, and loss of such control results in maladaptive synaptic signaling.


Subject(s)
Astrocytes/physiology , Chemoreceptor Cells/physiology , Excitatory Postsynaptic Potentials/physiology , Glutamate Plasma Membrane Transport Proteins/metabolism , Glutamic Acid/metabolism , Hypoxia , Signal Transduction/physiology , Sleep Apnea, Obstructive , Solitary Nucleus , Animals , Disease Models, Animal , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , Hypoxia/metabolism , Hypoxia/physiopathology , Male , Rats , Rats, Sprague-Dawley , Sleep Apnea, Obstructive/metabolism , Sleep Apnea, Obstructive/physiopathology , Solitary Nucleus/metabolism , Solitary Nucleus/physiopathology
3.
FEBS Open Bio ; 9(12): 2016-2024, 2019 12.
Article in English | MEDLINE | ID: mdl-31561285

ABSTRACT

The effect of sodium-glucose cotransporter 2 inhibitor (SGLT2I) on nonalcoholic steatohepatitis (NASH) has been reported, but there are few studies on its effect on NASH-related renal injury. In this study, we examined the effect of SGLT2I using a novel medaka fish model of NASH-related kidney disease, which was developed by feeding the d-rR/Tokyo strain a high-fat diet. SGLT2I was administered by dissolving it in water of the feeding tank. SGLT2I ameliorates macrophage accumulation and oxidative stress and maintained mitochondrial function in the kidney. The results demonstrate the effect of SGLT2I on NASH-related renal injury and the usefulness of this novel animal model for research into NASH-related complications.


Subject(s)
Benzhydryl Compounds/pharmacology , Glucosides/pharmacology , Glutamate Plasma Membrane Transport Proteins/metabolism , Kidney Diseases/metabolism , Animals , Diabetes Mellitus, Type 2 , Diet, High-Fat , Disease Models, Animal , Glucose/metabolism , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , Glutamate Plasma Membrane Transport Proteins/physiology , Hypoglycemic Agents , Kidney/pathology , Kidney Diseases/physiopathology , Non-alcoholic Fatty Liver Disease/complications , Non-alcoholic Fatty Liver Disease/metabolism , Oryzias/metabolism , Sodium/metabolism , Sodium-Glucose Transporter 2 Inhibitors/pharmacology
4.
J Med Chem ; 61(17): 7741-7753, 2018 09 13.
Article in English | MEDLINE | ID: mdl-30011368

ABSTRACT

Aspartate (Asp) derivatives are privileged compounds for investigating the roles governed by excitatory amino acid transporters (EAATs) in glutamatergic neurotransmission. Here, we report the synthesis of various Asp derivatives with (cyclo)alkyloxy and (hetero)aryloxy substituents at C-3. Their pharmacological properties were characterized at the EAAT1-4 subtypes. The l- threo-3-substituted Asp derivatives 13a-e and 13g-k were nonsubstrate inhibitors, exhibiting pan activity at EAAT1-4 with IC50 values ranging from 0.49 to 15 µM. Comparisons between (dl- threo)-19a-c and (dl- erythro)-19a-c Asp analogues confirmed that the threo configuration is crucial for the EAAT1-4 inhibitory activities. Analogues (3b-e) of l-TFB-TBOA (3a) were shown to be potent EAAT1-4 inhibitors, with IC50 values ranging from 5 to 530 nM. Hybridization of the nonselective EAAT inhibitor l-TBOA with EAAT2-selective inhibitor WAY-213613 or EAAT3-preferring inhibitor NBI-59159 yielded compounds 8 and 9, respectively, which were nonselective EAAT inhibitors displaying considerably lower IC50 values at EAAT1-4 (11-140 nM) than those displayed by the respective parent molecules.


Subject(s)
Ammonia-Lyases/metabolism , Aspartic Acid/analogs & derivatives , Aspartic Acid/pharmacology , Excitatory Amino Acid Transporter 1/antagonists & inhibitors , Excitatory Amino Acid Transporter 3/antagonists & inhibitors , Excitatory Amino Acid Transporter 4/antagonists & inhibitors , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , Aspartic Acid/chemical synthesis , Excitatory Amino Acid Transporter 2 , HEK293 Cells , Humans , Molecular Structure , Structure-Activity Relationship
5.
ACS Chem Biol ; 13(6): 1480-1486, 2018 06 15.
Article in English | MEDLINE | ID: mdl-29851333

ABSTRACT

Solute carriers (SLCs) are transmembrane proteins that transport various nutrients, metabolites, and drugs across cellular membranes. Despite the relevance of SLCs to cell homeostasis, metabolism, and disease states, for the majority of SLCs we lack experimental evidence regarding the nature of the cognate ligands, whether endobiotic or xenobiotic. Moreover, even for the roughly 20 SLCs for which inhibitors have been characterized, engagement assays in cells are limited to the accessibility of radiolabeled or fluorescent probes. The cellular thermal shift assay (CETSA) has been introduced as a powerful method to assess target engagement by monitoring ligand-induced changes in the thermal stability of cellular proteins. We addressed the question of whether CETSA could be modified to become routinely applicable to membrane transporters such as SLCs. We used SLC16A1 (MCT1) and SLC1A2 (EAAT2) as targets to establish robust conditions by which chemical engagement of SLCs can be detected. Using immunoblotting, we demonstrate that treatment with the SLC16A1 inhibitors AZD3965 and AR-C155858 stabilized endogenous SLC16A1 in HEK293 cell lysates as well as intact cells. In addition, the high-affinity ligand of SLC16A1, l-lactate, and the low-affinity ligand, formate, resulted in strong and weak stabilization of SLC16A1, respectively. Moreover, we observed stabilization of SLC1A2 upon treatment with the selective inhibitor WAY-213613. We propose that the experimental approach presented here should be generally and easily applicable for monitoring the engagement of chemical ligands by SLCs in cellular settings and thus assisting in their deorphanization.


Subject(s)
Biological Assay/methods , Glutamate Plasma Membrane Transport Proteins/metabolism , Monocarboxylic Acid Transporters/metabolism , Excitatory Amino Acid Transporter 2 , Formates/metabolism , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , HEK293 Cells , Heating , Humans , Lactic Acid/metabolism , Ligands , Monocarboxylic Acid Transporters/antagonists & inhibitors , Protein Binding , Protein Stability , Pyrimidinones/metabolism , Thiophenes/metabolism , Uracil/analogs & derivatives , Uracil/metabolism
6.
Endocr J ; 65(3): 335-344, 2018 Mar 28.
Article in English | MEDLINE | ID: mdl-29375082

ABSTRACT

This study evaluates the efficacy and safety of sodium-glucose cotransporter 2 (SGLT2) inhibitors as add-on to metformin and sulfonylurea treatment for type 2 diabetes management. The literature search was conducted in electronic databases and meta-analyses of mean differences in the changes from baseline in selected disease endpoints (efficacy endpoints) or odds ratios (for safety endpoints) were performed to compare outcomes between SGLT2 inhibitor- and placebo-/comparator-treatments. Seven studies (5,143 patients; age 56.75 years [95% CI: 56.19, 57.37]; body mass index 29.53 kg/m2 [28.23, 30.83]; and 51.87% [50.46, 53.57] males) were included. Compared to placebo, SGLT2 inhibitors significantly (p < 0.00001) reduced glycated hemoglobin (HbA1c; -0.79% [95% CI: -0.90, -0.68]), fasting plasma glucose (FPG; -1.73 mmol/L [-1.86, -1.60]) and body weight (-1.85 kg [-2.11, -1.59]) after 52-78 weeks of treatment. There were no significant differences in reduction of either HbA1c, FPG or body weight between 18-24 weeks and after 52-76 weeks of treatment. Treatment with SGLT2 inhibitors as add-on to metformin and sulfonylurea was also associated with significant reductions in blood pressure and triglycerides and increase in high-density lipoprotein-cholesterol. Incidence of hypoglycemia was significantly higher, but incidence of hyperglycemia was significantly lower in SGLT2 inhibitor group. Overall, drug-related adverse events were more common in SGLT2 group mainly due to higher incidence of genital tract infections.


Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , Hypoglycemic Agents/therapeutic use , Metformin/therapeutic use , Sulfonylurea Compounds/therapeutic use , Drug Therapy, Combination , Humans , Hypoglycemic Agents/adverse effects , Metformin/adverse effects , Sulfonylurea Compounds/adverse effects , Treatment Outcome
7.
CNS Neurosci Ther ; 23(7): 580-589, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28544775

ABSTRACT

AIMS: We previously demonstrated that intrathecal IL-1ß upregulated phosphorylation of p38 mitogen-activated protein kinase (P-p38 MAPK) and inducible nitric oxide synthase (iNOS) in microglia and astrocytes in spinal cord, increased nitric oxide (NO) release into cerebrospinal fluid, and induced thermal hyperalgesia in rats. This study investigated the role of spinal glutamatergic response in intrathecal IL-1ß-induced nociception in rats. METHODS: The pretreatment effects of MK-801 (5 µg), minocycline (20 µg), and SB203580 (5 µg) on intrathecal IL-1ß (100 ng) in rats were measured by behavior, Western blotting, CSF analysis, and immunofluorescence studies. RESULTS: IL-1ß increased phosphorylation of NR-1 (p-NR1) subunit of N-methyl-D-aspartate receptors in neurons and microglia, reduced glutamate transporters (GTs; glutamate/aspartate transporter by 60.9%, glutamate transporter-1 by 55.0%, excitatory amino acid carrier-1 by 39.8%; P<.05 for all), and increased glutamate (29%-133% increase from 1.5 to 12 hours; P<.05) and NO (44%-101% increase from 4 to 12 hours; P<.05) levels in cerebrospinal fluid. MK-801 significantly inhibited all the IL-1ß-induced responses; however, minocycline and SB203580 blocked the IL-1ß-downregulated GTs and elevated glutamate but not the upregulated p-NR1. CONCLUSION: The enhanced glutamatergic response and neuron-glia interaction potentiate the intrathecal IL-1ß-activated P-p38/iNOS/NO signaling and thermal hyperalgesia.


Subject(s)
Glutamic Acid/metabolism , Hyperalgesia/metabolism , Interleukin-1beta/metabolism , Microglia/metabolism , Neurons/metabolism , Spinal Cord/metabolism , Animals , Disease Models, Animal , Dizocilpine Maleate/pharmacology , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , Glutamate Plasma Membrane Transport Proteins/metabolism , Hot Temperature , Hyperalgesia/pathology , Imidazoles/pharmacology , Interleukin-1beta/administration & dosage , Male , Microglia/drug effects , Microglia/pathology , Minocycline/pharmacology , Neurons/drug effects , Neurons/pathology , Nitric Oxide/metabolism , Nociceptive Pain/metabolism , Nociceptive Pain/pathology , Phosphorylation/drug effects , Pyridines/pharmacology , Random Allocation , Rats, Wistar , Receptors, N-Methyl-D-Aspartate/metabolism , Spinal Cord/drug effects , Spinal Cord/pathology
8.
ACS Chem Neurosci ; 8(8): 1668-1672, 2017 08 16.
Article in English | MEDLINE | ID: mdl-28414419

ABSTRACT

Excitatory amino acid transporters clear glutamate from the synaptic cleft and play a critical role in glutamatergic neurotransmission. Their differential roles in astrocytes, microglia, and neurons are poorly understood due in part to a lack of pharmacological tools that can be targeted to specific cells and tissues. We now describe a photoswitchable inhibitor, termed ATT, that interacts with the major mammalian forebrain transporters EAAT1-3 in a manner that can be reversibly switched between trans (high-affinity) and cis (low-affinity) configurations using light of different colors. In the dark, ATT competitively inhibited the predominant glial transporter EAAT2 with ∼200-fold selectivity over the neuronal transporter EAAT3. Brief exposure to 350 nm light reduced the steady-state blocker affinity by more than an order of magnitude. Illumination of EAAT2 complexed with ATT induced a corresponding increase in the blocker off-rate monitored in the presence of glutamate. ATT can be used to reversibly manipulate glutamate transporter activity with light and may be useful to gain insights into the dynamic physiological roles of glutamate transporters in the brain, as well as to study the molecular interactions of transporters with ligands.


Subject(s)
Aspartic Acid/analogs & derivatives , Excitatory Amino Acid Transporter 1/antagonists & inhibitors , Excitatory Amino Acid Transporter 3/antagonists & inhibitors , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , Membrane Transport Modulators/pharmacology , Animals , Aspartic Acid/chemical synthesis , Aspartic Acid/chemistry , Aspartic Acid/pharmacology , Dose-Response Relationship, Drug , Excitatory Amino Acid Transporter 1/metabolism , Excitatory Amino Acid Transporter 2 , Excitatory Amino Acid Transporter 3/metabolism , Glutamate Plasma Membrane Transport Proteins/metabolism , Humans , Isomerism , Light , Membrane Potentials/drug effects , Membrane Transport Modulators/chemical synthesis , Membrane Transport Modulators/chemistry , Molecular Structure , Oocytes , Patch-Clamp Techniques , Photochemical Processes , Xenopus laevis
9.
Oncotarget ; 8(24): 39922-39934, 2017 Jun 13.
Article in English | MEDLINE | ID: mdl-28404980

ABSTRACT

During HIV-associated neurocognitive disorder (HAND), decreasing in excitatory amino acid transporter 2 (EAAT-2) in astrocyte plasma membranes leads to elevated levels of extracellular glutamate and, in turn, neuronal apoptosis. We used immunohistochemistry, western blot, qRT-PCR, and RNA interference to elucidate the molecular mechanisms underlying the decreased EAAT-2 expression during HAND at the tissue and cellular levels. We used simian immunodeficiency virus-human immunodeficiency virus chimeric virus (SHIV)-infected macaques as an in vivo model of HAND. Our results show that EAAT-2 expression was decreased in the cerebral cortex, while AEG-1 expression was increased, and the expression levels of these proteins were negatively correlated. In vitro analyses showed that HIV-1 Tat inhibited EAAT-2 expression by inducing overexpression of AEG-1. More specifically, HIV-1 Tat increased AEG-1 expression via the PI3-K signaling pathway, while increasing EAAT-2 inhibition by YinYan-1 (YY-1) via the NF-κB signaling pathway. These results warrant testing AEG-1 as a potential therapeutic target for treating HAND.


Subject(s)
Cell Adhesion Molecules/metabolism , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , HIV Infections/physiopathology , HIV-1/pathogenicity , Neurocognitive Disorders/pathology , tat Gene Products, Human Immunodeficiency Virus/pharmacology , Excitatory Amino Acid Transporter 2 , HIV Infections/virology , Humans , Membrane Proteins , Neurocognitive Disorders/epidemiology , Neurocognitive Disorders/metabolism , Neurocognitive Disorders/virology , RNA-Binding Proteins , Up-Regulation
10.
J Med Chem ; 59(19): 8771-8786, 2016 10 13.
Article in English | MEDLINE | ID: mdl-27636002

ABSTRACT

In this study inspired by previous work on 3-substituted Asp analogues, we designed and synthesized a total of 32 ß-sulfonamide Asp analogues and characterized their pharmacological properties at the excitatory amino acid transporter subtypes EAAT1, EAAT2, and EAAT3. In addition to several potent EAAT inhibitors displaying IC50 values ∼1 µM at all three subtypes, this elaborate structure-activity relationship also identified analogues exhibiting distinct preferences or selectivities for specific transporter subtypes. Introduction of two fluorine atoms on the phenyl ring yielded analogue 4y that displayed an IC50 of 0.8 µM at EAAT1 with a 14- and 9-fold preference over EAAT2 and EAAT3, respectively. Conversely, the m-CF3-phenyl analogue 4r was a potent selective EAAT2-inhibitor (IC50 = 2.8 µM) exhibiting 30- and 50-fold selectivity over EAAT1 and EAAT3, respectively. In conclusion, even small structural differences in these ß-sulfonamide Asp analogues provide analogues with diverse EAAT subtype selectivity profiles.


Subject(s)
Aspartic Acid/analogs & derivatives , Aspartic Acid/pharmacology , Excitatory Amino Acid Transporter 1/antagonists & inhibitors , Excitatory Amino Acid Transporter 3/antagonists & inhibitors , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , Sulfonamides/chemistry , Sulfonamides/pharmacology , Biological Transport/drug effects , Excitatory Amino Acid Transporter 1/metabolism , Excitatory Amino Acid Transporter 2 , Excitatory Amino Acid Transporter 3/metabolism , Glutamate Plasma Membrane Transport Proteins/metabolism , HEK293 Cells , Humans
11.
ACS Chem Neurosci ; 7(5): 534-9, 2016 05 18.
Article in English | MEDLINE | ID: mdl-26918289

ABSTRACT

Glutamate is the major excitatory neurotransmitter in the mammalian brain. Its rapid clearance after the release into the synaptic cleft is vital in order to avoid toxic effects and is ensured by several transmembrane transport proteins, so-called excitatory amino acid transporters (EAATs). Impairment of glutamate removal has been linked to several neurodegenerative diseases and EAATs have therefore received increased attention as therapeutic targets. O-Benzylated l-threo-ß-hydroxyaspartate derivatives have been developed previously as highly potent inhibitors of EAATs with TFB-TBOA ((2S,3S)-2-amino-3-((3-(4-(trifluoromethyl)benzamido)benzyl)oxy)succinic acid) standing out as low-nanomolar inhibitor. We report the stereoselective synthesis of all four stereoisomers of TFB-TBOA in less than a fifth of synthetic steps than the published route. For the first time, the inhibitory activity and isoform selectivity of these TFB-TBOA enantio- and diastereomers were assessed on human glutamate transporters EAAT1-3. Furthermore, we synthesized potent photoaffinity probes based on TFB-TBOA using our novel synthetic strategy.


Subject(s)
Aspartic Acid/analogs & derivatives , Excitatory Amino Acid Transporter 1/antagonists & inhibitors , Excitatory Amino Acid Transporter 3/antagonists & inhibitors , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , Photoaffinity Labels/chemical synthesis , Aspartic Acid/chemical synthesis , Aspartic Acid/pharmacology , Chemistry, Pharmaceutical , Dose-Response Relationship, Drug , Excitatory Amino Acid Transporter 1/metabolism , Excitatory Amino Acid Transporter 2 , Excitatory Amino Acid Transporter 3/metabolism , Glutamate Plasma Membrane Transport Proteins/metabolism , HEK293 Cells , Humans , Photoaffinity Labels/pharmacology , Stereoisomerism
12.
J Neurophysiol ; 115(3): 1691-702, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26719090

ABSTRACT

The nucleus tractus solitarii (nTS) is the initial central termination site for visceral afferents and is important for modulation and integration of multiple reflexes including cardiorespiratory reflexes. Glutamate is the primary excitatory neurotransmitter in the nTS and is removed from the extracellular milieu by excitatory amino acid transporters (EAATs). The goal of this study was to elucidate the role of EAATs in the nTS on basal synaptic and neuronal function and cardiorespiratory regulation. The majority of glutamate clearance in the central nervous system is believed to be mediated by astrocytic EAAT 1 and 2. We confirmed the presence of EAAT 1 and 2 within the nTS and their colocalization with astrocytic markers. EAAT blockade withdl-threo-ß-benzyloxyaspartic acid (TBOA) produced a concentration-related depolarization, increased spontaneous excitatory postsynaptic current (EPSC) frequency, and enhanced action potential discharge in nTS neurons. Solitary tract-evoked EPSCs were significantly reduced by EAAT blockade. Microinjection of TBOA into the nTS of anesthetized rats induced apneic, sympathoinhibitory, depressor, and bradycardic responses. These effects mimicked the response to microinjection of exogenous glutamate, and glutamate responses were enhanced by EAAT blockade. Together these data indicate that EAATs tonically restrain nTS excitability to modulate cardiorespiratory function.


Subject(s)
Glutamate Plasma Membrane Transport Proteins/metabolism , Glutamic Acid/metabolism , Heart Rate , Respiration , Solitary Nucleus/physiology , Synapses/physiology , Action Potentials , Animals , Excitatory Amino Acid Antagonists/pharmacology , Excitatory Postsynaptic Potentials , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , Male , Neurons/metabolism , Neurons/physiology , Rats , Rats, Sprague-Dawley , Solitary Nucleus/metabolism , Synapses/drug effects , Synapses/metabolism
13.
Curr Eye Res ; 41(3): 357-66, 2016.
Article in English | MEDLINE | ID: mdl-25897760

ABSTRACT

PURPOSE: Maintaining the high glutathione (GSH; tripeptide of glutamate, cysteine and glycine) levels in the lens cortex promotes lens health. The role of glutamate/aspartate (Glu/Asp) transporters and the cystine (Cys)/Glu exchanger (Xc(-) exchanger) in maintaining GSH in transformed human lens epithelial cells (SRA 01/04) was investigated. METHODS: Detection and differentiation of excitatory amino acid transporters (EAAT1-5) and the Xc(-) exchanger was performed by the uptake of radiolabeled l-Glu, d-Asp and l-Cys in the presence and absence of Na(+), substrate-specific inhibition studies and Western-blot analysis. Reductions in GSH levels post-inhibition of Xc(-) exchanger and EAAT activities by substrate inhibitors demonstrated the roles of EAAT and Xc(-) exchanger in maintaining GSH. RESULTS: Glu and d-Asp uptake in HLEC was Na(+)-dependent. Strong inhibition by substrate-specific Glu/Asp uptake inhibitors and weak inhibition by kainic acid (KA) was consistent with Na(+)-dependent EAAT1/3/4/5 activity and weak EAAT2 activity, respectively. Na(+)-independency and Glu inhibition of Cys uptake were consistent with Xc(-) exchanger activity, but inhibition of Na(+)-dependent Cys uptake by N-acetylcysteine suggests Cys uptake by EAAT3. EAAT1-5 and xCT (Xc(-) exchanger light chain) immunoreactive peptides were detected by Western-blot analysis of HLEC lysates. EAAT and Xc(-) exchanger inhibition by substrate antagonists depleted GSH concentrations by 15-28% (p's ≤ 0.02), while GSH synthesis inhibition by buthionine sulfoximine depleted GSH by 33% (p = 0.008). CONCLUSION: Inhibition of Glu and Cys uptake by EAAT and Xc(-) exchanger antagonists depletes GSH in human lens epithelial cells. These in vitro results support pivotal roles for EAAT and Xc(-) exchanger activities in maintaining GSH and protection against oxidative stress in cortical lens epithelium.


Subject(s)
Amino Acid Transport System y+/antagonists & inhibitors , Epithelial Cells/metabolism , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , Glutathione/metabolism , Lens, Crystalline/cytology , Aspartic Acid/analogs & derivatives , Aspartic Acid/pharmacology , Blotting, Western , Cell Line, Transformed , Dose-Response Relationship, Drug , Epithelial Cells/drug effects , Excitatory Amino Acid Antagonists/pharmacology , Humans , Kainic Acid/pharmacology
14.
Pharmacol Biochem Behav ; 127: 70-81, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25312503

ABSTRACT

L-Glutamate is the predominant excitatory neurotransmitter in the central nervous system (CNS) and is directly and indirectly involved in a variety of brain functions. Glutamate is released in the synaptic cleft at a particular concentration that further activates the various glutaminergic receptors. This concentration of glutamate in the synapse is maintained by either glutamine synthetase or excitatory amino acid proteins which reuptake the excessive glutamate from the synapse and named as excitatory amino acid transporters (EAATs). Out of all the subtypes GLT-1 (glutamate transporter 1) is abundantly distributed in the CNS. Down-regulation of GLT-1 is reported in various neurological diseases such as, epilepsy, stroke, Alzheimer's disease and movement disorders. Therefore, positive modulators of GLT-1 which up-regulate the GLT-1 expression can serve as a potential target for the treatment of neurological disorders. GLT-1 translational activators such as ceftriaxone are found to have significant protective effects in ALS and epilepsy animal models, suggesting that this translational activation approach works well in rodents and that these compounds are worth further pursuit for various neurological disorders. This drug is currently in human clinical trials for ALS. In addition, a thorough understanding of the mechanisms underlying translational regulation of GLT-1, such as identifying the molecular targets of the compounds, signaling pathways involved in the regulation, and translational activation processes, is very important for this novel drug-development effort. This review mainly emphasizes the role of glutamate and its transporter, GLT-1 subtype in excitotoxicity. Further, recent reports on GLT-1 transporters for the treatment of various neurological diseases, including a summary of the presumed physiologic mechanisms behind the pharmacology of these disorders are also explained.


Subject(s)
Drug Delivery Systems/methods , Glutamate Plasma Membrane Transport Proteins/metabolism , Nervous System Diseases/drug therapy , Nervous System Diseases/metabolism , Animals , Excitatory Amino Acid Agonists/administration & dosage , Excitatory Amino Acid Antagonists/administration & dosage , Excitatory Amino Acid Transporter 2 , Glutamate Plasma Membrane Transport Proteins/agonists , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , Glutamic Acid/metabolism , Humans , Signal Transduction/drug effects , Signal Transduction/physiology
15.
J Clin Invest ; 124(8): 3645-55, 2014 Aug.
Article in English | MEDLINE | ID: mdl-25036707

ABSTRACT

During brain ischemia, an excessive release of glutamate triggers neuronal death through the overactivation of NMDA receptors (NMDARs); however, the underlying pathways that alter glutamate homeostasis and whether synaptic or extrasynaptic sites are responsible for excess glutamate remain controversial. Here, we monitored ischemia-gated currents in pyramidal cortical neurons in brain slices from rodents in response to oxygen and glucose deprivation (OGD) as a real-time glutamate sensor to identify the source of glutamate release and determined the extent of neuronal damage. Blockade of excitatory amino acid transporters or vesicular glutamate release did not inhibit ischemia-gated currents or neuronal damage after OGD. In contrast, pharmacological inhibition of the cystine/glutamate antiporter dramatically attenuated ischemia-gated currents and cell death after OGD. Compared with control animals, mice lacking a functional cystine/glutamate antiporter exhibited reduced anoxic depolarization and neuronal death in response to OGD. Furthermore, glutamate released by the cystine/glutamate antiporter activated extrasynaptic, but not synaptic, NMDARs, and blockade of extrasynaptic NMDARs reduced ischemia-gated currents and cell damage after OGD. Finally, PET imaging showed increased cystine/glutamate antiporter function in ischemic rats. Altogether, these data suggest that cystine/glutamate antiporter function is increased in ischemia, contributing to elevated extracellular glutamate concentration, overactivation of extrasynaptic NMDARs, and ischemic neuronal death.


Subject(s)
Amino Acid Transport System y+/physiology , Brain Ischemia/etiology , Glutamic Acid/metabolism , Amino Acid Transport System y+/deficiency , Amino Acid Transport System y+/genetics , Animals , Benzoates/pharmacology , Brain Ischemia/pathology , Brain Ischemia/physiopathology , Cell Death , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , Glutamate Plasma Membrane Transport Proteins/metabolism , Glycine/analogs & derivatives , Glycine/pharmacology , Ion Channel Gating/physiology , Mice , Mice, Inbred C3H , Mice, Knockout , Pyramidal Cells/drug effects , Pyramidal Cells/pathology , Pyramidal Cells/physiology , Rats , Rats, Sprague-Dawley , Receptors, N-Methyl-D-Aspartate/metabolism , Vesicular Glutamate Transport Proteins/antagonists & inhibitors , Vesicular Glutamate Transport Proteins/physiology
16.
Neurochem Res ; 39(10): 1964-79, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24682739

ABSTRACT

Uptake of the major excitatory neurotransmitter in the CNS, (S)-glutamate, is mediated by a family of excitatory amino acid transporters (EAAT). Previously we have explored the structure-activity relationship (SAR) of a series of EAAT1 selective inhibitors, leading to the development of the potent inhibitors UCPH-101 and UCPH-102. In the present study, we set out to improve the solubility properties of these EAAT1 inhibitors with the objective to develop analogs more suited as pharmacological tools for in vivo studies of EAAT1 in terms of their bioavailability. A total of 23 novel UCPH-101/102 analogs were designed, synthesized and characterized pharmacologically at EAAT1-3 in a [(3)H]-D-aspartate uptake assay. Most notably, the potent EAAT1 inhibition displayed of UCPH-101 and UCPH-102 was retained in analog 1d in which the napht-1-yl group in the 7-position of UCPH-102 has been replaced by an o-biphenyl moiety. In contrast, EAAT1 activity was dramatically compromised in analogs 1e and 1f comprising m- and p-biphenyl groups as 7-substituents, respectively. Analog 1d displayed low bioavailability after oral administration in rats, and this problem was addressed by the synthesis of a series of analogs with different chloro, fluoro, methoxy, triflouromethyl and carboxy substitution patterns at the o-biphenyl group of 1d (1h-1s) and m- and p-pyridine analogs of 1d (1t and 1v). Unfortunately, all of the modifications resulted in substantial decreased EAAT1 inhibitory activity, which supports the notion of a very lipophilic binding pocket in EAAT1 for the aromatic 7-substituent in these ligands. In conclusion, while we have not succeeded in developing UCPH-101/102 analogs possessing improved bioavailability properties, this study does offer interesting SAR information about this inhibitor class, and analog 1d seems to be an interesting lead for future SAR studies with focus on the development of more potent EAAT1 inhibitors.


Subject(s)
Benzopyrans/pharmacology , Biphenyl Compounds/pharmacology , Coumarins/pharmacology , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , Benzopyrans/chemistry , Benzopyrans/pharmacokinetics , Biological Availability , Chromatography, High Pressure Liquid , Chromatography, Liquid , Coumarins/chemistry , Coumarins/pharmacokinetics , Magnetic Resonance Spectroscopy , Mass Spectrometry
17.
Mol Aspects Med ; 34(2-3): 108-20, 2013.
Article in English | MEDLINE | ID: mdl-23506861

ABSTRACT

Glutamate transporters play important roles in the termination of excitatory neurotransmission and in providing cells throughout the body with glutamate for metabolic purposes. The high-affinity glutamate transporters EAAC1 (SLC1A1), GLT1 (SLC1A2), GLAST (SLC1A3), EAAT4 (SLC1A6), and EAAT5 (SLC1A7) mediate the cellular uptake of glutamate by the co-transport of three sodium ions (Na(+)) and one proton (H(+)), with the counter-transport of one potassium ion (K(+)). Thereby, they protect the CNS from glutamate-induced neurotoxicity. Loss of function of glutamate transporters has been implicated in the pathogenesis of several diseases, including amyotrophic lateral sclerosis and Alzheimer's disease. In addition, glutamate transporters play a role in glutamate excitotoxicity following an ischemic stroke, due to reversed glutamate transport. Besides glutamate transporters, the SLC1 family encompasses two transporters of neutral amino acids, ASCT1 (SLC1A4) and ASCT2 (SLC1A5). Both transporters facilitate electroneutral exchange of amino acids in neurons and/or cells of the peripheral tissues. Some years ago, a high resolution structure of an archaeal homologue of the SLC1 family was determined, followed by the elucidation of its structure in the presence of the substrate aspartate and the inhibitor d,l-threo-benzyloxy aspartate (d,l-TBOA). Historically, the first few known inhibitors of SLC1 transporters were based on constrained glutamate analogs which were active in the high micromolar range but often also showed off-target activity at glutamate receptors. Further development led to the discovery of l-threo-ß-hydroxyaspartate derivatives, some of which effectively inhibited SLC1 transporters at nanomolar concentrations. More recently, small molecule inhibitors have been identified whose structures are not based on amino acids. Activators of SLC1 family members have also been discovered but there are only a few examples known.


Subject(s)
Amino Acids, Neutral/metabolism , Glutamate Plasma Membrane Transport Proteins/chemistry , Glutamate Plasma Membrane Transport Proteins/physiology , Models, Molecular , Multigene Family/genetics , Protein Conformation , Synapses/metabolism , Aspartic Acid/analogs & derivatives , Aspartic Acid/pharmacology , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , Glutamate Plasma Membrane Transport Proteins/metabolism , Humans , Models, Biological , Molecular Structure , Phylogeny
18.
ACS Chem Neurosci ; 3(2): 105-13, 2012 Feb 15.
Article in English | MEDLINE | ID: mdl-22860180

ABSTRACT

We recently found that tamoxifen suppresses l-glutamate transport activity of cultured astrocytes. Here, in an attempt to separate the l-glutamate transporter-inhibitory activity from the estrogen receptor-mediated genomic effects, we synthesized several compounds structurally related to tamoxifen. Among them, we identified two compounds, 1 (YAK01) and 3 (YAK037), which potently inhibited l-glutamate transporter activity. The inhibitory effect of 1 was found to be mediated through estrogen receptors and the mitogen-activated protein kinase (MAPK)/phosphatidylinositol 3-kinase (PI3K) pathway, though 1 showed greatly reduced transactivation activity compared with that of 17ß-estradiol. On the other hand, compound 3 exerted its inhibitory effect through an estrogen receptor-independent and MAPK-independent, but PI3K-dependent pathway, and showed no transactivation activity. Compound 3 may represent a new platform for developing novel l-glutamate transporter inhibitors with higher brain transfer rates and reduced adverse effects.


Subject(s)
Antineoplastic Agents, Hormonal/pharmacology , Glutamic Acid/metabolism , Neuroglia/metabolism , Receptors, Estrogen/drug effects , Tamoxifen/analogs & derivatives , Tamoxifen/pharmacology , Animals , Antineoplastic Agents, Hormonal/chemical synthesis , Biological Transport, Active/drug effects , Brain/drug effects , Brain/metabolism , Cells, Cultured , Drug Discovery , Excitatory Amino Acid Transporter 1/antagonists & inhibitors , Excitatory Amino Acid Transporter 2 , Extracellular Space/metabolism , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , HEK293 Cells , Humans , Magnetic Resonance Spectroscopy , Mitogen-Activated Protein Kinases/metabolism , Neuroglia/drug effects , Phosphatidylinositol 3-Kinases/metabolism , Rats , Rats, Wistar , Receptors, Estrogen/agonists , Tamoxifen/chemical synthesis
19.
J Neurosci ; 32(13): 4360-71, 2012 Mar 28.
Article in English | MEDLINE | ID: mdl-22457487

ABSTRACT

Excitatory amino acid transporters (EAATs) terminate signaling in the CNS by clearing released glutamate. Glutamate also evokes an EAAT-mediated Cl(-) current, but its role in CNS signaling is poorly understood. We show in mouse retina that EAAT-mediated Cl(-) currents that were evoked by light inhibit rod pathway signaling. EAATs reside on rod bipolar cell axon terminals where GABA and glycine receptors also mediate light-evoked inhibition. We found that the mode of inhibition depended on light intensity. Dim light evoked GABAergic and glycinergic inhibition with rapid kinetics and a large spatial extent. Bright light evoked predominantly EAAT-mediated inhibition with slow kinetics and a small spatial extent. The switch to EAAT-mediated signaling in bright light supplements receptor-mediated signaling to expand the dynamic range of inhibition and contributes to the transition from rod to cone signaling by suppressing rod pathway signaling in bright light conditions.


Subject(s)
Glutamate Plasma Membrane Transport Proteins/physiology , Light , Membrane Potentials/physiology , Neural Inhibition/physiology , Retinal Bipolar Cells/physiology , Retinal Rod Photoreceptor Cells/physiology , Synaptic Transmission/physiology , Animals , Exocytosis/physiology , Female , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , In Vitro Techniques , Male , Membrane Potentials/drug effects , Mice , Mice, Inbred C57BL , Neural Inhibition/drug effects , Presynaptic Terminals/drug effects , Presynaptic Terminals/physiology , Retinal Bipolar Cells/drug effects , Retinal Rod Photoreceptor Cells/drug effects , Synaptic Transmission/drug effects
20.
Proc Natl Acad Sci U S A ; 108(36): 14980-5, 2011 Sep 06.
Article in English | MEDLINE | ID: mdl-21873219

ABSTRACT

A prominent aqueous cavity is formed by the junction of three identical subunits in the excitatory amino acid transporter (EAAT) family. To investigate the effect of this structure on the interaction of ligands with the transporter, we recorded currents in voltage-clamped Xenopus oocytes expressing EAATs and used concentration jumps to measure binding and unbinding rates of a high-affinity aspartate analog that competitively blocks transport (ß-2-fluorenyl-aspartylamide; 2-FAA). The binding rates of the blocker were approximately one order of magnitude slower than l-Glu and were not significantly different for EAAT1, EAAT2, or EAAT3, but 2-FAA exhibited higher affinity for the neuronal transporter EAAT3 as a result of a slower dissociation rate. Unexpectedly, the rate of recovery from block was increased by l-Glu in a saturable and concentration-dependent manner, ruling out a first-order mechanism and suggesting that following unbinding, there is a significant probability of ligand rebinding to the same or neighboring subunits within a trimer. Consistent with such a mechanism, coexpression of wild-type subunits with mutant (R447C) subunits that do not bind glutamate or 2-FAA also increased the unblocking rate. The data suggest that electrostatic and steric factors result in an effective dissociation rate that is approximately sevenfold slower than the microscopic subunit unbinding rate. The quaternary structure, which has been conserved through evolution, is expected to increase the transporters' capture efficiency by increasing the probability that following unbinding, a ligand will rebind as opposed to being lost to diffusion.


Subject(s)
Aspartic Acid/chemistry , Glutamate Plasma Membrane Transport Proteins/chemistry , Animals , Aspartic Acid/analogs & derivatives , Aspartic Acid/metabolism , Binding Sites/physiology , Biological Transport/physiology , Glutamate Plasma Membrane Transport Proteins/antagonists & inhibitors , Glutamate Plasma Membrane Transport Proteins/metabolism , Humans , Ligands , Xenopus laevis
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