Extracellular Concentrations Of Excitatory Amino Acids Research Articles

Glutamate transporters: the regulatory proteins for excitatory/excitotoxic glutamate in brain

Excitatory aminoacids (EAAs) are stored in glutamatergic neurons and related into synaptic cleft, where they can activate inotropic or metabotropic receptors. Their action ends due to transport mechanisms performed by EAAT transporters (EAAT1/GLAST, EAAT2/GLT1, EAAT3/EAAC1, and EAAT4 or EAAT5). Glutamate neurotoxicity has been described in several neurodegenerative diseases such as Alzheimer’s disease (AD), Huntington’s disease (HD), Parkinson’s disease (PD) and amyotropic lateral sclerosis ALS). Some drugs, such as paclitaxel, are able to increase translation of microRNA and could be possible used as regulatory against glutamate neurotoxicity. Abbreviations: AD: Alzheimer’s disease; ALS: amyotrophic lateral sclerosis; AMPA: α-amino-3-hydroxy-5-methyl-isoxazole-4propionate; Asp: aspartate; CSF: cerebral spinal fluid; EAA: excitatory amino acid; EAAC1 (EAAT3): excitatory amino acid carrier; EAAT: excitatory amino acid transporter; GABA: gamma-aminobutyric acid; GDH: glutamate dehydrogenase; GLAST (EAAT1): glutamate – aspartate transporter; GLT1 (EAAT2): glutamate transporter; iGluR: ionotropic glutamate receptor; KA: kaninic acid; L-Glu: L-glutamate; mGluR: metabotropic glutamate receptor; miR: micro RNA; NMDA: N-methyl-D-aspartate; PD: Parkinson’s disease Introduction Some amino acids act as neurotransmitters in the nervous system, being glutamate and aspartate the common excitatory amino acids and GABA, glycine and taurine the inhibitory ones. Of these amino acids, glutamate and GABA are intimately associated, as their metabolism is associated through glutamic acid decarboxylase (E.C. 4.1.1.15.) (Figure 1). Furthermore, GABA and glutamic acid effects are antagonic and they are related with CO2 fixation (relevant to central ventilation). Glutamic acid metabolism is also related with NH3 detoxification (due to a reduction in α-ketoglutarate and glutamate contents and an increase in glutamine). The efflux of glutamate from brain across the hemato-encephalic barrier is much higher than the influx [1-3], meaning that metabolism of glutamate must play an important role in regulating the brain glutamate levels. Studies on metabolic generation of glutamate/glutamine by using radioactive substrates in brain shows that two pathways are involved. Glucose, glycerol, lactate, pyruvate, α-ketoglutarate and β-hydroxybutyrate seem to be metabolized to glutamate in neurons [4,5], as a low specific radioactivity of glutamine is obtained. In glial cells [6], where higher glutamine synthase is present [7,8], low radiolabelled glutamate and higher glutamine marked are obtained. This is the case of acetate, propionate, butyrate, citrate, leucine, GABA, aspartate and ammonia [9]. In order to decrease glutamic acid in synaptic cleft, excitatory amino acids transporters have an important effect. Therefore, in this paper we present some aspects of these proteins. Glutamatergic neurotransmission The excitatory amino acids (EAAs) are stored in synaptic vesicles in glutamatergic neurons and, upon an action potential, are released via exocytosis into the synaptic cleft where they can activate two different families of receptors: ionotropic (ligand-gated ion channels) and metabotropic (GTP-binding protein coupled) receptors. The ligandgated ion channels are further divided into three families: α-amino3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), kainate (KA) and N–methyl-D-aspartate (NMDA). While AMPA and kainate receptors mediate rapid depolarizing responses at most synapses in the mammalian central nervous system [10], the NMDA receptor participates in synaptic plasticity and synapse formation [11]. The family of metabotropic receptors consists of at least eight subtypes and are involved in the modulation of synaptic signaling by EAAs and other neurotransmitters [12]. The termination of the EAA action takes place by an uptake mechanism that uses the Na+, K+ and pH gradients as a driving force to translocate the neurotransmitters against their concentration gradients, keeping their concentration below the level that activates their receptors (~1 μM) [13-15] (Figure 2). Excessive activation of EAA receptors contributes to brain injury through a process known as excitotoxicity. Therefore, this transport mechanism is not only important for ensuring accurate synaptic signaling but also for limiting the EAA-mediated excitotoxicity Excitatory amino acid (EAA) transporters Three broad subtypes of EAA transport activities have been Correspondence to: Josep J Centelles, Departament de Bioquimica i Biologia Molecular (Biologia), Facultat de Biologia, Universitat de Barcelona, Avda Diagonal, 643. Edifici Prevosti, planta-2, 08028, Barcelona, Spain, Tel: 934021870; E-mail: josepcentelles@ub.edu Received: January 16, 2016; Accepted: January 27, 2016; Published: January 30, 2016 Centelles JJ (2016) Glutamate transporters: the regulatory proteins for excitatory/excitotoxic glutamate in brain J Transl Sci, 2016 doi: 10.15761/JTS.1000123 Volume 2(1): 92-99 extracellular concentrations of EAAs by reducing the driving force required to transport an EAA into the cytoplasm. The second activity is a chloride-dependent transport which exchanges amino acids identified in brain preparations. One type, which is directly coupled to ATP hydrolysis, introduces glutamate into vesicles for release upon depolarization of the synaptic terminal [16]. It indirectly ensures low Glutamine ADP + Pi Glutamine synthetase (GS) (E.C. 6.3.1.2.) Glutaminase (Gase) (E.C. 3.5.1.2.) NH4 H2O Glutamate GABA Glutamate decarboxylase (GD) (E.C. 4.1.1.15.) Oxalacetate Glutamate dehydrogenase (GDH) (E.C. 1.4.1.2.) Glutamate oxalacetate transaminase (GOT) (E.C. 2.6.1.1.) NH4 + NADH Aspartate α-Ketoglutarate CO2 NH4 + ATP

Preemptive analgesia with lidocaine prevents Failed Back Surgery Syndrome

Failed Back Surgery Syndrome (FBSS) is commonly encountered in pain-treatment settings in the United States. We tested whether potential key factors in this syndrome, such as extracellular concentrations of excitatory amino acids (EAAs), are increased in the dorsal horn by synaptic release due to unintentional stretch and/or deformation/compression/transection of dorsal spinal structures during surgery. We hypothesized that pharmacological nerve block as a form of preemptive analgesia prior to any insult to dorsal root neurons will prevent an abnormally high increase in extracellular concentrations of EAAs in the dorsal horn and ultimately the establishment of central sensitization during back surgery. The L4 and L5 dorsal roots were cut bilaterally near the spinal cord to provide an adequate model to test for preemptive analgesia. Amino acid concentrations were measured by dorsal horn microdialysis sampling; EAAs aspartate and glutamate were significantly increased by 80% and 65% respectively, as were other amino acids compared to sham control values. Topical application of 1% Lidocaine ®, a voltage-gated Na + channel blocker, for 10 min prior to L4 and L5 bilateral dorsal rhizotomy (BDR) significantly attenuated the increase in EAA concentrations such that their values were not different from sham controls. Behavioral tests demonstrated significant hindlimb mechanical allodynia after BDRs that was significantly attenuated by Lidocaine® pretreatment. Thus, Lidocaine® pretreatment could offer a safe measure for prevention of chronic pain for back surgical procedures if given by intramuscular injection, topical administration onto spinal nerves and/or the dorsal spinal surface during surgical procedures that include nerve entrapment release, intervertebral disc modification and laminectomies.

Group I metabotropic glutamate receptors in spinal cord injury: roles in neuroprotection and the development of chronic central pain.

Spinal cord injury (SCI) initiates a cascade of biochemical events that leads to an increase in extracellular excitatory amino acid (EAA) concentrations, which results in glutamate receptor-mediated excitotoxic events. An important division of these glutamate receptors is the metabotropic glutamate receptor (mGluR) class, which is divided into three groups. Of these three groups, group I (mGluR1 and mGluR5) activation can initiate a number of intracellular pathways that lead to increased extracellular EAA concentrations. To evaluate subtypes of group I mGluRs in SCI, we administered AIDA (group I antagonist), LY 367385 (mGluR1 specific antagonist), or MPEP (mGluR5 specific antagonist) by interspinal injection to adult male Sprague-Dawley rats (175-200 g) immediately following injury at T10 with an NYU impactor (12.5-mm drop, 10-g rod, 2 mm in diameter). AIDA- and LY 367385-treated subjects had improved locomotor scores and demonstrated an attenuation in the development of mechanical allodynia as measured by von Frey stimulation of the forelimbs; however, LY 367385 potentiated the development of thermal hyperalgesia. MPEP had no effect on locomotor recovery or mechanical allodynia, but attenuated the development of thermal hyperalgesia. AIDA and LY 367385 treatment resulted in a significant increase in tissue sparing compared to the vehicle-treated group at 4 weeks following SCI. These results suggest that mGluRs play an important role in EAA toxicity and have different acute pathophysiological roles following spinal cord injury.

Involvement of metabotropic glutamate receptors in excitatory amino acid and GABA release following spinal cord injury in rat.

Spinal cord injury (SCI) leads to an increase in extracellular excitatory amino acid (EAA) concentrations resulting in glutamate receptor-mediated excitotoxic events. The glutamate receptors include ionotropic (iGluRs) and metabotropic (mGluR) receptors. Of the three groups of mGluRs, group-I activation can initiate intracellular pathways that lead to further transmitter release. Groups II and III mGluRs function mainly as autoreceptors to regulate neurotransmitter release. In an effort to examine the role of mGluRs in the increase in EAAs following SCI, we administered AIDA, a potent group-I mGluR antagonist immediately after injury. To determine subtype specific roles of the group-I mGluRs, we evaluated EAA release following LY 367385 (mGluR1 antagonist) and MPEP (mGluR5 antagonist) administration. To evaluate group-II and -III mGluRs we administered APDC (group-II agonist) and L-AP4 (group-III agonist) immediately following injury; additionally, we initiated treatment with CPPG (group-II/-III antagonist) and LY 341495 (group-II antagonist) 5 min prior to injury. Subjects were adult male Sprague-Dawley rats (225-250 g), impact injured at T10 with an NYU impactor (12.5 mm drop). Agents were injected into the epicenter of injury, amino acids where collected by microdialysis fibers inserted 0.5 mm caudal from the edge of the impact region and quantified by HPLC. Treatment with AIDA significantly decreased extracellular EAA and GABA concentrations. MPEP reduced EAA concentrations without affecting GABA. Combining LY 367385 and MPEP resulted in a decrease in EAA and GABA concentrations greater than either agent alone. L-AP4 decreased EAA levels, while treatment with LY 341495 increased EAA levels. These results suggest that mGluRs play an important role in EAA toxicity following SCI.

Changes in Metabotropic Glutamate Receptor Expression Following Spinal Cord Injury

Spinal cord injury (SCI) initiates biochemical events that lead to an increase in extracellular excitatory amino acid concentrations, resulting in glutamate receptor-mediated excitotoxic events. These receptors include the three groups of metabotropic glutamate receptors (mGluRs). Group I mGluR activation can initiate a number of intracellular pathways that increase neuronal excitability. Group II and III mGluRs may function as autoreceptors to modulate neurotransmission. Thus, all three groups may contribute to the mechanisms of central sensitization and chronic central pain. To begin evaluating mGluRs in SCI, we quantified the changes in mGluR expression after SCI in control (naive), sham, and impact injured adult male Sprague–Dawley rats (200–250 g). SCI was produced at spinal segment T10 with a New York University impactor (12.5-mm drop, 10-g rod of 2-mm diameter). Expression levels were determined by Western blot and immunohistochemistry analyses at the epicenter of injury, as well as segments rostral and caudal. The group I subtype mGluR1 was increased over control levels in segments rostral and caudal by postsurgical day (PSD) 7 and remained elevated through PSD 60. The group I subtype mGluR5 was unchanged in all segments rostral and caudal to the injury at every time point measured. Group II mGluRs were decreased compared to control levels from PSD 7 through PSD 60 in all segments. These results suggest that different subtypes of mGluRs have different spatial and temporal expression patterns following SCI. The expression changes in mGluRs parallel the development of mechanical allodynia and thermal hyperalgesia following SCI; therefore, understanding the expression of mGluRs after SCI may give insight into mechanisms underlying the development of chronic central pain.

Coupling on-line brain microdialysis, precolumn derivatization and capillary electrophoresis for routine minute sampling of O-phosphoethanolamine and excitatory amino acids

In previous papers, we described the analysis of excitatory amino acids (EAAs) and catecholamines in microdialysis samples using capillary electrophoresis with laser-induced fluorescence detection (CE–LIFD). In the present paper, we report that an automated analysis of such samples can be easily achieved by on-line coupling of the microdialysis probe with a continuous flow derivatization system and a commercially available CE–LIFD apparatus. Because of the short analysis time (less than 2 min) and high separation efficiency (100–200 000 theoretical plates), high temporal resolution of microdialysis (minute range) is preserved as compared to off-line systems, while both EAAs and O-phosphoethanolamine (PEA) can be simultaneously detected. This new method has been applied to the measurement of these compounds in microdialysis samples from hippocampal slice cultures and striatum of anesthetized rats. Extracellular concentrations of EAAs, but not PEA, increased during perfusion of a solution containing high K + or a glutamate uptake inhibitor. However, after in vitro ischemia on hippocampal slices, both EAAs and PEA concentrations increased, but with different temporal patterns.

Effects of hypothermia on the rate of excitatory amino acid release after ischemic depolarization.

Hypothermia slows the increase in extracellular excitatory amino acid (EAA) concentrations during temporary cerebral ischemia. However, it is unclear whether hypothermia slows the rate of EAA release or just delays the time until the first sharp increase (which occurs coincident with terminal depolarization). Pericranial temperatures were adjusted to 38 degrees C, 34 degrees C, 31 degrees C, or 25 degrees C in halothane-anesthetized rats. The cortical DC voltage was recorded from a glass microelectrode while the cortical concentrations of glutamate, aspartate, glycine, and gamma-aminobutyric acid (GABA) were measured by microdialysis. A cardiac arrest was induced with intravenous KCl, and the times until electroencephalograph isoelectricity and terminal depolarization were recorded. Dialysate concentrations of the four compounds were measured at 10, 20, and 30 minutes after depolarization. The times to isoelectricity and depolarization varied inversely with temperature; depolarization time increased from 70 +/- 9 seconds at 38 degrees C (mean +/- SD) to 294 +/- 34 seconds at 25 degrees C. The dialysate concentrations of all four compounds increased during ischemia, and the rate of increase was inhibited by cooling. After 30 minutes of ischemia, glutamate concentration in 38 degrees C animals was 58.4 +/- 31.8 mumol/L; this decreased to 15.9 +/- 8.4 mumol/L at 25 degrees C. The magnitude of the effects of temperature on amino acid release differed with the compound measured. For glutamate, the calculated Q10 was 3.63. Corresponding values for aspartate and glycine were 3.68 and 1.95, respectively. By contrast, Q10 for GABA release was 6.31, indicating greater sensitivity to cooling. These results suggest that effects of hypothermia on EAA concentrations during cerebral ischemia may be the result of both a delay until initial EAA release as well as a direct effect of temperature on the rate of amino acid release. The observed temperature effects are more consistent with carrier-mediated processes controlling EAA release.