Concentrations Of L-glutamate Research Articles

Optical biosensor test strip for the screening and direct determination of l-glutamate in food samples

An optical glutamate biosensor test strip based on stacked membranes of nafion/sol–gel (bottom layer) and chitosan (uppermost layer) was fabricated on a piece of paper as support to form a test strip. The use of a stacked membrane system allows multiple immobilizations of sensing components directly without any covalent attachment via straight forward procedures. The uppermost membrane consisted of immobilised enzymes l-glutamate oxidase (GLOD) and horseradish peroxidase (HRP), which sensed the presence of l-glutamate and the bottom membrane contained the indicator dye, 3,3′,5,5′-tetramethylbenzidine. The test strip can be used to measure l-glutamate quantitatively by observing a colour change from light green to dark green with increasing l-glutamate concentrations. Quantitative analysis could be performed by measuring the reflectance intensity of the colour change at 550 nm. The glutamate biosensor test strip gave a linear response range of 0.01–0.30 mM to l-glutamate with a limit of detection of 5 μM. The strips were successfully applied for the estimation of l-glutamate in common food items such as sauces, soups, processed food and flavour enhancers. Results of analysis of l-glutamate in various food samples using the glutamate test strip were comparable to a standard procedure employing HPLC method.

A Conserved Aspartate Residue Located at the Extracellular End of the Binding Pocket Controls Cation Interactions in Brain Glutamate Transporters

In the brain, transporters of the major excitatory neurotransmitter glutamate remove their substrate from the synaptic cleft to allow optimal glutamatergic neurotransmission. Their transport cycle consists of two sequential translocation steps, namely cotransport of glutamic acid with three Na(+) ions, followed by countertransport of K(+). Recent studies, based on several crystal structures of the archeal homologue Glt(Ph), indicate that glutamate translocation occurs by an elevator-like mechanism. The resolution of these structures was not sufficiently high to unambiguously identify the sites of Na(+) binding, but functional and computational studies suggest some candidate sites. In the Glt(Ph) structure, a conserved aspartate residue (Asp-390) is located adjacent to a conserved tyrosine residue, previously shown to be a molecular determinant of ion selectivity in the brain glutamate transporter GLT-1. In this study, we characterize mutants of Asp-440 of the neuronal transporter EAAC1, which is the counterpart of Asp-390 of Glt(Ph). Except for substitution by glutamate, this residue is functionally irreplaceable. Using biochemical and electrophysiological approaches, we conclude that although D440E is intrinsically capable of net flux, this mutant behaves as an exchanger under physiological conditions, due to increased and decreased apparent affinities for Na(+) and K(+), respectively. Our present and previous data are compatible with the idea that the conserved tyrosine and aspartate residues, located at the external end of the binding pocket, may serve as a transient or stable cation binding site in the glutamate transporters.

Push–Pull Perfusion Sampling with Segmented Flow for High Temporal and Spatial Resolution in Vivo Chemical Monitoring

Low-flow push-pull perfusion is a sampling method that yields better spatial resolution than competitive methods like microdialysis. Because of the low flow rates used (50 nL/min), it is challenging to use this technique at high temporal resolution which requires methods of collecting, manipulating, and analyzing nanoliter samples. High temporal resolution also requires control of Taylor dispersion during sampling. To meet these challenges, push-pull perfusion was coupled with segmented flow to achieve in vivo sampling at 7 s temporal resolution at 50 nL/min flow rates. By further miniaturizing the probe inlet, sampling with 200 ms resolution at 30 nL/min (pull only) was demonstrated in vitro. Using this method, L-glutamate was monitored in the striatum of anesthetized rats. Up to 500 samples of 6 nL each were collected at 7 s intervals, segmented by an immiscible oil and stored in a capillary tube. The samples were assayed offline for L-glutamate at a rate of 15 samples/min by pumping them into a reagent addition tee fabricated from Teflon where reagents were added for a fluorescent enzyme assay. Fluorescence of the resulting plugs was monitored downstream. Microinjection of 70 mM potassium in physiological buffered saline evoked l-glutamate concentration transients that had an average maxima of 4.5 ± 1.1 μM (n = 6 animals, 3-4 injections each) and rise times of 22 ± 2 s. These results demonstrate that low-flow push-pull perfusion with segmented flow can be used for high temporal resolution chemical monitoring and in complex biological environments.

Simultaneous real-time measurement of EEG/EMG and l-glutamate in mice: A biosensor study of neuronal activity during sleep

We report on electroencephalograph (EEG) and electromyograph (EMG) measurements concurrently with real-time changes in l-glutamate concentration. These data reveal a link between sleep state and extracellular neurotransmitter changes in a freely-moving (tethered) mouse. This study reveals, for the first time in mice, that the extracellular l-glutamate concentration in the pre-frontal cortex (PFC) increases during periods of extended wakefulness, decreases during extended sleep episodes and spikes during periods of REM sleep. Individual sleep epochs (10 s in duration) were scored as wake, slow-wave (SW) sleep or rapid eye movement (REM) sleep, and then correlated as a function of time with measured changes in l-glutamate concentrations. The observed l-glutamate levels show a statistically significant increase of 0.86 ± 0.26 μM ( p < 0.05) over 37 wake episodes recorded from all mice ( n = 6). Over the course of 49 measured sleep periods longer than 15 min, l-glutamate concentrations decline by a similar amount (0.88 ± 0.37 μM, p < 0.08). The analysis of 163 individual REM sleep episodes greater than one min in length across all mice ( n = 6) demonstrates a significant rise in l-glutamate levels as compared to the 1 min preceding REM sleep onset (RM-ANOVA, DF = 20, F = 6.458, p < 0.001). The observed rapid changes in l-glutamate concentration during REM sleep last only between 1 and 3 min. The approach described can also be extended to other regions of the brain which are hypothesized to play a role in sleep. This study highlights the importance of obtaining simultaneous measurements of neurotransmitter levels in conjunction with sleep markers to help elucidate the underlying physiological and ultimately the genetic components of sleep.

Turnover and accessibility of a reentrant loop of the Na+-glutamate transporter GltS are modulated by the central cytoplasmic loop

GltS of Escherichia coli is a secondary transporter that catalyzes Na+-glutamate symport. The structural model of GltS shows two homologous domains with inverted membrane topology that are connected by a central loop that resides in the cytoplasm. Each domain contains a reentrant loop structure. Accessibility of the Cys residues in two GltS mutants in which Pro351 and Asn356 in the reentrant loop in the C-terminal domain were replaced by Cys is demonstrated to be sensitive to the catalytic state supporting a role for the reentrant loops in catalysis. Saturating concentrations of the substrate L-glutamate protected both mutants against inactivation by thiol reagents, while the presence of the co-ion Na+ stimulated the inactivation of both mutants. Insertion of the 10 kDa biotin acceptor domain (BAD) of oxaloacetate decarboxylase of Klebsiella pneumoniae in the central cytoplasmic loop blocked the access pathway from the periplasmic side of the membrane to the cysteine residues in mutants P351C and N356C in the reentrant loop. Kinetically, insertion of BAD increased the maximal rate of uptake 2.7-fold while leaving the apparent affinity constants for L-glutamate and Na+ unaltered. The data suggests that insertion of BAD in the central loop results in conformational changes at the translocation site that lower the activation energy of the translocation step without affecting the access pathway from the periplasmic side for substrate and co-ions. It is concluded that changes in the central loop that connects the two domains may have a regulatory function on the activity of the transporter.

Effect of l-glutamate and glycine incorporated in activation media, on sperm motility and fertilization rate of rainbow trout (Oncorhynchus mykiss) spermatozoa

Summary In terms of fish production, sperm activating substances are important in the controlled insemination. This holds also for salmonids, when being provided with an agent that energizes the spermatozoa. In this study two amino acids (l-glutamate and glycine) were evaluated in their effect on sperm motility and fertilizing capacity for rainbow trout (Oncorhynchus mykiss) spermatozoa. Different concentrations of l-glutamate and glycine were added to the sperm medium (T1 = 0.075 g per 100 ml of l-glutamate; T2 = 0.3 g per 100 ml of glycine; T3 = 0.075 g per 100 ml of l-glutamate + 0.3 g per 100 ml of glycine). A control treatment was also performed without the two amino acids. Results show the capacity of l-glutamate to increase the time period of sperm motility (s) significantly in comparison to other treatments, registering a maximum motility time of 57.7 ± 10.8 s. For fertilization issue, the results do not show significant differences, being the highest value for T1 = 98 ± 4.5%.

Role of Mechanical Stress-induced Glutamate Signaling-associated Molecules in Cytodifferentiation of Periodontal Ligament Cells

In this study, we analyzed the effects of tensile mechanical stress on the gene expression profile of in vitro-maintained human periodontal ligament (PDL) cells. A DNA chip analysis identified 17 up-regulated genes in human PDL cells under the mechanical stress, including HOMER1 (homer homolog 1) and GRIN3A (glutamate receptor ionotropic N-methyl-d-aspartate 3A), which are related to glutamate signaling. RT-PCR and real-time PCR analyses revealed that human PDL cells constitutively expressed glutamate signaling-associated genes and that mechanical stress increased the expression of these mRNAs, leading to release of glutamate from human PDL cells and intracellular glutamate signal transduction. Interestingly, exogenous glutamate increased the mRNAs of cytodifferentiation and mineralization-related genes as well as the ALP (alkaline phosphatase) activities during the cytodifferentiation of the PDL cells. On the other hand, the glutamate signaling inhibitors riluzole and (+)-MK801 maleate suppressed the alkaline phosphatase activities and mineralized nodule formation during the cytodifferentiation and mineralization. Riluzole inhibited the mechanical stress-induced glutamate signaling-associated gene expressions in human PDL cells. Moreover, in situ hybridization analyses showed up-regulation of glutamate signaling-associated gene expressions at tension sites in the PDL under orthodontic tooth movement in a mouse model. The present data demonstrate that the glutamate signaling induced by mechanical stress positively regulates the cytodifferentiation and mineralization of PDL cells.

A Conserved Aspartate Determines Pore Properties of Anion Channels Associated with Excitatory Amino Acid Transporter 4 (EAAT4)

Excitatory amino acid transporter (EAAT) glutamate transporters function not only as secondary active glutamate transporters but also as anion channels. Recently, a conserved aspartic acid (Asp(112)) within the intracellular loop near to the end of transmembrane domain 2 was proposed as a major determinant of substrate-dependent gating of the anion channel associated with the glial glutamate transporter EAAT1. We studied the corresponding mutation (D117A) in another EAAT isoform, EAAT4, using heterologous expression in mammalian cells, whole cell patch clamp, and noise analysis. In EAAT4, D117A modifies unitary conductances, relative anion permeabilities, as well as gating of associated anion channels. EAAT4 anion channel gating is characterized by two voltage-dependent gating processes with inverse voltage dependence. In wild type EAAT4, external l-glutamate modifies the voltage dependence as well as the minimum open probabilities of both gates, resulting in concentration-dependent changes of the number of open channels. Not only transport substrates but also anions affect wild type EAAT4 channel gating. External anions increase the open probability and slow down relaxation constants of one gating process that is activated by depolarization. D117A abolishes the anion and glutamate dependence of EAAT4 anion currents and shifts the voltage dependence of EAAT4 anion channel activation by more than 200 mV to more positive potentials. D117A is the first reported mutation that changes the unitary conductance of an EAAT anion channel. The finding that mutating a pore-forming residue modifies gating illustrates the close linkage between pore conformation and voltage- and substrate-dependent gating in EAAT4 anion channels.

Amitriptyline inhibits the activity of the rat glutamate transporter EAAT3 expressed in Xenopus oocytes

Abstract Objectives Evidence suggests that glutamatergic systems may be involved in the pathophysiology of major depression and the mechanism of action of antidepressants. We have investigated the effects of amitriptyline, a tricyclic antidepressant, on the activity of the excitatory amino acid transporter type 3 (EAAT3), a protein that can regulate extracellular glutamate concentrations in the brain. Methods EAAT3 was expressed in Xenopus oocytes. Using a two-electrode voltage clamp, membrane currents were recorded after application of 30 μM l-glutamate in the presence or absence of various concentrations of amitriptyline or after application of various concentrations of l-glutamate in the presence or absence of 0.64 μM amitriptyline. Key findings Amitriptyline concentration-dependently reduced EAAT3 activity. This inhibition reached statistical significance at 0.38–1.27 μM amitriptyline. Amitriptyline 0.64 μM reduced the pharmacokinetic parameter Vmax, but did not affect the pharmacokinetic parameter Km, of EAAT3 for l-glutamate. The amitriptyline inhibition disappeared after a 4-min washout. Phorbol-12-myristate-13-acetate, a protein kinase C activator, increased EAAT3 activity. However, 0.64 μM amitriptyline induced a similar degree of decrease in EAAT3 activity in the presence or absence of phorbol-12-myristate-13-acetate. Conclusions Our results suggested that amitriptyline at clinically relevant concentrations reversibly reduced EAAT3 activity via decreasing its maximal velocity of glutamate transporting function. The effects of amitriptyline on EAAT3 activity may have represented a novel site of action for amitriptyline to increase glutamatergic neuro-transmission. Protein kinase C may not have been involved in the effects of amitriptyline on EAAT3.

Symmetry in the Structure of the Glutamate Transporter GltPh Suggests Conformation of an Alternate State

Glutamate transporters regulate synaptic concentrations of L-glutamate to prevent excitotoxicity in nerve cells. Current crystal structures of GltPh, an archeal homologue of the Glutamate transporters, have an extracellular-facing binding site. The alternating access theory implies that a cytoplasm-facing state also exists. In order to model this state, we have identified two distinct sets of inverted-topology repeats, and used these repeats to model an inward-facing conformation of the protein. Specifically, we modeled the sequence of each repeat on the structure of its partner. In this model, a portion of the protein containing two transmembrane helices (TM7 and 8) and two helical hairpins (HP1 and HP2) is displaced relative to the crystal structure so that the binding site is exposed to the cytoplasm. In order to validate our model, pairs of cysteines were introduced into the neuronal glutamate transporter EAAC1 at positions that were greater than 27 Angstroms apart in the outward-facing crystal structure, but closer to 10 Angstroms apart in our model. Transport in these mutants was activated by pretreatment with the reducing agent dithithreitol. Once treated with the oxidizing agent copper(II)(1,10-phenantroline)3, however, activity ceased. Importantly, this inhibition was potentiated under conditions expected to promote the inward-facing conformation. This suggests that during the transport cycle these cysteines come within the range necessary to crosslink, as predicted by our inverted-topology repeat model of the cytoplasm-facing state. Previously, an alternative conformational state of the LeuT transporter was also modeled using inverted-topology repeats, suggesting that inverted-topology repeats may provide a general and elegant solution to the requirement for two symmetry-related states in a single protein.

Keratinocytes: a source of the transmitter l‐glutamate in the epidermis

Various glutamate receptors have been described in both keratinocytes and melanocytes. L-Glutamate is the physiological agonist of the glutamate receptor family. The source of this transmitter had not yet been identified. In normal human epidermal keratinocytes (NHEK) and HaCaT-keratinocytes, cell supernatants were sampled in various stages of cell density and the l-glutamate content photometrically determined. The following examination time-points were defined: non-confluent (ca. 33%), subconfluent (ca. 70%) and confluent (90-100%). The L-glutamate concentration originally in the culture medium was 14.7 mg/l (0.1 mm/l). The L-glutamate concentration in the cell supernatant increased in NHEK with increasing cell density: non-confluent 39.9 + or - 4 mg/l, subconfluent 60.6 + or - 15.8 mg/l, confluent 100.7 + or - 33.2 mg/l. A linear increase of L-glutamate concentration was also found for HaCaT cells. The investigations show that keratinocytes are capable of producing and releasing L-glutamate. Thus they are a source of L-glutamate which acts as a transmitter on epidermal glutamate receptors.

Metabotropic glutamate receptor type 1 in taste tissue

l-Glutamate confers cognitive discrimination for umami taste (delicious or savory) and dietary information to the brain through the activation of G protein–coupled receptors in specialized taste receptor cells of the tongue. The taste heterologous receptor T1R1 plus T1R3 is not sufficient to detect umami taste in mice. The lack of T1R3 diminished but did not abolish nerve and behavioral responses in null mice that still contained umami-sensitive taste receptor cells. The remnant umami responses in T1R3 knockout mice indicate that there are also T1R3 independent receptors. Metabotropic glutamate receptor 1 (mGluR1), which is widely expressed throughout the central nervous system and regulates synaptic signaling, is another l-glutamate receptor candidate. It is found within taste buds, although the amount of l-glutamate in the perisynaptic region is in the order of μmol/L, whereas free dietary l-glutamate is in the mmol/L range. We reexamined the expression of one mGluR1 variant with a lower affinity for l-glutamate that is found in fungiform and circumvallate papillae. This taste mGluR1 receptor responds in vitro to the concentration of l-glutamate usually found in foodstuffs.

CARDIOPROTECTIVE EFFECT OF l‐GLUTAMATE IN OBESE TYPE 2 DIABETIC ZUCKER FATTY RATS

1. Because diabetic hearts have an increased threshold for cardioprotection by ischaemic preconditioning (IPC), we hypothesized that protection by L-glutamate during reperfusion is restricted in Type 2 diabetic hearts. Previously, we found that L-glutamate-mediated postischaemic cardioprotection mimics IPC. 2. Rat hearts were studied in a Langendorff preparation perfused with Krebs'-Henseleit solution and subjected to 40 min global no-flow ischaemia, followed by 120 min reperfusion. L-Glutamate (0, 15 and 30 mmol/L) was added to the perfusate during reperfusion of hearts from non-diabetic (Wistar-Kyoto) and diabetic (Zucker diabetic fatty (ZDF)) rats, studied at 16 weeks of age. The infarct size (IS)/area-at-risk (AAR) ratio was the primary end-point. Expression of L-glutamate excitatory amino acid transporter (EAAT) 1 (mitochondrial) and EAAT3 (sarcolemmal) was determined by quantitative polymerase chain reaction and immunoblotting. 3. The ISS/AAR ratio did not differ between control hearts from Wistar-Kyoto and ZDF rats (0.52 ± 0.03 and 0.51 ± 0.04, respectively; P = 0.90). L-Glutamate (15 mmol/L) significantly reduced the IS/AAR ratio in non-diabetic hearts, but not in diabetic hearts, compared with their respective controls. The higher concentration of L-glutamate (30 mmol/L) reduced infarct size in diabetic hearts to the same degree as in non-diabetic hearts (IS/AAR 0.35 ± 0.03 (P = 0.002) and 0.34 ± 0.03 (P = 0.004), respectively). The mitochondrial L-glutamate transporter EAAT1 was downregulated in hearts from ZDF rats at both the mRNA and protein levels (P < 0.0005 and P < 0.0001, respectively). However, there was no change in EAAT3 expression at the protein level. Myocardial L-glutamate content was increased by 43% in diabetic hearts (P < 0.0001). 4. Hearts from obese diabetic rats have an elevated threshold for metabolic postischaemic cardioprotection by L-glutamate. These findings may reflect underlying mechanisms of inherent resistance against additional cardioprotection in the diabetic heart.

Effects of cryoprotectant treatments on bovine sperm function and osmolyte content

The hypothesis that addition and removal of cryoprotectants to and from spermatozoa would initiate regulatory volume decrease, and lead to osmolyte loss and reduced sperm function, was tested. Common cryoprotectants, in the absence of freezing and thawing, affected bovine ejaculated spermatozoa by lowering their total and progressive motility in medium, reducing their migration through surrogate cervical mucus, damaging sperm head membranes and inducing sperm tail coiling. Sperm function was slightly better maintained after cryoprotectants were added and removed in multiple small steps rather than in a single step. The intracellular content of the polyol osmolytes, D-sorbitol and myo-inositol, exceeded that of the zwitterion osmolytes, L-carnitine and L-glutamate. Certain cryoprotectants reduced intracellular L-carnitine and L-glutamate concentration but not that of myo-inositol or D-sorbitol. Multistep treatments with some cryoprotectants had advantages over one-step treatments in mucus penetration depending on the original amount of intracellular carnitine and glutamate in the spermatozoa. Overall, sperm quality was best maintained by multistep treatment with glycerol and propanediols that were associated with decreased intracellular glutamate concentration. Bovine spermatozoa seem to use glutamate to regulate cryoprotectant-induced cell swelling.

Elementary mode analysis and metabolic flux analysis of L-glutamate biosynthesis byCorynebacterium glutamicum

In this study we applied elementary mode analysis and metabolic flux analysis to L-glutamate biosynthesis by Corynebacterium glutamicum GDK-9. The theoretically optimal flux distribution with the maximal L-glutamate yield and the actual flux distribution of L-glutamate production were determined, which suggested that phosphoenol-pyruvate (PEP), 2-oxoglutarate (2-OXO) and iso-citric acid (ICI) were the key nodes of L-glutamate biosynthesis. It was also indicated that the carbon-fixation reaction from CO(2) had great influence on the flux distribution of the key nodes. Besides, the biosynthesis of lactate could greatly decrease the L-glutamate yield. Therefore we further developed a rational dissolved oxygen control strategy to improve the L-glutamate production by strengthening the carbon-fixation reaction from CO2 and reducing the lactate production. Under the optimized fermentation conditions, a final L-glutamate concentration of 141 g/L was obtained and the carbon yield of L-glutamate on glucose was 81.7%.

Vesicular glutamate filling and AMPA receptor occupancy at the calyx of Held synapse of immature rats

At central glutamatergic synapses, neurotransmitter often saturates postsynaptic AMPA receptors (AMPARs), thereby restricting the dynamic range of synaptic efficacy. Here, using simultaneous pre- and postsynaptic whole-cell recordings, at the calyx of Held synapse of immature rats, we have investigated the mechanism by which transmitter glutamate saturates postsynaptic AMPARs. When we loaded L-glutamate (1-100 mM) into presynaptic terminals, the quantal EPSC (qEPSC) amplitude changed in a concentration-dependent manner. At physiological temperature (36-37 degrees C), the qEPSC amplitude increased when intraterminal L-glutamate concentration was elevated from 1 mM to 10 mM, but it reached a plateau at 10 mM. This plateau persisted after bath-application of the low affinity AMPAR antagonist kynurenate, suggesting that it was caused by saturation of vesicular filling with glutamate rather than by saturation of postsynaptic AMPARs. In contrast to qEPSCs, action potential-evoked EPSCs remained unchanged by increasing intraterminal L-glutamate from 1 mM to 100 mM , even at room temperature, indicating that multi-quantal glutamate saturated postsynaptic AMPARs. This saturation could be relieved by blocking AMPAR desensitization using cyclothiazide (100 microM). The concentration of ambient glutamate in the slice, estimated from NMDA receptor current fluctuations, was 55 nM; this was far below the concentration required for AMPAR desensitization. We conclude that rapid AMPAR desensitization, caused by glutamate released from multiple vesicles during synaptic transmission, underlies postsynaptic AMPAR saturation at this immature calyceal synapse before the onset of hearing.

A microelectrode biosensor for real time monitoring of l-glutamate release

We have developed an amperometric microbiosensor for real time monitoring l-glutamate release in neural tissue, based on enzymatic oxidation catalyzed by the l-glutamate oxidase. By means of a sol–gel coating method, l-glutamate oxidase was entrapped in a biocompatible gel layer that provided a benign environment and retained enzyme activity on the surface of Pt microelectrode. Prior to gel layer formation, a modification on the surface of Pt microelectrode with poly(phenylene diamine) enabled the microbiosensor screen majority of common potential interfering substances existing in physiological samples. The miniaturized biosensor achieved a steady state response to l-glutamate within 10 s and exhibited a linear dependence on the concentration of l-glutamate from 0.5 to 100 μmol L −1 with a high sensitivity of 279.4 ± 2.0 μA (mmol L −1) −1 cm −2 ( n = 4, R.S.D. = 2.8%). The microbiosensor also exhibited excellent long-term stability in dry storage. We have successfully used the microbiosensor for real time measuring of l-glutamate in vivo.

Amitriptyline inhibits the activity of the rat glutamate transporter EAAT3 expressed in &lt;I&gt;Xenopus&lt;/I&gt; oocytes

Evidence suggests that glutamatergic systems may be involved in the pathophysiology of major depression and the mechanism of action of antidepressants. We have investigated the effects of amitriptyline, a tricyclic antidepressant, on the activity of the excitatory amino acid transporter type 3 (EAAT3), a protein that can regulate extracellular glutamate concentrations in the brain. EAAT3 was expressed in Xenopus oocytes. Using a two-electrode voltage clamp, membrane currents were recorded after application of 30 microM L-glutamate in the presence or absence of various concentrations of amitriptyline or after application of various concentrations of L-glutamate in the presence or absence of 0.64 microM amitriptyline. Amitriptyline concentration-dependently reduced EAAT3 activity. This inhibition reached statistical significance at 0.38-1.27 microM amitriptyline. Amitriptyline 0.64 microM reduced the pharmacokinetic parameter Vmax, but did not affect the pharmacokinetic parameter Km, of EAAT3 for L-glutamate. The amitriptyline inhibition disappeared after a 4-min washout. Phorbol-12-myristate-13-acetate, a protein kinase C activator, increased EAAT3 activity. However, 0.64 microM amitriptyline induced a similar degree of decrease in EAAT3 activity in the presence or absence of phorbol-12-myristate-13-acetate. Our results suggested that amitriptyline at clinically relevant concentrations reversibly reduced EAAT3 activity via decreasing its maximal velocity of glutamate transporting function. The effects of amitriptyline on EAAT3 activity may have represented a novel site of action for amitriptyline to increase glutamatergic neurotransmission. Protein kinase C may not have been involved in the effects of amitriptyline on EAAT3.

Glutarate and N-acetyl-l-glutamate buffers for cell-free synthesis of selectively 15N-labelled proteins

Cell-free protein synthesis provides rapid and economical access to selectively 15N-labelled proteins, greatly facilitating the assignment of 15N-HSQC spectra. While the best yields are usually obtained with buffers containing high concentrations of potassium L-glutamate, preparation of selectively 15N-Glu labelled samples requires non-standard conditions. Among many compounds tested to replace the L-Glu buffer, potassium N-acetyl-L-glutamate and potassium glutarate were found to perform best, delivering high yields for all proteins tested, with preserved selectivity of 15N-Glu labelling. Assessment of amino-transferase activity by combinatorial 15N-labelling revealed that glutarate and N-acetyl-L-glutamate suppress the transfer of the 15N-alpha-amino groups between amino acids less well than the conventional L-Glu buffer. On balance, the glutarate buffer appears most suitable for the preparation of samples containing 15N-L-Glu while the conventional L-Glu buffer is advantageous for all other samples.

Comparison of the l-Glutamate Level in Mouse Hippocampal Slices under Tetraethylammonium Chloride Stimulation as Measured with a Glass Capillary Sensor and a Patch Sensor

The concentration level of L-glutamate released from the region CA1 of mouse hippocampal slices under tetraethylammonium chloride (TEA) stimulation was measured by two independent methods, i.e., a glass capillary-based enzyme sensor and a patch sensor, and compared with each other for different slice preparations. In a submerged slice preparation, the sensors were positioned in bath solutions several tens microm above CA1, respectively. The sensors exhibited almost the same level of extra-slice L-glutamate concentration. When a capillary sensor was implanted in region CA1 at a depth of approximately 10 microm, the TEA-induced L-glutamate release pattern was very similar to those observed with the capillary sensor in a bath use. The concentration level of intra-slice (extracellular) L-glutamate was found to be in the range from 6 to 10 microM, which was significantly larger than that of the extra-slice one. These results demonstrate that L-glutamate released from each neuronal region inevitably diffuses out of the slices, and the extra-slice L-glutamate level reflects the extracellular one.