Modulation of amino acid neurotransmitter actions by other neurotransmitters: some examples.

Developments in the field of central neurotransmission indicate that amino acids serve as important and widespread transmitters throughout the central nervous system. There are increasing indications from recent experimental studies that several of the other central neurotransmitters may exert potent effects on central neurons by modulating the actions of amino acids. Noradrenaline and serotonin have received particular attention as potential modulators, and a wide variety of actions has been reported for them. Modulatory actions have been reported at both pre- and post-synaptic levels, including both short- and long-term effects and facilitation or inhibition of amino acid actions. Selectivity has been found both for specific receptor subtypes of the neuromodulator and for specific effects of amino acids. Examples of such selectivity are modification of actions of an amino acid with little effect on spontaneous activity or membrane properties of the target cell, or in comparison to the actions of other neurotransmitters, or even other selective amino acid analogs. Modulatory actions on amino acids have also been reported for several other neurotransmitters including acetylcholine and various peptides. Recent studies of angiotensin II demonstrate that when iontophoretically applied, it can potently and selectively block the depolarizing action of glutamate on locus coeruleus neurons. It is possible that physiological influences of these various transmitter substances are expressed through modification of amino acid actions, rather than through direct effects on central neurons.     

Possible role of cGMP in excitatory amino acid induced cytotoxicity in cultured cerebral cortical neurons

Using cultured cerebral cortical neurons at mature stages (9 days in culture, d.i.c.) it was demonstrated that glutamate, NMDA (N-methyl-D-aspartate) and to a lesser extent KA (kainate) increase the intracellular cGMP concentration ([cGMP]_i) whereas no such effect was observed after exposure of the cells of QA (quisqualate) and AMPA (2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionate). No effect of glutamate, NMDA and KA was observed in immature neurons (2 d.i.c.). The pharmacology of these cGMP responses was investigated using the glutamate antagonists APV (2-amino-5-phosphonovalerate) with selectivity for NMDA receptors, CNQX (6-cyano-7-nitro-quinoxaline-2,3-dione) with selectivity for non-NMDA receptors and the novel KA selective antagonists AMOA (2-amino-3-[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propionate) and AMNH (2-amino-3-[2-(3-hydroxy-5-methylisoxazol-4-yl)methyl-5-methyl-3-oxoisoxazolin-4-yl]propionate). In addition, the cytotoxicity of glutamate, NMDA and KA was studied and found to be enhanced by addition of the non-metabolizable cGMP analogue 8-Br-cGMP. On the contrary, the toxicity of QA and AMPA was not affected by 8-Br-cGMP. Pertussis toxin augmented the toxicity elicited by glutamate, NMDA, KA and QA but not that induced by AMPA. On the other hand, only glutamate and KA induced toxicity was potentiated by cholera toxin, which also enhanced the stimulatory effect of glutamate and NMDA but not that of KA on the cellular cGMP content. The toxicity as well as the effects on intracellular cGMP levels could be antagonized by the specific excitatory amino acid (EAA) antagonists. These results suggest that the mechanisms by which the various excitatory amino acids exert cytotoxicity are different, and that increased cGMP levels may participate in the mediation of glutamate, NMDA or KA induced toxicity but less likely in QA and AMPA mediated toxicity. Furthermore, G-proteins or other pertussis or cholera toxin sensitive entities seem to be involved in the cytotoxic action of all excitatory amino acids except AMPA.     

Glutamate Transport in Large Muscle Fibres of Balanus nubilus

The uptake of glutamate and other acidic amino acids into barnacle single muscle fibres has been characterized. The uptake of glutamate consists of two components, one Na-independent and one Na-dependent. The Na-dependent uptake is saturable (half-maximal at 250 microM external glutamate) and is inhibited by a variety of analogues of which L-cysteate and D- and L-aspartate are the most potent. These amino acids are also transported into the muscle in a Na-dependent manner. The excitatory agonists kainate, quisqualate, and N-methyl-D-aspartate do not inhibit or affect uptake in any way. Progressive replacement of external Na by choline reduces uptake with very little effect on the apparent affinity for glutamate, suggesting that Na and glutamate bind to the transporter independently. The kinetics of activation are consistent with a requirement for at least two Na ions. Na activation of glutamate uptake can be inhibited by guanidinium with kinetics that are consistent with competitive inhibition at the Na binding site. Studies on the efflux of L-glutamate and other analogues have shown that efflux rates are only slightly increased by the removal of Na and do not seem to be affected in any clear manner by external levels of acidic amino acids.     

Relationship of glutamate and aspartate to the periaqueductal gray-raphe magnus projection: analysis using immunocytochemistry and microdialysis.

This study tested the hypothesis that the excitatory amino acid transmitters glutamate and/or aspartate are associated with the periaqueductal gray (PAG)-raphe magnus (NRM) projection. Retrograde neuroanatomical tracing procedures utilizing the tracers WGA-HRP or D-[3H]-aspartate were combined with immunocytochemical localization of glutamate or aspartate to determine if glutamate and/or aspartate immunostained neurons projected to the NRM. Both glutamate- and aspartate-immunoreactive cells in the PAG were found to project to the NRM. Double labeling immunocytochemichemical procedures indicated that glutamate and aspartate are co-localized in many PAG neurons, suggesting the following possibilities: (a) one of these two amino acids may serve as a precursor to the other; (b) both amino acids may be co-released from the same PAG neuron; or (c) both amino acids are present in high levels in the perikarya for metabolic purposes. At the EM level, both glutamate- and aspartate-immunoreactive terminals were identified in the NRM, strengthening the concept that both amino acids participate in synaptic transmission in this medullary nucleus. To determine if glutamate and aspartate are in fact released from PAG-NRM axons, the PAG was stimulated chemically with homocysteic acid (HCA) and amino acids were collected from the NRM using a microdialysis probe. Microinjection of HCA, but not vehicle, into the PAG resulted in the release of both glutamate and aspartate in the nucleus raphe magnus. These data suggest that both glutamate and aspartate are released from PAG fibers terminating in the NRM and provide strong support for the hypothesis that excitatory amino acids play a neurotransmitter role in the PAG-NRM pathway.     

Inhibition by Diazepam and γ-Aminobutyric Acid of Depolarization-Induced Release of [14C]Cysteine Sulfinate and [3H]Glutamate in Rat Hippocampal Slices

Effects of diazepam and gamma-aminobutyric acid-related compounds on the release of [14C]cysteine sulfinate and [3H]glutamate from preloaded hippocampal slices of rat brain were examined by a superfusion method. Diazepam markedly inhibited the release of cysteine sulfinate and glutamate evoked either by high K+ or veratridine without affecting that of other neurotransmitter candidates, e.g., gamma-aminobutyric acid, acetylcholine, noradrenaline, and dopamine; IC50 values for the release of cysteine sulfinate and glutamate were about 20 and 7 microM, respectively. gamma-Aminobutyric acid (1 to 10 microM) and muscimol (100 microM) significantly reduced high K+-stimulated release of glutamate. Bicuculline, which had no effect on the release at a concentration of 50 microM by itself, antagonized the inhibitor effects of diazepam and gamma-aminobutyric acid on glutamate release. Similar results were obtained with the release of cysteine sulfinate except that a high concentration (100 microM) of gamma-aminobutyric acid was required for the inhibition. These results indicate the modulation by gamma-aminobutyric acid innervation of the release of excitatory amino acids in rat hippocampal formation, and also suggest that some of the pharmacological effects of diazepam may be a consequence of inhibition of excitatory amino acid transmission.     

The in vitro uptake of glutamate in GLAST and GLT-1 transfected mutant CHO-K1 cells is inhibited by manganese

In the central nervous system (CNS), extracellular concentrations of amino acids (e.g., aspartate, glutamate) and divalent metals (e.g., zinc, copper, manganese) are primarily regulated by astrocytes. Adequate glutamate homeostasis and control over extracellular concentrations of these excitotoxic amino acids are essential for the normal functioning of the brain. Not only is glutamate of central importance for nitrogen metabolism but, along with aspartate, it is the primary mediator of excitatory pathways in the brain. Similarly, the maintenance of proper Mn levels is important for normal brain function. Brain glutamate is removed from the extracellular fluid mainly by astrocytes via high affinity astroglial Na+-dependent excitatory amino acid transporters, glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1). The effects of Mn on specific glutamate transporters have yet to be determined. As a first step in this process, we examined the effects of Mn on the transport of [D-2, 3-3H]D-aspartate, a non-metabolizable glutamate analog, in Chinese hamster ovary cells (CHO) transfected with two glutamate transporter subtypes, GLAST (EAAT1) or GLT-1 (EAAT2). Mn-mediated inhibition of glutamate transport in the CHO-K1 cell line DdB7 was pronounced in both the GLT-1 and GLAST transfected cells. This resulted in a statistically significant inhibition (p<0.05) of glutamate uptake compared with transfected control in the absence of Mn treatment. These studies suggest that Mn accumulation in the CNS might contribute to dysregulation of glutamate homeostasis.     

Cooperative interaction of glutamate and aspartate with receptors in the neuromuscular excitatory membrane in walking limbs of the lobster.

When applied to lobster muscle fibers, L-glutamate, L-aspartate, and combinations of the two amino acids can induce membrane depolarization. Under normal conditions, a quantitative analysis of the depolarization response or change in membrane conductance was precluded by nonlinearities in the voltage-current relationship of the membrane. By including gamma-aminobutyrate (GABA) in the bathing medium, the voltage-current relationship was made linear in the depolarizing direction over a range of 15-20 mV from the resting potential. However, a meaningful examination of the increase in membrane conductance caused by glutamate and aspartate was still not possible. Therefore, the depolarization responses caused by the excitatory amino acids were taken as a quantitative reflection of receptor activation in the excitatory postsynaptic membrane. In the presence of GABA, aspartate by itself, at concentrations up to 10 mM, had little excitatory activity, whereas glutamate effected an appreciable membrane depolarization at concentrations of 0.1 to 0.2 mM. Aspartate, at concentrations which exhibited no activity alone, markedly enhanced the excitatory action of glutamate. Aspartate shifted the glutamate dose-response curve to the left, but did not appear to affect the maximum depolarization response elicited by glutamate. These observations are consistent with the concept that aspartate increases the affinity between glutamate and the glutamate binding sites. Limiting slopes of log-dose versus log-response curves for the excitatory action of glutamate suggest that the interaction of glutamate with excitatory receptors is a cooperative process. The possibility exists that individual receptors contain multiple and distinct glutamate and aspartate binding sites. These results support the view that neuromuscular excitation in the lobster is mediated by glutamate and aspartate functioning synergistically.     

Stress Preferentially Increases Extraneuronal Levels of Excitatory Amino Acids in the Prefrontal Cortex: Comparison to Hippocampus and Basal Ganglia

Abstract: The technique of intracerebral microdialysis was used to assess the effect of stress on the extracellular concentrations of excitatory amino acids, glutamate and aspartate, in the rat medial prefrontal cortex, hippocampus, striatum, and nucleus accumbens. A 20-min restraint procedure led to an increase in extracellular glutamate in all regions tested. The increase in glutamate levels was significantly higher in the prefrontal cortex than that observed in other regions. With the exception of the striatum, extracellular levels of aspartate were increased in all regions. Furthermore, the increase in aspartate levels was significantly higher in prefrontal cortex compared to hippocampus and nucleus accumbens. Local perfusion of tetrodotoxin during the restraint procedure significantly decreased the stress-induced increase in extracellular excitatory amino acids. In order to ensure that the above results were not an artifact of restraint not associated with stress (e.g., decreased mobility), we also examined the effect of swimming stress on the extracellular levels of excitatory amino acids in selected regions, i.e., striatum and medial prefrontal cortex. Both regions displayed a significant increase in extracellular levels of aspartate and glutamate following 20 min of swimming in room temperature water. This study provides direct evidence that stress increases the neuronal release of excitatory amino acids in a regionally selective manner. The implications of the present findings for stress-induced catecholamine release and/or hippocampal degeneration are discussed.     

Cooperative interaction of glutamate and aspartate with receptors in the neuromuscular excitatory membrane in walking limbs of the lobster

When applied to lobster muscle fibers, L-glutamate, L-aspartate, and combinations of the two amino acids can induce membrane depolarization. Under normal conditions, a quantitative analysis of the depolarization response or change in membrane conductance was precluded by nonlinearities in the voltage—current relationship of the membrane. By including γ-aminobutyrate (GABA) in the bathing medium, the voltage—current relationship was made linear in the depolarizing direction over a range of 15–20 mV from the resting potential. However, a meaningful examination of the increase in membrane conductance caused by glutamate and aspartate was still not possible. Therefore, the depolarization responses caused by the excitatory amino acids were taken as a quantitative reflection of receptor activation in the excitatory postsynaptic membrane. In the presence of GABA, aspartate by itself, at concentrations up to 10 mM, had little excitatory activity, whereas glutamate effected an appreciable membrane depolarization at concentrations of 0.1 to 0.2 mM. Aspartate, at concentrations which exhibited no activity alone, markedly enhanced the excitatory action of glutamate. Aspartate shifted the glutamate dose-response curve to the left, but did not appear to affect the maximum depolarization response elicited by glutamate. These observations are consistent with the concept that aspartate increases the affinity between glutamate and the glutamate binding sites. Limiting slopes of log-dose versus log-response curves for the excitatory action of glutamate suggest that the interaction of glutamate with excitatory receptors is a cooperative process. The possibility exists that individual receptors contain multiple and distinct glutamate and aspartate binding sites. These results support the view that neuromuscular excitation in the lobster is mediated by glutamate and asparate functioning synergistically.     

siRNA knock down of glutamate dehydrogenase in astrocytes affects glutamate metabolism leading to extensive accumulation of the neuroactive amino acids glutamate and aspartate

Glutamate is the most abundant excitatory neurotransmitter in the brain and astrocytes are key players in sustaining glutamate homeostasis. Astrocytes take up the predominant part of glutamate after neurotransmission and metabolism of glutamate is necessary for a continuous efficient removal of glutamate from the synaptic area. Glutamate may either be amidated by glutamine synthetase or oxidatively metabolized in the mitochondria, the latter being at least to some extent initiated by oxidative deamination by glutamate dehydrogenase (GDH). To explore the particular importance of GDH for astrocyte metabolism we have knocked down GDH in cultured cortical astrocytes employing small interfering RNA (siRNA) achieving a reduction of the enzyme activity by approximately 44%. The astrocytes were incubated for 2h in medium containing either 1.0mM [(15)NH(4)(+)] or 100μM [(15)N]glutamate. For those exposed to [(15)N]glutamate an additional 100μM was added after 1h. Metabolic mapping was performed from isotope incorporation measured by mass spectrometry into relevant amino acids of cell extracts and media. The contents of the amino acids were measured by HPLC. The (15)N incorporation from [(15)NH(4)(+)] into glutamate, aspartate and alanine was decreased in astrocytes exhibiting reduced GDH activity. However, the reduced GDH activity had no effect on the cellular contents of these amino acids. This supports existing in vivo and in vitro studies that GDH is predominantly working in the direction of oxidative deamination and not reductive amination. In contrast, when exposing the astrocytes to [(15)N]glutamate, the reduced GDH activity led to an increased (15)N incorporation into glutamate, aspartate and alanine and a large increase in the content of glutamate and aspartate. Surprisingly, this accumulation of glutamate and net-synthesis of aspartate were not reflected in any alterations in either the glutamine content or labeling, but a slight increase in mono labeling of glutamine in the medium. We suggest that this extensive net-synthesis of aspartate due to lack of GDH activity is occurring via the concerted action of AAT and the part of TCA cycle operating from α-ketoglutarate to oxaloacetate, i.e. the truncated TCA cycle.     

Expression of a heterologous glutamate dehydrogenase gene in Lactococcus lactis highly improves the conversion of amino acids to aroma compounds

The first step of amino acid degradation in lactococci is a transamination, which requires an alpha-keto acid as the amino group acceptor. We have previously shown that the level of available alpha-keto acid in semihard cheese is the first limiting factor for conversion of amino acids to aroma compounds, since aroma formation is greatly enhanced by adding alpha-ketoglutarate to cheese curd. In this study we introduced a heterologous catabolic glutamate dehydrogenase (GDH) gene into Lactococcus lactis so that this organism could produce alpha-ketoglutarate from glutamate, which is present at high levels in cheese. Then we evaluated the impact of GDH activity on amino acid conversion in in vitro tests and in a cheese model by using radiolabeled amino acids as tracers. The GDH-producing lactococcal strain degraded amino acids without added alpha-ketoglutarate to the same extent that the wild-type strain degraded amino acids with added alpha-ketoglutarate. Interestingly, the GDH-producing lactococcal strain produced a higher proportion of carboxylic acids, which are major aroma compounds. Our results demonstrated that a GDH-producing lactococcal strain could be used instead of adding alpha-ketoglutarate to improve aroma development in cheese.     

The osmotic/calcium stress theory of brain damage: Are free radicals involved?

This overview presents data showing that glucose use increases and that excitatory amino acids (i.e., glutamate, aspartate), taurine and ascorbate increase in the extracellular fluid during seizures. During the cellular hyperactive state taurine appears to serve as an osmoregulator and ascorbate may serve as either an antioxidant or as a pro-oxidant. Finally, a unifying hypothesis is given for seizure-induced brain damage. This unifying hypothesis states that during seizures there is a release of excitatory amino acids which act on glutamatergic receptors, increasing neuronal activity and thereby increasing glucose use. This hyperactivity of cells causes an influx, of calcium (i.e. calcium stress) and water movements (i.e., osmotic stress) into the cells that culminate in brain damage mediated by reactive oxygen species.Special issue dedicated to Dr. Frederick E. Samson     

Effect of Impact Trauma on Neurotransmitter and Nonneurotransmitter Amino Acids in Rat Spinal Cord

N-Methyl-D-aspartate (NMDA) administration exacerbates neurological dysfunction after traumatic spinal cord injury in rats, whereas NMDA antagonists improve outcome in this model. These observations suggest that release of excitatory amino acids contributes to secondary tissue damage after traumatic spinal cord injury. To further examine this hypothesis, concentrations of free amino acids were measured in spinal cord samples from anesthetized rats subjected to various degrees of impact trauma to the T9 spinal segment. Levels of excitatory and inhibitory neurotransmitter amino acids [gamma-aminobutyric acid (GABA), glutamate, aspartate, glycine, taurine] and levels of nonneurotransmitter amino acids (asparagine, glutamine, alanine, threonine, serine) were determined at 5 min, 4 h, and 24 h posttrauma. Uninjured surgical (laminectomy) control animals showed modest but significant declines in aspartate and glutamate levels, but not in other amino acids, at all time points. In injured animals, the excitatory amino acids glutamate and aspartate were significantly decreased by 5 min posttrauma, and remained depressed at 4 h and 24 h as compared with corresponding laminectomy controls. In contrast, the inhibitory amino acids, glycine, GABA, and taurine, were decreased at 5 min postinjury, had partially recovered at 4 h, and were almost fully recovered at 24 h. The nonneurotransmitter amino acids were unchanged at 5 min posttrauma and significantly increased at 4 h, with partial recovery at 24 h. At 4 h postinjury, severe trauma caused significantly greater decreases in aspartate and glutamate than did either mild or moderate injury. These findings are consistent with the postulated role of excitatory amino acids in CNS trauma.     

Neuromuscular synaptic transmission in Limulus polyphemus--II. Release of amino acid putative transmitters from the neuromuscular preparation

High-performance liquid chromatography with fluorescence detection was used to assay the release of putative amino acid transmitters from the Limulus neuromuscular preparation. Motor axon stimulation increased the concentrations of aspartate, glutamate and eight other amino acids in fluid bathing the neuromuscular preparation. Pentobarbital, which attenuates the excitatory postsynaptic potential of Limulus muscle, was used to block both synaptic activation of muscle fibers and any amino acid release that may have resulted from this activation. Stimulus-induced release of glutamate and five other amino acids was blocked by pentobarbital, while release of aspartate and three other amino acids was unaffected; a result which suggests that the latter group of amino acids was released presynaptically. Aspartate is the only physiologically active compound in this group. Consideration is given both to the difficulties involved in interpreting sites of amino acid release and to the problem of using pentobarbital as a presumed postsynaptic antagonist. The evidence concerning the relative merits of either aspartate or glutamate as the natural excitatory transmitter at the Limulus neuromuscular junction is discussed.     

Stress-Induced Dopamine Release in the Neostriatum: Evaluation of the Role of Action Potentials in Nigrostriatal Dopamine Neurons or Local Initiation by Endogenous Excitatory Amino Acids

Abstract: It has been hypothesized that excitatory amino acids can initiate dopamine release in neostriatum. We examined whether the increase in extracellular dopamine in neostriatum produced by acute stress reflects presynaptic initiation of dopamine release by endogenous excitatory amino acids. Thirty minutes of intermittent tail-shock stress significantly elevated extracellular concentrations of dopamine, glutamate, aspartate, and γ-aminobutyric acid in neostriatum of freely moving rats as measured with in vivo microdialysis. Local infusion of the N -methyl- d -aspartate receptor antagonist 2-amino-5-phosphonovaler-ate or the non- N -methyl- d -aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione via the dialysis probe did not attenuate the stress-induced increase in extra cellular dopamine. In fact, the increase was prolonged in rats treated with specific excitatory amino acid receptor antagonists. Infusion of tetrodotoxin into medial forebrain bundle increased extra cellular glutamate and aspartate in neostriatum yet reduced basal dopamine in extra cellular fluid to below the limit of detection of the assay and eliminated the stress-induced increase in extra cellular dopamine. These findings fail to support the hypothesis that the stress-induced increase in extra cellular dopamine in neostriatum is initiated locally by excitatory amino acids. Rather, the effects of stress on extra cellular dopamine seem to be determined by impulse propagation in dopamine neurons.     

Protein kinase C activation decreases cell surface expression of the GLT-1 subtype of glutamate transporter. Requirement of a carboxyl-terminal domain and partial dependence on serine 486

Na(+)-dependent glutamate transporters are required for the clearance of extracellular glutamate and influence both physiological and pathological effects of this excitatory amino acid. In the present study, the effects of a protein kinase C (PKC) activator on the cell surface expression and activity of the GLT-1 subtype of glutamate transporter were examined in two model systems, primary co-cultures of neurons and astrocytes that endogenously express GLT-1 and C6 glioma cells transfected with GLT-1. In both systems, activation of PKC with phorbol ester caused a decrease in GLT-1 cell surface expression. This effect is opposite to the one observed for the EAAC1 subtype of glutamate transporter (Davis, K. E., Straff, D. J., Weinstein, E. A., Bannerman, P. G., Correale, D. M., Rothstein, J. D., and Robinson, M. B. (1998) J. Neurosci. 18, 2475-2485). Several recombinant chimeric proteins between GLT-1 and EAAC1 transporter subtypes were generated to identify domains required for the subtype-specific redistribution of GLT-1. We identified a carboxyl-terminal domain consisting of 43 amino acids (amino acids 475-517) that is required for PKC-induced GLT-1 redistribution. Mutation of a non-conserved serine residue at position 486 partially attenuated but did not completely abolish the PKC-dependent redistribution of GLT-1. Although we observed a phorbol ester-dependent incorporation of (32)P into immunoprecipitable GLT-1, mutation of serine 486 did not reduce this signal. We also found that chimeras containing the first 446 amino acids of GLT-1 were not functional unless amino acids 475-517 of GLT-1 were also present. These non-functional transporters were not as efficiently expressed on the cell surface and migrated to a smaller molecular weight, suggesting that a subtype-specific interaction is required for the formation of functional transporters. These studies demonstrate a novel effect of PKC on GLT-1 activity and define a unique carboxyl-terminal domain as an important determinant in cellular localization and regulation of GLT-1.     

Disturbances of Amino Acids from Temporal Lobe Synaptosomes in Human Complex Partial Epilepsy

We have studied the levels of neuroactive amino acids in synaptosomes (P₂ fraction) isolated from brain tissue of ten patients with medically intractable epilepsy who were undergoing temporal lobectomy. First, lateral temporal tissue (nonfocal) was removed followed by medial temporal tissue (focal). A synaptosomal fraction (P₂) was immediately prepared from each tissue and analyzed for free amino acid concentrations. Statistically significant reductions were seen in glutamine and GABA concentrations in focal tissue compared to nonfocal tissue. The ratio of excitatory amino acids (aspartate and glutamate) to inhibitory amino acids (taurine and GABA) was significantly higher in focal tissue compared to nonfocal. The glutamine/glutamate ratio was significantly reduced. These data support the hypothesis that alterations in the balance between excitatory and inhibitory amino acids may be involved in the expression of epilepsy.     

Two distinct modes of hypoosmotic medium-induced release of excitatory amino acids and taurine in the rat brain in vivo

A variety of physiological and pathological factors induce cellular swelling in the brain. Changes in cell volume activate several types of ion channels, which mediate the release of inorganic and organic osmolytes and allow for compensatory cell volume decrease. Volume-regulated anion channels (VRAC) are thought to be responsible for the release of some of organic osmolytes, including the excitatory neurotransmitters glutamate and aspartate. In the present study, we compared the in vivo properties of the swelling-activated release of glutamate, aspartate, and another major brain osmolyte taurine. Cell swelling was induced by perfusion of hypoosmotic (low [NaCl]) medium via a microdialysis probe placed in the rat cortex. The hypoosmotic medium produced several-fold increases in the extracellular levels of glutamate, aspartate and taurine. However, the release of the excitatory amino acids differed from the release of taurine in several respects including: (i) kinetic properties, (ii) sensitivity to isoosmotic changes in [NaCl], and (iii) sensitivity to hydrogen peroxide, which is known to modulate VRAC. Consistent with the involvement of VRAC, hypoosmotic medium-induced release of the excitatory amino acids was inhibited by the anion channel blocker DNDS, but not by the glutamate transporter inhibitor TBOA or Cd2+, which inhibits exocytosis. In order to elucidate the mechanisms contributing to taurine release, we studied its release properties in cultured astrocytes and cortical synaptosomes. Similarities between the results obtained in vivo and in synaptosomes suggest that the swelling-activated release of taurine in vivo may be of neuronal origin. Taken together, our findings indicate that different transport mechanisms and/or distinct cellular sources mediate hypoosmotic medium-induced release of the excitatory amino acids and taurine in vivo.     

Functional analysis of Caenorhabditis elegans glutamate receptor subunits by domain transplantation

Glutamate receptors are not only abundant and important mediators of fast excitatory synaptic transmission in vertebrates, but they also serve a similar function in invertebrates such as Drosophila and the nematode Caenorhabditis elegans. In C. elegans, an animal with only 302 neurons, 10 different glutamate receptor subunits have been identified and cloned. To study the ion channel properties of these receptor subunits, we recorded glutamate-gated currents from Xenopus oocytes that expressed either C. elegans glutamate receptor subunits or chimeric rat/C. elegans glutamate receptor subunits. The chimeras were constructed between the C. elegans glutamate receptor pore domains and either the rat kainate receptor subunit GluR6, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit GluR1, or the N-methyl-d-aspartate (NMDA) receptor subunit NMDAR1-1a. Although native subunits were nonfunctional, 9 of 10 ion pores were found to conduct current upon transplantation into rat receptor subunits. A provisional classification of the C. elegans glutamate receptor subunits was attempted based on functionality of the chimeras. C. elegans glutamate receptor ion pores, at a position homologous to a highly conserved site critical for ion permeation properties in vertebrate glutamate receptor pores, contain amino acids not found in vertebrate glutamate receptors. We show that the pore-constricting Q/R site, which in vertebrate receptors determines calcium permeability and rectification properties of the ion channel, in C. elegans can be occupied by other amino acids, including, surprisingly, lysine and proline, without loss of these properties.