Presynaptic Glutamate/Quisqualate Receptors: Effects on Synaptosomal Free Calcium Concentrations

Intracellular free [Ca2+]i was measured using fura-2 in synaptosomes prepared from cerebral cortices of adult male rats (12 weeks). L-(+)-Glutamate, D-(-)-glutamate, and quisqualate produced similar dose-dependent increases in [Ca2+]i, with EC50 values of 0.38 microM, 0.74 microM, and 0.1 microM, respectively, and maximum increases of approximately 40%. Ibotenate showed less affinity (EC50 4.4 microM) but had a greater maximum effect (57%). N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) did not increase [Ca2+]i. The increases in [Ca2+]i induced by quisqualate and ibotenate were not diminished in the absence of extrasynaptosomal Ca2+. L-2-Amino-4-phosphonobutyrate (L-AP4) (1 microM) completely blocked the changes in [Ca2+]i induced by L-(+)-glutamate, D-(-)-glutamate, quisqualate, or ibotenate. The effects of quisqualate and ibotenate on [Ca2+]i were also blocked by coincubation of synaptosomes with L-(+)-serine-O-phosphate (L-SP) (1 mM) (which, like L-AP4, blocks the effects of quisqualate and ibotenate on inositol phospholipid metabolism). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) had no effect on agonist-mediated increases in [Ca2+]i when coincubated with either quisqualate or ibotenate. These data are consistent with the existence of presynaptic glutamate receptors (of the excitatory amino acid metabotropic type) which activate phospholipase C leading to the elevation of inositol 1,4,5-trisphosphate and release of Ca2+ from intracellular stores.     

Glutamate Inhibition of the Adrenergic-Stimulated Production of Melatonin in Rat Pineal Gland In Vitro

Abstract: The effect of l -glutamate on the adrenergic-stimulated release of melatonin in the rat pineal gland was examined using an in vitro perfusion system. l -Glutamate by itself had no effect on melatonin secretion whereas l -glutamate administered prior to (–)-isoproterenol (β-adrenergic agonist) and l -phenylephrine (α-adrenergic agonist) inhibited melatonin production by 42%. l -Glutamate did not inhibit melatonin secretion when glands were stimulated with (–)-isoproterenol alone. d -Glutamate, as well as the l -glutamate agonists kainate, N -methyl- d -aspartate, quisqualate, and trans -1-aminocyclopentane-1, 3-dicarboxylic acid, had no effect on the (–)-isoproterenol-and l -phenylephrine-stimulated secretion of melatonin, which suggests that the inhibitory effects of glutamate are not mediated via any of the known glutamate receptor subtypes. The possibility that l -glutamate may be converted to another neuroactive compound (GABA) prior to the addition of (–)-isoproterenol and l -phenylephrine is suggested by the observation that simultaneous administration of l -glutamate with (–)-isoproterenol and l -phenylephrine did not inhibit melatonin production.     

Pharmacology of Sodium-Dependent High-Affinity l-[3H]Glutamate Transport in Glial Cultures

Abstract: Pharmacological and molecular biological studies provide evidence for subtypes of sodium-dependent high-affinity glutamate (Glu) transport in the mammalian CNS. At least some of these transporters appear to be selectively expressed in different brain regions or by different cell types. In the present study, the properties of l -[³H]Glu transport were characterized using astrocyte-enriched cultures prepared from cerebellum and cortex. In both brain regions, the kinetic data for sodium-dependent transport were consistent with a single site with K_m values of 91 ± 17 µM in cortical glial cells and 66 ± 23 µM in cerebellar glial cells. The capacities were 6.1 ± 1.6 nmol/mg of protein/min in cortical glial cells and 8.4 ± 0.9 nmol/mg of protein/min in cerebellar glial cells. The potencies of ～40 excitatory amino acid analogues for inhibition of sodium-dependent transport into glial cells prepared from cortex and cerebellum were examined, including compounds that are selective inhibitors of transport in synaptosomes prepared from either cerebellum or cortex. Of the analogues tested, 14 inhibited transport activity by >50% at 1 mM concentrations. Unlike l -[³H]Glu transport in synaptosomes prepared from cerebellum or cortex, there were no large differences between the potencies of compounds for inhibition of transport measured in glial cells prepared from these two brain regions. With the exception of (2S,1′R,2′R)-2-(carboxycyclopropyl)glycine and l -α-aminoadipate, all of the compounds examined were ～10–200-fold less potent as inhibitors of l -[³H]Glu transport measured in glial cells than as inhibitors of transport measured in synaptosomes prepared from their respective brain regions. The pharmacology of transport measured in these glial cells differs from the reported pharmacology of the cloned Glu transporters, suggesting the existence of additional uncloned Glu transporters or Glu transporter subunits.     

Subtypes of Sodium-Dependent High-Affinity L-[3H]Glutamate Transport Activity: Pharmacologic Specificity and Regulation by Sodium and Potassium

Abstract: Some data suggest that the sodium-dependent, high-affinity L-glutamate (Glu) transport sites in forebrain are different from those in cerebellum. In the present study, sodium-dependent transport of L-[³H]Glu was characterized in cerebellum and cortex. In both cerebellar and cortical tissue, activity was enriched in synaptosomes. Approximately 100 excitatory amino acid analogues were tested as potential inhibitors of transport activity. Many of the compounds tested inhibited transport activity by <65% at 1 mM and were not studied further. One group of compounds exhibited inhibition conforming to theoretical curves with Hill coefficients of 1 and were <10-fold selective as inhibitors of transport activity. These included three of the putative endogenous substrates for transport: L-Glu, L-aspartate, and L-cysteate. Four of the compounds exhibited inhibition conforming to theoretical curves with Hill coefficients of 1 and were > 10-fold selective as inhibitors. These included β-N-oxalyl-L-α,β-diaminopropionate, α-methyl-DL-glutamate, (2S, 1′S,2′S)-2-(carboxycyclopropyl)glycine, and (2S, 1′S,2′S,3′S)-2-(2-carboxy-3-methoxymethylcyclopropyl)glycine. Data obtained with a few of the inhibitors were consistent with two sites in one or both of the brain regions. (2S, 1′R,2′R)-2-(Carboxycyclopropyl)glycine (L-CCG-II) was identified as the most potent (IC₅₀= 5.5 μM) and selective (60–100-fold) inhibitor of transport activity in cerebellum. One of the potential endogenous substrates, L-homocysteate, was also a selective inhibitor of cerebellar transport activity. The data for inhibition of transport activity in cortex by both L-CCG-II and L-homocysteate were best fit to two sites. Kainate was equipotent as an inhibitor of transport activity, and in both brain regions the data for inhibition were best fit to two sites. The possibility that there are four subtypes of excitatory amino acid transport is discussed. Altering sodium and potassium levels affects cerebellar and cortical transport activity differently, suggesting that the differences extend to other recognition sites on these transporters.     

Identification and Characterization of an N-Methyl-D-Aspartate-Specific L-[3H]Glutamate Recognition Site in Synaptic Plasma Membranes

Conditions have been developed for an L-[3H]glutamate binding assay in which 85-95% of the specific binding is to a site that corresponds to the N-methyl-D-aspartate subclass of acidic amino acid receptors. Incubation of synaptic plasma membranes with L-[3H]glutamate in 50 mM Tris/acetate, pH 7.4, for 2-20 min at 2 degrees C results in binding with pharmacological characteristics of the electrophysiologically defined N-methyl-D-aspartate receptor. The fraction of glutamate binding to this subclass of receptors, relative to the total, decreases with both increased time and temperature. This binding is reversible, is concentrated in the synaptic plasma membrane fraction, has a pH optimum of 7.0-7.4, and is linear with respect to tissue protein concentration. The binding is unaffected by 1 mM concentrations of the anions sulfate, chloride, bromide, thiocyanate, phosphate, acetate, nitrate, or carbonate and the monovalent cations potassium or ammonium. However sodium and the divalent cations copper, cobalt, zinc, cadmium, and manganese decrease binding to this N-methyl-D-aspartate site.     

Glutamate Neurotoxicity in Culture Depends on the Presence of Glutamine: Implications for the Role of Glial Cells in Normal and Pathological Brain Development

Glutamate toxicity was studied in neuronal (SC9), glial (WC5), and neuroblastoma-glioma hybrid cell lines. In all three cell types, glutamate had a dual effect, depending on the concentration of glutamine in the culture medium. An expected dose-dependent cytotoxicity of the amino acid was observed when cells were cultured in medium containing the standard glutamine concentration (1-4 mM), but when the culture's glutamine content was decreased to 0.15-0.5 mM, glutamate had an apparent opposite, growth-promoting effect. The specificity of glutamate effect was indicated by the following: (a) it was stereospecific, with the L and not the D isomer being active; (b) monosodium aspartate was inactive in the presence of either high or low glutamine; and (c) monosodium glutamate and monopotassium glutamate had a similar dual effect. Furthermore, the glutamate receptor antagonist gamma-glutamylglycine blocked the amino acid cytotoxicity in a dose-dependent fashion. As glial cells are a major source of glutamine in the brain, neuronal-glial co-cultures were used to analyze the possible role of glial cells in glutamate neurotoxicity. It was found that SC9 cells were more sensitive to glutamate when co-cultured with WC5 cells. Continuous depolarization of the SC9 cells with KCl decreased cell number, but glutamate had no additive neurotoxic effect when added with KCl. We suggest that glutamine, glial cells, and neuronal activation play roles in modulating glutamate neurotoxicity, in developing as well as aged brains. It is tempting to speculate also that alterations in the glutamate/glutamine ratio under pathological conditions may take part in the etiology of some neurodegenerative diseases.     

A Novel Chloride-Dependent L-[3H]Glutamate Binding Site in Astrocyte Membranes

Membrane fractions prepared from astrocytes grown in culture exhibit a specific binding site for L-[3H]glutamate that is Cl--dependent and Na+-independent. The binding site is a single saturable site with a KD of about 0.5 microM, is inhibited by L-aspartate, L-cysteate, and quisqualate, and is insensitive to kainate, N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate, and 2-amino-4-phosphonobutyrate. The pharmacological characteristics of the binding site indicate that it is distinct from any site previously described in synaptic membrane preparations. Comparisons of ionic requirements, ligand specificity, and inhibitor sensitivities, however, suggest the described binding is the first step in a Cl--dependent high-affinity glutamate uptake system. Such binding studies provide a useful model system in which to investigate the close association between excitatory amino acids, astrocytes, the termination of glutamate's excitatory action by high-affinity uptake, and the excitotoxic action of acidic amino acids in membranes of a single cell type.     

On the Origin of Extracellular Glutamate Levels Monitored in the Basal Ganglia of the Rat by In Vivo Microdialysis

Abstract: Several putative neurotransmitters and metabolites were monitored simultaneously in the extracellular space of neostriatum, substantia nigra, and cortex and in subcutaneous tissue of the rat by in vivo microdialysis. Glutamate (Glu) and aspartate (Asp) were at submicromolar and γ-aminobutyric acid (GABA) was at nanomolar concentrations in all brain regions. The highest concentration of dopamine (DA) was in the neostriatum. Dynorphin B (Dyn B) was in the picomolar range in all brain regions. Although no GABA, DA, or Dyn B could be detected in subcutaneous tissue, Glu and Asp levels were ≈5 and ≈0.4 µM, respectively. Lactate and pyruvate concentrations were ≈200 and ≈10 µM in all regions. The following criteria were applied to ascertain the neuronal origin of substances quantified by microdialysis: sensitivity to (a) K⁺ depolarization, (b) Na⁺ channel blockade, (c) removal of extracellular Ca²⁺, and (d) depletion of presynaptic vesicles by local administration of α-latrotoxin. DA, Dyn B, and GABA largely satisfied all these criteria. In contrast, Glu and Asp levels were not greatly affected by K⁺ depolarization and were increased by perfusing with tetrodotoxin or with Ca²⁺-free medium, arguing against a neuronal origin. However, Glu and Asp, as well as DA and GABA, levels were decreased under both basal and K⁺-depolarizing conditions by α-latrotoxin. Because the effect of K⁺ depolarization on Glu and Asp could be masked by reuptake into nerve terminals and glial cells, the reuptake blocker dihydrokainic acid (DHKA) or l -trans-pyrrolidine-2,4-dicarboxylic acid (PDC) was included in the microdialysis perfusion medium. The effect of K⁺ depolarization on Glu and Asp levels was increased by DHKA, but GABA levels were also affected. In contrast, PDC increased only Glu levels. It is concluded that there is a pool of releasable Glu and Asp in the rat brain. However, extracellular levels of amino acids monitored by in vivo microdialysis reflect the balance between neuronal release and reuptake into surrounding nerve terminals and glial elements.     

Binding Sites for [3H]Glutamate and [3H]Aspartate in Human Cerebellum

Abstract The binding of [³H]aspartate and [³H]glutamate to membranes prepared from frozen human cerebellar cortex was studied. The binding sites differed in their relative proportions, their inhibition by amino acids and analogues, and by the effects of cations. A proportion (about 30%) of [³H]glutamate binding was to sites similar to those labelled by [³H]aspartate. An additional component of [³H]gluta-mate binding (about 50%) was displaced by quisqualate and aL-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and may represent a “quisqualate-preferring” receptor. Neither N-methyl-d-aspartic acid-sensitive nor dl-2-amino-4-phosphonobutyric acid-sensitive [³H]glutamate binding was detected.     

Regulation of human lymphocyte proliferation by fatty acids

In the present study, the effect of increasing concentrations of palmitic (PA, C16:0), stearic (SA, C18:0), oleic (OA, C18:1, n-9), linoleic (LA, C18:2n-6), docosahexaenoic (DHA, C22:6 n-3) and eicosapentaenoic (EPA, C20:5 n-3) acids on lymphocyte proliferation was investigated. The maximal non-toxic concentrations of these fatty acids for human lymphocytes in vitro were determined. It was also evaluated whether these fatty acids at non-toxic concentrations affect IL-2 induced lymphocyte proliferation and cell cycle progression. OA and LA at 25 microM increased lymphocyte proliferation and at higher concentrations (75 microM and 100 microM) inhibited it. Both fatty acids promoted cell death at 200 microM concentration. PA and SA decreased lymphocyte proliferation at 50 microM and promoted cell death at concentrations of 100 microM and above. EPA and DHA decreased lymphocyte proliferation at 25 and 50 microM being toxic at 50 and 100 microM, respectively. PA, SA, DHA and EPA decreased the stimulatory effect of IL-2 on lymphocyte proliferation, increasing the percentage of cells in G1 phase and decreasing the proportion of cells in S and G2/M phases. OA and LA caused an even greater pronounced effect. The treatment with all fatty acids increased neutral lipid accumulation in the cells but the effect was more pronounced with PA and DHA. In conclusion, PA, SA, DHA and EPA decreased lymphocyte proliferation, whereas OA and LA stimulated it at non-toxic concentrations.     

Novel Glutamate Receptor Antagonists Selectively Protect Against Kainic Acid Neurotoxicity in Cultured Cerebral Cortex Neurons

The effect on excitatory amino acid (EAA)-induced toxicity of two novel non-N-methyl-D-aspartate (non-NMDA) antagonists 2-amino-3-[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propionic acid (AMOA) and 2-amino-3-[2-(3-hydroxy-5-methyl-isoxazol-4-yl)methyl-5-methyl-3- oxoisoxazolin-4-yl]propionic acid (AMNH) was tested in primary cultures of cerebral cortex neurons. Such cultures provide a useful model for the investigation of the toxicity of EAAs and a convenient screening system for potential neuroprotective activity of pharmacological agents. It was demonstrated that AMNH and AMOA abolished neurotoxicity induced by kainic acid with IC50 values of 62 +/- 10 and 120 +/- 19 microM, respectively. No effect on neuronal damage induced by NMDA or AMPA could be detected.     

Effect of Acute Stress on Hippocampal Glutamate Levels and Spectrin Proteolysis in Young and Aged Rats

Abstract: Aging in rats is associated with a loss of hippocampal neurons, which may contribute to age-related cognitive deficits. Several lines of evidence suggest that stress and glucocorticoids may contribute to age-related declines in hippocampal neuronal number. Excitatory amino acids (EAAs) have been implicated in the glucocorticoid endangerment and stress-induced morphological changes of hippocampal neurons of young rats. Previously, we have reported that acute immobilization stress can increase extracellular concentrations of the endogenous excitatory amino acid, glutamate, in the hippocampus. The present study examined the effect of an acute bout of immobilization stress on glutamate levels in the hippocampus and medial prefrontal cortex of young (3–4-month) and aged (22–24-month) Fischer 344 rats. In addition, the effect of stress on spectrin proteolysis in these two brain regions was also examined. Spectrin is a cytoskeleton protein that contributes to neuronal integrity and proteolysis of this protein has been proposed as an important component of EAA-induced neuronal death. There was no difference in basal glutamate levels between young and old rats in the hippocampus or medial prefrontal cortex. During the period of restraint stress a modest increase in glutamate levels in the hippocampus of young and aged rats was observed. After the termination of the stress procedure, hippocampal glutamate concentrations continued to rise in the aged rats, reaching a level approximately five times higher than the young rats, and remained elevated for at least 2 h after the termination of the stress. A similar pattern was also observed in the medial prefrontal cortex with an augmented post-stress-induced glutamate response observed in the aged rats. There was no increase in spectrin proteolysis in the hippocampus or medial prefrontal cortex of young or aged rats after stress or under basal nonstress conditions. The enhanced poststress glutamate response in the aged rats may contribute to the increased sensitivity of aged rats to neurotoxic insults.     

Effects of Ionotropic Excitatory Amino Acid Receptor Antagonists on Glutamate Transport and Transport-Mediated Changes in Extracellular Excitatory Amino Acids in the Rat Striatum

Abstract: This study examined the effects of intrastriatal administration of ionotropic excitatory amino acid receptor antagonists on biochemical markers of excitatory amino acid transmission in the rat striatum. High-affinity glutamate uptake was measured ex vivo on striatal homogenates 15 min after the local administration of either 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA receptor antagonist, or dl -2-amino-5-phosphonopentanoic acid (AP5), a competitive NMDA antagonist, at various doses (10–500 pmol injected). DNQX induced a dose-dependent increase in glutamate uptake rate, related to an increase in the V _max of the transport process, whereas no significant change in glutamate uptake was detected after AP5 administration. Similar results were obtained from animals subjected to excitotoxic lesion of striatal neurons by kainate administration 15 days before the injection of DNQX or AP5. In a parallel series of experiments using in vivo microdialysis we showed that DNQX (10⁻⁵ M ) in the dialysis probe diminished by ∼30–40% the increases in the concentrations of glutamate and aspartate elicited by l - trans -pyrrolidine-2,4-dicarboxylic acid (1 m M ). These data suggest that presynaptic glutamate transmission in the rat striatum may undergo facilitatory autoregulatory processes involving ionotropic non-NMDA receptors and highlight the view that transporters for glutamate may be potent regulatory sites for glutamatergic transmission.     

Glutamate Stimulation of [3H]Dopamine Release from Dissociated Cell Cultures of Rat Ventral Mesencephalon

In dissociated cell cultures of fetal rat ventral mesencephalon preloaded with [3H]dopamine, glutamate (10(-5)-10(-3) M) stimulated the release of [3H]dopamine. Glutamate stimulation of [3H]dopamine release was Ca2+ dependent and was blocked by the glutamate antagonist, cis-2,3-piperidine dicarboxylic acid. Glutamate stimulation of [3H]dopamine release was not due to glutamate neurotoxicity because (1) glutamate did not cause release of a cytosolic marker, lactate dehydrogenase, and (2) preincubation of cultures with glutamate did not impair subsequent ability of the cells to take up or release [3H]dopamine. Thus, these dissociated cell cultures appear to provide a good model system to characterize glutamate stimulation of dopamine release. Release of [3H]dopamine from these cultures was stimulated by veratridine, an activator of voltage-sensitive Na+ channels, and this stimulation was blocked by tetrodotoxin. However, glutamate-stimulated [3H]dopamine release was not blocked by tetrodotoxin or Zn2+. Substitution of NaCl in the extracellular medium by sucrose, LiCl, or Na2SO4 had no effect on glutamate stimulation of [3H]dopamine release; however, release was inhibited when NaCl was replaced by choline chloride or N-methyl-D-glucamine HCl. Glutamate-stimulated [3H]-dopamine release was well maintained (60-82% of control) in the presence of Co2+, which blocks Ca2+ action potentials, and was unaffected by the local anesthetic, lidocaine. These results are discussed in terms of the receptor and ionic mechanisms involved in the stimulation of dopamine release by excitatory amino acids.     

Glutamate-Evoked Release of Endogenous Adenosine from Rat Cortical Synaptosomes Is Mediated by Glutamate Uptake and Not by Receptors

L-Glutamate (10 microM-1 mM) released endogenous adenosine from rat cortical synaptosomes. Studies with excitatory amino acid antagonists, (+)-5-methyl-16,11,dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801), 6,7-dinitroquinoxaline-2,3-dione (DNQX), Mg2+, and agonists N-methyl-D-aspartate (NMDA), kainate, and quisqualate, indicated that this release was not receptor mediated. D,L-2-Amino-4-phosphonobutanoic acid (APB) also did not affect glutamate-evoked adenosine release. Inhibition of glutamate uptake by dihydrokainate or replacement of extracellular Na+ blocked glutamate-evoked adenosine release. D-aspartate, which is a substrate for the glutamate transporter but is not metabolized, also released adenosine, suggesting that release was due to amino acid transport and not to its subsequent metabolism. D-Glutamate, a relatively poor substrate for the transporter, was correspondingly less potent than L-glutamate at releasing adenosine. Glutamate-evoked adenosine release was not Ca2+ dependent or tetrodotoxin sensitive and did not appear to occur on the bidirectional nucleoside transporter. Inhibition of ecto-5'-nucleotidase virtually abolished glutamate-evoked adenosine release, indicating that adenosine was derived from extracellular metabolism of released nucleotide(s). However, L-glutamate did not release ATP and did not appear to release cyclic AMP. Therefore, transport of glutamate into presynaptic terminals releases some other nucleotide which is converted extracellularly to adenosine. This adenosine could act at P1-purinoceptors to modulate glutamatergic neurotransmission.     

Effects of L-arginine and L-cysteine on growth,and chlorophyll and mineral contents of shoots of the apple rootstock EM 26 cultured <Emphasis Type="Italic">in vitro</Emphasis>

1, 5, or 10 mM arginine and 25, 50, or 100 μM cysteine were added in the Murashige and Skoog medium. By increasing arginine concentration the number of shoots per explant increased. Inclusion of 50 μM cysteine in the medium resulted in maximum number of shoots but it was not significantly different in comparison to 10 mM arginine. The chlorophyll content was significantly increased in explants treated with 10 mM arginine in comparison to the control, 1 mM arginine and 25 μM cysteine. By increasing arginine and cysteine concentrations of the medium, N, K, and Ca contents of explants increased but no significant changes in P, Mg, Fe, Mn, Zn, and B contents were observed.     

Regulation of Glutamate and Aspartate Release from Slices of the Hippocampal CA1 Area: Effects of Adenosine and Baclofen

Glutamate and/or aspartate is the probable transmitter released from synaptic terminals of the CA3-derived Schaffer collateral, commissural, and ipsilateral associational fibers in area CA1 of the rat hippocampal formation. Slices of the CA1 area were employed to test the effects of adenosine- and gamma-aminobutyrate (GABA)-related compounds on the release of glutamate and aspartate from this projection. Under the conditions of these experiments, the release of glutamate and aspartate evoked by 50 mM K+ was more than 90% Ca2+-dependent and originated predominantly from the CA3-derived pathways. Adenosine reduced the K+-evoked release of glutamate and aspartate by a maximum of about 60%, but did not affect the release of GABA. This action was reversed by 1 microM 8-phenyltheophylline. The order of potency for adenosine analogues was as follows: L-N6-phenylisopropyladenosine greater than N6-cyclohexyladenosine greater than D-N6-phenylisopropyladenosine approximately equal to 2-chloroadenosine greater than adenosine much greater than 5'-N-ethylcarboxamidoadenosine. 8-Phenyltheophylline (10 microM) by itself enhanced glutamate/aspartate release, whereas dipyridamole alone depressed release. These results support the view that adenosine inhibits transmission at Schaffer collateral-commissural-ipsilateral associational synapses mainly by reducing transmitter release and that these effects involve the activation of an A1 receptor. Neither adenosine, L-N6-phenylisopropyladenosine, nor 8-phenyltheophylline affected the release of glutamate or aspartate evoked by 10 microM veratridine. The differing effects of adenosine compounds on release evoked by K+ and veratridine suggest that A1 receptor activation either inhibits Ca2+ influx through the voltage-sensitive channels or interferes with a step subsequent to Ca2+ entry that is coupled to the voltage-sensitive Ca2+ channels in an obligatory fashion. Neither baclofen nor any other agent active at GABAB or GABAA receptors affected glutamate or aspartate release evoked by elevated K+ or veratridine. Therefore, either baclofen does not inhibit transmission at these synapses by depressing transmitter release or else it does so in a way that cannot be detected when a chemical depolarizing agent is employed.     

Changes in Extracellular Concentrations of Glutamate, Aspartate, Glycine, Dopamine, Serotonin, and Dopamine Metabolites After Transient Global Ischemia in the Rabbit Brain

Although considerable evidence supports a role for excitatory amino acids in the pathogenesis of ischemic neuronal injury, few in vivo studies have examined the effect of increasing durations of ischemia on the extracellular concentrations of these agents. Recently, other neurotransmitters (e.g., glycine and dopamine) have been implicated in the mechanism of ischemic neuronal injury. Accordingly, this study was undertaken to examine the patterns of changes of extracellular glutamate, aspartate, glycine concentrations in the hippocampus, and dopamine, serotonin, and dopamine metabolites in the caudate nucleus with varying durations (5, 10, or 15 minutes) of transient global cerebral ischemia as evidence to support their pathogenetic roles. Microdialysis was used to sample the brain's extracellular space before, during, and after the ischemic period. Glutamate and aspartate concentrations in the dialysate increased from baseline by 1-, 5-, and 13-fold and by 4-, 9-, and 31-fold, respectively, for the three ischemic durations. The concentrations returned to baseline rapidly after reperfusion. The peak concentrations of glutamate and aspartate were significantly higher with increasing ischemic duration. Dopamine concentrations increased by approximately 700-fold in response to all three ischemic durations and returned to baseline within 10 min of reperfusion. Glycine, in contrast, increased during ischemia by a mean of 4-fold, but remained elevated throughout the 80-min period of reperfusion. The final concentrations of glycine were significantly higher than baseline levels (p = 0.0002, Mann-Whitney test). That glutamate and aspartate concentrations in the hippocampus co-vary with the duration of global ischemia is taken as supportive evidence of their pathogenetic role in ischemic neuronal injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spinal cord ischemia-induced elevation of amino acids: Extracellular measurement with microdialysis

Excitatory amino acids have been implicated in the production of calcium mediated neuronal death following central nervous system ischemia. We have used microdialysis to investigate changes in the extracellular concentrations of amino acids in the spinal cord after aortic occlusion in the rabbit. Glutamate, aspartate, glutamine, asparagine, glycine, taurine, valine, and leucine were measured in the micordialysis perfusate by high pressure liquid chromatography. The concentrations of glutamate, glycine, and taurine were significantly higher during ischemia and reperfusion than controls. Delayed elevations in the concentrations of asparagine and valine were also detected. The elevation of glutamate is consistent with the hypothesis that excitotoxins may mediate neuronal damage in the ischemic spinal cord. Increased extracellular concentrations of asparagine and valine may reflect preferential use of amino acids for energy metabolism under ischemic conditions. The significance of increased concentrations of inhibitory amino acid neurotransmitters is unclear.     

Cytokine and nitric oxide responses of monocyte-derived human macrophages to microsporidian spores

Microsporidia are obligate intracellular parasites that emerged as opportunistic pathogens since the onset of the AIDS pandemic. They are capable of disseminating through the body using macrophages as vehicles. We incubated human macrophages with spores of all three Encephalitozoon spp. as well as with Vittaforma corneae, and the number of intracellular spores per cell was determined by fluorescence microscopy. Cell culture supernatants were collected and the content of TNF-alpha, INF-gamma, IL-10, and of nitric oxide was determined. Microsporidian spores did not induce a nitric oxide response in macrophages and there was a negative correlation between the number of intracellular spores and the amount of nitric oxide. TNF-alpha, INF-gamma, and IL-10 increased after simulation of macrophages with microsporidian spores but for TNF-alpha and INF-gamma no clear correlation of cytokine levels with the number of intracellular spores could be observed. A modulation of the nitric oxide response by intracellular microsporidia may contribute to the survival of microsporidia within the macrophage by a mechanism yet unknown.