Release Of Endogenous Aspartate Research Articles

Release of d, l- threo-β-hydroxyaspartate as a false transmitter from excitatory amino acid-releasing nerve terminals

This study examined whether preaccumulated d, l- threo-ß-hydroxyaspartate (tHA), a competitive substrate for the high-affinity excitatory amino acid (EAA) transporter, is released as a false transmitter from EAA-releasing nerve terminals. Potassium-stimulation (50 mM for 1 min) evoked significant release of the endogenous EAAs (aspartate and glutamate) from superfused neocortical minislices. Endogenous EAA release was largely calcium-dependent and was inhibited by tetanus toxin, a neurotoxin which specifically blocks vesicular exocytosis. In parallel experiments, minislices were pre-incubated with 500 μM tHA. Potassium (50 mM) evoked significant release of tHA and this release was also calcium-dependent and reduced by tetanus toxin. Pre-accumulation of tHA did not affect the release of endogenous glutamate whereas the release of endogenous aspartate was significantly attenuated. These data suggest that tHA selectively accumulates in a vesicular aspartate pool and is released upon depolarization as a false transmitter from EAA nerve terminals.

Inhibition of evoked glutamate release by the neuroprotective 5-HT 1A receptor agonist BAY x 3702 in vitro and in vivo

Brain ischemia provoked by stroke or traumatic brain injury induces a massive increase in neurotransmitter release, in particular of the excitotoxin glutamate. Glutamate triggers a cascade of events finally leading to widespread neuronal cell damage and death. The aminomethylchroman derivative BAY x 3702 is a novel neuroprotectant which shows pronounced beneficial effects in various animal models of ischemic brain injury. As shown previously BAY x 3702 binds to 5-HT 1A receptors of different species in subnanomolar range and is characterized as a full receptor agonist. In this study we investigated the influence of BAY x 3702 on potassium-evoked glutamate release in vitro and ischemia-induced glutamate release in vivo. In rat hippocampal slices BAY x 3702 inhibited evoked glutamate release in a dose-dependent manner (IC 50=1 μM). This effect was blocked by the selective 5-HT 1A receptor antagonist WAY 100635, indicating that BAY x 3702 specifically acts via 5-HT 1A receptors. In vivo, release of endogenous aspartate and glutamate was measured in the cortex of rats by microdialysis before and after onset of permanent middle cerebral artery occlusion. Single dose administration of BAY x 3702 (1 μg/kg or 10 μg/kg i.v.) immediately after occlusion reduced the increase and total release of extracellular glutamate by about 50% compared to non-treated animals, whereas the extracellular aspartate levels were not significantly affected. Inhibition of glutamate release may therefore contribute to the pronounced neuroprotective efficacy of BAY x 3702 in various animal models of ischemic brain damage.

RELEASE OF GLUTAMATE IN THE NUCLEUS TRACTUS SOLITARII IN RESPONSE TO BAROREFLEX ACTIVATION IN RATS

Release of endogenous aspartate and glutamate from the region of the nucleus tractus solitarii was measured in vitro by perfusion methods and in vivo by microdialysis. Stimulation of the nucleus tractus solitarii with 35 mM potassium in vitro significantly increased extracellular concentrations of aspartate and glutamate. Glutamate and aspartate concentrations also increased with dialysis of 100 mM KCl into the nucleus tractus solitarii in vivo, but only changes in glutamate were significant. Experiments in vivo revealed that activation of the baroreflex by intravenous infusion of phenylephrine significantly increased glutamate in dialysates, while hypoventilation that accompanies baroreceptor activation and may activate chemoreceptors tended to increase aspartate but not glutamate.The demonstration that glutamate, but not aspartate, is released with activation of the baroreflex further supports the hypothesis that glutamate is a neurotransmitter of baroreceptor afferents terminating in the nucleus tractus solitarii.

Release of endogenous aspartate from rat cerebellum slices and synaptosomes: inhibition mediated by a 5-HT2 receptor and by a 5-HT1 receptor of a possibly novel subtype

The effects of 5-hydroxytryptamine (5-HT) and of a number of 5-HT receptor agonists and antagonists on the release of endogenous aspartate were investigated in rat cerebellum slices and synaptosomes depolarized with high K+. The release of endogenous aspartate evoked from slices by 35 mmol/l KCl and from synaptosomes by 15 mmol/l KCl was strongly (about 90%) calcium-dependent. In slices the release of aspartate was inhibited by exogenous 5-HT (0.1-100 nmol/l) in a concentration-dependent manner. The indoleamine was very potent, producing 30% inhibition at 0.1 nmol/l. The effect of 10 nmol/l 5-HT was partly but maximally counteracted by ketanserin (300-1000 nmol/l), a 5-HT2 receptor antagonist, but fully blocked by 300 nmol/l of the mixed 5-HT1/5-HT2 receptor antagonist methiothepin. The 5-HT1A receptor agonist 5-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT) and the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) inhibited the K(+)-evoked release of endogenous aspartate in a concentration-dependent manner. The effect of 8-OH-DPAT was antagonized by methiothepin, but not by ketanserin which fully antagonized the inhibition produced by DOI. In cerebellar synaptosomes the release of endogenous aspartate evoked by 15 mmol/l K+ was inhibited by exogenous 5-HT and by 8-OH-DPAT, but not by DOI. Methiothepin (100-300 nmol/l) antagonized the inhibitory effects of 100 nmol/l 5-HT or 8-OH-DPAT. However, 1000 nmol/l of various 5-HT receptor antagonists [ketanserin, methysergide, (--)-propranolol, spiperone or ICS 205-930] did not counteract the effect of 100 nmol/l 5-HT.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Climbing Fiber Deprivation on Release of Endogenous Aspartate, Glutamate, and Homocysteate in Slices of Rat Cerebellar Hemispheres and Vermis

Aspartate (Asp) and/or glutamate (Glu) have been proposed as putative excitatory transmitters released from synaptic terminals of the olivo-cerebellar climbing fiber afferents to the Purkinje cells. Investigations of the climbing fiber transmitter(s) separately for hemispheres and vermis were performed to examine whether the current controversy over the role of Asp as a neurotransmitter in the climbing fibers may be due to topographic differences. K(+)-induced Ca2(+)-dependent release of endogenous substances was investigated in slices of cerebellar hemisphere and vermis of control rats and those deprived of climbing fibers by 3-acetylpyridine (3-AP) treatment. A release of Asp and Glu, as well as a small but significant release of homocysteic acid (HCA) was confirmed in control rats. Climbing fiber deprivation by 3-AP treatment reduced the stimulated release of Asp by 48% in slices of cerebellar hemispheres, but not in vermis. Climbing fiber deprivation completely abolished the release of HCA in both hemispheres and vermis. The release of HCA, Asp, and Glu from slices of control and climbing fiber-deprived rats evoked by 50 mM K+ was greater than 90% Ca2(+)-dependent. These results support the hypothesis that Asp is a transmitter candidate of the climbing fibers projecting to the cerebellar hemispheres, but not to the vermis, and provide the first evidence that HCA can be linked to a specific pathway.

Characterization of the receptor-mediated sulphur amino acid-evoked release of [3H]d-aspartate from primary cultures of cerebellar granule cells

A continuous superfusion system was used to study the release of [(3)H]d-aspartate from primary cultures of mouse cerebellar granule cells. A number of neuroactive acidic sulphur amino acids (viz. cysteine sulphinate, cysteate, homocysteine sulphinate, homocysteate and S-sulpho-cysteine) of endogenous origin were shown to evoke a dose-dependent, saturable and stereospecific release of [(3)H]d-aspartate. Substitution of Ca(2+) in the superfusion medium by Co(2+) (which blocks voltage-gated Ca(2+)-channels) resulted in a marked attenuation of evoked release following stimulation by l-enantiomers of all sulphur amino acids tested, thus demonstrating a "Ca(2+)-dependent" release of [(3)H]d-aspartate. Cysteine sulphinate-evoked release of endogenous glutamate was shown to be Ca(2+)-dependent whereas negligible release of endogenous aspartate was observed. All sulphur amino acids inhibited to varying degrees, the intracellular accumulation of the [(3)H]tetraphenylphosphonium cation, an observation consistent with either partial or complete depolarization of the plasma membrane. The sulphur amino acid-evoked release of [(3)H]d-aspartate from the granule cells was inhibited by a variety of excitatory amino acid receptor antagonists. At a concentration range of antagonist between 0.1-500 ?M, the sulphur amino acid-evoked release of [(3)H]d-aspartate was inhibited by: (i) the selective, competitive N- methyl- d -aspartate receptor antagonists; 3-(+/-)-2-carboxypiperazin-4-yl) propyl-2-phosphonic acid and d-2-amino-5-phosphonopentanoic acid; (ii) the competitive quisqualate/kainate receptor antagonist, 6,7-dinitroquinoxalinedione; and (iii) the broad spectrum excitatory amino acid receptor antagonist, kynurenate. Such observations demonstrate that excitatory sulphur amino acid-evoked release of [(3)H]d-aspartate from primary cultures of cerebellar granule cells occurs primarily by a receptor-mediated event in which both N- methyl- d -aspartate and quisqualate/kainate receptors are involved. The findings of this study provide additional evidence to support the transmitter candidacy of certain endogenous neuroactive sulphur amino acids and furthermore, suggest the mechanism by which their epileptogenic and neurotoxic actions may be explained.

Spontaneous release of endogenous aspartate and glutamate from rat striatal slices is increased following destruction of local neurons by ibotenic acid.

We sought to determine in rat striatum whether the release of neurotransmitter amino acids aspartate (Asp), glutamate (Glu) and gamma-aminobutyric acid (GABA) were affected by local neurons. To do so, unilateral microinjections of ibotenic acid, and excitotoxin that destroys local neurons without affecting fibers of passage, were made into the striatum. Release of endogenous amino acids from lesioned and intact striatal slices were measured by HPLC one week later. The effectiveness and specificity of the lesion were confirmed by measuring the enzyme activity associated with extrinsic dopamine neurons (tyrosine hydroxylase; 111 +/- 14%), intrinsic GABA neurons (glutamic acid decarboxylase; 19 +/- 7%) and intrinsic acetylcholine neurons (choline acetyltranferase; 37 +/- 10%). Destruction of local striatal neurons markedly attenuated the release of GABA (41 +/- 12% of control) elicited by depolarization with K+ (35 mM), but did not significantly reduced the K+-evoked release of Asp (80 +/- 17%) and Glu (92 +/- 8%). However, spontaneous release of Asp and Glu was significantly greater than that observed in unlesioned tissue (159 +/- 18% and 209 +/- 27%, respectively), while the spontaneous release of GABA was not significantly reduced (75 +/- 43%). Although release of the neurotransmitter amino acids Asp, Glu and GABA were affected by the lesion, the release of the non-neurotransmitter amino acid tyrosine was unaffected. These data are consistent with the hypotheses that: 1) the predominant source of releasable stores of endogenous Asp and Glu in the striatum arises from extinsic neurons, and 2) that the spontaneous release of Asp and Glu from axon terminals in the striatum may be regulated, at least in part, by local inhibitory neurons.

Release of endogenous aspartate and glutamate induced by electrical stimulation in guinea pig cerebellar slices

Whether endogenous aspartate and glutamate, candidates for the excitatory neurotransmitter of cerebellar climbing and parallel fibers, are actually released from guinea pig cerebellar slices by electrical stimulation of the cerebellar white matter, was examined by means of mass fragmentography using gas chromatograph-mass spectrometer and thin layer chromatography. Both endogenous aspartate and glutamate were found to be significantly released in a Ca- and stimulus-frequency-dependent manner. Although the origin of each amino acid could not be specified in spite of pharmacological attempt to selectively block the mossy fiber-granule cell (parallel fiber) system, these results were at least in favor of the electrophysiologically and pharmacologically suggested candidacy of these amino acids for the transmitters of cerebellar climbing and parallel fibers.

Adenosine decreases aspartate and glutamate release from rat hippocampal slices

The effect of adenosine and related compounds on the release of endogenous aspartate and glutamate from isolated, superfused rat hippocampal slices was studied at rest and during electrical stimulation of the stratum radiatum in the CA 3/CA 2 region, using a sensitive mass-spectrometric technique. Evoked extracellular potentials were recorded from the CA 1 region. Adenosine, at 3×10 −4 M concentration, inhibited the stimulation-evoked potentials and prevented the stimulation-induced release of aspartate and glutamate. Similarly, 1-phenylisopropyladenosine (10 −6 M) and cyclohexyladenosine (10 −6 M) depressed both electrical and neurochemical responses to stimulation of the stratum radiatum. 8-Phenyltheophylline (5×10 −6 M) increased the release of aspartate and glutamate and antagonized the cyclohexyladenosine-induced inhibition of amino acid release. Our results support the hypothesis that adenosine modulates the electrophysiological responses to stimulation of stratum radiatum through a reduction of the release of the excitatory amino acids aspartate and glutamate.

Release of endogenous and accumulated exogenous amino acids from slices of normal and climbing fibre-deprived rat cerebellar slices.

Efflux of various amino acids from slices of rat cerebellar hemispheres was determined under resting or depolarizing conditions. It was increased under high K+(50 mM) as compared to low K+ (5 mM) conditions by 1258 pmol/mg protein for aspartate, 478 for gamma-aminobutyric acid (GABA), 44,693 for glutamate, and 615 for glycine. These were significantly higher than the corresponding values obtained under low-Ca2+ (0.1 mM), high-Mg2+ (12 mM) conditions, whereas for 11 other amino acids the K+-induced efflux was similar under normal and low-Ca2+ concentrations. The K+-induced efflux of exogenously accumulated L-[3H]aspartate, D-[3H]aspartate, and L-[3H]glutamate was higher by factors of 2, 5.8, and 6.3, respectively, under normal Ca2+ conditions, as compared with low-Ca2+, high-Mg2+ conditions. After climbing fibre degeneration induced by destruction of the inferior olive with 3-acetylpyridine, release of endogenous aspartate and exogenous L-[3H]glutamate and D-[3H]aspartate was significantly reduced, by 26%, 38%, and 27%, respectively. These results support the hypothesis that climbing fibres may use aspartate or a related compound as a neurotransmitter. In rat cerebellar tissue, L-[3H]glutamate and L-[3H]aspartate differ in several aspects: (1) L-[3H]glutamate uptake was 4 times higher than that of L-[3H]aspartate; (2) fractional rate constant of K+- evoked release of L-[3H]aspartate was 7% X 2.5 min-1, and of L-[3H]glutamate 36% X 2.5 min-1; and (3) specific activity of L-[3H]glutamate in the eluate collected during K+ stimulation was 3.5 times the value in the tissue, whereas for L-[3H]aspartate, specific activities in the eluate and tissue were similar.