Synaptic vesicles immunoisolated from rat cerebral cortex contain high levels of glutamate

L-Glutamate is regarded as the major excitatory neurotransmitter in the mammalian CNS. However, whether the released transmitter originates from a cytosolic pool or is discharged from synaptic vesicles by exocytosis (vesicle hypothesis) remains controversial. A problem with the general acceptance of the vesicle hypothesis is that the enrichment of glutamate in synaptic vesicles has not been convincingly demonstrated. In the present study, we have analyzed the glutamate content of synaptic vesicles isolated from rat cerebral cortex by a novel immunobead procedure. A large amount of glutamate was present in these vesicles when a proton electrochemical gradient was maintained across the vesicle membrane during isolation. Compared with the starting fraction, glutamate was enriched more than 10-fold relative to other amino acids. Addition of N-ethylmaleimide prevented glutamate loss during isolation. Isotope exchange experiments revealed that exchange or re-uptake of glutamate after homogenization is negligible. We conclude that rat brain synaptic vesicles contain high levels of glutamate in situ.     

ON THE SITE OF ORIGIN OF TRANSMITTER AMINO ACIDS RELEASED BY DEPOLARIZATION OF NERVE TERMINALS IN VITRO

Abstract— —The site of origin of transmitter amino acids released by depolarizing agents from nerve endings was studied. The model used was the incubated and depolarized synaptosome preparation from which the component soluble, synaptic vesicle, membrane and mitochondrial sub-fractions were obtained. Synaptosomal amino acids were radioactively labelled from D-[U-¹⁴C]glucose in vivo by intraventricular injection and in vitro during subsequent incubation. The specific radioactivities of amino acids released in response to K⁺ (56 mM) or veratrine (75 μM) were found to closely resemble those of the soluble cytoplasmic fraction, in most cases differing significantly from those of the other fractions. The specific radioactivity of the GABA and aspartate released by K⁺ stimulation and the GABA and glutamate released by veratrine were significantly different from that of the vesicles in each case. The specific radioactivities of glutamate released by both agents, and also GABA with K⁺ stimulation, were approximately double that of the amino acid released in control conditions. Depletion of the soluble cytoplasmic pools of glutamate, GABA and aspartate occurred following stimulation, corresponding to the induced-release of these compounds. Turnover of the amino acids in the other subfractions was too low to account for their participation in the release process in addition to the soluble cytoplasmic pool. A cytoplasmic origin of release of neurotransmitter amino acids from nerve endings is proposed.     

GLUCOSE AND AMINO ACID METABOLISM IN ISOLATED NEURONAL AND GLIAL CELL FRACTIONS IN VITRO

Abstract—

• 1 The metabolism of three substrates, [U-¹⁴C]glucose, [U-¹⁴C]pyruvate and [U-¹⁴C]glutamate has been studied in vitro in neuronal and glial cell fractions obtained from rat cerebral cortex by a density gradient technique.
• 2 The mixed cell suspension, after washing, metabolized glucose and glutamate in a manner essentially similar to the tissue slice. Exceptions were a reduced ability to generate lactate from glucose and alanine from glutamate, and a lowered effect of added glucose in suppressing the production of aspartate from glutamate.
• 3 After 2 hr incubation with [U-¹⁴C]glucose, the concentration of the amino acids glutamate, glutamine, GABA, aspartate and alanine were raised in the neuronal, compared to the glial fraction to 234 per cent, 176 per cent, 202 per cent, 167 per cent and 230 per cent respectively although both were lower than in the tissue slice. Incorporation of radio-activity was absolutely lower in the neuronal fraction, however, and the specific activities of the amino acids were: glutamate 12 per cent, GABA 18 per cent, aspartate 34 per cent, and alanine 33 per cent of those in the glial fraction.
• 4 After the incubation with [U-¹⁴C]pyruvate, the pool size of the amino acids were higher than after incubation with glucose, except for GABA, which was reduced to one-third. The concentrations of the amino acids glutamate, glutamine, GABA, aspartate, and alanine in the neuronal fraction were respectively 46 per cent, 143 per cent, 105 per cent, 97 per cent, and 57 per cent of those in the glial. Thus, with the exception of alanine, the specific activity of the neuronal amino acids compared to the glial was little increased when pyruvate replaced glucose as substrate.
• 5 After 2 hr incubation with [U-¹⁴C]glutamate in the presence of non-radioactive glucose, the pool sizes of all the amino acids were increased in both neuronal and glial fractions, with the exception of neuronal alanine and glial glutamine. The concentrations of the amino acids glutamine, GABA, aspartate and alanine were raised in the neuronal fraction, compared to the glial, to 425 per cent, 187 per cent, 222 per cent, and 133 per cent respectively. The specific activities of all the amino acids were higher than with glucose alone with the exception of alanine, and neuronal GABA. Neuronal glutamine and aspartate had specific activities respectively 102 per cent and 84 per cent of glial.
• 6 An unidentified amino acid, with R_F comparable to that of alanine and specific activity close to that of glutamate, was also present after incubation. It was relatively concentrated in the neuronal fraction.
• 7 The distribution of the enzymes glutamate dehydrogenase, aspartate aminotransferase, glutamate decarboxylase and glutamine synthetase between the cell fractions was studied. With the exception of glutamine synthetase, none of the enzymes was lost from the cell fractions during their preparation. Only 14 per cent of the glutamine synthetase, compared with 75 per cent of total protein, was recovered in the fractions. Of the enzymes, glutamate dehydrogenase activity was 406 per cent, and glutamate synthetase activity 177 per cent in the neuronal fraction compared to the glial in the absence of detergent. In the presence of detergent, glutamate dehydrogenase control was 261 per cent, aspartate aminotransferase activity 237 per cent is the neuronal as compared to the glial fraction.
• 8 Incorporation of radioactivity into acid-insoluble material from either glutamate or pyruvate was twice as high into the neuronal as the glial fraction.
• 9 The extent to which these differences may be extrapolated back to the intact tissue is considered, and certain correction factors calculated. The significance of the observations for an understanding of the compartmentation of amino acid pools and metabolism in the brain, and the possible identification of such compartments, is discussed.

     

EFFECT OF ELECTRICAL STIMULATION ON THE YIELD AND COMPOSITION OF SYNAPTIC VESICLES FROM THE CHOLINERGIC SYNAPSES OF THE ELECTRIC ORGAN OF TORPEDO: A COMBINED BIOCHEMICAL, ELECTROPHYSIOLOGICAL AND MORPHOLOGICAL STUDY

Abstract— The effect of stimulating the electric organ of Torpedo marmorata , anaesthetized with 0.01% Tricaine methane sulphonate, by means of electrical stimulation (5/s) administered via an electrode placed on the electric lobe has been studied electrophysiologically, biochemically and morphologically. The response of the organ declined to about 50 per cent of its initial value after about 500 stimuli, by a further 10 per cent after another 500 stimuli and then to about 12 per cent of the initial value after a further 1000 stimuli. Thereafter the response fell off progressively. However, even when the response was less than 1 per cent of its initial value, the organ had considerable powers of recuperation during a 30-s rest period, to 30–50 per cent of its initial value.
The fall in response was accompanied by a reduction in vesicle size and number, an increase in the area of the presynaptic membrane and a fall in the protein, total nucleotide, ATP and acetylcholine content of the vesicle fraction isolated from the stimulated tissue. However, whereas vesicle numbers and the protein and total nucleotide content of the vesicle fraction fell by only about 50 per cent, vesicular ATP and acetylcholine levels were reduced to about 10 per cent. An analysis of the covariance of vesicular ATP and acetylcholine showed an initial loss of an acetylcholine-rich (relative to ATP) population of vesicles. The early loss of vesicular protein and nucleotide and vesicle numbers as well as the morphological changes seen would be consistent with a loss of vesicles due to fusion with the external membrane. The preferential loss of acetylcholine and ATP from the vesicle fraction indicates that the vesicles surviving the stimulation procedure have been utilized in a number of cycles causing the progressive fall in vesicle volume, and acetylcholine and ATP content.     

Amino acid neurotransmission: Dynamics of vesicular uptake

Glutamate, GABA and glycine, the major neurotransmitters in CNS, are taken up and stored in synaptic vesicles by a Mg²⁺-ATP dependent process. The main driving force for vesicular glutamate uptake is the membrane potential, whereas both the membrane potential and the proton gradient contribute to the uptake of GABA and glycine. Glutamate is taken up by a specific transporter with no affinity for aspartate. Evans blue and related dyes are competitive inhibitors of the uptake of glutamate. GABA, β-alanine, and glycine are taken up by the same family of transporter molecules. Aspartate, taurine, and proline are not taken up by any synaptic vesicle preparations. It is suggested that vesicular uptake and release are characteristics that identify these amino acids as neurotransmitters. We also discuss that “quanta” in the brain are not necessarily related the content of neurotransmitter in the synaptic vesicles, but rather to postsynaptic events. Special issue dedicated to Dr. Herman Bachelard.     

Amino Acids in the Substantia Nigra of Rats with Striatal Lesions Produced by Kainic Acid

In an attempt to estimate the pool size of glutamate and other amino acids in γ-aminobutyric acid (GABA)-containing neurons, we determined the content of 12 amino acids in the bilateral substantia nigra of rats, in which unilateral striatal lesions had been made with kainic acid two weeks earlier. The assay of the amino acids (including glutamate, aspartate, glutamine, asparagine, glycine, and GABA) and ethanolamine was based on HPLC and fluorimetric detection after precolumn derivatization with o-phthaldialdehyde. The levels of all measured amino acids (except those of tyrosine, threonine, and ethanolamine) were decreased in the affected striatum, but only the levels of aspartate, taurine, and GABA were lowered in the ipsilateral substantia nigra. These results indicate that the pool size of the various amino acids in the striatonigral GABAergic pathway is small compared to their nigral content, and that in addition to GABA a significant fraction of aspartate and taurine may be confined to nerve terminals in the substantia nigra.     

Amino Acid Content of Nerve Endings (Synaptosomes) in Different Regions of Brain: Effects of Gabaculine and Isonicotinic Acid Hydrazide

Abstract: The amino acid content of synaptosomes was determined in six regions of rat brain, and in all regions the five predominant amino acids were glutamate, glutamine, aspartate, taurine, and GABA (γ-aminobutyrate). However, the proportions of the individual amino acids varied considerably from one region to another, the GABA content being particularly high and the taurine content low in synaptosomes from the diencephalon and mesencephalon. Administration of isonicotinic acid hydrazide to rats lowered the synaptosomal GABA level by similar amounts in all brain regions, but the administration of gabaculine resulted in a particularly long-acting elevation in GABA levels in the nerve endings of the diencephalon and mesencephalon. The possibility is raised that the high GABA levels in the nerve terminals of the diencephalon may be involved in the gabaculine-induced lowering of the body temperature of the rats. A constancy in the amount of the synaptosomal pool of "aspartate + glutamate + glutamine + GABA" was observed despite large changes in the relative amounts of the four amino acids brought about by gabaculine.     

Role of glutamine and neuronal glutamate uptake in glutamate homeostasis and synthesis during vesicular release in cultured glutamatergic neurons

Glutamate exists in a vesicular as well as a cytoplasmic pool and is metabolically closely related to the tricarboxylic acid (TCA) cycle. Glutamate released during neuronal activity is most likely to a large extent accumulated by astrocytes surrounding the synapse. A compensatory flux from astrocytes to neurons of suitable precursors is obligatory as neurons are incapable of performing a net synthesis of glutamate from glucose. Glutamine appears to play a major role in this context. Employing cultured cerebellar granule cells, as a model system for glutamatergic neurons, details of the biosynthetic machinery have been investigated during depolarizing conditions inducing vesicular release. [U-13C]Glucose and [U-13C]glutamine were used as labeled precursors for monitoring metabolic pathways by nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) technologies. To characterize release mechanisms and influence of glutamate transporters on maintenance of homeostasis in the glutamatergic synapse, a quantification was performed by HPLC analysis of the amounts of glutamate and aspartate released in response to depolarization by potassium (55 mM) in the absence and presence of DL-threo-beta-benzyloxyaspartate (TBOA) and in response to L-trans-pyrrolidine-2,4-dicarboxylate (t-2,4-PDC), a substrate for the glutamate transporter. Based on labeling patterns of glutamate the biosynthesis of the intracellular pool of glutamate from glutamine was found to involve the TCA cycle to a considerable extent (approximately 50%). Due to the mitochondrial localization of PAG this is unlikely only to reflect amino acid exchange via the cytosolic aspartate aminotransferase reaction. The involvement of the TCA cycle was significantly lower in the synthesis of the released vesicular pool of glutamate. However, in the presence of TBOA, inhibiting glutamate uptake, the difference between the intracellular and the vesicular pool with regard to the extent of involvement of the TCA cycle in glutamate synthesis from glutamine was eliminated. Surprisingly, the intracellular pool of glutamate was decreased after repetitive release from the vesicular pool in the presence of TBOA indicating that neuronal reuptake of released glutamate is involved in the maintenance of the neurotransmitter pool and that 0.5 mM glutamine exogenously supplied is inadequate to sustain this pool.     

Distinct changes in neuronal and astrocytic amino acid neurotransmitter metabolism in mice with reduced numbers of synaptic vesicles

The relations between glutamate and GABA concentrations and synaptic vesicle density in nerve terminals were examined in an animal model with 40–50% reduction in synaptic vesicle numbers caused by inactivation of the genes encoding synapsin I and II. Concentrations and synthesis of amino acids were measured in extracts from cerebrum and a crude synaptosomal fraction by HPLC and ¹³C nuclear magnetic resonance spectroscopy (NMRS), respectively. Analysis of cerebrum extracts, comprising both neurotransmitter and metabolic pools, showed decreased concentration of GABA, increased concentration of glutamine and unchanged concentration of glutamate in synapsin I and II double knockout (DKO) mice. In contrast, both glutamate and GABA concentrations were decreased in crude synaptosomes isolated from synapsin DKO mice, suggesting that the large metabolic pool of glutamate in the cerebral extracts may overshadow minor changes in the transmitter pool. ¹³C NMRS studies showed that the changes in amino acid concentrations in the synapsin DKO mice were caused by decreased synthesis of GABA (20–24%) in cerebral neurons and increased synthesis of glutamine (36%) in astrocytes. In a crude synaptosomal fraction, the glutamate synthesis was reduced (24%), but this reduction could not be detected in cerebrum extracts. We suggest that lack of synaptic vesicles causes down-regulation of neuronal GABA and glutamate synthesis, with a concomitant increase in astrocytic synthesis of glutamine, in order to maintain normal neurotransmitter concentrations in the nerve terminal cytosol.     

DISTRIBUTION OF AMINO ACIDS AND OF EXO- AND ENDOPEPTIDASES ALONG VERTEBRATE AND INVERTEBRATE NERVES

Abstract— The proximo-distal gradients for representative peptidases, peptidylpeptide hydrolases, and amino acids were measured in segments of peripheral nerve from invertebrates and vertebrates and in the lobster brain and ventral cord.
Crustacean nerve was characterized by a large pool of free amino acids totaling 100–200 μmoles/g wet wt. In lobster nerve, the principal free amino acid was aspartic acid which comprised 55 per cent of the free pool, whereas in the rat sciatic nerve it comprised only 5 per cent. The principal free amino acid in rat sciatic nerve was taurine (32 per cent of the pool) and in lobster brain glycine comprised 30 per cent of the pool. No consistent patterns emerged for the gradients along the nerves for amino acids and hydrolytic enzymes. In the leg nerve of the lobster, concentrations of aspartic acid and arginine were higher in the proximal region, and concentrations of proline and alanine were higher in the distal region. Concentrations of most amino acids were higher in the proximal regions of crab nerve, of lobster brain and ventral cord, and of rat sciatic nerve.
Rat sciatic nerve exhibited a pronounced proximo-distal increase in activity of aminopeptidase (Leu-Gly-Gly). In lobster leg nerve, activity of neutral proteinase was higher in the proximal segment, whereas activity of acid proteinase was higher in the distal segment. The best examples of proximo-distal gradients were found in lobster brain and ventral cord; activities of endopeptidases, arylamidases (Leu- and Arg-βNA), and aminopeptidase were higher in the supra-esophageal ganglia or cephalothorax segments than in the distal regions.     

In vivo release patterns and cardiovascular properties of inhibitory and excitatory amino acids in the hypothalamus

Summary The posterior hypothalamus of conscious, freely moving rats was superfused with artificial cerebrospinal fluid through a push-pull cannula and the release of amino acids was determined in the superfusate. Under basal conditions, the release rates of taurine, GABA and glutamate fluctuated according to ultradian rhythms with different frequencies. Hypothalamic superfusion with veratridine or high concentrations of potassium choride enhanced the release rates of taurine, GABA and glutamate in a concentration-dependent way. Tetrodotoxin decreased the basal release rates of the three amino acids. The release of arginine was not influenced significantly by these compounds. A fall of blood pressure elicited by intravenous infusion of nitroprusside decreased the release rates of GABA and taurine and enhanced the release of glutamate. Infusion of noradrenaline increased blood pressure and release rates of GABA and taurine, while the release of glutamate was not influenced. Neither the pressor, nor the depressor responses to drugs influenced the release of arginine in the hypothalamus. It is concluded that the inhibitory amino acids taurine and GABA released from hypothalamic neurons possess a tonic hypotensive function. The excitatory amino acid glutamate, released from glutamatergic neurons of the hypothalamus, seems to possess a hypertensive function in counteracting a fall of blood pressure.This work was supported by the Fonds zur Förderung der wissenschaftlichen Forschung. These results were presented at the Third International Congress on Amino Acids, Vienna, August 1993     

Salinity Stress and the Content of Proline in Roots of Pisum sativum and Tamarix tetragyna

Amino acid composition of the free amino acid pool and the TCA-insolubleprotein fraction were investigated in root tips of pea and Tamarixtetragyna plants grown at various levels of NaCl salinity. Salinitystress induced an increase of proline content, mainly in thefree amino acid pool in both plants, and of proline or hydroxyprolinecontent in the protein. Externally-supplied proline was absorbedand incorporated into protein, by pea roots, more effectivelythan by Tamarix roots. Salinity stress, apparently, stimulatedthe metabolism of externally-supplied labelled proline. Pearoots have a very large pool of free glutamic acid; however,70 per cent of the ¹⁴C from externally-supplied ¹⁴C-U-glutamicacid was released as CO₂. Very small amounts of it were incorporatedinto protein. No measurable amount of radioactivity could bedetected in any one of the individual amino acids, either ofprotein hydrolysate or the free amino acid pool. Proline very effectively counteracted the inhibitory effectof NaCl on pea seed germination and root growth. A similar effectbut to a lesser degree was achieved with phenylalanine and asparticacid. The feasibility of proline being a cytoplasmic osmoticumis discussed.     

GABA and glycine in synaptic vesicles: storage and transport characteristics.

gamma-Aminobutyric acid (GABA) and glycine are major inhibitory neurotransmitters that are released from nerve terminals by exocytosis via synaptic vesicles. Here we report that synaptic vesicles immunoisolated from rat cerebral cortex contain high amounts of GABA in addition to glutamate. Synaptic vesicles from the rat medulla oblongata also contain glycine and exhibit a higher GABA and a lower glutamate concentration than cortical vesicles. No other amino acids were detected. In addition, the uptake activities of synaptic vesicles for GABA and glycine were compared. Both were very similar with respect to substrate affinity and specificity, bioenergetic properties, and regional distribution. We conclude that GABA, glycine, and glutamate are the only major amino acid neurotransmitters stored in synaptic vesicles and that GABA and glycine are transported by similar, if not identical, transporters.     

Effect of amino acid levels on matrix vesicle formation by epiphyseal growth plate chondrocytes in primary culture

The effect of varying the amino acid concentrations of the culture medium on matrix vesicle formation was studied in primary cultures of chicken epiphyseal growth plate chondrocytes grown in Dulbecco's modified Eagle's medium (DME) supplemented with 10% fetal bovine serum (FBS). Decreasing the levels of free amino acids in the culture medium to levels of one-half, one quarter, and one eighth of the values normally present in DME caused a progressive decline in matrix vesicle (MV) formation. Increasing the level in the culture medium of those amino acids that are enriched in extracellular fluid (ECF) of growth plate cartilage significantly increased formation of matrix vesicles (MV), as assayed by the alkaline phosphatase (AP) activities present in high-speed sediments from spent culture media. However, adjusting the levels of all amino acids to match those of the ECF produced the greatest stimulation of MV formation. Of the amino acids that are notably enriched in ECF, glutamate (GLU), alanine (ALA), serine (SER), asparagine (ASN), and taurine (TAU) individually enhanced MV production, whereas proline (PRO), glycine (GLY), and aspartate (ASP) had essentially no effect. The simple combination of ECF levels of ALA and GLU resulted in a stimulation of MV formation equal to that observed when the eight aforementioned amino acids were elevated to ECF levels. Other combinations of ASP and GLY, or of TAU, SER, and ASN showed some stimulation, but at a lower level. Increasing the amino acid concentrations, alone or in combination, also increased the levels of cellular AP, and to a lesser extent cellular protein. While increases in cellular AP were generally correlated with increased formation of AP-rich MV, this was not uniformly true. These results indicate that in addition to hormones and growth factors, nutritional factors such as the levels of amino acids are also critical for normal phenotypic expression, growth, and matrix formation by epiphyseal chondrocytes.     

Release of Endogenous Amino Acids from the Striatum from Developing and Adult Mice in Ischemia

In most other studies the release of amino acid neurotransmitters and modulators in vitro has been studied mostly using labeled preloaded compounds. For several reasons the estimated release may not reliably reflect the release of endogenous compounds. The magnitudes of the release cannot thus be quite correctly estimated using radioactive labels. The basal and K⁺-evoked release of the neuroactive endogenous amino acids γ-aminobutyrate (GABA), glycine, taurine, glutamate and aspartate was now studied in slices from the striatum from 7-day-old to 3-month-old mice under control (normoxic) and ischemic conditions. The release of alanine, threonine and serine was assessed as control. GABA and glutamate release was much greater in 3-month-old than in 7-day-old mice, whereas with taurine the situation was the opposite. Ischemia markedly enhanced the release of all these three amino acids. The release of aspartate and glycine was markedly enhanced as well whereas no effects were discernible in the release of glutamine, alanine, serine and threonine. K⁺ stimulation (50 mM) enhanced the release of GABA, glutamate, taurine, aspartate and glycine in most cases, except with taurine in 3-month-old mice under the ischemic conditions and with aspartate in 7-day-old mice under the control conditions. K⁺ stimulation did not affect the release of glutamine, alanine, serine or threonine. The results on endogenous amino acids are qualitatively similar to those obtained in our earlier experiments with labeled preloaded amino acids. In conclusion, in developing mice only inhibitory taurine is released in such amounts that may counteract the harmful effects of excitatory amino acids in ischemia.     

The content of GABA and other amino acids in bovine cerebral cortex synaptic vesicles

The content of γ-amino butyric acid (GABA) and of other water soluble amino acids in bovine brain synaptic vesicles was determined by a modified automated amino acid analysis method. Following subcellular fractionation, GABA, glutamate and aspartate were distributed largely in the supernatant fractions and in the upper layer of the sucrose gradient. Only 10–20% of the total content was associated with the vesicular fraction. On the other hand, the other water soluble amino acids, such as serine, glycine and alanine, were evenly distributed between cytoplasmic and particulate fractions in a similar pattern to that observed with cytoplasmic enzyme markers. The results may indicate specific association of GABA, glutamate and aspartate with low density particles or cytoplasmic components.     

In Vivo Studies on the Extracellular, and Veratrine-Releasable, Pools of Endogenous Amino Acids in the Rat Striatum: Effects of Corticostriatal Deafferentation and Kainic Acid Lesion

The effects of corticostriatal deafferentation (decortication) and destruction of intrinsic neurons (intrastriatal kainate injection) on the extracellular concentration, and veratrine-releasable pools, of endogenous amino acids in the rat striatum were examined using the in vivo brain dialysis technique. Intracellular amino acid content was also determined. Decortication reduced selectively intra- and extracellular levels of glutamate (Glu) and aspartate (Asp). Extracellular changes were more pronounced than those in tissue content. gamma-Aminobutyric acid (GABA), taurine (Tau), and phosphoethanolamine (PEA) levels were not affected, whereas nonneuroactive amino acids were increased at 1 week but not at 1 month post-lesion. The intracellular pool of Glu and Asp was also reduced in kainate-lesioned striata. However, extracellular levels of these compounds were not affected significantly by this treatment. The tissue content of all other amino acids was decreased, the most prominent change being in the concentration of GABA. Extracellular GABA concentration was also reduced dramatically, whereas the concentrations of noneuroactive amino acids were increased to varying degrees. These data suggest that transmitter pools of neuroactive amino acids are an important supply for their extracellular pools. Lesion-induced alterations in nonneuroactive amino acids are discussed with regard to the loss of metabolic pools, glial reactivity, and changes in blood-brain barrier transport. Veratrine induced a massive release of neuroactive amino acids such as Glu, Asp, GABA, and Tau into the extracellular fluid, and a delayed increase in PEA. Extracellular levels of neuroactive amino acids were raised slightly. Decortication reduced, selectively, the amounts of Glu and Asp released by veratrine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ethanol extraction of free amino acids from soil

Summary A technique was developed for extracting and analyzing the free amino acid fraction of soil. Ethanol was used as an extracting agent. Ethanolextraction curves showed 20 per cent ethanol was the optimum percentage for extraction. Extraction-time curves indicated 18 to 20 hours of extraction with 20 per cent ethanol produced satisfactory results.The free amino acid fraction of soil was characterized and the limitations of the technique were determined. The naturally occurring amino acids extracted with 20 per cent ethanol were limited to acidic and neutral amino acids; basic amino acids were not extracted in sufficient quantities to permit detection. Based on the percent recovery of amino acids incorporated into soil and extracted with 20 per cent ethanol 90 to 95 per cent of the acidic, 80 to 85 per cent of the neutral and 1 to 5 per cent of the basic amino acids used were recovered with the technique.     

Effects of methylmercury on the spontaneous and potassium-evoked release of endogenous amino acids from mouse cerebellar slices

The effects of methylmercury on the spontaneous and potassium-evoked release of endogenous amino acids from mouse cerebellar slices have been examined. Methylmercury induced a concentration-dependent increase in the spontaneous release of glutamate, aspartate, gamma-aminobutyric acid, and taurine from mouse cerebellar slices. Glycine release was slightly increased, but not in a concentration-dependent manner. The spontaneous release of glutamine from mouse cerebellar slices was not altered by any concentration of methylmercury examined (10, 20, and 50 microM). The tissue content of glutamate, gamma-aminobutyric acid, glutamine, and taurine decreased after exposure to methylmercury. Exposure of cerebellar slices to 20 microM methylmercury resulted in a significant enhancement in glutamate release during stimulation with 35 mM K+. This increase could be accounted for by the methylmercury-induced increase in spontaneous glutamate release. The increase in spontaneous release of glutamate and gamma-aminobutyric acid was independent of the availability of extracellular calcium. These results suggest that methylmercury increases the release of neurotransmitter amino acids, particularly gamma-aminobutyric acid and glutamate, by acting at intracellular sites to increase release from a neurotransmitter pool. The increase in the potassium-stimulated release of glutamate may reflect an increased sensitivity of the cerebellar granule cell to the effects of methylmercury. It is suggested that alterations in amino acid neurotransmitter function in the cerebellum may contribute to some of the neurological symptoms of methylmercury intoxication.     

Ischemia and reoxygenation induced amino acid release release and tissue damage in the slices of rat corpus striatum

Summary. Ischemic incubation significantly increased amino acid release from rat striatal slices. Reoxygenation (REO) of the ischemic slices, however, enhanced only taurine and citrulline levels in the medium. Ischemia-induced increases in glutamate, taurine and GABA outputs were accompanied with a similar amount of decline in their tissue levels. Tissue final aspartic acid level, however, was doubled by ischemia. Lactate dehydrogenase (LDH) leakage was not altered by ischemia, but enhanced during REO. Presence of tetrodotoxine (TTX) during ischemic period caused significant decline in ischemia-induced glutamate output, but not altered REO-induced LDH leakage. Although omission of extracellular calcium ions from the medium during ischemic period protected the slices against REO-induced LDH leakage, this treatment failed to alter ischemia-induced glutamate and GABA outputs. The release of other amino acids, however, declined 50% in calcium-free medium. Blockade of the glutamate uptake transporter by L-trans-PDC, on the other hand, doubled ischemia induced glutamate and aspartic acid outputs. These results indicate that more than one mechanisms probably support the ischemia-evoked accumulation of glutamate and other amino acids in the extracellular space. Although LDH leakage enhanced during REO, processes involved in this increment were found to be dependent on extracellular calcium ions during ischemia but not REO period.