Metabolic disturbances of synaptosomes isolated from ischemic gerbil brain

The effects of cerebral ischemia, induced for 10 min by bilateral common carotid ligation in the Mongolian gerbil, on the brain and synaptosomal content of phospholipids and free fatty acids were measured. Moreover, the incorporation of arachidonic acid and oleoyl-CoA into phospholipids, as well as the respiration and the accumulation of⁴⁵Ca, norepinephrine, dopamine, choline, glutamate, and -aminobutyrate in the ischemic brain synaptosomal fraction were studied. Analyses of lipids showed a drop in phospholipids content with concomitant increase of lysocompounds and free fatty acids in ischemic cerebral cortex. Disturbances in lipid metabolism including rapid phospholipids hydrolysis and changes in the incorporation of arachidonic acid into inositol and choline phosphoglycerides were also shown in the synaptosomal fraction of ischemic brain. The uptake of neurotransmitter substances, expressed as a percent of control value, was reduced 21% for norepinephrine, 40% for dopamine, 20% for choline, 24% for glutamate and 13% for -aminobutyrate in ischemic synaptosomes. There was no significant effect of ischemia on synaptosomal respiration and⁴⁵Ca uptake in both control and high potassium media. the inhibition of neurotransmitter uptake in ischemic brain synaptosomes may be caused by the disturbance of fatty acid metabolism.     

β-N-Oxalylamino-l-Alanine: Action on High-Affinity Transport of Neurotransmitters in Rat Brain and Spinal Cord Synaptosomes

beta-N-Oxalylamino-L-alanine (BOAA) is a dicarboxylic diamino acid present in Lathyrus sativus (chickling pea). Excessive oral intake of this legume in remote areas of the world causes humans and animals to develop a type of spastic paraparesis known as lathyrism. BOAA is one of several neuroactive glutamate analogs reported to stimulate excitatory receptors and, in high concentrations, cause neuronal vacuolation and necrosis. The present study investigates the action of BOAA in vitro on CNS high-affinity transport systems for glutamate, gamma-aminobutyric acid (GABA), aspartate, glycine, and choline and in the activity of glutamate decarboxylase (GAD), the rate-limiting enzyme in the decarboxylation of glutamate to GABA. Crude synaptosomal fractions (P2) from rat brain and spinal cord were used for all studies. [3H]Aspartate transport in brain and spinal cord synaptosomes was reduced as a function of BOAA concentration, with reductions to 40 and 30% of control values, respectively, after 15-min preincubation with 1 mM BOAA. Under similar conditions, transport of [3H]glutamate was reduced to 74% (brain) and 60% (spinal cord) of control values. High-affinity transport of [3H]GABA, [3H]glycine, and [3H]choline, and the enzyme activity of GAD, were unaffected by 1 mM BOAA. While these data are consistent with the excitotoxic (convulsant) activity of BOAA, their relationship to the pathogenesis of lathyrism is unknown.     

Effects of spinal transection on presynaptic markers for glutamatergic neurons in the rat

To evaluate the hypothesis that glutamic acid may be the neurotransmitter of descending, excitatory supraspinal pathways, the uptake and release ofl-[³H] glutamate and the levels of endogenous glutamate were measured in preparations from rat lumbar spinal cord following complete mid-thoracic transection. Following transection, the activity of the synaptosomal high-affinty glutamate uptake process was increased in both dorsal and ventral halves of lumbar cord between 1 and 14 days after transection and returned to control levels by 21 days posttransection. At 7 days, the increased activity of the uptake process forl-[³H] glutamate resulted in elevation ofV _max with no significant alteration inK _t as compared to age-matched controls. Depolarization-induced release ofl-[³H]glutamate from prelabeled slices did not differ significantly from control in the lesioned rat except at 21 days after lesion when the amount of tritium release was significantly greater in the transected preparations than in control. Amino acid analysis of the lumbar cord from control and transected rats indicated only a 10% decrease in the level of endogenous glutamate and no alterations in the concentration of GABA and glycine 7 days after lesion. These findings do not support the hypothesis that glutamate serves as a major excitatory neurotransmitter in supraspinal pathways innervating the lumbar cord of the rat.     

Choline mustard: an irreversible ligand for use in studies of choline transport mechanisms at the cholinergic nerve terminal

It has been shown in our laboratory that choline mustard aziridinium ion is a potent and irreversible inhibitor of choline transport into rat brain synaptosomes; this compound showed selectivity for the sodium-dependent, high affinity carrier in that it was 30 times more potent as an inhibitor when compared with the effect on sodium-independent, low affinity choline uptake. In the present study, this mustard analogue did not inhibit synaptosomal uptake of 5-hydroxytryptamine, noradrenaline, or gamma-aminobutyric acid, thereby confirming further the specificity of this compound for the choline carrier. Studies of the effect of depolarization of the nerve terminals on the inactivation of choline carriers by choline mustard were performed. It was determined that alkylation of the carrier was significantly increased in nerve endings previously depolarized. The enhancing effect of depolarization on choline transport velocity and on the alkylation of choline carriers by choline mustard was dependent upon the presence of sodium in the external medium. Possible mechanisms for the enhanced inactivation of choline carriers by choline mustard aziridinium ion are proposed, and kinetic interactions of choline mustard with the high affinity choline carrier and with choline acetyltransferase are reviewed and discussed.     

Amplification by glycine of the effect of the GABA transport inhibitor THPO on synaptosomal GABA level

Mice were injected intramuscularly (2 mmol/kg) with the glia-selective GABA uptake inhibitor 4,5,6,7-tetrahydroisoxazolo[4,5-c]pyridin-3-ol (THPO) 60 min prior to sacrifice, or with glycine (10 mmol/kg) 45 min before death, or with a combination of both. After decapitation of the animals, the brains were removed and synaptosomes prepared and analyzed for content of GABA, taurine, glutamine, serine, glutamate and aspartate. While no differences as compared with control animals were found for aspartate, serine and glutamine, synaptosomal GABA levels were increased significantly after injections with either THPO or glycine. The individual effects of THPO and glycine were found to be additive. Taurine levels were decreased to a similar extent in animals which had received either THPO alone or THPO in conjunction with glycine. Treatment with THPO and glycine in combination led to a decrease in the synaptosomal glutamate content. The findings are consistent with the previously observed synergistic anticonvulsant actions of THPO and glycine being mediated via the GABA neurotransmitter system.     

Relationships between glutamine, glutamate, and GABA in nerve endings under Pb-toxicity conditions

Glutamine (Gln), glutamate (Glu) and gamma-amino butyric acid (GABA) are essential amino acids for brain metabolism and function. Astrocytic-derived glutamine is the precursor of the two most important neurotransmitters: glutamate, an excitatory neurotransmitter, and GABA, an inhibitory neurotransmitter. In addition to their roles in neurotransmission these neurotransmitters act as alternative metabolic substrates that enable metabolic coupling between astrocytes and neurons. The relationships between Gln, Glu and GABA were studied under lead (Pb) toxicity conditions using synaptosomal fractions obtained from adult rat brains to investigate the cause of Pb neurotoxicity-induced seizures. We have found that diminished transport of [(14)C]GABA occurs after Pb treatment. Both uptake and depolarization-evoked release decrease by 40% and 30%, respectively, relative to controls. Lower expression of glutamate decarboxylase (GAD), the GABA synthesizing enzyme, is also observed. In contrast to impaired synaptosomal GABA function, the GABA transporter GAT-1 protein is overexpressed (possibly as a compensative mechanism). Furthermore, similar decreases in synaptosomal uptake of radioactive glutamine and glutamate are observed. However, the K(+)-evoked release of Glu increases by 20% over control values and the quantity of neuronal EAAC1 transporter for glutamate reaches remarkably higher levels after Pb treatment. In addition, Pb induces decreased activity of phosphate-activated glutaminase (PAG), which plays a role in glutamate metabolism. Most noteworthy is that the overexpression and reversed action of the EAAC1 transporter may be the cause of the elevated extracellular glutamate levels. In addition to the impairment of synaptosomal processes of glutamatergic and GABAergic transport, the results indicate perturbed relationships between Gln, Glu and GABA that may be the cause of altered neuronal-astrocytic interactions under conditions of Pb neurotoxicity.     

Glutamate and γ-Aminobutyric Acid Neurotransmitter Systems in the Acute Phase of Maple Syrup Urine Disease and Citrullinemia Encephalopathies in Newborn Calves

Cerebral cortex tissue was obtained at autopsy from neonatal Poll Hereford calves with clinically confirmed maple syrup urine disease (MSUD), neonatal Holstein-Friesian calves with clinically confirmed citrullinemia, and matched controls. From this, synaptosomes were prepared for studies of neurotransmitter amino acid uptake and stimulus-induced release, and synaptic plasma membranes were obtained for studies of associated postsynaptic receptor binding sites. As well as having abnormal brain tissue concentrations of the pathognomic plasma amino acids (markedly increased levels of the branched-chain compounds valine, isoleucine, and leucine in MSUD; marked elevation of citrulline levels in citrullinemia), both groups of diseased animals showed reduced brain tissue concentrations of each of the transmitter amino acids glutamate, aspartate, and gamma-aminobutyric acid (GABA). Nontransmitter amino acids were generally unaffected in either disease. Citrullinemic calves showed a marked increase in brain glutamine concentration; in calves with MSUD, the glutamine concentration was raised, but to a much lesser extent. The Na(+)-dependent synaptosomal uptake of both glutamate and GABA was markedly reduced (to less than 50% of control values in both cases) in citrullinemic calves but was unaltered in calves with MSUD. Whereas synaptosomes from normal calves showed the expected stimulus-coupled release of transmitter amino acids, especially glutamate and aspartate, and no response to stimulus of nontransmitter amino acids, there was no increased release of transmitter amino acids in response to depolarization in synaptosomes from citrullinemic calves.(ABSTRACT TRUNCATED AT 250 WORDS)     

DEVELOPMENTAL TRANSITIONS IN UPTAKE OF AMINO ACIDS BY SYNAPTOSOMAL FRACTIONS ISOLATED FROM RAT CEREBRAL CORTEX

Developmental changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Well-defined changes over an age continuum could be observed in both the rates of amino acid accumulation and the effects of Na⁺ on the accumulation. The uptakes of five amino acids (threonine, serine and valine in Na⁺-free medium, aspartic acid and proline in Na⁺-containing medium) increased progressively with the age of the animal, whereas the uptakes of leucine and arginine (in Na⁺-free medium) decreased steadily. The uptake of serine or threonine by synaptosomal fractions prepared from newborn rats was markedly dependent on the presence of Na⁺in the incubation media. Na⁺exerted progressively less effect on the accumulation process with continuing postnatal development and to some extent inhibited uptake by fractions obtained from rats older than about 15 days. Na⁺significantly enhanced the accumulation of glycine in fractions from newborn and adult rats, but had only a slight effect in fractions prepared from 12 to 17-day old rats. A detailed study of the accumulation of glycine indicated that the synaptosomal transport of this amino acid proceeded by two independent systems, one of which was totally dependent on external Na⁺and the and adult animals than in fractions from 12 to 17-day-old rats, wheras the Na⁺-independent system was most active during this latter period of development. The decline in the Na⁺-independent accumulation of glycine from about the 15th day to adulthood was characterized by a decrease in the V_max. and an increase in the K_m.     

Depolarization-Induced Release of Amino Acids From the Vestibular Nuclear Complex

There is evidence from immunohistochemistry, quantitative microchemistry, and pharmacology for several amino acids as neurotransmitters in the vestibular nuclear complex (VNC), including glutamate, γ-aminobutyrate (GABA), and glycine. However, evidence from measurements of release has been limited. The purpose of this study was to measure depolarization-stimulated calcium-dependent release of amino acids from the VNC in brain slices. Coronal slices containing predominantly the VNC were prepared from rats and perfused with artificial cerebrospinal fluid (ACSF) in an interface chamber. Fluid was collected from the chamber just downstream from the VNC using a microsiphon. Depolarization was induced by 50 mM potassium in either control calcium and magnesium concentrations or reduced calcium and elevated magnesium. Amino acid concentrations in effluent fluid were measured by high performance liquid chromatography. Glutamate release increased fivefold during depolarization in control calcium concentration and twofold in low calcium/high magnesium. These same ratios were 6 and 1.5 for GABA, 2 and 1.3 for glycine, and 2 and 1.5 for aspartate. Differences between release in control and low calcium/high magnesium ACSF were statistically significant for glutamate, GABA, and glycine. Glutamine release decreased during and after depolarization, and taurine release slowly increased. No evidence for calcium-dependent release was found for serine, glutamine, alanine, threonine, arginine, taurine, or tyrosine. Our results support glutamate and GABA as major neurotransmitters in the VNC. They also support glycine as a neurotransmitter and some function for taurine.     

Effects of Kainic Acid in Rat Brain Synaptosomes: The Involvement of Calcium

Abstract: The effects of kainic acid were investigated in preparations of rat brain synaptosomes. It was found that kainic acid inhibited competitively the uptake of d -[³H]aspartate, with a K _i of approximately 0.3 m m . Kainic acid also caused release of two excitatory amino acid neurotranstnitters, aspartate and glutamate, in a time- and concentration-dependent manner, but had no effect on the content of γ-aminobutyric acid. Concomitant with the release of aspartate and glutamate, depolarization of the synaptosomal membrane and an increase in intracellular calcium were observed, with no measurable change in the concentration of internal sodium ions. The increase in intrasynaptosomal calcium and decrease in transmem-brane electrical potential were prevented by the addition of glutamate, whereas the kainate-induced release of ra-dioactive aspartate was substantially inhibited by lowering the concentration of calcium in the external medium. It is postulated that kainic acid reacts with a class of glutamate receptors located in a subpopulation of synaptosomes, presumably derived from the glutamatergic and aspartatergic neuronal pathways, which possesses high-affinity uptake system(s) for glutamate and/or aspartate. Activation of these receptors causes opening of calcium channels, influx of calcium into the synaptosomes, and depolarization of the synaptosomal plasma membrane with consequent release of amino acid neurotransmitters.     

MECHANISM OF ACTION OF NOTEXIN AND NOTECHIS 11-5 ON SYNAPTOSOMES

The neurochemical activity of notexin and notechis II-5 was investigated using a synaptosomal preparation of rat cerebral cortices. In preparations preincubated with [³H]choline in order to label acetylcholine, the toxins caused a rapid release of the transmitter which was calcium-dependent. The toxins were also potent inhibitors of high affinity choline uptake. Both agents produced a marked depolarization of the synaptosomal preparation as measured by a fluorescent dye and at high concentrations lysed the preparation. At a concentration of 0.1 μM, notexin and notechis II-5 caused a 50% increase in the efflux of lactate dehydrogenase activity. These results, together with electron microscopic observations, indicated that the toxins disrupt the synaptosomal membranes presumably by their inherent phospholipase activity. The release of acetylcholine and inhibition of choline uptake, together with the depolarization of synaptosomal membranes noted in this study, could explain the observed electrophysiological effects of these toxins.     

Synaptosomal Transport of Radiolabel from N-Acetyl-Aspartyl-[3H]Glutamate Suggests a Mechanism of Inactivation of an Excitatory Neuropeptide

This study was undertaken to explore in synaptosomal preparations the disposition of N-acetyl-aspartyl-glutamate (NAAG), an endogenous acidic dipeptide neurotransmitter candidate. Radiolabel from N-acetyl-aspartyl[3H]glutamate was taken up rapidly into an osmotically sensitive compartment by rat brain synaptosomal preparations in a sodium-, temperature-, and time-dependent manner. HPLC analysis of the accumulated radiolabel indicated that the bulk of the tritium cochromatographed with glutamic acid and not with NAAG. In contrast, [14C]NAAG, labeled on the N-terminal acetate, was not taken up by the synaptosomal preparation. All effective inhibitors of synaptosomal, Na+-dependent [3H]glutamate uptake were found to exhibit similar potency in inhibiting uptake of tritium derived from [3H]NAAG. However, certain alpha-linked acidic dipeptides, structurally similar to NAAG, as well as the potent convulsant quisqualic acid inhibited synaptosomal transport of [3H]NAAG but were ineffective as inhibitors of [3H]glutamate transport. Together with a demonstration of disparities between the regional accumulation of radiolabel from [3H]NAAG and high-affinity [3H]glutamate uptake, these data suggest the presence in brain of a specific peptidase targeting carboxy-terminal glutamate-containing dipeptides that may be coupled to the Na+-dependent glutamate transporter. These findings provide a possible mechanism for NAAG inactivation subsequent to its release from nerve endings.     

Regulation of phosphate-activated glutaminase (PAG) by glutamate analogues

The ability of structural analogues of glutamate (GLU) to modulate phosphate activated glutaminase (PAG) was assessed in the present series of studies. A number of GLU receptor agonists and antagonists were tested for their ability to inhibit synaptosomal PAG activity. PAG activity was determined by measuring GLU formation from 0.5mM glutamine (GLN) in the presence of 10 mM phosphate. GLU analogues at 5–10 mM were found to significantly inhibit PAG activity. It was determined that PAG inhibition occurred regardless of whether the GLU analogues were receptor agonists or antagonists, however, PAG inhibition was influenced by analogue chain length, isomeric form and substituent substitution. The glutamate uptake blockers, dihydrokainic acid and DL-threo--hydroxyaspartic acid were relatively weak inhibitors of PAG (<25% inhibition) as were the receptor agonists, ibotenic acid and (±)cis-2,3-piperidine-dicarboxylic acid. Other GLU analogues produced inhibition of PAG in the range of 40–70%. PAG inhibition by GLU analogues did not appear to differ substantially among the brain regions evaluated (cortex, striatum and hippocampus). The endogenous amino acids, glycine, taurine and N-acetylaspartic acid, also significantly inhibited PAG activity in the 5–10 mM range. The noncompetitive NMDA antagonists, (+)MK801 and ketamine, at a concentration of 5 mM, significantly stimulated PAG activity 1.5–2 fold over control values. The activation of PAG by (+)MK801 was dose-related, stereoselective and appeared to result from a synergistic interaction with phosphate to enhance substrate (GLN) binding to PAG. The results of these studies suggest that GLU analogues could potentially alter neurotransmitter GLU synthesis if sufficient concentrations of these drugs are used in in vitro or in vivo studies. Furthermore, preliminary evidence suggests that other endogenous amino acids (glycine, taurine, N-acetylaspartic acid) may modulate PAG activity. These studies have further characterized the structural requirements for the allosteric regulation of PAG by glutamate and its analogues.     

EFFECTS OF RETINAL ABLATION ON UPTAKE OF GLUTAMATE, GLYCINE, GABA, PROLINE AND CHOLINE IN PIGEON TECTUM

—The effect of retinal ablation on the high and low affinity uptake of choline, GABA, glutamate, glycine and proline into the crude mitochondrial fraction of the pigeon optic tectum was studied. After 4–8 weeks of degeneration the uptake for glutamate, and to a lesser degree the uptake for GABA, at both the high and the low affinity substrate concentration, decreased. In contrast, the uptake of glycine increased. Kinetic analysis showed that the reduced uptake of glutamate was due to a decrease in the V_max. By intraocular injection of [³H]proline the retinal endings in the optic tectum were labelled with the fast axoplasmic transport fraction. Optic terminals labelled in this way have the same sedimentation characteristics in continous sucrose gradients as the particles accumulating giutamate in the uptake assay.     

A model of the sodium dependence of dopamine uptake in rat striatal synaptosomes

Initial velocity of uptake of dopamine (DA) has been measured in rat striatal synaptosomes as a function of both [DA] and [Na]. Carrier mediated uptake is totally dependent on external sodium. The data were fitted to a rapid equilibrium model which has been found in previous studies to fit, with appropriate simplification, uptake data for glutamate, GABA, and choline in several brain regions under varying conditions. This model also gives a good fit to the dopamine data. The minimal best fit simplification of this model allows for DA uptake along with two sodium ions and predicts that apparent maximal velocity of uptake should increase with [Na], while the Michaelis-Menten constant should decrease. The minimal best fit model for DA, and a number of kinetic parameters which quantitate the model, are compared to those for the GABA, glutamate, and choline transporters. The results are consistent with a symmetrical, rapid equilibrium model, which has been presented previously for other neurotransmitters and precursors (18). This model offers a unifying basis for understanding the sodium and membrane potential dependence of neurotransmitter transport and the possible participation of transporters in depolarization induced release throughout the CNS.     

Adaptive enhancement of cystine and glutamate uptake in human diploid fibroblasts in culture

The effect of cystine starvation on the transport system of cystine and glutamate was examined in cultures of human diploid fibroblasts. The 2-min uptake of cystine and glutamate increased progressively after a lag of 6 h of cystine starvation. There was approx. 2–3-fold increase, and the increased rate of uptake was accompanied by an increase in the V_max and unchanged K_m. The cystine starvation-induced enhancement appeared specific for the uptake of cystine and glutamate. Actinomycin D or cycloheximide completely blocked the time-related increase in the uptake. Depletion of glutamate did not lead to the enhanced uptake, whereas depletion of glycine and serine caused as much increase in the uptake as depletion of cystine did. The intracellular pool of glutathione was extremely reduced by depletion of cystine, or of glycine and serine, but to a far less extent by depletion of glutamate. The results indicate that the transport system for cystine and glutamate appears to undergo adaptive regulation. It is suggested that glutathione may function as a regulatory signal to this transport system.     

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.     

Effects of dorsal root section and occlusion of dorsal spinal artery on the neurotransmitter candidates in rat spinal cord

In order to obtain further evidence of putative neurotransmitters in primary sensory neurons and interneurons in the dorsal spinal cord, we have studied the effects of unilateral section of dorsal roots and unilateral occlusion of the dorsal spinal artery on cholinergic enzyme activity and on selected amino acid levels in the spinal cord. One week after sectioning dorsal roots from caudal cervical (C₇) to cranial thoracic (T₂) levels, the specific activity of choline acetyltransferase (ChAT) was significantly decreased and acetylcholinesterase (AChE) showed a tendency to decrease in the dorsal quadrant on the operated side of the spinal cord. Dorsal root sectioning had little effect on the levels of free glutamic acid or other amino acids in the dorsal spinal cord. These results suggest that primary sensory neurons may include some cholinergic axons, and that levels of putative amino acid transmitters are not regulated by materials supplied by axonal transport from the dorsal root ganglia. By contrast, one week following unilateral occlusion of the dorsal spinal artery, the activities of ChAT and AChE were unchanged in the operated quadrant of the spinal cord, while decreases of Asp, Glu, and GABA, and an increase in Tau were detected. These findings are consistent with the proposals that such amino acids, but not ACh, may function as neurotransmitter candidates in interneurons of the dorsal spinal cord.Abbreviation used ACh acetylcholine - AChE acetylcholinesterase - Asp aspartic acid - ChAT choline acetyltransferase - GABA -aminobutyric acid - Glu glutamic acid - Gly glycine - SP substance P - Tau taurine     

Enhancement in dopamine uptake and release induced by monosodium l-glutamate from caudate nucleus under in vitro conditions

l-Glutamate has an excitatory and cytotoxic effect on the central nervous system. It was shown previously that norepinephrine and dopamine uptake and release were affected by in vivo administration of glutamate to adult rats. The kinetic parameters, K_m and V_max of [¹⁴C]DA uptake and release were measured on synaptosomal and slices from caudate nucleus under in vitro conditions at different glutamate concentrations. Results showed an important increase in [¹⁴C]DA uptake on synaptosomal (> 100%) and slices by lower glutamate concentrations, the affinity for transport system was increased (100%) and its release of high potassium evoked was also increased at 0.5 μM of glutamate. The results suggest the possibility that glutamate may modify DA uptake and release interacting with the DA transporter complex at the synaptic level.     

Calcium-Dependent Release of Tyrosine in Brain Elicited by Stimulation of Neuropeptide Receptors

We sought to establish whether the endogenous opiate-receptor agonist Met-enkephalin (m-ENK) selectively modulates the release of endogenous tyrosine (Tyr) from brain slices prepared from the corpus striatum (CS). Amino acids (AAs) released from slices of CS and, for comparison, cerebral cortex (Cx) were measured by HPLC. Incubation of slices with m-ENK (1-10 microM) increased the basal release of Tyr (up to 293% of control) from CS, but not Cx, whereas other nonneurotransmitter AAs, phenylalanine (Phe) and valine (Val), were unchanged. The release of the putative neurotransmitter AAs glutamate (Glu), taurine (Tau), and glycine (Gly) were similarly increased by 50-150% with m-ENK in slices of CS, but not Cx. The enhanced release of AAs by m-ENK was prevented by removal of extracellular Ca2+ or by preincubation with the opiate receptor antagonist naloxone. Neuronal depolarization by potassium (5-55 mM) in the presence of Ca2+ did not affect the release of Tyr, whereas release of neurotransmitter AAs such as gamma-aminobutyric acid (GABA) were markedly increased. The increase in basal Tyr release by m-ENK was not the result of a decreased uptake of Tyr. Relative to slices, the basal release of Tyr, Phe, and Val from a synaptosomal (P2) preparation of CS was small (8-51%) compared to that of GABA, Gly, Glu, and Tau (49-123%). Nonetheless, m-ENK (10 microM) markedly increased the release of Tyr (to 833%), but not Glu, Gly, and Tau from the P2 fraction.(ABSTRACT TRUNCATED AT 250 WORDS)