HIGH AFFINITY UPTAKE OF TRANSMITTERS: STUDIES ON THE UPTAKE OF l-ASPARTATE, GABA, l-GLUTAMATE AND GLYCINE IN CAT SPINAL CORD

Abstract— The uptake of l -aspartate, l -glutamate and glycine each appeared to be mediated by two kinetically distinct systems with apparent K_m's of the order of 10 ('high affinity') and 100 μM ('low affinity') in slices of cat spinal cord, whereas the uptake of GABA appeared to be mediated by a single system of high affinity. The high affinity uptake of these amino acids in slices of spinal grey matter was approximately 5 times faster than that in slices of spinal white matter. The high affinity uptake systems in the cord slices survived homogenisation of the tissue under conditions known to preserve nerve terminals. Subcellular fractionation studies indicated that osmotically-sensitive particles of equilibrium density equivalent to that of 1.0 m -sucrose were at least in part responsible for the uptake of these amino acids. Inhibition studies indicated that three structurally specific systems of high affinity transported these amino acids:

• 1 specific for glycine—not inhibited by GABA or any of the other depressant amino acids found in cat spinal cord;
• 2 specific for GABA—not inhibited by glycine, taurine, l -aspartate or l -glutamate and (3) specific for l -aspartate and l -glutamate—not inhibited by glycine or GABA but strongly inhibited by various acidic amino acids such as l -cysteate and l -cysteine sulphinate.

The high affinity uptake of these amino acids was not inhibited by any of the known antagonists of the postsynaptic actions of these amino acids—strychnine (glycine), bicuculline and benzyl penicillin (GABA), methioninesulphoximine and l -glutamate diethyl ester (l -aspartate and l -glutamate). p-Chloromercuriphenylsulphonate strongly inhibited the high affinity uptake of glycine and GABA but was much less effective as an inhibitor of l -aspartate/l -glutamate high affinity uptake. This is in good agreement with microelectrophoretic studies in which this mercurial was found to potentiate depression of neuronal firing induced by glycine and GABA much more readily than excitation induced by l -aspartate or l -glutamate. These findings suggest the importance of high affinity transport processes in the removal of amino acids from the synaptic environment.     

SODIUM AND POTASSIUM IONS AND ACCUMULATION OF LABELLED d-ASPARTATE AND GABA IN CRUDE SYNAPTOSOMAL FRACTION FROM RAT CEREBRAL CORTEX

The accumulation of labelled d -aspartate into crude synaptosomal fraction (P₂) prepared from the rat cerebral cortex proceeded by a ‘high affinity’ system (K_m= 15.1 μm The maximal velocity of d -aspartate uptake was higher than that of the ‘high affinity’ component of l -aspartate uptake and almost equal to that of l -glutamate under the same incubation conditions. Negligible metabolism of labelled d -aspartate was observed in the P₂ fraction. These findings are in accord with those which have been reported for rat cerebral cortical slices. The following observations were made on d -aspartate uptake into rat cerebral P₂ fraction. (1) The requirement of sodium is almost absolute and obligatory. (2) The affinity of the carrier for the substrate is increased by increasing sodium concentration in the medium, but the maximal velocity is not altered. (3) It is suggested that sodium ion is co-transported mole for mole with the substrate molecule. (4) Omission of potassium from the medium inhibits the uptake competitively. (5) Ouabain is a competitive inhibitor on the uptake. (6) Whereas thallium, rubidium and ammonium are efficient substitutes for potassium in exhibiting Na–K ATPase activity of the P₂ fraction, the uptake is activated only by rubidium in the absence of potassium. These observations were in common with the uptake of l -aspartate as well as of l - and d -glutamate, but not with GABA uptake. The requirement of sodium for the uptake of d -glutamate was indicated to be higher than that in the uptake of the other amino acids. Mutual inhibitions of the uptake among l - and d -isomers of glutamate and aspartate suggested that a common carrier is involved in the transport. Mechanisms of the transport of these amino acids in the crude synaptosomal fraction were discussed.     

ACTIONS OF l-GLUTAMATE AND RELATED AMINO ACIDS ON OXYGEN UPTAKE, LACTATE PRODUCTION AND NADH LEVELS OF RAT BRAIN IN VITRO

Abstract— A range of acidic amino acids differing in (i) their potency as neuronal excitants, (ii) their transport properties and (iii) their ability to act as substrates for metabolism have been compared with respect to their effects on energy metabolism of rat cerebral cortex in vitro. l -Glutamate, and d - and l -homocysteate, increased tissue slice NADH levels, and the same three amino acids, together with d -glutamate and kainate, increased oxygen uptake by the slices. It was concluded that these effects were predominantly due to neuronal depolarization and the ensuing activation of ion pump mechanisms. l -Glutamate, d -glutamate and l -homocysteate increased lactate production by the slices, whereas d -homocysteate and kainate did not. Since the two latter amino acids are the strongest neuroexcitants but probably the least rapidly transported, it is suggested that stimulation of lactate production in slices by amino acid excitants is a consequence of the energy requirements of active uptake of the amino acids, and probably occurs mainly in glial cells. Although the metabolism of l -glutamate appeared not to be an essential requirement for the effects observed with this amino acid in the present work, such metabolism may make a proportionately greater contribution under sub-optimal conditions of slice preparation and incubation, where electrical activity of the tissue may be impaired.     

Characterization of uptake and release processes ford- andl-aspartate in primary cultures of astrocytes and cerebellar granule cells

The uptake ofl-andd-aspartate was studied in astrocytes cultured from prefrontal cortex and in granule cells cultured from cerebellum. A high affinity uptake system forl- andd-aspartate was found in both cell types, and the two stereoisomers exhibited essentially the sameK _m - andV _max -values in bouth astrocytes (l-aspartate:K _m 77 μM;V _max 11.8 nmol×min^?1×mg^?1;d-aspartate:K _m 83 μM;V _max 14.0 nmol×min^?1×mg^?1) and granule cells (l-aspartate:K _m 32 μM;V _max 2.8 nmol ×min^?1×mg^?1;d-aspartate:K _m 26 μM;V _max 3.0 nmol×min^?1×mg^?1). To investigate whetherl-glutamate,l-aspartate andd-aspartate use the same uptake system a detailed kenetic analysis was performed. The uptake kinetics of each one of the three amino acids was studied in the presence of the two other amino acids, and no essential differences between the uptake characteristics of the amino acids were found. In addition to the uptake studies the release ofD-aspartate from cerebellar granule cells was investigated and compared withl-glutamate release. A Ca²⁺-dependent, K⁺-induced release was found for both amino acids.     

IONIC BASIS FOR THE DEPOLARIZATION OF CEREBRAL TISSUES BY EXCITATORY ACIDIC AMINO ACIDS

1. Membrane potentials were measured in samples of guinea pig cerebral cortex, before and after small localized additions of neutral solutions of a number of acidic amino acids. 2. Prompt depolarization from resting membrane potentials of about ?60 mv to values of ?30 to ?45 mv occurred in response to l -aspartate, l -cysteate, l -glutamate, dl -homocysteate and l -α-aminoadipate. Slower, smaller and less frequent depolarization followed N-methyl-d -aspartate, N-methyl-dl -aspartate and N-ethyl-D-aspartate. No change was observed to follow N-n-propyl-d -aspartate, N-methyl-dl -glutamate, β-alanine or glutamine. 3. The five amino acids which promptly depolarized, were found also to lower the phosphocreatine of cerebral tissues to which they were added during in vitro metabolism. They also increased the tissues’inorganic phosphate and in some cases diminished its acid-labile nucleotide phosphate. 4. The five amino acids which promptly depolarized increased the intracellular sodium of the tissues within a minute of their addition; two of the N-alkylated acids also had this effect. None of the acids had a comparably prompt effect on the tissues’potassium content. 5. Calculation from the data presented suggests (i) that glutamate increases fivefold the tissues’permeability to sodium relative to that to potassium; and (ii) that the additional sodium entering the tissue could accelerate its Na,K-dependent adenosine triphosphatase sufficiently to account for the observed loss of energy-rich phosphate and appearance of inorganic phosphate.     

Na+-dependent medium-affinity uptake of L-glutamate in the insect epidermis

It is proposed that the activity of an epidermal cotransport system for Na⁺ and dicarboxylic amino acids accounts for the small amounts of L-glutamate and L-aspartate in the otherwise amino-acid-rich blood plasma of insects. This Na⁺-dependent transport system is responsible for more than 95% of the uptake of these amino acids into the larval epidermis of the beetle Tenebrio molitor. Kinetic analysis of uptake showed that the Na⁺-dependent co-transporter has medium affinity for L-glutamate and L-aspartate. The K _m for L-glutamate uptake was 146 mol·l^-1, and the maximum velocity of uptake (V _max) was 12.1 pmol·mm^-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 mol·l^-1 and 8.4 pmol·mm^-2·min^-1. The Na⁺/L-glutamate co-transporter has a stoichiometry of at least two Na⁺ ions for each L-glutamate-ion transported (n=217). The co-transporter has an affinity for Na⁺ equivalent to a K _m of 21 mmol · l^-1 Na⁺. Na⁺ is the only external ion apparently required to drive L-glutamate uptake. Li⁺ substitutes weakly for Na⁺. Removal of external K⁺ or addition of ouabain decreases uptake slowly over 1 h, suggesting that these treatments dissipate the Na⁺/K⁺ gradient by inhibiting epidermal Na⁺/K⁺ ATPase. Several structural analogues of L-glutamate inhibit the medium-affinity uptake of L-glutamate. The order of potency with which these competitive inhibitors block glutamate uptake is L-cysteatethreo-3-hydroxy-Dl-aspartate > D-aspartateL-aspartate> L-cysteine sulphinate > L-homocysteateD-glutamate. L-trans-Pyrrolidine-2,4-dicarboxylate, a potent inhibitor of L-glutamate uptake in mammalian synaptosomes, is a relatively weak blocker of epidermal uptake. The epidermis takes up substantially more L-glutamate by this Na⁺-dependent system than tissues such as skeletal muscle and ventral nerve cord. The epidermis may be a main site regulating blood L-glutamate levels in insects with high blood [Na⁺]. Because L-glutamate and L-aspartate stimulate skeletal muscle in insects, a likely role for epidermal L-glutamate/L-aspartate transporter is to keep the level of these excitatory amino acids in the blood below the postsynaptic activation thresholds.Abbreviation ac acetate - Ch choline - CNS central nervous system - cpm counts per minute - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acids - HPLC high performance liquid chromatography - K _m Michaelis constant - n _app apparent number - NMG N-methyl-D-glucamine - Pipes Piperazine-N,N-bis-[2-ethanesulfonic acid] - SD standard deviation - TEA tetraethyl-ammonium - V velocity of uptake - V _max maximum velocity of uptake     

THE INFLUENCE OF INTRAVENTRICULARLY INJECTED AMINO ACID EXCITANTS ON THE LABELLING OF ENDOGENOUS BRAIN AMINO ACIDS FROM [U-14C]ACETATE IN NEMBUTALIZED MICE

Mice were anaesthetized with nembutal and the effects of intraventricularly injected excitant amino acids on [U-¹⁴C]acetate metabolism were investigated. The natural excitant amino acids, l -glutamate and l -aspartate, reduced the incorporation of ¹⁴C from [U-¹⁴C]acetate into glutamine, GAB A and possibly alanine. The synthetic excitant amino acid, N-methyl-d -aspartate caused a reduction in the incorporation of ¹⁴C from intraventricularly injected [U-¹⁴C]acetate into all of the brain amino acids labelled by [U-¹⁴C]acetate within 5 min. It is suggested that these effects may be due to changes in pool sizes of tricarboxylic cycle intermediates, to inhibition of acetyl-CoA formation, or both. Differences in the metabolic effects of the synthetic and natural excitants are interpreted in terms of the uptake of the natural amino acids into glutamine-forming pool(s) of glutamate metabolism.     

SYNTHETIC AMINO ACIDS AND THE NATURE OF l-DOPA TRANSPORT AT THE BLOOD-BRAIN BARRIER

—The blood-brain barrier transport of amino acids has been measured using the carotid injection technique in the rat. The synthetic amino acids, 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) and α-(methylamino)isobutyric acid (MeAIB), were model substrates in the Ehrlich cell for the leucine (L) and alanine (A) neutral amino acid transport mechanisms, respectively. The uptake (±)b-[carboxyl-¹⁴C]BCH at the same rate for the five brain regions tested suggested a similarity between regions for the L transport mechanism. At injectant concentrations of 0·1 mm (similar to naturally occurring aromatic neutral amino acids), BCH was mainly taken up by a saturable mediated transport mechanism (K₁, 0·16 mm and V_max, 0·03/μmol/g per min). At higher concentrations, uptake by a nonsaturable or diffusional mechanism could be demonstrated. When BCH was added as a second amino acid to l -[3-¹⁴C]DOPA, the saturable component of l -DOPA transport was significantly inhibited. MeAIB had no measurable effect on the rate of l -DOPA transport. These results suggested that the mediated transport mechanism for l -DOPA at the cerebral capillaries is similar to the l -neutral amino acid transport system.     

THE STRUCTURAL SPECIFICITY OF THE HIGH AFFINITY UPTAKE OF l-GLUTAMATE AND l-ASPARTATE BY RAT BRAIN SLICES

Abstract— The high affinity uptake system for l -glutamate and l -aspartate in rat cerebral cortex may not be specific for these likely excitatory synaptic transmitters, as threo-3-hydroxy- dl -aspartate, l -cysteinesulphinate, l -cysteate and d -aspartate strongly inhibit the observed high affinity uptake of l -[³H]glutamate by rat brain slices in a manner consistent with linear competitive inhibition. These substances should therefore be considered as possible substrates for the transport system. Each of these four acidic amino acids excites central neurones in a manner similar to excitation induced by l -glutamate, and as each might occur in brain tissue, their possible synaptic role should be investigated.
l -Glutamate high affinity uptake was shown to be sodium-dependent, but under certain conditions appeared to be less sensitive than GABA uptake to changes in the external sodium ion concentration, and to drugs which modify sodium ion movements. This may be relevant to the efficiency of the glutamate uptake process during synaptic depolarization induced by glutamate.
l -Glutamate high affinity uptake was inhibited in a relatively nonspecific manner by a variety of drugs including mercurials and some electron transport inhibitors.     

CEREBRAL UPTAKES AND EXCHANGE DIFFUSION IN VITRO OF l- AND d-GLUTAMATES

Abstract— 1. Whereas exogenous l -glutamate enters rat brain cortex slices incubated in a glucose-physiological saline medium by both low affinity (K_m= 0.7 mm ) and high affinity (K_m= 27?30 μM) processes, the uptake of d -glutamate occurs only by a low affinity (K_m= 2mm ) system. 2. d -glutamate appears to release l -glutamate from incubated rat brain cortex slices only to a very small extent, whether the tissue l -glutamate is of endogenous or exogenous origin. 3. Competitive inhibition takes place between l - and d -glutamates at the low affinity carrier. This indicates that a common carrier exists for l - and d -glutamates for the low affinity uptake process. 4. Apparently non-competitive inhibition by d -glutamate of l -glutamate uptake occurs at the high affinity carrier, but the affinity of d -glutamate for this carrier is about 0.4% of that of l -glutamate. 5. Both d -, and l -glutamate exchange freely with labelled d -glutamate taken up by preliminary incubation of the brain slices with this amino acid. Whereas l -glutamate exchanges freely with labelled l -glutamate taken up by preliminary incubation, d -glutamate shows little or no exchange. 6. The uptake of labelled d -glutamate by exchange diffusion into brain slices previously loaded with unlabelled d -glutamate proceeds by a low affinity system. Therefore, the process of exchange diffusion does not necessarily involve a high affinity uptake component. 7. Whereas ouabain suppresses both high and low affinity concentrative uptakes of l - and d -glutamate it has little apparent effect on the exchange diffusion process. 8. Sensitivity to tetrodotoxin of evoked release of l - and d -glutamates, taken up by brain slices by preliminary incubation with these amino acids, indicates that the major proportion of the uptake of exogenous l - or d -glutamate proceeds into non-neuronal structures (presumably the glia). 9. At 0°C non-carrier mediated (passive) diffusion of labelled d - and l -glutamate takes place in brain slices.     

COUPLED TRANSPORT OF GLUTAMATE AND SODIUM IN A CEREBELLAR NERVE CELL LINE

The cerebellar nerve cell line ε¹ has a very effective active transport system for glutamate. Glutamate uptake is dependent on extracellular Na⁺ and furthermore, ²²Na⁺ uptake is stimulated by glutamate, indicating that glutamate uptake and Na⁺ uptake are coupled. Two molecules of Na ⁺ are transported for each molecule of glutamate. The K_m for glutamate is found to be 5 × 10^?5M in both the glutamate uptake assay and the ²²Na⁺ uptake assay, providing additional evidence for glutamate-Na⁺ coupling. Pre-incubation with ouabain, which inhibits the Na⁺-K⁺ ATPase, results in a gradual inhibition of glutamate uptake due to the deterioration of the Na⁺ gradient. Tetrodotoxin, however, has no effect on glutamate-induced ²²Na⁺ uptake, showing that this Na⁺ flux does not occur via voltage-dependent Na⁺ channels. Studies on the specificity of the ε¹ glutamate transport system show that it is distinct from systems that transport alanine and glycine. l -Glutamate, d -aspartate, l -cysteate, and l -cysteine sulfinate are able to utilize the transport system efficiently. d -Glutamate, l -homocysteate, N-methyl-d , l -aspartate, and kainic acid are very poor substrates for the glutamate transport system, and in addition do not stimulate ²²Na⁺ uptake. These data allow us to distinguish the glutamate transport system from the glutamate receptor which is known to mediate depolarization in response to all nine of the above compounds. Thus, ε¹ does not have an excitatory glutamate receptor.     

Adenosine and Glutamate Modulate Each Other's Release from Rat Hippocampal Synaptosomes

Abstract: In rat hippocampal synaptosomes, adenosine decreased the K⁺ (15 mM) or the kainate (1 mM) evoked release of glutamate and aspartate. An even more pronounced effect was observed in the presence of the stable adenosine analogue, R-phenylisopropyladenosine. All these effects were reversed by the selective adenosine A₁ receptor antagonist 8-cyclo-pentyltheophylline. In the same synaptosomal preparation, K⁺ (30 mM) strongly stimulated the release of the preloaded [³H]adenosine in a partially Ca²⁺-dependent and tetrodotoxin (TTX)-sensitive manner. Moreover, in the same experimental conditions, both l -glutamate and l -aspartate enhanced the release of [³H]adenosine derivatives ([³H]ADD). The gluta-mate-evoked release was dose dependent and appeared to be Ca²⁺ independent and tetrodotoxin insensitive. This effect was not due to metabolism because even the nonmetabolizable isomers d -glutamate and d -aspartate were able to stimulate [³H]ADD release. In contrast, the specific glutamate agonists N-methyl-d -aspartate, kainate, and quisqualate failed to stimulate [³H]ADD release, suggesting that glutamate and aspartate effects were not mediated by known excitatory amino acid receptors. Moreover, NMDA was also ineffective in the absence of Mg²⁺ and l -glutamate-evoked release was not inhibited by adding the specific antagonists 2-amino-5-phosphonovaleric acid or 6–7-dinitroquinoxaline-2, 3-dione. The stimulatory effect did not appear specific for only excitatory amino acids, as γ-anunobutyric acid stimulated [³H]ADD release in a dose-related manner. These results suggest that, at least in synaptosomal preparations from rat hippocampus, adenosine and glutamate modulate each other's release. The exact mechanism of such interplay, although still, unknown, could help in the understanding of excitatory amino acid neurotoxicity.     

Amino acid and monoamine transport in primary astroglial cultures from defined brain regions

The uptake ofl-[³H]glutamate,l-[³H]aspartate, -[³H]aminobutric acid (GABA), [³H]dopamine,dl-[³H]norepinephrine and [³H]5-hydroxytryptamine (5-HT) was studied in astrocytes cultured from the cerebral cortex, striatum and brain stem of newborn rat and grown for 2 weeks in primary cultures. The astrocytes exhibited a high-affinityl-glutamate uptake withK _m values ranging from 11 to 110 M.V _max values were 4.5 in cerebral cortex, 39.1 in striatum, and 0.4 in brain stem, nmol per mg cell protein per min. There was a less prominent high-affinity uptake ofl-aspartate withK _m values from 88 to 187 M.V _max values were 7.4 in cerebral cortex, 37.1 in striatum, and 3.1 in brain stem, nmol per mg cell protein per min. The high-affinity GABA uptake exhibitedK _m values ranging from 5 to 17 M andV _max values were 0.01 for cerebral cortex, 0.04 for striatum, and 0.1 for brain stem, nmol per mg cell protein per min. No high-affinity, high-capacity uptake was found for the monoamines. The results demonstrate a heterogeneity among the astroglial cells cultivated from the different brain regions concerning the uptake capacity of amino acid neurotransmitters. Furthermore, amino acid transmitters and monoamines are taken up by the cells in different ways.     

Transport of monoamine and amino acid neurotransmitters by primary astroglial cultures

The transport of [³H]l-glutamate, [³H]l-aspartate, [³H]-aminobutyric acid ([³H]GABA), [³H]dopamine, [³H]norepinephrine and [³H]5-hydroxytryptamine (³H-5-HT) was measured in primary astroglial cultures from newborn rat cerebral hemispheres. There was a high-affinity uptake with aK _m of 69.0 M for L-glutamate, 12.3 M forl-aspartate and 3.1 M for GABA. The uptake showed properties of high capacity with aV _max of 17.0 nmol·mg prot^–1·min^–1 forl-glutamate, 1.1 nmol·mg prot^–1·min^–1 forl-aspartate and 0.04 nmol·mg prot^–1·min^–1 for GABA. No high-affinity high capacity transport system was found for the monoamines studies. Autoradiographic examination demonstrated a heavy deposit of grains suggesting a prominent accumulation of [³H]l-glutamate and [³H]l-aspartate in the astroglial-like cells of the cultures, while the [³H]GABA accumulation was less intense. On the other hand, there was only a weak accumulation of grains after incubating the cultures with [³H]dopamine, [³H]norepinephrine or [³H]5-HT. Thus, astroglial cells in culture accumulate amino acid neurotransmitters and monoamines in different ways with a high-affinity high-capacity uptake of glutamate, aspartate and GABA and a diffusion-uptake of dopamine, norepinephrine and 5-HT.     

KINETICS OF AMINO ACID INFLUX INTO NITELLA FLEXILIS1

The uptake of amino acids by Nitella flexilis has been investigated. Influx of glycine, alanine, and valine appears to be a diffusive process. Influx ranged from 0.14 to 0.06 and 0.04 pmoles/(cm)(sec), respectively. Aspartic acid uptake is an active transport mechanism. The V_max is 2.8 pmoles/(cm)(sec); the transport constant (Michaelis constant) K_m, 7.8 × 10^?3 M. The uptake of arginine is apparently due to 2 transport systems, one with a V_max and K_m of 3.1 pmoles/(cm)(sec) and 3.2 × 10^?3M, respectively. The second system has a V_max of 1.4 pmoles/(cm)(sec) and a K_m of 2.1 × 10^?4 M. The possibility that the second system is diffusive has been considered.     

Free Radicals Enhance Basal Release of D-[3H]Aspartate from Cerebral Cortical Synaptosomes

Abstract: Excessive generation of free radicals has been implicated in several pathological conditions. We demonstrated previously that peroxide-generated free radicals decrease calcium-dependent high K⁺-evoked l -[³H]-glutamate release from synaptosomes while increasing calcium-independent basal release. The present study evaluates the nonyesicular release of excitatory amino acid neurotransmitters, using d -[³H]aspartate as an exogenous label of the cytoplasmic pool of l -glutamate and l -aspartate. Isolated presynaptic nerve terminals from the guinea pig cerebral cortex were used to examine the actions and interactions of peroxide, iron, and desferrioxamine. Pretreatment with peroxide, iron alone, or peroxide with iron significantly increased the calcium-independent basal release of d -[³H]aspartate. Pretreatment with desferrioxamine had little effect on its own but significantly limited the enhancement by peroxide. High K⁺-evoked release in the presence of Ca²⁺ was enhanced by peroxide but not by iron. These data suggest that peroxide increases nonvesicular basal release of excitatory amino acids through Fenton-generated hydroxyl radicals. This release could cause accumulation of extracellular excitatory amino acids and contribute to the excitotoxicity associated with some pathologies.     

Effects of excitatory neurotransmitter amino acids on swelling of rat brain cortical slices.

Abstract— With the single rat brain cortical slice serving as an in vitro bio-assay system, the effects of neurotransmitter amino acids (1 mm ) on brain swelling, water, sodium and potassium content, inulin space, and lactate production were studied. The putative dicarboxylic amino acid neurotransmitters, l -glutamic acid and l -aspartic acids, greatly increased intracellular brain swelling with increased intracellular Na⁺, water content and lactate production, and decreased inulin space and intracellular K⁺. Equimolar GABA, taurine, glycine, the putative inhibitory neurotransmitter amino acids, and equimolar α-amino-isobutyric acid had no effect. Brain swelling and intracellular Na⁺/K⁺ ratios were greatly increased by l -glutamate and l -aspartate at a concentration of 10 mm . However, l -aspartate at these concentrations greatly depleted the K⁺ content and lactate production as compared to l -glutamate. Further studies indicated that only the structural analogs and isomers of the dicarboxylic amino acids possessing two acidic groups and an α-amino group had a similar effect on the induction of brain swelling. Among the analogs of glutamic acid, dl -homocysteic acid and kainic acid had a greater effect on brain swelling, as observed from the total adenosine 5′-triphosphate (ATP) levels and the time-course and dose-response. A biphasic response in lactate production was induced by dl -homocysteic acid and kainic acid, suggesting that these analogs had a neurotoxic effect on cellular metabolism at higher concentrations.     

TRANSPORT OF TYROSINE, PHENYLALANINE, TRYPTOPHAN AND GLYCINE IN NEUROBLASTOMA CLONES

Amino acid transport was studied in three neuroblastoma clones, N-TD6, which synthesizes norepinephrine, N-T16, which synthesizes small amounts of serotonin, and N-S20Y, which synthesizes acetylcholine. All three clones exhibited high-affinity saturable transport systems for tyrosine, phenylalanine, tryptophan and glycine as well as systems unsaturated at amino acid concentrations of 1 mM in the external medium. Tyrosine, phenylalanine and tryptophan enter all three clones by rapidly exchanging transport systems which appear to be relatively insensitive to lowered external [Na⁺] or to the presence of 2,4-dinitrophenol (DNP). Glycine uptake was slower and was much more sensitive to lowered external [Na⁺] and to the presence of DNP in the medium. Glycine transport in N-T16 cells was decreased more markedly at low temperature than was transport of the three aromatic amino acids. K_m and V_max values found for saturable transport of tyrosine, phenylalanine and tryptophan were sufficiently low to suggest that, if similar amino acid transport systems exist in neuronal membranes, and if amino acid levels in brain extracellular fluid are similar to levels in plasma, such systems may serve, in conjunction with transport systems in cerebral capillaries, to limit the entry of amino acids into brain cells when blood amino levels are near the normal physiological range.     

l-Glutamate Uptake Inhibitors May Stimulate Phosphoinositide Hydrolysis in Baby Hamster Kidney Cells Expressing mGluR1a via Heteroexchange with l-Glutamate Without Direct Activation of mGluR1a

Abstract: The functional efficacies of inhibitors of l -glutamate uptake for altering second messenger formation in baby hamster kidney cells expressing subtypes mGluR1a, mGluR2, and mGluR4 of the metabotropic glutamate receptor family were examined. l -Serine-O-sulfate was an agonist at mGluR1a (EC₅₀ = 70 µM), mGluR2 (EC₅₀ = 25 µM), and mGluR4 (EC₅₀ = 324 µM). l -Cysteine sulfinate, 1-aminocyclobutane-trans-1,3-dicarboxylate, l -cysteine, and dl -threo-3-methylaspartate stimulated phosphoinositide hydrolysis in mGluR1a cells with EC₅₀ values of 43, 64, 463, and 488 µM, respectively, and displaced l -[³H]glutamate binding from membranes prepared from these cells with respective IC₅₀ values of 48, 44, 79, and 139 µM. However, d -aspartate,l -trans-pyrrolidine-2,4-dicarboxylate, l -threo-3-hydroxyaspartate, and l -aspartate-β-hydroxamate stimulated phosphoinositide hydrolysis in mGluR1a cells (respective EC₅₀ values of 73, 54, 57, and 430 µM) but did not displace l -[³H]glutamate binding. These compounds inhibited Na⁺-dependent l -glutamate uptake into baby hamster kidney cells with IC₅₀ values similar to those for stimulation of phosphoinositide hydrolysis in mGluR1a cells. Phosphoinositide hydrolysis in mGluR1a cells, as stimulated by inhibitors of (or substrates for) this l -glutamate transporter, was significantly attenuated in the presence of l -glutamate decarboxylase (EC 4.1.1.15) or l -alanine aminotransferase (EC 2.6.1.2). Furthermore, incubation with 1 mMl -trans-pyrrolidine-2,4-dicarboxylate for 30 min increased the basal levels of free glutamate (1.5 ± 0.2 µM) in the assay buffer four- to fivefold as measured by HPLC analysis. Thus, heteroexchange with endogenous l -glutamate may lead to erroneous estimations of the functional efficacies at mGluR1a.     

INCORPORATION OF PHENYLALANINE, TYROSINE AND TRYPTOPHAN INTO PROTEIN OF HOMOGENATES FROM DEVELOPING RAT BRAIN: KINETICS OF INCORPORATION AND RECIPROCAL INHIBITION

The kinetics of the incorporation into protein of [³H]phenylalanine, [³H]tyrosine and [³H]tryptophan were studied with homogenates prepared from whole brain of 1-, 7-, 21- and 60-day-old rats. The maximal velocities (V_max)of incorporation of phenylalanine and tyrosine decreased and the apparent Michaelis-constants (K_m) for all three amino acids increased with increasing age of the rats. Tyrosine had the smallest and tryptophan the largest K_m values in all age groups. Phenylalanine competitively inhibited the incorporation of tyrosine, but tyrosine inhibited non-competitively the incorporation of phenylalanine. Tryptophan inhibited competitively the incorporation of phenylalanine, but at least partially non-competitively the incorporation of tyrosine. Phenylalanine and tyrosine did not significantly affect the incorporation of tryptophan in homogenates from 60-day-old rats. In 1-day-old rats only a very large excess of phenylalanine or tyrosine inhibited detectably. The K_i for phenylalanine in the incorporation of tyrosine was significantly smaller in 1- than in 60-day-old rats. In every case the inhibition presumably occurred at a single rate-limiting step in the complicated process of incorporation of amino acids into protein.