ACTION OF THE NEUROTOXIN KAINIC ACID ON HIGH AFFINITY UPTAKE OF l-GLUTAMIC ACID IN RAT BRAIN SLICES

Kainic acid is a linear competitive inhibitor (K_is 250 μm ) of the ‘high affinity’ uptake of l -glutamic acid into rat brain slices. Kainic acid inhibits the ‘high affinity’ uptake of l -glutamic, d -aspartic and l -aspartic acids to a similar extent. Kainic acid is not actively taken up into rat brain slices and is thus not a substrate for the ‘high affinity’ acidic amino acid transport system or any other transport system in rat brain slices. Kainic acid (300 μm ) does not influence the steady-state release or potassium-stimulated release of preloaded d -aspartic acid from rat brain slices. Kainic acid binds to rat brain membranes in the absence of sodium ions in a manner indicating binding to a population of receptor sites for l -glutamic acid. Only quisqualic and l -glutamic acid inhibit kainic acid binding in a potent manner. The affinity of kainic acid for these receptor sites appears to be some 4 orders of magnitude higher than for the ‘high affinity’l -glutamic acid transport carrier. Dihydrokainic acid is approximately twice as potent as kainic acid as an inhibitor of ‘high affinity’l -glutamic acid uptake but is some 500 times less potent as an inhibitor of kainic acid binding and at least 1000 times less potent as a convulsant of immature rats on intraperitoneal injection. Dihydrokainic acid might be useful as a ‘control uptake inhibitor’ for the effects of kainic acid on ‘high affinity’l -glutamic acid uptake since it appears to have little action on excitatory receptors. N-Methyl-d -aspartic acid is a potent convulsant of immature rats, but does not inhibit kainic acid binding or ‘high affinity’l -glutamic acid uptake. N-Methyl-d -aspartic acid might be useful as a ‘control excitant’ that activates different excitatory receptors to kainic acid and does not influence ‘high affinity’l -glutamic acid uptake.     

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.     

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.     

MOBILIZATION OF SYNAPTIC MEMBRANE-BOUND CALCIUM BY ACIDIC AMINO ACIDS1

A fluorescent chelate probe (chlorotetracycline) and radioactive ⁴⁵Ca were used to study the effects of amino acids on the calcium bound to external synaptosomal membranes isolated from guineapig brain. Acidic amino acids released some of the membrane-bound calcium. On the basis of ⁴⁵Ca studies, the order of mobilization potency-DL-homocysteic acid and l -cysteic acid > l -aspartic acid, l -glutamic acid, d -glutamic acid > N-methyl-dl -glutamic acid and dl -cyteic acid-is in general agreement with that found by fluorescent chelate method with the exception of N-methyl-dl -aspartic acid and N-methyl-dl -glutamic acid, which are at least as potent as dl -homocysteic acid. This order of potency is observed only with a fraction enriched in external synaptosomal membranes, but not with microsomes, myelin and mitochondria. Neutral and basic amino acids, including glutamine. glycine and γ-aminobutyric acid are ineffective. These results suggest that acidic amino acids have a specific ability to mobilize membranebound calcium; this is consistent with the proposed role of some of these compounds as excitatory transmitters in the central nervous system.     

THE UPTAKE OF [3H]GABA BY SLICES OF RAT CEREBRAL CORTEX

—A rapid accumulation of [³H]GABA occurs in slices of rat cerebral cortex incubated at 25° or 37° in a medium containing [³H]GABA. Tissue medium ratios of almost 100:1 are attained after a 60 min incubation at 25°. At the same temperature no labelled metabolites of GABA were found in the tissue or the medium. The process responsible for [³H]GABA uptake has many of the properties of an active transport mechanism: it is temperature sensitive, requires the presence of sodium ions in the external medium, is inhibited by dinitrophenol and ouabain, and shows saturation kinetics. The estimated K_m value for GABA is 2·2 × 10^?5m , and V_max is 0·115 μmoles/min/g cortex. There is only negligible efflux of the accumulated [³H]GABA when cortical slices are exposed to a GABA-free medium. [³H]GABA uptake was not affected by the presence of large molar excesses of glycine, l -glutamic acid, l -aspartic acid, or β-aminobutyrate, but was inhibited in the presence of l -alanine, l -histidine, β-hydroxy-GABA and β-guanidinopropionate. It is suggested that the GABA uptake system may represent a possible mechanism for the inactivation of GABA or some related substance at inhibitory synapses in the cortex.     

ACTIONS OF l- AND d-HOMOCYSTEATE IN RAT CNS: A CORRELATION BETWEEN LOW-AFFINITY UPTAKE AND THE TIME COURSES OF EXCITATION BY MICROELECTROPHORETICALLY APPLIED L-GLUTAMATE ANALOGUES

Abstract— A correlation has been attempted between the uptake characteristics of l - and d -homocysteate and the time courses of neuronal excitation by these and other amino acids related to l -glutamate. The uptake of l - and d -homocysteate and of l -[³⁵S]homocysteate was studied in individual slices of rat cerebral cortex at 37°C. Tissue: medium ratios attained over l0 min for the unlabelled enantiomers at 2.5 mM were 3.7 for l -homocysteate but only 0.8 for the d -isomer. The uptake of l -[³⁵S]homocysteate over the concentration range 0.09 μm -2 mm can be attributed mainly to a low-affinity transport process with K_m approx 3 mm and V_max 1.7 μmol/g/min, but a high-affinity process of low V_max may make a minor contribution at the lower concentrations within this range. In terms of dependence on energy metabolism and [Na⁺], and on inhibition by p-chloromercuriphenylsulphonate, ouabain and structural analogues of the amino acid, the main uptake system for L-[³⁵S]homocysteate appears to be similar to that mediating low-affinity uptake of l -glutamate and other acidic amino acids. d -Homocysteate was but a weak inhibitor of this uptake system compared with other structural analogues. The time courses of excitation by 6 amino acids were determined by microelectrophoretic application to rat spinal neurones. d -Homocysteate induced responses with recovery times considerably longer than those of the other amino acids; this correlates with the absence of rapid uptake systems demonstrated for this amino acid in cortical tissue. d -Glutamate and l -homocysteate, which are only accumulated by low-affinity transport mechanisms, induced responses with recovery periods similar to those of l -glutamate, l -aspartate and d -aspartate, which are accumulated by both high- and low-affinity uptake systems. Although contributions of other factors to the observed time courses, such as rates of association and dissociation of the amino acid-receptor complexes, cannot be excluded, the present results are consistent with the hypothesis that low-affinity uptake systems of high V_max play an important role in the rapid termination of the effects of amino acid excitants.     

HIGH AFFINITY UPTAKE OF l-GLUTAMINE IN RAT BRAIN SLICES

—l -Glutamine is taken up into rat brain slices by a specific‘high affinity’uptake system (K_m 52 μm ) which is not influenced by high concentrations of l -glutamate and l -asparagine. The uptake system appears to be associated with cellular structures that do not survive homogenization under conditions which yield synaptosomes. The‘high affinity’uptake of glutamine is dependent on the external sodium ion concentration and can be inhibited by p-chloromercuriphenylsulphonate, amino-oxyacetic acid, ouabain, dibenamine and allylglycine. The effects of several inhibitors indicate that l -asparagine uptake is mediated by a system different from the‘high affinity’system mediating l -glutamine uptake.     

INFLUX OF GLUTAMIC ACID IN PERIPHERAL NERVE—CHARACTERISTICS OF INFLUX1

Abstract— —The influx of glutamic acid in frog sciatic nerve has been studied by monitoring the disappearance of ¹⁴C labelled compound from the bathing medium. After 5hr of incubation in 10 ⁻⁶m non-labelled l -glutamic acid and 0·01, μc/ml labelled isotope, the intracellular concentration of labelled glutamic acid is about 15 times the concentration in the bathing medium; however, there appears to be a net loss of non-labelled compound with incubation. Uptake of L,-glutamic acid is accompanied by conversion of significant amounts of labelled E-glutamic acid to carbon dioxide and glutamine; small amounts of γ-aminobutyric acid and aspartic acid are also formed. The rate of disappearance of labelled l -glutamic acid decreases with increasing concentration of non-labelled isotope in the bathing medium. Construction of a Lineweaver-Burk plot from initial velocities of influx yields an average V_m of 4·02 × 10⁻⁹ moles/g/min and an average K_m. of 3·23 × 10 ⁻⁵ moles/l. The influx of glutamic acid is highly specific with regard to molecular structure; of the compounds tested, only l -glutamine, l -glutamic acid, GABA, l -lysine, and l -aspartic acid are taken up, and only l -aspartic acid will compete with l -glutamic acid for uptake.     

UPTAKE OF [3H-METHYL]CHOLINE BY MICROSOMAL, SYNAPTOSOMAL, MITOCHONDRIAL AND SYNAPTIC VESICLE FRACTIONS OF RAT BRAIN

Abstract— Microsomal, mitochondrial, synaptosomal and synaptic vesicle fractions of rat brain took up [³H-methyl]choline by a similar carrier-mediated transport system. The apparent K_m for the uptake of [³H-methyl]choline in these subcellular fractions was about 5 × 10^?5 M. Choline uptake was also observed in microsomal fractions prepared from liver and skeletal muscle. Virtually identical kinetic properties for [³H-methyl]choline transport were found in the synaptosomal fractions prepared from the whole brain, cerebellum or basal ganglia. Countertransport of [³H-methyl]choline from the synaptosomal fraction was demonstrated against a concentration gradient. HC-3 was a competitive inhibitor of the uptake of [³H-methyl]choline in brain microsomal, synaptosomal and mitochondria] fractions with respective values for K_i of 4.0, 2.1 and 2.3 × 10^?5 M. HC-15 was a competitive inhibitor of the transport of [³H-methyl]choline in the synaptosomal fraction, with a K_i of 1.7 × 10^?4 M. Upon entry into the microsomal fraction, 74 per cent of the radioactivity could be recovered as unaltered choline, 10 per cent as phosphorylcholine, 1.5 per cent as acetylcholine and 2.5 per cent as phospholipid. Choline acetyltransferase (EC 2.3.1.6) was assayed with [¹⁴C]acetylCoA in synaptosomal fractions prepared from basal ganglia and cerebellum, and in the 31,000 g supernatant fraction of a rat brain homogenate. Enzyme activity was 11-fold greater in the synaptosomal fraction from the basal ganglia than in that from the cerebellum. HC-3 did not inhibit choline acetyltransferase and there was no evidence for acetylation of HC-3. Our findings suggest that choline uptake is a ubiquitous property of membranes in the CNS and cannot serve to distinguish cholinergic nerve endings and their synaptic vesicles.     

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.     

HIGH AFFINITY AMINO ACID TRANSPORT BY FROG SPINAL CORD SLICES

The characteristics of amino acid uptake by frog spinal cord slices was studied by in vitro incubations in appropriate media. The uptake mechanisms exhibited saturation; kinetic analysis demonstrated 2 distinct systems for the influx of the possible neurotransmitters: GABA, glycine, L-glutamic acid and L-aspartic acid. One system showed a comparatively high substrate affinity (K_m values, 10-26 μM) while the other system had a lower affinity (K_m, 0.4-1.6 mM).-Leucine, an amino acid presumably not a transmitter, was accumulated only by a low affinity mechanism (K_m 1.6 mM). The process responsible for high affinity uptake had many of the properties of an active transport mechanism. These included temperature sensitivity, energy dependence, requirement for Na⁺ ions and inhibition by ouabain. GABA and glycine uptake was inhibited only by closely related amino acids or structural analogues. The influx of L-glutamic acid was competitively inhibited by the presence of L-aspartic acid in the medium; the converse was also demonstrated. Thus, the high affinity uptake system for possible transmitter amino acids in the frog spinal cord closely resembles that described for mammalian CNS tissue. These results are compatible with the assumption that GABA, glycine, L-glutamic acid and L-aspartic acid are neurotransmitters in the amphibian spinal cord.     

Uptake and release of possible false transmitter amino acids by rat brain tissue

—The uptake of l [¹⁴C]glutamine by a crude isolated nerve ending fraction of rat brain was found to be linear with time for at least 5 min, profoundly temperature-dependent, apparently half-saturated at a substrate concentration of 0·26 mm , partially inhibited by dinitrophenol and ouabain and elevated [K⁺], weakly Na⁺-dependent, poorly inhibited by drugs which block uptake of biogenic amines and more strongly inhibited by glutamic acid (IC₅₀= 0·5mm ) than by aspartic acid, GABA, glycine or methionine. The [¹⁴C]glutamine taken up appeared to be associated with nerve endings and was released by membrane-disruption; about 20 per cent was associated with free mitochondria. Glutamine, δ-aminolevulinic acid and several other amino acids were poor inhibitors of [³H]GABA-uptake; δ-aminolevulinic acid was a poor inhibitor of [³H]glutamine-uptake, whereas glutamine was a moderately effective competitive inhibitor (K_i= 1 mm ). [¹⁴C]glutamine and [³H]GABA were released from brain slices by electrical stimulation or 50 mm K⁺, while labeled δ-aminolevulinic acid, leucine, urea, amphetamine and tyramine were poorly released. [¹⁴C]glutamine was not released by unlabeled glutamate or several aromatic amines. We conclude that the neuropsychiatric features of porphyria are not likely due to a ‘false transmitter’ role for δ-aminolevulinic acid although such a role for glutamine in hepatic encephalopathy or other neuropsychiatric diseases should be considered.     

Presence of an X AG − carrier in frog (Rana esculenta) red blood cells

Evidence is presented that the high levels of internal l-glutamic and l-aspartic acid in frog Rana esculenta red blood cells are due to the existence of a specific carrier for acidic amino acids of high affinity K _m = 3 m and low capacity (V_max) 0.4 mol l-Glu · Kg^–1 dry cell mass · 10 min^–1. It is Na+ dependent and the incorporation of l-glutamic acid can be inhibited by l and d-aspartate and l-cysteic acid, while d-glutamic does not inhibit. Moreover, this glutamic uptake shows a bell-shaped dependence on the external pH. All these properties show that this carrier belongs to the system X _AG ^– family. Besides the incorporation through this system, l-glutamic acid is also taken up through the ASC system, although, under physiological conditions, this transport is far less important, since it has relatively low affinity K _m 39 m but high capacity (V _max) 1.8 mol l-Glu · Kg^–1 dry cell mass · 10 min^–1.     

Uptake and K+-stimulated release of [14C]glycine from frog retinal synaptosomal fractions

The uptake of [¹⁴C]glycine and the effect of depolarizing potassium concentrations on its release was investigated in the whole frog retina and its synaptosomal fractions. The uptake of [¹⁴C]glycine in retina and synaptosomal fractions was found to be saturable as well as energy and Na⁺-dependent. TheK _m value for glycine uptake was found to be 46 M for P₂ fraction and 100 M for P₁ fraction, with aV _max of 3.5 and 3.8 nmol/mg protein/min respectively. The release of [¹⁴C]glycine from P₁ and P₂ synaptosomal fractions was markedly increased by raising potassium concentration in the medium, in a partially Ca²⁺-dependent manner. Evoked glycine release was 50% reduced when calcium was omitted from the medium. The K⁺-stimulated release of glycine from P₂ fraction was significantly reduced in the presence of TTX. The cellular origin of the P₁ and P₂ synaptosomal fractions releasing glycine is discussed.     

Taurine uptake in synaptosomal fractions of rat cerebral cortex.

Abstract— [³⁵S]Taurine was found to be accumulated in synaptosomal fractions of rat cerebral cortex. Kinetic analysis in the range of 1–800 μm -[³⁵S]taurine revealed at least two different uptake processes. A high affinity uptake with a K_m of 20 μM and a low affinity uptake with a K_m of about 450 μM. The high affinity component was dependent on temperature and energy, and virtually abolished in the absence of sodium. Examination of the influence of structural analogues and putative transmitter substances indicates that the high affinity uptake of taurine into synaptosomal fractions of rat cerebral cortex is unique and highly specific. No specific actions of several centrally acting drugs on taurine uptake could be observed.     

Uptake of Amino Acids by Barley Leaf Slices: Kinetics, Specificity, and Energetics

Uptake of ¹⁴C-labelled _L-lysine. _L-arginine, _L-glutamic acid, _L-aspartic acid, and glycine was studied in 0.75 mm wide barley (Hordeum vulgare L. cv. Lise) leaf slices. After an initial period (10 min) of rapid accumulation amino acid uptake proceeded at a steady, lower rate for several hours. Uptake was stimulated by 10^?4M Ca^?2+ ions. Uptake was strongly _pH dependent with the following optima: aspartic acid _pH 3.5. glutamic acid _pH 4.1. glycine _pH 5.8, lysine _pH 6–7, and arginine _pH 5–8 (a broad plateau). The optimal temperature was about 30°C. and the temperature coefficient in the range 0–20gGC was 2.3–2.5. Concentration-dependence data gave uptake isotherms which appeared to be multiphasic for all the amino acids used. The amino acids inhibited each other in a competitive fashion, indicating that they were all transported by a single carrier system. Uptake of lysinc was strongly inhibited by 10^?4M 2.4-dinitrophenol. Lysine uptake was not stimulated by light under aerobic conditions. However, it was much reduced in the dark under anaerobic conditions. This reduction was almost compensated for by light. The light-stimulation of uptake under anaerobic conditions was abolished by 10^?5M 3-(3,4-dichlorophenyl) 1.1-dimethylurea.     

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.     

High affinity transport of choline into synaptosomes of rat brain

—The accumulation of [³H]choline into synaptosome-enriched homogenates of rat corpus striatum, cerebral cortex and cerebellum was studied at [³H]choline concentrations varying from 0.5 to 100 μm . The accumulation of [³H]choline in these brain regions was saturable. Kinetic analysis of the accumulation of the radiolabel was performed by double-reciprocal plots and by least squares iterative fitting of a substrate-velocity curve to the data. With both of these techniques, the data were best satisfied by two transport components, a high affinity uptake system with K_m. values of 1.4 μM (corpus striatum), and 3.1 μM (ceμ(cerebral cortex) and a low affinity uptake system with respective K_m. values of 93 and 33 μM for these two brain regions. In the cerebellum choline was accumulated only by the low affinity system. When striatal homogenates were fractionated further into synaptosomes and mitochondria and incubated with varying concentrations of [³H]choline, the high affinity component of choline uptake was localized to the synaptosomal fraction. The high affinity uptake system required sodium, was sensitive to various metabolic inhibitors and was associated with considerable formation of [³H]acetylcholine. The low affinity uptake system was much less dependent on sodium, and was not associated with a marked degree of [³H]acetylcholine formation. Hemicholinium-3 and acetylcholine were potent inhibitors of the high affinity uptake system. A variety of evidence suggests that the high affinity transport represents a selective accumulation of choline by cholinergic neurons, while the low affinity uptake system has some less specific function.     

RAT BRAIN SYNAPTIC VESICLES: UPTAKE SPECIFICITIES OF [3H]NOREPINEPHRINE AND [3H]SEROTONIN IN PREPARATIONS FROM WHOLE BRAIN AND BRAIN REGIONS1

A fraction containing neurotransmitter storage vesicles was isolated from rat whole brain and brain regions, and the uptakes of [³H]norepinephrine and [³H]serotonin were determined in vitro. Norepinephrine uptake in vesicle preparations from corpus striatum was higher than in prep arations from cerebral cortex, and uptake in vesicles from the remainder (midbrain + brainstem + cerebellum) was intermediate. The K_m for norepinephrine uptake was the same in the three brain regions, but the regions differed in maximal uptake capacity by factors which paralleled total catecholamine concentration rather than content of norepinephrine alone. Intracisternal administration of 6-hydroxydopamine, but not of 5,6-dihydroxytryptamine, reduced vesicular norepinephrine uptake, and pretreat-ment with desmethylimipramine (which protects specifically norepinephrine neurons but not dopamine neurons from the 6-hydroxydopamine) only partially prevented the loss of vesicular norepinephrine uptake. These studies indicate that uptake of norepinephrine by rat brain vesicle preparations occurs in vesicles from norepinephrine and dopamine neurons, but probably not in vesicles from serotonin neurons. Uptake of serotonin by brain vesicle preparations exhibited time, temperature and ATP-Mg²⁺ requirements nearly identical to those of norepinephrine uptake. The affinity of serotonin uptake matched that of serotonin for inhibition of norepinephrine uptake, and the maximal capacity was the same for serotonin as for norepinephrine. Norepinephrine, dopamine and reserpine inhibited serotonin uptake in a purely competitive fashion, with K_is similar to those for inhibition of norepinephrine uptake. Whereas 5,6-dihydroxytryptamine treatment reduced synaptosomal serotonin uptake but not vesicular serotonin uptake, 6-hydroxydopamine reduced vesicular serotonin uptake in the absence of reductions in synaptosomal serotonin uptake. Thus, in this preparation, serotonin appears to be taken up in vitro into catecholamine vesicles, rather than into serotonin vesicles.     

A KINETIC ANALYSIS OF SODIUM DEPENDENT GLUTAMIC ACID TRANSPORT IN PERIPHERAL NERVE

—The kinetics of sodium dependent glutamic acid transport have been studied in desheathed frog sciatic nerve. Initial velocities have been measured as a function of both glulamic acid and sodium concentration. Lineweaver–Burk plots are constructed from these data, and the kinetic constants describing uptake are estimated. V_max is unaffected by sodium concentration, which implies that translocation is not directly affected by sodium. K₁ is sodium dependent, which implies that sodium affects the affinity of the carrier for glutamic acid. Reciprocal plots of velocity vs [Na] or K₁ vs 1/[Na] are linear, suggesting that glutamic acid and sodium are co-transported on a one-to-one basis. _t, the sodium concentration giving half maximal velocity of uptake, was found to vary from about 57 mm to 48 mm at glutamic acid concentrations of 1.0–10.0 ± 10^?6m . A model of a mechanism by which sodium and glutamate could be co-transported is presented; the model is in very good agreement with the experimental data.