Ca2+-Dependent Enhancement of [3H]Dopamine Uptake in Rat Striatum: Possible Involvement of Calmodulin-Dependent Kinases

Abstract: We studied effects of Ca²⁺ in the incubation medium on [³H]dopamine ([³H]DA) uptake by rat striatal synaptosomes. Both the duration of the preincubation period with Ca²⁺ (0–30 min) and Ca²⁺ concentration (0–10 m M ) in Krebs-Ringer medium affected [³H]DA uptake by the synaptosomes. The increase was maximal at a concentration of 1 m M Ca²⁺ after a 10-min preincubation (2.4 times larger than the uptake measured without preincubation), which reflected an increase in V _max of the [³H]DA uptake process. On the other hand, [³H]DA uptake decreased rapidly after addition of ionomycin in the presence of 1 m M Ca²⁺. The Ca²⁺-dependent enhancement of the uptake was still maintained after washing synaptosomes with Ca²⁺-free medium following preincubation with 1 m M Ca²⁺. Protein kinase C inhibitors did not affect apparently Ca²⁺-dependent enhancement of the uptake, whereas 1-[ N,O -bis(1,5-isoquinolinesulfonyl)- N -methyl- l -tyrosyl]-4-phenylpiperazine (KN-62; a Ca²⁺/calmodulin-dependent kinase II inhibitor) and wortmannin (a myosin light chain kinase inhibitor) significantly reduced it. Inhibitory effects of KN-62 and wortmannin appeared to be additive. N -(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7; a calmodulin antagonist) also remarkably inhibited the enhancement. These results suggest that Ca²⁺-dependent enhancement of [³H]DA uptake is mediated by activation of calmodulin-dependent protein kinases.     

Potassium-Stimulated Release of Radiolabelled Taurine and Glycine from the Isolated Rat Retina

Abstract: The release of preloaded [³H]glycine and [³H]taurine in response to a depolarising stimulus (12.5-50 m M KCl) has been studied in the superfused rat retina. High external potassium concentration immediately increased the spontaneous efflux of [3H]glycine, the effect of 50 m M K⁺ apparently being abolished by omitting calcium from the superfusing medium. In contrast, although high potassium concentrations increased the spontaneous emux of [³H]taurine from the superfused rat retina, this release was not evident until the depolarising stimulus was removed from the superfusing medium. The magnitude of this "late" release of [³H]taurine was dependent on external K⁺ concentrations, and appeared immediately after cessation of the stimulus irrespective of whether it was applied for 4, 8, or 12 min. Potassium (50 m M )-induced release of taurine appeared partially calcium-dependent, being significantly reduced (p < 0.01) but not abolished by replacing calcium with 1 mM EDTA in the superfusate. High-affinity uptake systems for both [³H]glycine and [³H]taurine were demonstrated in the rat retina in vitro ( K _m values, 1.67 μ M and 2.97 μ M ; V_max values, 19.3 and 23.1 nmol/g wet weight tissue/h, respectively). The results are discussed with respect to the possible neuro-transmitter roles of both amino acids in the rat retina.     

ON THE MECHANISM OF OUABAIN INHIBITION OF SYNAPTOSOME PROTEIN SYNTHESIS

Abstract— Ouabain (200μ m ) inhibited incorporation of radiolabelled leucine or glycine into the protein of neonatal synaptosome fractions but had minimal effect on preparations from adult rats. Leucine uptake into synaptosomes was rapid but not influenced by 200μ m -ouabain in contrast to ouabain inhibition of [¹⁴C]glycine and [¹⁴C]γ-aminobutyric acid uptake. Ouabain blocked the Na⁺ -dependent (stimulated) component of synaptosome fraction protein synthesis in the presence of 25m m -K⁺. Ouabain inhibition was not alleviated by addition of ADP or ATP. 100μ m -atractylate failed to influence [³H]leucine uptake or incorporation. Synergistic inhibition by ouabain was observed with the cycloheximide-sensitive component of protein synthesis and the chloramphenicol sensitive phase. Increasing the medium Ca²⁺ concentration stimulated protein synthesis and this stimulated component was inhibited by ouabain. Ouabain inhibition was associated with decreasing intraterminal K⁺ concentration and [K]_i was linearly related to the protein synthesis rate in control and ouabain treated preparations.     

Opioid Effects on [3H]Norepinephrine Release from Dissociated Embryonic Locus Coeruleus Cell Cultures

Abstract: The acute and chronic effects of opioid exposure on [³H]norepinephrine ([³H]NE) release were examined in cell cultures of embryonic rat locus coeruleus (LC). Initial morphological and biochemical characterization of the cultures indicated that the cells exhibited properties similar to those observed in situ. Specific [³H]NE uptake was saturable with a K _m value of 222 ± 52 n M . [³H]NE accumulated by LC cells was released in response to 20 m M K⁺ stimulation, in a calcium-dependent manner. Both components of neurotransmitter release, spontaneous and K⁺ evoked, were significantly inhibited by β-endorphin, with the latter being maintained in the presence of tetrodotoxin. The pharmacology of the opioid effect was consistent with that of μ-receptor activation. The effect of chronic exposure to the μ-selective agonist fentanyl (1 μ M ) was examined following 4 days of drug treatment. Although there was no significant effect of fentanyl on K⁺-evoked [³H]NE release, these cells were tolerant to the acute inhibitory effect of β-endorphin. These results indicate that this is an appropriate system for examining the effects of acute and chronic opioid treatment on noradrenergic cells in vitro. In addition, this system may be useful as a CNS model for examining mechanisms that underlie tolerance and dependence following chronic opioid exposure.     

Melatonin Effect on [3H]Glutamate Uptake and Release in the Golden Hamster Retina

Abstract: The effect of melatonin on [³H]glutamate uptake and release in the golden hamster retina was studied. In retinas excised in the middle of the dark phase, i.e., at 2400 h, melatonin (0.1 and 10 n M ) significantly increased [³H]glutamate uptake, and this effect persisted in a Ca²⁺-free medium. On the other hand, melatonin significantly increased [³H]glutamate release in retinas excised at 2400 h, but this effect was Ca²⁺ sensitive. Melatonin significantly increased ⁴⁵Ca²⁺ uptake by a crude synaptosomal fraction from retinas of hamsters killed at 2400 h. In retinas excised at 1200 h, melatonin had no effect on [³H]glutamate uptake, [³H]glutamate release, or ⁴⁵Ca²⁺ uptake at any concentration tested. Cyclic GMP analogues, i.e., 8-bromoguanosine 3',5'-cyclic monophosphate and 2'- O -dibutyrylguanosine 3',5'-cyclic monophosphate, significantly increased [³H]glutamate uptake, [³H]glutamate release, and ⁴⁵Ca²⁺ uptake by tissue removed at 1200 and 2400 h, suggesting that the effects of melatonin could correlate with a previously described effect of melatonin on cyclic GMP levels in the golden hamster retina. Taking into account the key role of glutamate in visual mechanisms, the results suggest the participation of melatonin in retinal physiology.     

RELEASE OF [3H]GAMMA-AMINOBUTYRIC ACID FROM GLIAL CELLS IN RAT DORSAL ROOT GANGLIA.

Abstract— Desheathed rat dorsal root ganglia were incubated in a medium containing amino-oxyacetic acid and [³H]GABA. Under these conditions, [³H]GABA is taken up exclusively by the satellite glial cells in the ganglia. Efflux of [³H]GABA from the tissue was measured after passing the ganglia through a series of wash solutions. The spontaneous efflux of radioactivity, mostly [³H]GABA, was more rapid in the absence of amino-oxyacetic acid in the incubation and wash media.
Raising the potassium concentration in the wash media caused an increase in the efflux of [³H]GABA. This increase was sigmoidally related to the potassium concentration in the wash media, reaching a maximum at 64 m m -K⁺. The releasing effect of K⁺ was inhibited by removing calcium from the media. Reducing the calcium and raising the magnesium concentration in the wash solutions inhibited the increased efflux of [³H]GABA due to 64 m m -K⁺ by 48 per cent, while 5 mM-La³⁺ and diphenylhydantoin (0·005 and 0·5 m m ) had no effect on this increase.
Only a small increase in the efflux of [¹⁴C]glutamate was produced by 64 m m -K⁺ and it had no effect upon the effluxes of [³H]glycine, [³H]alanine or [³H]leucine. The efflux of lactate dehydrogenase was similarly unaffected by 64 mM-K⁺. The results suggest that glial cells in spinal ganglia can respond to depolarizing concentrations of potassium by releasing GABA in a calcium-dependent process.     

Elevated Levels of Glucose and L-Fucose Reduce 22Na+ Uptake and Whole Cell Na+ Current in Cultured Neuroblastoma Cells

Abstract: Na⁺ flux was studied in cultured neuroblastoma cells grown in medium containing increased glucose or L - fucose concentrations. Chronic exposure of neuroblastoma cells to 30 m M glucose or 30 m M L-fucose caused a decrease in ouabain-sensitive and veratridine-stimulated ²²Na⁺ uptake compared with cells cultured in unsupplemented medium. The Na⁺ current, determined by using whole-cell configuration of the patch clamp, was also decreased in these cells. Tetrodotoxin (3 μ M ), which blocked whole cell Na⁺ currents, also blocked veratridine-stimulated ²²Na⁺ accumulation. Culturing cells in medium containing 30 m M fructose as an osmotic control had no effect on Na⁺ flux. Specific [³H] saxitoxin binding was not affected by 30 m M glucose or 30 m M L-fucose compared with cells grown in unsupplemented medium, suggesting that the number of Na⁺ channels was not decreased. These studies suggest that exposing cultured neuronal cells to conditions that occur in the diabetic milieu alters Na⁺ transport and Na⁺-channel activity.     

Transport of G AB A, β-Alanine and Glutamate into Perikarya of Postnatal Rat Cerebellum

Abstract: Cells dissociated from the postnatally developing rat cerebellum retain their high-affinity carrier-mediated transport systems for [³H]GABA ( K _t=1.9 μM, V = 1.8 pmol/10⁶ cells/min) and [³H]glutamate ( K _t= 10 μM, V = 7.9 pmol/10⁶ cells/min). Using a unit gravity sedimentation technique it was demonstrated that [3H]GABA was taken principally into fractions that were enriched in inhibitory neurons (Purkinje, stellate and basket cells). [³H]β-alanine (which is taken up specifically by the glial GABA transport system) and [³H]glutamate were concentrated by glial-enriched fractions. However [³H]glutamate uptake was minimal in fractions enriched in precursors of granule cells, which may utilise this amino acid as their neurotransmitter. These results are discussed in relation to reports of high-affinity [³H]glutamate uptake by glia. The role of glutamate transport in glutamatergic cells is also considered. The data suggest that high-affinity glutamate transport is a property of glial cells but not granule neurons.     

Sodium and Chloride Ion-Dependent Transport of β-Alanine Across the Blood-Brain Barrier

Abstract: The characteristics of β-alanine transport at the blood-brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the β-[³H]alanine transport indicated that the transporter for β-alanine functions with K_t of 25.3 ± 2.5 µ M and J _max of 6.90 ± 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. β-[³H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4-dinitrophenol and NaN₃) and low temperature reduced the uptake significantly. Furthermore, the uptake of β-[³H]alanine required Na⁺ and Cl⁻ in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one β-alanine molecule. The Na⁺ and Cl⁻-dependent uptake of β-[³H]alanine was stimulated by a valinomycin-induced inside-negative K⁺-diffusion potential. β-Amino acids (β-alanine, taurine, and hypotaurine) inhibited strongly the uptake of β-[³H]alanine, whereas α- and γ-amino acids had little or no inhibitory effect. In ATP-depleted cells, the uptake of β-[³H]alanine was stimulated by preloading of β-alanine or taurine but not l -leucine. These results show that β-alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to β-amino acids.     

Uptake of [3H]Dopamine into Dopaminergic and Noradrenergic Neurones of the Isolated Neurointermediate Lobe of the Rat Hypophysis. Effects of Desipramine and Nomifensine

Abstract: The isolated neurointermediate lobe (NIL) of the rat hypophysis accumulates [³H]dopamine from the incubation medium. Column chromatographic analysis showed that 92% of the tissue radioactivity was contained in the catecholamine fraction. [³H]Dopamine represented 70% and [³H]noradrenaline 30% of the [³H]catecholamines. Desipramine (1 μM) prevented the formation of [³H]noradrenaline without affecting the storage of [³H]dopamine. Nomifensine (10 μM) blocked the storage of [³H]dopamine and [³H]noradrenaline. Thus, in the NIL, [³H]dopamine is taken up into dopaminergic and noradrenergic neurones. In the latter, [³H]dopamine is converted to [³H]noradrenaline, indicating a significant dopamine β-hydroxylase activity in the NIL tissue. A selective labeling of the dopamine stores with [³H]dopamine can be achieved in the presence of desipramine.     

THE UPTAKE OF [3H]CHOLINE BY SNAIL (HELIX POMATIA) NERVOUS TISSUE IN VITRO

Abstract— When suboesophageal ganglia of the snail Helix comalia were incubated at 25°C in a medium containing [³H]choline, tissue: medium ratios of about 14:1 were obtained after 20 min incubation, and only 15°, of the accumulated choline was metabolized to form [³H]acetylcholine. The uptake of [³H]choline showed saturation kinetics and was dependent upon temperature and sodium ions. Kinetic analysis suggested the existence of a high affinity uptake process (K_m= 1.7 μM, V_max= 0.21 nmol/g/min) and a low affinity process (K_m= 100 μM, V_max= 1.2 nmol/g/min). The high affinity uptake differed from the low affinity system in that it was sensitive to various metabolic inhibitors and was competitively inhibited by low concentrations of hemicholinium- and acetylcholine. Neither uptake system was greatly influenced by the absence of calcium, potassium or magnesium ions or by the presence of low concentrations of 5-HT, dopamine. tetrabenazine, chlorpromazine, decamethonium, nalaxone or imipramine. The high affinity uptake process may be important in supplying choline for the biosynthesis of acetylcholine in cholinergic neurons.     

SYNTHESIS AND RELEASE OF [14C]ACETYLCH0LINE IN SYNAPTOSOMES

Abstract— Synaptosomes took up [¹⁴C]choline, about half or more of which was converted to [^I4C]acetylcholine when incubated in an appropriate medium containing 1 to 5 μ M-[¹⁴C] choline and neostigmine. The amount of [¹⁴C]acetylcholine synthesized in synaptosomes increased in parallel with the increase of Na⁺ concentration in the incubation medium. The effect of Na⁺ on the uptake of [^I4C]choline into synaptosomes was dependent on the concentration of choline in the incubation medium.
About 25 per cent of [¹⁴C]acetylcholine synthesized in synaptosomes was released rapidly into the medium by increasing the K⁺ concentration in the medium from 5 m m to 35 m m . The change of Na⁺ concentration hardly affected the release of [¹⁴C]acetylcholine. The effect of K⁺ on the release of [¹⁴C]choline was rather small compared to that on [¹⁴C] acetylcholine. Ouabain promoted the release of [¹⁴C]acetylcholine.     

RELEASE AND EXCHANGE STUDIES RELATING TO THE SYNAPTOSOMAL UPTAKE OF GABA

Abstract— Synaptosomal release and exchange of [³H]GABA were studied by a superfusion technique which minimizes reuptake. The release of [³H]GABA was increased by depolarizing concentrations of KCl and showed calcium-dependence. Superfusion with 1-1000 μ m unlabelled GABA caused a dose dependent, saturable increase in the release of radioactivity by homoexchange. The exchange process showed high substrate specificity: among the various amino acids and putative neurotransmitters tested, only γ-amino-β-hydroxybutyric acid was a good stimulator of [³H]GABA release. Superfusion with sodium-free medium (NaCl replaced by sucrose) virtually abolished homoexchange. Ouabain also increased the release of [³H]GABA, and its action was additive to that of unlabelled GABA.
The presence of exchange at concentrations that are in the range of the high affinity uptake system, the apparent similarity between calculated rates of exchange and initial uptake rates, the non-detectability of exchange in a condition (Na⁺ deprivation) which inhibits high affinity uptake, and the lack of decrease of actual GABA concentration in incubation media used for uptake experiments, all suggest that homoexchange accounts for a substantial part of the synaptosomal accumulation of [³H]GABA generally interpreted as high affinity uptake.     

Comparison of DABA and GABA Transport into Plasma Membrane Vesicles Derived from Synaptosomes

Abstract: Transport of GABA by a high-affinity transport system ( K _m≃ 10⁻⁵ M) is thought to terminate the action of this postulated neurotransmitter. 2,4-Diaminobutyric acid (DABA), a structural analogue, is taken up by neuronal elements and inhibits GABA uptake. Localization of [³H]DABA by auto-radiography has been used to identify neurons with the GABA high-affinity transport system. After reconstitution of lysed synaptosomal fractions in potassium salts, transfer of these membrane vesicles to sodium salts produces sodium and potassium ion gradients which drive [³H]GABA and [³H]DABA transport. For each, transport requires external sodium, is abolished by ionophores that dissipate the Na⁺ gradient, and is enhanced by conditions which make the intravesicular electromotive force more negative. Some characteristics of the transport of these substances, however, differ. For example, external chloride is required for GABA, but not DABA, transport. Internal potassium is required for DABA, but not GABA, transport. DABA is a competitive inhibitor ( K _i≃ 0.6 MM) of GABA transport into membrane vesicle and synaptosomes. GABA, however, is a feeble inhibitor of DABA uptake into the membrane vesicles. These differences suggest that the two substances are transported by different mechanisms and possibly by different carriers. In addition to these experiments, using enzymatic-fluorometric techniques, it was shown that the artificially imposed ion gradients drive net chemical transport of GABA into the vesicles.     

THE EFFECT OF ELECTRICAL STIMULATION AND HIGH POTASSIUM CONCENTRATIONS ON THE EFFLUX OF [3H] γ-AMINOBUTYRIC ACID FROM BRAIN SLICES

Abstract— Brain slices were incubated with [³H]GABA in a medium containing aminooxyacetic acid to prevent metabolism of [³H]GABA by GABA-glutamate transaminase. The slices, which rapidly accumulated radioactivity, were then continuously perfused and the efflux of [³H]GABA from the tissue was measured. The spontaneous efflux of [³H]GABA consisted of an initial rapid phase followed by a much slower release of [³[H]GABA. After 40 min perfusion 90 per cent of the radioactivity remained in the tissue.
The slices were depolarized by electrical stimulation or by perfusion with a medium containing a high potassium concentration (40 mM). These procedures caused a striking increase in the efflux of [³H]GABA. The increased efflux produced by potassium, but not that produced by electrical stimulation, was dependent on calcium ions in the medium. The effect of electrical stimulation on [³H]GABA release was considerably reduced by a raised concentration (10 mM) of magnesium in the medium.
High potassium concentrations and electrical stimulation did not cause an increase in the efflux of [¹⁴C]urea, L-[³H]leucine or [¹⁴C]α-amino-isobutyric acid from brain slices. These results are consistent with the suggestion that GABA may be an inhibitory transmitter in the cerebral cortex.     

Phosphatidylserine Enhancement of [3H]γ-Aminobutyric Acid Uptake by Rat Whole Brain Synaptosomes

Abstract: [³H] γ -Aminobutyric acid ([³H]GABA) binding to purified lipids was examined in an organic solvent-aqueous partition system. In addition, the [³H]GABA binding capacity in the partition system was compared with the capacity of lipids to alter sodium-dependent [³H]GABA uptake into synaptosomes isolated from rat whole brains. [³H]GABA was found to bind to all of the lipids studied in the organic solvent-aqueous partition system [phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), gangliosides, and sulfatide], although PS exhibited the greatest binding capacity. [³H]GABA uptake into synaptosomes was enhanced by PS (48.0%) but was not altered by any other lipid. PS enhancement of [³H]GABA uptake required the presence of sodium and was blocked by nipecotic acid (10 μ m ). These results suggest that PS may play a role in the sodium-dependent GABA reuptake process in the presynaptic nerve end.     

[3H]Xylamine Accumulation by Two Sympathetically Innervated Tissues

Abstract: The accumulation of [³H]xylamine ([ring-G-³H]- N -2'-chloroethyl- N -ethyl-2-methylbenzylamine; [³H]XYL) by rabbit thoracic aorta and rat vas deferens was studied in vitro . [³H]XYL uptake in both tissues saturated at the same concentrations and displayed the same time course as that for maximal inhibition of [³H]noradrenaline ([³H]NA) accumulation by XYL. Hydrolysis of [³H]XYL or coincubation with Na₂S₂O₃ reduced tissue accumulation of tritium by 80%. In aorta and vas deferens, about 45% and 65%, respectively, of the total [³H]XYL accumulation was blocked by 100 μmol/L desmethylimipramine (DMI), l -NA, or bretylium. In the absence of sodium ion, the uptake of [³H]XYL was reduced by approximately these same amounts. In both tissues nearly 70% of the [³H]XYL taken up was protein bound when measured as trichloroacetic acid-precipitated tritium. Of this radioactivity, the same proportion was sensitive to 100 μmol/L DMI or the absence of sodium ion as found for total tissue accumulation of [³H]XYL. Hydrolysis of [³H]XYL prior to incubation with vas deferens reduced the protein-bound tritium by more than 95%. The studies indicate that [³H]XYL interacts with NA uptake carrier in both rabbit aorta and rat vas deferens and that a substantial portion of the resulting protein-bound radioactivity is carrier-dependent.     

45Ca2+ Uptake into Rat Whole Brain Synaptosomes Unaltered by Dihydropyridine Calcium Antagonists

Abstract: Voltage-dependent ⁴⁵Ca²⁺ uptake into rat whole brain synaptosomes was measured after 3-s KCl-induced depolarization to investigate possible inhibitory effects of calcium antagonists, nitrendipine, nimodipine, and nisoldipine. At a Ca²⁺ concentration of 1.2 m M , nitrendipine, in concentrations ranging from 0.1 n M to 10 μ M , had no effect on ⁴⁵Ca²⁺ uptake. When the Ca²⁺ concentration was lowered to 0.06 and 0.12 m M , nitrendipine, 10 μ M , inhibited ⁴⁵Ca²⁺ uptake in response to 109 m M KCl depolarization. However, in a separate concentration response study, nitrendipine, nimodipine, and nisoldipine, 0.1 n M to 10 μ M , failed to alter the uptake of ⁴⁵Ca²⁺ (0.06 m M Ca²⁺) into 30 m M KCl-depolarized synaptosomes. The high concentrations of these agents required to depress ⁴⁵Ca²⁺ uptake indicate that the dihydropyridine calcium antagonists are considerably less potent in brain tissue than in peripheral tissue.     

Cationic and Anionic Requirements for the Binding of 2β-Carbomethoxy-3β-(4-Fluorophenyl)[3H]tropane to the Dopamine Uptake Carrier

Abstract: The present study reports the ion dependency of 2β-carbomethoxy-3β-(4-fluorophenyl)[³H]tropane ([³H]- CFT) binding to the dopamine transporter in the rat striaturn. The results indicate that [³H]CFT binding to synaptosomal P₂ membranes requires low concentrations of Na⁺ (peak binding between 20 and 50 m M Na⁺), is stimulated by phosphate anion or l^-, but is unaffected or only slightly affected by F^-, Cl^-, Br^-, NO₃^-, or SO₄^2-, Concentrations of Na⁺ of >50 m M become inhibitory except in the presence of l^-, which shifts peak binding levels toward higher Na⁺ concentrations and also elevates the peak binding level. K⁺ strongly decreased [³H]CFT binding with a shallow inhibition curve, and Na⁺ could not overcome this effect. Saturation analysis of [³H]CFT binding revealed a single binding site changing its affinity for CFT depending on the concentration of sodium phosphate buffer (6, 10, 30, 50, 130, or 200 m M ; 1 mM plus 49 mM NaCIversus 10 m M plus 40 m M NaCI; or 1 mM plus 129 m M Nal versus 10 m M plus 120 m M Nal). No differences were observed in the density of CFT binding sites between any of the conditions examined.     

Increase in Chloride-Dependent l-Glutamate Transport Activity in Synaptic Membrane After In Vitro Ischemic Treatment

Abstract: The effect of energy failure on Cl⁻-dependent l -glutamate ( l -Glu) transport was examined with an in vitro preparation. Rat brain slices were incubated in low oxygen and glucose-deprived medium (in vitro ischemia), and a synaptic membrane fraction was prepared from the slices. Cl⁻-dependent l -[³H]Glu uptake into vesicles increased about twofold after 20 min of in vitro ischemia. The increased l -[³H]Glu uptake was inhibited by l -Glu, dl -2-amino-4-phosphonobutyrate, l -homocysteic acid, l -cystine, 4,4'-diisothiocyano-2,2'-disulfonic stilbene, and removal of Cl⁻. Uptakes of Na⁺-dependent l -[³H]-Glu, [³H]GABA, and [³H]taurine were not changed by the in vitro ischemia. In vitro ischemia increased the V _max value without affecting the K _m value. The increased l -[³H]Glu uptake by in vitro ischemia was reduced by subsequent incubation in a normoxic glucose-containing solution. ATP content in brain slices decreased to <10% of control values by in vitro ischemia for 10 min. The decrease in ATP content was restored by subsequent incubation in normoxic glucose-containing solution. Treatment with veratrine, 2,4-dinitrophenol, carbonyl cyanide m -chlorophenylhydrazone, and NaCN in normoxic conditions increased l -[³H]Glu uptake with a concomitant decrease in ATP content in slices. These results suggest that Cl⁻-dependent l -Glu transport activity in synaptic membranes increases in ischemia- or hypoxia-induced brain energy failures.