Tryptophan and phenylalanine transport in rat cerebral cortex slices as influenced by sodium ions

Tryptophan and phenylalanine transport in rat cerebral cortex slices was studied in sodium-free media and during influx and efflux of sodium ions. Choline as a substitute for sodium in incubation media increased efflux and decreased influx of tryptophan and phenylalanine. Exchange of intracellular [³H]tryptophan and [³H]phenylalanine with extracellular unlabeled histidine, phenylalanine, and tryptophan was sodium-independent. Efflux of sodium ions from the slices had no immediate effects on phenylalanine and tryptophan efflux, but influx decreased. Influx of sodium into the sodium-depleted slices provoked a transient increase in tryptophan and phenylalanine efflux and also enhanced influx. The results are interpreted to indicate that sodium ions may possibly affect the function of the primary transport sites for aromatic amino acids at cerebral membranes by controlling the orientation of their reactive sites towards the intracellular and extracellular sides, rather than by being directly involved in the binding of amino acids to the carriers.     

Na- and Cl-dependent glycine transport in human red blood cells and ghosts. A study of the binding of substrates to the outward-facing carrier

Na- and Cl-dependent glycine transport was investigated in human red blood cells. The effects of the carrier substrates (Na, Cl, and glycine) on the glycine transport kinetics were studied with the goal of learning more about the mechanism of transport. The K1/2-gly was 100 microM and the Vmax-gly was 109 mumol/kg Hb.h. When cis Na was lowered (50 mM) the K1/2-gly increased and the Vmax-gly decreased, which was consistent with a preferred order of rapid equilibrium loading of glycine before Na. Na-dependent glycine influx as a function of Na concentration was sigmoidal, and direct measurement of glycine and Na uptake indicated a stoichiometry of 2 Na:1 glycine transported. The sigmoidal response of glycine influx to Na concentration was best fit by a model with ordered binding of Na, the first Na with a high K1/2 (greater than 250 mM), and the second Na with a low K1/2 (less than 10.3 mM). In the presence of low Cl (cis and trans 5 mM), the K1/2-gly increased and the Vmax-gly increased. The Cl dependence displayed Michaelis-Menten kinetics with a K1/2-Cl of 9.5 mM. At low Cl (5 mM Cl balanced with NO3), the glycine influx as a function of Na showed the same stoichiometry and Vmax-Na but a decreased affinity of the carrier for Na. These data suggested that Cl binds to the carrier before Na. Experiments comparing influx and efflux rates of transport using red blood cell ghosts indicated a functional asymmetry of the transporter. Under the same gradient conditions, Na- and Cl-dependent glycine transport functioned in both directions across the membrane but rates of efflux were 50% greater than rates of influx. In addition, the presence of trans substrates modified influx and efflux differently. Trans glycine largely inhibited glycine efflux in the absence or presence of trans Na; trans Na largely inhibited glycine influx and this inhibition was partially reversed when trans glycine was also present. A model for the binding of these substrates to the outward-facing carrier is presented.     

EXCHANGE OF TAURINE IN BRAIN SLICES OF ADULT and 7-DAY-OLD RATS

Abstract— The influx or efflux of taurine in brain slices prepared from adult and 7-day-old rats was studied in Krebs-Ringer bicarbonate-glucose medium with 0,2 and 10 m m -taurine. The exchange of taurine between the slices and the medium was slow, and no steady-state concentration was reached within the experimental period of 150 min. In both experimental groups there was a net influx of taurine into the slices from 10 m m -taurine and a slight net efflux from the slices into 2 m m -taurine. The rate of influx from 10 m m -taurine was about the same in the two groups after an initial period of faster influx into the slices of adult rats. There was some rapid initial efflux into 0 and 2 m m -taurine solutions from the slices from 7-day-old rats, but with prolonged incubation these slices were better able to maintain their intracellular taurine than the slices from adult rats. The reasons and significance of the high cerebral concentration of taurine in immature brain in vivo are briefly discussed in the light of the present and earlier studies.     

Efflux and exchange of glycine by plasma membrane vesicles isolated from glioblastoma cells

The efflux and exchange of glycine were studied in plasma membrane vesicles isolated from cultured glioblastoma cells. The mechanism of glycine translocation has been probed by comparing the ion dependence of net efflux to that of exchange. Dilution-induced efflux requires the simultaneous presence of internal sodium and chloride, while influx is dependent on the presence of these two ions on the outside (Zafra, F. and Giménez, C. (1986) Brain Res. 397, 108-116). Glycine efflux from the membrane vesicles is stimulated by external glycine, this exchange being dependent on external sodium, but not on external chloride. The parallelism observed in influx and efflux processes suggests that glycine is translocated in both directions across the membrane, probably by interacting with the carrier. To account for all the observed effects of external ions, glycine concentrations and membrane potential on glycine influx and efflux, a kinetic model of the Na+/Cl-/glycine cotransport system is discussed.     

The effect of diamide on amino acid transport by rat renal cortex slices.

Diamide directly added to renal cortical slices inhibits the uptake of amino acids. Steady-state kinetic analysis indicates an inhibition of alpha-amino acid influx without effect on efflux. The effect could be reversed by addition of pyruvate to the incubation medium. Although there was a good correlation of the transport effect of diamide with its ability to decrease cellular reduced glutathione concentration, there did not appear to be a necessary connection between them. This was shown by the fact that renal cortical slices stored at 4 degrees C have no alteration in amino acid uptake despite the fact that GSH concentration is as low as that seen with diamide. Diamide was shown to have a direct effect on the uptake of glycine by isolated renal brush border membrane vesicles.     

The effect of diamide on amino acid transport by rat renal cortex slices

Diamide directly added to renal cortical slices inhibits the uptake of amino acids. Steady-state kinetic analysis indicates an inhibition of α-amino acid influx without effect on efflux. The effect could be reversed by addition of pyruvate to the incubation medium. Although there was a good correlation of the transport effect of diamide with its ability to decrease cellular reduced glutathione concentration, there did not appear to be a necessary connection between them. This was shown by the fact that renal cortical slices stored at 4°C have no alteration in amino acid uptake despite the fact that GSH concentration is as low as that seen with diamide. Diamide was shown to have a direct effect on the uptake of glycine by isolated renal brush border membrane vesicles.     

Effects of tetrodotoxin and anaesthetics on brain metabolism and transport during anoxia

1. Tetrodotoxin, at concentrations at which it abolishes generation of action potentials in the nervous system, enhances by about 300% the rate of anaerobic glycolysis of brain-cortex slices from adult rats, or from adult and infant guinea pigs. This occurs to a greater extent in Ca(2+)-deficient incubation media than in Ca(2+)-rich media. Tetrodotoxin has no accelerative effect on cerebral aerobic glycolysis. 2. Tetrodotoxin does not affect the rate of anaerobic glycolysis of 2-day-old rat brain-cortex slices, nor that of adult rat kidney medulla, nor that of an extract of an acetone-dried powder of brain. 3. Tetrodotoxin does not affect the rate of penetration of glucose into brain slices. 4. Its effect is not apparent if it is added 10min or later after the onset of anoxia. 5. Its effect diminishes as the concentration of K(+) in the incubation medium is increased while that of Na(+) is decreased. 6. Its salient effect, at the onset of anoxia, is to diminish influx of Na(+) into, and efflux of K(+) from, the brain slices. 7. Substances that promote cerebral influx of Na(+), e.g. protoveratrine, sodium l-glutamate, diminish the accelerative action of tetrodotoxin. 8. It is concluded that tetrodotoxin exerts its effect on anaerobic glycolysis by suppressing, at the onset of anoxia, the generation of action potentials and thereby the accompanying influx of Na(+) and efflux of K(+). It is suggested that glycolytic stimulation occurs because a rate-limiting step, e.g. operation of pyruvate kinase, is stimulated by K(+) and depressed by Na(+). 9. Local anaesthetics behave in a manner similar to that of tetrodotoxin in enhancing cerebral anaerobic glycolysis. 10. Sodium Amytal has a marked effect at relatively high concentration. 11. Tetrodotoxin diminishes efflux of amino acids, particularly glutamate and aspartate, at the onset of anoxia.     

The effect of extracellular sodium concentration on α-methyl-d-glucoside transprot by rat kidney cortex slices

The kinetics of α-methyl-d-glucoside accumulation by rat kidney cortex slices under conditions of varying extracellular sodium concentration were investigated. Extracellular sodium reduction below 144 mequivi/l resulted in a diminished initial uptake and reduced influx calculated by steady-state analysis of a two-compartment system. At 72 mequiv/l extracellular sodium efflux was decreased to the same extent as influx resulting in the same steady state concentration that was observed at 144 mequiv/l sodium. At 36 mequiv/l the steady state concentration was below that observed at 144 mequiv/l sodium because of a disproportionate decrease of influx. In the complete absence of extracellular sodium, no concentration gradient was achieved and efflux had become increased. Low extracellular sodium was associated with an increase in the apparent K_m of transport without affecting the V. The apparent K_i for sodium appeared to be in th e 40 to 59 mequiv/l range. The presence of 20 mM α-methyl-d-glucose prompted the accelerated efflux of the sugar from the tubule cells in a normal fashion despite a low extracellular sodium concentration.     

Anomalous kinetics of sucrose uptake in maize scutellum slices1

Abstract The kinetics of sucrose uptake into maize scutellum slices showed that the uptake mechanism had a saturable component with a Km of l.5mol m^?3 sucrose. Nevertheless, uptake rate was constant (zero order) over extended periods of time until the bathing solution was nearly depleted of sucrose. It is concluded that these anomalous uptake kinetics reflect sucrose influx across the plasmalemma because of the following results: (a) Efflux of sucrose into buffer was negligible compared with uptake rate, (b) When slices were incubated in fructose, sucrose was synthesized and there was a net release of sucrose to the bathing solution until a steady-state was reached when influx and efflux were equal in magnitude. After the steady-state was reached, efflux of sucrose from the slices was nearly the same in magnitude as the estimated rate of uptake that would have occurred from bathing solutions initially containing the steady-state sucrose concentration, (c) Exchange of sucrose between bathing solution and slices was negligible compared with uptake rate, (d) Pretreatment of slices with uranyl nitrate abolished sucrose uptake, but uptake rate was re-established in these slices after treatment with HCl (pH 2). Uptake rate was set by the initial sucrose concentration of the bathing solution, and was not influenced by the level of endogenous sucrose or by the rate at which the sucrose concentration of the bathing solution declined. Abrupt increases in sucrose concentration during the uptake period increased the rate of uptake only if the concentration was increased above that at the start of the uptake period. Following abrupt decreases in sucrose concentration, there was a lag of about 30 min before uptake rate decreased greatly. If slices were washed and replaced in a fresh sucrose solution during the uptake period, a new uptake rate was set to correspond to the new initial sucrose concentration. It is suggested that the sucrose carrier has a transport site with a relatively low Km (much below 1.5mol m^?3) and that the measured Km (1.5mol m^?3) is that of a site that binds sucrose and thereby controls the rate of uptake. The low Km suggested for the transport site would explain the zero order kinetics but a model of the uptake mechanism that includes the control site cannot, as yet, be constructed from the data.     

SODIUM-DEPENDENT EFFLUX AND EXCHANGE OF GABA IN SYNAPTOSOMES

Abstract— The influx and efflux of [³H]GABA were investigated in synaptosomes. Two efflux components were detected. The first, termed spontaneous efflux, was not affected by the external sodium chloride concentration. The second, termed GABA-stimulated efflux, was observed when low levels of GABA were added to the incubation medium and was found to require external sodium chloride. The rate of spontaneous efflux at 0°C was about 37 per cent of the rate at 27°C but both GABA-stimulated efflux and GABA influx were completely inhibited at 0°C. The stimulation of efflux by external GABA followed simple Michaelis–Menten kinetics with respect to external GABA. The concentration of external GABA required for half-maximal stimulation was 4·9 ± 1·4 μm and the V_max for efflux was 1·0 ± 0·6 nmol. min^-1.mg^-1 of protein. A similar stimulation of efflux was observed with GABA analogue l -2,4-diamino-butyric acid which is a competitive inhibitor of influx. The concentration of external l -2,4-diaminobutyric acid required for half-maximal stimulation of efflux was 51 ± 12 μm and the V_max for efflux was 0·8 ± 0·5 nmol.min^-1.mg^-1 of protein. Since the sodium-dependency, temperature sensitivity, and kinetic properties of the GABA-stimulated efflux system were similar to the influx system, GABA-stimulated efflux was attributed to carrier-mediated exchange diffusion. Measurement of efflux and influx in the same preparation showed there was a net efflux when total fluxes were considered and that the exchange ratio (influx to GABA-stimulated efflux) was 0·9 when carrier-mediated fluxes were considered. The effect of the temperature of the fluid used to rinse synaptosomes collected on filters in influx experiments was investigated. There was no detectable difference in measured values of influx between samples rinsed with cold fluid (0°C) and warm fluid (27°C). The endogenous GABA content of synaptosomes was found to be 20·3 ± 2·5 nmol GABA per mg of protein. From this value, the cytoplasmic concentration of GABA in synaptosomes was estimated to be a maximum of 40 mm . About 5 per cent of total cerebral cortical GABA was found in the synaptosomal fraction.     

CALCIUM MOVEMENTS IN BRAIN SLICES IN LOW SODIUM OR CALCIUM MEDIA

Abstract— Movement of ⁴⁵Ca into and out of cerebral slices in vitro was altered by incubation in Na-free medium or in the presence of ouabain. ⁴⁵Ca uptake into the 'non-inulin' space was more than doubled in slices that were losing Na into Na-deficient media. Replacement of Na in the incubation medium by choline chloride reduced the efflux of ⁴⁵Ca into Ca-free medium. In the presence of ouabain (10^-4M), the rate of efflux was also reduced. NaCN or NaCN plus ethacrynic acid increased the rate of ⁴⁵Ca efflux. The data are compatible with a sodium-calcium exchange mechanism in cerebral slices.     

Application of purging biotinylated liposomes from plasma to elucidate influx and efflux processes associated with accumulation of liposomes in solid tumors

Although small, 100-nm liposomes are known to selectively accumulate in solid tumors, the individual contributions of liposome influx and egress rates are not well understood. The aim of this work was to determine influx and efflux kinetics for 100-nm, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/cholesterol (Chol) liposomes by inducing aggregate formation of biotinylated liposomes upon administering avidin. Injecting 50 microg of neutravidin intravenously to mice that had previously been administered 100 mg/kg DPSC/Chol liposomes containing 0.5 mol% biotin-conjugated lipid resulted in >90% elimination of the liposomes from plasma within 1 h. This rapid removal by the reticuloendothelial system (RES) permitted the determination of the tumor efflux kinetics due to negligible tumor influx after neutravidin injection. The tumor efflux rate constant (k(-1)) was determined to be 0.041 h(-1) when neutravidin was injected 4 h after liposome injection. This allowed the determination of the tumor influx rate constant (k(1)), which under these conditions was 0.022 h(-1). Therefore, DSPC/Chol liposomal accumulation, in LS180 solid tumors, is dictated primarily by plasma liposome concentrations and liposome egress is comparable or slightly faster than influx into the tumors. This method is applicable for a wide range of lipid doses, and can be used to characterize influx and efflux parameters at different time points after accumulation. The application, therefore, has the potential to be used to fully characterize the impact of different liposome parameters such as lipid composition, steric stabilization, size and dose on tumor accumulation kinetics.     

Effect of Leaf Age on Potassium Efflux and Net Flux in Tomato Leaf Slices

The effects on leaf age on K (⁸⁶Rb) efflux, influx and net fluxinto lamina slices from leaf 7 on a tomato plant (Lycopersiconesculentum Mill.) were determined. The ontogenetic trend inK efflux was dependent on the external K concentration. At externalKCI concentrations between 0.5 and 10.0 mM, K efflux rates increasedduring leaf elongation. Only a small increase in efflux occurredin mature leaves with increasing age. It is suggested that thetonoplast retains its structural integrity through the initialstages of leaf senescence. In fully expanded leaves, a zeronet K flux (a balance between influx and efflux) was achievedat external KCI concentrations between 1.0 and 3.5 mM. The Kcontent of lamina slices from leaves 5 and 13 remained constantwhen bathed in a solution containing 2 to 3 mM K. It is suggestedthat the decline in K concentration in mature tomato leaf tissueis due to a decline in leaf free space K concentrations below1 to 3 mM which would result in a net efflux out of leaf cells. Lycopersicon esculentum Mill., tor ato, free space, ion fluxes, leaf age, leaf ontogeny, potassium     

Active efflux of lithium from erythrocytes of manic-depressive subjects

Lithium transport kinetics, studied under physiological conditions in erythrocytes obtained from manic-depressive patients, are characterized by asymmetric rate constants of efflux and influx. The efflux constants, which are more than twice as large as the influx constants, correlate well with the $\text{in vivo}$ distribution of lithium between erythrocytes and plasma. The efflux process, which is not inhibited by ouabain and is therefore distinct from the sodium-potassium pump, is characterized by Michaelis - Menten kinetics and a large energy of activation. There appears to be a normal endogenous inhibitor which regulates the activity of the postulated lithium pump.     

Bile-salt inhibition of sodium ion-coupled D-glucose and L-alanine accumulation by brush-border-membrane vesicles from hamster jejunum.

The effects of bile salts on Na+-coupled accumulation of D-glucose and L-alanine by brush-border-membrane vesicles isolated from hamster jejunum were investigated. The approximate percentage inhibition of Na+-coupled D-glucose accumulation produced by various bile salts at a concentration of 1 mM were: deoxycholate and chenodeoxycholate, 60%; glycine and taurine conjugates of deoxycholate and chenodeoxycholate, 40--50%; lithocholate, 45%; cholate and its glycine and taurine conjugates, less than 10%. Inhibition of Na+-coupled accumulation of D-glucose was rapid, reversible and not due to dissolution of the vesicles. Na+-coupled accumulation of L-alanine was also inhibited by deoxycholate. Deoxycholate but not cholate enhanced (1) the rate of Na+ influx, (2) the rate of influx of D-glucose and L-alanine in the absence of a Na+ gradient and (3) the rate of efflux of D-glucose and L-alanine from vesicles preloaded with this sugar or amino acid. Deoxycholate-stimulated efflux of D-glucose was not blocked by phlorizin, which completely prevented efflux in the absence of this bile salt. These results suggest that selected bile salts inhibit Na+-coupled accumulation of D-glucose and L-alanine by enhancing the rate of dissipation of the Na+ gradient required for substrate accumulation. In addition, bile salts may also decrease D-glucose and L-alanine accumulation by increasing the rate of efflux of these substrates across the brush-border plasma membrane.     

Kinetics of Tryptophan Influx into Brain Slices Depleted of Sodium and Loaded with l-Histidine

Abstract: The kinetics of tryptophan influx were studied with rat brain slices preloaded with l -histidine and/or depleted of sodium ions. The best fits of the data (velocity of influx versus tryptophan concentration) were computed by use of a model consisting of a saturable (Michaelis-Menten type) and an unsaturable (diffusional) component with an iterative nonlinear regression analysis. Sodium depletion of the slices reduced the maximal velocity of saturable influx. In histidine-preloaded slices, depleted or not depleted of sodium ions, the most marked alteration again occurred in the maximal velocity, which more than doubled. Slices preloaded with histidine contained greatly elevated levels of glutamine and histidine, which may have stimulated the influx by exchange with extracellular tryptophan even in the absence of sodium ions. The maximal velocity was higher with increasing concentration of large neutral amino acids in slices at the start of the influx measurements. The influx of tryptophan in brain cells is apparently modified by changes in the intracellular amino acid pool, which, when increased, also counteracts the effect of sodium depletion on the tryptophan influx.     

MUSCIMOL UPTAKE, RELEASE AND BINDING IN RAT BRAIN SLICES

Abstract— The GABA analogue, muscimol, was taken up relatively inefficiently compared to GABA by slices of rat cerebral cortex at 37 C. Muscimol uptake followed saturation kinetics (K_m ImM. V_m 0.1 μmol g mini and showed an absolute dependence on sodium ions. The relative susceptibilities of muscimol uptake and GABA high affinity uptake to a variety of inhibitors, including (-)-nipecotic acid. (+)-2.4-diaminobutyric acid and arecaidine, and the stimulation of muscimol efflux by 50μM-GABA, suggest that muscimol and GABA share some common transport carriers. Since L-histidine inhibited muscimol uptake hut not GABA high affinity uptake, at least part of the observed muscimol uptake may be mediated by the 'small basic'amino acid transport system. Muscimol appeared to he taken up into nerve terminals, since uptake was inhibited by the neuronal uptake inhibitor cis -3-aminocyclohexanecarboxylic acid but not by the glial uptake inhibitor β-alanine. Muscimol efflux was stimulated in a calcium-dependent manner by an increased potassium ion concentration.
Sodium-independent binding of muscimol was observed in slices of rat cerebral cortex at 4 C. Binding could be inhibited by a variety of substances. including GABA, isoguvacine and (+)-bicuculline methochloride, which are known to inhibit the binding of muscimol to putative GABA receptors associated with synaptic membranes purified from rat brain.     

Intracellular chloride and calcium transients evoked by gamma-aminobutyric acid and glycine in neurons of the rat inferior colliculus.

Microfluorometric recordings showed that the inhibitory neurotransmitters gamma-aminobutyric acid (GABA) and glycine activated transient increases in the intracellular Cl- concentration in neurons of the inferior colliculus (IC) from acutely isolated slices of the rat auditory midbrain. Current recordings in gramicidin-perforated patch mode disclosed that GABA and glycine mainly evoked inward or biphasic currents. These currents were dependent on HCO3- and characterized by a continuous shift of their reversal potential (E(GABA/gly)) in the positive direction. In HCO3- -buffered saline, GABA and glycine could also evoke an increase in the intracellular Ca2+ concentration. Ca2+ transients occurred only with large depolarizations and were blocked by Cd2+, suggesting an activation of voltage-gated Ca2+ channels. However, in the absence of HCO3-, only a small rise, if any, in the intracellular Ca2+ concentration could be evoked by GABA or glycine. We suggest that the activation of GABAA or glycine receptors results in an acute accumulation of Cl- that is enhanced by the depolarization owing to HCO3- efflux, thus shifting E(GABA/gly) to more positive values. A subsequent activation of these receptors would result in a strenghtened depolarization and an enlarged Ca2+ influx that might play a role in the stabilization of inhibitory synapses in the auditory pathway.     

Influx and efflux kinetics of cationic dye binding to respiring mitochondria.

We have investigated the kinetics of interaction of cationic fluorescent lipophiles (dyes) rhodamine 123, rhodamine 6G, tetramethyl rhodamine ethyl ester, safranine O, 1,1'-diethyloxacarbocyanine, 1,1'-diethyloxadicarbocyanine, and 1,1'-diethylthiadicarbocyanine iodide with isolated respiring rat-liver mitochondria (RLM). Dye flux across the RLM inner membrane was measured by following the kinetics of fluorescence signal change after mixing of dye and RLM. The time course of fluorescence was analysed in terms of a kinetic model of the binding and transport processes involved. The rate constants of dye influx and efflux were extracted from the observed effect on the apparent time constant of fluorescence change to equilibrium intensity upon mixing dye with increasing concentrations of RLM. From the influx rate constants obtained, the apparent permeability constants for dye influx (at zero potential) across the membrane were calculated and ranged from 3 to 140 x 10(-4) cm/s. The influx rate constant was found to be linearly related to relative dye lipophilicity, as predicted by the model. As another test of the model, from the ratio of the influx and efflux rate constants, the apparent trans-membrane potential, psi, was calculated and found generally to agree with reported values, but to depend on the lipophilicity of the dye used. Not predicted by the simple model was a dissymmtry observed in the influx and efflux time constants for fluorescence change to equilibrium intensity. Inferences are made relating to the utility of these dyes as probes of psi.     

Expression of human equilibrative nucleoside transporter 1 in mouse neurons regulates adenosine levels in physiological and hypoxic-ischemic conditions

Activation of adenosine A(1) receptors inhibits excitatory synaptic transmission. Equilibrative nucleoside transporters (ENTs) regulate extracellular adenosine levels; however, the role of neuronal ENTs in adenosine influx and efflux during cerebral ischemia has not been determined. We used mice with neuronal expression of human ENT type 1 and wild type (Wt) littermates to compare responses to in vitro hypoxic or ischemic conditions. Extracellular recordings in the CA1 region of hippocampal slices from transgenic (Tg) mice revealed increased basal synaptic transmission, relative to Wt slices, and an absence of 8-cyclopentyl-1,3-dipropyl-xanthine mediated augmentation of excitatory neurotransmission. Adenosine (10-100 μM) had a reduced potency for inhibiting synaptic transmission in slices from Tg mice; inhibitory concentration 50% values were approximately 25 and 50 μM in Wt and Tg slices, respectively. Potency of the A(1) receptor agonist N(6) -cyclopentyladenosine (1 nM-1 μM) was unchanged. Transient hypoxia or oxygen-glucose deprivation produced greater inhibition of excitatory neurotransmission in slices from Wt than Tg, mice. The ENT1 inhibitor S-(4-nitrobenzyl)-6-thioinosine abolished these differences. Taken together, our data provide evidence that neuronal ENTs reduce hypoxia- and ischemia-induced increases in extracellular adenosine levels and suggest that inhibition of neuronal adenosine transporters may be a target for the treatment of cerebral ischemia.