Dopamine Uptake In Synaptosomes Research Articles

Modulation of dopamine uptake and release in rat striatal synaptosomes by glycine and glutamate.

The uptake and release of dopamine (DA) by rat brain striatal synaptosomes were studied in presence of glycine or glutamate (0.5-20 mM) in incubation media containing 5 or 55 mM KCl. In low K+ medium glycine, up to 5 mM had little effect on DA uptake, but higher concentrations of the amino acid inhibited the uptake. High K+ medium resulted in a decrease in DA uptake as compared to that of basal K+ medium. Glycine at 1 microM, but not at higher concentrations, prevented the inhibition induced by high K+ depolarization. However, glutamate in the low K+ medium and at a 0.5 mM concentration, stimulated DA uptake, but at higher concentrations it inhibited the uptake. In the high K+ medium, glutamate in all concentrations potentiated the inhibition of DA uptake induced by K+ depolarization. The DA release response of the synaptosomes to glycine concentrations (1-20 mM) in low K+ medium was a biphasic pattern, with a stimulation at 1 mM and an inhibition at higher concentrations. This pattern was reversed when DA release was measured in the high K+ medium. The pattern of DA release in the presence of glutamate concentrations (1-20 mM) was a triphasic one, with an inhibition at 1 mM, stimulation at 5 mM, and a less effective inhibition at higher concentrations. In the high K+ medium, glutamate at 1 mM concentration prevented the stimulation induced by K+ depolarization, but at 5 mM reversed the rate of release to the depolarization state. Results of this study suggest that glycine and glutamate have more than a simple inhibitory or excitatory transmitter role in the striatum, respectively. The identical effects of glycine with that of glutamate in certain concentrations is consistent with previous reports that glycine and N-methyl-D-aspartate (NMDA) act as coagonists of a common excitatory amino acid receptor.

Nitric oxide inhibits [ 3H]dopamine uptake

Sodium nitroprusside (SNP), a generator of nitric oxide (NO), decreased [ 3H]dopamine uptake into rat striatal synaptosomal preparations in a dose-, time- and temperature-dependent fashion. Reduced hemoglobin, a substance that binds NO, prevented the SNP-induced decrease in uptake. Potassium ferri- and ferrocyanide, compounds similar to SNP that do not produce NO, were without effect on uptake. SNP inhibited [ 3H]dopamine uptake in synaptosomes from nucleus accumbens and olfactory tubercle as well but with a lower potency. SNP inhibited [ 3H]serotonin and [ 3H]glutamate uptake but had no effect on [ 3H]norepinephrine uptake. S-Nitroso- N-acetylpenicillamine (SNAP), another generator of NO, had effects similar to those of SNP. The SNP-induced decrease in [ 3H]dopamine uptake was due to a V max decrease at 100 μM SNP and to both a V max and K m change at 300 μM SNP. Depletion of calcium by omission of calcium from buffers and addition of EGTA increased the potency of SNP in inhibiting uptake. There was no change in [ 3H]WIN 35428 binding to the dopamine transporter with doses of SNP that inhibited uptake. These data indicate that NO can decrease [ 3H]dopamine transporter function.

Cocaine binding sites in fetal rat brain: implications for prenatal cocaine action.

Binding of [3H]cocaine to membrane preparations from whole fetal rat brain was studied. High-affinity binding (10 nM cocaine) was detected as early as gestational day (GD) 15 and steadily increased across subsequent development. Saturation studies comparing [3H]cocaine binding at GD20 and adulthood yielded similar KD values, and LIGAND analyses favored a two-site model if an extended range of [3H]cocaine concentrations was used. Various monoamine uptake inhibitors displaced labeled cocaine with potencies consistent with the idea that [3H]cocaine labels the dopamine (DA), serotonin (5-HT), and possibly also the norepinephrine (NE) transporters in whole fetal brain preparations. Synaptosomal DA uptake was well developed by GD20, as was the potency of cocaine to inhibit such uptake. The results indicate that functional, monoamine transporter related cocaine binding sites are present in the fetal rat brain. Such sites are likely to play an important role in mediating the direct interactions of prenatally-administered cocaine with developing monoaminergic systems in both animals and humans.

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

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

Arylcyclohexylamines derived from BTCP are potent indirect catecholamine agonists

N-[1-(2-Benzo[ b]thiophenyl)cyclohexyl]piperidine (BTCP)-related molecules were prepared by means of chemical modulation of the 3 rings which constitute this dopamine (DA) and norepinephrine (NE) uptake inhibitor. Tested in vitro (binding to the DA uptake complex and to the PCP receptor sites, inhibition of the synaptosomal uptake of DA and NE) and in vivo (locomotor activity in mice) these molecules showed good homogeneity of action. The newly prepared structures, although formally related to the phencyclidine (PCP) structural model, no longer display significant affinity for the PCP receptor. These compounds behave like a new series of indirect potent stimulants of the dopaminergic and noradrenergic systems leading to potential antidepressive compounds.

LY248686, A New Inhibitor of Serotonin and Norepinephrine Uptake

LY248686 is an inhibitor of serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine (NE) uptake in synaptosomal preparations of hypothalamus and cerebral cortex, and 5-HT uptake in human blood platelets, with inhibitor constants near nanomolar concentrations. Upon administration to rats 1 hour before sacrifice, LY248686 caused dose-dependent and parallel decreases of 5-HT and NE uptake in hypothalamus homogenates ex vivo. LY248686 is a positive enantiomer and was slightly more potent than its negative isomer, LY248685, as an inhibitor of 5-HT uptake. Both isomers were only weak inhibitors of dopamine (DA) uptake in striatal synaptosomes. The inhibitory effects on 5-HT and NE uptake after a single administration of LY248686 followed similar time courses and simultaneously persisted for as long as 6 hours. LY248686 in vivo could effectively antagonize the p-chloroamphetamine-induced decreases of 5-HT uptake and levels of 5-HT and 5-hydroxyindoleacetic acid in cerebral cortex, and block the accumulation of 14C-NE in rat hearts. In food deprived rats, LY248686 suppressed food intake synergistically with 5-hydroxytryptophan, a precursor amino acid of 5-HT. Because of its lack of affinity for receptors of 5-HT, NE, DA, acetylcholine, histamine and naloxone, and its ability to inhibit 5-HT and NE uptake simultaneously, LY248686 has a favorable pharmacological profile as a potential antidepressant drug.

Molecular basis of discrepancies in neurotoxic properties among 1-methyl-4-aryl-1,2,3,6-tetrahydropyridines.

The relationship between structural specificity of the main stages of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) action and the display of parkinsonogenic properties among homologous structures in a number of 4-tolyl derivatives of MPTP has been studied. All the compounds are better substrates for monoamine oxidase (MAO) than MPTP. MAO is inactivated during the reaction according to a mechanism of irreversible inhibition by 2,3-dihydropyridinium metabolite. All the tolyl derivatives are stronger inhibitors of MAO than 1-methyl-2,3-dihydropyridinium (MPDP). A significant contribution of enzyme inhibition to the catalytic conversion of the substrate leads to the fact that substrates having equal (para isomer) or even higher (meta isomer) values of catalytic parameters are oxidized by MAO to a lesser extent than MPTP. It has been found that all 4-arylpyridiniums (final products of MATP bioconversion) competitively and reversibly inhibit [14C]dopamine (DA) uptake in mouse brain synaptosomes. Affinity toward DA transporter characterized by KI (microM) is 0.37 +/- 0.04, 0.7 +/- 0.1, 2.0 +/- 0.15, 2.0 +/- 0.35 for MPP, and its o-, m-, and p-tolyl derivatives, respectively. Joint calculation of specificity factors for the processes discussed define the following rank order for the bio-delivery of MATP's metabolic produces into DA nerve terminals: o-tolyl > MPTP >> m-tolyl > p-tolyl. The regularity revealed is in good agreement with the observed relative potency of these compounds to cause dopaminergic neurodegeneration.

Prevention by Ginkgo biloba extract (EGb 761) and trolox C of the decrease in synaptosomal dopamine or serotonin uptake following incubation

Prolonged incubation of synaptosomes in Krebs-Ringer oxygenated medium in the presence of ascorbic acid (10 −4M) led, after 20 min, to a decrease in [ 3H]dopamine (DA) (synaptosomes prepared from the striatum) and [ 3H]serotonin (5HT) (synaptosomes prepared from the cortex) uptake. The decrease was progressive and uptake was virtually abolished after a 60 min incubation period. A concentration-dependent (from 5 × 10 −6 M) role of ascorbic acid in the decrease of [ 3H]DA or [ 3H]5HT uptake was demonstrated. This decrease was potentiated by Fe 2+ ions and prevented by the ferrous chelating agent desferrioxamine. Thus, the progressive decrease in synaptosomal uptake of either [ 3H]DA or [ 3H]5HT could depend on the generation of free radicals by the association of ascorbic acid with Fe 2+ ions. The decrease in synaptosomal uptake was prevented, in a concentration-dependent manner, by the Ginkgo biloba extract EGb 761 (4–16 μg/mL) and the vitamin E analog trolox C (10 −4 M). The terpenic fraction of EGb 761, Bn 52063 (up to 0.5 μg/mL), did not prevent the reduction of [ 3H]amine uptake. In contrast, the flavonoidic fraction, Cp 202, was effective (from 1 μg/mL) and its efficacy was shared by the flavonoid quercetin (from 0.1 μg/mL). The prolongation of the ability of synaptosomes to take up [ 3H]amine elicited by EGb 761, in particular its flavonoidic fraction, as well as by trolox C could be due to their free radical scavenger properties.

Investigations about a direct neurotensin-dopamine interaction by nuclear magnetic resonance study, synaptosomal uptake of dopamine, and binding of neurotensin to its receptors.

Interactions between dopamine and neurotensin can occur at various levels of the dopaminergic pathways. By using different approaches in vitro, we investigated the proposed hypothesis that neurotensin might bind to dopamine in the synaptic cleft. Nuclear magnetic resonance spectra of neurotensin were not modified by the addition of dopamine, and no nuclear Overhauser effect was detected. Synaptosomal uptake of [3H]dopamine in the presence of neurotensin did not lead to any modifications of the kinetic constants of the uptake. Neurotensin binding was not modified by the addition of dopamine. These results did not confirm the suggestion that neurotensin can form a complex with dopamine.

Inhibition of high affinity choline uptake in the rat brain by neurotoxins: Effect of monosialoganglioside GM1

Mustard derivatives of ethyl-choline and hemicholinium-3 have been suggested as possible specific cholinergic neurotoxins. In this study a structural analog of hemicholinium-3, a,a′-bis[di(2-chloroethyl)amino]-4,4′-2-biacetophenone (toxin 7), was added to synaptosomes prepared from the cortex, striatum or hippocampus of rat brain. Synaptosomal high affinity choline uptake (HACU) was significantly decreased in a dose-dependent manner by addition of toxin 7, while synaptosomal uptake of GABA or dopamine was not changed. Incubation of cortical synaptosomes with the monosialoganglioside GM1 prevented the decrease in HACU seen following administration of toxin 7. This preventative effect of GM1 was greater if GM1 was added prior to or concomitant with toxin 7, than if GM1 was added following toxin 7. Two newly synthesized hemicholinium-3 analogs, 4-[3′-di(2-chloroethyl)aminopropionyl] biphenyl (toxin 5) and 4-[3′-di(2-bromoethyl)aminopropionyl]biphenyl (toxin 6) caused a large decrease in HACU when added to cortical synaptosomes, this decrease was significantly greater than that seen with the same dose of toxin 7 or ethyl-choline aziridinium (AF64A). Ultrastructural changes in the synaptosomal membrane following incubation with toxin 7 or toxin 7 with GM1 were examined by electron microscopy. Development of a compound which is both a potent neurotoxin, and is specific for cholinergic neurons will allow new insights into the normal function of the cholinergic system in the CNS and provide animal models of disease states in which cholinergic degeneration is an important element.

Biochemical and morphological changes in rat brain synaptosomes after exposure to normobaric hyperoxia in vivo.

Adult rats were submitted to normobaric hyperoxia for 1 to 24 h, then the brain synaptosomes were isolated and their metabolic and morphologic properties were studied. Hyperoxia lasting 1-2 h significantly increased the content of thiobarbituric acid-reactive material (TBAR) and decreased the level of protein thid groups. During the next 5-8 h of hyperoxia SH groups as well as TBAR content became almost normal, reflecting adaptation of the animals to an elevated oxygen tension. After 24 h of hyperoxia a maximal increase in the TBAR content and parallel fall in protein thiol groups were noted. Simultaneously, significant morphological differences between control synaptosomes and synaptosomes isolated from rats exposed to 24 h oxygenation were observed in electron microscopy. The high-affinity dopamine uptake in hyperoxic synaptosomes was significantly increased in all experimental groups. A specific high sensitivity of the dopamine uptake system in synaptoplasmatic membranes to the free radical modification of the membrane structure is suggested.

Pharmacological effects of Hoe 249: A new potential antidepressant

AbstractThe 1‐(2‐hydroxy‐1‐phenylethyl)‐imidazole derivative Hoe 249 has a profile which suggests the compound is a promising candidate for clinical evaluation as an antidepressant drug. In pharmacological tests commonly considered predictive of antidepressant activity, e.g., reserpine‐ and tetrabenazine‐antagonism, reversal of oxotremorine‐ and apomorphine‐induced hypothermia, and potentiation of yohimbine toxicity, Hoe 249 is equipotent to nomifensine, but does not meet excitation and stereotypic behavior in a comparable dose range. Hoe 249 shows weak anticonvulsant activity against pentylenetetrazole‐induced seizures and it causes a slight dose‐dependent prolongation of hexobarbital narcosis. Biochemical studies characterize Hoe 249 as an inhibitor of synaptosomal dopamine and norepinephrine uptake, whereas serotonin uptake is not influenced. © 1992 Wiley‐Liss, Inc.

Inhibition of [ 3H]dopamine uptake by flunarizine

The effect of different calcium channel blockers was studied on basal and cocaine-inhibited [ 3H]dopamine uptake in rat striatal synaptosomes. Isradipine (dihydropyridine calcium antagonist) and diltiazem (L-type calaium antagonist) were devoid of effect on [ 3H]dopamine uptake, while flunarizine (T-type calcium antagonist) inhibited [ 3H]dopamine uptake. Flunarizine inhibition was competitive and the inhibitory curve was biphasic with a Hill coefficient of 2.1. The high Hill number suggested a mechanism of positive cooperativity between two sites. Flunarizine inhibition showed a complex interaction with cocaine inhibition. While flunarizine at low concentrations interacts with a distinct site, at higher concentrations it interacts with the same site as cocaine. The relevance of this finding for the potentiation by flunarizine of cocaine-induced dopamine release in vivo is discussed.

Relationship Between [3H]Mazindol Binding to Dopamine Uptake Sites and [3H]Dopamine Uptake in Rat Striatum During Aging

Certain drugs exhibit a remarkable correlation between their ability to inhibit synaptosomal uptake of dopamine and the binding of [3H]mazindol to striatal membranes. To investigate the role of mazindol binding sites in the dopamine uptake process and the fate of these sites (labeling dopaminergic neurons) during aging, we have examined the properties of mazindol binding and dopamine uptake in individual young and old rats. There was a 48% decrease (p = 0.0001) in the Bmax of mazindol binding and a 23% decrease (p = 0.0166) in the Vmax of dopamine uptake with no apparent change in their affinities with age. Regression analysis of the relationship between Bmax and Vmax exhibited a significant correlation in old (p = 0.0156) but not young rats (p = 0.1398). These data suggest that the number of mazindol binding sites decreases with age and that the number of sites on the dopamine transporter complex far exceeds the number required to elicit maximal dopamine uptake.

Evidence for Functional Activity of Up‐Regulated Nicotine Binding Sites in Rat Striatal Synaptosomes

A number of studies have found that the chronic administration of nicotine causes an increase in the density of nicotinic binding sites in the brain, but it is not known whether these additional binding sites are functionally active receptors. In this study, the effects of 1-week administration of the potent nicotinic agonist, (+)-anatoxin-a (96 nmol/day via osmotic minipumps), was assessed on [3H]nicotine binding and [3H]dopamine uptake and release in rat striatal synaptosomes. Chronic (+)-anatoxin-a treatment resulted in a 32% increase in the Bmax of [3H]nicotine binding in anatoxin-treated animals compared to control. There was a 43% increase in the activity of 3 microM nicotine to release [3H]dopamine from synaptosomes of anatoxin-treated animals, but the release induced by 20 mM K+ depolarization was unaffected. There was no effect of chronic (+)-anatoxin-a treatment on the uptake of [3H]dopamine. A strong positive correlation (r = 0.64) was found between the density of [3H]nicotine binding sites and the nicotine-induced stimulation of [3H]dopamine release in individual animals. These results indicate that (+)-anatoxin-a, like nicotine, produces an up-regulation of nicotine binding sites following chronic administration, and that these additional sites are functional receptors capable of mediating the release of dopamine from striatal synaptosomes.

Plant‐Derived Neurotoxic Amino Acids (β‐N‐Oxalylamino‐l‐Alanine and β‐N‐Methylamino‐l‐Alanine): Effects on Central Monoamine Neurons

In the present study the subacute effects of beta-N-oxalylamino-L-alanine (BOAA) and beta-N-methylamino-L-alanine (BMAA) on CNS monoamine neurons in rats were investigated following intracisternal injections or local intracerebral administration into substantia nigra. In vitro effects of BOAA and BMAA on high-affinity synaptosomal uptake of dopamine (DA), noradrenaline (NA), and serotonin (5-HT) were also examined. Intracisternal administration of BMAA decreased NA levels in hypothalamus, whereas no effects were seen on DA or 5-HT levels. Following intranigral injections of BOAA, NA levels tended to decrease in several regions, whereas the DA levels and the levels of DA metabolites were unaffected in all regions analyzed. Loss of tyrosine hydroxylase (TH) immunoreactivity in the intranigral injection sites and the presence of TH-immunoreactive pyknotic neurons near the borders of the injection sites were observed following both BOAA and BMAA treatments. Furthermore, substance P-immunoreactive terminals in substantia nigra pars reticulata were also found to have disappeared within the lesioned area following either BOAA or BMAA injections. Incubations with both BOAA and BMAA (10(-5) M) reduced high-affinity [3H]NA uptake in cortical synaptosomes to 69% and 41% of controls, respectively, whereas the striatal high-affinity [3H]DA uptake and the cortical high-affinity [3H]5-HT uptake were unaffected by BOAA or BMAA. The results demonstrate that both BOAA and BMAA can affect central monoamine neurons, although the potency and specificity of these substances on monoamine neurons when administered acutely into cerebral tissue or liquor cerebri seem to be low. However, the in vitro studies indicate selective effects of both compounds on NA neurons in synaptosomal preparations.

Kinetics and Block of Dopamine Uptake in Synaptosomes from Rat Caudate Nucleus

The dopamine (DA) uptake system in mammalian nerve terminals was studied by measuring the unidirectional influx of tritiated DA into synaptosomes prepared from rat caudate nucleus. Two distinct time-dependent components of DA uptake were observed. The principal component was saturable with respect to DA concentration, required both external Na and Cl, and was competitively blocked by micromolar concentrations of the psychotropic agents cocaine, benztropine, nomifensine, amphetamine, and methamphetamine. This principal component of uptake has the properties expected for a carrier-mediated transport system. The second component, which accounted for about 10-30% of the DA uptake at 2 microM DA, was not saturable, and was independent of external Na, Cl, and blockers of the carrier-mediated system. The saturable, Na-dependent component had an apparent Km(DA) of about 0.5 microM. The dependence of DA uptake on external Na was sigmoid [Hill coefficient = 2; Ka(Na) = 45 mM] whereas the dependence on Cl was best described by a rectangular hyperbola [Ka(Cl) = 15 mM]. Depolarizing conditions (elevated external K) reduced the rate of DA influx. The data are consistent with a carrier-mediated DA transport mechanism in which each DA molecule entering the nerve terminal via the carrier is accompanied by two or more Na ions and one Cl ion in a rheogenic process carrying one or more net positive charges into the cell. Net, concentrative accumulation of DA inside nerve terminals may be accomplished by utilizing the Na electrochemical gradient to drive DA against its electrochemical gradient via this carrier system.

Dopamine uptake inhibitory capacities of β-carboline and 3,4-dihydro-β-carboline analogs of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) oxidation products

Potentially endogenous β-carboline and 3,4-dihydro-β-carboline alkaloidal compounds were compared, generally as 2-methylated (quaternary) and normethylated pairs, to the neurotoxin, 1-methyl-4-phenyl-dihydropyridinium ion (MPP +), with respect to inhibition of [ 3H]dopamine uptake into rat striatal synaptosomal preparations. Although less potent than MPP +, several compounds displayed IC 50 values for inhibition in the moderate range (12–24 μM). Notably, quaternization generally did not improve inhibitory potency, and the 3,4-dihydro-compounds often were more effective inhibitors than their heteroaromatic analogs. The partially competetive nature of inhibition by one of the more effective pairs, 2-methyl-harmine and harmine, was consistent with uptake of the beta-carbolines by the synaptosomal dopamine uptake system, as was the fact that the accumulation of 2-[ 14C]methyl-harmine was significantly reduced by low Na + media and by nomifensine, a potent inhibitor of the dopamine transporter. When viewed with reports that certain 2-methyl-β-carbolines show MPP +-like toxicity in vitro and in vivo, these studies support the proposal that a mammalian β-carbolinium compound may be taken up by nigrostriatal neurons and provoke the neuronal degeneration underlying Parkinson's disease.

Structure-neurotoxicity trends of analogues of 1-methyl-4-phenylpyridinium (MPP +), the cytotoxic metabolite of the dopaminergic neurotoxin MPTP

The dopaminergic neurotoxicity of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) derives from its metabolism to 1-methyl-4-phenylpyridinium cation (MPP +), which is then selectively accumulated in dopaminergic neurons. In an effort to assess the structural requirements governing MPP + cytotoxicity, we evaluated dopaminergic toxicity of MPP + analogues 3 weeks after their microinfusion into rat substantia nigra. We also evaluated the substrate suitability of MPP + analogues for high-affinity dopamine uptake in striatal synaptosomes by measuring their ability to induce specific dopamine release. The intranigral neurotoxicity of MPP + analogues in vivo correlates mainly with their in vitro inhibitory activity on mitochondrial respiration, consistent with a compromise in cellular energy production as the principal mechanism of MPTP-induced cell death. This study extends the structure-neurotoxicity data base beyond that obtainable using MPTP analogues, since many of these are not metabolized to pyridinium compounds. Such information is crucial to assess which possible endogenous or exogenous compounds may exert MPTP/MPP +-like toxicity.

The dopamine uptake inhibitor GBR 12909: selectivity and molecular mechanism of action

The neurochemical profile of GBR 12909 (1-(2-(bis(4-fluorphenyl)-methoxy)-ethyl)-4-(3-phenyl-propyl)piperazine) was investigated. GBR 12909 was a potent and selective inhibitor of synaptosomal dopamine uptake (K I = 1 nM), with a 20-fold lower affinity for the histamine H 1-receptor and a more than 100-fold lower affinity for the noradrenaline and 5-HT uptake carriers, the dopamine D-1, D-2, 5-HT 1A and α 1-receptors and voltage-dependent sodium channels. GBR 12909 (3 μM) was without effect on muscarinic, α 2, β 1+2, γ-aminobutyric acid (GABA) and benzodiazepine receptors, and on choline and GABA uptake carriers. The selective dopamine uptake inhibitory profile of GBR 12909 was confirmed by ex vivo uptake experiments. GBR 12909 inhibited dopamine uptake in vitro in a competitive manner as did cocaine and methylphenidate. [ 3H]GBR 12935 binding was competitively inhibited by GBR 12909 as well as by dopamine, cocaine and methylphenidate. Off-rate analysis of the [ 3H]GBR 12935 binding excluded the presence of allosteric binding sites on the dopamine carrier complex. Instead, the data favored the notion that GBR 12909 inhibits dopamine uptake by binding to the dopamine binding site on the carrier protein itself, thereby blocking the carrier process. In conclusion, GBR 12909 is a highly selective inhibitor of dopamine uptake, both in vivo and in vitro. At the moment GBR 12909 is the only compound with this neurochemical profile. The selective effect of GBR 12909 on this neuronal system makes it an interesting experimental tool and a potential antidepressant agent.