Rate of binding of various inhibitors at the dopamine transporter in vivo

The rate of entry of drugs into brain is thought to be a factor in their abuse liability. In this investigation, we have examined the rate of entry and binding at dopamine transporters in mouse striatum for a variety of dopamine transporter inhibitors. The method utilized was based on measuring the displacement of³H-WIN 35,428 from striatal dopamine transporter sites in vivo at different times. Eleven cocaine analogs (RTI-31, RTI-32, RTI-51, RTI-55, RTI-113, RTI-114, RTI-117, RTI-120, RTI-121, WIN 35,065-2, and WIN 35,428) as well as other dopamine uptake site blockers (bupropion, nomifensine, and methylphenidate) were compared with (–) cocaine for their rates of displacement of³H-WIN 35,428 binding in vivo. The drugs that displayed the fastest occupancy rates were bupropion, (–) cocaine, nomifensine, and methylphenidate. RTI-51, RTI-121, RTI-114, RTI-117, RTI-120, RTI-32, RTI-55, and RTI-113, showed intermediate rates, whereas RTI-31, WIN 35,065-2, and WIN 35,428 exhibited the slowest rates of displacement. While many of the cocaine analogs have proven to be behaviorally and pharmacologically more potent than (–) cocaine, their rates of entry and binding site occupancy were slower than that for (–) cocaine. Earliest times of transporter occupancy by the different drugs were correlated (although weakly) with their degree of lipophilicity (r=0.59;P<0.02). Kinetic effects and metabolism of the compounds could complicate the interpretations of these data. There was no obvious correlation between rate of occupancy in this animal model and abuse liability in humans, which is consistent with the notion that other factors are critical as well.     

Spermine interacts with cocaine binding sites on dopamine transporters

These studies were designed to assess the potential interaction of the polyamine spermine with cocaine binding to dopamine and serotonin transporters. The results of the experiments presented here indicate that spermine inhibits binding of the cocaine congener [³H] CFT to striatal synaptosomal membranes. Further, although [³H] CFT is known to interact with both dopamine and serotonin transporters, our results indicate that the observed inhibition of [³H] CFT binding is likely to reflect a specific inhibition of binding to dopamine transporters. Spermine significantly inhibited the binding of both [³H] CFT and [³H] mazindol to dopamine transporters, while it had no apparent effects on the binding of the potent serotonin uptake inhibitor [³H] paroxetine. Finally, saturation experiments show that the inhibition of ligand binding to the cocaine binding site on dopamine transporters appears not to be due to a modification of ligand affinity for the transporter, but to a decrease in the apparent density of ligand binding sites. The results of these experiments indicate that endogenously produced polyamines can alter cocaine binding to the dopamine transporter. The results are discussed in terms of possible impact on novel approaches for pharmacologically manipulating cocaine reinforcement and craving in clinical treatments for cocaine addiction, as well as for emergency treatment of cocaine overdose.     

Translocation of dopamine and binding of WIN 35,428 measured under identical conditions in cells expressing the cloned human dopamine transporter

Translocation of [³H]dopamine and binding of [³H]WIN 35,428 were measured in intact C6 glioma cells expressing the cloned human dopamine transporter (hDAT) under identical conditions of assay buffer (phosphate-Krebs) and temperature (25°C) with uptake at initial velocity and binding at equilibrium. In the intact cells, [³H]dopamine uptake was a one-component process; in contrast, [³H]WIN 35,428 binding included both a high-affinity component, inhibitable by micromolar concentrations of dopamine, and a low-affinity component only partially inhibited by millimolar concentrations of dopamine. Binding (high-affinity) over uptake Ki ratios were on the average 2.3 for the inhibitors WIN 35,428, cocaine, GBR 12909, and BTCP. The potency of dopamine in inhibiting its own translocation was close to that in inhibiting [³H]WIN 35,428 binding consonant with a more rapid reorientation step of the DAT in the C6-hDAT system than in rat striatal synaptosomes. The similarity in turnover values of the DAT estimated in the current experiments with the C6-hDAT system and in our previous study on rat striatal synaptosomes, performed under comparable conditions, suggest that all DAT's inserted into the C6 cell membrane are functionally active.     

The promiscuity of the dopamine transporter: implications for the kinetic analysis of [3H]serotonin uptake in rat hippocampal and striatal synaptosomes

Evidence indicates that monoaminergic neurotransmitter transporters are promiscuous, transporting substrates other than their cognate neurotransmitters. For example, serotonin is transported by the dopamine transporter (DAT) under conditions in which serotonin transporter (SERT) activity is eliminated (e.g., pharmacological inhibition). We performed a kinetic analysis of [³H]serotonin uptake in rat striatal synaptosomes (expressing DAT and SERT) and hippocampal synaptosomes (expressing SERT, but not DAT). Nonspecific [³H]serotonin uptake was defined as the amount of uptake remaining in the presence of fluoxetine (10 μM) or paroxetine (0.05 μM). In hippocampal synaptosomes, K_m and V_max values for [³H]serotonin uptake did not differ whether fluoxetine or paroxetine was used to define nonspecific uptake. However, in striatal synaptosomes, both K_m and V_max values for [³H]serotonin uptake were greater when fluoxetine, rather than paroxetine, was used to define nonspecific uptake. These data suggest that, at the concentrations employed, fluoxetine inhibits serotonin uptake at both DAT and SERT, whereas paroxetine only inhibits serotonin uptake at SERT. Thus, when DAT is inhibited by GBR 12909, kinetic parameters for serotonin uptake via SERT in striatum are not different from those obtained in hippocampus. These findings have important implications regarding the analysis of monoaminergic reuptake in brain regions exhibiting heterogeneous transporter expression.     

Characterization of conditioned place preference to cocaine in congenic dopamine transporter knockout female mice

Rationale The dopamine transporter (DAT) is thought to play a major role in the rewarding effects of cocaine. Therefore, it is surprising that cocaine reveals conditioned effects in DAT knockout (DAT-KO) mice.Objectives To examine these findings further, we obtained complete dose–effect curves for DAT-KO and DAT wild-type (DAT-WT) mice in a cocaine conditioned place preference (CPP) procedure.Methods Congenic C57BL6 background female DAT-KO and DAT-WT mice were conditioned in a three-compartment place preference apparatus. Conditioning consisted of three 30-min sessions with cocaine (2.5, 5.0, 10.0, 20.0, or 40.0 mg/kg) and three 30-min sessions with saline. The distribution of time in each choice compartment was determined after each pair of conditioning sessions (one cocaine and one saline session).Results DAT-WT mice revealed CPP over a wide range of cocaine doses (5.0–40 mg/kg), whereas DAT-KO mice revealed CPP over a more restricted range of doses, with consistent CPP only occurring with 10 mg/kg of cocaine.Conclusions CPP for cocaine develops in both DAT-KO and DAT-WT mice; however, the dose range at which CPP develops is much more restricted in DAT-KO mice than in DAT-WT mice. These observations corroborate the significant role of DAT inhibition in cocaines conditioned effects.     

The dopamine transporter: role in neurotoxicity and human disease

The dopamine transporter (DAT) is a plasma membrane transport protein expressed exclusively within a small subset of CNS neurons. It plays a crucial role in controlling dopamine-mediated neurotransmission and a number of associated behaviors. This review focuses on recent data elucidating the role of the dopamine transporter in neurotoxicity and a number of CNS disorders, including Parkinson disease, drug abuse, and attention deficit hyperactivity disorder (ADHD).     

Dopamine transporter binding without cocaine-like behavioral effects: synthesis and evaluation of benztropine analogs alone and in combination with cocaine in rodents

Rationale: Previous SAR studies demonstrated that small halogen substitutions on the diphenylether system of benztropine (BZT), such as a para-Cl group, retained high affinity at the cocaine binding site on the dopamine transporter. Despite this high affinity, the compounds generally had behavioral effects different from those of cocaine. However, compounds with meta-Cl substitutions had effects more similar to those of cocaine. Objectives: A series of phenyl-ring analogs of benztropine (BZT) substituted with 3′-, 4′-, 3′,4′′- and 4′,4′′-position Cl-groups were synthesized and their pharmacology was evaluated in order to assess more fully the contributions to pharmacological activity of substituents in these positions. Methods: Compounds were synthesized and their pharmacological activity was assessed by examining radioligand binding and behavioral techniques. Results: All of the compounds displaced [³H]WIN 35,428 binding with affinities ranging from 20 to 32.5 nM. Affinities at norepinephrine ([³H]nisoxetine) and serotonin ([³H]citalopram) transporters, respectively, ranged from 259 to 5120 and 451 to 2980 nM. Each of the compounds also inhibited [³H]pirenzepine binding to muscarinic M₁ receptors, with affinities ranging from 0.98 to 47.9 nM. Cocaine and the BZT analogs produced dose-related increases in locomotor activity in mice. However, maximal effects of the BZT analogs were uniformly less than those produced by cocaine, and were obtained 2–3 h after injection compared to the relatively rapid onset (within 30 min) of cocaine effects. In rats trained to discriminate IP saline from 29 μmol/kg cocaine (10 mg/kg), cocaine produced a dose-related increase in responding on the cocaine lever, reaching 100% at the training dose; however, none of the BZT analogs fully substituted for cocaine, with maximum cocaine responding from 20 to 69%. Despite their reduced efficacy compared to cocaine in cocaine discrimination, none of the analogs antagonized the effects of cocaine. As has been reported previously for 4′-Cl-BZT, the cocaine discriminative-stimulus effects were shifted leftward by co-administration of the present BZT analogs. Conclusions: The present results indicate that although the BZT analogs bind with relatively high affinity and selectivity at the dopamine transporter, their behavioral profile is distinct from that of cocaine. The present results suggest that analogs of BZT may be useful as treatments for cocaine abuse in situations in which an agonist treatment is indicated. These compounds possess features such as reduced efficacy compared to cocaine and a long duration of action that may render them particularly useful leads for the development of therapeutics for cocaine abusers. Electronic Publication     

多巴胺转运体基因多态性与精神分裂症的相关研究

精神分裂症是在精神病中最具代表性和最常见的一组症状群。研究显示遗传因素对于精神分裂症的发病起主要作用。多巴胺转运体生化研究结果和临床实验的证据支持多巴胺转运体在精神分裂症病理机制中起重要作用,但具体机制尚不清楚。本文就多巴胺转运体基因多态性与精神分裂症的相关研究及其临床意义做一综述。     

The substituted aspartate analogue L-beta-threo-benzyl-aspartate preferentially inhibits the neuronal excitatory amino acid transporter EAAT3

The excitatory amino acid transporters (EAATs) play key roles in the regulation of CNS L-glutamate, especially related to synthesis, signal termination, synaptic spillover, and excitotoxic protection. Inhibitors available to delineate EAAT pharmacology and function are essentially limited to those that non-selectively block all EAATs or those that exhibit a substantial preference for EAAT2. Thus, it is difficult to selectively study the other subtypes, particularly EAAT1 and EAAT3. Structure activity studies on a series of beta-substituted aspartate analogues identify L-beta-benzyl-aspartate (L-beta-BA) as among the first blockers that potently and preferentially inhibits the neuronal EAAT3 subtype. Kinetic analysis of D-[(3)H]aspartate uptake into C17.2 cells expressing the hEAATs demonstrate that L-beta-threo-BA is the more potent diastereomer, acts competitively, and exhibits a 10-fold preference for EAAT3 compared to EAAT1 and EAAT2. Electrophysiological recordings of EAAT-mediated currents in Xenopus oocytes identify L-beta-BA as a non-substrate inhibitor. Analyzing L-beta-threo-BA within the context of a novel EAAT2 pharmacophore model suggests: (1) a highly conserved positioning of the electrostatic carboxyl and amino groups; (2) nearby regions that accommodate select structural modifications (cyclopropyl rings, methyl groups, oxygen atoms); and (3) a unique region L-beta-threo-BA occupied by the benzyl moieties of L-TBOA, L-beta-threo-BA and related analogues. It is plausible that the preference of L-beta-threo-BA and L-TBOA for EAAT3 and EAAT2, respectively, could reside in the latter two pharmacophore regions.     

Low and high cocaine locomotor responding male Sprague-Dawley rats differ in rapid cocaine-induced regulation of striatal dopamine transporter function

Adult outbred Sprague-Dawley rats can be classified as either low or high cocaine responders (LCRs or HCRs, respectively). Importantly, LCRs and HCRs are distinguished by their differential responsiveness to acute cocaine-induced (but not baseline) locomotor activity, inhibition of the dopamine transporter (DAT) and resulting extracellular DA (HCR > LCR), as well as by repeated cocaine-induced locomotor sensitization and measures of cocaine's rewarding and reinforcing effects (LCR > HCR). Curiously, 30 min after acute cocaine HCRs exhibit greater DAT-mediated [³H]DA uptake into striatal synaptosomes than LCRs. To investigate this finding further, we measured locomotor activity, striatal [³H]DA uptake kinetics and DAT cell surface expression in LCRs and HCRs over an extended period (25-180 min) after a single relatively low-dose of cocaine (10 mg/kg, i.p.). HCRs exhibited the “predicted” locomotor response: a marked initial activation that returned to baseline by 120 min post-injection. While LCRs exhibited a >50% lower maximal locomotor response, this increase was sustained, lasting ∼33% longer than in HCRs. At 25 min post-cocaine, maximal velocity (V_max) of [³H]DA uptake was significantly higher by 25% in HCRs than LCRs, with no difference in affinity (K_m). Despite the DAT V_max difference, however, DAT surface expression did not differ between LCRs and HCRs. There was a similar trend (HCR > LCR) for DAT V_max at 40 min, but not at 150 or 180 min. These findings suggest that, compared to LCRs, HCRs have an enhanced ability to rapidly up-regulate DAT function in response to acute cocaine, which may contribute to their more “normal” cocaine-induced locomotor activation.     

Relationships among dopamine transporter affinities and cocaine-like discriminative-stimulus effects

Rationale: The discriminative-stimulus effects of cocaine have been reported to be mediated by indirect agonist actions initiated by the blockade of dopamine uptake, and the potencies of drugs that have discriminative-stimulus effects like cocaine are directly related to their dopamine transporter binding affinities. The binding to the dopamine transporter by cocaine and many of its analogs has been reported to fit better using a two-site model than a one-site model. Objectives: The present study examined the relationship among binding affinities of dopamine uptake inhibitors at these two sites and their potencies to produce discriminative-stimulus effects. Methods: The inhibition constants (K _i values) were derived for unlabeled dopamine uptake inhibitors for displacement of [³H]WIN 35,428 from rat caudate putamen membranes. These K _i values were related to the ED₅₀ values obtained in rats trained to discriminate 10 mg/kg cocaine from saline injections under a fixed-ratio 20 schedule of food reinforcement. Results: Among the dopamine uptake inhibitors studied, the binding data for eight compounds (WIN 35,428, nomifensine, WIN 35,981, WIN 35,065-2, methylphenidate, cocaine, cocaethylene, and bupropion) were better fit by a two-site model than a one-site model. The data for the remaining eleven compounds (RTI-31, RTI-55, RTI-121, RTI-32, LU19-005, BTCP, GBR12909, GBR12935, mazindol, LU17-133, and EXP561) were better fit by a one-site model. Of the drugs that were fit best by a two-site model, there was a higher correlation among the K _i values for the high-affinity site and the ED₅₀ values (R²=0.655; P=0.015) than there was for the low-affinity site (R²=0.543; P=0.037). Of the remaining drugs, there was a high correlation among the K _i values and the ED₅₀ values for the discriminative-stimulus effects (R²=0.523; P=0.012). Conclusions: These data suggest that the discriminative-stimulus effects of cocaine are more closely related to actions mediated by high-affinity binding to the dopamine transporter than they are to actions mediated by the low-affinity site. The further assessment of the respective contributions of high- and low-affinity binding to the behavioral effects of cocaine will be greatly enhanced with the development of pharmacological tools that have a high degree of selectivity for one of these components. Received: 24 May 1999 / Final version: 30 July 1999     

Antipsychotic-induced extrapyramidal symptoms in patients with schizophrenia: associations with dopamine and serotonin receptor and transporter polymorphisms

Background Little is known about the influence of polymorphisms of the dopamine and serotonin system on the risk for extrapyramidal symptoms (EPS) during treatment with antipsychotic drugs. Methods Of 119 subjects with schizophrenia treated with antipsychotics, 63 had current or previous EPS (acute dystonia, parkinsonism, tardive dyskinesia), and 56 had no such symptoms. All subjects were genotyped for a total of eight dopamine and serotonin receptor and transporter polymorphisms: the Taq1A polymorphism of the dopamine D₂ receptor (DRD2) gene, the Msc1 polymorphism of the dopamine D₃ receptor (DRD3) gene, the variable number of tandem repeat (VNTR) polymorphism of the dopamine transporter (DAT1) gene, four polymorphisms (102T/C, His452Tyr, 516 C/T, and Thr25Asn) of the serotonin 5-HT_2A receptor (5HTR2A) gene, and the 5HTTLPR polymorphism of the serotonin transporter (5HTT) gene. Results The frequency of the A1 allele of the DRD2 Taq1A polymorphism was significantly higher in the EPS group than in the control group [16% vs. 7%, P = 0.040; odds ratio (OR) 2.4; 95% confidence interval (CI) 1.1–5.7]. Also, the 9 repeat allele of the DAT1 VNTR polymorphism was significantly more common in the EPS group (42% vs. 28%, P = 0.030; OR 1.9; 95% CI 1.1–3.3). Being a carrier of both DRD2 Taq1A A1 and DAT1 VNTR 9 repeat alleles was also significantly associated with the occurrence of EPS (19% vs. 6%, P = 0.040; OR 4.0; 95% CI 1.05–15.2) No significant differences in allele frequencies were found for the other polymorphisms. Conclusion Presence of the Taq1A A1 allele of the DRD2 and the 9 repeat allele of the DAT1 VNTR polymorphisms might be risk factors for EPS caused by antipsychotic drugs.     

D-161, a novel pyran-based triple monoamine transporter blocker: behavioral pharmacological evidence for antidepressant-like action

Pinocembrin is the most abundant flavonoids in propolis, and has been proven to have antioxidant, antibacterial and anti-inflammatory property. To assess the protective effects of pinocembrin on neurons, SH-SY5Y neuronal cells were pretreated with pinocembrin for 2 h followed by co-treatment with glutamate (2 mM) for 12 h. Cell viability was determined by(3,4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide assay, and apoptosis was confirmed by cell morphology, capillary zone electrophoresis and flow cytometry assay. Cell morphology was evaluated with Hoechst33258/PI dye. Treatment with pinocembrin (10(-5), 10(-6), 10(-7) mol/l) increased cell viability dose-dependently, inhibited LDH release and attenuated apoptosis. Intracellular free [Ca(2+)] was increased after glutamate exposure, and this increase was attenuated in cells treated with pinocembrin. bax mRNA expression increased remarkably following glutamate exposure and pinocembrin treatment manifested a reduction effect. bcl-2 mRNA expression changes were not detected in groups with or without pinocembrin. Western blotting results indicated that pinocembrin treatment reduced the expression of Bax and had no effect on Bcl-2, thus decreased the Bax-Bcl-2 ratio, which is in consistent with the gene expression result. Pinocembrin could also down-regulate the expression of p53 protein, and inhibit the release of cytochrome c from mitochondria to cytosol. Thus we conclude that pinocembrin exerts its neuroprotective effects in glutamate injury model partly by inhibiting p53 expression, thus Bax-Bcl-2 ratio, and the release of cytochrome c.     

Pyrethroid pesticide-induced alterations in dopamine transporter function

Parkinson's disease (PD) is a progressive neurodegenerative disease affecting the nigrostriatal dopaminergic pathway. Several epidemiological studies have demonstrated an association between pesticide exposure and the incidence of PD. Studies from our laboratory and others have demonstrated that certain pesticides increase levels of the dopamine transporter (DAT), an integral component of dopaminergic neurotransmission and a gateway for dopaminergic neurotoxins. Here, we report that repeated exposure (3 injections over 2 weeks) of mice to two commonly used pyrethroid pesticides, deltamethrin (3 mg/kg) and permethrin (0.8 mg/kg), increases DAT-mediated dopamine uptake by 31 and 28%, respectively. Using cells stably expressing DAT, we determined that exposure (10 min) to deltamethrin and permethrin (1 nM-100 microM) had no effect on DAT-mediated dopamine uptake. Extending exposures to both pesticides for 30 min (10 microM) or 24 h (1, 5, and 10 microM) resulted in significant decrease in dopamine uptake. This reduction was not the result of competitive inhibition, loss of DAT protein, or cytotoxicity. However, there was an increase in DNA fragmentation, an index of apoptosis, in cells exhibiting reduced uptake at 30 min and 24 h. These data suggest that up-regulation of DAT by in vivo pyrethroid exposure is an indirect effect and that longer-term exposure of cells results in apoptosis. Since DAT can greatly affect the vulnerability of dopamine neurons to neurotoxicants, up-regulation of DAT by deltamethrin and permethrin may increase the susceptibility of dopamine neurons to toxic insult, which may provide insight into the association between pesticide exposure and PD.     

Low dopamine transporter occupancy by methylphenidate as a possible reason for reduced treatment effectiveness in ADHD patients with cocaine dependence

Methylphenidate (MPH) occupies brain striatal dopamine transporters (DATs) and is an effective treatment for attention deficit hyperactivity disorder (ADHD). However, patients with ADHD and comorbid cocaine dependence do not benefit significantly from treatment with MPH. To better understand the neurobiology of this phenomenon, we examined DAT availability and the effects of MPH treatment on DAT occupancy in ADHD patients with and without cocaine dependence. ADHD patients without a comorbid substance use disorder (N=16) and ADHD patients with comorbid cocaine dependence (N=8) were imaged at baseline and after two weeks MPH treatment using single photon emission computed tomography (SPECT) with the DAT tracer [¹²³I]FP-CIT. Changes in ADHD symptoms were measured with the ADHD symptom rating scale (ASRS). At baseline, we observed lower striatal DAT availability in ADHD patients with cocaine dependence. Following fixed MPH treatment, MPH occupied significantly less striatal DATs in cocaine-dependent than in non-cocaine dependent ADHD patients. There were no significant correlations between baseline DAT availability or DAT occupancy by MPH and ADHD symptom improvement. However, we did find significant correlations between DAT occupancy by MPH and decreases in impulsivity scores and years of cocaine use. These preliminary findings suggest that low DAT occupancy is not the reason why ADHD patients with cocaine dependence do not benefit from MPH treatment. It also suggests that higher dosages of MPH in these patients are probably not the solution and that medications directed at other pharmacological targets should be considered in these comorbid ADHD patients.This trial is registered at the Dutch Trial Register, www.trialregister.nl, under Trial ID number NTR3127.     

Hypolocomotor effects of acute and daily d-amphetamine in mice lacking the dopamine transporter

RATIONALE: Mice lacking the dopamine transporter (DAT(-/-)) exhibit high extracellular dopamine levels and marked hyperactivity. This hyperlocomotion is paradoxically decreased by acute administration of amphetamine-like psychostimulants, an effect that has been previously related to the activation of serotonergic neurotransmission. OBJECTIVES: The goal of the present study was to investigate the effects of acute and daily administration of d-amphetamine on the locomotor activity of DAT(-/-) mice and examine the development of behavioral sensitization. In addition, we tested the implication of the serotonin system in the observed effects. METHODS: DAT(+/+), DAT(+/-), and DAT(-/-) mice were injected with acute amphetamine (0, 0.3, 1, 3, or 10 mg/kg, SC), repeated amphetamine (1 mg/kg for 8 days, SC), or with the serotonin reuptake inhibitor fluoxetine (0, 5, 10, or 20 mg/kg, SC) and their locomotor activity was evaluated. Moreover, the expression of the serotonin transporter and 5-HT(1A) receptors in the brain of DAT(-/-) mice was studied using autoradiography. RESULTS: Acute and repeated d-amphetamine injection (1 mg/kg) induced an hypolocomotor response in DAT(-/-) and DAT(+/-) mice, but only DAT(+/-) mice developed locomotor sensitization to the drug. Acute treatment with fluoxetine decreased locomotion in DAT(-/-) mice in a dose-dependent manner. The common hypolocomotor effect induced by d-amphetamine and fluoxetine in DAT(-/-) mice suggests an action on the serotonin transporter. However, autoradiography of the serotonin transporter and 5-HT(1A) receptors showed normal density and distribution in the brain, suggesting no compensatory effects due to the deletion of the DAT. CONCLUSIONS: These findings indicate that partial or total DAT gene deletion result in decreased locomotion in response to d-amphetamine and modify behavioral sensitization depending on the proportion of DAT removed, suggesting that inhibition of the DAT is necessary for the development of sensitization to psychostimulant drugs.     

Lanthanides inhibit the human noradrenaline, 5-hydroxytryptamine and dopamine transporters

Transporters for the monoamine neurotransmitters, including noradrenaline, 5-hydroxytryptamine [5-HT] and dopamine, have twelve transmembrane spanning regions and cotransport Na⁺ and Cl^– ions. Another family of Na⁺-dependent transporters is that containing the Na⁺/glucose and Na⁺/proline cotransporters that are found in the epithelial cells of renal and intestinal brush border membranes. It has been shown that various trivalent lanthanides can substitute for Na⁺ for transport of glucose and proline. The aim of this study was to determine the effects of lanthanides on the activities of the human noradrenaline, 5-HT and dopamine transporters. Cultured cells were incubated for 2min with 10nM ³H-noradrenaline (SK-N-SH-SY5Y human neuroblastoma cells), ³H-5-HT (JAR human placental choriocarcinoma cells) or ³H-dopamine (COS-7 cells transfected with the cDNA of the human dopamine transporter). Specific amine uptake was determined as the difference between accumulation of the amine in the cells in the absence and presence of a corresponding uptake inhibitor. Under both isotonic (150mM NaCl or LiCl or 90mM lanthanide salt) and hypertonic (150mM NaCl + 100mM LiCl, 250mM LiCl or 150mM lanthanide salt) conditions, replacement of Na⁺ by Li⁺, La³⁺, Eu³⁺ or Sm³⁺ abolished the specific uptake of noradrenaline in SK-N-SH-SY5Y cells and replacement of Na⁺ by Li⁺ or Eu³⁺ decreased the specific uptake of 5-HT in JAR cells by 94–100% and that of dopamine in transfected COS-7 cells by 95–99%. The direct effects of Eu³⁺ (with Na⁺ present) on the human noradrenaline transporter in SK-N-SH-SY5Y cells were also examined. Eu³⁺ inhibited noradrenaline uptake into the cells (IC₅₀ 2.6mM) and nisoxetine binding to crude membranes of SK-N-SH-SY5Y cells (IC₅₀ 4.7mM) with similar potencies. Further experiments showed that 4.5mM EuCl₃ in the presence of 150mM Na⁺ caused a 3.5-fold increase in the K_m of noradrenaline and no change in the maximal rate of noradrenaline uptake. EuCl₃ (4.5mM) also caused a pronounced inhibition of the Na⁺-dependent stimulation of noradrenaline uptake by SK-N-SH-SY5Y cells. It can be concluded from these data that, in contrast with the Na⁺/glucose and Na⁺/proline cotransporters, the lanthanides cannot substitute for Na⁺ in the transport of substrates by the monoamine neurotransmitter transporters and that the lanthanides inhibit the latter transporters by interacting with sites of the transporters involved in amine and Na⁺ binding. Received: 15 July 1996 / Accepted: 28 January 1997     

Choline transporter hemizygosity results in diminished basal extracellular dopamine levels in nucleus accumbens and blunts dopamine elevations following cocaine or nicotine

Dopamine (DA) signaling in the central nervous system mediates the addictive capacities of multiple commonly abused substances, including cocaine, amphetamine, heroin and nicotine. The firing of DA neurons residing in the ventral tegmental area (VTA), and the release of DA by the projections of these neurons in the nucleus accumbens (NAc), is under tight control by cholinergic signaling mediated by nicotinic acetylcholine (ACh) receptors (nAChRs). The capacity for cholinergic signaling is dictated by the availability and activity of the presynaptic, high-affinity, choline transporter (CHT, SLC5A7) that acquires choline in an activity-dependent matter to sustain ACh synthesis. Here, we present evidence that a constitutive loss of CHT expression, mediated by genetic elimination of one copy of the Slc5a7 gene in mice (CHT+/−), leads to a significant reduction in basal extracellular DA levels in the NAc, as measured by in vivo microdialysis. Moreover, CHT heterozygosity results in blunted DA elevations following systemic nicotine or cocaine administration. These findings reinforce a critical role of ACh signaling capacity in both tonic and drug-modulated DA signaling and argue that genetically imposed reductions in CHT that lead to diminished DA signaling may lead to poor responses to reinforcing stimuli, possibly contributing to disorders linked to perturbed cholinergic signaling including depression and attention-deficit hyperactivity disorder (ADHD).     

Methamphetamine-induced locomotor activity and sensitization in dopamine transporter and vesicular monoamine transporter 2 double mutant mice

Rationale The dopamine transporter (DAT) and the vesicular monoamine transporter 2 (VMAT2) play pivotal roles in the action of methamphetamine (MAP), including acute locomotor effects and behavioral sensitization. However, the relative impact of heterozygous DAT and VMAT2 knockouts (KOs) on the behavioral effects of MAP remains unknown. Objectives To evaluate the roles of DAT and VMAT2 in MAP-induced locomotor behavior, we examined locomotor activity and sensitization in heterozygous DAT KO (DAT+/−), heterozygous VMAT2 KO (VMAT2+/−), double heterozygous DAT/VMAT2 KO (DAT+/−VMAT2+/−), and wild-type (WT) mice. Results Acute 1 mg/kg MAP injection induced significant locomotor increases in WT and VMAT2+/− mice but not in DAT+/− and DAT+/−VMAT2+/− mice. Acute 2 mg/kg MAP significantly increased locomotor activity in all genotypes. Repeated 1 mg/kg MAP injections revealed a delayed and attenuated development of sensitization in DAT+/− and DAT+/−VMAT2+/− mice compared to WT mice and delayed development in VMAT2+/− mice. In repeated 2 mg/kg MAP injections, DAT+/− and DAT+/−VMAT2+/− mice showed delayed but not attenuated development of sensitization, while there was no difference in the onset of sensitization between VMAT2+/− and WT mice. In DAT+/−VMAT2+/− mice, all of MAP-induced behavioral responses were similar to those in DAT+/− but not VMAT2+/− mice. Conclusions Heterozygous deletion of DAT attenuates the locomotor effects of MAP and may play larger role in behavioral responses to MAP compared to heterozygous deletion of VMAT2.