Maarten Reith, Ph.D.

Ibogaine and the inhibition of acetylcholinesterase
Alper, Kenneth; Reith, Maarten E A; Sershen, Henry
2012 Feb 15;139(3):879-882, Journal of ethnopharmacology
ETHNOPHARMACOLOGICAL RELEVANCE: Ibogaine is a psychoactive monoterpine indole alkaloid extracted from the root bark of Tabernanthe iboga Baill. that is used globally in medical and nonmedical settings to treat drug and alcohol addiction, and is of interest as an ethnopharmacological prototype for experimental investigation and pharmaceutical development. The question of whether ibogaine inhibits acetylcholinesterase (AChE) is of pharmacological and toxicological significance. MATERIALS AND METHODS: AChE activity was evaluated utilizing reaction with Ellman's reagent with physostigmine as a control. RESULTS: Ibogaine inhibited AChE with an IC(50) of 520+/-40muM. CONCLUSIONS: Ibogaine's inhibition of AChE is physiologically negligible, and does not appear to account for observations of functional effects in animals and humans that might otherwise suggest the possible involvement of pathways linked to muscarinic acetylcholine transmission
— id: 150565, year: 2012, vol: 139, page: 879, stat: Journal Article,

Correction: the atypical stimulant and nootropic modafinil interacts with the dopamine transporter in a different manner than classical cocaine-like inhibitors
Schmitt, Kyle C; Reith, Maarten E A
2012 ;7(1):19-27, PLoS One
[This corrects the article on p. e25790 in vol. 6.].
— id: 155599, year: 2012, vol: 7, page: 19, stat: Journal Article,

Characterization of [(3)H]CFT binding to the norepinephrine transporter suggests that binding of CFT and nisoxetine is not mutually exclusive
Zhen, Juan; Ali, Solav; Dutta, Aloke K; Reith, Maarten E A
2012 Jan 15;203(1):19-27, Journal of neuroscience methods
The norepinephrine transporter (NET) is an important target for a wide variety of antidepressants and psychostimulants. Despite its prominence as a drug target, there is only one radioligand in use for NET competitive binding assays, [(3)H]nisoxetine. However, traditional [(3)H]nisoxetine binding protocols often give an underestimation for the affinity of certain classes of NET ligands, particularly cocaine and other tropanes. Here, we explore the feasibility of using the phenyltropane [(3)H]CFT for labeling human NET (hNET) in heterologous cell-based binding studies. Assays were optimized for time and protein content and specific, one-site binding was observed. Potencies of tested NET ligands for inhibition of [(3)H]CFT binding to whole cells (at physiological [Na(+)] and 25 degrees C) were similar to potencies observed in the [(3)H]NE uptake assay. Inhibition constants (K(i)) for binding assays were highly correlated with uptake inhibition constants for all compounds tested (R(2)=0.99, p<0.0001). Cell-free membrane preparations did not display the same pharmacological profile. Under conditions routinely used for measuring [(3)H]nisoxetine binding to membrane preparations (4 degrees C for 3h, [Na(+)] at 295mM), the potency of nisoxetine and desipramine in inhibiting [(3)H]CFT binding became greater than that measured in a functional assay of [(3)H]NE uptake at physiological [Na(+)]. However, the opposite was true for CFT and cocaine. Interestingly, while investigating [(3)H]CFT as a potential NET radioligand, we uncovered evidence suggesting that CFT and nisoxetine are not mutually exclusive in binding to the NET. Dixon plots of the interaction between nisoxetine and CFT in inhibition of [(3)H]dopamine uptake by the NET indicate that the two compounds can simultaneously bind to the transporter
— id: 141695, year: 2012, vol: 203, page: 19, stat: Journal Article,

The novel trisubstituted pyran derivative D-142 has triple monoamine reuptake inhibitory activity and exerts potent antidepressant-like activity in rodents
Dutta, Aloke K; Gopishetty, Bhaskar; Gogoi, Sanjib; Ali, Solav; Zhen, Juan; Reith, Maarten
2011 Dec;671(1-3):39-44, European journal of pharmacology
Major depression disorder is a significant health problem with 10-20% of all adults suffering from this disease. The underlying causes of depression are still unclear and 15% of depressed patients are resistant to all known therapies. Monoamine therapies have so far been the most successful approach for treating depression. Triple monoamine reuptake inhibitors have recently been implicated in generation of potent antidepressant activity while possibly exhibiting a low side-effect profile in addition to treating anhedonia. The additional, previously under-appreciated involvement of dopaminergic systems in depression prompted our efforts to develop novel asymmetric trisubstituted and disubstituted pyran derivatives as triple reuptake inhibitors. One of the lead compounds, D-142, exhibited uptake inhibition (K(i)) values of 29.3 nM, 14.7 nM and 59.3 +/- 13.7 nM for norepinephrine, serotonin and dopamine transporters, respectively. Its affinity for serotonin transporter was comparable to fluoxetine, a well known SSRI. In the rat forced swimming test, compound D-142 exhibited potent antidepressant activity in the dose range tested (2.5, 5 and 10mg/kg) and was far more efficacious than the reference compound imipramine. In the mouse tail suspension test, compound D-142 reduced immobility in a dose (2.5, 5 and 10mg/kg) dependent manner, indicating a potent antidepressant effect. In locomotor activity tests, compound D-142 did not exhibit any stimulation in the same dose ranges. In the extended CNS receptors screening assay this molecule exhibited little or no non-specific interaction in the CNS, indicating high specificity for monoamine transporters. These results advance D-142 as a potential potent antidepressant.
— id: 155600, year: 2011, vol: 671, page: 39, stat: Journal Article,

Dopamine D/D agonists with potent iron chelation, antioxidant and neuroprotective properties: potential implication in symptomatic and neuroprotective treatment of Parkinson's disease
Gogoi, Sanjib; Antonio, Tamara; Rajagopalan, Subramanian; Reith, Maarten; Andersen, Julie; Dutta, Aloke K
2011 Jun;6(6):991-995, ChemMedChem
— id: 155601, year: 2011, vol: 6, page: 991, stat: Journal Article,

Further Structure-Activity Relationship Studies on 4-((((3S,6S)-6-Benzhydryltetrahydro-2H-pyran-3-yl)amino)methyl)phenol: Identification of Compounds with Triple Uptake Inhibitory Activity as Potential Antidepressant Agents
Gopishetty B; Hazeldine S; Santra S; Johnson M; Modi G; Ali S; Zhen J; Reith M; Dutta A
2011 Apr 28;54(8):2924-2932, Journal of medicinal chemistry
To investigate structural alterations of the lead triple uptake inhibitor molecule, disubstituted 4-((((3S,6S)-6-benzhydryltetrahydro-2H-pyran-3-yl)amino)methyl)phenol, we have carried out structure-activity relationship (SAR) studies to investigate the effect of alteration of aromatic substitutions and introduction of heterocyclic aromatic moieties on this molecular template. The novel compounds were tested for their affinities for the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in inhibiting the uptake of [(3)H]DA, [(3)H]5-HT, and [(3)H]NE, respectively. SAR results indicate dopamine norepinephrine reuptake inhibitory (DNRI) type activity in thiophene (10g) and pyrrole (10i) derivatives. On the other hand, 3-hydroxyphenyl derivative 10f and 4-methoxyphenyl derivative 10j exhibited a triple reuptake inhibitory (TUI) activity profile, as these molecules exhibited potent uptake inhibition for all the monoamine transporters (K(i) of 31.3, 40, 38.5 and K(i) of 15.9, 12.9, 29.3 for DAT, SERT, and NET for 10f and 10g, respectively). Compound 10f was further evaluated in the rat forced swim test to evaluate its potential antidepressant effect. The results show significant reduction of immobility by TUI 10f at 10 mg/kg dose, indicating potential antidepressant activity
— id: 129546, year: 2011, vol: 54, page: 2924, stat: Journal Article,

Interaction of novel hybrid compounds with the D3 dopamine receptor: Site-directed mutagenesis and homology modeling studies
Kortagere, Sandhya; Cheng, Shu-Yuan; Antonio, Tamara; Zhen, Juan; Reith, Maarten E A; Dutta, Aloke K
2011 Jan 1;81(1):157-163, Biochemical pharmacology
The dopamine D3 receptor has been implicated as a potential target for drug development in various complex psychiatric disorders including psychosis, drug dependence, and Parkinson's disease. In our overall goal to develop molecules with preferential affinity at D3 receptors, we undertook a hybrid drug development approach by combining a known dopamine agonist moiety with a substituted piperazine fragment. In the present study, three compounds produced this way with preferential D3 agonist activity, were tested at D3 receptors with mutations in the agonist binding pocket of three residues known to be important for agonist binding activity. At S192A and T369V, the hybrid agonist compounds produced an interaction profile in [(3)H]spiperone binding assays similar to that of the parent 5-OH-DPAT and 7-OH-DPAT molecules. The loss of affinity at the S192A mutant was most prominent for 5-OH-DPAT and its corresponding hybrid compound D237. D110N did not show any radioligand binding. Homology modeling indicated that 7-OH-DPAT-derived D315 uniquely shares H-bonding with Tyr365 which produced favorable interaction and no loss of H-bonding in the S192A mutant, suggesting that agonist activity may not be solely controlled by residues in the binding pocket
— id: 120619, year: 2011, vol: 81, page: 157, stat: Journal Article,

Clinical and molecular characterisation of hereditary dopamine transporter deficiency syndrome: an observational cohort and experimental study
Kurian, Manju A; Li, Yan; Zhen, Juan; Meyer, Esther; Hai, Nebula; Christen, Hans-Jurgen; Hoffmann, Georg F; Jardine, Philip; von Moers, Arpad; Mordekar, Santosh R; O'Callaghan, Finbar; Wassmer, Evangeline; Wraige, Elizabeth; Dietrich, Christa; Lewis, Timothy; Hyland, Keith; Heales, Simon Jr; Sanger, Terence; Gissen, Paul; Assmann, Birgit E; Reith, Maarten E A; Maher, Eamonn R
2011 Jan;10(1):54-62, Lancet neurology
BACKGROUND: dopamine transporter deficiency syndrome is the first identified parkinsonian disorder caused by genetic alterations of the dopamine transporter. We describe a cohort of children with mutations in the gene encoding the dopamine transporter (SLC6A3) with the aim to improve clinical and molecular characterisation, reduce diagnostic delay and misdiagnosis, and provide insights into the pathophysiological mechanisms. METHODS: 11 children with a biochemical profile suggestive of dopamine transporter deficiency syndrome were enrolled from seven paediatric neurology centres in the UK, Germany, and the USA from February, 2009, and studied until June, 2010. The syndrome was characterised by detailed clinical phenotyping, biochemical and neuroradiological studies, and SLC6A3 mutation analysis. Mutant constructs of human dopamine transporter were used for in-vitro functional analysis of dopamine uptake and cocaine-analogue binding. FINDINGS: children presented in infancy (median age 2.5 months, range 0.5-7) with either hyperkinesia (n=5), parkinsonism (n=4), or a mixed hyperkinetic and hypokinetic movement disorder (n=2). Seven children had been initially misdiagnosed with cerebral palsy. During childhood, patients developed severe parkinsonism-dystonia associated with an eye movement disorder and pyramidal tract features. All children had raised ratios of homovanillic acid to 5-hydroxyindoleacetic acid in cerebrospinal fluid, of range 5.0-13.2 (normal range 1.3-4.0). Homozygous or compound heterozygous SLC6A3 mutations were detected in all cases. Loss of function in all missense variants was recorded from in-vitro functional studies, and was supported by the findings of single photon emission CT DaTSCAN imaging in one patient, which showed complete loss of dopamine transporter activity in the basal nuclei. INTERPRETATION: dopamine transporter deficiency syndrome is a newly recognised, autosomal recessive disorder related to impaired dopamine transporter function. Careful characterisation of patients with this disorder should provide novel insights into the complex role of dopamine homoeostasis in human disease, and understanding of the pathophysiology could help to drive drug development
— id: 120618, year: 2011, vol: 10, page: 54, stat: Journal Article,

The atypical stimulant and nootropic modafinil interacts with the dopamine transporter in a different manner than classical cocaine-like inhibitors
Schmitt, Kyle C; Reith, Maarten E A
2011 ;6(10):e25790-e25790, PLoS ONE
Modafinil is a mild psychostimulant with pro-cognitive and antidepressant effects. Unlike many conventional stimulants, modafinil has little appreciable potential for abuse, making it a promising therapeutic agent for cocaine addiction. The chief molecular target of modafinil is the dopamine transporter (DAT); however, the mechanistic details underlying modafinil's unique effects remain unknown. Recent studies suggest that the conformational effects of a given DAT ligand influence the magnitude of the ligand's reinforcing properties. For example, the atypical DAT inhibitors benztropine and GBR12909 do not share cocaine's notorious addictive liability, despite having greater binding affinity. Here, we show that the binding mechanism of modafinil is different than cocaine and similar to other atypical inhibitors. We previously established two mutations (W84L and D313N) that increase the likelihood that the DAT will adopt an outward-facing conformational state-these mutations increase the affinity of cocaine-like inhibitors considerably, but have little or opposite effect on atypical inhibitor binding. Thus, a compound's WT/mutant affinity ratio can indicate whether the compound preferentially interacts with a more outward- or inward-facing conformational state. Modafinil displayed affinity ratios similar to those of benztropine, GBR12909 and bupropion (which lack cocaine-like effects in humans), but far different than those of cocaine, beta-CFT or methylphenidate. Whereas treatment with zinc (known to stabilize an outward-facing transporter state) increased the affinity of cocaine and methylphenidate two-fold, it had little or no effect on the binding of modafinil, benztropine, bupropion or GBR12909. Additionally, computational modeling of inhibitor binding indicated that while beta-CFT and methylphenidate stabilize an 'open-to-out' conformation, binding of either modafinil or bupropion gives rise to a more closed conformation. Our findings highlight a mechanistic difference between modafinil and cocaine-like stimulants and further demonstrate that the conformational effects of a given DAT inhibitor influence its phenomenological effects
— id: 140536, year: 2011, vol: 6, page: e25790, stat: Journal Article,

SKF-83566, a D(1) -dopamine receptor antagonist, inhibits the dopamine transporter
Stouffer, Melissa A; Ali, Solav; Reith, Maarten E A; Patel, Jyoti C; Sarti, Federica; Carr, Kenneth D; Rice, Margaret E
2011 Sep;118(5):714-720, Journal of neurochemistry
J. Neurochem. (2011) 118, 714-720. ABSTRACT: Dopamine (DA) is an important transmitter in both motor and limbic pathways. We sought to investigate the role of D(1) -receptor activation in axonal DA release regulation in dorsal striatum using a D(1) -receptor antagonist, SKF-83566. Evoked DA release was monitored in rat striatal slices using fast-scan cyclic voltammetry. SKF-83566 caused a concentration-dependent increase in peak single-pulse evoked extracellular DA concentration, with a maximum increase of approximately 65% in 5 muM SKF-83566. This was accompanied by a concentration-dependent increase in extracellular DA concentration clearance time. Both effects were occluded by nomifensine (1 muM), a dopamine transporter (DAT) inhibitor, suggesting that SKF-83566 acted via the DAT. We tested this by examining [(3) H]DA uptake into LLc-PK cells expressing rat DAT, and confirmed that SKF-83566 is a competitive DAT inhibitor with an IC(50) of 5.7 muM. Binding studies with [(3) H]CFT, a cocaine analog, showed even more potent action of SKF-83566 at the DAT cocaine binding site (IC(50) = 0.51 muM). Thus, data obtained using SKF-83566 as a D(1) DA-receptor antagonist may be confounded by concurrent DAT inhibition. More positively, however, SKF-83566 might be a candidate to attenuate cocaine effects in vivo because of the greater potency of this drug at the cocaine versus DA binding site of the DAT
— id: 136635, year: 2011, vol: 118, page: 714, stat: Journal Article,

The selective dopamine uptake inhibitor, D-84, suppresses cocaine self-administration, but does not occasion cocaine-like levels of generalization
Batman, Angela M; Dutta, Aloke K; Reith, Maarten E A; Beardsley, Patrick M
2010 Dec 1;648(1-3):127-132, European journal of pharmacology
A successful replacement pharmacotherapy for treating cocaine dependency would likely reduce cocaine's abuse, support a low abuse liability, overlap cocaine's subjective effects, and have a long duration of action. Inhibitors with varying selectivity at the dopamine transporter (DAT) have approximated these properties. The objective of the present study was to characterize the behavioural effects of an extremely selective DAT inhibitor, (+) trans-4-(2-Benzhydryloxyethyl)-1-(4-fluorobenzyl) piperadin-3-ol (D-84), a 3-hydroxy substituted piperidine derivative of GBR-12935, for its cocaine-like discriminative stimulus effects, its effects on cocaine self-administration, and for its own self-administration. During cocaine discrimination tests, cocaine occasioned the 10 mg/kg cocaine training stimulus with an ED(50) value of 3.13 (1.54-6.34) mg/kg, and reduced response rates with an ED(50) value of 20.39 (7.24-57.44) mg/kg. D-84 incompletely generalized to the cocaine stimulus occasioning a maximal 76% cocaine-lever responding, while reducing response rates with lower potency than cocaine (ED(50)=30.94 (12.34-77.60) mg/kg). Pretreatment with D-84 (9.6-30.4 mg/kg) significantly (P<0.05) reduced cocaine intake at 17.1 mg/kg D-84 when cocaine was self-administered at 0.5 mg/kg/infusion, and at 30.4 mg/kg D-84 when cocaine was self-administered at 0.1, 0.5 .and 1.0 mg/kg/infusion. During self-administration tests with D-84 (0.1-1 mg/kg/infusion), numbers of infusions significantly exceeded vehicle levels at 0.3 mg/kg/infusion. These results show that D-84 pretreatment can decrease cocaine intake especially when high doses of cocaine are being self-administered. This observation, combined with its incomplete generalization to the cocaine discriminative stimulus and its reported long duration of action, provides a profile consistent with a potential replacement therapy for treating cocaine-abusing patients
— id: 119204, year: 2010, vol: 648, page: 127, stat: Journal Article,

Further delineation of hydrophobic binding sites in dopamine D(2)/D(3) receptors for N-4 substituents on the piperazine ring of the hybrid template 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-na phthalen-2-ol
Ghosh, Balaram; Antonio, Tamara; Gopishetty, Bhaskar; Reith, Maarten; Dutta, Aloke
2010 Aug 1;18(15):5661-5674, Bioorganic & medicinal chemistry
Here we report a structure-activity relationship (SAR) study of analogues of 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-na phthalen-2-ol. Our SAR is focused on introduction of various substitutions in the piperazine ring of the hybrid template. The goal behind this study is to delineate the nature of the binding pocket for N-aryl substitution in the piperazine ring by observing the effect of various hydrophobic and other heteroaromatic substitutions on binding affinity (K(i)), as measured with tritiated spiperone and HEK-293 cells expressing either D(2) or D(3) receptors. Functional activity of selected compounds was assessed with the GTPgammaS binding assay. Compound 8d was the most selective for the D(3) receptor in the spiperone binding assay. An interesting similarity in binding affinity was observed between isoquinoline derivative D-301 and the 2-substituted pyridine derivative 8d, suggesting the importance of relative spatial relationships between the N-atom of the ligand and the molecular determinants of the binding pocket in D(2)/D(3) receptors. Functional activity assays demonstrated high potency and selectivity of (+)-8a and (-)-28b (D(2)/D(3) (ratio of EC(50)): 105 and 202, respectively) for the D(3) receptor and both compounds were more selective compared to the reference drug ropinirole (D(2)/D(3) (ratio of EC(50)): 29.5)
— id: 120620, year: 2010, vol: 18, page: 5661, stat: Journal Article,

Discovery of 4-(4-(2-((5-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino)ethyl) piperazin-1-yl)quinolin-8-ol and its analogues as highly potent dopamine D2/D3 agonists and as iron chelator: in vivo activity indicates potential application in symptomatic and neuroprotective therapy for Parkinson's disease
Ghosh, Balaram; Antonio, Tamara; Reith, Maarten E A; Dutta, Aloke K
2010 Mar 11;53(5):2114-2125, Journal of medicinal chemistry
The role of iron in the pathogenesis of Parkinson's disease (PD) has been implicated strongly because of generation of oxidative stress leading to dopamine cell death. In our overall goal to develop bifunctional/multifunctional drugs, we designed dopamine D2/D3 agonist molecules with a capacity to bind to iron. Binding assays were carried out with HEK-293 cells expressing either D2 or D3 receptor with tritiated spiperone to evaluate inhibition constants (K(i)). Functional activity of selected compounds was carried out with GTPgammaS binding assay. SAR results identified compounds (+)-19a and (-)-19b as two potent agonists for both D2 and D3 receptors (EC(50) (GTPgammaS); D2 = 4.51 and 1.69 nM and D3 = 1.58 and 0.74 nM for (-)-19b and (+)-19a, respectively). In vitro complexation studies with 19b demonstrated efficient chelation with iron. Furthermore, the deoxyribose assay with 19b demonstrated potent antioxidant activity. In PD animal model study, (-)-19b exhibited potent in vivo activity in reversing locomotor activity in reserpinized rats and also in producing potent rotational activity in 6-OHDA lesioned rats. This reports initial development of unique lead molecules that might find potential use in symptomatic and neuroprotective treatment of PD
— id: 120621, year: 2010, vol: 53, page: 2114, stat: Journal Article,

Development of (S)-N6-(2-(4-(isoquinolin-1-yl)piperazin-1-yl)ethyl)-N6-propyl-4,5,6,7-tet rahydrobenzo[d]-thiazole-2,6-diamine and its analogue as a D3 receptor preferring agonist: potent in vivo activity in Parkinson's disease animal models
Ghosh, Balaram; Antonio, Tamara; Zhen, Juan; Kharkar, Prashant; Reith, Maarten E A; Dutta, Aloke K
2010 Feb 11;53(3):1023-1037, Journal of medicinal chemistry
Here we report structure-activity relationship study of a novel hybrid series of compounds where structural alteration of aromatic hydrophobic moieties connected to the piperazine ring and bioisosteric replacement of the aromatic tetralin moieties were carried out. Binding assays were carried out with HEK-293 cells expressing either D2 or D3 receptors with tritiated spiperone to evaluate inhibition constants (K(i)). Functional activity of selected compounds in stimulating GTPgammaS binding was assessed with CHO cells expressing human D2 receptors and AtT-20 cells expressing human D3 receptors. SAR results identified compound (-)-24c (D-301) as one of the lead molecules with preferential agonist activity for D3 receptor (EC(50) (GTP gamma S); D3 = 0.52 nM; D2/D3 (EC(50)): 223). Compounds (-)-24b and (-)-24c exhibited potent radical scavenging activity. The two lead compounds, (-)-24b and (-)-24c, exhibited high in vivo activity in two Parkinson's disease (PD) animal models, reserpinized rat model and 6-OHDA induced unilaterally lesioned rat model. Future studies will explore potential use of these compounds in the neuroprotective therapy for PD
— id: 120622, year: 2010, vol: 53, page: 1023, stat: Journal Article,

Interrelation of dopamine transporter oligomerization and surface presence as studied with mutant transporter proteins and amphetamine
Li, Yan; Cheng, Shu-Yuan; Chen, Nianhang; Reith, Maarten E A
2010 Aug;114(3):873-885, Journal of neurochemistry
Our previous work suggested a role for oligomerization in regulating dopamine transporter (DAT) internalization, with d-amphetamine dissociating DAT oligomers and monomers being endocytosed. This model was put to detailed testing in the present work with the use of DAT constructs differentially tagged with Myc or Flag, reversal of tags in co-immunoprecipitation and cross-linking assays, and application of antibodies against different tags in biotinylation experiments. Upon pairing wild-type (WT) DAT with W84L mutant, effects of d-amphetamine on oligomerization (decrease) but not surface DAT are observed. Internalization of W84L monomers appears to be slow as inferred from the inability of d-amphetamine to reduce surface Myc upon co-expressing Flag-WT with Myc-W84L but not Myc-WT with Flag-W84L, and from the sluggish Myc-W84L endocytosis rate (both with or without d-amphetamine). Results obtained for D313N, D345N, or D436N mutants can all be accommodated by a model in which D-amphetamine is unable to dissociate mutant protomers from oligomers (tetramers or higher-order assemblies) that contain them; this interpretation is confirmed in experiments with both tag reversal in co-expression and antibody reversal in western blotting. Upon co-transfecting Myc- and Flag-tagged constructs, resulting tetramers can be calculated to be composed of different species (MycMycMycMyc, MycMycMycFlag, MycMycFlagFlag, MycFlagFlagFlag, and FlagFlagFlagFlag), but it is shown that outcomes predicted by models based on MycMycFlagFlag oligomers are not changed in a major way by the occurrence of the additional species
— id: 111351, year: 2010, vol: 114, page: 873, stat: Journal Article,

Substrate and drug binding sites in LeuT
Nyola, Ajeeta; Karpowich, Nathan K; Zhen, Juan; Marden, Jennifer; Reith, Maarten E; Wang, Da-Neng
2010 Aug;20(4):415-422, Current opinion in structural biology
LeuT is a member of the neurotransmitter/sodium symporter family, which includes the neuronal transporters for serotonin, norepinephrine, and dopamine. The original crystal structure of LeuT shows a primary leucine-binding site at the center of the protein. LeuT is inhibited by different classes of antidepressants that act as potent inhibitors of the serotonin transporter. The newly determined crystal structures of LeuT-antidepressant complexes provide opportunities to probe drug binding in the serotonin transporter, of which the exact position remains controversial. Structure of a LeuT-tryptophan complex shows an overlapping binding site with the primary substrate site. A secondary substrate binding site was recently identified, where the binding of a leucine triggers the cytoplasmic release of the primary substrate. This two binding site model presents opportunities for a better understanding of drug binding and the mechanism of inhibition for mammalian transporters
— id: 112042, year: 2010, vol: 20, page: 415, stat: Journal Article,

Bivalent phenethylamines as novel dopamine transporter inhibitors: evidence for multiple substrate-binding sites in a single transporter
Schmitt, Kyle C; Mamidyala, Sreeman; Biswas, Swati; Dutta, Aloke K; Reith, Maarten E A
2010 Mar;112(6):1605-1618, Journal of neurochemistry
Bivalent ligands--compounds incorporating two receptor-interacting moieties linked by a flexible chain--often exhibit profoundly enhanced binding affinity compared with their monovalent components, implying concurrent binding to multiple sites on the target protein. It is generally assumed that neurotransmitter sodium symporter (NSS) proteins, such as the dopamine transporter (DAT), contain a single domain responsible for recognition of substrate molecules. In this report, we show that molecules possessing two substrate-like phenylalkylamine moieties linked by a progressively longer aliphatic spacer act as progressively more potent DAT inhibitors (rather than substrates). One compound bearing two dopamine (DA)-like pharmacophoric 'heads' separated by an 8-carbon linker achieved an 82-fold gain in inhibition of [(3)H] 2beta-carbomethoxy-3beta-(4-fluorophenyl)-tropane (CFT) binding compared with DA itself; bivalent compounds with a 6-carbon linker and heterologous combinations of DA-, amphetamine- and beta-phenethylamine-like heads all resulted in considerable and comparable gains in DAT affinity. A series of short-chain bivalent-like compounds with a single N-linkage was also identified, the most potent of which displayed a 74-fold gain in binding affinity. Computational modelling of the DAT protein and docking of the two most potent bivalent (-like) ligands suggested simultaneous occupancy of two discrete substrate-binding domains. Assays with the DAT mutants W84L and D313N--previously employed by our laboratory to probe conformation-specific binding of different structural classes of DAT inhibitors--indicated a bias of the bivalent ligands for inward-facing transporters. Our results strongly indicate the existence of multiple DAT substrate-interaction sites, implying that it is possible to design novel types of DAT inhibitors based upon the 'multivalent ligand' strategy
— id: 109199, year: 2010, vol: 112, page: 1605, stat: Journal Article,

Regulation of the dopamine transporter
Schmitt, Kyle C; Reith, Maarten E A
2010 Feb;1187:316-340, Annals of the New York Academy of Sciences
Dopaminergic signaling in the brain is primarily modulated by dopamine transporters (DATs), which actively translocate extraneuronal dopamine back into dopaminergic neurons. Transporter proteins are highly dynamic, continuously trafficking between plasmalemmal and endosomal membranes. Changes in DAT membrane trafficking kinetics can rapidly regulate dopaminergic tone by altering the number of transporters present at the cell surface. Various psychostimulant DAT ligands-acting either as amphetamine-like substrates or cocaine-like nontranslocated inhibitors-affect transporter trafficking, triggering rapid insertion or removal of plasmalemmal DATs. In this review, we focus on the effects of psychostimulants of addiction (particularly d-methamphetamine and cocaine) on DAT regulation and membrane trafficking, with an emphasis on how these drugs may influence intracellular signaling cascades and transporter-associated scaffolding proteins to affect DAT regulation. In addition, we consider involvement of presynaptic receptors for dopamine and other ligands in DAT regulation. Finally, we discuss possible implications of transporter regulation to the putative toxicity of several substituted amphetamine derivatives commonly used as recreational drugs, as well as to the design of therapeutics for cocaine addiction
— id: 107930, year: 2010, vol: 1187, page: 316, stat: Journal Article,

Concentration of receptor and ligand revisited in a modified receptor binding protocol for high-affinity radioligands: [3H]Spiperone binding to D2 and D3 dopamine receptors
Zhen, Juan; Antonio, Tamara; Dutta, Aloke K; Reith, Maarten E A
2010 Apr 30;188(1):32-38, Journal of neuroscience methods
In receptor binding assays with ultra-high-affinity radioligands, it is difficult, in practice, to adhere the golden rule that the receptor concentration in the assay should be substantially (at least 10-fold) lower than the dissociation constant (K(d)) of the radioligand and inhibition constant (K(i)) of compound. Especially for low specific activity radioligands (usually tritiated ligands of a couple of TBq/mmol), routinely applied in concentrations at around or below the K(d), the use of extremely small amounts of receptor protein per assay will result in low levels of bound radioactivity; the alternative use of larger assay volumes will make it difficult to apply 96-well filtration devices. For assessing the inhibition constant (K(i)) of competitive inhibitors under conditions violating the above golden rule, equations are available incorporating both [receptor] and [ligand] versus K(d); however, their application requires precise knowledge of [receptor] or initial bound/free [radioligand] ratio. In this study, we present the theoretical basis for determining the K(i) for a competitive inhibitor in a new protocol at high [protein] and high [radioligand] with the simple Cheng-Prusoff correction without the need to correct for [receptor] or initial bound/free [radioligand] ratio. In addition, we present results on the binding of the ultra-high-affinity ligand [(3)H]spiperone to dopamine D(2) and D(3) receptors validating the K(i) values calculated with the new protocol for competitive inhibitors as compared with those calculated with the most comprehensive equation available to date, that of Munson and Rodbard (1988). Binding was measured at varying [radioligand] and [receptor], test compounds (including (-)5-OH-DPAT, (+/-)7-OH-DPAT, and ropinirole) were run with varying [receptor], and simulations were done at vastly varying [radioligand] for inhibitors with vastly different K(i)s. The modified high [radioligand] protocol presented here removes a major hindrance in the proper execution of binding assays with ultra-high-affinity tritiated ligands with K(d) values in the sub-nanomolar range, allowing the use of 96-well plates with small volumes of 100-200 microl per binding assay
— id: 108921, year: 2010, vol: 188, page: 32, stat: Journal Article,

Investigation of various N-heterocyclic substituted piperazine versions of 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-na phthalen-2-ol: effect on affinity and selectivity for dopamine D3 receptor
Brown, Dennis A; Mishra, Manoj; Zhang, Suhong; Biswas, Swati; Parrington, Ingrid; Antonio, Tamara; Reith, Maarten E A; Dutta, Aloke K
2009 Jun 1;17(11):3923-3933, Bioorganic & medicinal chemistry
Here we report on the design and synthesis of several heterocyclic analogues belonging to the 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-na phthalen-2-ol series of molecules. Compounds were subjected to [(3)H]spiperone binding assays, carried out with HEK-293 cells expressing either D2 or D3 dopamine receptors, in order to evaluate their inhibition constant (K(i)) at these receptors. Results indicate that N-substitution on the piperazine ring can accommodate various substituted indole rings. The results also show that in order to maintain high affinity and selectivity for the D3 receptor the heterocyclic ring does not need to be connected directly to the piperazine ring as the majority of compounds included here are linked either via an amide or a methylene linker to the heterocyclic moiety. The enantiomers of the most potent racemic compound 10e exhibited differential activity with (-)-10e (K(i); D2=47.5 nM, D3=0.57 nM) displaying higher affinity at both D2 and D3 receptors compared to its enantiomer (+)-10e (K(i); D2=113 nM, D3=3.73 nM). Additionally, compound (-)-10e was more potent and selective for the D3 receptor compared to either 7-OH-DPAT or 5-OH-DPAT. Among the bioisosteric derivatives, the indazole derivative 10g and benzo[b]thiophene derivative 10i exhibited the highest affinity for D2 and D3 receptors. In the functional GTPgammaS binding study, one of the lead molecules, (-)-15, exhibited potent agonist activity at both D2 and D3 receptors with preferential affinity at D3
— id: 120625, year: 2009, vol: 17, page: 3923, stat: Journal Article,

Synthesis and biological characterization of (3R,4R)-4-(2-(benzhydryloxy)ethyl)-1-((R)-2-hydroxy-2-phenylethyl)-piperid in-3-ol and its stereoisomers for activity toward monoamine transporters
Kharkar, Prashant S; Batman, Angela M; Zhen, Juan; Beardsley, Patrick M; Reith, Maarten E A; Dutta, Aloke K
2009 Jul;4(7):1075-1085, ChemMedChem
A novel series of optically active molecules based on a 4-(2-(benzhydryloxy)ethyl)-1-((R)-2-hydroxy-2-phenylethyl)-piperidin-3-ol template were developed. Depending on stereochemistry, the compounds exhibit various degrees of affinity for three dopamine, serotonin, and norepinephrine transporters. These molecules have the potential for treating several neurological disorders such as drug abuse, depression, and attention deficit hyperactivity disorder.Herein we describe the synthesis and biological evaluation of a series of asymmetric 4-(2-(benzhydryloxy)ethyl)-1-((R)-2-hydroxy-2-phenylethyl)-piperidin-3-ol- based dihydroxy compounds in which the hydroxy groups are located on both the piperidine ring and the N-phenylethyl side chain. In vitro uptake inhibition data of these molecules indicate high affinity for the dopamine transporter (DAT) in addition to moderate to high affinity for the norepinephrine transporter (NET). Interestingly, compounds 9 b and 9 d exhibit affinities for all three monoamine transporters, with highest potency at DAT and NET, and moderate potency at the serotonin transporter (SERT) (K(i): 2.29, 78.4, and 155 nM for 9 b and 1.55, 14.1, and 259 nM for 9 d, respectively). Selected compounds 9 a, 9 d, and 9 d' were tested for their locomotor activity effects in mice and for their ability to occasion the cocaine-discriminative stimulus in rats. These test compounds generally exhibit a much longer duration of action than cocaine for elevating locomotor activity, and completely generalize the cocaine-discriminative stimulus in a dose-dependent manner
— id: 120624, year: 2009, vol: 4, page: 1075, stat: Journal Article,

Homozygous Loss-of-Function Mutations in the Dopamine Transporter (DAT), SLC6A3 Cause Infantile Parkinsonism-Dystonia (IPD)
Kurian, M; Zhen, J; Cheng, SY; Li, Y; Mordekar, S; Jardine, P; Morgan, NV; Meyer, E; Tee, L; Pasha, S; Wassmer, E; Assmann, B; Heales, SJR; Gissen, P; Reith, MEA; Maher, ER
2009 SEP ;46(47):S16-S16, Journal of medical genetics
— id: 105632, year: 2009, vol: 46, page: S16, stat: Journal Article,

Homozygous loss-of-function mutations in the gene encoding the dopamine transporter are associated with infantile parkinsonism-dystonia
Kurian, Manju A; Zhen, Juan; Cheng, Shu-Yuan; Li, Yan; Mordekar, Santosh R; Jardine, Philip; Morgan, Neil V; Meyer, Esther; Tee, Louise; Pasha, Shanaz; Wassmer, Evangeline; Heales, Simon J R; Gissen, Paul; Reith, Maarten E A; Maher, Eamonn R
2009 Jun;119(6):1595-1603, Journal of clinical investigation
Genetic variants of the SLC6A3 gene that encodes the human dopamine transporter (DAT) have been linked to a variety of neuropsychiatric disorders, particularly attention deficit hyperactivity disorder. In addition, the homozygous Slc6a3 knockout mouse displays a hyperactivity phenotype. Here, we analyzed 2 unrelated consanguineous families with infantile parkinsonism-dystonia (IPD) syndrome and identified homozygous missense SLC6A3 mutations (p.L368Q and p.P395L) in both families. Functional studies demonstrated that both mutations were loss-of-function mutations that severely reduced levels of mature (85-kDa) DAT while having a differential effect on the apparent binding affinity of dopamine. Thus, in humans, loss-of-function SLC6A3 mutations that impair DAT-mediated dopamine transport activity are associated with an early-onset complex movement disorder. Identification of the molecular basis of IPD suggests SLC6A3 as a candidate susceptibility gene for other movement disorders associated with parkinsonism and/or dystonic features
— id: 120623, year: 2009, vol: 119, page: 1595, stat: Journal Article,

Interaction of catechol and non-catechol substrates with externally or internally facing dopamine transporters
Liang, Ying-Jian; Zhen, Juan; Chen, Nianhang; Reith, Maarten E A
2009 May;109(4):981-994, Journal of neurochemistry
Our previous work suggested that collapsing the Na(+) gradient and membrane potential converts the dopamine (DA) transporter (DAT) to an inward-facing conformation with a different substrate binding profile. Here, DAT expressing human embryonic kidney 293 cells were permeabilized with digitonin, disrupting ion/voltage gradients and allowing passage of DAT substrates. The potency of p-tyramine and other non-catechols (d-amphetamine, beta-phenethylamine, MPP(+)) in inhibiting cocaine analog binding to DAT in digitonin-treated cells was markedly weakened to a level similar to that observed in cell-free membranes. In contrast, the potency of DA and another catechol, norepinephrine, was not significantly changed by the same treatment, whereas epinephrine showed only a modest reduction. These findings suggest that catechol substrates interact symmetrically with both sides of DAT and non-catechol substrates, favoring binding to outward-facing transporter. In the cocaine analog binding assay, the mutant W84L displayed enhanced intrinsic binding affinity for substrates in interacting with both outward- and inward-facing states; D313N showed wild-type-like symmetric binding; but D267L and E428Q showed an apparent improvement in the permeation pathway from the external face towards the substrate site. Thus, the structure of both substrate and transporter play a role in the sidedness and mode of interaction between them
— id: 100191, year: 2009, vol: 109, page: 981, stat: Journal Article,

Antidepressant specificity of serotonin transporter suggested by three LeuT-SSRI structures
Zhou, Zheng; Zhen, Juan; Karpowich, Nathan K; Law, Christopher J; Reith, Maarten E A; Wang, Da-Neng
2009 Jun;16(6):652-657, Nature structural & molecular biology
Sertraline and fluoxetine are selective serotonin re-uptake inhibitors (SSRIs) that are widely prescribed to treat depression. They exert their effects by inhibiting the presynaptic plasma membrane serotonin transporter (SERT). All SSRIs possess halogen atoms at specific positions, which are key determinants for the drugs' specificity for SERT. For the SERT protein, however, the structural basis of its specificity for SSRIs is poorly understood. Here we report the crystal structures of LeuT, a bacterial SERT homolog, in complex with sertraline, R-fluoxetine or S-fluoxetine. The SSRI halogens all bind to exactly the same pocket within LeuT. Mutation at this halogen-binding pocket (HBP) in SERT markedly reduces the transporter's affinity for SSRIs but not for tricyclic antidepressants. Conversely, when the only nonconserved HBP residue in both norepinephrine and dopamine transporters is mutated into that found in SERT, their affinities for all the three SSRIs increase uniformly. Thus, the specificity of SERT for SSRIs is dependent largely on interaction of the drug halogens with the protein's HBP
— id: 103150, year: 2009, vol: 16, page: 652, stat: Journal Article,

Bioisosteric heterocyclic versions of 7-{[2-(4-phenyl-piperazin-1-yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphth alen-2-ol: identification of highly potent and selective agonists for dopamine D3 receptor with potent in vivo activity
Biswas, Swati; Hazeldine, Stuart; Ghosh, Balaram; Parrington, Ingrid; Kuzhikandathil, Eldo; Reith, Maarten E A; Dutta, Aloke K
2008 May 22;51(10):3005-3019, Journal of medicinal chemistry
In the current report, we extend the SAR study on our hybrid structure 7-{[2-(4-phenyl-piperazin-1-yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphth alen-2-ol further to include heterocyclic bioisosteric analogues. Binding assays were carried out with HEK-293 cells expressing either D2 or D3 receptors with tritiated spiperone to evaluate inhibition constants (Ki). Functional activity of selected compounds in stimulating GTPgammaS binding was assessed with CHO cells expressing human D2 receptors and AtT-20 cells expressing human D3 receptors. The highest binding affinity and selectivity for D3 receptors were exhibited by (-)-34 (Ki=0.92 nM and D2/D3=253). In the functional GTPgammaS binding assay, (-)-34 exhibited full agonist activity with picomolar affinity for D3 receptor with high selectivity (EC50=0.08 nM and D2/D3=248). In the in vivo rotational study, (-)-34 exhibited potent rotational activity in 6-OH-DA unilaterally lesioned rats with long duration of action, which indicates its potential application in neuroprotective treatment of Parkinson's disease
— id: 86156, year: 2008, vol: 51, page: 3005, stat: Journal Article,

Further structure-activity relationships study of hybrid 7-{[2-(4-phenylpiperazin-1-yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphtha len-2-ol analogues: identification of a high-Affinity D3-preferring agonist with potent in vivo activity with long duration of action
Biswas, Swati; Zhang, Suhong; Fernandez, Fernando; Ghosh, Balaram; Zhen, Juan; Kuzhikandathil, Eldo; Reith, Maarten E A; Dutta, Aloke K
2008 Jan 10;51(1):101-117, Journal of medicinal chemistry
This paper describes an extended structure-activity relationships study of aminotetralin-piperazine-based hybrid molecules developed earlier for dopamine D2/D3 receptors. Various analogues as positional isomers have been developed where location of the phenolic hydroxyl group has been varied on the aromatic ring. Between two catechol derivatives, compound 6e with a two methylene linker length exhibited higher affinity and selectivity for D3 over D2 receptors over compound 6f with four methylene linkers (D2/D3 = 50.6 vs 7.51 for 6e and 6f, respectively). In general, the (-)-isomer was more potent than the (+)-isomeric counterpart. Binding results indicated highest selectivity for D3 receptors in compound (-)- 10 ( K i = 0.35 nM; D2/D3 = 71). In the 5-hydroxy series, highest selectivity for D3 receptors was exhibited by compound (-)- 25 ( K i = 0.82 nM; D2/D3 = 31.5). Most potent compounds exhibited binding and functional affinities at the sub-nanomolar level for the D3 receptor. Binding assays were carried out with HEK-293 cells expressing either D2 or D3 receptors by using tritiated spiperone as radioligand for competition studies to evaluate inhibition constants ( K i). A functional assay of selected compounds for stimulating GTPgammaS binding was carried out with CHO cells expressing human D2 receptors and AtT-20 cells expressing human D3 receptors. The functional assay results indicated partial to full agonist characteristics of test compounds. Compound (-)- 25 was selected further for in vivo study to evaluate its potency in producing contralateral rotations in rats with unilateral lesion in the nigrostriatal region induced by neurotoxin 6-OHDA, a Parkinsonian animal model. Compound (-)- 25 at 5 micromol/kg exhibited rotational activity that lasted beyond 12 h, whereas at a 1 micromol/kg dose the rotations lasted beyond 8 h
— id: 75684, year: 2008, vol: 51, page: 101, stat: Journal Article,

MEDI 266-Design, synthesis, and biological evaluation of both conformationally flexible and rigid analogs of 7-((2-(4-phenylpiperazin-1-yl)ethyl)(propyl)amino)-5,6,7,8-tetrahydronap hthalenols as dopamine D2/D3 receptor ligands: Development of a four point pharmacophore model for interaction with the D2 and D3 receptor subtypes
Brown, DA; Kharkar, P; Parrington, I; Reith, M; Dutta, A
2008 APR 6 ;235(12):-, Abstracts of papers (American Chemical Society)
— id: 110159, year: 2008, vol: 235, page: , stat: Journal Article,

Structurally constrained hybrid derivatives containing octahydrobenzo[g or f]quinoline moieties for dopamine D2 and D3 receptors: binding characterization at D2/D3 receptors and elucidation of a pharmacophore model
Brown, Dennis A; Kharkar, Prashant S; Parrington, Ingrid; Reith, Maarten E A; Dutta, Aloke K
2008 Dec 25;51(24):7806-7819, Journal of medicinal chemistry
A series of structurally constrained analogues based on hybrid compounds containing octahydrobenzo[g or f]quinoline moieties were designed, synthesized, and characterized for their binding to dopamine D2 and D3 receptors expressed in HEK-293 cells. Among the newly developed constrained molecules, trans-octahydrobenzo[f]quinolin-7-ol (8) exhibited the highest affinity for D2 and D3 receptors, the (-)-isomer being the eutomer. Interestingly, this hybrid constrained version 8 showed significant affinity over the corresponding nonhybrid version 1 (representing a constrained version of the aminotetralin structure only) when assayed under same conditions (K(i) of 49.1 and 14.9 nM for 8 vs 380 and 96.0 nM for 1 at D2 and D3, respectively). Similar results were found with other lead hybrid compounds, indicating a contribution of the piperazine moiety in the observed enhanced affinity. On the basis of the data of new lead constrained derivatives and other lead hybrid derivatives developed by us, a unique pharmacophore model was proposed consisting of three pharmacophoric centers, two with aromatic/hydrophobic and one with cationic features
— id: 94432, year: 2008, vol: 51, page: 7806, stat: Journal Article,

Substrates dissociate dopamine transporter oligomers
Chen, Nianhang; Reith, Maarten E A
2008 May;105(3):910-920, Journal of neurochemistry
Substrate-induced endocytic trafficking of dopamine transporter (DAT) has been observed, but little is known about the regulation of DAT oligomerization by substrate. The present study investigates the effect on substrates on DAT oligomerization and explores a potential link with the presence of DAT at the cell surface in human embryonic kidney cells transiently or stably expressing N-terminal tagged DAT constructs. Dopamine (100 muM) or amphetamine (2-10 muM) reduced Myc-DAT coimmunoprecipitated along with Flag-DAT (oligomeric DAT) in tandem with a reduction in surface DAT determined by biotinylation. Dopamine (10-1000 muM) and amphetamine (0.2-200 muM) reduced DAT oligomerization as assessed by cross-linking with copper sulfate phenanthroline or Cu(2+). Inhibition of endocytosis by 10 muM phenylarsine oxide or 450 mM sucrose counteracted the effect of 10 muM DA or 2 muM amphetamine in reducing DAT cross-linking. In addition to overall similarities between the results with the two cross-linking agents and between the results with the two different endocytosis inhibitors, some differences were noted as well, likely related to the efficiency of the cross-linking process and the sulfhydryl-reactive properties of phenylarsine oxide, respectively. The present results are the first to indicate regulation of oligomerization of an solute carrier family 6 transporter, the DAT, by substrates that act at DAT. In addition, the present study opens up the possibility of an important linkage between oligomerization of DAT and endocytic or other modulatory mechanisms impacting surface DAT
— id: 75683, year: 2008, vol: 105, page: 910, stat: Journal Article,

D-161, a novel pyran-based triple monoamine transporter blocker: behavioral pharmacological evidence for antidepressant-like action
Dutta, Aloke K; Ghosh, Balaram; Biswas, Swati; Reith, Maarten E A
2008 Jul 28;589(1-3):73-79, European journal of pharmacology
Deficiency in dopaminergic activity has been linked to a depressed state in pharmacological and clinical studies. Current pharmacological treatment for depression primarily involves modulation of serotonergic and noradrenergic systems but not dopaminergic neurotransmission. Available pharmacotherapy for depression has a number of drawbacks as a significant number of people are either refractory or develop tolerance to the antidepressant agents resulting in relapse. Furthermore, the slow onset of action of current therapies often poses a challenge for effective treatment. In our effort to develop novel molecules impacting all three above mentioned monoamine systems, we discovered structurally unique pyran derivatives with various profiles in inhibiting monoamine transporters. One of our lead molecules, D-161 exhibited triple monoamine transporter inhibitory activity with the highest affinity for norepinephrine transporter (NET) followed by its affinity for serotonin transporter (SERT) and dopamine transporter (DAT). D-161 exhibited potent activity in reducing immobility significantly in the rat forced swim test as well as in the mouse tail suspension test. Moreover, results from locomotor activity tests indicated that the reduction of immobility by D-161 was not due to motor activation as no significant motor activation was observed when the rats were subjected to the same doses of drug under the same conditions as in the forced swim test. These results suggest that the novel asymmetric pyran derivative D-161 with unique molecular structure exhibiting triple monoamine transporter inhibitory activity could possess potent antidepressant activity
— id: 86155, year: 2008, vol: 589, page: 73, stat: Journal Article,

Three-dimensional quantitative structure-activity relationship (3D QSAR) and pharmacophore elucidation of tetrahydropyran derivatives as serotonin and norepinephrine transporter inhibitors
Kharkar, Prashant S; Reith, Maarten E A; Dutta, Aloke K
2008 Jan;22(1):1-17, Journal of computer-aided molecular design
Three-dimensional quantitative structure-activity relationship (3D QSAR) using comparative molecular field analysis (CoMFA) was performed on a series of substituted tetrahydropyran (THP) derivatives possessing serotonin (SERT) and norepinephrine (NET) transporter inhibitory activities. The study aimed to rationalize the potency of these inhibitors for SERT and NET as well as the observed selectivity differences for NET over SERT. The dataset consisted of 29 molecules, of which 23 molecules were used as the training set for deriving CoMFA models for SERT and NET uptake inhibitory activities. Superimpositions were performed using atom-based fitting and 3-point pharmacophore-based alignment. Two charge calculation methods, Gasteiger-Huckel and semiempirical PM3, were tried. Both alignment methods were analyzed in terms of their predictive abilities and produced comparable results with high internal and external predictivities. The models obtained using the 3-point pharmacophore-based alignment outperformed the models with atom-based fitting in terms of relevant statistics and interpretability of the generated contour maps. Steric fields dominated electrostatic fields in terms of contribution. The selectivity analysis (NET over SERT), though yielded models with good internal predictivity, showed very poor external test set predictions. The analysis was repeated with 24 molecules after systematically excluding so-called outliers (5 out of 29) from the model derivation process. The resulting CoMFA model using the atom-based fitting exhibited good statistics and was able to explain most of the selectivity (NET over SERT)-discriminating factors. The presence of -OH substituent on the THP ring was found to be one of the most important factors governing the NET selectivity over SERT. Thus, a 4-point NET-selective pharmacophore, after introducing this newly found H-bond donor/acceptor feature in addition to the initial 3-point pharmacophore, was proposed
— id: 75685, year: 2008, vol: 22, page: 1, stat: Journal Article,

Further structural optimization of cis-(6-benzhydryl-piperidin-3-yl)-benzylamine and 1,4-diazabicyclo[3.3.1]nonane derivatives by introducing an exocyclic hydroxyl group: interaction with dopamine, serotonin, and norepinephrine transporters
Mishra, Manoj; Kolhatkar, Rohit; Zhen, Juan; Parrington, Ingrid; Reith, Maarten E A; Dutta, Aloke K
2008 Mar 15;16(6):2769-2778, Bioorganic & medicinal chemistry
Our earlier effort to develop constrained analogues of flexible piperidine derivatives for monoamine transporters led to the development of a series of 3,6-disubstituted piperidine derivatives, and a series of 4,8-disubstituted 1,4-diazabicyclo[3.3.1]nonane derivatives. In further structure-activity relationship (SAR) studies on these constrained derivatives, several novel analogues were developed where an exocyclic hydroxyl group was introduced on the N-alkyl-aryl side chain. All synthesized derivatives were tested for their affinities for the dopamine transporter (DAT), serotonin (5-HT) transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in inhibiting the uptake of [(3)H]DA, [(3)H]5-HT, and [(3)H]NE, respectively. Compounds were also tested for their binding potency at the DAT by their ability to inhibit binding of [(3)H]WIN 35,428. The results indicated that position of the hydroxyl group on the N-alkyl side chain is important along with the length of the side chain. In general, hydroxyl derivatives derived from more constrained bicyclic diamines exhibited greater selectivity for interaction with DAT compared to the corresponding 3,6-disubstituted diamines. In the current series of molecules, compound 11b with N-propyl side chain with the hydroxyl group attached in the benzylic position was the most potent and selective for DAT (K(i)=8.63nM; SERT/DAT=172 and NET/DAT=48.4)
— id: 86154, year: 2008, vol: 16, page: 2769, stat: Journal Article,

Interaction of cocaine-, benztropine-, and GBR12909-like compounds with wild-type and mutant human dopamine transporters: molecular features that differentially determine antagonist-binding properties
Schmitt, Kyle C; Zhen, Juan; Kharkar, Prashant; Mishra, Manoj; Chen, Nianhang; Dutta, Aloke K; Reith, Maarten E A
2008 Nov;107(4):928-940, Journal of neurochemistry
The widely abused psychostimulant cocaine is thought to elicit its reinforcing effects primarily via inhibition of the neuronal dopamine transporter (DAT). However, not all DAT inhibitors share cocaine's behavioral profile, despite similar or greater affinity for the DAT. This may be due to differential molecular interactions with the DAT. Our previous work using transporter mutants with altered conformational equilibrium (W84L and D313N) indicated that benztropine and GBR12909 interact with the DAT in a different manner than cocaine. Here, we expand upon these previous findings, studying a number of structurally different DAT inhibitors for their ability to inhibit [(3)H]CFT binding to wild-type, W84L and D313N transporters. We systematically tested structural intermediates between cocaine and benztropine, structural hybrids of benztropine and GBR12909 and a number of other structurally heterologous inhibitors. Derivatives of the stimulant desoxypipradrol (2-benzhydrylpiperidine) exhibited a cocaine-like binding profile with respect to mutation, whereas compounds possessing the diphenylmethoxy moiety of benztropine and GBR12909 were dissimilar to cocaine-like compounds. In tests with specific isomers of cocaine and tropane analogues, compounds with 3alpha stereochemistry tended to exhibit benztropine-like binding, whereas those with 3beta stereochemistry were more cocaine-like. Our results point to the importance of specific molecular features--most notably the presence of a diphenylmethoxy moiety--in determining a compound's binding profile. This study furthers the concept of using DAT mutants to differentiate cocaine-like inhibitors from atypical inhibitors in vitro. Further studies of the molecular features that define inhibitor-transporter interaction could lead to the development of DAT inhibitors with differential clinical utility
— id: 92696, year: 2008, vol: 107, page: 928, stat: Journal Article,

Substrates and inhibitors display different sensitivity to expression level of the dopamine transporter in heterologously expressing cells
Chen, Nianhang; Reith, Maarten E A
2007 Apr;101(2):377-388, Journal of neurochemistry
The use of heterologous expression systems for studying dopamine (DA) transporter (DAT) function has provided important information corroborating and complementing in situ obtained knowledge. Preliminary experiments with human embryonic kidney cells (HEK293) heterologously expressing varying amounts of DAT suggested fluctuations in the potency of cocaine in inhibiting DA uptake and led to the present systematic assessment of the impact of the density of DAT on its function. Transiently expressing intact HEK293 cells, transfected with increasing amounts of DAT cDNA, displayed increasing levels of surface DAT, binding of the cocaine analog [(3)H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane ([(3)H]CFT), and uptake of [(3)H]DA, [(3)H]N-methyl-4-phenylpyridinium ([(3)H]MPP(+)), [(3)H]norepinephrine, and [(3)H]serotonin. However, the amount of DAT cDNA and the DAT expression level required to produce 50% of maximal activity was threefold higher for CFT binding than for DA uptake. Increased DAT expression was accompanied by weakened potency in inhibiting [(3)H]DA uptake for cocaine, CFT, benztropine, and its analog JHW025, GBR 12909 and mazindol; their potency in inhibiting [(3)H]CFT binding was unaffected. Inhibition of uptake by the substrates DA, m-tyramine, d-amphetamine, or MPP(+) was also unaffected. Increasing DAT in stably expressing HEK293 cells by stimulation of gene expression with sodium butyrate also decreased the uptake inhibitory potency of a number of the above blockers without affecting the interaction between substrates and DAT. The present results prompt discussion of models explaining how factors regulating DAT expression at the plasma membrane can regulate DAT function and pharmacology.
— id: 72807, year: 2007, vol: 101, page: 377, stat: Journal Article,

Regulation of biogenic amine transporters
Jayanthi LD; Samuvel DJ; Buck E; Reith MEA; Ramamoorthy S
Handbook of neurochemistry and molecular neurobiology : neural membranes and transport New York : Springer, 2007,
— id: 4629, year: 2007, vol: , page: ?, stat: Chapter,

Handbook of neurochemistry and molecular neurobiology : neural membranes and transport
Reith MEA
New York : Springer, 2007,
— id: 1367, year: 2007, vol: , page: , stat: ,

LeuT-Desipramine Structure Reveals How Antidepressants Block Neurotransmitter Reuptake
Zhou, Zheng; Zhen, Juan; Karpowich, Nathan K; Goetz, Regina M; Law, Christopher J; Reith, Maarten E A; Wang, Da-Neng
2007 Sep 7;317(5843):1390-1393, Science
Tricyclic antidepressants exert their pharmacological effect-inhibiting the reuptake of serotonin, norepinephrine, and dopamine-by directly blocking neurotransmitter transporters (SERT, NET, and DAT, respectively) in the presynaptic membrane. The drug-binding site and the mechanism of this inhibition are poorly understood. We determined the crystal structure at 2.9 A of the bacterial leucine transporter (LeuT), a homolog of SERT, NET, and DAT, in complex with leucine and the antidepressant desipramine. Desipramine binds at the inner end of the extracellular cavity of the transporter and is held in place by a hairpin loop and by a salt bridge. This binding site is separated from the leucine-binding site by the extracellular gate of the transporter. By directly locking the gate, desipramine prevents conformational changes and blocks substrate transport. Mutagenesis experiments on human SERT and DAT indicate that both the desipramine-binding site and its inhibition mechanism are probably conserved in the human neurotransmitter transporters
— id: 73794, year: 2007, vol: 317, page: 1390, stat: Journal Article,

The importance of company - Na+ and Cl- influence substrate interaction with SLC6 transporters and other proteins
Reith MEA; Zhen J; Chen N
Neurotransmitter transporters Berlin : Springer, 2006,
— id: 4628, year: 2006, vol: , page: 76, stat: Chapter,

The importance of company: Na+ and Cl- influence substrate interaction with SLC6 transporters and other proteins
Reith, M E A; Zhen, J; Chen, N
2006 ;(175):75-93, Handbook of experimental pharmacology
SLC6 transporters, which include transporters for gamma-aminobutyric acid (GABA), norepinephrine, dopamine, serotonin, glycine, taurine, L-proline, creatine, betaine, and neutral cationic amino acids, require Na+ and Cl- for their function, and this review covers the interaction between transporters of this family with Na+ and Cl- from a structure-function standpoint. Because detailed structure-function information regarding ion interactions with SLC6 transporters is limited, we cover other proteins cotransporting Na+ or Cl- with substrate (SLClA2, PutP, SLC5A1, melB), or ion binding to proteins in general (rhodanese, ATPase, LacY, thermolysine, angiotensin-converting enzyme, halorhodopsin, CFTR). Residues can be involved in directly binding Na+ or Cl-, in coupling ion binding to conformational changes in transporter, in coupling Na+ or Cl- movement to transport, or in conferring ion selectivity. Coordination of ions can involve a number of residues, and portions of the substrate and coupling ion binding sites can be distal in space in the tertiary structure of the transporter, with other portions that are close in space thought to be crucial for the coupling process. The reactivity with methanethiosulfonate reagents of cysteines placed in strategic positions in the transporter provides a readout for conformational changes upon ion or substrate binding. More work is needed to establish the relationships between ion interactions and oligomerization of SLC6 transporters
— id: 111684, year: 2006, vol: , page: 75, stat: Journal Article,

Further structural exploration of trisubstituted asymmetric pyran derivatives (2S,4R,5R)-2-benzhydryl-5-benzylamino-tetrahydropyran-4-ol and their corresponding disubstituted (3S,6S) pyran derivatives: a proposed pharmacophore model for high-affinity interaction with the dopamine, serotonin, and norepinephrine transporters
Zhang, Shijun; Fernandez, Fernando; Hazeldine, Stuart; Deschamps, Jeffrey; Zhen, Juan; Reith, Maarten E A; Dutta, Aloke K
2006 Jul 13;49(14):4239-4247, Journal of medicinal chemistry
In our previous report, we described a novel series of asymmetric pyran derivatives (2S,4R,5R)-2-benzhydryl-5-benzylamino-tetrahydropyran-4-ol and their enantiomers as blockers of monoamine transporters in the brain. In this report, we describe the further exploration of this series of molecules by incorporating functional groups in the molecular template, which should promote the formation of H bonds with the transporters. In addition, a new synthetic scheme for the asymmetric synthesis of disubstituted cis-(6-benzhydryl-tetrahydro-pyran-3-yl)-benzylamine analogues and their biological characterization is reported. All synthesized derivatives were tested for their affinities for the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in inhibiting the uptake of [(3)H]DA, [(3)H]5-HT, and [(3)H]NE, respectively. The compounds were also tested for their binding potency at the DAT by their ability to inhibit binding of [(3)H]WIN 35, 428. The results indicated that the presence of functional groups, such as -OH, -NH(2), and the bioisosteric 5-substituted indole moiety in both di and trisubstituted compounds, significantly increased their potencies for the SERT and NET, especially for the NET. Among the trisubstituted compounds, (-)-4b exhibited the highest potency for the NET and the SERT (K(i) of 2.13 and 15.3 nM, respectively) and was a serotonin norepinephrine reuptake inhibitor (SNRI). Compound (-)-4a exhibited the highest selectivity for the NET. Among the disubstituted compounds, a number of compounds, such as (-)-9a, (+)-9b, (-)-9b, and (+)-9d, exhibited significant low-nanomolar potencies for the SERT and the NET. Interestingly, compound (-)-9d exhibited appreciable potencies at all three transporters. On the basis of our present and past findings, we propose a qualitative model for the interaction of these compounds with monoamine transporters, which will be refined further in the future
— id: 68806, year: 2006, vol: 49, page: 4239, stat: Journal Article,

Design, synthesis, and preliminary SAR study of 3- and 6-side-chain-extended tetrahydro-pyran analogues of cis- and trans-(6-benzhydryl-tetrahydropyran-3-yl)-benzylamine
Zhang, Shijun; Zhen, Juan; Reith, Maarten E A; Dutta, Aloke K
2006 Jun 1;14(11):3953-3966, Bioorganic & medicinal chemistry
In our effort to further understand interaction of novel pyran derivatives with monoamine transporters, we have designed, synthesized, and biologically characterized side-chain-extended derivatives of our earlier developed cis- and trans-(6-benzhydryl-tetrahydro-pyran-3-yl)-benzylamine derivatives. Both 3- and 6-position extensions were explored. All synthesized derivatives were tested for their affinities for the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in inhibiting the uptake of [(3)H]DA, [(3)H]5-HT, and [(3)H]NE, respectively. Compounds were also tested for their binding affinity at the DAT by their ability to inhibit binding of [(3)H]WIN 35, 428. The results indicated that extension at the 3-position resulted in loss of activity compared to the original compound I. On the other hand, extension at the 6-position resulted in improvement of activity in the compound cis-12 by 2-fold over the parent compound I indicating favorable interaction. In addition, two glycoside derivatives were designed, synthesized, and biologically characterized. The glycosidic trans-isomer 24 exhibited highest potency for the NET in the current series of compounds
— id: 68808, year: 2006, vol: 14, page: 3953, stat: Journal Article,

Chronic food restriction and dopamine transporter function in rat striatum
Zhen, Juan; Reith, Maarten E A; Carr, Kenneth D
2006 Apr 12;1082(1):98-101, Brain research
The present communication reports on DA uptake in rat striatum in a model of chronic food restriction. The K(m) for DA uptake was unaltered, but the V(max) was reduced by 32%, not supporting the idea that the enhanced behavioral sensitivity to cocaine or d-amphetamine upon chronic food restriction is due to a greater density of DAT at the plasma membrane for drug interaction. Chronic food restriction did not alter the potency of cocaine or D-amphetamine in inhibiting DA uptake in the striatum, suggesting that the enhanced behavioral sensitivity to these drugs upon chronic food restriction is not due to their enhanced affinity for DAT. These results point to factors other than DAT density or affinity underlying the sensitized response to psychostimulants in food restriction
— id: 68807, year: 2006, vol: 1082, page: 98, stat: Journal Article,

Synthesis and monoamine transporter affinity of 3-aryl substituted trop-2-enes
Krunic, A; Mariappan, SVS; Reith, MEA; Dunn, WJ
2005 DEC 15 ;15(24):5488-5493, Bioorganic & medicinal chemistry letters
A series of new 3-aryl-tropanes have been synthesized, and their affinities and selectivities were evaluated for monoamine transporters. (1RS)-3-(Fluoren-2-yl)-8-methyl-8-azabicyclo[3.2.1]oct-2-ene exhibited the highest affinity for the human serotonin transporter IC50 = 14.5 nM). It is also 52-fold and 230-fold selective over human dopamine and norepinephrine transporters, respectively. (c) 2005 Elsevier Ltd. All rights reserved
— id: 58998, year: 2005, vol: 15, page: 5488, stat: Journal Article,

Studying monoamine transporters: beyond hypermonoaminaemia
Reith, Maarten E A
2005 Apr 15;143(1):1-2, Journal of neuroscience methods
— id: 57941, year: 2005, vol: 143, page: 1, stat: Journal Article,

Pharmacological profile of radioligand binding to the norepinephrine transporter: instances of poor indication of functional activity
Reith, Maarten E A; Wang, Lijuan C; Dutta, Aloke K
2005 Apr 15;143(1):87-94, Journal of neuroscience methods
Binding assays for the norepinephrine (NE) transporter (NET) with [3H]nisoxetine have generally yielded weak potencies for compounds related to cocaine and 1-(2-(di(4-fluorophenyl)-methoxy)-ethyl)-4-(3-phenylpropyl)piperazine (GBR 12909), as compared with their functional activity in inhibiting NE uptake. In the present work with HEK-293 cells expressing the human NET (hNET), potential underlying causes for this discrepancy have been addressed: ambient temperature of the binding assay, buffer in the assay, preparation used for source of the NET, and radioligand. The results indicate that the standard [3H]nisoxetine binding assay at 0 degrees C with cell membrane preparations in high Na+ buffer underestimates the functional potency of compounds related to cocaine and GBR 12909; in drug development studies it is advisable to either carry out [3H]nisoxetine binding assays with intact cells under uptake conditions, or perform classical [3H]NE uptake studies with intact cells (or with synaptosomes from brain tissue)
— id: 56142, year: 2005, vol: 143, page: 87, stat: Journal Article,

Dopamine and glutamate in psychiatric disorders
Schmidt WJ; Reith MEA
Totowa NJ : Humana Press, 2005,
— id: 1368, year: 2005, vol: , page: , stat: ,

Discovery of novel trisubstituted asymmetric derivatives of (2S,4R,5R)-2-benzhydryl-5-benzylaminotetrahydropyran-4-ol, exhibiting high affinity for serotonin and norepinephrine transporters in a stereospecific manner
Zhang, Shijun; Zhen, Juan; Reith, Maarten E A; Dutta, Aloke K
2005 Jul 28;48(15):4962-4971, Journal of medicinal chemistry
In our structure-activity relationship study on 3,6-disubstituted pyran derivatives, we have carried out asymmetric synthesis and biological characterization of trisubstituted (2S,4R,5R)-2-benzhydryl-5-benzylaminotetrahydropyran-4-ol and (3S,4R,6S)-6-benzhydryl-4-benzylaminotetrahydropyran-3-ol derivatives and their enantiomers. All synthesized derivatives were tested for their affinities for the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in inhibiting the uptake of [(3)H]DA, [(3)H]-5-HT, and [(3)H]NE, respectively. Compounds were also tested for their binding affinity at the DAT by their inhibition of [(3)H]WIN 35,428. Biological results indicated that regioselectivity and stereoselectivity played important roles in determining activity for monoamine transporters as only (-)-isomers of 2-benzhydryl-5-benzylaminotetrahydropyran-4-ol derivatives exhibited appreciable potency for the monoamine transporters, in particular for the SERT and NET. Among the active analogues, (-)-9d exhibited potent and selective affinity at the NET (K(i), [(3)H]NE = 4.92 nM; DAT/NET = 91 and SERT/NET = 140). One of the derivatives with p-methoxybenzyl substitution, (-)-9a, was potent at both SERT and NET (K(i), [(3)H]-5-HT = 25.9 and [(3)H]NE = 15.8 nM, respectively). In the active analogue series ((-)-9a-(-)-9e), a cis-relationship between the biphenyl and the amino moiety was maintained for the SERT and NET interactions, as was observed with our earlier 3,6-disubstituted pyran compounds for the DAT interaction. To the best of our knowledge, this current series of compounds represents a novel class of pyran derivatives as blockers for monoamine transporters
— id: 57940, year: 2005, vol: 48, page: 4962, stat: Journal Article,

Further structural exploration of novel (2,4,5)-trisubstituted asymmetric pyran derivatives: Exploration of different N-benzyl substitutions on activity for monoamine transporters, Part-II
Zhang, SJ; Fernandez, F; Zhen, JA; Reith, M; Dutta, AK
2005 AUG 28 ;230(11):U2532-U2532, Abstracts of papers (American Chemical Society)
— id: 64634, year: 2005, vol: 230, page: U2532, stat: Journal Article,

Discovery of novel (2,4,5)-trisubstituted asymmetric pyran derivatives exhibiting selective affinity for the norepinephrine transporter: Importance of regioselectivity and stereoselectivity in binding activity, Part-I
Zhang, SJ; Zhen, LA; Reith, M; Dutta, AK
2005 AUG 28 ;230(11):U2531-U2531, Abstracts of papers (American Chemical Society)
— id: 64633, year: 2005, vol: 230, page: U2531, stat: Journal Article,

Differences in interactions with the dopamine transporter as revealed by diminishment of Na(+) gradient and membrane potential: Dopamine versus other substrates
Zhen, Juan; Chen, Nianhang; Reith, Maarten E A
2005 Nov;49(6):769-779, Neuropharmacology
In heterologous cells expressing the dopamine transporter (DAT), simultaneous elevation of intracellular Na(+) and depolarization of the membrane with gramicidin reduced the potency of various DAT substrates, including dopamine, d-amphetamine, beta-phenethylamine, p-tyramine, and MPP(+), in inhibiting binding of the cocaine analog [(3)H]CFT, with the greatest reduction observed for d-amphetamine. In rat striatal synaptosomes, gramicidin exerted similar effects; in addition, the potency of d-amphetamine was reduced by the Na(+)-channel activator veratridine. The latter effect was counteracted by the Na(+)-channel blocker tetrodotoxin. In broken membranes, where, as the situation with gramicidin, both sides of the non-polarized membrane were exposed to 130mM Na(+), gramicidin was ineffective. Dopamine had a potency for membrane preparations that was not significantly different from that for control cells or synaptosomes, while other substrates had potencies for membrane preparations that were reduced to a level similar to those observed in gramicidin-treated cells or synaptosomes. These results suggest that diminishing Na(+) gradient and membrane potential may convert DAT to a conformational state that dopamine could easily bind to when gaining free access to its intracellular portion. In contrast, non-dopamine substrates may not be able to readily interact with this state from either side of the membrane
— id: 57939, year: 2005, vol: 49, page: 769, stat: Journal Article,

Structure-activity relationships for substrate recognition by the human dopamine transporter
Appell, Michael; Berfield, Janet L; Wang, Lijuan C; Dunn, William J 3rd; Chen, Nianhang; Reith, Maarten E A
2004 Jan 15;67(2):293-302, Biochemical pharmacology
Information is available on the structure-activity relationships for dopamine as a substrate for uptake by the dopamine transporter. However, dopamine transport is a complex process involving substrate binding, translocation, release as well as transporter reorientation. The present study examines only the substrate recognition step by assessment of the potency of various dopamine-related compounds in inhibiting the binding of the cocaine analog [3H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane ([3H]WIN 35,428) to human dopamine transporters expressed in HEK-293 cells. alpha-Methylation of the side chain, the presence of the amine, and the 2-carbon-length of the side chain were found to be important for binding affinity, whereas beta-hydroxylation of the side chain and methoxylation at the phenyl ring generated weaker compounds. In addition, the presence of both m- and p-OH at the phenyl ring bestowed an increase in potency but the presence of p-OH alone a decrease. N-alkylation (propylation or methylation) had little or an even slightly beneficial effect on affinity, whereas alpha-carbonylation and alpha-methanoylation reduced affinity. Amino naphthalene compounds with a fused benzenoid ring system retained some potency consonant with the extended (i.e. beta-rotameric) trans (=anti) form of the side chain in dopamine when interacting with the transporter. In a second series of experiments, the interaction between dopamine and structural variants was assessed by monitoring the capability of a compound to shift the dopamine inhibition curve to the right as expected for a competitive inhibitor acting at the same site. Appreciable deviation from competitive interaction was observed by removal of the amine from the side chain, by alpha-carbonylation, and by alpha-methanoylation. Two blocker-type compounds, semi-rigid variants of cocaine, also displayed significant deviation. A substrate-based compound, inhibiting cocaine analog binding without interfering with dopamine recognition, could be a cocaine antagonist allowing conformational changes to occur during dopamine uptake
— id: 57947, year: 2004, vol: 67, page: 293, stat: Journal Article,

Synaptic uptake and beyond: the sodium- and chloride-dependent neurotransmitter transporter family SLC6
Chen, Nian-Hang; Reith, Maarten E A; Quick, Michael W
2004 Feb;447(5):519-531, Pflugers archiv = European journal of physiology
The SLC6 family is a diverse set of transporters that mediate solute translocation across cell plasma membranes by coupling solute transport to the cotransport of sodium and chloride down their electrochemical gradients. These transporters probably have 12 transmembrane domains, with cytoplasmic N- and C-terminal tails, and at least some may function as homo-oligomers. Family members include the transporters for the inhibitory neurotransmitters GABA and glycine, the aminergic transmitters norepinephrine, serotonin, and dopamine, the osmolytes betaine and taurine, the amino acid proline, and the metabolic compound creatine. In addition, this family includes a system B(0+) cationic and neutral amino acid transporter, and two transporters for which the solutes are unknown. In general, SLC6 transporters act to regulate the level of extracellular solute concentrations. In the central and the peripheral nervous system, these transporters can regulate signaling among neurons, are the sites of action of various drugs of abuse, and naturally occurring mutations in several of these proteins are associated with a variety of neurological disorders. For example, transgenic animals lacking specific aminergic transporters show profoundly disturbed behavioral phenotypes and probably represent excellent systems for investigating psychiatric disease. SLC6 transporters are also found in many non-neural tissues, including kidney, intestine, and testis, consistent with their diverse physiological roles. Transporters in this family represent attractive therapeutic targets because they are subject to multiple forms of regulation by many different signaling cascades, and because a number of pharmacological agents have been identified that act specifically on these proteins
— id: 57955, year: 2004, vol: 447, page: 519, stat: Journal Article,

Interaction between dopamine and its transporter: role of intracellular sodium ions and membrane potential
Chen, Nianhang; Reith, Maarten E A
2004 Jun;89(3):750-765, Journal of neurochemistry
The present study addresses the effect of intracellular Na(+) and membrane potential on the binding of dopamine (DA) to the dopamine transporter (DAT). Perforation of plasma membranes of DAT-expressing cells with gramicidin diminished DA uptake and decreased the potency (increases K(i)) of DA in inhibiting the binding of cocaine analog [(3)H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT). It also compromised the ability of external Na(+) to reduce DA K(i). No substantial effect on DA K(i) was observed upon gramicidin treatment in Na(+)-free buffer, membrane depolarization with high [K(+)](o), or elevation of [Na(+)](i) with monensin under non-depolarizing conditions. Elevation of DA K(i) was greater at more positive potentials when [Na(+)](i) was raised to a similar level, or at higher [Na(+)](i) when the membrane was depolarized to a similar level. In cells expressing D313N DAT, DA K(i) was significantly higher but less sensitive to gramicidin than that in wild-type (WT) cells. In contrast, DA K(i) in cell-free membranes was insensitive to Na(+), gramicidin, and D313N mutation. The data suggest that (i) intracellular Na(+) plays a role in affecting the external access to DA binding sites at DAT on depolarized plasma membranes of cells, and (ii) access to DA binding sites in cell-free membranes may occur from the intracellular side of the membrane. Unlike DA binding, CFT binding to both cells and membranes was sensitive to Na(+) and D313N mutation but insensitive to gramicidin, consistent with exclusively external access to sites that are different from but conformationally linked to those for DA
— id: 46216, year: 2004, vol: 89, page: 750, stat: Journal Article,

Aspartate 345 of the dopamine transporter is critical for conformational changes in substrate translocation and cocaine binding
Chen, Nianhang; Rickey, Judy; Berfield, Janet L; Reith, Maarten E A
2004 Feb 13;279(7):5508-5519, Journal of biological chemistry
The present study elucidated the role of aspartate 345, a residue conserved in the third intracellular loop of all Na+/Cl(-)-dependent neurotransmitter transporters, in conformational changes of the dopamine (DA) transporter. Asparagine substitution (D345N) resulted in near normal transporter expression on the cell surface but caused extremely low Vmax and Km values for DA uptake, converted the inhibitory effect of Zn2+ on DA uptake to a stimulatory one, and eliminated reverse transport. The cocaine-like inhibitor 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane or the selective DA transporter inhibitor GBR12935 bound to D345N with a normal affinity and still inhibited DA uptake potently. However, the mutation reduced the binding capacity of the surface transporter for these two inhibitors by 90% or more. Moreover, the binding activity of D345N can be significantly improved by Zn2+ but not by Na+. These results are consistent with a defect in reorientation of the substrate-binding site to the extracellular side, leading to a loss of the outward-facing conformational state where external DA binds to initiate uptake and the inhibitors bind to initiate uptake inhibition. Alanine or glutamate substitution produced a similar phenotype, suggesting that both the negative charge and the residue volume at position 345 are vital. Furthermore, in intact cells, cocaine potentiated the reaction of the membrane-impermeant sulfhydryl reagent methanethiosulfonate ethyltrimethylammonium with the extracellularly located endogenous cysteines of D345N but not those of wild type, and this potentiation was blocked upon K+ substitution for Na+. Thus, cocaine binding to D345N likely induces a different and Na(+)-dependent conformational change, which may contribute to its Na(+)-dependent uptake inhibitory activity
— id: 42638, year: 2004, vol: 279, page: 5508, stat: Journal Article,

Mutation of Trp84 and Asp313 of the dopamine transporter reveals similar mode of binding interaction for GBR12909 and benztropine as opposed to cocaine
Chen, Nianhang; Zhen, Juan; Reith, Maarten E A
2004 May;89(4):853-864, Journal of neurochemistry
The different psychomotor-stimulant effects of cocaine, GBR12909, and benztropine may partially stem from their different molecular actions on the dopamine transporter (DAT). To explore this possibility, we examined binding of these inhibitors to mutated DATs with altered Na(+) dependence of DAT activities and with enhanced binding of a cocaine analog, [(3)H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (CFT). In [(3)H]CFT competition assays with intact cells, the mutation-induced change in the ability of Na(+) to enhance the apparent affinity of CFT, cocaine, GBR12909, and benztropine was inhibitor-independent. Thus, for the four inhibitors, the curve of [Na(+)] versus apparent ligand affinity was steeper at W84L compared with wild type, shallower at D313N, and flat at W84LD313N. At each mutant, the apparent affinity of CFT and cocaine was enhanced regardless of whether Na(+) was present. However, the apparent affinity of GBR12909 and benztropine for W84L was reduced in the absence of Na(+) but near normal in the presence of 130 mm Na(+), and that for D313N and W84LD313N was barely changed. At the single mutants, the alterations in Na(+) dependence and apparent affinity of the four inhibitors were comparable between [(3)H]CFT competition assays and [(3)H]dopamine uptake inhibition assays. These results demonstrate that DAT inhibitors producing different behavioral profiles can respond in an opposite way when residues of the DAT protein are mutated. For GBR12909 and benztropine, their cocaine-like changes in Na(+) dependence suggest that they prefer a DAT state similar to that for cocaine. However, their cocaine-unlike changes in apparent affinity argue that they, likely via their diphenylmethoxy moiety, share DAT binding epitopes that are different from those for cocaine
— id: 48721, year: 2004, vol: 89, page: 853, stat: Journal Article,

Synthesis and biological characterization of novel hybrid 7-[[2-(4-phenyl-piperazin-1-yl)-ethyl]-propyl-amino]-5,6,7,8-tetrahydro-na phthalen-2-ol and their heterocyclic bioisosteric analogues for dopamine D2 and D3 receptors
Dutta, Aloke K; Venkataraman, Sylesh K; Fei, Xiang-Shu; Kolhatkar, Rohit; Zhang, Shijun; Reith, Maarten E A
2004 Aug 15;12(16):4361-4373, Bioorganic & medicinal chemistry
In a recent preliminary communication we described the development of a series of hybrid molecules for the dopamine D2 and D3 receptor subtypes. The design of these compounds was based on combining pharmacophoric elements of aminotetralin and piperazine molecular fragments derived from known dopamine receptor agonist and antagonist molecules. Molecules developed from this approach exhibited high affinity and selectivity for the D3 receptor as judged from preliminary [(3)H]spiperone binding data. In this report, we have expanded our previous finding by developing additional novel molecules and additionally evaluated functional activities of these novel molecules in the [(3)H]thymidine incorporation mitogenesis assay. The binding results indicated highest selectivity in the bioisosteric benzothiazole derivative N6-[2-(4-phenyl-piperazin-1-yl)-ethyl]-N6-propyl-4,5,6,7-tetrahydro-benzot hiazole-2,6-diamine (14) for the D3 receptor whereas the racemic compound 7-([2-[4-(2,3-dichloro-phenyl)-piperazin-1-yl]-ethyl]-propyl-amino)-5,6,7, 8-tetrahydro-naphthalen-2-ol (10c) showed the strongest potency. Mitogenesis studies to evaluate functional activity demonstrated potent agonist properties in these novel derivatives for both D2 and D3 receptors. In this regard, compound 7-[[4-(4-phenyl-piperazin-1-yl)-butyl]-prop-2-ynyl-amino]-5,6,7,8-tetrahyd ro-naphthalen-2-ol (7b) exhibited the most potent agonist activity at the D3 receptor, 10 times more potent than quinpirole and was also the most selective compound for the D3 receptor in this series. Racemic compound 10a was resolved; however, little separation of activity was found between the two enantiomers of 10a. The marginally more active enantiomer (-)-10a was examined in vivo using the 6-OH-DA induced unilaterally lesioned rat model to evaluate its activity in producing contralateral rotations. The results demonstrated that in comparison to the reference compound apomorphine, (-)-10a was quite potent in inducing contralateral rotations and exhibited longer duration of action
— id: 57945, year: 2004, vol: 12, page: 4361, stat: Journal Article,

Further structurally constrained analogues of cis-(6-benzhydrylpiperidin-3-yl)benzylamine with elucidation of bioactive conformation: discovery of 1,4-diazabicyclo[3.3.1]nonane derivatives and evaluation of their biological properties for the monoamine transporters
Kolhatkar, Rohit; Cook, Charles D; Ghorai, Sujit K; Deschamps, Jeffrey; Beardsley, Patrick M; Reith, Maarten E A; Dutta, Aloke K
2004 Oct 7;47(21):5101-5113, Journal of medicinal chemistry
Our structure-activity relationship (SAR) study on piperidine analogues for monoamine transporters led to the development of a series of 3,6-disubstituted piperidine derivatives, structurally constrained versions of flexible piperidine analogues, with preferential affinity for the dopamine transporter (DAT). In our attempt to further rigidify this structure to study influence of rigidity on binding and in vivo activity, we have developed a series of 4,8-disubstituted 1,4-diazabicyclo[3.3.1]nonane derivatives. All synthesized derivatives were tested for their affinity at the DAT, serotonin transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in competing for the binding of [(3)H]WIN 35, 428, [(3)H]citalopram, and [(3)H]nisoxetine, respectively. Selected compounds were also tested for their ability to inhibit uptake of [(3)H]DA. The SAR study led to the discovery of a potent lead compound (-)-S,S-10c which exhibited high affinity and selectivity for the DAT (IC(50) = 22.5 nM; SERT/DAT = 384 and NET/DAT > 444). It is interesting to note that both (-)-10c and the lead compound from the 3,6-disubstituted series (-)-2 exhibited highest activity in their (S,S) isomer indicating similar requirement of regiospecificity for maximum interaction. Overall, our current SAR results corresponded well with the results from less constrained 3,6-disubstituted versions of these molecules albeit the former class exhibited more stringent requirement in molecular structure for activity. However, the potent compounds in the current series exhibited greater selectivity for the DAT compared to their corresponding lesser constrained 3,6-disubstituted versions indicating an effect of rigidity in selective interaction with the transporter proteins. In an effort to elucidate the bioactive conformational structure of the lead molecules in the current and the 3,6-disubstituted series, a preliminary molecular modeling study was carried out where the most rigid derivative (-)-10c was used as a template structure. Compounds (-)-2 and (-)-10c exhibited stimulant activity in locomotor tests in mice in which (-)-2 exhibited a slower onset and longer duration of action compared to (-)-10c. Both compounds occasioned complete cocaine-like responding in mice trained to discriminate 10 mg/kg ip cocaine from vehicle
— id: 57944, year: 2004, vol: 47, page: 5101, stat: Journal Article,

The role of N-glycosylation in function and surface trafficking of the human dopamine transporter
Li, Li-Bin; Chen, Nianhang; Ramamoorthy, Sammanda; Chi, Limen; Cui, Xiao-Nan; Wang, Lijuan C; Reith, Maarten E A
2004 May 14;279(20):21012-21020, Journal of biological chemistry
The present study addressed the role of N-linked glycosylation of the human dopamine transporter (DAT) in its function with the help of mutants, in which canonical N-glycosylation sites have been removed (N181Q, N181Q,N188Q, and N181Q,N188Q,N205Q), expressed in human embryonic kidney-293 cells. Removal of canonical sites produced lower molecular weight species as did enzymatic deglycosylation or blockade of glycosylation, and all three canonical sites were found to carry sugars. Prevention of N-glycosylation reduced both surface and intracellular DAT. Although partially or non-glycosylated DAT was somewhat less represented at the surface, no evidence was found for preferential exclusion of such material from the plasma membrane, indicating that glycosylation is not essential for DAT expression. Non-glycosylated DAT was less stable at the surface as revealed by apparently enhanced endocytosis, consonant with weaker DAT immunofluorescence at the cell surface and stronger presence in cytosol in confocal analysis of the double and triple mutant. Non-glycosylated DAT did not transport dopamine as efficiently as wild-type DAT as judged from the sharp reduction in uptake V(max), and prevention of N-glycosylation enhanced the potency of cocaine-like drugs in inhibiting dopamine uptake into intact cells without changing their affinity for DAT when measured in membrane preparations prepared from these cells. Thus, non-glycosylated DAT at the cell surface displays appreciably reduced catalytic activity and altered inhibitor sensitivity compared with wild type
— id: 57946, year: 2004, vol: 279, page: 21012, stat: Journal Article,

Structural requirements for 2,4- and 3,6-disubstituted pyran biomimetics of cis-(6-benzhydryl-piperidin-3-yl)-benzylamine compounds to interact with monoamine transporters
Zhang, Shijun; Zhen, Juan; Reith, Maarten E A; Dutta, Aloke K
2004 Dec 1;12(23):6301-6315, Bioorganic & medicinal chemistry
In our effort to delineate novel pharmacophoric configuration of bioisosteric pyran versions of cis-(6-benzhydryl-piperidin-3-yl)-benzylamine derivatives in interacting with the monoamine transporter, further structure-activity relationship study was carried out. Both cis and trans 2,4- and 3,6-disubstituted derivatives were synthesized to determine the positional importance of N-substitution on affinity for monoamine transporters, that is the dopamine transporter (DAT), the serotonin transporter (SERT), and the norepinephrine transporter (NET) in rat brain. For that purpose, the potency of compounds was determined in competing for the binding of [(3)H]WIN 35,428, [(3)H]citalopram, and [(3)H]nisoxetine, respectively. Selected compounds were also evaluated for their activity in inhibiting the uptake of [(3)H]DA by DAT. Our binding results demonstrated potency in 3,6-disubstituted derivatives while 2,4-disubstituted derivatives failed to exhibit any appreciable binding affinity. Further structural exploration of the exocyclic N-atom in 3,6-disubstituted derivatives produced compounds potent at both DAT and NET. Compounds 16h and 16o with hydroxyl and amino groups in the phenyl moiety of the benzyl group produced the highest activity for the NET. In this regard, compound 16e with a methoxy substituent produced weak affinity at NET, which upon conversion into a hydroxyl functionality as in 16h produced potent affinity for the NET. Various indole derivatives displayed different interactions; the 5-substituted indole derivative 16n exerted potent affinity for NET, confirming the bioisosteric equivalence between this indole moiety and the phenyl-4-hydroxy group in 16h
— id: 57943, year: 2004, vol: 12, page: 6301, stat: Journal Article,

Interaction between a hydroxypiperidine analogue of 4-(2-benzhydryloxy-ethyl)-1-(4-fluorobenzyl)piperidine and Aspartate 68 in the human dopamine transporter
Zhen, Juan; Maiti, Soumen; Chen, Nianhang; Dutta, Aloke K; Reith, Maarten E A
2004 Dec 3;506(1):17-26, European journal of pharmacology
Compound (+)-R,R-D-84 is an optically active trans-hydroxy-substituted derivative of 4-(2-benzhydryloxy-ethyl)-1-(4-fluorobenzyl)piperidine (D-164). As a hydroxypiperidine analog of GBR 12935, (+)-R,R-D-84 is a candidate dopamine transporter compound for the treatment of cocaine dependence. The present work addresses the functional activity of (+)-R,R-D-84 at monoamine transporters and its potential molecular mechanism involving acidic amino acids (D and E). The selectivity for the dopamine vs. serotonin transporter of (+)-R,R-D-84 was greater than that of (-)-S,S-D-83, its enantiomer, and the selectivity of both compounds was greater than that of GBR 12909 (diphenyl-fluorinated GBR 12935). Only (+)-R,R-D-84 displayed improved selectivity vs. the norepinephrine transporter. D313N or E215Q mutation did not alter the pattern of affinities (measured by membrane binding of the cocaine analog [3H]CFT) for the dopamine transporter of (+)-R,R-D-84, (-)-S,S-D-83, D-164 (non-hydroxylated analog), or GBR 12909. In contrast, D68N mutation specifically lowered the affinity of (+)-R,R-D-84, pointing to a role for D68 in the interaction with (+)-R,R-D-84, possibly through hydrogen bonding between the hydroxyl and the carboxyl group of D68 which is lacking in N68. The present results, combined with behavioral data, implicate D68 in the dopamine transporter in cocaine antagonist activity of (+)-R,R-D-84
— id: 57942, year: 2004, vol: 506, page: 17, stat: Journal Article,

Inhibition by arachidonic acid and other fatty acids of dopamine uptake at the human dopamine transporter
Chen, Nianhang; Appell, Michael; Berfield, Janet L; Reith, Maarten E A
2003 Oct 8;478(2-3):89-95, European journal of pharmacology
It is known that arachidonic acid, in addition to promoting release of dopamine, can inhibit its transport. The present study provides preliminary information on structure-activity relationships for uptake inhibition by rotating disk voltammetry in human embryonic kidney-293 cells expressing the human dopamine transporter. Except for anandamide, all other fatty acids studied at a pretreatment concentration of 80 microM caused significant reductions in Vmax but not Km. Increasing saturation of the hydrocarbon tails (partial saturation: oleic acid, linoleic acid; full saturation: arachidic acid, stearic acid, stearic acid ethyl ester) removed inhibitory activity incrementally, suggesting a role for cis-unsaturation (folding/bending of hydrocarbon tails). The relative lack of effect of 5,8,11,14-eicosatetraynoic acid also supports the idea that less linear structures are less inhibitory on dopamine uptake. Esterification of the free carboxylic group (arachidonic acid ethyl ester) prevented most of the inhibitory activity, arguing against mere membrane lipid disruption. Finally, the endogenous cannabinoid anandamide greatly reduced uptake Vmax accompanied by a small decrease in Km, a potentially important effect on dopaminergic neurotransmission
— id: 57951, year: 2003, vol: 478, page: 89, stat: Journal Article,

Na+ and the substrate permeation pathway in dopamine transporters
Chen, Nianhang; Reith, Maarten E A
2003 Oct 31;479(1-3):213-221, European journal of pharmacology
Advances have been made in characterizing the relationship between Na+ and the substrate permeation pathway in the dopamine transporter. This review covers the role of Na+ in co-transport with dopamine as well as in the recognition of dopamine. Apparent recognition depends on the preparation studied: it differs between intact cells heterologously expressing the dopamine transporter and membranes prepared from these cells. In our search for amino acid residues in the transporter involved in Na+ action, W84 and D313 were found to play a special role in cation interaction, with evidence for regulation of both Na+ and H+ sensitivity. Mutation of D313 to N appeared to decrease the affinity for the dopamine transporter in intact cells, not by altering recognition per se. A model is proposed in which access of dopamine, not recognition itself, is regulated by D313 and Na+. Thus, the role of external Na+ in intact cell preparations is to turn dopamine transporters to the externally facing form, allowing access of dopamine to its binding site
— id: 57949, year: 2003, vol: 479, page: 213, stat: Journal Article,

Na+ stimulates binding of dopamine to the dopamine transporter in cells but not in cell-free preparations
Chen, Nianhang; Rickey, Judy; Reith, Maarten E A
2003 Aug;86(3):678-686, Journal of neurochemistry
Although Na+ is crucial for the function of the dopamine (DA) transporter (DAT), its role in the substrate binding step has been questioned. To address this issue, we investigated the effect of Na+ on DA binding by measuring the potency of DA in inhibiting the binding of the cocaine analogue [3H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) in intact cells expressing DAT in their plasma membranes and in membranes isolated from these cells. In cells, Na+ substantially enhanced the potency of DA in inhibiting CFT binding. This effect of Na+ was independent of buffer compositions and substitutes (sucrose vs. NMDG), more pronounced at 4 degrees C than 25 degrees C, and correlated with its stimulatory effect on DA uptake Km. Removing extracellular Na+ had little effect on intracellular concentrations of Na+ and K+, or on membrane potential. These data suggest that extracellular Na+ most likely acts at the transporter level to enhance the binding of external DA during the transport cycle. In contrast, in cell-free membrane preparations the Na+ stimulation was abolished without impairment of the potency of DA in inhibiting CFT binding, regardless of whether sucrose was used to maintain the buffer osmolarity. The difference in Na+ dependence for DA to inhibit CFT binding between plasma membranes of intact cells and isolated membranes raises the possibility that intracellular ion environment, alone or in combination with other cellular factors, plays a critical role in determining DA-DAT interaction and the integration of Na+ modulation in this interaction
— id: 57953, year: 2003, vol: 86, page: 678, stat: Journal Article,

Substrate-induced trafficking of the dopamine transporter in heterologously expressing cells and in rat striatal synaptosomal preparations
Chi, Limen; Reith, Maarten E A
2003 Nov;307(2):729-736, Journal of pharmacology & experimental therapeutics
Dopamine transporter (DAT) trafficking was assessed by functional measurements of dopamine uptake and by biotinylation of surface proteins followed by gel electrophoresis and Western blotting. In human embryonic kidney (HEK)-293 cells expressing human DAT (HEK-hDAT), pretreatment with dopamine (0.1-100 microM) followed by washout caused reductions in subsequent dopamine uptake (reflected in Vmax) with effective dopamine concentrations in the 10 to 100 microM range and pretreatment times of 10 to 60 min. Reductions assessed after 60-min pretreatment with 100 microM dopamine corresponded with decreases measured in surface DAT by the noncleavable biotin method, which were caused, at least in part, by enhanced endocytosis as monitored with cleavable biotin. Pretreatment of rat striatal synaptosomes with dopamine (10 and 100 microM) also caused reductions in DAT uptake activity (Vmax), and again the underlying mechanism seemed to be a diminished presence of DAT at the surface of synaptosomes as measured by the noncleavable biotin method. The copresence of cocaine during pretreatment with dopamine prevented the down-regulation of surface DAT. The present results show that DAT surface residency can be regulated by substrate acting on it, not only in cells heterologously expressing DAT but also in situ in rat brain tissue
— id: 57952, year: 2003, vol: 307, page: 729, stat: Journal Article,

Dopamine transporter as target for drug development of cocaine dependence medications
Dutta, Aloke K; Zhang, Shijun; Kolhatkar, Rohit; Reith, Maarten E A
2003 Oct 31;479(1-3):93-106, European journal of pharmacology
Because much evidence implicates the dopamine transporter in the reinforcing effects of cocaine, development of potential medications for cocaine dependence has included the dopamine transporter as a target. The present overview covers progress in the drug development area regarding several classes of dopamine uptake inhibitors, with an emphasis on structure-activity relationships that enhance potency and selectivity at transporters for dopamine compared with those for serotonin or norepinephrine. The following categories of compounds are covered: tropane, benztropine, 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR), methylphenidate, mazindol, and phencyclidine analogs. Activity at transporters as well as on behavior is highlighted
— id: 57950, year: 2003, vol: 479, page: 93, stat: Journal Article,

High affinity hydroxypiperidine analogues of 4-(2-benzhydryloxyethyl)-1-(4-fluorobenzyl)piperidine for the dopamine transporter: stereospecific interactions in vitro and in vivo
Ghorai, Sujit K; Cook, Charles; Davis, Matthew; Venkataraman, Sylesh K; George, Clifford; Beardsley, Patrick M; Reith, Maarten E A; Dutta, Aloke K
2003 Mar 27;46(7):1220-1228, Journal of medicinal chemistry
In our effort to develop high-affinity ligands for the dopamine transporter which might find potential use as cocaine medication, a polar hydroxy substituent was introduced into the piperidine ring of one of our disubstituted lead analogues derived from 1-[2-(diphenylmethoxy)-ethyl]-4-(3-phenylpropyl)piperazine (GBR 12935). Both cis- and trans-3-hydroxy derivatives were synthesized and the racemic trans isomer, (+/-)-5, was further resolved into two enantiomers. Newly synthesized compounds were characterized for their binding affinity at the dopamine, serotonin, and norepinephrine transporter systems in rat brain. The two enantiomers (+)-5 and (-)-5 exhibited marked differential affinities at the dopamine transporter with (+)-5 being 122-fold more potent than (-)-5 in inhibiting radiolabeled cocaine analogue binding (IC(50); 0.46 vs 56.7 nM) and 9-fold more active for inhibiting dopamine uptake (IC(50); 4.05 vs 38.0 nM). Furthermore, the most active (+)-5 was 22-fold more potent at the dopamine transporter compared to the standard GBR 12909. Absolute configuration of one of the enantiomers was determined unambiguously by X-ray structural analysis. In in vivo locomotor activity studies, the enantiomer (+)-5 and the racemic (+/-)-5, but not (-)-5, exhibited stimulant activity with a long duration of effect. All three compounds, (+)-5, (-)-5, and (+/-)-5, within the dose range tested, partially (50%) but incompletely (80%) produced cocaine-like responses in mice trained to discriminate 10 mg/kg ip cocaine from vehicle. Compound (-)-5 was distinctive in this regard in that, unlike (+)-5 and (+/-)-5, it did not affect locomotor activity yet, but similar to them, was able to engender (albeit incompletely) cocaine-like responses
— id: 57956, year: 2003, vol: 46, page: 1220, stat: Journal Article,

Interaction of cis-(6-benzhydrylpiperidin-3-yl)benzylamine analogues with monoamine transporters: structure-activity relationship study of structurally constrained 3,6-disubstituted piperidine analogues of (2,2-diphenylethyl)-[1-(4-fluorobenzyl)piperidin-4-ylmethyl]amine
Kolhatkar, Rohit B; Ghorai, Sujit K; George, Clifford; Reith, Maarten E A; Dutta, Aloke K
2003 May 22;46(11):2205-2215, Journal of medicinal chemistry
To explore structure-activity relationships (SAR) of a novel conformationally constrained lead cis-3,6-disubstituted piperidine derivative derived from (2,2-diphenylethyl)-[1-(4-fluorobenzyl)piperidine-4-ylmethyl]amine (I), a series of compounds was synthesized by derivatizing the exocyclic N-atom at the 3-position of the lead. This study led to the formation of substituted phenyl and heterocyclic derivatives. All novel compounds were tested for their affinity at the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in competing for the binding of [3H]WIN 35 428, [3H]citalopram, and [3H]nisoxetine, respectively. Selected compounds were also evaluated for their activity in inhibiting the uptake of [3H]DA. The SAR results demonstrated that the nature of substitutions on the phenyl ring is important in activity at the DAT with the presence of an electron-withdrawing group having the maximum effect on potency. Replacement of the phenyl ring in the benzyl group by heterocyclic moieties resulted in the development of compounds with moderate activity for the DAT. Two most potent racemic compounds were separated by a diastereoisomeric separation procedure, and differential affinities were observed for the enantiomers. Absolute configuration of the enantiomers was obtained unambiguously by X-ray crystal structural study. One of the enantiomers, compound S,S-(-)-19a, exhibited the highest potency for the DAT (IC50 = 11.3 nM) among all the compounds tested and was as potent as GBR 12909 (1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine). However, the compound (-)-19a was more selective than GBR 12909 in binding to the DAT compared with binding to the SERT and NET. The present results establish the newly developed 3,6-disubstituted piperidine derivatives as a novel template for high-affinity inhibitors of DAT. Structurally these molecules are more constrained compared to our earlier flexible piperidine molecules and, thus, should provide more insights about their bioactive conformations
— id: 57954, year: 2003, vol: 46, page: 2205, stat: Journal Article,

Transporters as targets for drugs and endogenous compounds - Preface
Reith, MEA
2003 OCT 31 ;479(1-3):1-1, European journal of pharmacology
— id: 98221, year: 2003, vol: 479, page: 1, stat: Journal Article,

Binding of cocaine-like radioligands to the dopamine transporter at 37 degrees C: effect of Na+ and substrates
Wang, Lijuan C; Cui, Xiao-Nan; Chen, Nianhang; Reith, Maarten E A
2003 Dec 30;131(1-2):27-33, Journal of neuroscience methods
Although dopamine (DA) translocation by the DA transporter (DAT) requires Na+, a role for Na+ in the DA recognition step in the translocation cycle has been questioned. Thus, when binding techniques were used to indirectly measure the affinity of DA for DAT via its potency in inhibiting cocaine analog binding, no stimulation of DA binding was observed when assay temperature was at or below room temperature. The present work describes the use of 3H-labeled cocaine analogs for assays at 37 degrees C. When there is sufficient Na+ in the medium (> or =25 mM), [3H]2beta-carbomethoxy-3beta-(4-iodophenyl)tropane ([3H]CIT) is an excellent radioligand to label human DAT with high affinity in membrane preparations of HEK-293 cells expressing the transporter. However, at 0 and 5 mM of Na+, appreciable binding of [3H]CIT occurs to proteins other than DAT, hampering accurate assessment of DAT-associated binding. No such problems occur with the binding of the 4-fluoro homolog of [3H]CIT, [3H]CFT at 37 degrees C, and this radioligand can be used at low [Na+], provided enough protein is present in the assay. The application of these assays show that, in contrast to the strong Na+ dependency of the binding of CFT, the substrates DA, D-amphetamine, p-tyramine, and DL-octopamine are not stimulated by Na+. This demonstrates that lack of Na+ stimulation of binding of substrates, including DA to DAT, in membrane preparations at room temperature is not caused by the reduced fluidity of the frozen state of the hydrocarbon membrane interior at this temperature as compared with the liquid-expanded state at 37 degrees C
— id: 57948, year: 2003, vol: 131, page: 27, stat: Journal Article,

An analysis of the binding of cocaine analogues to the monoamine transporters using tensor decomposition 3-d QSAR
Appell, Michael; Dunn, William J 3rd; Reith, Maarten E A; Miller, Larry; Flippen-Anderson, Judith L
2002 May;10(5):1197-1206, Bioorganic & medicinal chemistry
The conformation and alignment of cocaine analogues bound to the monoamine transporter proteins were explored using the tensor decomposition 3-D QSAR method. It is proposed from these calculations that the bound conformation of these ligands to the three transporter proteins has the 3beta-aryl substituent in a conformation in which the aryl group is orthogonal or approximately orthogonal to the tropane ring. Based on these results, rigid and semi-rigid tropane analogues were designed, synthesized and their affinities for the monoamine transporters were determined
— id: 57962, year: 2002, vol: 10, page: 1197, stat: Journal Article,

Nitric oxide inhibits uptake of dopamine and N-methyl-4-phenylpyridinium (MPP+) but not release of MPP+ in rat C6 glioma cells expressing human dopamine transporter
Cao, Bo-Jin; Reith, Maarten E A
2002 Dec;137(8):1155-1162, British journal of pharmacology
1. Conflicting results have been reported regarding the influence of nitric oxide (NO) and peroxynitrite on dopamine (DA) uptake and release. In the present study, effects of NO donors were studied in rat C6 glioma cells expressing human DA transporter. 2. [(3)H]-DA uptake was inhibited by S-nitroso-thiol S-nitroso-N-acetylpenicillamine, spermine/NO, diethylamine/NO (DEA/NO), (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)-amino]/NO (PAPA/NO), and 3-morphosynodiomine (SIN-1) in a rank order correlating with their half lives as NO donors, whereas no effect was observed for diethylenetriamine/NO and dipropylenetriamine/NO, which release NO very slowly. 3. Hydroxycobalamin, a NO scavenger, but not superoxide dismutase and catalase, enzymes that metabolize superoxide and hydrogen peroxide, respectively, abolished the inhibitory effect of DEA/NO and SIN-1, indicating that they inhibit DA uptake through a mechanism related to the production of NO but unrelated to the formation of peroxynitrite. In consonance, peroxynitrite did not alter DA uptake in the present system. 4. DEA/NO and PAPA/NO reduced [(3)H]-MPP(+) uptake, whereas the release of [(3)H]-MPP(+) was not modified, demonstrating that NO can inhibit uptake of DA transporter substrate without accelerating DA transporter-mediated reverse transport of substrate under the same conditions
— id: 57957, year: 2002, vol: 137, page: 1155, stat: Journal Article,

Nitric oxide scavenger carboxy-PTIO potentiates the inhibition of dopamine uptake by nitric oxide donors
Cao, Bo-Jin; Reith, Maarten E A
2002 Jul 12;448(1):27-30, European journal of pharmacology
2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO) has been increasingly used as nitric oxide (NO) scavenger. Carboxy-PTIO reacts with NO to form nitric dioxide and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl (carboxy-PTI). In rat C6 glioma cells expressing human dopamine transporter, carboxy-PTIO paradoxically potentiated the inhibition of [(3)H]dopamine uptake by two NO donors, diethylamine/NO and (Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)-amino]/NO. Further examinations revealed that carboxy-PTI concentration-dependently reduced dopamine uptake, indicating that the formation of carboxy-PTI may account for the failure of carboxy-PTIO to abolish NO elicited effects. These results suggest that caution should be taken in interpreting data obtained using carboxy-PTIO and probably other NO scavengers
— id: 57958, year: 2002, vol: 448, page: 27, stat: Journal Article,

Cationic interactions at the human dopamine transporter reveal binding conformations for dopamine distinguishable from those for the cocaine analog 2 alpha-carbomethoxy-3 alpha-(4-fluorophenyl)tropane
Chen, Nianhang; Sun, LingLing; Reith, Maarten E A
2002 Jun;81(6):1383-1393, Journal of neurochemistry
In membrane preparations, CFT, a phenyltropane cocaine analog, and dopamine (DA) interact with the recombinant human dopamine transporter (hDAT) in Na+ -free medium. Na+ markedly increased the transporter's affinity for CFT, but had little or no effect on DA potency for inhibiting CFT binding. Raising [Na+ ] from 20 to 155 mm reduced Li+ -induced increase in DA K (i), but not CFT K (d). The presence of 155 mm Na+ enhanced the tolerance to low pH of CFT Kd but not DA Ki. Leucine substitution for tryptophan 84 (W84L) in transmembrane domain (TM) 1 or asparagine substitution for aspartate 313 (D313N) in TM 6 did not or only modestly enhance the affinity of Na+ -independent CFT binding, and retained the near normal ability of DA, Li+, K+, or H+ to inhibit this binding. However, the mutations significantly enhanced the Na+ stimulation of CFT binding as well as the Na+ antagonism against Li+ and H+ inhibition of CFT binding. In contrast, the mutations neither changed the Na+ -insensitive feature of DA Ki nor enhanced the Na+ protection of DA Ki against Li+ 's inhibitory effect, though they caused Na+ protection of DA Ki against H+ 's inhibitory action. These results are consistent with the existence of binding conformations for DA that are distinguishable from those for CFT, and with a differential association of cation interactions with DA and CFT binding. The mutations likely alter Na+ -bound state(s) of hDAT, preferentially strengthening the positive allosteric coupling between Na+ and CFT binding, and reducing the impact of Li+ or H+ on the CFT binding
— id: 57960, year: 2002, vol: 81, page: 1383, stat: Journal Article,

Expansion of structure-activity studies of piperidine analogues of 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine (GBR 12935) compounds by altering substitutions in the N-benzyl moiety and behavioral pharmacology of selected molecules
Dutta, Aloke K; Davis, Matthew C; Fei, Xiang-Shu; Beardsley, Patrick M; Cook, Charles D; Reith, Maarten E A
2002 Jan 31;45(3):654-662, Journal of medicinal chemistry
A series of substituted N-benzyl analogues of the dopamine transporter (DAT) specific compound, 4-[2-(diphenylmethoxy)ethyl]-1-benzylpiperidine were synthesized and biologically characterized. Different 4'-alkyl, 4'-alkenyl, and 4'-alkynyl substituents were introduced in the phenyl ring of the benzyl moiety along with the replacement of the same phenyl ring by the isomeric alpha- and beta-naphthyl groups. Different polar substitutions at the 3'- and 4'-position were also introduced. Novel compounds were tested for their binding affinity at the dopamine, serotonin, and norepinephrine transporter systems in the brain by competing for [(3)H]WIN 35 428, [(3)H]citalopram, and [(3)H]nisoxetine, respectively. Selected compounds were also evaluated for their activity in inhibiting the uptake of [(3)H]dopamine. Binding results demonstrated that alkenyl and alkynyl substitutions at the 4'-position produced potent compounds in which compound 6 with a vinyl substitution was the most potent. In vivo evaluation of three selected compounds indicated that despite their high potency at the DAT, these compounds stimulated locomotor activity (LMA) less than cocaine when tested across similar dose ranges. In a drug discrimination study procedure, none of these three compounds generalized from cocaine in mice trained to discriminate 10 mg/kg cocaine from vehicle. In a 4 h time course LMA experiment, one of our previous lead piperidine derivatives (1a) showed considerable prolonged action. Thus, in this report, we describe a structure-activity relationship study of novel piperidine analogues assessed by both in vitro transporter assays and in vivo behavioral activity measurements
— id: 57964, year: 2002, vol: 45, page: 654, stat: Journal Article,

A novel series of hybrid compounds derived by combining 2-aminotetralin and piperazine fragments: binding activity at D2 and D3 receptors
Dutta, Aloke K; Fei, Xiang-Shu; Reith, Maarten E A
2002 Feb 25;12(4):619-622, Bioorganic & medicinal chemistry letters
A series of 7-hydroxy-2-[N-alkyl-(N-(4-phenylpiperazine)-alkyl)amino]tetralins was developed based on a novel hybrid approach that combined 2-aminotetralin and arylpiperazine pharmacophoric moieties. Our preliminary study revealed that a four-methylene butyl linker produced very potent compounds for both the D2 and D3 receptors. Further structure-activity studies led to a novel template showing 50- to 100-fold selectivity for the D3 receptor
— id: 57963, year: 2002, vol: 12, page: 619, stat: Journal Article,

Is Na(+) required for the binding of dopamine, amphetamine, tyramine, and octopamine to the human dopamine transporter?
Li, Li-Bin; Cui, Xiao-Nan; Reith, Maarten A
2002 Apr;365(4):303-311, Naunyn-Schmiedeberg's archives of pharamacology
The role of Na(+) in the recognition of blockers by the dopamine transporter is accomodated by a model with a cation site that overlaps with the blocker binding domain, and a distal Na(+) site that interacts with this cation site and perhaps with the blocker binding domain itself. The present study addresses the application of this model to the recognition of substrates by the dopamine transporter, focusing on conditions that should reveal a stimulatory effect, if present, of Na(+) on substrate binding. Recognition was studied via the inhibition of binding of [(3)H]WIN 35,428 (2beta-carbomethoxy-3beta-(4-fluorophenyl) [(3)H]tropane), a cocaine analog, to the human dopamine transporter in human embryonic kidney 293 cells. Little or no changes in binding were noted for dopamine, d-amphetamine, p-tyramine, or dl-octopamine by increasing [Na(+)] from 2 mM to 20 mM with co-varying Br(-), both at pH 7.4 and 7.0. In 74-mM Tris-HBr or -HCl, only dopamine and d-amphetamine showed binding increases upon raising Na(+), leveling off with NO(3)(-) or SO(4)(2-) but not Br(-) as anion at approximately 60 mM Na(+), consonant with a partly stimulatory action of Br(-). An Na(+) free, low 5-mM Tris-HEPES buffer was used for studying Na(+) curves truly starting at 0 mM, and, with SO(4)(2-) as the anion, no stimulation of binding by Na(+) was observed. This suggested that the stimulations observed in high (74 mM) Tris(+) buffer by Na(+) were not a direct effect of Na(+) but rather a disinhibitory effect of Na(+) in removing Tris(+) inhibition that depended upon substrate. Tris(+) IC(50) values in Na(+) free buffer were not lower for dopamine and d-amphetamine than p-tyramine and dl-octopamine. No evidence was found for a stronger inhibitory effect of Na(+) for dopamine and dl-octopamine potentially offsetting Tris(+) disinhibition. All results together support the existence of a substrate domain overlapping with a cation site that also binds Tris(+); a distal Na(+) site interacts with this cation site and with the substrate domain by negative allosterism and is additionally impacted by Cl(-). Importantly, interactions between sites vary with the type of substrate, and, in membrane preparations, Na(+) is not required for, or stimulatory to, the binding of any of the four substrates studied unlike the binding of the cocaine analog WIN 35,428
— id: 57961, year: 2002, vol: 365, page: 303, stat: Journal Article,

Neurotransmitter transporters : structure, function, and regulation
Reith MEA
Totowa NJ : Humana Press, 2002,
— id: 1370, year: 2002, vol: , page: , stat: ,

Concurrent autoreceptor-mediated control of dopamine release and uptake during neurotransmission: an in vivo voltammetric study
Wu, Qun; Reith, Maarten E A; Walker, Q David; Kuhn, Cynthia M; Carroll, F Ivy; Garris, Paul A
2002 Jul 15;22(14):6272-6281, Journal of neuroscience
Receptor-mediated feedback control plays an important role in dopamine (DA) neurotransmission. Recent evidence suggests that release and uptake, key mechanisms determining brain extracellular levels of the neurotransmitter, are governed by presynaptic autoreceptors. The goal of this study was to investigate whether autoreceptors regulate both mechanisms concurrently. Extracellular DA in the caudate-putamen and nucleus accumbens, evoked by electrical stimulation of the medial forebrain bundle, was monitored in the anesthetized rat by real-time voltammetry. Effects of the D2 antagonist haloperidol (0.5 mg/kg, i.p.) on evoked DA levels were measured to evaluate autoreceptor control mechanisms. Two strategies were used to resolve individual contributions of release and uptake to the robust increases in DA signals observed after acute haloperidol challenge in naive animals: pretreatment with 3beta-(p-chlorophenyl)tropan-2beta-carboxylic acid p-isothiocyanatophenylmethyl ester hydrochloride (RTI-76; 100 nmol, i.c.v.), an irreversible inhibitor of the DA transporter, and kinetic analysis of extracellular DA dynamics. RTI-76 effectively removed the uptake component from recorded signals. In RTI-76-pretreated rats, haloperidol induced only modest increases in DA elicited by low frequencies and had little or no effect at high frequencies. These results suggest that D2 antagonism alters uptake at all frequencies but only release at low frequencies. Kinetic analysis similarly demonstrated that haloperidol decreased V(max) for DA uptake and increased DA release at low (10-30 Hz) but not high (40-60 Hz) stimulus frequencies. We conclude that presynaptic DA autoreceptors concurrently downregulate release and upregulate uptake, and that the mechanisms are also independently controlled during neurotransmission
— id: 57959, year: 2002, vol: 22, page: 6272, stat: Journal Article,

Effect of metaphit on GABA transport into the brain
Lipovac, MN; Zlokovic, BV; Reith, MEA; Lajtha, A
2001 ;78(8-9):64-64, Journal of neurochemistry
— id: 115474, year: 2001, vol: 78, page: 64, stat: Journal Article,

Preferential increases in nucleus accumbens dopamine after systemic cocaine administration are caused by unique characteristics of dopamine neurotransmission. [References]
Wu, Qun; Reith, Maarten EA; Kuhar, Michael J; Carroll, FIvy; Garris, Paul A.
2001 ;21(16):6338-6347 Aug, Journal of neuroscience
Investigated the preferential increase of extracellular dopamine (DA) in the nucleus accumbens (NA) relative to the caudate-putamen after systemic cocaine administration in male rats. Cocaine was compared with 2 other blockers of
— id: 45418, year: 2001, vol: 21, page: 6338, stat: Journal Article,

Cerebral signal transduction from first to fourth messengers
Reith MEA
Totowa NJ : Humana Press, 2000,
— id: 1369, year: 2000, vol: , page: , stat: ,

From first to fourth messengers in the brain : an overview
Reith MEA
Cerebral signal transduction from first to fourth messengers Totowa NJ : Humana Press, 2000,
— id: 4630, year: 2000, vol: , page: ?, stat: Chapter,

Enhanced accumbal dopamine release following 5-HT-sub(2A ) receptor stimulation in rats pretreated with intermittent cocaine
Yan, Qing-Shan; Reith, Maarten EA; Yan, Shue.
2000 ;863(1-2):254-258 Apr, Brain research
Examined whether dopamine (DA) release in the nucleus accumbens (NACC) following 5-hydroxytryptamine (5-HT) type 2A receptor stimulation is potentiated by intermittent cocaine administration in male rats. Ss received daily injections of either cocaine or saline for 14 days. On the 7th day of withdrawal, Ss were administered the 5-HT receptor agonist (+or-)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI) with or without the 5-HT receptor antagonist ketanserin. Results showed that activation of 5-HT-sub(2A ) receptors within the NACC produced greater and longer-lasting increases in extracellular DA in Ss pretreated with cocaine than in those pretreated with saline. The DOI-induced increases in NACC DA were attenuated by co-perfusion with ketanserin. It is concluded that intermittent cocaine may cause enhanced sensitivity of 5-HT-sub(2A ) receptors within the NACC.
— id: 45419, year: 2000, vol: 863, page: 254, stat: Journal Article,

Intracellular messengers in drug addiction
Reith MEA
Introduction to cellular signal transduction Boston : Birkhauser, 1999,
— id: 4631, year: 1999, vol: , page: ?, stat: Chapter,

Neurotransmitter transporters : structure, function, and regulation
Reith MEA
Totowa NJ : Humana Press, 1997,
— id: 1371, year: 1997, vol: , page: , stat: ,

Role of axonal and somatodendritic monoamine transporters in action of uptake blockers
Reith MEA
Neurotransmitter transporters : structure, function, and regulation Totowa NJ : Humana Press, 1997,
— id: 4632, year: 1997, vol: , page: ?, stat: Chapter,

Dizocilpine (MK-801) increases not only dopamine but also serotonin and norepinephrine transmissions in the nucleus accumbens as measured by microdialysis in freely moving rats
Yan, Qing-Shan; Reith, Maarten EA; Jobe, Phillip C; Dailey, John W.
1997 ;765(1):149-158 Aug, Brain research
The extracellular concentrations of dopamine (DA), norepinephrine (NE), and serotonin 5-hydroxytryptamine (5-HT) in the nucleus accumbens (NACC) of freely moving rats were monitored simultaneously via intracerebral microdialysis. Local infusion of the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 (dizocilpine) produced significant increases in extracellular levels of DA,
— id: 45420, year: 1997, vol: 765, page: 149, stat: Journal Article,

The role of serotonin in the actions of psychostimulants: Molecular and pharmacological analyses
Cunningham, Kathryn A; Bradberry, Charles W; Chang, Albert S; Reith, Maarten E.
1995 ;73(1-2):93-102 Dec, Behavioural brain research
Discusses the interaction of cocaine with 5-hydroxytryptamine (5-HT) systems, to elucidate involvement of 5-HT in behavioral effects of psychostimulants. Interaction of cocaine with 5-HT systems has a major impact on brain function. Cessation of neuronal activity in raphe 5-HT cells self-limits 5-HT rise, due to cocaine-induced terminal reuptake inhibition. Cocaethylene, a cocaine metabolite formed in the presence of ethanol, increases dopamine levels equipotently to cocaine, but increases 5-HT levels to a much lesser extent. Cocaine-induced suppression of 5-HT raphe neuron firing is mediated by indirect activation of 5-HT-sub(1A ) autoreceptors and longloop mesohabenular feedback pathways, which may include a dopamine component. Acute cocaine also affects cellular activity of amygdaloid neurons, which are mediated in part by potentiation of normal actions of 5-HT.
— id: 45421, year: 1995, vol: 73, page: 93, stat: Journal Article,

Dopamine and serotonin release-regulating autoreceptor sensitivity in A9/A10 cell body and terminal areas after withdrawal of rats from continuous infusion of cocaine
Chen, N H; Reith, M E
1993 Dec;267(3):1445-1453, Journal of pharmacology & experimental therapeutics
The effects of dopamine (DA) and 5-hydroxytryptamine (5-HT) autoreceptor agents on electrically induced [3H]DA and [3H]5-HT release from superfused slices of striatum, nucleus accumbens and ventral mesencephalon (VM) containing A9 and A10 neurons were investigated in rats made tolerant to the stimulatory effect of cocaine on locomotor behavior by a 14-day continuous infusion of cocaine (29 mg/kg/day) by s.c. implanted osmotic minipumps followed by a 7-day drug-free period. In VM, electrically induced [3H]DA was increased, the ability of pergolide to inhibit this release was abolished, but the ability of sulpiride to facilitate the release was potentiated, implicating a higher concentration of synaptic DA modifying the responsiveness of somatodendritic D2 autoreceptors to D2 agents. Both electrically induced [3H]5-HT release from VM and the stimulatory effect of in vitro cocaine on this release were enhanced whereas the effects of both 5-methoxytryptamine and methiothepin were attenuated, indicating that subsensitivity of 5-HT autoreceptors developed in DA cell body regions. In striatum and nucleus accumbens, no significant changes were observed in [3H]DA and [3H]5-HT release, except for a modest reduction in the effects of both pergolide and sulpiride on electrically induced [3H]DA release from striatum. These results emphasize the importance of pretreatment-induced changes in DA cell body regions, rather than terminal areas, under the present conditions. The observed increase in DA autoinhibitory tone and subsensitivity of 5-HT release-regulating autoreceptors in the VM may contribute to the locomotor tolerance upon cocaine challenge after continuous cocaine.
— id: 155598, year: 1993, vol: 267, page: 1445, stat: Journal Article,

Metaphit-induced audiogenic seizures in mice: I. Pharmacologic characterization
Debler EA; Lipovac MN; Lajtha A; Zlokovic BV; Dunlop DS; Jacobson AE; Rice KC; de Costa B; Reith ME
1993 Mar-Apr;34(2):201-210, Epilepsia
Metaphit [an analogue of phencyclidine (PCP) with an acylating isothiocyanate group] induced audiogenic clonic to clonic-tonic seizures in mice exposed to audio stimulation 24 h after metaphit administration. The incidence of seizures was reduced by treatment 30 min before audio stimulation with specific PCP-like compounds [5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-imine maleate (MK-801), and PCP itself], competitive N-methyl-D-aspartate antagonists 2-amino-5-phosphonopentanoic acid (AP-5 and NPC-12626), antiepileptic drugs [phenobarbital (PB), phenytoin (PHT)], and gamma-aminobutyric acid (GABA) agonists (muscimol and diazepam). In contrast, when given in conjunction with metaphit, most of these drugs were ineffective in protecting animals from audiogenic seizures 24 h later. Only compounds with long half-lives (t1/2) such as MK-801, PB, and PHT had a protective effect. High-performance liquid chromatography (HPLC) determination of [3H]MK-801 showed its long-term presence in the brain after intraperitoneal (i.p.) administration of [3H]MK-801. Audiogenic seizures observed 24 h after metaphit administration were potentiated by administration of the GABA antagonist picrotoxin 15 min before audio stimulation, and picrotoxin-induced spontaneous seizures were enhanced by pretreatment (24 h earlier) with a dose of metaphit that in itself did not produce spontaneous seizures at the time of the picrotoxin test. Similar observations were made with N-methyl D-aspartic acid (NMDA) instead of picrotoxin. Thus, an interplay exists between excitatory glutaminergic and inhibitory GABAergic circuitries in the metaphit seizure model
— id: 60484, year: 1993, vol: 34, page: 201, stat: Journal Article,

Cocaine binding sites in mouse striatum, dopamine autoreceptors, and cocaine-induced locomotion
Reith, Maarten E; Selmeci, Gabor.
1992 ;41(1):227-230 Jan, Pharmacology biochemistry & behavior
BALB/cByJ mice received cocaine (25 mg/kg, ip) once a day for 3 days, resulting in a greater locomotor response to cocaine on Day 3 than on Day 1. On Day 4, a dose (0.03 mg/kg, sc) of apomorphine, targeted at dopamine autoreceptors, caused the same degree of locomotor depression in cocaine- as in saline-pretreated mice. C57BL/6ByJ mice displayed a greater locomotor response to cocaine than BALB/cByJ mice but had the same number of striatal [-3H]C
— id: 45423, year: 1992, vol: 41, page: 227, stat: Journal Article,

Carrier-mediated efflux of [3H]dopamine and [3H]1-methyl-4-phenylpyridine: effect of ascorbic acid
Debler EA; Sershen H; Hashim A; Lajtha A; Reith ME
1991 Feb;7(2):99-105, Synapse
The carrier-mediated efflux of [3H]1-methyl-4-phenylpyridine (MPP+) and [3H]dopamine was examined in mouse striatal synaptosomal P2 fractions. Although the two compounds are transported by the same carrier, the translocation of the carrier-ligand complex is more rapid with MPP+ than with dopamine. With dopamine-stimulated efflux of preloaded [3H]dopamine, externally present dopamine at a concentration of 1.3 microM reduced the intrasynaptosomal concentration of [3H]dopamine by 50% (the ECR value) with 8 min of incubation. The ECR value of dopamine in promoting the efflux of [3H]MPP+, however, was only 0.15 microM. Similarly, ascorbic acid was weaker in enhancing the efflux of [3H]dopamine (ECR greater than 2000 microM) than that of [3H]MPP+ (ECR = 567 microM). This effect of ascorbic acid on the efflux of [3H]MPP+ was attenuated by mazindol, a blocker of dopamine uptake. It is proposed that ascorbic acid has a neuromodulatory role involving changes at the level of carrier-membrane translocation and/or orientation
— id: 60510, year: 1991, vol: 7, page: 99, stat: Journal Article,

Sertraline and cocaine-induced locomotion in mice: II. Chronic studies
Reith, Maarten E; Fischette, Christine T.
1991 ;103(3):306-313 Mar, Psychopharmacology
Examined the effects of repeated treatment with the serotonin 5-hydroxytryptamine (5-HT) uptake blocker sertraline on cocaine-induced locomotion in female C57BL/6ByJ mice in 3 paradigms. When Ss were treated for 2 wks with a daily injection of sertraline (or placebo) and challenged with cocaine 1 hr after the final sertraline injection, their cocaine-induced locomotion was the same as that of placebo-pretreated controls. Ss were treated for 2 wks with cocaine (or saline) and then for 2 wks with sertraline (or placebo). Locomotion induced by cocaine administered 1 hr after the final sertraline (placebo) injection was higher in cocaine- than saline-pretreated Ss. (PsycIN
— id: 45425, year: 1991, vol: 103, page: 306, stat: Journal Article,

Sertraline and cocaine-induced locomotion in mice: I. Acute studies
Reith, Maarten E; Wiener, Harvey L; Fischette, Christine T.
1991 ;103(3):297-305 Mar, Psychopharmacology
Assessed the behavioral and pharmacokinetic interaction between the serotonin 5-hydroxytryptamine (5-HT) uptake blocker sertraline and cocaine in C57BL/6ByJ mice. Pretreatment with sertraline did not affect the total amount of spontaneous locomotor activity during 50 min following administration of cocaine. At doses of sertraline higher than those found to inhibit ex vivo neuronal uptake of serotonin by 50%, the peak of cocaine-induced locomotor activity was shifted toward a later time. A similar effect was seen after pretreatment with serotonin uptake blockers other than sertraline, and also after desipramine. (PsycIN
— id: 45424, year: 1991, vol: 103, page: 297, stat: Journal Article,

Electroencephalographic characteristics of audiogenic seizures induced in metaphit-treated small rodents
Susic V; Reith ME; Zlokovic BV; Lajtha A; Jacobson AE; Rice KC; Lipovac MN
1991 Nov-Dec;32(6):783-790, Epilepsia
Adult male mice, rats, and guinea pigs were subjected to intense sound stimulation of an electric bell (100 dB, 12 kHz for 60 s) after a single intraperitoneal (i.p.) injection of metaphit (1-(1-(3 isothiocyanatophenyl)-cyclohexyl)piperidine) (50 mg/kg). When the animals were tested 24 h after administration of metaphit, audiogenic seizures were observed. None of the control saline-injected animals had convulsions. EEG recordings demonstrated the appearance of paroxysmal activity and spike-wave complexes in the trace from cortical and hippocampal electrodes, with frequency and amplitude increasing with time. Behaviorally, myoclonic jerks of facial muscles, ears, and neck appeared, but no correlation was noted between EEG and the motor phenomena. Auditory stimulation was necessary to elicit the full-blown sequence of seizure responses consisting of wild running followed by clonic and then tonic extension. At the time of seizures, repetitive high-amplitude spikes and waves appeared in the EEG, followed by profound EEG and behavioral depression. None of the animals died during or immediately after seizures. The seizure response to sound stimulation of mice, rats, and guinea pigs was phenomenologically similar, with minor differences in quantitative pattern of convulsive components, which suggests that all three animal species share the common property of extreme susceptibility to audiogenic stimulation caused by metaphit administration
— id: 60505, year: 1991, vol: 32, page: 783, stat: Journal Article,

D-tartrate alters uptake of [3H]dopamine into brain synaptic vesicles
Reith ME; Kramer HK; Sershen H; Lajtha A
1990 Feb;31(2):133-136, Journal of neuroscience methods
The use of D-tartrate containing media for measuring uptake of catecholamines into brain synaptic vesicles alters the properties of transport. Absolute concentrations of inhibitors determined in competition studies should be viewed with caution
— id: 60521, year: 1990, vol: 31, page: 133, stat: Journal Article,

Correlation between cocaine-induced locomotion and cocaine disposition in the brain among four inbred strains of mice
Wiener, Harvey L; Reith, Maarten E.
1990 ;36(3):699-701 Jul, Pharmacology biochemistry & behavior
BALB/cByJ, C57BL/6ByJ, CXBH/By, and CXBK/By mice differed in their locomotor response to cocaine measured 1-20 min after ip administration. These differences were paralleled by differences in the disposition of cocaine (measured at 12 min) in the brain. Among all individual Ss taken together, there was a significant correlation between locomotor stimulation and the brain concentration of cocaine. The differences between strains in their locomotor responsiveness to cocaine are determined, in part, by the disposition of cocaine in the brain following ip administration of cocaine. (PsycIN
— id: 45426, year: 1990, vol: 36, page: 699, stat: Journal Article,

Evidence for the involvement of Na+/Ca2+ exchange in the stimulation of inositol phospholipid hydrolysis by sodium channel activation and depolarization
Benuck M; Reith ME; Lajtha A
1989 Jan 10;159(2):187-190, European journal of pharmacology
Amiloride, an inhibitor of the Na+/Ca2+ exchanger, blocked the hydrolysis of inositol phospholipids in mouse cerebrocortical slices induced by the sodium channel activator veratridine, by KCl, or by the sodium ionophore monensin; there was no inhibition by A 23187, a Ca2+ ionophore, or by serotonin. It is concluded that agents that increase intracellular Na2+ stimulate inositide hydrolysis by an indirect effect via Na+/Ca2+ exchange
— id: 60543, year: 1989, vol: 159, page: 187, stat: Journal Article,

Phosphoinositide hydrolysis induced by depolarization and sodium channel activation in mouse cerebrocortical slices
Benuck M; Reith ME; Lajtha A
1989 Aug;28(8):847-854, Neuropharmacology
Carbachol, a muscarinic receptor agonist and the sodium channel-activating agents, scorpion venom, veratridine, batrachotoxin and aconitine, were shown to stimulate the formation of [3H]inositol phosphates in [3H]inositol-labelled miniprisms, obtained from the cerebral cortex of the mouse. The inositol response to the Na+ channel-activating agents was inhibited by the sodium channel blocker tetrodotoxin (TTX), while the response induced by carbachol was partially resistant to TTX. The response to scorpion venom and the TTX-insensitive portion of the response to carbachol was additive, indicating different mechanisms. The presence of high potassium (K+) induced hydrolysis of inositide in a TTX-insensitive manner and was not additive with that resulting from sodium channel activators, thus indicating a common mechanism. The addition of large concentrations of magnesium to block the release of acetylcholine, did not inhibit the inositol response to high K+ or to veratridine. Calcium channel blockers such as nickel or cobalt, or the dihydropyridine calcium (Ca2+) channel activator BAY K 8644 and the calcium channel blocker nifedipine, nimodipine or PN-200 110 had little effect. Monensin, a sodium ionophore, stimulated the turnover of phosphatidylinositol at non-depolarizing concentrations and the omission of Na+ ions inhibited the response to sodium channel agents and to high K+. Thus, membrane potential and gradients of K+, Na+ and Ca2+ are all important factors determining the final effect on the turnover of phosphatidylinositol. The data are consistent with a model in which all these factors impinge on the Na+/Ca2+ exchanger regulating internal Ca2+ that, in turn, activates phospholipase C
— id: 60531, year: 1989, vol: 28, page: 847, stat: Journal Article,

Oxidative metabolism of cocaine: comparison of brain and liver
Benuck M; Reith ME; Sershen H; Wiener HL; Lajtha A
1989 Jan;190(1):7-13, Proceedings of the Society for Experimental Biology & Medicine
Norcocaine (NC) and N-hydroxynorcocaine (NHNC), products of the oxidative metabolism of cocaine, were examined in plasma, brain, and liver of mice injected intraperitoneally with cocaine. Plasma levels of NHNC were altered in vivo by inhibiting esterase activity with diazinon and chloral hydrate or activating esterase activity with phenobarbital, and activating the microsomal P-450 system with phenobarbital. Changes in plasma concentrations of NHNC resulted in similar changes in brain, which were often different from those in liver. After intracisternal administration of cocaine to mice, no appreciable amount of NC or NHNC could be detected in brain; the same results were obtained upon intracisternal and intraventricular administration to rats. Microsomal preparations from mouse brain were found to be considerably less active than those from liver in converting NC to NHNC. We conclude that the cerebral oxidative metabolism of cocaine is not appreciable and that most of the NC and NHNC found in the brain after systemic cocaine administration is derived from plasma rather than formed centrally by brain microsomal enzymes
— id: 60544, year: 1989, vol: 190, page: 7, stat: Journal Article,

Metaphit, an isothiocyanate analog of PCP, induces audiogenic seizures in mice
Debler EA; Lipovac MN; Lajtha A; Zlokovic BV; Jacobson AE; Rice KC; Reith ME
1989 Jun 8;165(1):155-159, European journal of pharmacology
Metaphit induces audiogenic seizures in mice. The most severe clonic/tonic seizures occur 18-24 h after metaphit administration. After 48 h the incidence of the seizure episodes begin to diminish. These audiogenic seizures can be prevented by the administration of either PCP or MK-801 24 h after metaphit and 30 min prior to audio stimulation. These seizures may be due to a modulation of the PCP recognition site by metaphit which results in an enhanced probability that the NMDA/PCP ion channels are open
— id: 60534, year: 1989, vol: 165, page: 155, stat: Journal Article,

The effect of nicotine on catecholaminergic storage vesicles
Kramer HK; Sershen H; Lajtha A; Reith ME
1989 Dec 4;503(2):296-298, Brain research
The present study examined the action of nicotine on the accumulation of [3H]dopamine into synaptic vesicles prepared from mouse cerebral cortex or bovine striatum. Nicotine was shown to be a weak inhibitor of [3H]dopamine accumulation, with an IC50 of approximately 0.2-0.4 mM. In addition, repeated nicotine administration (1.2 mg (-)-nicotine di-(+)tartrate/kg s.c., twice daily for 10 days) in vivo in BALB/cBy male mice did not alter the potency of reserpine in inhibiting [3H]dopamine accumulation into synaptic vesicles, nor did it change the slight shift induced by nicotine in the potency of reserpine in inhibiting [3H]dopamine accumulation. The present results show that nicotine is an inhibitor of vesicular dopamine accumulation at high concentrations
— id: 60525, year: 1989, vol: 503, page: 296, stat: Journal Article,

COCAINE COMPETITIVELY INHIBITS CATECHOLAMINE UPTAKE INTO BRAIN SYNAPTIC VESICLES
REITH, MEA; KRAMER, HK; SERSHEN, H; LAJTHA, A
1989 ;10(3):205-208, Research communications in substance abuse
— id: 115533, year: 1989, vol: 10, page: 205, stat: Journal Article,

Long-term blockade of the dopamine uptake complex by metaphit, an isothiocyanate derivative of phencyclidine
Zimanyi I; Jacobson AE; Rice KC; Lajtha A; Reith ME
1989 ;3(3):239-245, Synapse
[3H]Mazindol was used to label the dopamine uptake complex in mouse striatum in vitro in the presence and absence of metaphit, an isothiocyanate derivative of phencyclidine. In some experiments, metaphit was present in the incubation fluid throughout the procedure; in other experiments it was eliminated by several washings and centrifugations. It was found that after removal of the metaphit by washing and centrifugation, the mazindol binding was not restored. Membranes that were pretreated with metaphit and washed had a lower density of mazindol binding sites than control membranes; the remaining mazindol sites had the same afinity for [3H]mazindol. These findings are in agreement with the previous studies on [3H]cocaine and [3H]methylphenidate binding. The following observations support that metaphit is irreversibly acting and not slowly dissociating from the mazindol recognition sites of the dopamine uptake carrier complex: 1) Metaphit did not change the off-rate of [3H]mazindol binding, arguing against an allosteric action at a distinct site. 2) The presence of cocaine protected the mazindol binding sites from the action of metaphit, supporting binding of metaphit and mazindol to the same site. 3) Nine hours after metaphit pretreatment and removal, the degree of inhibition of mazindol binding was the same as immediately after pretreatment, consonant with an irreversible effect of metaphit. 4) The potency of metaphit in inhibiting mazindol binding was greater under slightly alkaline conditions, consistent with acylation of the mazindol sites. Furthermore, it was found that intracerebroventricular application of metaphit did not result in a decrease in the binding of [3H]mazindol 5 hr after the administration
— id: 60545, year: 1989, vol: 3, page: 239, stat: Journal Article,

Comparison of characteristics of dopamine uptake and mazindol binding in mouse striatum
Zimanyi I; Lajtha A; Reith ME
1989 Dec;340(6):626-632, Naunyn-Schmiedeberg's archives of pharamacology
Biochemical and pharmacological studies suggest that the binding of [3H]mazindol is functionally related to the dopamine uptake carrier complex in rodent striatum. In order to study further the relationship between the substrate recognition site for dopamine uptake and the high-affinity binding site for mazindol the uptake of [3H]dopamine and the binding of [3H]mazindol was studied in BALB/cBy mouse striatum in various buffers (Tris, HEPES, bicarbonate-phosphate). Kinetic analysis showed that the Kd of the binding of [3H]mazindol and the Km of the uptake of [3H]dopamine was changed by different sodium concentrations and/or by the presence of Tris, while the Bmax and the Vmax remained essentially the same. However, the shape of the Na+ dependency curves was not the same for mazindol binding and dopamine uptake in the various buffers. The inhibitory effect of other cations such as K+ and Tris was also different on binding and uptake under similar experimental circumstances. Dopamine did not slow down the dissociation of mazindol from its site and this effect was not sodium-sensitive. These complexities can be accommodated by a model that involves overlapping sites for mazindol and dopamine on the dopamine uptake carrier complex, and translocation-reorientation steps
— id: 60526, year: 1989, vol: 340, page: 626, stat: Journal Article,

Effect of cocaine and cocaine congeners on veratridine-induced depolarization in mouse cerebrocortical synaptoneurosomes
Zimanyi I; Wang E; Lajtha A; Reith ME
1989 Feb;22(2):201-208, Journal of neuroscience research
Structure-activity relationships were determined for cocaine congeners in counteracting the depolarization induced by the action of veratridine on voltage-dependent sodium channels of synaptoneurosomes from mouse brain cortex, and their potency was compared to those determined previously on Na+ uptake and batrachotoxinin binding. Cocaine, norcocaine, (+)-pseudococaine, (-)-pseudococaine, (+)-neopseudococaine, benzoyltropine, benzoylpseudotropine, ecgonine methylester, atropine, WIN-35,428, WIN-35,140, WIN-35,065-3, WIN-35,004, and procaine were tested for their potency in inhibiting depolarization as measured by the distribution of the lypophilic cation [3H]triphenylmethylphosphonium across the membrane. All of the tested compounds inhibited the veratridine-induced depolarization in a competitive manner. The structure-activity relationships were similar to those for inhibition of 22Na+ uptake in mouse brain homogenates, and the potency of these local anesthetics in inhibiting veratridine-induced uptake of [3H]triphenylmethylphosphonium correlated well with their potency in inhibiting [3H]batrachotoxinin A 20-alpha-benzoate binding in mouse brain synaptosomes
— id: 60541, year: 1989, vol: 22, page: 201, stat: Journal Article,

Evidence for a common site of action of lidocaine and carbamazepine in voltage-dependent sodium channels
Zimanyi I; Weiss SR; Lajtha A; Post RM; Reith ME
1989 Aug 29;167(3):419-422, European journal of pharmacology
The finding that the development of lidocaine-kindled seizures is blocked by carbamazepine suggests an interaction of carbamazepine with local anesthetic mechanisms. To study the site of interaction, the effects of lidocaine, carbamazepine and another anticonvulsant drug, phenytoin on scorpion venom-enhanced specific binding of [3H]batrachotoxinin A 20-alpha-benzoate to the sodium channel gating complex were examined in vitro in a rat brain hippocampus preparation. Lidocaine shifted the concentration inhibition curve of carbamazepine to the right and vice versa. Carbamazepine shifted the concentration inhibition curve of phenytoin to the right and vice versa. The experimentally determined apparent dissociation constants were in a good agreement with the dissociation constants calculated for a one-site model, suggesting that the interaction occurs because lidocaine shares a common binding site with carbamazepine and phenytoin in the voltage-dependent sodium channels
— id: 60529, year: 1989, vol: 167, page: 419, stat: Journal Article,

Presence of the toxic metabolite N-hydroxy-norcocaine in brain and liver of the mouse
Benuck M; Reith ME; Lajtha A
1988 Mar 15;37(6):1169-1172, Biochemical pharmacology
— id: 60556, year: 1988, vol: 37, page: 1169, stat: Journal Article,

Synaptosomal sodium channels in rat lines selected for alcohol-related behaviors
Korpi ER; Reith ME; Lajtha A
1988 Jan-Feb;5(1):81-84, Alcohol
Alcohol has been shown to inhibit veratridine- and batrachotoxin-stimulated sodium flux in rodent brain synaptosomes. In this study, binding of [3H]batrachotoxinin A 20-alpha-benzoate ([3H]BTX-B) and uptake of [14C]guanidine in cerebrocortical synaptosomes were measured in naive rats belonging to lines with high or low acute sensitivity to ethanol (ANT, Alcohol Non-Tolerant, vs. AT, Alcohol Tolerant, lines) and to lines with high or low voluntary alcohol consumption (AA, Alko Alcohol, vs. ANA, Alko Non-Alcohol, lines). Our rat line pairs did not differ in the equilibrium binding characteristics of [3H]BTX-B nor in the properties of [14C]guanidine uptake, suggesting that the genetic selection has not modulated the genes or the expression of the genes associated with the voltage-sensitive sodium channels
— id: 60561, year: 1988, vol: 5, page: 81, stat: Journal Article,

Metaphit prevents locomotor activation induced by various psychostimulants and interferes with the dopaminergic system in mice
Sershen H; Berger P; Jacobson AE; Rice KC; Reith ME
1988 Jan;27(1):23-30, Neuropharmacology
Metaphit, an isothiocyanate analog of phencyclidine and a proposed phencyclidine receptor acylator, inactivated the carrier involved in the neuronal uptake of dopamine in in vitro experiments with preparations of the striatum in the mouse. In ex vivo experiments 2 and 24 hr after the intravenous administration of metaphit, no changes were observed either in the binding of [3H]cocaine to striatal membranes or in the uptake of [3H]dopamine into synaptosomes or slices. In in vivo experiments 24 hr after pretreatment with metaphit, selective labelling of uptake sites for dopamine in the striatum of the mouse with [3H]GBR 12935 was unaffected. In these in vivo experiments, however, metaphit antagonized the locomotor stimulation induced by blockers of the uptake of dopamine (methylphenidate, mazindol, cocaine, GBR 12909) but not that induced by drugs that affect locomotion by other mechanisms (amphetamine, phencyclidine). Twenty-four hours after treatment with metaphit there was an increase in homovanillic acid in all regions of the brain studied (striatum, olfactory tubercle, cerebral cortex). There was no effect of metaphit on the disappearance rate of 3,4-dihydroxyphenylacetic acid and homovanillic acid from the striatum during the inhibition of monoamine oxidase with pargyline. If the increase in homovanillic acid reflected a greater rate of dopamine catabolism in metaphit-treated mice, it could explain the lack of locomotor stimulation of blockers uptake of the dopamine in these animals, resulting from a rapid breakdown of extracellularly accumulated dopamine
— id: 60889, year: 1988, vol: 27, page: 23, stat: Journal Article,

The mode of action of ethanol on batrachotoxinin-A benzoate binding to sodium channels in mouse brain cortex
Zimanyi I; Lajtha A; Reith ME
1988 Jan 27;146(1):7-16, European journal of pharmacology
Since ethanol has local anesthetic activity its effect was examined in vitro on the scorpion toxin-enhanced specific binding of [3H]batrachotoxinin A 20-alpha-benzoate ([3H]BTX-B) to the gating complex in sodium channel preparations from mouse brain cortex by equilibrium and kinetic experiments. Ethanol inhibited the specific binding of [3H]BTX-B in a vesicular preparation with an IC50 value of 310 mM and a Hill number of 1.0. Ethanol increased the equilibrium dissociation constant of batrachotoxin in a concentration dependent manner without changing the maximal binding capacity and decreased the half-time of the aconitine-induced dissociation of [3H]BTX-B. Thus, ethanol acts as an apparent competitive inhibitor, allosterically affecting the [3H]BTX-B binding, like other local anesthetics. Results of competition experiments in the presence of different concentrations of ethanol and fixed concentration of tetracaine (and vice versa) are consonant with an interaction of ethanol and tetracaine with the same binding sites. Experiment carried out at 32, 37 and 42 degrees C indicated that the effect of ethanol is not mimicked by increasing the temperature
— id: 60559, year: 1988, vol: 146, page: 7, stat: Journal Article,

Interaction of yohimbine with batrachotoxinin binding to mouse brain sodium channels
Zimanyi I; Lajtha A; Vizi ES; Reith ME
1988 Feb 15;37(4):641-645, Biochemical pharmacology
To study the local anesthetic properties of yohimbine in more detail, its effect was examined in vitro on the scorpion toxin-enhanced specific binding of [3H]batrachotoxinin A 20-alpha-benzoate [( 3H]BTX-B) to the gating complex in sodium channel preparations from mouse brain cortex. Both equilibrium and kinetic experiments were carried out. Yohimbine inhibited the specific binding of [3H]BTX-B in the vesicular preparation with an IC50 value of 2.2 X 10(-5) M. This is about one order of magnitude higher than the concentration required for antagonism via the alpha 2-adrenoceptors; however, yohimbine is 7-fold more potent in inhibiting [3H]BTX-B binding than lidocaine. In a concentration-dependent manner, yohimbine increased the dissociation constant (Kd) of high-affinity [3H]BTX-B binding without changing the maximal binding capacity (Bmax). The dissociation rate constant was not affected by yohimbine, suggesting competitive inhibition as opposed to the action of local anesthetics involving an allosteric action via receptor sites distinct from the BTX site. Alpha 2-adrenoceptors are apparently not involved because clonidine and alpha-methyl-noradrenaline had no appreciable effect on [3H]BTX-B binding and did not antagonize the inhibitory effect of yohimbine. The present findings indicate a mechanism of local anesthetic action of yohimbine that differs from that of other local anesthetics such as tetracaine and lidocaine involving direct binding to the BTX site, thereby stabilizing a non-permeable form of the sodium channel
— id: 60558, year: 1988, vol: 37, page: 641, stat: Journal Article,

Evidence that there is no direct correlation between alpha 2-adrenoceptor antagonism and inhibition of voltage-dependent sodium channels
Zimanyi I; Lajtha A; Vizi ES; Wang E; Reith ME
1988 Nov;27(11):1205-1208, Neuropharmacology
Electrophysiological and biochemical evidence suggests that the voltage-dependent sodium channel is the site of local anesthetic action, and that there is pharmacological similarity between alpha-adrenoceptors and Na+-channels. Yohimbine, a non-selective alpha 2-adrenoceptor antagonist, with a structure similar to that of cocaine affects the sodium channel by a mechanism different from that of other local anesthetics including cocaine. Some structural analogues of yohimbine -berbane compounds- were found to be potent and selective alpha 2-adrenoceptor antagonists. In this work the local anesthetic properties of two berbane compounds (6c and 6d (CH-38083) from the paper of Vizi, Toth, Somogyi, Szabo, Harsing and Szantay, 1987) were examined and compared to those of yohimbine in vitro on scorpion venom-enhanced specific binding of [3H]batrachotoxinin A 20-alpha-benzoate [( 3H]BTX-B) to the voltage-sensitive sodium channel and on the veratridine-induced depolarization measured by the uptake of [3H]trimethylphenylphosphonium ion [( 3H]TPMP+) in mouse brain cortex. Both of the compounds inhibited the [3H]BTX-B binding with an IC50 of (approximately) 150 microM, which is more than four orders of magnitude higher than the concentration required for antagonism of a presynaptic alpha 2-adrenoceptor (7 nM). They are 15 times less potent in inhibiting [3H]BTX-B binding and 2.5 times less potent in inhibiting veratridine-induced depolarization than yohimbine
— id: 60550, year: 1988, vol: 27, page: 1205, stat: Journal Article,

Pharmacokinetics of systemically administered cocaine and locomotor stimulation in mice
Benuck M; Lajtha A; Reith ME
1987 Oct;243(1):144-149, Journal of pharmacology & experimental therapeutics
Cocaine and its metabolites were measured in plasma and brain from mice injected i.p. with cocaine and monitored for spontaneous locomotor behavior. Cocaine concentrations in the brain reached peak values within 5 min after administration of cocaine. At all time points between 5 and 60 min the concentrations of cocaine in the brain were 7-fold higher, on the average, than those in plasma. The opposite was true for the concentrations of benzoylecgonine; brain to plasma ratios of benzoylecgonine were approximately 0.1 from 5 to 30 min after i.p. cocaine injection. After i.p. injection of either 10 or 25 mg/kg of cocaine, cocaine disappeared from plasma and brain with a half-life of 16 min and benzoylecgonine disappeared from plasma with a half-life of 62 min. There was good correspondence between locomotor stimulation and concentration of cocaine in the brain measured at 12, 22 and 32 min after i.p. administration of 25 mg/kg of cocaine. Among individual animals there was a significant correlation between their locomotor stimulation and their brain cocaine concentration, indicating that differences in cocaine levels in the brain between animals contribute to their different behavioral response; however, the correlation analysis also indicated the role of other factors determining the locomotor response to cocaine
— id: 60565, year: 1987, vol: 243, page: 144, stat: Journal Article,

Cocaine disposition in the brain after continuous or intermittent treatment and locomotor stimulation in mice
Reith ME; Benuck M; Lajtha A
1987 Oct;243(1):281-287, Journal of pharmacology & experimental therapeutics
Intermittent s.c. and i.p. injections of cocaine (20 mg/kg; total 430 mg/kg for each animal) for 18 days resulted in locomotor stimulation of mice upon challenge with cocaine on the 25th or 26th day, compared with no locomotor stimulation in a saline-pretreated group. In contrast to the sensitization by intermittent cocaine administration, tolerance was found upon challenge after continuous administration of cocaine by minipumps (25 mg/kg/day; total 450 mg/kg for each animal) on a similar schedule. No differences were found between the sensitized and tolerant groups in the levels of cocaine and benzoylecgonine in plasma and brain 12 min after i.p. administration of a challenge dose of cocaine, suggesting that in these chronic experiments the changes in the locomotor response are not accounted for by dispositional effects. In contrast, in animals treated daily for 2 or 3 days i.p. with cocaine and challenged with cocaine 1 day later, there was both a greater locomotor stimulation and a higher level of brain cocaine than in saline-pretreated animals, suggesting a dispositional effect. Among individual animals there was a positive correlation between their locomotor stimulation by the challenge dose and their brain cocaine concentration
— id: 60564, year: 1987, vol: 243, page: 281, stat: Journal Article,

Binding sites for [3H]tetracaine in synaptosomal sodium channel preparations from mouse brain
Reith ME; Kim SS; Lajtha A
1987 Nov 10;143(2):171-178, European journal of pharmacology
The present study was an attempt to answer the question whether the local anesthetic [3H]tetracaine labels sodium channels in mouse brain synaptosomes. Binding of [3H]tetracaine had a Kd of 0.19 microM and a Bmax ranging from 3.7 to 5.2 pmol/mg of protein. Local anesthetics other than tetracaine and cocaine-related compounds inhibited [3H]tetracaine binding with Hill numbers between 0.3 and 0.6. Initiation of the dissociation of [3H]tetracaine binding by an excess of unlabeled tetracaine resulted in non-linear curves. These results are consonant with site heterogeneity, negative cooperativity, or complexities arising from the use of synaptosomal preparations instead of broken membranes. There were important differences between the absolute values of the potency of various local anesthetic drugs and cocaine-related compounds in inhibiting binding of [3H]tetracaine and those in inhibiting binding of [3H]batrachotoxinin A 20-alpha-benzoate. Although there were some effects of manipulating the state of the channel with activating toxins on the apparent rate of association and dissociation of [3H]tetracaine binding, the equilibrium binding was not much affected by the toxins. The results indicate that further characterization is necessary before accepting [3H]tetracaine binding as a valid tool for studying sodium channels
— id: 60562, year: 1987, vol: 143, page: 171, stat: Journal Article,

Effects of caffeine on monoaminergic systems in mouse brain
Reith ME; Sershen H; Lajtha A
1987 ;22(2-3):149-163, Acta biochimica et biophysica Hungarica
Effects of caffeine on monoamine systems in the mouse brain were studied in three lines of experiments. First, concentrations of 10(-7) M to 10(-2) M of caffeine were tested for their potency in inhibiting the carriers involved in the neuronal uptake of dopamine, norepinephrine, and serotonin. The IC50 of caffeine in inhibiting the first two carriers was approximately 10(-2) M, and that in inhibiting the serotonin was 2 x 10(-3) M. Second, concentrations of 10(-5) M to 10(-3) M of caffeine were tested for their potency in affecting the in vitro KC1-induced release of [3H]dopamine from dopamine terminals in the striatum and from norepinephrine terminals in the hypothalamus, and the release of [3H]serotonin from serotonin terminals in the striatum. Little or no effect was observed. Third, caffeine was administered for 3 weeks to mice via their drinking water at 73, 123, and 162 mg/kg per day. No changes were found in their D2-dopaminergic, 5-HT2-serotonergic, or alpha 1-adrenergic receptors in the striatum or cerebral cortex as compared with animals on normal drinking water. All these neurochemical results are consonant with the interpretation of behavioral studies suggesting that caffeine is an only mildly stimulatory drug that should not be grouped with other psycho-stimulant drugs such as amphetamine and cocaine that do affect monoamine systems in the brain
— id: 60578, year: 1987, vol: 22, page: 149, stat: Journal Article,

Nicotine-induced changes in the metabolism of specific brain proteins
Sershen H; Banay-Schwartz M; Dunlop DS; Debler EA; Reith ME
1987 Feb;12(2):197-202, Neurochemical research
The effect of acute and chronic nicotine on the metabolism of specific brain proteins was examined by measuring incorporation of labeled valine into protein, with densitometric scanning of proteins resolved by gel electrophoresis. Acute and chronic administration of nicotine (0.4 mg/kg per 30 min for 2 hours, s.c., or 0.5 mg/kg per 30 min for 5 days (Alzet mini-pump implanted subcutaneously] reduced incorporation of [14C]valine administered by approximately 6-7%. The results with chronic nicotine administration indicated a lack of tolerance for this effect of nicotine. Mecamylamine, a nicotinic ganglionic antagonist, does not seem to block the inhibition of protein synthesis. Small increases in protein content were observed in a high- and a low-molecular-weight region of SDS-polyacrylamide gel, used to separate proteins from newborn brain. In adult brain after chronic nicotine administration, selective increases and a decrease were seen in selective bands. Results are consonant with selective effects of nicotine on the synthesis or degradation of specific brain proteins
— id: 60890, year: 1987, vol: 12, page: 197, stat: Journal Article,

METAPHIT, A RECEPTOR ACYLATOR, INACTIVATES COCAINE BINDING-SITES IN STRIATUM AND ANTAGONIZES COCAINE-INDUCED LOCOMOTOR STIMULATION IN RODENTS
BERGER, P; JACOBSEN, AE; RICE, KC; LESSOR, RA; REITH, MAE
1986 AUG ;25(8):931-933, Neuropharmacology
— id: 98555, year: 1986, vol: 25, page: 931, stat: Journal Article,

Structural requirements for cocaine congeners to interact with [3H]batrachotoxinin A 20-alpha-benzoate binding sites on sodium channels in mouse brain synaptosomes
Reith ME; Kim SS; Lajtha A
1986 Jun 5;261(16):7300-7305, Journal of biological chemistry
The present study examines the possible role of sodium channels in the behavioral effects of cocaine. Cocaine congeners are apparent competitive inhibitors of the scorpion toxin-enhanced binding of [3H]batrachotoxinin A 20-alpha-benzoate to sodium channels in mouse cerebrocortical synaptosomes. However, in agreement with the allosteric model for heterotropic cooperative interactions, the compounds produce a concentration-dependent increase in the rate of dissociation of binding. Concentrations that give a 2-fold increase of k-1 are close to Ki values for inhibiting equilibrium binding of [3H]bactrachotoxinin A 20-alpha-benzoate, suggesting that the inhibitory effect on binding results mostly from an increase of the apparent dissociation rate constant. The ester linkage between the tropane and benzoyl ring of cocaine is not essential for the inhibitory potency, and for both the C-2 and C-3 substituents the equatorial position results in a higher potency than the axial position. There is reasonable agreement between the rank order of potencies in blocking the sodium channel and in inhibiting locomotor behavior. The present results do not support a relationship between the capability of cocaine congeners in blocking sodium flux and in inhibiting uptake of dopamine into striatal synaptosomes. However, peak levels of cocaine in the brain of cocaine addicts could be high enough to interfere with sodium channel functioning, possibly contributing to some of cocaine's actions
— id: 60586, year: 1986, vol: 261, page: 7300, stat: Journal Article,

Locomotor depression in mice by norcocaine does not involve central alpha 2-adrenergic or presynaptic dopamine receptors
Reith ME; Lajtha A
1986 Feb;24(2):305-307, Pharmacology biochemistry & behavior
The inhibition of spontaneous locomotor behavior of mice by norcocaine was antagonized neither by the adrenoceptor antagonists yohimbine and phentolamine, nor by the neuroleptics haloperidol and spiperone, at low doses aimed at presynaptic dopamine receptors. In contrast, the antagonists were effective in reducing the hypomotility induced by clonidine and apomorphine, respectively. These results make it unlikely that central alpha 2-adrenergic or presynaptic dopamine receptors are involved in the hypomotive effect of norcocaine
— id: 60590, year: 1986, vol: 24, page: 305, stat: Journal Article,

Structural requirements for cocaine congeners to interact with dopamine and serotonin uptake sites in mouse brain and to induce stereotyped behavior
Reith ME; Meisler BE; Sershen H; Lajtha A
1986 Apr 1;35(7):1123-1129, Biochemical pharmacology
We report here saturation analysis of [3H]cocaine binding in various mouse brain regions, and the necessary structure-activity relationships for cocaine congeners to inhibit Na+-dependent [3H]cocaine binding and [3H]dopamine uptake in the mouse striatum, and to inhibit [3H]cocaine binding that cannot be stimulated by Na+ and [3H]serotonin uptake in the mouse cerebral cortex. Generally similar structure-activity relationships were noted for all these processes. The ester linkage between the tropane and phenyl rings was not required for activity, in contrast to the configuration of the groups on C2, and to a lesser extent C3, in the tropane ring. Stereospecificity was evident from the differences between cocaine and (+)-pseudococaine, and between WIN 35,065-2 and WIN 35,065-3. There were remarkable differences between the above structure-activity relationships and those for local anesthetic activity of cocaine congeners, indicating that sodium channels were not labeled to a measurable extent with [3H]cocaine under the present conditions. Preliminary data indicated a significant correlation between the potencies of cocaine congeners in inhibiting the Na+-dependent binding of [3H]cocaine and their potencies in inducing stereotyped sniffing upon intraventricular administration
— id: 60588, year: 1986, vol: 35, page: 1123, stat: Journal Article,

Binding sites for [3H]cocaine in mouse striatum and cerebral cortex have different dissociation kinetics
Reith ME; Sershen H; Lajtha A
1986 Jan;46(1):309-312, Journal of neurochemistry
[3H]Cocaine dissociates from its binding sites in the mouse cerebral cortex with a half-time of 25 s. The dissociation kinetics in the striatum is consonant with the presence of two populations of sites with dissociation half times of 2 s and 27 s, comprising 88% and 12%, respectively, of the total binding sites. On the basis of previous pharmacological characterization of [3H]cocaine binding, we propose that the slowly dissociating component represents the sites associated with 5-hydroxytryptamine (serotonin) uptake, and the rapidly dissociating component the 3,4-dihydroxyphenylethylamine (dopamine)-related sites. Evidence is presented that the extremely high dissociation rates do not preclude the measurement of [3H]cocaine binding by rapid filtration. The dissociation of [3H]cocaine from cerebrocortical membranes is slowed to a small but statistically significant extent by serotonin
— id: 60591, year: 1986, vol: 46, page: 309, stat: Journal Article,

Effect of amphetamine on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity in mice
Sershen H; Mason MF; Reith ME; Hashim A; Lajtha A
1986 Aug;25(8):927-930, Neuropharmacology
Amphetamine has been shown to either potentiate or protect against MPTP neurotoxicity. The time course of changes in dopamine and its metabolites was examined after MPTP, amphetamine, or MPTP plus amphetamine administration. Results suggest that under conditions of granular depletion and release of dopamine by 10 mg/kg amphetamine, increased MPTP neurotoxicity occurs. Amphetamine injections at 2-5 mg/kg prevents the decline in dopamine possibly by blockade of the uptake of MPP+, rather than by an inhibition of monoamine oxidase
— id: 60582, year: 1986, vol: 25, page: 927, stat: Journal Article,

Effect of nicotine and amphetamine on the neurotoxicity of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in mice
Sershen H; Mason MF; Reith ME; Hashim A; Lajtha A
1986 Nov;25(11):1231-1234, Neuropharmacology
The present results show the potentiating effect of amphetamine on the ability of MPTP to destroy dopaminergic neurons in striatum of the mouse. A single injection of MPTP (8 mg/kg, retro-orbital) reduced the binding of [3H]mazindol, a marker for dopamine terminals, by 24%. When D-amphetamine (10 mg/kg, s.c.) was given 20 min prior to MPTP, the binding of [3H]mazindol, measured 3-5 days later, was reduced by 58%. It is proposed that the mechanism of this potentiation primarily involves an increased release of dopamine by D-amphetamine, and free radical-mediated processes. Although nicotine also releases dopamine from the striatum, no effect was observed when it was administered prior to MPTP. The lack of effect is probably related to short duration of action of nicotine and the modest effect on release of dopamine as compared to that of amphetamine
— id: 60581, year: 1986, vol: 25, page: 1231, stat: Journal Article,

Locomotor effects of cocaine, cocaine congeners, and local anesthetics in mice
Reith ME; Meisler BE; Lajtha A
1985 Nov;23(5):831-836, Pharmacology biochemistry & behavior
Spontaneous locomotor activity of mice was stimulated by IP administration of cocaine and its closely related phenyltropane analogs. In contrast, locomotion was inhibited by IP administration of cocaine congeners such as norcocaine, (+)-pseudococaine, and tropacocaine, and of isomers of phenyltropane analogs. Also inhibitory were the local anesthetics procaine, tetracaine, benzocaine, lidocaine, and prilocaine. The locomotor inhibition induced by IP norcocaine or tetracaine could be reversed by subsequent treatment with cocaine. Both cocaine and norcocaine were centrally stimulatory when injected intracerebroventricularly. The rank order of potencies of cocaine congeners and local anesthetics in depressing locomotion was similar to that of their potencies in interacting with sodium channels. From these results we infer that the locomotor depression induced by systemic administration of cocaine congeners results from a local anesthetic action involving inhibition of the ion conductance of sodium channels
— id: 60596, year: 1985, vol: 23, page: 831, stat: Journal Article,

Sodium-independent binding of [3H]cocaine in mouse striatum is serotonin related
Reith ME; Meisler BE; Sershen H; Lajtha A
1985 Sep 2;342(1):145-148, Brain research
There was a highly significant correlation between IC50 values of various drugs in inhibiting the Na+-independent [3H]cocaine binding in the mouse striatum and their values in inhibiting the synaptosomal uptake of [3H]serotonin. In contrast, there was no correlation between the inhibition of binding in the absence of Na+ and the inhibition of [3H]dopamine uptake. Lesioning of serotonergic nerve terminals with 5,7-dihydroxytryptamine reduced the Na+-independent [3H]cocaine binding, without affecting the Na+-dependent binding. These results indicate that the bulk of the Na+-independent [3H]cocaine binding in the mouse striatum is associated with serotonergic nerve terminals
— id: 60597, year: 1985, vol: 342, page: 145, stat: Journal Article,

Comparison of [3H]nicotine and [3H]acetylcholine binding in mouse brain: regional distribution
Sershen H; Reith ME; Hashim A; Lajtha A
1985 Jun;48(3):345-352, Research communications in chemical pathology & pharmacology
In a continuing study of nicotine binding sites, we determined the relative amount of nicotine binding and acetylcholine binding in various brain regions of C57/BL and of DBA mice. Although midbrain showed the highest and cerebellum the lowest binding for both [3H]nicotine and [3H]acetylcholine, the ratio of nicotine to acetylcholine binding showed a three-fold regional variation. Acetylcholine inhibition of [3H]nicotine binding indicated that a portion of nicotine binding was not inhibited by acetylcholine. These results indicate important differences between the binding of (+/-)-[3H]nicotine and that of [3H]acetylcholine
— id: 60599, year: 1985, vol: 48, page: 345, stat: Journal Article,

Protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity by the antioxidant ascorbic acid
Sershen H; Reith ME; Hashim A; Lajtha A
1985 Dec;24(12):1257-1259, Neuropharmacology
Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 2 X 8 mg/kg retro-orbital) to BALB/cBy mice reduced [3H]mazindol binding to striatal membranes by 50%. Reactive oxygen derivatives have been suggested to be involved in MPTP neurotoxicity; therefore we examined the effects of ascorbic acid (an antioxidant). Ascorbic acid (100 mg/kg) given 20 min prior to MPTP administration appreciably prevented the reduction of [3H]mazindol binding. The involvement of oxidative processes in the mechanism of MPTP neurotoxicity may suggest a relationship to the etiology of Parkinson's disease, and the possible benefit of treatment with ascorbic acid
— id: 60594, year: 1985, vol: 24, page: 1257, stat: Journal Article,

Similarities and differences between high-affinity binding sites for cocaine and imipramine in mouse cerebral cortex
Reith ME; Allen DL; Sershen H; Lajtha A
1984 Jul;43(1):249-255, Journal of neurochemistry
Previously we found close similarities between high-affinity binding sites for [3H]cocaine and those for [3H]imipramine in the mouse cerebral cortex in regard to their association with neuronal uptake of serotonin. In the present study we investigated whether the two ligands bind to the same site. The two ligands had the following high-affinity binding properties in common: localization in both synaptosomal and microsomal fractions; vulnerability to treatment with N-ethylmaleimide, trypsin, and phospholipase A2; and resistance to exposure to dithiothreitol. In contrast, cocaine binding in the cerebral cortex was more sensitive to heat inactivation than imipramine binding. In addition, the mechanism by which cocaine inhibited [3H]imipramine binding differed from that by which imipramine inhibited [3H]cocaine binding. These data suggest that the high-affinity binding sites for [3H]cocaine and [3H]imipramine in the cerebral cortex are distinct entities
— id: 60606, year: 1984, vol: 43, page: 249, stat: Journal Article,

[3H]cocaine binding in brain is inhibited by Tris (hydroxymethyl) aminomethane
Reith ME; Meisler BE; Sershen H; Lajtha A
1984 Dec;12(2):151-154, Journal of neuroscience methods
High-affinity binding of [3H]cocaine to membranes of mouse cerebral cortex is inhibited by Tris (hydroxymethyl) aminomethane, the buffer commonly used in receptor binding assays. This inhibition is not due to an effect of ionic strength in general. Comparison of binding in Tris buffer with that in sodium phosphate buffer indicates a more than 4-fold higher Kd in the former buffer, with no differences in the Bmax values
— id: 60602, year: 1984, vol: 12, page: 151, stat: Journal Article,

Binding of imipramine and cocaine to a model lipid membrane: comparison with binding to brain membranes
Reith ME; Sershen H; Lajtha A
1984 Jul;9(7):965-977, Neurochemical research
[3H]Imipramine and [3H]cocaine were concentrated at membranes of liposomes prepared from phosphatidylcholine, cholesterol, and dicetylphosphate. This 'binding' has an apparent dissociation constant in the micromolar range and a density close to 2 pmol/micrograms of phosphatidylcholine. The potencies of various drugs in inhibiting the binding to liposomes correlated only weakly with those in inhibiting the high-affinity binding of [3H]imipramine and [3H]cocaine to brain membranes. However, there was a highly significant correlation between the potencies of drugs in inhibiting binding to liposomes and their lipophilic character, indicating the involvement of hydrophobic bonding. Although the amounts of phosphatidylcholine and cholesterol in brain preparations in assays for high-affinity binding to brain membranes were in the same range as those used in our assays with liposomes, the inhibition of the high-affinity binding to brain membranes was only weakly dependent upon the lipophilicity of the inhibiting drug. These results indicate that lipophilicity is but one of the factors in the complex binding interactions between lipophilic substances and integral brain membranes. In addition, the results are in agreement with the suggestion that phosphatidylcholine and cholesterol are not the primary sites of high-affinity binding [3H]imipramine and [3H]cocaine to brain membranes, although it cannot be ruled out that these lipids have different properties in natural biological membranes and in artificial liposome membranes
— id: 60607, year: 1984, vol: 9, page: 965, stat: Journal Article,

Thermodynamics of the interactions of tricyclic drugs with binding sites for [3H]imipramine in mouse cerebral cortex
Reith ME; Sershen H; Lajtha A
1984 Dec 15;33(24):4101-4104, Biochemical pharmacology
— id: 60601, year: 1984, vol: 33, page: 4101, stat: Journal Article,

Endogenous material in brain inhibiting [3H]nicotine and [3H]acetylcholine binding
Sershen H; Reith ME; Hashim A; Lajtha A
1984 ;12(4):563-569, Journal of neuroscience research
The supernatant obtained from mouse brain homogenates contains material that inhibits the saturable binding of [3H]nicotine in mouse cerebral cortex. This inhibitory material was further purified by heat denaturation, ultrafiltration through an Amicon PM-10 membrane filter, and gel chromatography on Sephadex G-10. The material inhibited the binding of [3H]acetylcholine with the same potency as it did that of [3H]nicotine. It also had some affinity for the sites that specifically bind [3H]D-Ala, D-Leu enkephalin, but had much lower affinity for the binding sites for [3H]quinuclidinyl benzilate (QNB), [3H]spiroperidol, [3H]naloxone, or [3H]imipramine. Acid hydrolysis destroyed the activity. These preliminary results suggest the presence in brain of 'nicotinelike' substances, one of which may be the endogenous ligand for the sites that specifically bind [3H]nicotine
— id: 60613, year: 1984, vol: 12, page: 563, stat: Journal Article,

Reduction of dopamine uptake and cocaine binding in mouse striatum by N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
Sershen H; Reith ME; Hashim A; Lajtha A
1984 Jun 15;102(1):175-178, European journal of pharmacology
Administration of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (NMPTP) (daily injections of 8 mg/kg for 5 days via tail vein) reduced [3H]dopamine uptake in striatal synaptosomes by 63% and reduced [3H]cocaine binding to striatal membranes by 61%. [3H]Cocaine binding was not affected in olfactory tubercle, suggesting a selective effect of NMPTP on the nigro-striatal but not on the mesolimbic dopaminergic system. The destruction of dopamine terminals in the striatum did not alter (up-regulate) [3H]spiroperidol binding. The results suggest that NMPTP causes a degenerative destruction of the striatal dopamine pathway and that NMPTP may be useful in developing a rodent model of Parkinson's disease
— id: 60608, year: 1984, vol: 102, page: 175, stat: Journal Article,

Imipramine does not act as a false transmitter in the rat hypothalamus
Allen DL; Reith ME; Sershen H; Lajtha A
1983 May 9;267(1):161-164, Brain research
[3H]Imipramine was accumulated by rat hypothalamic slices mostly by a passive low-affinity process. Accumulated [3H]imipramine was released by 60 mM potassium, but the release was small and was not Ca2+-dependent. Imipramine induced some release of [3H]norepinephrine. None of these events were abolished by pretreatment of the rats with reserpine. These results do not favor the view that imipramine acts as a false transmitter in the rat hypothalamus. Rather, imipramine appears to be accumulated in an extragranular pool from which it releases extragranular norepinephrine
— id: 60619, year: 1983, vol: 267, page: 161, stat: Journal Article,

High- and low-affinity binding of [3H]imipramine in mouse cerebral cortex
Reith ME; Sershen H; Allen D; Lajtha A
1983 Feb;40(2):389-395, Journal of neurochemistry
Binding of [3H]imipramine in mouse cerebral cortex was found to be nonhomogeneous. Competition experiments, Scatchard analysis, and Hill plots are compatible with the existence of binding with high (nanomolar) and low (micromolar) affinity. Low-affinity binding could be eliminated by the use of low concentrations of imipramine as the competing ligand. In contrast to the high-affinity binding, the low-affinity binding was found to be unrelated to the neuronal uptake system for serotonin
— id: 60624, year: 1983, vol: 40, page: 389, stat: Journal Article,

A portion of [3H]cocaine binding in brain is associated with serotonergic neurons
Reith ME; Sershen H; Allen DL; Lajtha A
1983 May;23(3):600-606, Molecular pharmacology
Three lines of evidence are brought forward in support of an association in the brain cortex of some, but not all, of the cocaine binding sites with serotonergic nerve terminals. The first is based upon the significant correlation observed between the inhibition of cocaine binding by various drugs and the inhibition of neuronal uptake of serotonin in the mouse cerebral cortex. The second is based upon the demonstration of cocaine binding in human blood platelets, a model system for central serotonergic neurons. The third comes from experiments in which rats were treated with p-chloroamphetamine and 5,7-dihydroxytryptamine (serotonin neurotoxins), 6-hydroxydopamine (catecholamine neurotoxin), or p-chlorophenylalanine (inhibitor of tryptophan hydroxylase). Only the serotonin neurotoxins decreased the binding of [3H]cocaine in the rat cerebral cortex, but to a lower extent than the binding of [3]imipramine, which is known to be associated with serotonergic terminals. In contrast to the cocaine binding in the mouse cerebral cortex, the binding in the rat cerebral cortex included a considerable portion of low-affinity binding that was relatively unaffected by lesions of serotonergic neurons
— id: 60620, year: 1983, vol: 23, page: 600, stat: Journal Article,

Effects of prenatal administration of nicotine on amino acid pools, protein metabolism, and nicotine binding in the brain
Sershen H; Reith ME; Banay-Schwartz M; Lajtha A
1982 Dec;7(12):1515-1522, Neurochemical research
The effects of nicotine on brain protein metabolism and on the properties of the nicotine binding site were investigated in newborn animals exposed to nicotine during gestation. Brain protein synthesis rates measured in vivo were lower by 18% in newborn of treated animals. Protein degradation rates measured in vitro in the presence of nicotine were lower by 13%. The effect was specific for L-(-)nicotine, since D-(+)nicotine, nicotinic acid, or nicotinamide had no effect on degradation rates. Newborn brain amino acid levels, mainly nonessential amino acids and amino acids of putative neurotransmitter function, were changed somewhat; an increase in the level of taurine (13%), threonine (21%), serine (35%) and glycine (35%), and a decrease in lysine (14%) was observed in the offspring of nicotine treated animals (0.5 mg/kg, s.c., 2 x daily throughout gestation). These changes could not account for the decrease in protein metabolism. Nicotine binding was higher by 25% in the offspring of animals exposed to nicotine during gestation. No such increase was found after treatment of adult rats with nicotine, indicating that the properties of the nicotine binding site change with age
— id: 60630, year: 1982, vol: 7, page: 1515, stat: Journal Article,

Comparison of the properties of central and peripheral binding sites for cocaine
Sershen H; Reith ME; Lajtha A
1982 May;21(5):469-474, Neuropharmacology
Cocaine and its analogs bound saturably to membranes of brain and liver of mice. The binding sites on membranes of liver had a lower affinity for cocaine than those of brain. In addition, there were striking differences between the two tissues in regard to the relative potencies of cocaine analogs in competing with [3H]cocaine for binding. In comparison with the binding sites in brain, those in liver had only moderate stereospecificity, and they discriminated less between the centrally active compounds and the centrally inert analogs
— id: 60635, year: 1982, vol: 21, page: 469, stat: Journal Article,

On the interaction between nicotine and cycloheximide
Sershen H; Reith ME; Lajtha A
1982 Nov 11;251(1):183-185, Brain research
This study examined whether the nicotine-induced reversal of the amnestic effect of cycloheximide is due to an interaction between nicotine and cycloheximide on brain protein synthesis, and whether it involves the sites in brain that saturably bind [3H]nicotine. Nicotine did not reverse the cycloheximide-induced inhibition of protein synthesis, both in in vivo (intact animal) and in vitro (brain slice), suggesting that on-going protein synthesis is not necessarily involved in memory consolidation. The nicotine binding sites were not affected by in vivo or in vitro treatment with cycloheximide in the presence or absence of nicotine
— id: 60631, year: 1982, vol: 251, page: 183, stat: Journal Article,

Binding of [3H]cocaine in mouse brain: kinetics and saturability
Reith ME; Sershen H; Lajtha A
1981 ;2(3):233-243, Journal of receptor research
Kinetic experiments indicate that association of [3H]-cocaine to its binding site in brain occurs rapidly (seconds). Dissociation of membrane-bound cocaine is also rapid, with a dissociation half-life in seconds; this raises the question of whether membrane-bound cocaine is released during the time required for rapid filtration of tissue-containing filters. Results from experiments with increasing numbers of filter-washes indicate that there is no significant loss of [3H]-cocaine saturably bound to brain membranes within the time-scale of the rapid filtration procedure. In addition, saturation analysis of binding data obtained with the filtration procedure and with the centrifugation method give similar estimates of the affinity (dissociation constant: 0.8 microM) and the maximal binding (5 pmol/mg of protein) of cocaine. However, the nonspecific binding and the experimental error in the saturable binding are considerably greater in centrifugation assays than in filtration assays
— id: 60647, year: 1981, vol: 2, page: 233, stat: Journal Article,

Strain differences in opiate receptors in mouse brain
Reith ME; Sershen H; Vadasz C; Lajtha A
1981 Sep 24;74(4):377-380, European journal of pharmacology
Various opiate ligands were bound to brain membranes of mice of the Recombinant Inbred System. The specific binding of low levels of [3H]naloxone, [3H]dihydromorphine and [3H]ethylketocyclazocine was disturbed in a similar fashion among the inbred strains, and in a pattern different from that observed for [3H](D-Ala2,D-Leu5)-enkephalin. The results indicate that the inbred strains differ in mu- and delta-type binding and support the concept of multiple opiate receptors in mouse brain
— id: 60641, year: 1981, vol: 74, page: 377, stat: Journal Article,

Effect of cigarette smoke on protein synthesis in brain and liver
Sershen H; Reith ME; Lajtha A; Gennaro J Jr
1981 May;20(5):451-456, Neuropharmacology
— id: 60644, year: 1981, vol: 20, page: 451, stat: Journal Article,

Noncholinergic, saturable binding of (+/-)-[3H]nicotine to mouse brain
Sershen H; Reith ME; Lajtha A; Gennaro J Jr
1981 ;2(1):1-15, Journal of receptor research
(+/-)-[3H]Nicotine was bound saturably to crude particulate, and synaptosomal-mitochondrial fraction from mouse brain. Scatchard and Hill plots of the binding data are in agreement with the existence of two independent classes of binding sites with high (Kd of 0.1-0.4 microM) and low(Kd is 20 microM) affinities, although negative cooperativity or a two-step model of ligand-receptor interaction cannot be ruled out. Nicotinic or muscarinic agonists and antagonists had little or no affinity for the nicotine binding sites, suggesting that nicotine binds in brain to noncholinergic sites. The binding did not display stereospecificity; this is consistent with the similarity in the pharmacological effects of (-)- and (+)-nicotine. Our results indicate that binding studies with [3H]nicotine should be interpreted with extreme caution
— id: 60648, year: 1981, vol: 2, page: 1, stat: Journal Article,

Endogenous peptide(s) inhibiting [3H]cocaine binding in mouse brain
Reith ME; Sershen H; Lajtha A
1980 Dec;5(12):1291-1299, Neurochemical research
The supernatant fraction of centrifuged homogenate of brain tissue contains material that inhibits the saturable binding of [3H]cocaine to crude mouse brain membranes. This material was subjected to heat treatment to remove protein; further purification was achieved by filtering through an Amicon UM-10 membrane ultrafilter and gel filtration of the ultrafiltrate on Sephadex G-25. Sensitivity to acid hydrolysis and peptidase action indicates that the inhibitory activity resides in peptide material with low molecular weight. The partially purified inhibitor has similar effects to that of cocaine on the specific binding of various ligands to opiate and nonopiate receptors in mouse brain membranes
— id: 60652, year: 1980, vol: 5, page: 1291, stat: Journal Article,

Saturable (3H)cocaine binding in central nervous system of mouse
Reith ME; Sershen H; Lajtha A
1980 Sep 22;27(12):1055-1062, Life sciences
— id: 60654, year: 1980, vol: 27, page: 1055, stat: Journal Article,

The pharmacological relevance of the cocaine binding site in mouse brain
Sershen H; Reith ME; Lajtha A
1980 Nov;19(11):1145-1148, Neuropharmacology
— id: 60653, year: 1980, vol: 19, page: 1145, stat: Journal Article,

Breakdown of corticotropin-(1-24) by mouse brain extracts
Reith ME; Neidle A; Lajtha A
1979 Jul;195(2):478-484, Archives of biochemistry & biophysics. ABB
— id: 60669, year: 1979, vol: 195, page: 478, stat: Journal Article,

The effect of behaviorally active ACTH-like peptides on protein biosynthesis in the brain stem of hypophysectomized rats [Thesis]
Reith MEA
[S.l. : s.n.], 1975,
— id: 1372, year: 1975, vol: , page: , stat: ,

Metabolism and enzymology of fluorosuccinic acids. I. Interactions with the succinate oxidase system
Brodie, J D; Nicholls, P
1970 Mar 18;198(3):423-437, Biochimica & biophysica acta
— id: 105391, year: 1970, vol: 198, page: 423, stat: Journal Article,