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.

doi:10.1016/j.jneumeth.2004.09.015

Cited by 13 publications

(27 citation statements)

References 36 publications

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“…Passage number and expression level in transfected cells caused variability in the inhibition potency of cocaine for DA uptake (Chen and Reith, 2007;Ukairo et al, 2007). (Tables 3 and 4) were comparable with values reported previously (Reith et al, 2005), possibly because of the similarity of the assay conditions and reagents used. ϭ 3), T58A-hNET (n ϭ 5), and T58D-hNET (n ϭ 5) were 3.3 Ϯ 1, 4.6 Ϯ 0.6, and 7.0 Ϯ 1 nM (Ϯ S.E.M.…”

Section: Resultssupporting

confidence: 83%

Site-Directed Mutations near Transmembrane Domain 1 Alter Conformation and Function of Norepinephrine and Dopamine Transporters

et al. 2010

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The human dopamine and norepinephrine transporters (hDAT and hNET, respectively) control neurotransmitter levels within the synaptic cleft and are the site of action for amphetamine (AMPH) and cocaine. We investigated the role of a threonine residue within the highly conserved and putative phosphorylation sequence RETW, located just before transmembrane domain 1, in regulating hNET and hDAT function. The Thr residue was mutated to either alanine or aspartate. Similar to the inward facing T62D-hDAT, T58D-hNET demonstrated reduced [ 3 H](Ϫ)-2-␤-carbomethoxy-3-␤-(4-fluorophenyl)tropane-1,5-napthalenedisulfonate (WIN35,428) binding was not increased, demonstrating a discordance between functional and binding site effects. Taken together, these results concur with the notion that the T3 D mutation in RETW alters the preferred conformation of both hNET and hDAT to favor one that is more inward facing. Although substrate activity and binding are primarily altered in this conformation, the function of inhibitors with distinct structural characteristics may also be affected.

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Section: Resultssupporting

confidence: 83%

Site-Directed Mutations near Transmembrane Domain 1 Alter Conformation and Function of Norepinephrine and Dopamine Transporters

et al. 2010

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“…The low potency of HD-205 at NET, approximately 300 nM, suggests that this ligand may not be ideal as a probe for norepinephrine transporters. On the other hand, tropane analogs in general have been shown to be relatively weak in displacing [ 3 H]nisoxetine binding to NET [42]. For this reason, HD-205 may actually be more potent in irreversibly blocking norepinephrine uptake than predicted from these [ 3 H]nisoxetine binding results.…”

Section: Discussionmentioning

confidence: 98%

Irreversible binding of a novel phenylisothiocyanate tropane analog to monoamine transporters in rat brain

Murthy

Davies

Hedley

et al. 2007

Biochemical Pharmacology

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Irreversible tropane analogs have been useful in identifying binding sites of cocaine on biogenic amine transporters, including transporters for dopamine (DAT), serotonin (SERT) and norepinephrine (NET). The present study characterizes the properties of the novel phenylisothiocyanate tropane HD-205, synthesized from the highly potent 2-napthyl tropane analog WF-23. [ 125 I]HD-244 was synthesized with chloramine-T, purified on HPLC, reacted with rat striatal membranes, and proteins were separated by SDS-PAGE. Results showed several non-specific labeled bands, but only a single specific band of radioactivity co-migrating with an immunoreactive DAT band at approx. 80 kDa was detected, suggesting that [ 125 I]HD-244 covalently labeled DAT protein in striatal membranes. These results demonstrate that phenylisothiocyanate analogs of WF-23 can be used as potential ligands to map distinct binding sites of cocaine analogs at DAT.

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“…Cultured cells expressing the human DAT were as described previously Dutta et al, 2008). The human SERT-expressing cells were those described previously by Eshleman et al (1995), and the human NET-expressing cells used were those described previously by Reith et al (2005). Voltage-gated sodium channel inhibition assays were performed with cultured neocortical neurons obtained from Swiss-Webster mice, as described briefly in the section below and in further detail elsewhere Jabba et al, 2010).…”

Section: Methodsmentioning

confidence: 99%

“…The ability of test compounds to inhibit radiolabeled [ 3 H]substrate uptake via human monoamine transporters was monitored as described previously (Reith et al, 2005). In brief, DAT and SERT cells were grown in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum, 5% bovine calf serum, 2 mM L-glutamine, 100 units/ml penicillin/streptomycin, and 2 g/ml puromycin at 37°C and 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

“…All buffers and solutions were prepared with purified water (Nanopure Diamond, Barnstead; Thermo Fisher Scientific). Cell suspensions were prepared and incubated as described previously (Reith et al, 2005) in 122 mM NaCl, 5 mM KCl, 1.2 mM MgSO 4 , 10 mM glucose, 1 mM CaCl 2 , 15 mM NaH 2 PO 4 , and 7.5 mM Na 2 HPO 4 , mixed to achieve pH 7.4 at room temperature (21°C) and containing 0.1 mM tropolone for the inhibition of catechol-O-methyltransferase. In 96-well plates, cells and test inhibitor compound were first preincubated at room temperature, tritiated substrate was added, incubation was continued, and the assay was terminated by rapid filtration through Whatman (Clifton, NJ) GF/C glass fiber filter mats with a 96-pin Brandel harvester (Brandel Inc., Gaithersburg, MD For each assay, test inhibitor ligands were preincubated with cells for 6 min, followed by addition of radiolabeled substrate and further incubation for 5, 8, or 7 min, respectively.…”

Section: Methodsmentioning

confidence: 99%

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Novel C-1 Substituted Cocaine Analogs Unlike Cocaine or Benztropine

Reith

Ali

Hashim

et al. 2012

J Pharmacol Exp Ther

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Despite a wealth of information on cocaine-like compounds, there is no information on cocaine analogs with substitutions at C-1. Here, we report on (R)-(Ϫ)-cocaine analogs with various C-1 substituents: methyl (2), ethyl (3), n-propyl (4), n-pentyl (5), and phenyl (6). Analog 2 was equipotent to cocaine as an inhibitor of the dopamine transporter (DAT), whereas 3 and 6 were 3-and 10-fold more potent, respectively. None of the analogs, however, stimulated mouse locomotor activity, in contrast to cocaine. Pharmacokinetic assays showed compound 2 occupied mouse brain rapidly, as cocaine itself; moreover, 2 and 6 were behaviorally active in mice in the forced-swim test model of depression and the conditioned place preference test. Analog 2 was a weaker inhibitor of voltage-dependent Na ϩ channels than cocaine, although 6 was more potent than cocaine, highlighting the need to assay future C-1 analogs for this activity. Receptorome screening indicated few significant binding targets other than the monoamine transporters. Benztropine-like "atypical" DAT inhibitors are known to display reduced cocaine-like locomotor stimulation, presumably by their propensity to interact with an inward-facing transporter conformation. However, 2 and 6, like cocaine, but unlike benztropine, exhibited preferential interaction with an outward-facing conformation upon docking in our DAT homology model. In summary, C-1 cocaine analogs are not cocaine-like in that they are not stimulatory in vivo. However, they are not benztropine-like in binding mechanism and seem to interact with the DAT similarly to cocaine. The present data warrant further consideration of these novel cocaine analogs for antidepressant or cocaine substitution potential.

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Pharmacological profile of radioligand binding to the norepinephrine transporter: instances of poor indication of functional activity

Cited by 13 publications

References 36 publications

Site-Directed Mutations near Transmembrane Domain 1 Alter Conformation and Function of Norepinephrine and Dopamine Transporters

Site-Directed Mutations near Transmembrane Domain 1 Alter Conformation and Function of Norepinephrine and Dopamine Transporters

Irreversible binding of a novel phenylisothiocyanate tropane analog to monoamine transporters in rat brain

Novel C-1 Substituted Cocaine Analogs Unlike Cocaine or Benztropine

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