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1.
J Neural Transm (Vienna) ; 109(10): 1229-40, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12373557

ABSTRACT

Meta-hydroxyephedrine (HED) comprises four stereoisomers consisting of two enantiomeric pairs related to ephedrine and pseudoephedrine. HED is transported into adrenergic neurons and radiolabeled HED has been employed in positron emission tomography (PET) to image adrenergic neurons in vivo. To extend structure-activity analyses of binding sites within monoamine transporters and to determine which stereoisomer displayed the best selectivity for PET imaging applications, we tested the HED compounds for their abilities to inhibit [(3)H]neurotransmitter uptake into platelets, transfected cells, and chromaffin vesicles. We hypothesized that the HED compounds would be most potent at the norepinephrine transporter (NET) compared to the serotonin or dopamine transporters and that the 1R diastereomers would be more effective than 1S diastereomers. Supporting the hypotheses, all stereoisomers were most potent at the NET and the 1R,2S stereoisomer was the most potent inhibitor overall. However, the 1S,2R isomer may be preferred for PET applications because of better selectivity among the transporters and reduced neuronal recycling.


Subject(s)
Biogenic Monoamines/metabolism , Carrier Proteins/antagonists & inhibitors , Ephedrine/analogs & derivatives , Ephedrine/pharmacology , Membrane Transport Proteins , Neuropeptides , Animals , Blood Platelets/drug effects , Blood Platelets/metabolism , Carrier Proteins/chemistry , Cattle , Cell Membrane/metabolism , Cells, Cultured , Chromaffin Granules/drug effects , Chromaffin Granules/metabolism , Dopamine/metabolism , Membrane Glycoproteins/metabolism , Norepinephrine/metabolism , Serotonin/metabolism , Stereoisomerism , Transfection , Vesicular Biogenic Amine Transport Proteins
3.
Eur J Pharmacol ; 381(1): 63-9, 1999 Sep 17.
Article in English | MEDLINE | ID: mdl-10528135

ABSTRACT

Methcathinone and methylone, the beta-ketone analogues of methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA), respectively, were tested for neurotransmitter uptake inhibition in vitro. The beta-ketones were threefold less potent than the nonketo drugs at inhibiting platelet serotonin accumulation, with IC(50)'s of 34.6+/-4.8 microM and 5.8+/-0.7 microM, respectively. Methcathinone and methylone were similar in potency to methamphetamine and MDMA at catecholamine transporters individually expressed in transfected glial cells. For dopamine uptake, IC(50)'s were 0.36+/-0.06 microM and 0.82+/-0.17 microM, respectively; for noradrenaline uptake, IC(50) values were 0.51+/-0.10 microM and 1. 2+/-0.1 microM, respectively. In chromaffin granules, IC(50)'s for serotonin accumulation were 112+/-8.0 microM for methcathinone and 166+/-12 microM for methylone, 10-fold higher than the respective values for methamphetamine and MDMA. Our results indicate that methcathinone and methylone potently inhibit plasma membrane catecholamine transporters but only weakly inhibit the vesicle transporter.


Subject(s)
Cell Membrane/drug effects , Dopamine Uptake Inhibitors/pharmacology , Membrane Glycoproteins/drug effects , Membrane Transport Proteins , Methamphetamine/pharmacology , Neuropeptides , 3,4-Methylenedioxyamphetamine/pharmacology , Animals , Cattle , Cell Membrane/metabolism , Chromaffin Cells/drug effects , Chromaffin Cells/metabolism , Dopamine/pharmacokinetics , Humans , Methamphetamine/analogs & derivatives , Norepinephrine/pharmacokinetics , Propiophenones/pharmacology , Serotonin/pharmacokinetics , Tritium , Tumor Cells, Cultured , Vesicular Biogenic Amine Transport Proteins
4.
Biochem J ; 330 ( Pt 2): 959-66, 1998 Mar 01.
Article in English | MEDLINE | ID: mdl-9480916

ABSTRACT

The full-length cDNA for the rat synaptic-vesicle monoamine transporter (VMAT2) containing a C-terminal polyhistidine epitope has been engineered into baculovirus DNA for expression in Spodoptera frugiperda (Sf9) insect cells. Using this recombinant baculovirus and cultured Sf9 cells, rVMAT2 has been expressed at levels of 7.8x10(6) transporters per cell, as assessed by [3H]dihydrotetrabenazine binding. A 1l culture of infected cells produced approx. 15 nmol (900 microg) of transporter. rVMAT2 expressed in the Sf9 cells bound [3H]dihydrotetrabenazine with a KD of 31.2 nM and a Bmax of 19.9 pmol/mg. Two polypeptides of 55 and 63 kDa were identified using the photolabel, 7-azido-8-[125I]iodoketanserin ([125I]AZIK). Photoaffinity labelling of rVMAT2 by 1 nM [125I]AZIK was protectable by 10 microM tetrabenazine and 10 microM 7-aminoketanserin. Digitonin-solubilized VMAT2 was purified to greater than 95% homogeneity using immobilized Ni2+-affinity chromatography, followed by lectin (Concanavalin A) chromatography. The purified transporter migrates as a single broad band with a molecular mass of approx. 63kDa, as analyzed by SDS/PAGE. The purified transporter retained the ability to bind ligands ([125I]AZIK and [3H]dihydrotetrabenazine). The purified VMAT2 bound [3H]dihydrotetrabenazine with a KD of 86.2 nM. As is the case with the monoamine transporter from bovine chromaffin granule membranes, purified VMAT2 is covalently modified by dicyclohexylcarbodi-imide (DCCD) and is specifically labelled by [14C]DCCD. This labelling is inhibited by tetrabenazine and ketanserin. These data indicate that VMAT2 can be overexpressed using the baculovirus expression system and purified.


Subject(s)
Membrane Glycoproteins/genetics , Membrane Transport Proteins , Neuropeptides , Synaptic Vesicles/metabolism , Animals , Baculoviridae , Biological Transport , Cattle , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/isolation & purification , Photochemistry , Rats , Spodoptera , Tetrabenazine/analogs & derivatives , Tetrabenazine/metabolism , Vesicular Biogenic Amine Transport Proteins , Vesicular Monoamine Transport Proteins
5.
J Biol Chem ; 272(41): 26049-55, 1997 Oct 10.
Article in English | MEDLINE | ID: mdl-9325342

ABSTRACT

The full-length cDNA for the rat recombinant synaptic vesicle monoamine transporter (rVMAT2) containing a COOH-terminal polyhistidine epitope was engineered into baculovirus DNA for expression in Spodoptera frugiperda (Sf9) cells. Using this recombinant baculovirus and cultured Sf9 cells, rVMAT2 has been expressed to high levels and purified to >95% homogeneity using immobilized Ni2+-affinity chromatography followed by lectin (concanavalin A) chromatography. Purified transporter was photolabeled using [125I]-7-azido-8-iodoketanserin ([125I]AZIK) and [125I]2-N-[(3'-iodo-4'-azidophenyl)propionyl]tetrabenazine ([125I]TBZ-AIPP). Both [125I]AZIK and [125I]TBZ-AIPP photoaffinity labeling of purified rVMAT2 were protectable by 10 microM tetrabenazine (TBZ), 10 microM 7-aminoketanserin, and 1 mM concentrations of the transporter substrates dopamine, norepinephrine, and serotonin. Radiolabeled peptides were generated using enzymatic and chemical methods, purified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NH2-terminal microsequenced. Radiosequencing of [125I]AZIK-labeled rVMAT2 indicated derivatization of Lys-20 in the NH2 terminus, just prior to putative transmembrane domain 1 (TMD1). [125I]TBZ-AIPP derivatized a segment of rVMAT2 between Gly-408 and Cys-431 in TMD10 and 11. These data implicate juxtaposition of TMD1 and 10/11.


Subject(s)
Affinity Labels/metabolism , Azides/metabolism , Ketanserin/analogs & derivatives , Membrane Glycoproteins/metabolism , Membrane Transport Proteins , Neuropeptides , Neurotransmitter Agents/metabolism , Synaptic Vesicles/metabolism , Tetrabenazine/analogs & derivatives , Affinity Labels/chemistry , Animals , Azides/chemistry , Binding Sites , Biological Transport , Iodine Radioisotopes , Ketanserin/chemistry , Ketanserin/metabolism , Membrane Glycoproteins/chemistry , Models, Molecular , Neurotransmitter Agents/chemistry , Peptide Mapping , Protein Binding , Rats , Recombinant Proteins/metabolism , Spodoptera , Synaptic Vesicles/chemistry , Tetrabenazine/chemistry , Tetrabenazine/metabolism , Vesicular Biogenic Amine Transport Proteins
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