Validation of a fluorescence-based high-throughput assay for the measurement of neurotransmitter transporter uptake activity.
Pre-synaptic dopamine, norepinephrine and serotonin transporters (DAT, NET and SERT) terminate synaptic catecholamine transmission through reuptake of released neurotransmitter. Common approaches for studying these transporters involve radiolabeled substrates or inhibitors which, however, have several limitations. In this study we have used a novel neurotransmitter transporter uptake assay kit. The assay employs a fluorescent substrate that mimics the biogenic amine neurotransmitters and is taken up by the cell through the specific transporters, resulting in increased fluorescence intensity. In order to validate the assay, a variety of reference and proprietary neurotransmitter transporter ligands from a number of chemical and pharmacological classes were tested. The ability of these compounds to inhibit the selective transporter-mediated uptake demonstrated a similar rank order of potency and IC(50) values close to those obtained in radiolabeled neurotransmitter uptake assays. The described assay enables monitoring of dynamic transport activity of DAT, NET and SERT and is amenable for high-throughput screening and compound characterization.
Jørgensen
Susanne
S
NeuroSearch A/S, Drug Discovery, 93 Pederstrupvej, Ballerup DK-2750, Denmark.
Nielsen
Elsebet Østergaard
EØ
Peters
Dan
D
Dyhring
Tino
T
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D050483:Dopamine Plasma Membrane Transport Proteins / Q000032:analysis / Q000187:drug effects / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D008024:Ligands; D008856:Microscopy, Fluorescence / Q000379:methods*; D015394:Molecular Structure; D009448:Neurochemistry / Q000379:methods*; D018377:Neurotransmitter Agents / Q000819:agonists*; D050479:Neurotransmitter Transport Proteins / Q000032:analysis* / Q000187:drug effects* / Q000378:metabolism; D050484:Norepinephrine Plasma Membrane Transport Proteins / Q000032:analysis / Q000187:drug effects / Q000378:metabolism; D011619:Psychotropic Drugs / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000032:analysis / Q000187:drug effects / Q000378:metabolism
D050483:Dopamine Plasma Membrane Transport Proteins; D005456:Fluorescent Dyes; D008024:Ligands; D018377:Neurotransmitter Agents; D050479:Neurotransmitter Transport Proteins; D050484:Norepinephrine Plasma Membrane Transport Proteins; D011619:Psychotropic Drugs; D050486:Serotonin Plasma Membrane Transport Proteins