title: Pharmacological profile of radioligand binding to the norepinephrine transporter: instances of poor indication of functional activity.
abstract: 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).
authors.affiliation: Department of Psychiatry, Millhauser Laboratories, New York University School of Medicine, Room MHL-604, New York, NY 10016, USA. maarten.reith@med.nyu.edu
authors.forename: Maarten E A
authors.initials: ME
authors.lastname: Reith
authors.forename: Lijuan C
authors.initials: LC
authors.lastname: Wang
authors.forename: Aloke K
authors.initials: AK
authors.lastname: Dutta
mesh_terms: D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D001667:Binding, Competitive / Q000187:drug effects* / Q000502:physiology; D001923:Brain Chemistry / Q000187:drug effects* / Q000502:physiology; D002021:Buffers; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003042:Cocaine / Q000378:metabolism / Q000493:pharmacokinetics; D018765:Dopamine Uptake Inhibitors / Q000378:metabolism / Q000493:pharmacokinetics; D005473:Fluoxetine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000493:pharmacokinetics; D006801:Humans; D009638:Norepinephrine / Q000378:metabolism*; D050484:Norepinephrine Plasma Membrane Transport Proteins; D010879:Piperazines / Q000378:metabolism / Q000493:pharmacokinetics; D011869:Radioligand Assay / Q000379:methods*; D015203:Reproducibility of Results; D027981:Symporters / Q000187:drug effects* / Q000378:metabolism; D013696:Temperature
chemical_list: D002021:Buffers; D018765:Dopamine Uptake Inhibitors; D050484:Norepinephrine Plasma Membrane Transport Proteins; D010879:Piperazines; C493227:SLC6A2 protein, human; D027981:Symporters; D005473:Fluoxetine; C011386:nisoxetine; C043425:vanoxerine; D003042:Cocaine; D009638:Norepinephrine