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

J Neurosci Methods. 2005 Apr 15;143(1):87-94. doi: 10.1016/j.jneumeth.2004.09.015.


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).

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Binding Sites / drug effects
  • Binding Sites / physiology
  • Binding, Competitive / drug effects*
  • Binding, Competitive / physiology
  • Brain Chemistry / drug effects*
  • Brain Chemistry / physiology
  • Buffers
  • Cell Line
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism
  • Cocaine / metabolism
  • Cocaine / pharmacokinetics
  • Dopamine Uptake Inhibitors / metabolism
  • Dopamine Uptake Inhibitors / pharmacokinetics
  • Fluoxetine / analogs & derivatives*
  • Fluoxetine / metabolism
  • Fluoxetine / pharmacokinetics
  • Humans
  • Norepinephrine / metabolism*
  • Norepinephrine Plasma Membrane Transport Proteins
  • Piperazines / metabolism
  • Piperazines / pharmacokinetics
  • Radioligand Assay / methods*
  • Reproducibility of Results
  • Symporters / drug effects*
  • Symporters / metabolism
  • Temperature


  • Buffers
  • Dopamine Uptake Inhibitors
  • Norepinephrine Plasma Membrane Transport Proteins
  • Piperazines
  • SLC6A2 protein, human
  • Symporters
  • Fluoxetine
  • nisoxetine
  • vanoxerine
  • Cocaine
  • Norepinephrine