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).
Publication types
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Research Support, N.I.H., Extramural
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Binding Sites / drug effects
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Binding Sites / physiology
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Binding, Competitive / drug effects*
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Binding, Competitive / physiology
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Brain Chemistry / drug effects*
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Brain Chemistry / physiology
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Buffers
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Cell Line
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Cell Membrane / drug effects
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Cell Membrane / metabolism
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Cocaine / metabolism
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Cocaine / pharmacokinetics
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Dopamine Uptake Inhibitors / metabolism
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Dopamine Uptake Inhibitors / pharmacokinetics
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Fluoxetine / analogs & derivatives*
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Fluoxetine / metabolism
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Fluoxetine / pharmacokinetics
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Humans
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Norepinephrine / metabolism*
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Norepinephrine Plasma Membrane Transport Proteins
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Piperazines / metabolism
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Piperazines / pharmacokinetics
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Radioligand Assay / methods*
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Reproducibility of Results
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Symporters / drug effects*
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Symporters / metabolism
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Temperature
Substances
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Buffers
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Dopamine Uptake Inhibitors
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Norepinephrine Plasma Membrane Transport Proteins
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Piperazines
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SLC6A2 protein, human
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Symporters
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Fluoxetine
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nisoxetine
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vanoxerine
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Cocaine
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Norepinephrine