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Artifactual High‐Affinity and Saturable Binding of [ 3 H]5‐Hydroxytryptamine Induced by Radioligand Oxidation
Author(s) -
Peroutka Stephen J.,
Ison Pamela J.,
Liu David U.,
Barrett Ronald W.
Publication year - 1986
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1986.tb02828.x
Subject(s) - chemistry , binding site , sodium metabisulfite , sodium ascorbate , sodium , mole , serotonin , ascorbic acid , biochemistry , stereochemistry , receptor , food science , organic chemistry
The binding of [ 3 H]5‐hydroxytryptamine (5‐HT, serotonin) to cerebellar membranes was examined after preincubation of [ 3 H]5‐HT in the presence or absence of ascorbate. The tissue preparation was identical in all experiments and consisted of rat cerebellar homog‐enates in Tris‐HCl buffer with 0.1 % ascorbate. Cerebellar membranes were used because of their low density of 5‐HT, binding sites. In the presence of ascorbate during a 4‐h preincubation period, minimal specific binding of 2 n M [ 3 H]5‐HT is detected. Similar results are obtained with equimolar concentrations of other antioxidants (bu‐tylated hydroxytoluene, sodium dithionite, and sodium metabisulfite). Apparent specific binding increases 14‐fold following a 4‐h preincubation of [ 3 H]5‐HT in the absence of ascorbate. The increase in apparent specific [ 3 H]5‐HT binding is time‐dependent and plateaus after 4–6 h of preincubation. When ascorbate is present during the 4‐h preincubation, Scatchard analysis of [ 3 H]5‐HT binding reveals a K D value of 3.0 ± 0.3 nM and a B max value of 1.9 ± 0.2 pmol/g tissue. When ascorbate is absent during the preincubation, the K D is essentially un changed at 3.6 ± 0.1 nM but the B max is significantly increased to 36.5 ± 7 pmol/g tissue. Drug competition studies reveal that the apparent specific “[ 3 H]5‐HT binding” in the absence of ascorbate appears to be displaced by nanomolar concentrations of hydroxylated tryptamines (5‐HT, bufotenine) but not by nonhydroxy‐lated tryptamines (5‐methoxytryptamine, tryptamine). HPLC analysis demonstrates that [ 3 H]5‐HT is essentially destroyed by a 4‐h incubation at 22°C in the absence of ascorbate. We conclude that oxidation of [ 3 H]5‐HT prior to the addition of membrane homogenates leads to the detection of an artifactual high‐affinity and saturable “[ 3 H]5‐HT binding site” that appears to have a distinct pharmacological profile. The capacity of other ligands to compete for this binding site is related to their potential as antioxidants and not to their affinity for a novel membrane recognition site labeled by [ 3 H]5‐HT.