Artifactual High‐Affinity and Saturable Binding of [3H]5‐Hydroxytryptamine Induced by Radioligand Oxidation

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Abstract
The binding of [3H]5‐hydroxytryptamine (5‐HT, serotonin) to cerebellar membranes was examined after preincubation of [3H]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 nM [3H]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 [3H]5‐HT in the absence of ascorbate. The increase in apparent specific [3H]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 [3H]5‐HT binding reveals a KD value of 3.0 ± 0.3 nM and a Bmax value of 1.9 ± 0.2 pmol/g tissue. When ascorbate is absent during the preincubation, the KD is essentially un changed at 3.6 ± 0.1 nM but the Bmax is significantly increased to 36.5 ± 7 pmol/g tissue. Drug competition studies reveal that the apparent specific “[3H]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 [3H]5‐HT is essentially destroyed by a 4‐h incubation at 22°C in the absence of ascorbate. We conclude that oxidation of [3H]5‐HT prior to the addition of membrane homogenates leads to the detection of an artifactual high‐affinity and saturable “[3H]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 [3H]5‐HT.