Artifactual high-affinity and saturable binding of [3H]5-hydroxytryptamine induced by radioligand oxidation.
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 homogenates in Tris-HCl buffer with 0.1% ascorbate. Cerebellar membranes were used because of their low density of 5-HT1 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 (butylated 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 unchanged 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 nonhydroxylated tryptamines (5-methoxytryptamine, tryptamine). HPLC analysis demonstrates that [3H]5-HT is essentially destroyed by a 4-h incubation at 22 degrees C in the absence of ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)
Peroutka
S J
SJ
Ison
P J
PJ
Liu
D U
DU
Barrett
R W
RW
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology; D001667:Binding, Competitive; D002021:Buffers; D002525:Cerebellar Cortex / Q000378:metabolism; D002531:Cerebellum / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D008024:Ligands / Q000378:metabolism*; D008566:Membranes / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D012701:Serotonin / Q000378:metabolism*; D013223:Statistics as Topic; D013997:Time Factors; D014316:Tritium
D000975:Antioxidants; D002021:Buffers; D008024:Ligands; D014316:Tritium; D012701:Serotonin; D001205:Ascorbic Acid