Characterization of [3H]paroxetine binding in rat brain.
The binding of the 5-hydroxytryptamine (5-HT, serotonin) uptake inhibitor [3H]paroxetine to rat cortical homogenates has been characterized. The effect of tissue concentration was examined and, with 0.75 mg wet weight tissue/ml in a total volume of 1,600 microliter, the binding was optimized with an apparent dissociation constant (KD) of 0.03-0.05 nM. Competition experiments with 5-HT, citalopram, norzimeldine, and desipramine revealed a high (90%) proportion of displaceable binding that fitted a single-site binding model. Fluoxetine and imipramine revealed, in addition to a high-affinity (nanomolar) site, also a low-affinity (micromolar) site representing approximately 10% of the displaceable binding. The specificity of the [3H]paroxetine binding was emphasized by the fact that 5-HT was the only active neurotransmitter bound and that the serotonin S1 and S2 antagonist methysergide was without effect on the binding. Both 5-HT- and fluoxetine-sensitive [3H]paroxetine binding was completely abolished after protease treatment, suggesting that the binding site is of protein nature. Saturation studies with 5-HT (100 microM) sensitive [3H]paroxetine binding were also consistent with a single-site binding model, and the binding was competitively inhibited by 5-HT and imipramine. The number of binding sites (Bmax) for 5-HT-sensitive [3H]paroxetine and [3H]imipramine binding was the same, indicating that the radioligands bind to the same sites. Lesion experiments with p-chloroamphetamine resulted in a binding in frontal and parietal cortices becoming undetectable and a greater than 60% reduction in the striatum and hypothalamus, indicating a selective localization on 5-HT terminals. Together these findings suggest that [3H]paroxetine specifically and selectively labels the substrate recognition site for 5-HT uptake in rat brain.
Marcusson
J O
JO
Department of Geriatric Medicine, University of Umeå, Sweden.
Bergström
M
M
Eriksson
K
K
Ross
S B
SB
D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism; D015283:Citalopram; D005473:Fluoxetine / Q000378:metabolism; D007099:Imipramine / Q000378:metabolism / Q000494:pharmacology; D008297:Male; D017374:Paroxetine; D010447:Peptide Hydrolases / Q000494:pharmacology; D010880:Piperidines / Q000378:metabolism*; D011437:Propylamines / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012701:Serotonin / Q000378:metabolism / Q000494:pharmacology; D012702:Serotonin Antagonists / Q000378:metabolism; D014316:Tritium
D010880:Piperidines; D011437:Propylamines; D012702:Serotonin Antagonists; D005473:Fluoxetine; D015283:Citalopram; D014316:Tritium; D012701:Serotonin; D017374:Paroxetine; D010447:Peptide Hydrolases; D007099:Imipramine