Methodologic pitfalls in measurement of 5-hydroxytriptamine uptake transporters in human platelets by [ 3H]-paroxetine binding assay

Abstract

Background Studies in platelet of 5-HT uptake transporters have been performed using binding assay methodology designed for ligand-receptor interactions; however, uptake transporters present requirements that may question the validity of these particular binding assays. Methods To explore methodologic aspects that may be crucial to the validity of these assays, we studied the binding of [ 3H]-paroxetine to platelet membranes of healthy subjects under different conditions of time, temperature, and protein concentrations. Results A correlation between protein concentration in incubation media and percentage of specific binding of [ 3H]-paroxetine was found: the lower the protein concentrations (10 and 20 μg/mL) in incubation media, the lower the percentage of specific [ 3H]-paroxetine binding. Moreover, low specificity in [ 3H]-paroxetine binding affected Bmax values obtained in saturation binding experiments. Conclusions The use of low protein concentrations could affect Bmax values in binding assays of 5-HT uptake transporters. This may induce confusing interpretation of data in clinical experiments that use human platelets to explore the participation of serotonin in depressed patients.