A Mu-opioid Receptor-filter Assay. Rapid Estimation of Binding Affinity of Ligands and Reversibility of Long-lasting Ligand-receptor Complexes

A filter-associated binding technique, originally described by Leysen and Gommeren [J. Receptor Res. 4, 817 (1984); Drug Dev. Res. 8, 119 (1986)], was focused on the study of mu-opioid receptor sites. The interesting binding features of 3H-Sufentanil, a mu-selective radioligand, permitted such a filter procedure to be performed. Its application was 3-fold. A 5-min binding assay allowed us to verify that specific 3H-Sufentanil binding to filter-absorbed rat brain membranes was endowed with the known kinetic and equilibrium binding properties of membrane-bound mu-opioid receptor sites. Moreover, the filtration technique allowed us to get rapid information about the dissociation rates of unlabelled compounds from the receptor sites. In practice, membranes, preincubated with high concentrations of cold drugs, were absorbed to filters. Dissociation was achieved by repeatedly applying buffer samples on the filter, and monitored by the recovery in free specific 3H-Sufentanil binding sites. Finally, such a dissociation procedure, improved by a washing buffer at high ionic strength, was found to be much more efficient and attractive than the classical dilution-centrifugation procedure, especially for slowly-dissociating compounds. Special attention was paid to the discrimination between pseudo-irreversible binding of drugs and stable covalent labelling of mu-opioid receptor sites (either by affinity or photoaffinity probes), particularly when unlabelled ligands were the only tools available.