INTACT CELL-BINDING AND THE RELATION TO OPIOID ACTIVITIES IN SH-SY5Y CELLS

Binding studies were conducted with intact SH-SY5Y cells, using the mu -opioid-selective antagonist [H-3]D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr- NH2. Previous studies had demonstrated equilibrium binding constants g enerally higher than those found when binding to membrane preparations for most opioid agonists. Studies on various other G protein-coupled receptors had shown that receptor internalization led to low apparent affinities for agonists and that short or low-temperature incubations could reveal high-affinity binding components. In SH-SY5Y cells, short (1-min) and 4 degrees C incubations did not reveal high-affinity bind ing components. Nonequilibrium binding experiments could also be used to compare binding affinities of opiate agonists with activity at mu r eceptors in the cells. SH-SY5Y cells have functional mu and delta rece ptors. mu Receptors can be measured independently of delta receptors i f activity is determined in the presence of the delta antagonist ICI 1 74,864. When both binding and activity, as measured by inhibition of f orskolin-stimulated cAMP accumulation, were conducted for 10 min, the ratio of binding IC50 to activity IC50 could give an indication of the relative efficacy of opioid agonists. These studies indicated that mo rphine has slightly lower efficacy than etorphine and the peptides DAM GO and DADLE. Each of these compounds has significantly higher efficac y than ethylketocyclazocine and the partial agonist buprenorphine.