Relationship Between Opioid-Receptor Occupancy and Stimulation of Low-KmGTPase in Brain Membranes

Abstract

Treatment of rat brain membranes with the irreversible opioid ligand cis-3-methylfentanylisothiocyanate (Superfit) was used to reduce gradually the number of available binding sites for the .delta.-selective agaonist [3H][D-Ser2,Leu5]enkephalin-Thr6 ([3H]DSLET). Subsequently, the correlation between ligand binding and low-Km GTPase was investigated. Alkylation with 10 .mu.M nd 25 .mu.M Superfit inactivated 66% and 71% of high-affinity (KD, 1 nM) binding sites without decreasing the affinity of the remaining sites and the stimulation of low-Km GTPase by DSLET. Following exposure of the membranes to 50 .mu.M and 75 .mu.M Superfit, ligand binding was confined to the low-affiity (KD 20) sites. In these membranes, the .delta.-agonists DSLET and [D-Pen2,D-Pen5]enkephalin still stimulated low-Km GTPase, and these effects were blocked by ICI 174864 (N,N-diallyl-Tyr-AIB-AIB-Phe-Leu-OH, AIB, .alpha.-aminoisobutyric acid) a .delta.-selective antagonist. A similar relationship between low-affinity ligand binding and GTPase stimulation was observed following alkylation of the .delta.-opioid receptor with the nonselective irreversible antagonist .beta.-chlornaltrexamine in the presence of protective concentrations of DSLET. The results reveal spare receptor sites in the coupling of the .delta.-opioid receptor to low-Km GTPase i brain and identify low-affiity ligand binding as a functional component in the process.