Effects of the affinity ligands 14-beta-chloroacetylnaltrexone and 14-beta-bromoacetamidomorphine on [3H]-dihydromorphine binding sites in rat brain.

The aim of the present study was to examine the inhibitory effects in vitro of the affinity ligands 14-beta-chloroacetylnaltrexone (CAN) and 14-beta-bromoacetamidomorphine (BAM) to characterize the pharmacological specificity of the ligands for high and low affinity opioid binding sites. Rat brain membranes were incubated with 2.0 microM BAM or CAN, or their parent compounds (morphine and naltrexone, respectively) at 37 degrees for 45 min, and the membranes were washed extensively to remove the unbound ligand. The specific binding of 0.3 nM [3H]dihydromorphine ([3H]DHM) was reduced 32 +/- 7% in membranes treated with CAN and BAM, whereas specific binding in preparations treated with morphine and naltrexone was not significantly different from controls. An increased affinity of BAM and CAN relative to morphine and naltrexone could not account for the observed irreversible inhibition, since the relative affinity of CAN was similar to that of naltrexone and that of BAM was 10-fold less active than morphine. Saturation binding assays revealed that the affinity ligands selectively abolished a high affinity binding site (Kd = 0.3 nM, Bmax 95 fmoles/mg protein), which comprised approximately one-third of the total number of sites. The affinity of the remaining site (Kd = 4.0 nM) was not altered significantly. The results indicate that the inhibition caused by the affinity ligands is irreversible and represents inactivation of high affinity opioid binding sites in a relatively selective manner.