Abstract

Chlornaltrexamine irreversibly inactivates opiate receptors in intact NG108-15 cells in a concentration- and time-dependent fashion. This inactivation is seen as a loss in the number of receptors per cell (decrease in B_max), and is quantitatively the same whether based on the number of specific binding sites for the agonist opioid peptide [D-Ala²-Met⁵] enkephalinamide or those for the opiate antagonist naltrexone. Inactivation appears to require direct interaction of chlornaltrexamine with these receptors as naltrexone and [D-Ala²-Met⁵]enkephalinamide protect against the irreversible effects of chlornaltrexamine. Furthermore, treatment with chlornaltrexamine does not decrease muscarinic or prostaglandin receptors or diazepam binding sites in NG108-15. In intact NG108-15 cells, the opioid peptide [D-Ala²-Met⁵]enkephalinamide can inhibit 80 ± 10% of the accumulation of cyclic AMP that is stimulated by prostaglandin E₁. In control cells, the action of this peptide is cooperative and the half-maximum effect (K_inh) occurs with 3.3 nM peptide. Under identical conditions, the half-maximum receptor occupancy by the peptide (K_D) is 23 nM. After the opiate receptor number is significantly decreased by previous incubation with chlornaltrexamine, both the maximal effects of prostaglandin E₁, as stimulator of cyclic AMP accumulation, and the opioid peptide, as inhibitor of this accumulation, are maintained. In the case where the receptor number has been reduced by 95%, although there is a small change (≤3-fold) in the K_D and K_inh values for this opioid peptide, there is no significant change in the K_D/K_inh ratio which remains ≃7. These results indicate that the high density of opiate receptors which exist in intact NG108-15 cells is not necessary for the maximum inhibitory effect of this enkephalinamide on adenylate cyclase. In addition, the mode of action of this peptide also appears to be relatively independent of the density of the opiate receptors in these cells.