Potent analgesics and their opioid receptor interaction 30: Regulation of opioid receptor binding; Possible mechanism of sulfhydryl groups in the binding site

Abstract

We have reported that a marked decrease of the receptor binding of opioid agonist but not antagonist was observed by treating the brain preparation with 5, 5’-dithio-bis-(2-nitrobenzoic acid), DTNB. In this paper, the receptor binding of four different ligands was investigated with the treatment of DTNB or 5, 5’-dithiobis-(2-nitro-N-2’-hydroxyethylbenzamide), DTNHEB, as a relatively positive charged analog of DTNB. DTNB (1 to 1000 μM) and DTNHEB (0.5 to 100 μM) inhibited the binding of 3H-dihydromorphine (DHM) and 3H-D-Ala2-D-Leu 5-enkephalin (DADLE). Despite the presence of maximally effective concentrations of DTNB for DHM and DADLE, the receptor binding of 3H-ethylketocyclazocine (EKC) decreased intermediately, like the effect of a partial agonist. DTNHEB inactivated the binding of EKC in a similar fashion to that of DHM. DTNB did not alter the intensity of the decrease of EKC binding by DTNHEB, even given concurrently. It suggests that an anionic center of the receptor has multiple active sulfhydryl sites. Attachment at one point (DTNB-sensitive) may define the direction of receptor interaction, and the other point (DTNHEB-sensitive) may play an important role for the formation of receptor-1igand complex. The ability of GTP to inhibit DADLE binding to the receptor disappeared by the pretreatment with DTNB, but DTNB-induced inactivation of opioid agonist binding was potentiated in the presence of NaCI. DTNB-sensitive site may couple a mechanism of ligand binding that GTP regulates. It seems that sodium ion itself is concerned with the control for the whole receptor system and does not involve the function of the sulfhydryl groups. DTNB and/or DTNHEB did not affect the binding of 3H-phencyclidine.