PROTECTION BY OPIOID LIGANDS AGAINST MODIFICATION OF THE OPIOID RECEPTOR BY A CARBODIIMIDE

Opioid receptors in membranes prepared from guinea-pig cerebellum were modified irreversibly by treatment with a water soluble carbodiimide, 1-ethyl,3-(3-dimethylaminoethyl)carbodiimide (EDAC). This decreased the number of [H-3]bremazocine binding sites (B(max) reduced from 140 to 100 fmol/mg by 1 mM EDAC) without changing their affinity. When the EDAC concentration used was sufficient (500 mM) to inactivate almost all of the opioid receptors, the modification was partly prevented by inclusion of high concentrations (100-mu-M) of opioid agonists ([D-Ala2, MePhe4, Glyol5]-enkephalin, [D-Ala2, D-Leu5]-enkephalin, (+)-trans-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzo[b]thiophene-4-acetamide hydrochloride), although they exhibited equal efficacy irrespective of their mu, delta or kappa-type selectivity. However, almost all of the opioid binding sites were protected when a guanine nucleotide analogue (GppNHp, 100-mu-M) was also included with the agonists during carbodiimide treatment.