Preparation of brain membranes containing a single type of opioid receptor highly selective for dynorphin.

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Abstract
Opioid receptors on guinea pig brain membranes were alkylated by the naltrexone analog .beta.-chlornaltrexamine. Binding of the prototypical .mu. and .kappa. ligands, [3H]dihydromorphine and [3H]ethylketocyclazocine, was more readily affected by the reagent than was binding of the .delta. ligand, 3H-l[D-Ala2, D-Leu5]enkephalin. Treatment of membranes with .beta.-chlornaltrexamine in the presence of dynorphin resulted in significant protection of [3H]ethylketocyclazocine binding sites, without protection of [3H]dihydromorphine or 3H[D-Ala2, D-Leu5]enkephalin sites. Similarly, [D-Ala2, D-Leu5]enkephalin and sufentanil selectively protected binding sites for 3H-[D-Ala2, D-Leu5]enkephalin and [3H]dihydromorphine, respectively. Scatchard analysis of [3H]ethylketocyclazocine binding to untreated membranes suggested 2 types of binding site with 40-fold difference in affinities. Membranes treated with .beta.-chlornaltrexamine in the presence of dynorphin retained about 40% of the high-affinity sites and lost the low-affinity sites. Selective protection of sites with high affinity for dynorphin and ethylketocyclazocine was confirmed in competition binding assays. Evidently the 3 types of opioid receptor are not interconvertible and the endogenous peptide dynorphin is a highly selective ligand of the K opioid receptor.