Interaction of Opiates with Opioid Binding Sites in the Bovine Adrenal Medulla: II. Interaction with K Sites

Abstract

In this study we examined the interaction of opiates with K binding sites in the bovine adrenal medulla. [³H]Ethylketocyclazocine (EKC), [³H]etorphine, and [³H]bremazocine stereoselective bindings were used to assay these interactions. The K sites were found to be heterogeneous: [³H]bremazocine identified with high affinity all subtypes of these sites. [³H]EKC, in the presence of saturating concentrations of [D-Ala², D-Leut]-enkephalin (DADLE) (5μM), was used to identify K¹ sites, on which dynorphin A (1–13) bound with high affinity. Either [³H]EKC or [³H]etorphine in the presence of 5μM DADLE identified the K² subtype. This subtype was found to interact with β-endorphin and especially with the octapeptide Met⁵-enkephalyl-Arg⁶-Gly⁷-Leu⁸. Furthermore, [³H]etorphine identified in the bovine adrenal medulla a third high-affinity component, in the presence of 5 μM DADLE. This residual interaction was found to be equally stereoselective and presenting K selectivity. Met⁵-enkephalyl-Arg⁶-Phe⁷ interacted preferentially with this site. The three K subtypes interacted differentially with monovalent (Na⁺, K⁺, and Li⁺) and divalent (Ca²⁺, Mg²⁺, and Mn²⁺) ions by modification of the apparent concentration of the accessible sites and/or by changes of the apparent K_D for radioligands. Modifying agents (proteolytic enzymes, thiol-modifying reagents, and A₂-phospholipase) produced different effects on each subtype of the K site, suggesting a different protein (or protein-lipid?) composition.