Differences in Binding Properties of μ and δ Opioid Receptor Subtypes from Rat Brain: Kinetic Analysis and Effects of Ions and Nucleotides

Abstract

Differences in binding properties of μ and δ opioid receptors were investigated using DAGO (Tyr-d-Ala-Gly-MePhe-Gly-ol) and DTLET (Tyr-d-Thr-Gly-Phe-Leu-Thr), which occur, respectively, as the most selective μ and δ radioligands available. At high concentration, each agonist is able to interact with its nonspecific sites. Competition experiments indicated that a two-site competitive model was adequate to explain the interactions of DAGO and DTLET with [³H]DTLET and [³H]DAGO binding sites, respectively. The weak cross-reactivity (≃10%) of DTLET for μ sites was taken into account in these experiments. On the other hand, DAGO and DTLET exhibit differential binding kinetics. Thus, at 35°C, the lifetime of DTLET within its receptor site about 14 times longer than that of the μ agonist. Sodium and manganese ions decrease the maximal number of high affinity μ and δ sites, but the sensitivity of μ receptors is three times higher towards Na⁺ and 20-fold higher towards Mn²⁺ than that of δ receptors. GTP reduces similarly the μ and δ binding whereas only the DAGO binding was modified by the nonhydrolyzable analogue guanylylimidodiphosphate [GMP-P(NH)P]. However, in the presence of Na⁺ ions, GMP-P(NH)P inhibits the DTLET binding in a concentration-dependent manner. The effects of Na⁺ and GMP-P(NH)P could be explained by a sequential transformation of δ receptors to low-affinity states. This model predicts that Na⁺, by lowering the affinity of a fraction of sites, produces a decrease in the maximal number of high-affinity δ receptors and that GMP-P(NH)P enhances the Na⁺, effect. Moreover, the binding kinetic to this high-affinity state was also modified by Na⁺ and nucleotides. All of these data support the existence of two independent μ and δ binding sites, the properties of which are differentially regulated by these endogenous effectors.