In the present study, we examined the binding of [3H][D-Ala2,D-Leu5]enkephalin ([3H]DADL) to bovine thalamic membranes. Scatchard plots were linear with a K(D) of 0.7 nM. However, competition experiments suggested binding heterogeneity. Approximately 20% of [3H]DADL binding was easily inhibited by [D-Pen2,D-Pen5]enkephalin (DPDPE) and was insensitive to morphine, implying labeling of δ receptors. The remaining 80% of binding was quite sensitive to both morphine and [D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAGO) and insensitive to DPDPE, consistent with a μ receptor. However, this binding did not correspond to classical morphine-selective μ receptors. Unlike morphine-selective receptors, this binding had similar affinities for morphine, DAGO, DADL and [D-Ser2,Leu5]enkephalin Thr6 (DSLET). In addition, it was far more sensitive to naloxonazine's wash-resistant inhibition and magnesium-induced enhancement of binding than either the morphine-selective (μ2) or δ sites. [3H]DSLET binding yielded results very similar to those using [3H]DADL. In conclusion, approximately 80% of [3H]DADL binding in thalamus corresponds to a μ receptor distinct from the classical morphine-selective site. Based upon the results of our studies, we feel that this binding represents μ1 receptors. DPDPE (10 nM) can effectively inhibit the binding of [3H]DADL to δ receptors, leaving a relatively homogeneous labeling of μ1 sites. The availability of this selective binding assay should facilitate additional studies of μ1 receptors.
