Enhancement of delta- but not mu-opiate agonist binding by calcium

Present evidence for distinction of 2 types of opiate receptor sites in rat brain homogenates originates from different relative affinities of morphine-like alkaloids and enkephalins to delta- or enkephalin and mu- or morphine-receptor sites. We now report that Ca2+ in a physiological dose range (0.5-3 mM) enhances the binding of 3H-enkephalin in hypotonically treated rat brain membranes, whereas specific binding of 3H-morphine-like alkaloids is not affected. Furthermore, the potency of [D-Ala2,D-Leu5]-enkephalin to inhibit [3H]-diprenorphine and [3H]-ethylketazocine binding increased in the presence of Ca2+, whereas an increase in potency of [D-Ala2,D-Leu5]-enkephalin to inhibit binding of mu-receptor ligands was not observed. Kinetic analysis revealed that Ca2+ decreased the rate of dissociation of [D-Ala2,D-Leu5]-enkephalin without affecting the rate of association, thereby increasing the affinity. However, in saturation binding studies, performed in diencephalic membranes, in which [D-Ala2,D-Leu5]-enkephalin binds predominantly to mu-receptors, Ca2+ also increased the binding affinity of [3H]- [D-Ala2,D-Leu5]-enkephalin. Double reciprocal analysis suggested a mixed competitive-noncompetitive type of inhibition of [D-Ala2,D-Leu5]-enkephalin binding by dihydromorphine. Thus, the interaction of delta- and mu-opiate ligands with mu-receptors may involve topographically different, but closely related binding sites, located on a single receptor molecule.