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Notes: Abstract: The opiate antagonist [3H]diprenorphine ([3H]dip), a universal ligand at the μ, δ, and k opiate receptor subtypes, was used to study the effects of Ca-II, Cu-II, Mg-II, Mn-II, and Na+ on the rat cerebral opiate receptor. Two categories of effects were observed: (a) those on the binding rate constants and (b) those on binding capacity. (a) Sodium ions increased on- and off-rates on [3H]dip with a rather small net change in receptor affinity. The effects of Na+ and the divalent ions Ca-II, Mg-II, and Mn-II were antagonistic to each other. Ca-II, Mg-II, and the more effective Mn-II decreased receptor association and dissociation rates, again with minimal changes in the overall binding affinity in washed membrane homogenates. Previous studies using equilibrium binding analysis alone failed to detect changes in [3H]dip binding kinetics caused by these metal ions. In untreated rat brain homogenates, however, Ca-II (and to a lesser extent Mg-II) decreased [3H]dip binding, an effect distinct from that on the binding rate constants in washed membrane homogenates. (b) In untreated, Tris-buffer homogenates not containing external metal ions, a gradual decline in [3H]dip binding was observed. Cu-II or an equivalent endogenous divalent metal ion was identified as a causative factor, and Mn-II partially reversed this effect. Moreover, the addition of Mn-II stabilized the [3H]dip binding sites at very low concentrations of the metal (nM to μM range) that did not change the binding rate constants and that were in the physiological range of Mn-II in rat brain. This unique effect of Mn-II may represent a physiological function in the regulation of the opiate receptor that is not shared by Mg-II and Ca-II. The opposite effects of Cu-II and Mn-II on the in vitro receptor stability may be related to their opposite pharmacological effect in vivo. Finally, multiple changes of the effects of the tested metal ions on [3H]dip binding were observed during in vitro membrane homogenate dilution, centrifugation, and washing. These changes indicate that the opiate receptor complex as it exists in vivo may lose some of its functions and control mechanisms in vitro.