Showing papers by "Andreas Pfeiffer published in 1982"

Discrimination of three opiate receptor binding sites with the use of a computerized curve-fitting technique.

Abstract: The presence of different types of opiate binding sites was investigated with the use of a computerized, weighted, nonlinear least-squares regression program. The experimental data were obtained from four groups. Each of three labeled opiate ligands was displaced using each of the same unlabeled ligands. The resulting nine different ligand combinations of each group were evaluated by use of a curve-fitting program. The four groups consisted of the kappa ligand ethylketocyclazocine, the sigma ligand SKF 10047, and the oripavine derivatives etorphine and diprenorphine, each in conjunction with the delta opiate receptor ligand (D-Ala2,D-Leu5)-enkephalin and the mu opiate receptor ligand dihydromorphine. The binding model which best fitted each of the four groups suggested the existence of three different binding sites in the rat brain homogenate. Two of these sites conform to the previously described mu and delta sites. A third site (R3) displayed high affinity for ethylketocyclazocine, SKF 10047, etorphine, and diprenorphine but very low affinity for dihydromorphine and [D-Ala2,D-Leu5]enkephalin. Naloxone, cyclazocine, and dynorphin-(1--13) had high affinity for R3. Behavioral data support the interpretation that the R3 site may represent a kappa site at which SKF 10047 acts antagonistically.

Opiate receptor: multiple effects of metal ions.

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.

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

Abstract: 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 δ-or enkephalin and μ- 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 μ-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 μ-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 interactions of δ- and μ-opiate ligands with μ-receptors may involve topographically different, but closely related binding sites, located on a single receptor molecule.