Computer modeling of subtypes of κ opioid receptors in adrenal medulla

Abstract

The existence of multiple subtypes of κ opioid receptors in brain and adrenal medulla, has been controversial. We have characterized opioid receptors in frozen membranes from bovine adrenal medulla by use of objective mathematical modeling. [ 3H]Etorphine, [ 3H]ethylketocyclazocine (EKC) and [ 3H][D-Ala 2, D-Leu 5]enkephalin (DADL) were utilized as labeled ligands. Self- and cross-displacement curves were constructed using the three corresponding unlabeled ligands in the presence or absence of increasing concentrations of DADL. Results indicated: (1) each of the three ligands studied individually showed the presence of heterogeneity of binding sites; (2) sites labeled by etorphine were heterogeneous: 84% of etorphine binding was displaceable by 10 −4 M DADL, while the remaining 16% was DADL non-suppressible; (3) 75% of the binding of EKC was displaceable by DADL while 25% was non-suppressible; (4) mathematical modeling showed the presence of three subtypes of κ binding sites (a) κ 1, showing slight selectivity for EKC relative to etorphine; (b) κ 2, with K d ≈ 1 nM for etorphine, and sufficiently high affinity for DADL (K d ≈ 150 nM) so that it is suppressible by 100 μM DADL; and (c) K 3, with no measurable affinity for DADL and a 27-fold selectivity for etorphine relative to EKC. The three subtypes of κ sites were present at concentrations of 7.4, 75, and 55 fmol/mg protein, respectively. The relative affinities of a series of κ agonists for the etorphine-binding sites were characterized. The present studies confirm the existence of three subtypes of κ opioid receptors in bovine adrenal medulla, and indicate the utility of mathematical modeling for characterization of complex receptor systems.