Opiate Receptor Subtypes in the Rat Hypothalamus and Neurointermediate Lobe

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Abstract
The potent opiate radioligands [3H]etorphine, [3H]ethylketocyclazocine (EKC), and [3H]naloxone, bound specifically and saturably to a single class of membrane-binding sites in rat neurointermediate lobe (NIL), with Kd values of 3.7, 24, and 51 mM, respectively. In the hypothalamus (Ht), [3H] etorphine bound to specific and saturable sites with a Kd of 2.9 nM. Binding-inhibition studies with [3H]etorpine and unlabeled etorphine-HCl as well as [3H]EKC and unlabeled EKC, revealed high and low affinity binding sites in rat Ht and NIL as well as in the neural lobe of the bovine pituitary gland. [3H] naloxone also bound specifically to two classes of sites in Ht membranes, but to only a single class of low affinity sites in NIL membranes. Specific binding represented 80-90% of total [3H] etorphine binding, about 75% of total [3H]EKC binding, and 45-55% of total [3H]naloxone binding at 22 C in NIL and Ht, respectively. Relative binding potencies derived from Ki values for binding-inhibition studies of [3H]etorphine with opioid peptides and opiates were: 1) NIL, etorphine-HCl > dynorphin A > naloxone-HCl > dynorphin-(1-9) > .beta.-endorphin .mchgt. .alpha.-neoendorphin .apprxeq. (Leu5)enkephalin .apprxeq. DAGO (Tyr-D-Ala-Gly-NMe-Phe-Gly-ol); 2) Ht, etorphine HCl > naloxone-HCl > .beta.-endorphin > dynorphin A .mchgt. DAGO > morphiceptin .mchgt. (Leu5)enkephalin. Specific [3H]etorphine binding was also demonstrable after preincubation of NIL membranes with DAGO and (Leu5)enkephalin and after preincubation of Ht membranes with morphiceptin and (Leu5)enkephalin; such binding could be displaced by nonradioactive dynorphin A. In addition, [3H]etorphine binding to bovine neural lobe was displaceable by naloxone-HCl, with an ED50 of 43 nM. Specific ligands for .delta.-opiate receptors, such as (+)-SKF 10,047 (N-allylnorcyclazocine), phencyclidine (PCP), and (-)cyclazocine, displaced specifically bound [3H]etorphine and [3H]EKC from NIL membranes only at high (micromolar) concentrations. However, specific [3H]PCP sites were of higher affinity in NIL and Ht membranes, with similar Kd values of 102 and 190 nM respectively, and different concentrations (0.15 and 1.32 pmol/mg protein, respectively). These data have revealed several differences in the opiate-binding properties of rat Ht and NIL membranes. Opiate receptor binding is about 4-fold greater in the Ht than in the NIL and includes both high and low affinity .mu.- and .kappa.-opiate sites, as well as .delta./PCP receptor sites. In contrast, the NIL contains only low affinity .kappa.-opiate sites and .delta./PCP sites.