CHARACTERIZATION AND VISUALIZATION OF [H-3] DERMORPHIN BINDING TO MU-OPIOID RECEPTORS IN THE RAT-BRAIN - COMBINED HIGH SELECTIVITY AND AFFINITY IN A NATURAL PEPTIDE AGONIST FOR THE MORPHINE (MU) RECEPTOR

Dermorphin, Tyr‐DAla‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH2, a potent opioid peptide isolated from amphibian skin, is endowed with outstanding structural and biological features. It has no common structure with mammalian opioid peptides and is a unique example of a peptide, synthesized by an animal cell, which contains a D‐amino acid in its native sequence. We have undertaken a complete evaluation of the receptor selectivity of dermorphin, together with the binding characteristics and receptor distribution of [3H]dermorphin in the rat brain. Dermorphin was tested for its relative affinity to μ‐, δ‐and χ‐opioid receptors by determining its potency in displacing the selective μ‐receptor ligand [3H]Tyr‐DAla‐Gly‐MePhe‐Gly‐ol (where Gly‐ol=glycinol), the prototypic δ‐receptor ligand, [3H]Tyr‐DPen‐Gly‐Phe‐DPen (where DPen =β,β‐dimethylcysteine) and the χ ligand [3H]ethylketocyclazocine from rat brain and/or guinea pig cerebellum membrane preparations. Inhibitory constant (Ki) values of dermorphin were 0.7 nM. 62 nM and > 5000 nM respectively for μ, δ and χ sites, indicating a selectivity ratio Ki(δ)/Ki(μ) = 88. Under similar conditions, Tyr‐DAla‐Gly‐MePhe‐Gly‐ol, which is regarded as one of the most selective high‐affinity μ‐agonist available, exhibited a selectivity ratio of 84. Specific binding properties of tritium‐labeled dermorphin (52 Ci/mmol) were characterized in the rat brain. Equilibrium measurements performed over a large range of concentrations revealed a single homogeneous population of high‐affinity binding sites (Kd= 0.46 nM; Bmax= 92 fmol/mg membrane protein). Profound differences were observed in the potencies displayed by various selective opiates and opioids ligands in inhibiting the specific binding of [3H]dermorphin. The rank order of potency was in good agreement with that obtained with other μ‐selective radiolabeled ligands. Receptor autoradiography in vitro was used to visualize the distribution of [3H]dermorphin binding sites in rat brain. The labeling pattern paralleled that observed using other μ probes. Binding parameters and selectivity profile of [3H]dermorphin on slide‐mounted sections were similar to those obtained with membrane homogenates. Finally, intracerebroventricular administration of synthetic dermorphin into mice showed that this peptide is the most potent analgesic known to date, being up to 5 and 670 times more active than β‐endorphin and morphine, respectively. Higher doses induced catalepsy. The overall data collected demonstrate that dermorphin is the first among the naturally occurring peptides to be highly potent and nearly specific super‐agonist towards the morphine (μ) receptor. The high binding specificity and affinity of dermorphin together with its very low non‐specific binding, its high resistance to enzymatic degradation and its ability to cross the blood brain barrier make this natural peptide very attractive for dissecting the role(s) and for identifying molecular and conformational determinants of high‐affinity binding to the morphine receptor. Copyright © 1990, Wiley Blackwell. All rights reserved