Molecular Pharmacology, Vol 14, 961-970, Copyright © 1978 by the American Society for Pharmacology and Experimental Therapeutics

Interaction of Enkephalin with Opiate Receptors in Intact Cultured Cells

¹ Department of Molecular Biology, The Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709

The interaction of enkephalins with opiate receptors in intact neuroblastoma cells (N4TG1) was studied by the use of a derivative of high specific activity (¹²⁵I-labeled) of the metabolically stable enkephalin analogue, (D-Ala², D-Leu⁵)-enkephalin. The binding is specifically inhibited by low concentrations of the natural (Leu⁵)- and (Met⁵)-enkephalins and by -endorphin, but not by -lipotropin. The binding data clearly show a single class of homogeneous binding sites without evidence for cooperative interactions at any ligand concentration. The association and dissociation rate constants of binding to cells are 3 x 10⁷ M^-1min^-1 and 0.035 min^-1, respectively, at 24°. In cells, the binding of [¹²⁵I] (D-Ala², D-Leu⁵)-enkephalin is saturated when 30 fmoles of the peptide are bound per 10⁶ cells. The K_d is 1 to 2 nM. No evidence for receptor or ligand-receptor complex internalization could be demonstrated on incubating the cells at 37°. The receptors in intact cells are very resistant to digestion by proteases and phospholipase A, in sharp contrast to the results observed in membrane preparations which show exquisite sensitivity to these enzymes. This resistance is apparently not due to the intracellular localization of a pool of receptors since cell homogenates have the same number of receptor sites as intact cells. In cells, the affinity of the receptor for enkephalin is very sensitive to the cationic composition of the medium. Removal of the divalent cations, Mg²⁺ and Ca²⁺, in the presence of Na⁺ reduces the affinity by about 3.5-fold. Narcotic agonists and antagonists are more potent in inhibiting [³H]naloxone binding than [¹²⁵I] (D-Ala², D-Leu⁵)-enkephalin binding. The opposite is true for enkephalins, although etorphine and -endorphin compete with both labeled ligands equally well. These results suggest that opiates and enkephalins bind differently to the same receptor or bind to different receptors with overlapping specificity.

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ACKNOWLEDGMENTS We gratefully acknowledge technical assistance of Mark Collins and Joanna T. Rogers.

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