Interaction of enkephalin with opiate receptors in intact cultured cells

K. J. Chang, R. J. Miller, P. Cuatrecasas

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71 Scopus citations

Abstract

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 β-endorphin, but not by β-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-Leu)-enkephalin binding. The opposite is true for enkephalins, although etorphine and β-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.

Original languageEnglish (US)
Pages (from-to)961-970
Number of pages10
JournalMolecular Pharmacology
Volume14
Issue number6
StatePublished - Dec 1 1978

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

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