Demonstration and characterization of zeta (ζ), a growth-related opioid receptor, in a neuroblastoma cell line

Abstract

Endogenous opioids and opioid receptors (i.e. endogenous opioid systems) are involved in carcinogenesis. Using homogenates of S20Y neuroblastoma (NB) cells grown in culture, the binding of a growth-selective ligand, [Me 5]enkephalin, was examined to ascertain the zeta (ζ) opioid receptor. Specific and saturable binding of [ 3H]-[Met 5]enkephalin was detected in NB cells; the data were consistent with a single binding site. Scatchard analysis yielded a K d of 1.6 nM and a binding capacity B max off 48.1 fmol/mg protein; 14,000 receptors per cell were estimated. Binding was dependent on protein concentration, time, temperature, and pH, and was sensitive to 100 nM, but not 5 nM, Na +, Ca 2+, and Mg 2+; GppNHp at concentrations of 100–500 mM had little effect on binding. Optimal binding required protease inhibitors, and pretreatment of the tumor cell homogenates with trypsin markedly reduced [ 3H]-[Met 5]enkphalin winding, suggesting that the binding site was proteinaceous in character. Displacement experiments indicated that [Met 5]enkephalin was the most potent displacer of [ 3H]-[Met 5]enkephalin. Cell density (log, confluence, postconfluence) did not alter the K d or B max. This study serves as the first demonstration and characterization of the zeta (ζ) opioid receptor in tissue culture cells. The homogenous nature of NB cell cultures, along with the enrichment in receptor number, provides an excellent model system to isolate and purify the ζ receptor.