Naloxazone irreversibly inhibits the high affinity binding of [125I]D-ala2-D-leu5-enkephalin

Scatchard analyses of ¹²⁵I]D-ala²-D-leu⁵-enkephalin binding in rat brain membranes are curvilinear, suggesting low and high affinity sites. Treating the membranes with naloxazone abolishes the high affinity binding with slight effect on low affinity binding. Displacement of ¹²⁵I]-D-ala²-D-leu⁵-enkephalin binding by morphine in untreated membranes is biphasic. Displacement by morphine in naloxazone-treated tissue is monophasic, with no inhibition by low concentrations of morphine. Naloxazone treatment has little effect on displacements by unlabeled D-ala²-D-leu⁵-enkephalin. Binding in N4TG1 neuroblastoma cells, which demonstrates a linear Scatchard plot with single affinity constant similar to that of the low affinity binding in brain, is less sensitive to naloxazone's actions. Naloxazone treatment $\text{in vivo}$ inhibits D-ala²-D-leu⁵-enkephalin analgesia.