DEMONSTRATION AND CHARACTERIZATION OF A STEREOSPECIFIC OPIATE RECEPTOR IN THE NEUROBLASTOMA N18TG2 CELLS

Abstract— Morphine has been observed to have only a minor effect on the prostaglandin E₁ (PGE₁) stimulated adenylate cyclase or the basal cyclase activity in the neuroblastoma N18TG2 calls. However, this ineffectiveness of the opiates was not due to the absence of opiate receptor in this cell line. Contrary to previous observations, neuroblastoma N18TG2 cells possessed a high affinity, stereospecific opiate receptor. When [³H]dihydromorphine and [³H]naloxone binding were determined, a single component receptor with K_diss= 25‐31 nm and with a capacity of 165 fmol/mg protein could be observed. This receptor has similar properties to those observed in the brain homogenates. The naloxone specific binding was dependent on the pH of the incubation medium and maximal binding occurred at pH 7.6. The agonist binding was inhibited by the alkali metal cations and divalent cations, while the antagonist binding was not affected by the cations significantly. There was no observable reversal of the Na⁺ inhibitory effect on agonist binding by the addition of Mn²⁺ to the incubation mixtures. Opiate binding to the neuroblastoma N18TG2 cells could be attenuated by pretreating the cells with N‐ethylmaleimide or proteolytic enzymes. Of the lipases tested, only phospholipase A₂ has an inhibitory effect on the naloxone binding. Fractionation of the cell homogenates with differential centrifugation and purification of the membrane fractions by sucrose gradients suggested the localization of the receptor at the plasma membranes. Thus, the receptor in the neuroblastoma N18TG2 cells closely resembles those observed in the brain homogenates