Down-regulation of opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells. Chloroquine promotes accumulation of tritiated enkephalin in the lysosomes

Abstract

Opiate receptor down-regulation in neuroblastoma X glioma NG108-15 hybrid cells possibly involved the internalization of ligand-receptor complexes during chronic treatment. However, receptor internalization was not supported by the observed decrease in [3H] enkephalin(D-Ala2,D-Leu5) ( [3H]DADLE) associated with the hybrid cells during prolonged incubation with 10 nM [3H]DADLE at 37 degrees C. This decrease in [3H]DADLE bound was determined to be due to degradation of the ligand-receptor complexes, for a time-dependent increase in [3H]DADLE bound was observed when the incubations were carried out in the presence of 0.1 mM chloroquine. The increase did not exceed the amount of down-regulated receptor, could be blocked by naloxone, and was not observed at 24 degrees C. The [3H]DADLE bound in the presence of chloroquine was not sensitive to trypsin or to 20 microM diprenorphine. The accumulated [3H]DADLE was demonstrated to be intracellularly located by the fractionation of the homogenates in self-generating Percoll gradients. In the presence of chloroquine, a time-dependent translocation of [3H]DADLE from the plasma membrane-enriched fractions to the lysosome-enriched fractions was observed. The translocation was not observed at 24 degrees C in the presence of chloroquine or at 37 degrees C in the absence of chloroquine. The [3H]DADLE in the lysosome-enriched fractions was not sensitive to trypsin and remained bound in the presence of chloroquine. With the removal of chloroquine, an increase in the release of [3H]DADLE into the medium was observed. Sephadex G-50 column chromatography of the sodium deoxycholate extracts of the lysosome-enriched fractions suggested that the [3H]DADLE was bound to macromolecules intracellularly. Thus, chronic [3H]DADLE treatment of the hybrid cells resulted in an internalization of ligand-receptor complexes which were degraded in the lysosomes. Subsequently, the [3H]DADLE was regurgitated by the hybrid cells.