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

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°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 observed at 24°C. The [³H]DADLE bound in the presence of chloroquine was not sensitive to trypsin or to 20 μM 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°C in the presence of chloroquine or at 37° 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.