Opioid peptides induce reduction of enkephalin receptors in cultured neuroblastoma cells

Abstract

Cultured mouse neuroblastoma cells (N4TG1)^1,2 and neuroblastoma–glioma hybrid cells (NG108-15)^3–5 contain enkephalin (δ) receptors². Studies on hybrid cells have indicated that occupation of enkephalin receptors by opiates and opioid peptides leads to a time-dependent inhibition of adeny-late cyclase activity^3–5. If exposure of these cells to agonists is continued, the activity of adenylate cyclase gradually returns to normal and increases above normal on removal of opioids, a phenomenon thought to resemble tolerance and dependence in animals. Direct receptor binding studies on neuroblastoma cells have shown no internalization of receptors¹. Using rhodamine-conjugated [D-Ala²,Lys⁶]Leu-enkephalin and image-intensified fluorescence microscopy, enkephalin (δ) receptors in neuroblastoma cells were found slowly to form clusters which do not appear to be internalized⁶. This is in contrast to many polypeptide receptors^7–12 which are internalized after the initial receptor–ligand interactions. This internalization is believed to be related to the so-called down-regulation or desensitization. We now report that after one to several hours exposure of monolayer cell cultures to enkephalin, the number of enkephalin receptors measured subsequently in the extensively washed membrane preparations is greatly reduced. This reduction in receptor number seems to be selective for opioid peptides and is antagonized by a variety of opiate agonists and antagonists. These observations may be important in elucidating the phenomenon of opioid-induced desensitization.