Cluster formation of opiate (enkephalin) receptors in neuroblastoma cells: differences between agonists and antagonists and possible relationships to biological functions.

Neuroblastoma cells were used to study the surface distribution and organization of opiate (enkephalin) receptors and the possible relevance of changes in these variables to biological functions. Opiate receptors readily form clusters that are visible by image-intensifier fluorescent microscopy and are localized on both the cell body and processes. These clusters do not become internalized even during prolonged incubation periods. The receptors appear to pre-exist largely in a diffuse state, with only a very small number pre-existing as clusters. The clusters are induced within 1 hr and they are stable for prolonged (7-9 hr) periods, even after removal of the receptor-bound ligand. Agonists and antagonists are both equally capable of inducing receptor clustering. However, the clusters induced by agonists are different from those induced by antagonists; the former can be dispersed by treatment with dithiothreitol. This dispersion requires removal of the receptor-bound agonist, indicating that the hormone protects or stabilizes disulfide bonds which are critical for maintenance of the clustered state. Pretreatment of cells with sulfhydryl-blocking reagents (iodoacetate, iodoacetamide, and N-ethylmaleimide) prevents cluster formation but does not alter the ability of agonists to inhibit adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity. Neither the number nor the affinity of binding sites is altered by pretreatment with opiates. These studies suggest that at least the acute, immediate biological effects of opiates and enkephalins occur prior to and are independent of the formation of gross receptor clusters. The possible relationship of cluster formation to the actions of opiates remains to be determined.