NCB-20, a cloned hybrid cell line of mouse neuroblastoma and fetal Chinese hamster brain cell, has been used as a model system for the study of receptor-receptor interactions in the same cell. NCB-20 cells express 5-HT1 receptors which are linked to adenylate cyclase; these 5-HT sensitive adenylate cyclase-can be potently blocked by a 5-HT antagonist ketanserin in the low nanomolar concentration ranges. The plasma membranes of these cells also contain high affinity binding sites for 3H-ketanserin and 3H-mianserin. Moreover intact NCB-20 cells bind specifically 125-1-LSD; pharmacological characterization indicates that LSD is preferentially labeling the 5-HT2 receptor sites. We found that these cells are also equipped with 5-HT presynaptic components. These include a 5-HT uptake system and a very high Bmax of 3H-imipramine binding sites (50-times the Bmax in the CNS). We have succeeded in the solubilization of imipramine binding sites from the plasma membrane of these cells by using selective detergents and are attempting to develop useful techniques for the purification of these binding sites. It has been shown that acetylcholine is synthesized in NCB-20 cells and can be released by stimulation of 5-HT1 receptors. We found that these cells possess muscarinic cholinergic receptors which are linked to phosphoinositide specific phospholipase C. The carbachol-induced phosphoinositide hydrolysis can be blocked by atropine and pirenzepine, a selective M1 cholinergic receptor antagonist. The functional role of this receptor messenger is currently under investigation. In addition, we have initiated our attempt to prepare a cDNA library for the messenger RNA of NCB-20 cells in order to clone the imipramine binding sites as well as other receptor sites in these cells. This study should lead to a better understanding of molecular mechanisms of receptor regulation in a neuron.
