Heterologous up-regulation of Ni-coupled receptors in cultured neural hybrid cells by a transferable factor, whose expression is inhibited in a cyclic AMP-dependent, cell-specific manner

L Noronha-Blob, VC Lowe, WJ Kinnier and DC U'Prichard

Nova Pharmaceutical Corporation, Baltimore, Maryland 21224.

Opiate, muscarinic, and alpha 2-adrenergic receptors on NCB-20 and NG108-15 neuroblastoma hybrid cells were up-regulated by treatment of the cells with media (CM) conditioned by previous incubation with either cell type. NG cells treated with CM from both NCB and NG cells (NCB-CM or NG-CM) showed a 2-fold increase in opiate receptor density relative to untreated cells, with no change in ligand affinities. Opiate receptor density on NG cells was also enhanced approximately 2- fold by CM derived from dibutyryl cyclic AMP (dBc)-treated NG cells (NG- dBc-CM) but not by CM from dBc-treated NCB cells, (NCB-dBc-CM). The data suggest that a transferable factor that up-regulates NG opiate receptors is produced by untreated NCB and NG cells, and is either suppressed or inactivated in dBc-treated NCB cells but not in dBc- treated NG cells. Muscarinic and alpha 2-adrenergic receptor site densities on NG cells were also up-regulated approximately 2-fold by NCB-CM but not by NCB-dBc-CM. Thus, the factor induced a heterologous up-regulation of three classes of Ni-coupled receptor sites on NG cells. The up-regulating factor, which accumulates in the media with time in culture, also acts directly upon cells that are synthesizing/secreting the factor (auto-up-regulation). Thus, opiate receptor density increased in untreated NCB and NG cells, as well as in dBc-treated NG cells, as a function of cell growth, but did not increase on dBc-treated NCB cells. Coupling of NG opiate receptors to adenylate cyclase (AC) was not altered by CM. Prostaglandin E1- stimulated AC was maximally inhibited by (approximately 40%) by 1 microM DADLE with the same efficiency and potency in untreated as in CM- treated NG cell membranes. Furthermore, NCB-dBc-CM which does not induce NG opiate receptor up-regulation and NCB-CM, which does induce it, had no effect on inhibition of AC by DADLE. The up-regulating factor is a relatively small molecule (molecular weight = 3000-6000), whose synthesis and/or secretion is suppressed by dBc in NCB but not in the related NG hybrid. This unique cell specificity may be exploited to study the mechanism of opiate, muscarinic, and alpha 2-adrenergic receptor expression and turnover in cultured neural hybrid cells.

Volume 32, Issue 5, pp. 669-677, 11/01/1987
Copyright © 1987 by American Society for Pharmacology and Experimental Therapeutics