Ni-coupled receptors in cultured neural hybrid cells: cell specificity for dibutyryl cyclic AMP-induced down-regulation but not morphological differentiation.Mol Pharmacol. 1986 Dec; 30(6):526-36.MP
Opiate, muscarinic, and alpha 2-adrenergic receptors and the Ni-coupled response of adenylate cyclase (AC) inhibition were examined in neuroblastoma X glioma NG108-15 (108 CC15) and neuroblastoma X Chinese hamster brain NCB-20 clonal hybrid cells, induced to differentiate with 1.0 mM dibutyryl cAMP (dBcAMP). Scatchard analysis of binding of the opiate agonist 3H-(D-Ala2,D-Leu5)enkephalin (DADLE) and the antagonist [3H] diprenorphine to dBcAMP-treated NCB-20 cell membranes indicated an 80% reduction in opiate receptor density relative to untreated cells (Bmax = 47 +/- 11 fmol/mg of protein versus 220 +/- 48 fmol/mg of protein), with no change in ligand affinities. Binding of the muscarinic cholinergic antagonist [3H]quinuclidinyl benzilate and the alpha 2-adrenergic agonist [3H]-p-aminoclonidine to dBcAMP-treated NCB-20 membranes was also reduced by 50% and 28%, respectively. In contrast, treatment of NG108-15 cells with dBcAMP did not down-regulate opiate, muscarinic, or alpha 2-adrenergic receptor sites. Opiate and alpha 2-adrenergic receptor sites were not down-regulated in the N18TG2 neuroblastoma clone, the common parent of both the hybrid cells, and the apparent source of these receptors. The C6BU-1 parent of the NG108-15 hybrid showed poor specific binding of all ligands examined. dBcAMP was very potent in inducing opiate receptor site down-regulation of NCB-20 cells, with an ED50 after 4 days treatment of 8 microM. The time course of loss of [3H]DADLE and [3H]quinuclidinyl benzilate specific binding was similar, and maximum down-regulation was achieved after 2 days. In contrast, neither higher concentrations of dBcAMP (5.0 mM) nor longer treatment times (7 days) resulted in down-regulation of receptor sites on NG108-15 cells. Coupling of opiate receptors to AC was also selectively altered in differentiated NCB-20 cells. Prostaglandin E1-stimulated AC was maximally inhibited by 1 microM DADLE in membranes from undifferentiated cells to different degrees (30% in NCB-20 and 54% in NG108-15). dBcAMP treatment had no effect on opiate inhibition of AC in NG108-15 cells but reduced by 50% the maximum opiate inhibition of AC in NCB-20 cells. These data indicate that the signal for receptor down-regulation which was triggered by dBcAMP in the NCB-20 cell comes uniquely from the Chinese hamster brain cell NCB-20 parent. The differences between NCB-20 and NG108-15 cells in the regulation of Ni-coupled receptors provides an example of dBcAMP-induced heterologous down-regulation with unique cell specificity, which is unrelated to the morphological differentiation process triggered by dBcAMP, which is common to both cells.