REGULATION OF HUMAN ALPHA-4-BETA-2 NEURONAL NICOTINIC ACETYLCHOLINE-RECEPTORS BY CHOLINERGIC CHANNEL LIGANDS AND 2ND-MESSENGER PATHWAYS

The alpha 4 beta 2 nicotinic acetylcholine receptors (nAChRs), a major subtype in the brain, have been shown to be modulated by chronic trea tment with nicotine. In this study, the regulation of recombinant huma n alpha 4 beta 2 nAChR subtype by (-)-nicotine and other cholinergic c hannel modulators was studied using human embryonic kidney 293 cells s tably expressing this subunit combination. The treatment of transfecte d cells with (-)-nicotine and other activator ligands, including (-)-c ytisine, 1,1-dimethyl-4-phenylpiperazinium, -5-(1-methyl-5-(1-methyl-2 -pyrrolidinyl)isoxazole, and (+/-)-epibatidine, resulted in concentrat ion-dependent increases in the levels of alpha 4 beta 2 nAChRs. The in crease in [H-3]cytisine binding sites was initiated by low concentrati ons of (-)-nicotine (<100 nM); was maximal at 10 mu M (15-fold), rapid (t(0.5) = 4.0 +/- 0.5 hr), and totally reversible (t(0.5) = 11.7 +/- 0.1 hr); and occurred with no change in ligand binding affinity. Antag onists, including dihydro-beta-erythroidine, d-tubocurarine, and methy llycaconitine, also elicited significant increases in receptor levels. A good correlation was observed between the K-i values for binding in hibition and the EC50 values for receptor up-regulation. Treatment of cells with mecamylamine, a noncompetitive antagonist, did not change r eceptor levels or alter (-)-nicotine-evoked up-regulation. (-)-Nicotin e-evoked up-regulation was blocked by cycloheximide, suggesting a role for protein synthesis. Treatment of cells with (-)nicotine or dihydro -beta-erythroidine differentially modulated the efficacy of acetylchol ine to activate cation efflux. Both 6-beta-[beta'(piperidino)propionyl ]forskolin and phorbol-12-myristate-13-acetate increased [H-3]cytisine binding sites and nAChR function and enhanced the effects of chronic (-)-nicotine treatment in a synergistic manner. These results collecti vely demonstrate that human alpha 4 beta 2 nAChRs can be differentiall y up-regulated by chronic treatment with nAChR ligands and activation of protein kinase A- and protein kinase C-dependent mechanisms.