Regulation of human alpha 4 beta 2 neuronal nicotinic acetylcholine receptors by cholinergic channel ligands and second 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 treatment with nicotine. In this study, the regulation of recombinant human alpha 4 beta 2 nAChR subtype by (-)-nicotine and other cholinergic channel modulators was studied using human embryonic kidney 293 cells stably expressing this subunit combination. The treatment of transfected cells with (-)-nicotine and other activator ligands, including (-)-cytisine, 1,1-dimethyl-4-phenylpiperazinium, (S)-3-methyl-5-(1-methyl-5-(1-methyl-2-pyrrolidinyl)isoxazole, and (+/-)-epibatidine, resulted in concentration-dependent increases in the levels of alpha 4 beta 2 nAChRs. The increase in [H-3]cytisine binding sites was initiated by low concentrations 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. Antagonists, including dihydro-beta-erythroidine, d-tubocurarine, and methyllycaconitine, also elicited significant increases in receptor levels. A good correlation was observed between the K-i values for binding inhibition and the EC50 values for receptor up-regulation. Treatment of cells with mecamylamine, a noncompetitive antagonist, did not change receptor levels or alter (-)-nicotine-evoked up-regulation. (-)-Nicotine-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 acetylcholine 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 collectively demonstrate that human alpha 4 beta 2 nAChRs can be differentially up-regulated by chronic treatment with nAChR ligands and activation of protein kinase A- and protein kinase C-dependent mechanisms.