Regulation of human alpha4beta2 neuronal nicotinic acetylcholine receptors by cholinergic channel ligands and second messenger pathways.
The alpha4beta2 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 alpha4beta2 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-2-pyrrolidinyl)isoxazole, and (+/-)-epibatidine, resulted in concentration-dependent increases in the levels of alpha4beta2 nAChRs. The increase in [3H]cytisine binding sites was initiated by low concentrations of (-)-nicotine (<100 nM); was maximal at 10 microM (15-fold), rapid (t0.5 = 4.0 +/- 0.5 hr), and totally reversible (t0.5 = 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 Ki 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 [3H]cytisine binding sites and nAChR function and enhanced the effects of chronic (-)-nicotine treatment in a synergistic manner. These results collectively demonstrate that human alpha4beta2 nAChRs can be differentially up-regulated by chronic treatment with nAChR ligands and activation of protein kinase A- and protein kinase C-dependent mechanisms.
Gopalakrishnan
M
M
Neuroscience Research, Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois 60064-3500, USA. murali.gopalakrishnan@abbott.com
Molinari
E J
EJ
Sullivan
J P
JP
D000470:Alkaloids / Q000378:metabolism / Q000494:pharmacology; D001392:Azocines; D001665:Binding Sites; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D006801:Humans; D007473:Ion Channels / Q000187:drug effects* / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism / Q000648:ultrastructure; D008024:Ligands; D009538:Nicotine / Q000494:pharmacology; D018733:Nicotinic Antagonists / Q000378:metabolism / Q000494:pharmacology; D011493:Protein Kinase C / Q000502:physiology; D011807:Quinolizines; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D015290:Second Messenger Systems / Q000187:drug effects / Q000502:physiology*; D014162:Transfection; D014316:Tritium; D015854:Up-Regulation / Q000187:drug effects; D000093843:Quinolizidine Alkaloids
D000470:Alkaloids; D001392:Azocines; D017868:Cyclic AMP-Dependent Protein Kinases; D007473:Ion Channels; D008024:Ligands; D009538:Nicotine; D018733:Nicotinic Antagonists; D011493:Protein Kinase C; D011807:Quinolizines; D011978:Receptors, Nicotinic; D014316:Tritium; C004712:cytisine; D000093843:Quinolizidine Alkaloids