Increased Nicotinic Acetylcholine Receptor Protein Underlies Chronic Nicotine-Induced Up-Regulation of Nicotinic Agonist Binding Sites in Mouse Brain

Chronic nicotine treatment elicits a brain region-selective increase in the number of high-affinity agonist binding sites, a phenomenon termed up-regulation. Nicotine-induced up-regulation of alpha 4 beta 2-nicotinic acetylcholine receptors (nAChRs) in cell cultures results from increased assembly and/or decreased degradation of nAChRs, leading to increased nAChR protein levels. To evaluate whether the increased binding in mouse brain results from an increase in nAChR subunit proteins, C57BL/6 mice were treated with nicotine by chronic intravenous infusion. Tissue sections were prepared, and binding of [(125)I]3-((2S)-azetidinylmethoxy)-5-iodo-pyridine (A85380) to beta 2*-nAChR sites, [(125)I] monoclonal antibody (mAb) 299 to alpha 4 nAChR subunits, and [(125)I]mAb 270 to beta 2 nAChR subunits was determined by quantitative autoradiography. Chronic nicotine treatment dose-dependently increased binding of all three ligands. In regions that express alpha 4 beta 2-nAChR almost exclusively, binding of all three ligands increased coordinately. However, in brain regions containing significant beta 2*-nAChR without alpha 4 subunits, relatively less increase in mAb 270 binding to beta 2 subunits was observed. Signal intensity measured with the mAbs was lower than that with [(125)I] A85380, perhaps because the small ligand penetrated deeply into the sections, whereas the much larger mAbs encountered permeability barriers. Immunoprecipitation of [(125)I] epibatidine binding sites with mAb 270 in select regions of nicotine-treated mice was nearly quantitative, although somewhat less so with mAb 299, confirming that the mAbs effectively recognize their targets. The patterns of change measured using immunoprecipitation were comparable with those determined autoradiographically. Thus, increases in alpha 4 beta 2*-nAChR binding sites after chronic nicotine treatment reflect increased nAChR protein.