PT  - JOURNAL ARTICLE
AU  - Michael J. Marks
AU  - Tristan D. McClure-Begley
AU  - Paul Whiteaker
AU  - Outi Salminen
AU  - Robert W. B. Brown
AU  - John Cooper
AU  - Allan C. Collins
AU  - Jon M. Lindstrom
TI  - Increased Nicotinic Acetylcholine Receptor Protein Underlies Chronic Nicotine-Induced Up-Regulation of Nicotinic Agonist Binding Sites in Mouse Brain
AID  - 10.1124/jpet.110.178236
DP  - 2011 Apr 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 187--200
VI  - 337
IP  - 1
4099  - http://jpet.aspetjournals.org/content/337/1/187.short
4100  - http://jpet.aspetjournals.org/content/337/1/187.full
SO  - J Pharmacol Exp Ther2011 Apr 01; 337
AB  - 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 α4β2-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 [125I]3-((2S)-azetidinylmethoxy)-5-iodo-pyridine (A85380) to β2*-nAChR sites, [125I]monoclonal antibody (mAb) 299 to α4 nAChR subunits, and [125I]mAb 270 to β2 nAChR subunits was determined by quantitative autoradiography. Chronic nicotine treatment dose-dependently increased binding of all three ligands. In regions that express α4β2-nAChR almost exclusively, binding of all three ligands increased coordinately. However, in brain regions containing significant β2*-nAChR without α4 subunits, relatively less increase in mAb 270 binding to β2 subunits was observed. Signal intensity measured with the mAbs was lower than that with [125I]A85380, perhaps because the small ligand penetrated deeply into the sections, whereas the much larger mAbs encountered permeability barriers. Immunoprecipitation of [125I]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 α4β2*-nAChR binding sites after chronic nicotine treatment reflect increased nAChR protein.