title: Agonist-induced up-regulation of alpha4beta2 nicotinic acetylcholine receptors in M10 cells: pharmacological and spatial definition.
abstract: Chronic nicotine up-regulates the number of high affinity nicotinic acetylcholine receptors (nAChRs) in mammalian brain. Here, we studied up-regulation of the nAChR composed of alpha4 and beta2 subunits in the M10 cell line by using [3H]epibatidine to measure nAChR in cells in situ and in membrane preparations. Cultures were exposed to drugs for 2 days before assay. All agonists up-regulated [3H]epibatidine binding sites with EC50 values typically 10-100-fold higher than their respective Ki values from competition binding assays. Maximum up-regulation ranged from 40% to 250% above control values. Maximally effective concentrations of the less efficacious agonists methylcarbamylcholine or (+/-)-epibatidine together with nicotine resulted in less up-regulation than that produced by nicotine alone, showing that they are partial up-regulatory agonists. The antagonists dihydro-beta-erythroidine, methyllycaconitine, d-tubocurarine, hexamethonium, decamethonium, and mecamylamine either failed to up-regulate [3H]epibatidine binding sites or up-regulated mildly at high concentrations. When tested at non-up-regulating concentrations, only d-tubocurarine significantly inhibited agonist-induced up-regulation; this inhibition seemed to be noncompetitive. Comparison of [3H]epibatidine displacement in intact M10 cells and membrane preparations by membrane-impermeant ligands indicated that 85% of [3H]epibatidine binding sites are intracellular. On chronic treatment with agonist, the proportion of surface receptors did not change significantly, indicating that most up-regulated [3H]epibatidine binding sites are internal. However, up-regulation is mediated at the cell surface because the impermeant ligand tetramethylammonium was as efficacious as nicotine in eliciting up-regulation, and methylcarbamylcholine (i.e., impermeant but with low efficacy) blocked nicotine induced up-regulation. Thus, agonists elicit up-regulation (mainly of intracellular receptors) by interacting with cell surface nAChRs that are not compatible with either an active or high affinity desensitized conformation.
authors.affiliation: Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK.
authors.forename: P
authors.initials: P
authors.lastname: Whiteaker
authors.forename: C G
authors.initials: CG
authors.lastname: Sharples
authors.forename: S
authors.initials: S
authors.lastname: Wonnacott
mesh_terms: D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism / Q000494:pharmacology*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D018722:Nicotinic Agonists / Q000494:pharmacology*; D018733:Nicotinic Antagonists / Q000494:pharmacology; D011725:Pyridines / Q000378:metabolism / Q000494:pharmacology*; D011978:Receptors, Nicotinic / Q000187:drug effects* / Q000378:metabolism; D014316:Tritium; D015854:Up-Regulation / Q000187:drug effects*
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D011725:Pyridines; D011978:Receptors, Nicotinic; D014316:Tritium; C082748:epibatidine