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 α4 and β2 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-β-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.
Footnotes
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Send reprint requests to: Dr. S. Wonnacott, Dept. of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, United Kingdom. E-mail: s.wonnacott{at}bath.ac.uk
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↵1 Current affiliation: Institute for Behavioral Genetics, University of Colorado, Denver, CO 80309-0447.
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This work was supported by Medical Research Council Grant 9619434 (S.W.). C.G.V.S. is the recipient of a postgraduate studentship from the Biotechnology and Biological Sciences Research Council.
- Abbreviations:
- nAChR
- nicotinic acetylcholine receptor
- DEC
- decamethonium
- DHβE
- dihydro-β-erythroidine
- dTC
- d-tubocurarine
- HEX
- hexamethonium
- MCC
- methylcarbamylcholine
- MLA
- methyllycaconitine
- TMA
- tetramethylammonium
- mAb
- monoclonal antibody
- DMEM
- Dulbecco’s modified Eagle’s medium
- PBS
- phosphate-buffered saline
- Received October 9, 1997.
- Accepted January 29, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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