Mechanisms of up-regulation of neuronal nicotinic acetylcholine receptors in clonal cell lines and primary cultures of fetal rat brain
Bencherif, M.; Fowler, K.; Lukas, R.J.; Lippiello, P.M.
Journal of Pharmacology and Experimental Therapeutics 275(2): 987-994
1995
ISSN/ISBN: 0022-3565 PMID: 7473192 Document Number: 446246
There is a consensus that high-affinity (3H)-L-nicotine binding sites in the mammalian brain, which are thought to represent a predominant form of central nervous system nicotinic acetylcholine receptor (nAChR) composed of alpha-4 and beta-2 subunits, are increased in number after chronic nicotine exposure. However, mechanisms responsible for this effect have not yet been elucidated. To evaluate this issue, we have used, as models, primary cell cultures of fetal rat brain cortex, in which high-affinity (3H)-L-nicotine binding sites are naturally expressed, and clonal cell cultures of fibroblasts stably transfected to express nAChR composed of transgenic chick alpha-4 and beta-2 subunits under control of dexamethasone-inducible promoters. Chronic nicotine exposure induced an -2.5-fold increase in high-affinity (3H)-L-nicotine binding sites in M10 cells maintained in the presence of dexamethasone or in primary fetal rat brain cortical cultures. Up-regulation of (3 H)-L-nicotine binding sites was evident for M10 cells treated at nicotine concentrations as low as 10 nM (EC-50 and EC-100 values were 100 nM and 10 mu-M, respectively). Scatchard analyses of (3H)-L-nicotine binding data in M10 cells indicated a change in B-max with no significant change in affinity for radioligand (K-D = 2.5-0.5 nM in control cells vs. 2.0 +- 0.4 nM in nicotine-treated cells). Northern blot analyses indicated that nicotine treatment alone had no direct effect on the promoter driving transgenic nAChR subunit gene transcription in M10 cells and that steady state levels of fetal rat brain cortical cell or M10 cell nAChR alpha-4 or beta-2 mRNAs were unaffected under conditions of chronic nicotine treatment that produced up-regulation of high-affinity (3H)-L-nicotine binding sites. Kinetic studies conducted at 4 degree C indicated no significant changes in the rate constants for fast (k-f = 0.2 min-1) or slow (k-s = 0.02 min-1) phases of (3H)-L-nicotine association with specific binding sites on control or nicotine-treated M10 cells. When dexamethasone was removed from control M10 cultures to terminate synthesis of nAChR, numbers of high-affinity (3 H)-L-nicotine binding sites decreased by approximately 50% after 1 day. However, if nicotine was added at the time of dexamethasone removal, levels of high-affinity binding sites remained near control levels (EC-50 = 100 nM). Nevertheless, when cells were pretreated with nicotine to induce up-regulation before dexamethasone removal, the rate constant for the decrease in high-affinity (3H)-L-nicotine binding sites was not significantly different from that for control cells and was unaffected by continued presence of nicotine. The results are consistent with the existence of a pool of high-affinity nAChR conformers (R-o) that degrade with a half-life of apprx 1 day and that exist in equilibrium with a low-affinity pool of nAChR (R) that is acutely converted into the high-affinity conformer in the presence of agonist (t(1/2) = 35 min at 4 degree C). We also hypothesize that there exist an additional, degradable, reserve pool of nAChR (R degree ) that are undetected in (3H)-L-nicotine binding assays under control conditions but can be converted into R-plus-R' after chronic nicotine exposure. We conclude that nicotine up-regulates high-affinity (3H)-L-nicotine-binding, neuronal alpha-4-beta-2-nAChR through post-translational mechanisms.