Bencherif M, Fowler K, Lukas R J, Lippiello P M
Pharmacology Division, R.J. Reynolds Research & Development, Winston-Salem, North Carolina, USA.
J Pharmacol Exp Ther. 1995 Nov;275(2):987-94.
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 approximately 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 [3H]-L-nicotine binding sites was evident for M10 cells treated at nicotine concentrations as low as 10 nM (EC50 and EC100 values were 100 nM and 10 microM, respectively). Scatchard analyses of [3H]-L-nicotine binding data in M10 cells indicated a change in Bmax with no significant change in affinity for radioligand (KD = 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.(ABSTRACT TRUNCATED AT 250 WORDS)
人们普遍认为，哺乳动物脑中高亲和力的[3H]-L-尼古丁结合位点在慢性尼古丁暴露后数量会增加，这些位点被认为代表了由α4和β2亚基组成的中枢神经系统烟碱型乙酰胆碱受体（nAChR）的主要形式。然而，导致这种效应的机制尚未阐明。为了评估这个问题，我们使用了胎鼠脑皮质原代细胞培养物（其中天然表达高亲和力的[3H]-L-尼古丁结合位点）和成纤维细胞克隆细胞培养物作为模型，这些成纤维细胞经过稳定转染，可在地塞米松诱导型启动子的控制下表达由转基因鸡α4和β2亚基组成的nAChR。在存在地塞米松的情况下维持培养的M10细胞或原代胎鼠脑皮质培养物中，慢性尼古丁暴露导致高亲和力的[3H]-L-尼古丁结合位点增加了约2.5倍。对于尼古丁浓度低至10 nM（EC50和EC100值分别为100 nM和10 μM）处理的M10细胞，[3H]-L-尼古丁结合位点的上调很明显。对M10细胞中[3H]-L-尼古丁结合数据的Scatchard分析表明Bmax发生了变化，而对放射性配体的亲和力没有显著变化（对照细胞中的KD = 2.5±0.5 nM，尼古丁处理细胞中的KD = 2.0±0.4 nM）。Northern印迹分析表明，单独的尼古丁处理对驱动M10细胞中转基因nAChR亚基基因转录的启动子没有直接影响，并且在产生高亲和力的[3H]-L-尼古丁结合位点上调的慢性尼古丁处理条件下，胎鼠脑皮质细胞或M10细胞nAChRα4或β2 mRNA的稳态水平不受影响。（摘要截短于250字）
