Ambrose Vikki, Miller John H, Dickson Stuart J, Hampton Scott, Truman Penelope, Lea Rodney A, Fowles Jefferson
Institute of Environmental Science and Research Ltd., Porirua, New Zealand.
Nicotine Tob Res. 2007 Aug;9(8):793-9. doi: 10.1080/14622200701485117.
The effect of total particulate matter (TPM) from cigarette smoke on the expression and binding properties of nicotinic acetylcholine receptors (nAChRs) was investigated using a human neuroblastoma cell line (SH-SY5Y). TPM but not nicotine on its own inhibited cell growth at nicotine concentrations above 5 microM. To examine effects on nAChR expression, intact cells were incubated with 3H-epibatidine, and a Bmax of 13 fmoles/10(5) cells (7.8 x 10(4) binding sites/cell) was measured in unexposed cells as well as in cells treated with 2 microM nicotine alone or with TPM containing 2 microM nicotine. Using Scatchard analysis, we measured a Kd of 0.3 nM for 3H-epibatidine binding to nAChRs. This Kd was increased to 1.3 nM by addition of nicotine or TPM extract, both at 2 microM nicotine. Bmax, however, was unaffected, suggesting competitive binding of nicotine to its receptor. Short-term and prolonged 3-day exposures of SH-SY5Y cells to either TPM or nicotine at nicotine concentrations ranging from 0.2 microM to 20 microM increased specific binding, suggesting upregulation of nAChR expression. Most significant, binding was consistently greater in cells pretreated with TPM than in cells pretreated with nicotine. We conclude that TPM contains compounds that are toxic to cells at high concentrations (cell growth inhibition) but that do not compete with nicotine for binding to nAChRs (Scatchard analysis). These non-nicotinic compounds are capable of increasing the expression of one or more of the nAChR subunits. Furthermore, our cell culture assay provides a useful in vitro model for assessing the relative addictiveness of different tobacco products, including that of non-nicotine components.
使用人神经母细胞瘤细胞系（SH-SY5Y）研究了香烟烟雾中的总颗粒物（TPM）对烟碱型乙酰胆碱受体（nAChRs）表达和结合特性的影响。在尼古丁浓度高于5 microM时，TPM而非尼古丁本身会抑制细胞生长。为了检查对nAChR表达的影响，将完整细胞与3H-埃博霉素一起孵育，在未暴露的细胞以及单独用2 microM尼古丁或含2 microM尼古丁的TPM处理的细胞中，测得的Bmax为13 fmol/10(5)个细胞（7.8 x 10(4)个结合位点/细胞）。使用Scatchard分析，我们测得3H-埃博霉素与nAChRs结合的Kd为0.3 nM。在2 microM尼古丁浓度下，添加尼古丁或TPM提取物后，该Kd增加到1.3 nM。然而，Bmax不受影响，表明尼古丁与其受体存在竞争性结合。将SH-SY5Y细胞短期和长期（3天）暴露于尼古丁浓度范围为0.2 microM至20 microM的TPM或尼古丁中，会增加特异性结合，表明nAChR表达上调。最重要的是，用TPM预处理的细胞中的结合始终比用尼古丁预处理的细胞中的结合更强。我们得出结论，TPM含有在高浓度下对细胞有毒的化合物（细胞生长抑制），但不与尼古丁竞争与nAChRs的结合（Scatchard分析）。这些非尼古丁化合物能够增加一种或多种nAChR亚基的表达。此外，我们的细胞培养试验为评估不同烟草产品（包括非尼古丁成分）的相对成瘾性提供了一个有用的体外模型。
