Nicotine-induced Ca 2+ signaling and down-regulation of nicotinic acetylcholine receptor subunit expression in the CEM human leukemic T-cell line

Abstract

We previously showed that T- and B-lymphocytes express both muscarinic and nicotinic acetylcholine (ACh) receptors (mAChR and nAChR, respectively), and that stimulation of M 3 mAChRs on lymphocytes increases the intracellular free Ca 2+ concentration ([Ca 2+] i) and up-regulates c-fos gene expression. Little is known about the effects of nicotinic stimulation on lymphocyte function, however. We therefore investigated the acute effect of nicotine on [Ca 2+] i in CEM cells, a model of T-lymphocytes, using confocal laser scanning microscopy with fluo-3, a Ca 2+-sensitive fluorescent indicator. In addition, we examined the long-term effect of nicotine on the expression of selected nAChR subunits using semiquantitative reverse transcription-polymerase chain reaction analysis. In the presence of extracellular Ca 2+, nicotine (30 μM) evoked rapid, transient increases in [Ca 2+] i. This effect was concentration-dependently inhibited by the α7 nAChR subunit antagonists, α-bungarotoxin (0.01-10 μM) and methyllycaconitine (0.01-10 mM), suggesting that the α7 nAChR subunit mediates Ca 2+ signaling in T-lymphocytes. Nicotine (0.01–10 μM) also concentration-dependently down-regulated expression of mRNAs for all the nAChR subunits tested: expression of the α6 and α7 subunits was down-regulated within 1 week, while expression of the α3 and α5 subunits declined gradually throughout the 8-week experimental period. These findings indicate that nicotine — and therefore likely smoking — affects immune function by suppressing expression of the neuronal nAChR subtype involved in Ca 2+ signaling in lymphocytes.