The Effects of Tobacco Smoke Condensate on Nicotinic Acetylcholine Receptors in Vitro

Abstract

The effects of total particulate matter (TPM) from cigarette smoke, which contains tar plus nicotine, on the expression and ligand binding of nicotinic acetylcholine receptors (nAChrs) were investigated in vitro, using the human neuroblastoma cell line, SH-SY5Y. As far as could be determined, research on the TPM effects in vitro, using a human cell line bioassay, is completely novel. There has, however, been TPM research in vivo, using humans and in vitro on bacterial cells. Expression of the main nAChr subunits was confirmed in the SH-SY5Y cells, using RT-PCR. Effects on the intact SH-SY5Y cells of exposure to TPM, containing a known amount of nicotine, or an equivalent nicotine standard were compared to each other and to unexposed intact SH-SY5Y control cells. Specifically, nAChrs were always up-regulated in response to both nicotine and TPM exposure. The peak induction of increased binding occurred after 24 hr incubation, of cells incubated with either TPM or nicotine at normal smokers’ blood concentrations of nicotine (0.2 - 2 μM), resulted in a large upregulation of nAChrs. This was measured by 3H-epibatidine binding to intact cells (nicotine exposed compared to control p = 0.0002, TPM exposed compared to control p = 0.0005 and TPM exposed compared to nicotine exposed p = 0.004). While the timing of specific nicotine receptor binding was similar with both exposures, the magnitude of increase was generally greater with TPM compared to nicotine at the same nicotine concentration. These results suggest that cigarette tar contains non-nicotine compounds that do not directly compete with nicotine for binding nAChrs, since TPM and nicotine alone had the same Kd value in Scatchard analysis, but that they are capable of increasing the expression of one or more of the nAChr subunits upon chronic exposure to neural cells in vitro. We conclude that TPM contains compounds that are toxic to cells at high concentrations (cell growth inhibition) but do not compete with nicotine for binding to nAChr. Furthermore this cell culture bioassay provides a useful in vitro model for assessing the relative addictiveness of different tobacco products, including that of non-nicotine components.