The Expression and Pharmacological Characterization of Nicotinic Acetylcholine Receptor Subunits in HBE16 Airway Epithelial Cells

This study characterizes the expression and the biological effects of the nicotinic acetylcholine receptor (nAChR) on human airway epithelial cells. Cultured HBE16 airway epithelial cells were incubated with either nicotine or cigarette smoke extract (CSE). The nAChR gene and protein expression in cells were detected by reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR, and western blot. The protein expression of the nAChR subunits, alpha 1, alpha 5, and alpha 7, were evaluated by immunohistochemistry. Cells were subsequently transfected with alpha 1-, alpha 5-, and alpha 7-specific siRNAs, and the effects of nicotine on the production of the pro-inflammatory factors, TNF-alpha, IL-8, and IL-6 in transfected cells were analyzed using an enzyme-linked immunosorbent assay and real-time PCR. We detected alpha 1, alpha 5, alpha 7, and beta 2 subunits in untreated HBE16 cells, and their expression was elevated after nicotinic incubation. Importantly, the most significant increase in expression was observed in the alpha 5 and alpha 7 subunits. However, CSE did not cause a significant enhancement in the expression of these genes and proteins. Cells pretreated with nicotine prior to lipopolysaccharide (LPS) stimulation exhibited a lower production of TNF-alpha, IL-8, and IL-6 compared to LPS-treated (only) cells. Cells that were transfected with alpha 7 siRNA and subsequently incubated with nicotine and LPS, exhibited a higher expression of TNF-alpha, IL-8, and IL-6 compared with non-transfected cells or alpha 1 and alpha 5 siRNA-transfected cells. In alpha 1- and alpha 5-siRNA-transfected cells, the expression of TNF-alpha, IL-8, and IL-6 showed no significant difference compared with non-transfected cells. Therefore, we concluded that alpha 1, alpha 5, alpha 7, and beta 2 nAChR subunits are highly expressed in human bronchial epithelial cells (HBE16) after nicotinic incubation and that the alpha 7 subunit is involved in the nicotine-induced inhibitory effect on the production of inflammatory factors. Moreover, alpha 1, alpha 5, and beta 2 subunits did not play an important role in this process.