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

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Abstract

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, α1, α5, and α7, were evaluated by immunohistochemistry. Cells were subsequently transfected with α1-, α5-, and α7-specific siRNAs, and the effects of nicotine on the production of the pro-inflammatory factors, TNF-α, IL-8, and IL-6 in transfected cells were analyzed using an enzyme-linked immunosorbent assay and real-time PCR. We detected α1, α5, α7, and β2 subunits in untreated HBE16 cells, and their expression was elevated after nicotinic incubation. Importantly, the most significant increase in expression was observed in the α5 and α7 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-α, IL-8, and IL-6 compared to LPS-treated (only) cells. Cells that were transfected with α7 siRNA and subsequently incubated with nicotine and LPS, exhibited a higher expression of TNF-α, IL-8, and IL-6 compared with non-transfected cells or α1 and α5 siRNA-transfected cells. In α1- and α5-siRNA-transfected cells, the expression of TNF-α, IL-8, and IL-6 showed no significant difference compared with non-transfected cells. Therefore, we concluded that α1, α5, α7, and β2 nAChR subunits are highly expressed in human bronchial epithelial cells (HBE16) after nicotinic incubation and that the α7 subunit is involved in the nicotine-induced inhibitory effect on the production of inflammatory factors. Moreover, α1, α5, and β2 subunits did not play an important role in this process.

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Abbreviations

BSA:

Bovine serum albumin IL interleukin

CE:

Cigarette smoke chloroform extract

CSE:

Cigarette smoke extract

FITC:

Fluorescein isothiocyanate

HRP:

Horseradish peroxidase

NF-κB:

Nuclear factor-κB

IL:

Interleukin

IFN-γ:

Interferon-γ

LPS:

Lipopolysaccharide

MUC5AC:

Mucin 5AC

PBS:

Phosphate-buffered saline

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Acknowledgments

This study was supported by the National Nature Science Foundation of China (No. 81070031 and No. 81000003) and the China-Russia Cooperation Research Program (81011120108).

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Authors and Affiliations

  1. Division of Respiratory Medicine, Second Affiliated Hospital, Chongqing Medical University, No.74, Linjiang Road, Yuzhong District, Chongqing, 400010, China

    Qi Li & Xiangdong Zhou

  2. Far Eastern Scientific Center of Physiology and Pathology of Respiration, Siberian Branch, Russian Academy of Medical Sciences, Blagoveschensk, Russia

    Victor P. Kolosov & Juliy M. Perelman

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  1. Qi Li
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  2. Xiangdong Zhou
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  4. Juliy M. Perelman
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Correspondence to Xiangdong Zhou.

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Li, Q., Zhou, X., Kolosov, V.P. et al. The Expression and Pharmacological Characterization of Nicotinic Acetylcholine Receptor Subunits in HBE16 Airway Epithelial Cells. Cell Biochem Biophys 62, 421–431 (2012). https://doi.org/10.1007/s12013-011-9324-z

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