Nicotinic acetylcholine receptor (CHRN) expression and function in cultured human adult fungiform (HBO) taste cells

In rodents, CHRNs are involved in bitter taste transduction of nicotine and ethanol. Currently, it is not clear if CHRNs are expressed in human taste cells and if they play a role in transducing the bitter taste of nicotine and ethanol or in the synthesis and release of neurohumoral peptides. Accordingly, we investigated the expression and functional role of CHRNs in HBO cells. Using molecular techniques, we demonstrate that a subset of HBO cells express CHRNs that also co-express TRPM5, T1R3 or T2R38. Exposing HBO cells to nicotine or ethanol acutely or to nicotine chronically induced a differential increase in the expression of CHRN mRNA and protein in a dose- and time-dependent manner. Acutely exposing HBO cells to a mixture containing nicotine plus ethanol induced a smaller increase in CHRN mRNAs relative to nicotine or ethanol treatment alone. A subset of HBO cells responded to nicotine, acetylcholine and ATP with a transient increase in [Ca2+]i. Nicotine effects on [Ca2+]i were mecamylamine sensitive. Brain-derived neurotrophic factor (BDNF) protein was detected in HBO cells using ELISA. Acute nicotine exposure decreased BDNF in HBO cells and increased BDNF release in the medium. CHRNs were also detected in HEK293 cells by RT-PCR. Unlike HBO cells, CHRNs were localized in most of HEK293 cells and majority of HEK293 cells responded to nicotine and ethanol stimulation with a transient increase in [Ca2+]i. BDNF levels in HEK293 cells were significantly higher than in HBO cells but the nicotine induced release of BDNF in the media was a fraction of the BDNF cellular content. We conclude that CHRNs are expressed in TRPM5 positive HBO cells. CHRN mRNA expression is modulated by exposure to nicotine and ethanol in a dose- and time-dependent manner. Nicotine induces the synthesis and release of BDNF in HBO cells.在啮齿类动物中，烟碱型乙酰胆碱受体（CHRNs）参与尼古丁与乙醇的苦味信号转导过程。目前尚不明确CHRNs是否表达于人类味觉细胞，是否参与尼古丁与乙醇的苦味信号转导，亦或参与神经体液肽的合成与释放。基于此，本研究针对HBO细胞中CHRNs的表达情况与功能角色展开了探究。本研究借助分子生物学技术证实，部分HBO细胞可表达CHRNs，且此类细胞同时共表达TRPM5、T1R3或T2R38。对HBO细胞进行急性尼古丁、乙醇暴露或慢性尼古丁处理后，CHRN的mRNA与蛋白表达水平呈现剂量和时间依赖性的差异性上调。与单独使用尼古丁或乙醇处理相比，将HBO细胞暴露于尼古丁与乙醇的混合溶液时，其CHRN mRNA的上调幅度更小。部分HBO细胞可对尼古丁、乙酰胆碱与三磷酸腺苷（ATP）产生响应，表现为细胞内游离Ca2+浓度的一过性升高。尼古丁对细胞内游离Ca2+浓度的调控作用可被美加明所阻断。本研究通过酶联免疫吸附测定（ELISA）在HBO细胞中检测到了脑源性神经营养因子（BDNF）蛋白。急性尼古丁暴露可降低HBO细胞内的BDNF水平，同时增加培养基中BDNF的释放量。本研究还通过逆转录聚合酶链式反应（RT-PCR）在人胚肾293（HEK293）细胞中检测到了CHRNs。与HBO细胞不同，HEK293细胞中绝大多数均表达CHRNs，且大部分HEK293细胞可在尼古丁与乙醇的刺激下出现细胞内游离Ca2+浓度的一过性升高。HEK293细胞内的BDNF水平显著高于HBO细胞，但尼古丁诱导的培养基中BDNF释放量仅占其细胞内BDNF总量的一小部分。本研究最终得出结论：CHRNs表达于TRPM5阳性的HBO细胞中。CHRN的mRNA表达可通过尼古丁与乙醇暴露呈现剂量和时间依赖性的调控。尼古丁可诱导HBO细胞内BDNF的合成与释放。