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口腔外组织中人类苦味受体表达的分析。

Analysis of the expression of human bitter taste receptors in extraoral tissues.

作者信息

Jaggupilli Appalaraju, Singh Nisha, Upadhyaya Jasbir, Sikarwar Anurag S, Arakawa Makoto, Dakshinamurti Shyamala, Bhullar Rajinder P, Duan Kangmin, Chelikani Prashen

机构信息

Manitoba Chemosensory Biology (MCSB) Research Group, University of Manitoba, Winnipeg, MB, R3E 0W4, Canada.

Department of Oral Biology, University of Manitoba, Winnipeg, MB, R3E 0W4, Canada.

出版信息

Mol Cell Biochem. 2017 Feb;426(1-2):137-147. doi: 10.1007/s11010-016-2902-z. Epub 2016 Dec 23.

DOI:10.1007/s11010-016-2902-z
PMID:28012014
Abstract

The 25 bitter taste receptors (T2Rs) in humans perform a chemosensory function. However, very little is known about the level of expression of these receptors in different tissues. In this study, using nCounter gene expression we analyzed the expression patterns of human TAS2R transcripts in cystic fibrosis bronchial epithelial (CuFi-1), normal bronchial epithelial (NuLi-1), airway smooth muscle (ASM), pulmonary artery smooth muscle (PASM), mammary epithelial, and breast cancer cells. Our results suggest a specific pattern of TAS2R expression with TAS2R3, 4, 5, 10, 13, 19, and 50 transcripts expressed at moderate levels and TAS2R14 and TAS2R20 (or TASR49) at high levels in the various tissues analyzed. This pattern of expression is mostly independent of tissue origin and the pathological state, except in cancer cells. To elucidate the expression at the protein level, we pursued flow cytometry analysis of select T2Rs from CuFi-1 and NuLi-1 cells. The expression levels observed at the gene level by nCounter analysis correlate with the protein levels for the T2Rs analyzed. Next, to assess the functionality of the expressed T2Rs in these cells, we pursued functional assays measuring intracellular calcium mobilization after stimulation with the bitter compound quinine. Using PLC inhibitor, U-73122, we show that the calcium mobilized in these cells predominantly takes place through the Quinine-T2R-Gαβγ-PLC pathway. This report will accelerate studies aimed at analyzing the pathophysiological function of T2Rs in different extraoral tissues.

摘要

人类的25种苦味受体(T2Rs)执行化学感应功能。然而,对于这些受体在不同组织中的表达水平却知之甚少。在本研究中,我们使用nCounter基因表达技术分析了人TAS2R转录本在囊性纤维化支气管上皮细胞(CuFi-1)、正常支气管上皮细胞(NuLi-1)、气道平滑肌(ASM)、肺动脉平滑肌(PASM)、乳腺上皮细胞和乳腺癌细胞中的表达模式。我们的结果表明,TAS2R存在特定的表达模式,在所分析的各种组织中,TAS2R3、4、5、10、13、19和50转录本以中等水平表达,而TAS2R14和TAS2R20(或TASR49)以高水平表达。这种表达模式大多独立于组织来源和病理状态,但癌细胞除外。为了阐明蛋白质水平的表达情况,我们对CuFi-1和NuLi-1细胞中选定的T2Rs进行了流式细胞术分析。通过nCounter分析在基因水平观察到的表达水平与所分析的T2Rs的蛋白质水平相关。接下来,为了评估这些细胞中表达的T2Rs的功能,我们进行了功能测定,测量用苦味化合物奎宁刺激后细胞内钙的动员情况。使用PLC抑制剂U-73122,我们发现这些细胞中动员的钙主要通过奎宁-T2R-Gαβγ-PLC途径发生。本报告将加速旨在分析T2Rs在不同口腔外组织中的病理生理功能的研究。

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