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激动剂作用下α_(1)-肾上腺素受体亚型在HEK293A细胞中的分布变化

王淑一, 宋峣, 徐明, 郝天袍, 韩启德, 张幼怡

北京大学第三医院血管医学研究所，分子心血管学教育部重点实验室. 北京 100083

摘要

为了明确α1-肾上腺素受体(α_(1) -adrenergic receptor, α_(1)-AR)三种亚型在人胚胎肾(human embryonic kidney, HEK) 293A细胞株中的分布特点，及其在激动剂作用下在细胞内的定位改变，本研究采用放射配体结合实验、实时荧光共聚焦成像 和Western blot方法检测α1-AR三种亚型在细胞中的定位及蛋白质表达的变化。α1-AR三种亚型在HEK293A细胞株转染效率相同，均达 90%以上。三株细胞的粗制膜上α1B-AR表达量最高， α1D-AR最低，α1A-AR居中，但三者 的解离常数(Kd)相等；在无激动剂作用时， α1A-AR均匀地分布在HEK293A细胞的胞膜和胞浆， α1B-AR主要位于胞膜，而α1D-AR则主要分布在胞浆中；用α1-AR激动剂苯肾上腺素(phenylephrine, PE)刺激细胞1 h后，α1A-和α1B-AR在胞膜上分布明显减少，而在胞浆中分布增加，其中 α1B-AR变化更为显著，α1D-AR的分布在 PE作用下无明显变化。以上结果提示，在激动剂作用下，α1-AR三种亚型在HEK293A细胞中的定位特点和分布变化各有不同。

关键词： α_(1)-肾上腺素受体; 分布; 定位; 细胞株

Redistribution of three α_(1)-adrenergic receptor subtypes in the stably transfected HEK 293A cells upon agonist stimulation

Wang Shuyi, Song Yao, Xu Ming, Hao Tianpao, Han Qide, Zhang Youyi

Institute of Vascular Medicine, Peking University Third Hospital and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education. Beijing 100083, China

Abstract

To investigate the subcellular distribution of three α1-adrenergic receptor subtypes and their internalization and trafficking upon agonist stimulation in human embryonic kidney (HEK) 293A cell line, saturation radioligand binding assay, laser confocal imaging, and Western blot were applied to examine the distribution and changes in localization of three α1-AR subtypes in transfected HEK 293A cells prior to and after treatment with phenylephrine. The results are as follows: (1) The transfection efficiency was over 90% and was equal among three α1-AR subtypes. α1B-AR expression in cell membrane was the highest, and α1D-AR was the lowest, as determined by 125I-BE2254 binding assay, however, Kds were not significantly different among the three receptor subtypes. (2) Without agonist stimulation, α1A-AR was detected not only on the cell surface but also in the cytosol, α1B-AR was predominantly located on the cell surface, whereas a1D-AR was mostly detected in the cytosol. (3) After 1 h of stimulation with phenylephrine, as observed using confocal microscope, less α1A- and α1B-AR were detected on the cell surface but more in the cytosol. The change was more remarkable in α1B-AR than that in α1A-AR, whereas no change of distribution was detected in a1D-AR in response to phenylephrine. However, when examined by Western blot, no change in distribution was detected in α1A- and α1D-AR, only α1B-AR showed the same change as that shown in confocal imaging. It is suggested that the characteristics of localization and changes of distribution are different among three α1-AR subtypes in HEK293A cells upon phenylephrine stimulation.

Key words: α_(1)-adrenergic receptor;Distribution;Localization;Cell line

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引用本文：

王淑一, 宋峣, 徐明, 郝天袍, 韩启德, 张幼怡. 激动剂作用下α_(1)-肾上腺素受体亚型在HEK293A细胞中的分布变化[J]. 生理学报 2005; 57 (4): .

Wang Shuyi, Song Yao, Xu Ming, Hao Tianpao, Han Qide, Zhang Youyi. Redistribution of three α_(1)-adrenergic receptor subtypes in the stably transfected HEK 293A cells upon agonist stimulation. Acta Physiol Sin 2005; 57 (4): (in Chinese with English abstract).