Detection of Nitric Oxide Induced by Angiotensin II Receptor Type 1 Using Soluble Guanylate Cyclase beta1 Subunit Fused to a Yellow Fluorescent Protein, Venus
Detection of Nitric Oxide Induced by Angiotensin II Receptor Type 1 Using Soluble Guanylate Cyclase beta1 Subunit Fused to a Yellow Fluorescent Protein, Venus
复制标题
使用与黄色荧光蛋白 Venus 融合的可溶性鸟苷酸环化酶 beta1 亚基检测 1 型血管紧张素 II 受体诱导的一氧化氮
DOI：
10.1007/s10895-016-1968-z
复制
发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Masanori Yoshizumi
中科院分区：
文献类型：
--
作者：
Yuichi Tsuji;Kentaro Ozawa;Akira T. Komatsubara;Jing Zhao;Mayumi Nishi;Masanori Yoshizumi
一氧化氮（NO）是一种重要的气体分子，参与许多生理和病理生理过程，包括G蛋白偶联受体（GPCRs）的调节。在这里，我们报告了一个高亲和力的方法来检测NO使用可溶性鸟苷酸环化酶β 1亚基融合金星，黄色荧光蛋白（sGC-Venus）的变体的发展。我们使用纯化的探针测量了具有和不具有NO供体的sGC-Venus的荧光强度。在560 nm发射下，sGC-Venus在405 nm激发下的荧光强度通过NO供体增加约2.5倍，但通过其他波长激发的sGC-Venus的荧光强度显示出小得多的增加或没有显著增加。为了测量活细胞中的NO，将sGC-Venus在405 nm激发下的荧光强度归一化为在488 nm激发下的荧光强度，因为其在有或没有NO供体的情况下没有显示出显著差异。在过表达血管紧张素II受体1型（AT 1受体）的HEK 293细胞中，使用sGC-Venus检测由AT 1受体激活诱导的NO产生。这些数据表明，sGC-Venus将是一个有用的工具，用于可视化活细胞中的细胞内NO，NO可能是一种常见的工具，以调节GPCR。
Nitric oxide (NO) is an important gaseous molecule involved in many physiological and pathophysiological processes, including the regulation of G protein-coupled receptors (GPCRs). Here, we report the development of a high-affinity method to detect NO using soluble guanylate cyclase beta1 subunit fused to Venus, a variant of yellow fluorescent protein (sGC-Venus). We measured the fluorescence intensity of sGC-Venus with and without an NO donor using purified probes. At 560 nm emission, the fluorescence intensity of sGC-Venus at 405 nm excitation was increased by approximately 2.5-fold by the NO donor, but the fluorescence intensities of sGC-Venus excited by other wavelengths showed much less of an increase or no significant increase. To measure NO in living cells, the fluorescence intensity of sGC-Venus at 405 nm excitation was normalized to that at 488 nm excitation because it showed no significant difference with or without the NO donor. In HEK293 cells overexpressing the angiotensin II receptor type 1 (AT1 receptor), the production of NO induced by activation of the AT1 receptor was detected using sGC-Venus. These data indicate that sGC-Venus will be a useful tool for visualizing intracellular NO in living cells and that NO might be a common tool to regulate GPCRs.