title: Detection of Nitric Oxide Induced by Angiotensin II Receptor Type 1 Using Soluble Guanylate Cyclase beta1 Subunit Fused to a Yellow Fluorescent Protein, Venus.
abstract: 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.
authors: Department of Pharmacology, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara, 634-0813, Japan.|Kyouto-Katsura Hospital, 17 Yamada-Hirao-cho, Nishi-kyoku, Kyoto, Japan.
authors: Yuichi
authors: Y
authors: Tsuji
authors: Department of Pharmacology, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara, 634-0813, Japan. ko12@naramed-u.ac.jp.
authors: Kentaro
authors: http://orcid.org/0000-0002-3648-4639
authors: K
authors: Ozawa
authors: Department of Pharmacology, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara, 634-0813, Japan.
authors: Akira T
authors: AT
authors: Komatsubara
authors: Department of Pharmacology, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara, 634-0813, Japan.
authors: Jing
authors: J
authors: Zhao
authors: Department of Anatomy and Cell Biology, Nara Medical University School of Medicine, Kashihara, Japan.
authors: Mayumi
authors: M
authors: Nishi
authors: Department of Pharmacology, Nara Medical University School of Medicine, 840 Shijo-cho, Kashihara, Nara, 634-0813, Japan.
authors: Masanori
authors: M
authors: Yoshizumi
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000032:analysis*; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D000071756:Soluble Guanylyl Cyclase / Q000235:genetics / Q000378:metabolism*
chemical_list: C544065:AGTR1 protein, human; D001426:Bacterial Proteins; D008164:Luminescent Proteins; D044140:Receptor, Angiotensin, Type 1; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D009569:Nitric Oxide; D000071756:Soluble Guanylyl Cyclase
keywords: Angiotensin II receptor type 1; Fluorescent protein; GPCR; Nitric oxide; S-nitrosylation