Detection of Nitric Oxide Induced by Angiotensin II Receptor Type 1 Using Soluble Guanylate Cyclase beta1 Subunit Fused to a Yellow Fluorescent Protein, Venus

摘要

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.