期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2019
卷号：116
期号：40
页码：20210-20217
DOI：10.1073/pnas.1910942116
出版社：The National Academy of Sciences of the United States of America
摘要：Nitric oxide (NO) synthesized by the endothelial isoform of nitric oxide synthase (eNOS) is a critical determinant of vascular homeostasis. However, the real-time detection of intracellular NO—a free radical gas—has been difficult, and surrogate markers for eNOS activation are widely utilized. eNOS phosphorylation can be easily measured in cells by probing immunoblots with phosphospecific antibodies. Here, we pursued multispectral imaging approaches using biosensors to visualize intracellular NO and Ca 2+ and exploited chemogenetic approaches to define the relationships between NO synthesis and eNOS phosphorylation in cultured endothelial cells. We found that the G protein-coupled receptor agonists adenosine triphosphate (ATP) and histamine promoted rapid increases in eNOS phosphorylation, as did the receptor tyrosine kinase agonists insulin and Vascular Endothelial Growth Factor (VEGF). Histamine and ATP also promoted robust NO formation and increased intracellular Ca 2+ . By contrast, neither insulin nor VEGF caused any increase whatsoever in intracellular NO or Ca 2+ —despite eliciting strong eNOS phosphorylation responses. Our findings demonstrate an unexpected and striking discordance between receptor-modulated eNOS phosphorylation and NO formation in endothelial cells. Previous reports in which phosphorylation of eNOS has been studied as a surrogate for enzyme activation may need to be reassessed..
