Discordance between eNOS phosphorylation and activation revealed by multispectral imaging and chemogenetic methods

Proc Natl Acad Sci U S A. 2019 Oct 1;116(40):20210-20217. doi: 10.1073/pnas.1910942116. Epub 2019 Sep 16.

Abstract

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 Ca2+ 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 Ca2+ By contrast, neither insulin nor VEGF caused any increase whatsoever in intracellular NO or Ca2+-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.

Keywords: endothelial cells; nitric oxide synthase; phosphorylation; signal transduction.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • AMP-Activated Protein Kinases / antagonists & inhibitors
  • AMP-Activated Protein Kinases / metabolism
  • Biosensing Techniques*
  • Calcium / metabolism
  • Cells, Cultured
  • Cytosol
  • Endothelial Cells / metabolism
  • Enzyme Activation
  • Molecular Imaging* / methods
  • Nitric Oxide / metabolism
  • Nitric Oxide Synthase / metabolism
  • Nitric Oxide Synthase Type III / metabolism*
  • Phosphorylation
  • Protein Kinase Inhibitors / pharmacology
  • Signal Transduction

Substances

  • Protein Kinase Inhibitors
  • Nitric Oxide
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type III
  • AMP-Activated Protein Kinases
  • Calcium