Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
SHR Neuro Krebs Kardio Lipid Stoffw Microb
Eroglu, E; Saravi, SSS; Sorrentino, A; Steinhorn, B; Michel, T.
Discordance between eNOS phosphorylation and activation revealed by multispectral imaging and chemogenetic methods.
Proc Natl Acad Sci U S A. 2019; 116(40):20210-20217
Doi: 10.1073/pnas.1910942116
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- 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.
- Find related publications in this database (using NLM MeSH Indexing)
- 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 -
- Find related publications in this database (Keywords)
- signal transduction
- nitric oxide synthase
- endothelial cells
- phosphorylation