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Significance Nitric oxide (NO) is synthesized by the endothelial isoform of nitric oxide synthase (eNOS) in vascular endothelial cells. The high reactivity of NO undermines its accurate detection in living cells. The direct detection of the low physiological concentrations of NO that are present in endothelial cells represents a significant analytical challenge. In these studies, we have used powerful biosensors to measure NO and other key signaling molecules in living endothelial cells in real time using fluorescence microscopy. These approaches have provided insights into the mechanisms whereby NO synthesis is regulated and call into question many prior conclusions about the intracellular pathways that regulate eNOS.

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