Imaging the Nanomolar Range of Nitric Oxide with an Amplifier-Coupled Fluorescent Indicator in Living Cells

Nitric oxide (NO) is a small uncharged free radical that is involved in diverse physiological and pathophysiological mechanisms. NO is generated by three isoforms of NO synthase, endothelial, neuronal, and inducible ones. When generated in vascular endothelial cells, NO plays a key role in vascular...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 102; no. 41; pp. 14515 - 14520
Main Authors Moritoshi Sato, Naoki Hida, Umezawa, Yoshio
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 11.10.2005
National Acad Sciences
Subjects
Animals
Biological Sciences
Cells
CHO Cells
Complementary DNA
Cricetinae
Cricetulus
Cultured cells
DNA Primers
Dyes
Endothelial cells
Endothelial Cells - metabolism
Fluorescence
Imaging
Indicators and Reagents - metabolism
Kidney cells
Microscopy, Fluorescence - methods
Models, Molecular
Molecules
Nitric oxide
Nitric Oxide - chemistry
Oxides
Plasmids - genetics
Shear stress
Transfection
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Summary:Nitric oxide (NO) is a small uncharged free radical that is involved in diverse physiological and pathophysiological mechanisms. NO is generated by three isoforms of NO synthase, endothelial, neuronal, and inducible ones. When generated in vascular endothelial cells, NO plays a key role in vascular tone regulation, in particular. Here, we describe an amplifier-coupled fluorescent indicator for NO to visualize physiological nanomolar dynamics of NO in living cells (detection limit of 0.1 nM). This genetically encoded high-sensitive indicator revealed that ≈1 nM of NO, which is enough to relax blood vessels, is generated in vascular endothelial cells even in the absence of shear stress. The nanomolar range of basal endothelial NO thus revealed appears to be fundamental to vascular homeostasis.
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This paper was submitted directly (Track II) to the PNAS office.
M.S. and N.H. contributed equally to this work.
To whom correspondence should be addressed. E-mail: umezawa@chem.s.u-tokyo.ac.jp.
Abbreviations: 8-Br-cGMP, 8-bromoguanosine-cGMP; l-NAME, Nω-nitro-l-arginine methyl ester; NOC-7, 3-(2-hydroxy-1-methylethyl-2-nitrosohydrazino)-N-methyl-1-propanamine; sGC, soluble guanylate cyclase; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; NOS, NO synthase; eNOS, endothelial NOS.
Edited by Solomon H. Snyder, The Johns Hopkins University School of Medicine, Baltimore, MD
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0505136102