Visualizing Inducible Nitric-Oxide Synthase in Living Cells with a Heme-Binding Fluorescent Inhibitor

The study of nitric-oxide synthase (NOS) physiology is constrained by the lack of suitable probes to detect NOS in living cells or animals. Here, we characterized a fluorescent inducible NOS (iNOS) inhibitor called PIF (pyrimidine imidazole FITC) and examined its utility for microscopic imaging of i...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 102; no. 29; pp. 10117 - 10122
Main Authors Panda, Koustubh, Chawla-Sarkar, Mamta, Santos, Cecile, Koeck, Thomas, Erzurum, Serpil C., Parkinson, John F., Stuehr, Dennis J., Snyder, Solomon H.
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 19.07.2005
National Acad Sciences
Subjects
Animals
Biochemistry
Biological Sciences
Blotting, Western
Cell culture techniques
Cell lines
Cells
Cells, Cultured
Chromatography, Gel
Cloning, Molecular
Epithelial cells
Fluorescein-5-isothiocyanate - metabolism
Fluorescence
Fluorescent Dyes - metabolism
Gene expression
Half-Life
HEK293 cells
Heme - metabolism
Humans
Imaging
Imidazoles
Imidazoles - metabolism
Immunohistochemistry
Luminescent Proteins
Memory interference
Microscopy, Confocal
Molecular Probe Techniques
Monomers
Nitric oxide
Nitric Oxide Synthase - metabolism
Nitric Oxide Synthase - physiology
Nitric Oxide Synthase Type II
Red Fluorescent Protein
Spectrometry, Fluorescence
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Summary:The study of nitric-oxide synthase (NOS) physiology is constrained by the lack of suitable probes to detect NOS in living cells or animals. Here, we characterized a fluorescent inducible NOS (iNOS) inhibitor called PIF (pyrimidine imidazole FITC) and examined its utility for microscopic imaging of iNOS in living cells. PIF binding to iNOS displayed high affinity, isoform selectivity, and heme specificity, and was essentially irreversible. PIF was used to successfully image iNOS expressed in RAW264.7 cells, HEK293T cells, human A549 epithelial cells, and freshly obtained human lung epithelium. PIF was used to estimate a half-life for iNOS of 1.8 h in HEK293T cells. Our work reveals that fluorescent probes like PIF will be valuable for studying iNOS cell biology and in understanding the pathophysiology of diseases that involve dysfunctional iNOS expression.
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Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved May 9, 2005
To whom correspondence may be addressed at: Department of Immunology, NB3, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail: pandak@ccf.org or stuehrd@ccf.org.
Abbreviations: H4B, (6R)-5,6,7,8-tetrahydro-l-biopterin; NO, nitric oxide; NOS, nitric-oxide synthase; eNOS, endothelial NOS; iNOS, inducible NOS; nNOS, neuronal NOS; PI, pyrimidine imidazole; PIF, PI FITC; RAW, RAW264.7; RFP, red fluorescent protein; SA, succinyl acetone.
This paper was submitted directly (Track II) to the PNAS office.
Author contributions: K.P. and D.J.S. designed research; K.P., M.C.-S., and T.K. performed research; S.C.E. and J.F.P. contributed new reagents/analytic tools; K.P., M.C.-S., and D.J.S. analyzed data; and K.P. and D.J.S. wrote the paper.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0408972102