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 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.
Panda
Koustubh
K
Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA. pandak@ccf.org
Chawla-Sarkar
Mamta
M
Santos
Cecile
C
Koeck
Thomas
T
Erzurum
Serpil C
SC
Parkinson
John F
JF
Stuehr
Dennis J
DJ
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D006207:Half-Life; D006418:Heme / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D007150:Immunohistochemistry; D008164:Luminescent Proteins; D018613:Microscopy, Confocal; D015336:Molecular Probe Techniques; D019001:Nitric Oxide Synthase / Q000378:metabolism / Q000502:physiology*; D052247:Nitric Oxide Synthase Type II; D013050:Spectrometry, Fluorescence; D000097573:Red Fluorescent Protein
D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006418:Heme; D007093:Imidazoles; D008164:Luminescent Proteins; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D000097573:Red Fluorescent Protein; C496319:NOS2 protein, human