Imaging and Quantification of Secreted Peroxynitrite at the Cell Surface by a Streptavidin–Biotin‐Controlled Binding Probe

Abstract

The ability to detect and image secreted peroxynitrite (ONOO−) along the extracellular surface of a single cell is biologically significant, as ONOO− generally exerts its function for host defense and signal transductions at the plasma membrane. However, as a result of the short lifetime and fast diffusion rate of small ONOO−, precise determination of the ONOO− level at the cell surface remains a challenging task. In this paper, the use of a membrane‐anchored streptavidin–biotin‐controlled binding probe (CBP), ONOO‐CBP, to determine quantitatively the ONOO− level at the cell surface and to investigate the effect of different stimulants on the production of ONOO− along the plasma membrane of macrophages is reported. Our results revealed that the combination of NO synthase (iNOS) and NADPH oxidase (NOX) activators was highly effective in inducing ONOO− secretion, achieving more than a 25‐fold increase in ONOO− relative to untreated cells. After 1 h of phorbol‐12‐myristate‐13‐acetate (PMA) stimulation, the amount of ONOO− secreted by RAW264.7 macrophages was similar to the condition treated with 25 μm 3‐morpholinosydnonimine hydrochloride (SIN‐1), which was estimated to release about 20 μm of ONOO− into Dulbecco's modified Eagle's medium (DMEM) in 1 h. This novel approach should open up new opportunities to image various reactive oxygen and nitrogen species secreted at the plasma membrane that cannot be simply achieved by conventional analytical methods.

High Salt Intake Impairs Erectile Function in Salt-Sensitive Rats Through Mineralocorticoid Receptor Pathway Beyond Its Effect on Blood Pressure

“…Increased ROS generation leads to oxidative stress and converts NO into its inactive form, peroxynitrite (ONOO À ). 37 Peroxynitrite oxidizes tetrahydrobiopterine, a critical cofactor for NO synthases, which, in turn, impairs the function of endothelial NO synthase and reduces NO production. 38 These reactions could be responsible for the decreased NO production in the CC of HS Dahl-S rats.…”

Boronate-Based Probes for Biological Oxidants: A Novel Class of Molecular Tools for Redox Biology

“…The typical procedure to introduce such a residue exploits the 4-hydroxymethylphenylboronic acid pinacol ester in which the hydroxyl group is converted, often in situ , into an acid chloride in a reaction with triphosgene (Srikun et al, 2008 ; Chung et al, 2011 ; Zhu D. et al, 2013 ) in the presence of a base in various solvents, which reacts further with the target amino group. There are some examples in which this approach is also used to mask the nitrogen atom on biotin (Chung et al, 2018 ; Wu Y. P. et al, 2018 ) or methylene blue (Hu et al, 2019 ) or in aliphatic systems (Lippert et al, 2010 ; Zhu D. et al, 2013 ; Yik-Sham Chung et al, 2018 ).…”

Mito-Specific Ratiometric Terbium(III)-Complex-Based Luminescent Probe for Accurate Detection of Endogenous Peroxynitrite by Time-Resolved Luminescence Assay

“…The luminescence in the red channel was quenched thoroughly, whereas the luminescence in the green channel increased obviously , (Figure D). To prove the phenomenon is caused by ONOO – , the A-375 cells were further treated with TEMPO, which is a radical scavenger, to eliminate the endogenous ONOO – , (Figure E). The intensity signal was also calculated and showed a 2.9-fold enhancement (Figure F).…”