Imaging and Quantification of Secreted Peroxynitrite at the Cell Surface by a Streptavidin-Biotin-Controlled Binding...

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.