Targeting Fluorescent Sensors to Endoplasmic Reticulum Membranes Enables Detection of Peroxynitrite During Cellular Phagocytosis

Abstract

Peroxynitrite is a highly reactive oxidant derived from superoxide and nitric oxide. In normal vertebrate physiology, some phagocytes deploy this oxidant as a cytotoxin against foreign pathogens. To provide a new approach for detection of endogenous cellular peroxynitrite, we synthesized fluorescent sensors targeted to membranes of the endoplasmic reticulum (ER). The very high surface area of these membranes, approximately 30 times greater than the cellular plasma membrane, was envisioned as a vast intracellular platform for the display of sensors to transient reactive species. By linking an ER-targeted profluorophore to reactive phenols, sensors were designed to be cleaved by peroxynitrite and release a highly fluorescent ER-associated rhodol. Studies of kinetics in aqueous buffer revealed a linear free energy relationship where electron-donating substituents accelerate this reaction. However, in living cells, the efficiency of detection of endogenous cellular peroxynitrite was directly proportional to association with ER membranes. By incorporating a 2,6-dimethylphenol to accelerate the reaction and enhance this subcellular targeting, endogenous peroxynitrite in living RAW 264.7 macrophage cells could be readily detected after addition of antibody-opsonized tentagel microspheres, without additional stimulation, a process undetectable with other known fluorescent sensors. This approach provides uniquely sensitive tools for studies of transient reactive species in living mammalian cells.

Carnosine Protects Macrophages against the Toxicity of Aβ1-42 Oligomers by Decreasing Oxidative Stress

“…Given that carnosine has been shown to increase the phagocytic activity of macrophages in vivo [ 35 , 63 ], we wondered whether the well-known antioxidant activity of carnosine was also paralleled by the ability of this dipeptide to increase the phagocytic activity of RAW 264.7 cells. In order to validate this hypothesis, we used a recently developed protocol that allowed us to measure phagocytosis in RAW 264.7 cells by using antibody-bound tentagel beads and PS3 [ 52 , 53 ]. As clearly depicted in Figure 8 , the addition of antibody-opsonized beads to RAW 264.7 macrophages resulted in a significant increase of cellular fluorescence compared to resting cells ( p < 0.001).…”

“…The day prior to treatment, cells were seeded in 96-well plates at a density of 2 × 10 4 cells per well and incubated in a humidified environment (5% CO 2 , 37 °C) to allow for complete cell attachment. Cells were left untreated (control) or treated with Aβ1-42 oligomers (2 µM) in the absence or in the presence of carnosine (20 mM; 1 h pre-treatment) for 24 h. The production of peroxynitrite under all our experimental conditions was carried out by using a recently designed and optimized probe for peroxynitrite detection, Peroxynitrite Sensor 3 (PS3) [ 52 , 53 ]. During the 24 h treatment, PS3 was added to the medium at the final concentration of 10 µM [ 52 ].…”

“…Measurement of phagocytic activity in macrophages was carried out through applying a recently developed protocol that allows to measure phagocytosis in RAW 264.7 cells by using antibody-bound tentagel beads and PS3 [ 52 , 53 ].…”

Visualizing Autophagic Flux during Endothelial Injury with a Pathway-Inspired Tandem-Reaction Based Fluorogenic Probe

“…The nitrosative stress marker, peroxynitrite (ONOO - ), has been implicated in the initiation of autophagy in endothelial cells and may work as the first target for imaging autophagy 15,18. Among the various probes reported to image ROS 19-21, those that are designed based on the dearylation of electron-rich diphenylamines into mono-aryl-anilines attracted our interest due to their specifcity towards ONOO - 22-25. Another merit of employing a diphenylamino group as the sensing trigger lies in its function as a good fluorescence quencher.…”

Physicochemical-property guided design of a highly sensitive probe to image nitrosative stress in the pathology of stroke

“…Given the reactivity of ONOO À to oxidize electron-rich phenols into quinines which has been utilized with a high degree of success for ONOO À probe design (Fig. 1b), [25][26][27][28][29][30] the phydroxyl aniline moiety was selected as a reaction trigger for ONOO À sensing, and probes B545a and B545b were designed (Fig. 1c).…”