Abstract
Production of reactive oxygen species (ROS) has been implicated in the pathology of many conditions, including cardiovascular, inflammatory and degenerative diseases, aging, muscular dystrophy, and muscle fatigue. NADPH oxidases (Nox) have recently gained attention as an important source of ROS involved in redox signaling. However, our knowledge of the source of ROS has been limited by the relatively impoverished array of tools available to study them and the limitations of all imaging probes to provide meaningful spatial resolution. By linking redox-sensitive GFP (roGFP) to the Nox organizer protein, p47phox, we have developed a redox sensitive protein to specifically assess Nox activity (p47-roGFP). Stimulation of murine macrophages with endotoxin resulted in rapid, reversible oxidation of p47-roGFP. In murine skeletal muscle, both passive stretch and repetitive electrical stimulation resulted in oxidation of p47-roGFP. The oxidation of p47-roGFP in both macrophages and skeletal muscle was blocked by a Nox specific peptide inhibitor. Furthermore, expression of p47-roGFP in p47phox deficient cells restored Nox activity. As Nox has been linked to pathological redox signaling, our newly developed Nox biosensor will allow for the direct assessment of Nox activity and the development of therapeutic Nox inhibitors.
Highlights
Free radicals and other reactive oxygen species (ROS) are produced in a wide range of physiological processes and have long been associated with inflicting biological damage
Using redox-sensitive GFP targeted to the mitochondria we have previously shown that increased contractile activity promotes ROS formation not from the mitochondria but potentially via Nox2 in skeletal muscle [7]
Using a redox sensitive GFP targeted to the mitochondria along with pharmacological inhibitors we have previously shown that the source of ROS during electrical stimulation is likely NADPH oxidase (Nox) [7]
Summary
Free radicals and other reactive oxygen species (ROS) are produced in a wide range of physiological processes and have long been associated with inflicting biological damage. Generation of superoxide and other downstream ROS by NADPH oxidase (Nox) has long been ascribed to phagocytes. Increased production of ROS from the non-phagocytic Noxs has been implicated in ischemia reperfusion, hypertension, heart failure, atrial fibrillation, Alzheimer’s and Parkinson’s disease, muscular dystrophy and muscle fatigue. Redox-sensitive fluorescent dyes such as DCFH have been used to detect oxidant generation within living cells. These dyes are prone to movement and bleaching artifacts, are non-reversible, lack specificity for the site of ROS generation, display low sensitivity, and even promote artificial ROS formation [4,5]. Due to the potential participation of Nox in a variety of diseases, there is a need to selectively measure ROS production from the Nox enzyme complex
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