Abdel-Rahman 2016 Oxid Med Cell Longev
|Abdel-Rahman EA, Mokhtar A, Aaliya A, Radwan Y, Yasseen B, Al-Okda A, Atwa A, Elhanafy E, Habashy M, Ali SS (2016) Resolving contributions of oxygen-consuming and ROS-generating enzymes at the synapse. Oxid Med Cell Longev p19.|
Abstract: Disruption of cellular redox homeostasis is implicated in a wide variety of pathologic conditions and aging. A fundamental factor that dictates such balance is the ratio between mitochondria-mediated complete oxygen reduction into water and incomplete reduction into superoxide radical by mitochondria and NADPH oxidase (NOX) enzymatic activity. Here we determined mitochondrial as well as NOX-dependent rates of oxygen consumption in parallel with H2O2 generation in freshly isolated synaptosomes using high-resolution respirometry combined with fluorescence or electrochemical sensory. Our results indicate that, although synaptic mitochondria exhibit substantially higher respiratory activities (8-82 folds greater than NOX oxygen consumption depending on mitochondrial respiratory state), NADPH-dependent oxygen consumption is associated with greater H2O2 production (6-7 folds higher NOX-H2O2). We also show that, in terms of the consumed oxygen, while synaptic mitochondria ‘leaked’ 0.71% ± 0.12 H2O2 during NAD+-linked resting, 0.21% ± 0.04 during NAD+-linked active, and 0.07% ± 0.02 during FAD+-linked active respirations, NOX converted 38% ± 13 of O2 into H2O2. Our results indicate that NOX rather than mitochondria is the major source of synaptic H2O2. The present approach may assist in the identification of redox-modulating synaptic factors that underlie a variety of physiological and pathological processes in neurons.
• Keywords: ROS, Mitochondria, NADPH Oxidase, Synaptosomes, High-resolution respirometry
• O2k-Network Lab: EG Cairo Ali SS
Labels: MiParea: Respiration
Stress:Oxidative stress;RONS Organism: Mouse Tissue;cell: Nervous system Preparation: Permeabilized cells
Coupling state: LEAK, OXPHOS Pathway: N, NS, ROX HRR: Oxygraph-2k, O2k-Fluorometer
2016-11, Amplex Ultra Red
H2O2 was detected by both fluomometry with the AmplexRed assay and electrochemically by using an HPO-ISO-2mm sensor (WPI, Sarasota, U.S.A.) which is compatible with the O2k-NO Amp-Module.