Cadenas 2006 Biochim Biophys Acta
|Aguirre E, Rodriguez-Juarez F, Gnaiger E, Cadenas S (2006) Measurement of the control of cellular respiration by nitric oxide under normoxia and hypoxia: instrumental comparison including high-resolution respirometry. Biochim Biophys Acta, EBEC Short Reports Suppl 14:136-7.|
Abstract: At low oxygen levels, mitochondrial respiration is controlled by the nitric oxide (NO)-cytochrome c oxidase (COX) signaling pathway, since NO is a membrane-permeant second messenger and competitive inhibitor of COX (1). It is now well established that oxygraphs, with Teflon-coated stirrer bars and other plastic materials of high oxygen solubility, yield high rates of oxygen back-diffusion into the chamber when oxygen levels decline, causing artefacts of respiratory measurements. High-resolution respirometry with the Oroboros O2k reduces such back-diffusion by at least an order of magnitude, and incorporates automatic instrumental background corrections, treating the ‘closed’ chamber essentially as an open system with oxygen transport between the aqueous phase and the system boundary (2). For measurement of NO in experimental chambers, however, the same instrumental problem of gas exchange between hydrophobic plastic materials and the aqueous medium has not been addressed, despite the high partition coefficient of NO between aqueous and organic phases (3). To address these problems, we incorporated an NO sensor (ISO-NOP, WPI) into a Hansatech oxygraph chamber and a high-resolution respirometer (O2k), for simultaneous recording of respiration and NO. The NO sensor was calibrated by addition of known concentrations of KNO2 under reducing conditions (KI/H2SO4) at 37 ºC and the response of the NO sensor in terms of accuracy, stability and reproducibility of the signal was compared between the two chambers. Measurements were taken in 1 ml (Hansatech) or 2 ml (O2k) closed chambers at 37 ºC, using their standard Teflon- or PEEK-coated stirrer bars, respectively. The titanium stopper of the O2k chamber was replaced by a polyvinylidenfluorid (PVDF) stopper, including a second inlet (2 mm diameter) for the NO sensor in addition to the capillary used for extrusion of gas bubbles and titration of chemicals. The PVDF stopper showed identical characteristics to titanium in terms of minimum back-diffusion of oxygen in aerobic-anaerobic transitions, can be cleaned with 70 % and pure ethanol, and offers increased flexibility for accommodation of various additional electrodes for multi-sensor applications. We compared the response of the NO sensor in the determination of the release of NO from a chemical source (DETA-NO) and the endogenous release from controlled intracellular NO production. We determined the inhibition of respiration caused by NO under physiological oxygen concentrations using conventional and high-resolution respirometry (2).
• Keywords: Nitric oxide, NO sensor, PVDF stopper, High-resolution respirometry
Labels: MiParea: Respiration, Genetic knockout;overexpression
Stress:Oxidative stress;RONS Organism: Human Tissue;cell: HEK Preparation: Intact cells, Enzyme, Oxidase;biochemical oxidation
Regulation: Inhibitor, Oxygen kinetics Coupling state: ROUTINE
HRR: Oxygraph-2k, NO