Mitochondrial Physiology Network 10.9: 26-27 (2005) - download pdf

Oxygen- and flux-dependence of ROS-formation of lung alveolar epithelial cells.

Heimo Mairbäurl, L Hotz, N Chaudhuri, P Bärtsch

Medical Clinic VII, Sports Medicine, University of Heidelberg, Germany. - heimo_mairbaeurl@med.uni-heidelberg.de

    ROS are produced in the cytoplasm and by mitochondria [3]. However, results are divergent on changes in ROS production upon varying cellular oxygen supply [1,2]. In the present study we measured ROS-formation during 15 min (37 °C) exposure of lung alveolar A549 cells to hypoxia (2% O₂), normoxia (21 % O₂) and hyperoxia (35 % O₂) by chemiluminescence (CL; 30 µM lucigenin) and ESR-spectroscopy (CMH, CPH, ACP, PPH; 500 µM). Both, CL and ESR indicate the lowest accumulation of ROS during exposure to hypoxia and an increase in ROS-formation with increasing oxygenation. It has to be noted, however, that decreasing the concentration of CMH also decreases the rate of ROS-formation in normoxic cells. In contrast to results obtained by CL and ESR, DCF-fluorescence was increased in hypoxia. In all cases, return to 21 % O₂ increased ROS-formation in hypoxic but decreased ROS in previously hyperoxic cells. N-acetyl-cysteine decreased CL in all states of oxygenation but oxygen-dependency of CL was still apparent. Decreased ROS-formation in hypoxia was associated with slightly increased NADH, measured by fluorescence microscopy, and a decrease in cellular oxygen consumption, measured by high-resolution respirometry (OROBOROS Oxygraph). In intact cells, addition of rotenone and antimycin A increased ROS-formation, whereas stimulation of complex I and II respiration by increasing ADP decreased ROS measured by CL in digitonin-permeabilized cells. Both, in the non-stimulated and ADP-stimulated state, ROS-formation was lower in hypoxia than normoxia. Our results confirm apparent discrepancies on changes in ROS formation upon changing oxygenation that can only be explained on the basis of different species of ROS that are seen by different detection methods. It might also be, however, that different ROS-indicators affect the rate of cellular ROS-formation in different ways, which needs further exploration.

1. Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, Schumacker PT (1998) Mitochondrial reactive oxygen species trigger hypoxia- induced transcription. Proc. Natl. Acad. Sci. USA 95: 11715-11720.

2. Michelakis ED, Hampl V, Nsair A, Wu XC, Harry G, Haromy A, Gurtu R, Archer SL (2002) Diversity in mitochondrial function explains differences in vascular oxygen sensing. Circ. Res. 90: 1307-1315.

3. Nohl H (1994) Generation of superoxide radicals as byproduct of cellular respiration. Ann. Biol. Clin. (Paris) 52: 199-204.