MiP2005: Session 11

Mitochondrial Physiology Network 10.9: 123-124 (2005) - download pdf

 

Mitochondrial respiratory control and oxygen dependence of ROS production in aging endothelial cells.

Eveline Hütter1, H Unterluggauer1, A Garedew2, P Jansen-Dürr1, E Gnaiger2

1Inst. Biomedical Aging Research, Austrian Academy of Sciences; 2D. Swarovski Research Lab., Dept. Transplant Surgery, Innsbruck Medical University, 6020 Innsbruck, Austria. -  eveline.huetter@oeaw.ac.at

    Oxidative stress is a major determinant of cellular aging. Mitochondria are frequently considered as the major source of ROS production, but the relative contributions and oxygen dependence of mitochondrial and non-mitochondrial ROS remain controversial. In contrast to low oxygen levels in the mitochondrial microenvironment of many tissues, isolated mitochondria are routinely exposed to air-level oxygen concentrations [1, 2]. Using human umbilical vein endothelial cells (HUVEC) as an in vitro model of cellular aging [3], the present study was aimed at the evaluation of mitochondrial integrity and quantitative assessment of ROS production as a function of oxygen concentration.

    Cellular oxygen consumption was measured by high-resolution respirometry with the OROBOROS Oxygraph-2k [1, 4]. Mitochondrial respiratory capacity and coupling of oxidative phosphorylation were preserved in senescent cells, as shown by oligomycin inhibition and FCCP stimulation in intact cells, and by substrate/inhibitor titrations after permeabilization with digitonin. Respiration through cytochrome c oxidase constituted a hyperbolic high-affinity component of oxygen kinetics in the low-oxygen range (up to 10 µM), independent of age. Enhanced oxidative stress was observed in aged endothelial cells by staining with the oxidant sensitive dye dihydroethidium. In direct agreement, a linear component of oxygen kinetics was pronounced in senescent cells suspended in culture medium at increasing oxygen levels. Hypoxia-induced down-regulation of ATP turnover could be excluded by performing oxygen kinetics of not only coupled, but also uncoupled respiration, which also showed linear oxygen dependence in the high oxygen range in senescent HUVEC. This indicates predominantly non-mitochondrial sources of ROS production. While the quantitative importance of several non-mitochondrial oxidases was excluded by application of specific inhibitors, the enzyme family of NADPH oxidases is being studied as one of the potential sources of ROS in senescent HUVEC.

1.  Gnaiger E, Méndez G, Hand SC (2000) High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. Proc. Natl. Acad. Sci. USA 97: 11080-11085.

2.  Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv. Exp. Med. Biol. 543: 39-55.

3.  Unterluggauer H, Hampel B, Zwerschke W, Jansen-Dürr P (2003) Senescence-associated cell death of human endothelial cells: the role of oxidative stress. Exp. Geront. 38: 1149-1160.

4. Hütter E, Renner K, Jansen-Dürr P, Gnaiger E (2002) Biphasic oxygen kinetics of cellular respiration and linear oxygen dependence of antimycin A inhibited oxygen consumption. Molec. Biol. Rep. 29: 83-87.

 

New reference:

  • Hutter E, Unterluggauer H, Garedew A, Jansen-Durr P, Gnaiger E (2006) High-resolution respirometry - a modern tool in aging research. Exp. Gerontol. 41: 103-109.

 


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