title: Respiratory uncoupling by UCP1 and UCP2 and superoxide generation in endothelial cell mitochondria.
abstract: Mitochondria represent a major source of reactive oxygen species (ROS), particularly during resting or state 4 respiration wherein ATP is not generated. One proposed role for respiratory mitochondrial uncoupling proteins (UCPs) is to decrease mitochondrial membrane potential and thereby protect cells from damage due to ROS. This work was designed to examine superoxide production during state 4 (no ATP production) and state 3 (active ATP synthesis) respiration and to determine whether uncoupling reduced the specific production of this radical species, whether this occurred in endothelial mitochondria per se, and whether this could be modulated by UCPs. Superoxide formation by isolated bovine aortic endothelial cell (BAE) mitochondria, determined using electron paramagnetic resonance spectroscopy, was approximately fourfold greater during state 4 compared with state 3 respiration. UCP1 and UCP2 overexpression both increased the proton conductance of endothelial cell mitochondria, as rigorously determined by the kinetic relationship of respiration to inner membrane potential. However, despite uncoupling, neither UCP1 nor UCP2 altered superoxide formation. Antimycin, known to increase mitochondrial superoxide, was studied as a positive control and markedly enhanced the superoxide spin adduct in our mitochondrial preparations, whereas the signal was markedly impaired by the powerful chemical uncoupler p-(trifluoromethoxyl)-phenyl-hydrazone. In summary, we show that UCPs do have uncoupling properties when expressed in BAE mitochondria but that uncoupling by UCP1 or UCP2 does not prevent acute substrate-driven endothelial cell superoxide as effluxed from mitochondria respiring in vitro.
authors.affiliation: Department of Internal Medicine, Division of Endocrinology and Metabolism, Iowa City Veterans Affairs Medical Center, University of Iowa, Iowa City, Iowa, USA.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Fink
authors.forename: Krzysztof J
authors.initials: KJ
authors.lastname: Reszka
authors.forename: Judy A
authors.initials: JA
authors.lastname: Herlein
authors.forename: Mary M
authors.initials: MM
authors.lastname: Mathahs
authors.forename: William I
authors.initials: WI
authors.lastname: Sivitz
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000166:cytology; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D019069:Cell Respiration / Q000502:physiology; D002478:Cells, Cultured; D004578:Electron Spin Resonance Spectroscopy; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism; D007473:Ion Channels; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology; D013481:Superoxides / Q000378:metabolism*; D000071256:Uncoupling Protein 1; D000071246:Uncoupling Protein 2
chemical_list: D002352:Carrier Proteins; D005227:Fatty Acids; D007473:Ion Channels; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; D000071256:Uncoupling Protein 1; D000071246:Uncoupling Protein 2; D013481:Superoxides; D000255:Adenosine Triphosphate