title: Type I cell ROS kinetics under hypoxia in the intact mouse carotid body ex vivo: a FRET-based study.
abstract: Reactive oxygen species (ROS) mainly originating from NADPH oxidases have been shown to be involved in the carotid body (CB) oxygen-sensing cascade. For measuring ROS kinetics, type I cells of the mouse CB in an ex vivo preparation were transfected with the ROS sensor construct FRET-HSP33. After 2 days of tissue culture, type I cells expressed FRET-HSP33 as shown by immunohistochemistry. In one population of CBs, 5 min of hypoxia induced a significant and reversible decrease of type I cell ROS levels (n = 9 CBs; P < 0.015), which could be inhibited by 4-(2-aminoethyl)benzensulfonylfluorid (AEBSF), a highly specific inhibitor of the NADPH oxidase subunits p47(phox) and p67(phox). In another population of CBs, however, 5 min of hypoxia induced a significant and reversible increase of ROS levels in type I cells (n = 8 CBs; P < 0.05), which was slightly enhanced by administration of 3 mM AEBSF. These different ROS kinetics seemed to coincide with different mice breeding conditions. Type I cells of both populations showed a typical hypoxia-induced membrane potential (MP) depolarization, which could be inhibited by 3 mM AEBSF. ROS and MP closely followed the hypoxic decrease in CB tissue oxygen as measured with an O2-sensitive dye. We conclude that attenuated p47(phox) subunit activity of the NADPH oxidase under hypoxia is the physiological trigger for type I cell MP depolarization probably due to ROS decrease, whereas the observed ROS increase has no influence on type I cell MP kinetics under hypoxia.
authors.affiliation: Institute of Physiology, University of Duisburg-Essen, Essen, Germany;
authors.forename: A
authors.initials: A
authors.lastname: Bernardini
authors.affiliation: Institute of Physiology, University of Duisburg-Essen, Essen, Germany;
authors.forename: U
authors.initials: U
authors.lastname: Brockmeier
authors.affiliation: Institute of Physiology, University of Duisburg-Essen, Essen, Germany;
authors.forename: E
authors.initials: E
authors.lastname: Metzen
authors.affiliation: Department of Ophthalmology, University of Duisburg-Essen, Essen, Germany;
authors.forename: U
authors.initials: U
authors.lastname: Berchner-Pfannschmidt
authors.affiliation: Institute for Cell Biology and Neuroscience and Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt, Germany; and.
authors.forename: E
authors.initials: E
authors.lastname: Harde
authors.affiliation: Institute for Cell Biology and Neuroscience and Buchmann Institute for Molecular Life Sciences, Goethe University, Frankfurt, Germany; and.
authors.forename: A
authors.initials: A
authors.lastname: Acker-Palmer
authors.affiliation: Biochemistry Department, University College Cork, Cork, Ireland.
authors.forename: D
authors.initials: D
authors.lastname: Papkovsky
authors.affiliation: Institute of Physiology, University of Duisburg-Essen, Essen, Germany; helmut.acker@uni-due.de.
authors.forename: H
authors.initials: H
authors.lastname: Acker
authors.affiliation: Institute of Physiology, University of Duisburg-Essen, Essen, Germany;
authors.forename: J
authors.initials: J
authors.lastname: Fandrey
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D015374:Biosensing Techniques; D002344:Carotid Body / Q000187:drug effects / Q000378:metabolism*; D015687:Cell Hypoxia; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006360:Heat-Shock Proteins / Q000235:genetics; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008564:Membrane Potentials; D008810:Mice, Inbred C57BL; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010641:Phenotype; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020218:Response Elements; D015398:Signal Transduction; D046509:Tissue Culture Techniques; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D006360:Heat-Shock Proteins; D008164:Luminescent Proteins; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; C527701:enhanced cyan fluorescent protein; C079789:neutrophil cytosol factor 67K; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1
keywords: FRET-HSP33; NADPH oxidase; ROS; carotid body; hypoxia; membrane potential; tissue oxygen