title: Detection of reactive oxygen species in isolated, perfused lungs by electron spin resonance spectroscopy.
abstract: BACKGROUND
  The sources and measurement of reactive oxygen species (ROS) in intact organs are largely unresolved. This may be related to methodological problems associated with the techniques currently employed for ROS detection. Electron spin resonance (ESR) with spin trapping is a specific method for ROS detection, and may address some these technical problems.
  
  
  METHODS
  We have established a protocol for the measurement of intravascular ROS release from isolated buffer-perfused and ventilated rabbit and mouse lungs, combining lung perfusion with the spin probe 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) and ESR spectroscopy. We then employed this technique to characterize hypoxia-dependent ROS release, with specific attention paid to NADPH oxidase-dependent superoxide formation as a possible vasoconstrictor pathway.
  
  
  RESULTS
  While perfusing lungs with CPH over a range of inspired oxygen concentrations (1-21 %), the rate of CP* formation exhibited an oxygen-dependence, with a minimum at 2.5 % O2. Addition of superoxide dismutase (SOD) to the buffer fluid illustrated that a minor proportion of this intravascular ROS leak was attributable to superoxide. Stimulation of the lungs by injection of phorbol-12-myristate-13-acetate (PMA) into the pulmonary artery caused a rapid increase in CP* formation, concomitant with pulmonary vasoconstriction. Both the PMA-induced CPH oxidation and the vasoconstrictor response were largely suppressed by SOD. When the PMA challenge was performed at different oxygen concentrations, maximum superoxide liberation and pulmonary vasoconstriction occurred at 5% O2. Using a NADPH oxidase inhibitor and NADPH-oxidase deficient mice, we illustrated that the PMA-induced superoxide release was attributable to the stimulation of NADPH oxidases.
  
  
  CONCLUSIONS
  The perfusion of isolated lungs with CPH is suitable for detection of intravascular ROS release by ESR spectroscopy. We employed this technique to demonstrate that 1) PMA-induced vasoconstriction is caused "directly" by superoxide generated from NADPH oxidases and 2) this pathway is pronounced in hypoxia. NADPH oxidases thus may contribute to the hypoxia-dependent regulation of pulmonary vascular tone.
authors.affiliation: Department of Internal Medicine II, Klinikstrasse 36, Justus-Liebig University, 35392 Giessen, Germany. Norbert.Weissmann@innere.med.uni-giessen.de
authors.forename: Norbert
authors.initials: N
authors.lastname: Weissmann
authors.forename: Nermin
authors.initials: N
authors.lastname: Kuzkaya
authors.forename: Beate
authors.initials: B
authors.lastname: Fuchs
authors.forename: Vedat
authors.initials: V
authors.lastname: Tiyerili
authors.forename: Rolf U
authors.initials: RU
authors.lastname: Schäfer
authors.forename: Hartwig
authors.initials: H
authors.lastname: Schütte
authors.forename: Hossein A
authors.initials: HA
authors.lastname: Ghofrani
authors.forename: Ralph T
authors.initials: RT
authors.lastname: Schermuly
authors.forename: Christian
authors.initials: C
authors.lastname: Schudt
authors.forename: Akylbek
authors.initials: A
authors.lastname: Sydykov
authors.forename: Bakytbek
authors.initials: B
authors.lastname: Egemnazarow
authors.forename: Werner
authors.initials: W
authors.lastname: Seeger
authors.forename: Friedrich
authors.initials: F
authors.lastname: Grimminger
mesh_terms: D000818:Animals; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D005260:Female; D066298:In Vitro Techniques; D008168:Lung / Q000378:metabolism*; D008297:Male; D010477:Perfusion / Q000379:methods; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000032:analysis*
chemical_list: D017382:Reactive Oxygen Species