Research

Detection of reactive oxygen species in isolated, perfused lungs by electron spin resonance spectroscopy

Norbert Weissmann¹ , Nermin Kuzkaya¹ , Beate Fuchs¹ , Vedat Tiyerili¹ , Rolf U Schäfer¹ , Hartwig Schütte² , Hossein A Ghofrani¹ , Ralph T Schermuly¹ , Christian Schudt³ , Akylbek Sydykov¹ , Bakytbek Egemnazarow¹ , Werner Seeger¹ and Friedrich Grimminger¹

¹  Justus-Liebig University, Department of Internal Medicine II, Klinikstrasse 36, 35392 Giessen, Germany

²  Charite, Department of Internal Medicine, Humboldt-University, 13353 Berlin, Germany

³  ALTANA Pharma, 78467 Konstanz, Germany

author email corresponding author email

Respiratory Research 2005, 6:86doi:10.1186/1465-9921-6-86

Published:	31 July 2005

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 l-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.

Conclusion

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.