Abstract
BackgroundThe 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.MethodsWe 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.ResultsWhile 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.ConclusionThe 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.
Highlights
The sources and measurement of reactive oxygen species (ROS) in intact organs are largely unresolved
In the context of regulatory processes of the lung, a growing body of evidence is emerging indicating that ROS may contribute to signaling events such as underlying hypoxic pulmonary vasoconstriction (HPV), an essential mechanism matching lung perfusion to ventilation in order to optimize pulmonary gas exchange [10,11]
After having proven the feasibility of CPH for ROS detection in the isolated perfused and ventilated rabbit lungs, with particular attention being paid to the autoxidation of CPH, we investigated the oxygen-dependence of intravascular ROS release in the intact lungs
Summary
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. Substantial evidence exists suggesting both decreases as well as increases, in lung ROS generation during alveolar hypoxia [13,14,15]. Both mitochondria and NADPH oxidases have been proposed as source(s) of ROS generation underlying HPV [12,13,15,16,17,18,19,20,21]
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