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.

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.

Figure 1

The effect of the iron chelating agent deferoxamine (DFO) on CP^• nitroxide signal intensity in vitro. (A) Typical ESR spectrum of CP^• nitroxide resulting from the reaction of the hydroxylamine spin probe CPH with ROS. The height of the first field component of the triple-line spectrum was used for quantification of signal intensity. (B) In-vitro incubation of CPH (1 mM) in Krebs-Henseleit buffer. Signal intensity is given in arbitrary units (AU). Data are shown for CPH oxidation in the absence (-DFO) or in the presence of either 20 μM or 2 mM deferoxamine (DFO). Asterisks indicate significant differences when compared to the-DFO group.

Figure 2

The ESR signal intensity in isolated perfused and ventilatedrabbit lungs during baseline conditions and in the presence of FeCl₂/H₂O₂. Lungs were either perfused with Krebs-Henseleit buffer containing 1 mM CPH only (control) or in the presence of FeCl₂ (1 μM, added at 0.5 h, FeCl₂/H₂O₂). After 1.5 h, H₂O₂ was added to the buffer fluid by continuous infusion (8 μmol/min) in the FeCl₂-perfused lungs. Bar indicates significant differences between the FeCl₂/H₂O₂ group and control.

Figure 3

Hypoxia-dependent superoxide release and vasoconstrictor responses in isolated perfused and ventilated rabbit lungs. During a total 3 h period of perfusion, the ventilator gas supply was switched to different oxygen concentrations every 30 min, using 1 %, 2.5 %, 5 %, 10 %, 16 % or 21 % O₂ in a randomized mode. (A) Superoxide release. The rate of increase in ESR signal intensity turned out to be linear during the last 20 min of each ventilation period. Therefore, changes in the ESR signal intensity/min during the last 20 min of each ventilation period are given. In the +SOD group, 150 mU/ml superoxide dismutase (SOD) was present throughout the experiments. * significant differences between the +SOD and the -SOD group for the respective oxygen concentration. ** significant differences as compared to 21 % O₂. (B) Vasoconstrictor response. The maximum increases in PAP (ΔPAP) are given for the different oxygen concentrations and are referenced to baseline (=normoxic) PAP values, which were 8.8 ± 0.6 mmHg

Figure 4

Superoxide release from isolated perfused rabbit lungs afteraddition of phorbol-12-myristate-13-acetate (PMA) during normoxic ventilation. (A) ESR signal intensity during normoxic ventilation of rabbit lungs in the presence (+SOD) and the absence of SOD (-SOD). After 3 h, PMA was injected into the pulmonary artery, resulting in a concentration of 1 μM in the recirculating buffer fluid. The increase in ESR signal intensity was linear before and after addition of PMA. The insert shows the PMA effect with higher time resolution. (B) Changes in the increase rate of the ESR signal intensity (%) by comparison of values prior to and after addition of PMA to isolated rabbit lungs. In the +SOD group, SOD was present throughout the experiments. In two separate sets of experiments, a fiber oxygenator was used instead of the lung for oxygenation of the recirculating buffer fluid ("fiber oxygenator"). The fibre oxygenator experiments were performed either in the absence ("fiber oxygenator") or in the presence of 1 μM FeCl₂ ("fiber oxygenator + FeCl₂") in the buffer fluid. * significant difference between the +SOD and the -SOD group

Figure 5

Effects of the NADPH oxidase and mitochondrial inhibitors, as well as of phagocytic NADPH oxidase gene deletion on the ESR signal intensity in isolated perfused lungs after addition of phorbol-12-myristate-13-acetate (PMA) during normoxic ventilation. (A) Effect of the NADPH oxidase inhibitor apocynin (500 μM) and the inhibitor of mitochondrial complex I, rotenone (350 nM) on the increase rate in ESR signal intensity after addition of PMA. Each inhibitor was added to the buffer fluid 30 min before addition of PMA. In the +SOD group, SOD was present throughout the experiments. * significant difference as compared to control. (B) Comparison of the increase rate in ESR signal intensity after addition of PMA in wildtype (WT) and gp91phox-deficient (gp91phox^-/-) mice. Lungs were perfused for 120 min prior to PMA addition (10 μM) either in the presence or absence of 150 U/ml SOD. * significant difference as compared to WT. Data are given as changes in the increase rate of the ESR signal intensity after PMA addition, as compared to the values before PMA addition (set as 100%). Data are from n = 4–5 experiments for each group.

Figure 6

Oxygen-dependency of PMA-induced changes in ESR signal intensity, lung superoxide release, and pulmonary artery pressure. Lungs were ventilated for 30 minutes with either 1 %, 2.5 %, 5 %, 10 %, 16 % or 21 % O₂, followed by injection of PMA into the pulmonary artery, resulting in a concentration of 1 μM in the recirculating buffer fluid. (A) Changes in the increase rate of the ESR signal intensity after PMA addition, as compared to the values before PMA addition (set as 100 %) are given. Experiments were performed in the presence (+SOD) or the absence (-SOD) of SOD. In the control group, lungs were replaced by a fiber oxygenator for equilibration of the buffer fluid with the different oxygen concentrations. The fibre oxygenator experiments were performed either in the absence or in the presence of 1 μM FeCl₂ in the buffer fluid. * significant differences between +SOD and -SOD groups. ** significant differences between different oxygen concentrations. (B) Increase in pulmonary artery pressure per minute (ΔPAP/min) within 7 min after PMA addition to the buffer fluid. In experiments without addition of PMA no significant change in PAP was observed. Data are from n = 4–9 experiments for each group.