Investigation of Reactive Oxygen Species Production in the Rat Lung Using Optical Imaging

Oxidative stress, the imbalance between production of oxidants or reactive oxygen species (ROS) and antioxidant activity, plays a key role in the pathogenesis of acute and chronic lung diseases. The objective of this thesis was to develop a robust protocol for measuring the rate of H2O2 production in isolated perfused rat lungs and to determine the cellular sources of that rate using Amplex Red (AR). For a given lung, AR (25 mM) along with horseradish peroxidase (5 U/ml) and ascorbate oxidase (1U/ml) were added to a perfusate reservoir that was recirculated through the lungs and sampled at 5 minute intervals to measure the emission signal (454 nm/610 nm). Experiments were carried without and with the inhibitors rotenone (40 M), thenoyltrifluoroacetone (20 M), antimycin A (3.76 M), potassium cyanide (2 mM), or diohenylene iodonium (5M) added to the recirculating perfusate. In addition, we evaluated the effect of %O2 ventilation on H2O2 production. For lungs from control rats, the results show that inhibiting mitochondrial complex II reduced this rate by 76 ± 3%, and inhibiting NOX reduced it by another 23 ± 2%. The results also show that inhibiting complex I had a small (13% ± 4%), but significant effect on the rate, whereas inhibiting complex III had no significant effect on this rate. Inhibition of complex IV increased the rate or ROS production by 310% ± 43%. Furthermore, the results show that increasing % O2 in the ventilation gas mixture from 15% to 95% O2 had a relatively small (27± 3 %), but significant effect on this rate, and that this O2-dependent increase was mostly non-mitochondrial. The results of this study suggest complex II as a potentially important source of ROS and a potential target for mitigating oxidative stress, and that most of the hyperoxia-enhanced lung rate H2O2 release is from NAD(P)H oxidase rather than mitochondrial sources. To the best of our knowledge, this is the first study measuring the rate of H2O2 release from isolated perfused rat lungs, identifying the main sources of this rate under physiological conditions, and evaluating the effect of acute hyperoxia on this rate

Detection of Hydrogen Peroxide Production in the Isolated Rat Lung Using Amplex Red

The objectives of this study were to develop a robust protocol to measure the rate of hydrogen peroxide (H2O2) production in isolated perfused rat lungs, as an index of oxidative stress, and to determine the cellular sources of the measured H2O2 using the extracellular probe Amplex red (AR). AR was added to the recirculating perfusate in an isolated perfused rat lung. AR’s highly fluorescent oxidation product resorufin was measured in the perfusate. Experiments were carried out without and with rotenone (complex I inhibitor), thenoyltrifluoroacetone (complex II inhibitor), antimycin A (complex III inhibitor), potassium cyanide (complex IV inhibitor), or diohenylene iodonium (inhibitor of flavin-containing enzymes, e.g. NAD(P)H oxidase or NOX) added to the perfusate. We also evaluated the effect of acute changes in oxygen (O2) concentration of ventilation gas on lung rate of H2O2 release into the perfusate. Baseline lung rate of H2O2 release was 8.45 ± 0.31 (SEM) nmol/min/g dry wt. Inhibiting mitochondrial complex II reduced this rate by 76%, and inhibiting flavin-containing enzymes reduced it by another 23%. Inhibiting complex I had a small (13%) effect on the rate, whereas inhibiting complex III had no effect. Inhibiting complex IV increased this rate by 310%. Increasing %O2 in the ventilation gas mixture from 15 to 95% had a small (27%) effect on this rate, and this O2-dependent increase was mostly nonmitochondrial. Results suggest complex II as a potentially important source and/or regulator of mitochondrial H2O2, and that most of acute hyperoxia-enhanced lung rate of H2O2 release is from nonmitochondrial rather than mitochondrial sources