Coenzyme Q(1) as a probe for mitochondrial complex I activity in the intact perfused hyperoxia-exposed wild-type and Nqo1-null mouse lung

Abstract

Previous studies showed that coenzyme Q(1) (CoQ(1)) reduction on passage through the rat pulmonary circulation was catalyzed by NAD(P)H:quinone oxidoreductase 1 (NQO1) and mitochondrial complex I, but that NQO1 genotype was not a factor in CoQ(1) reduction on passage through the mouse lung. The aim of the present study was to evaluate the complex I contribution to CoQ(1) reduction in the isolated perfused wild-type (NQO1(+/+)) and Nqo1-null (NQO1(-)/(-)) mouse lung. CoQ(1) reduction was measured as the steady-state pulmonary venous CoQ(1) hydroquinone (CoQ(1)H(2)) efflux rate during infusion of CoQ(1) into the pulmonary arterial inflow. CoQ(1)H(2) efflux rates during infusion of 50 μM CoQ(1) were not significantly different for NQO1(+/+) and NQO1(-/-) lungs (0.80 ± 0.03 and 0.68 ± 0.07 μmol·min(-1)·g lung dry wt(-1), respectively, P > 0.05). The mitochondrial complex I inhibitor rotenone depressed CoQ(1)H(2) efflux rates for both genotypes (0.19 ± 0.08 and 0.08 ± 0.04 μmol·min(-1)·g lung dry wt(-1) for NQO1(+/+) and NQO1(-/-), respectively, P < 0.05). Exposure of mice to 100% O(2) for 48 h also depressed CoQ(1)H(2) efflux rates in NQO1(+/+) and NQO1(-/-) lungs (0.43 ± 0.03 and 0.11 ± 0.04 μmol·min(-1)·g lung dry wt(-1), respectively, P < 0.05 by ANOVA). The impact of rotenone or hyperoxia on CoQ(1) redox metabolism could not be attributed to effects on lung wet-to-dry weight ratios, perfusion pressures, perfused surface areas, or total venous effluent CoQ(1) recoveries, the latter measured by spectrophotometry or mass spectrometry. Complex I activity in mitochondria-enriched lung fractions was depressed in hyperoxia-exposed lungs for both genotypes. This study provides new evidence for the potential utility of CoQ(1) as a nondestructive indicator of the impact of pharmacological or pathological exposures on complex I activity in the intact perfused mouse lung.

Fig. 1.

Coenzyme Q₁ (CoQ₁) and CoQ₁ hydroquinone (CoQ₁H₂) venous effluent fractional concentration vs. time curves following CoQ₁ (50 μM) infusion into room air- or hyperoxia-exposed NQO1^+/+ mouse lungs. A and B: CoQ₁, CoQ₁H₂, and CoQ₁ + CoQ₁H₂ concentrations [expressed as fractions of infused pulmonary arterial CoQ₁ concentration (50 μM)] and FITC-dextran concentrations (expressed as fraction of infused FITC-dextran concentration) in NQO1^+/+ lung from animals exposed to room air or hyperoxia (100% O₂ for 48 h). C: CoQ₁ and FITC-dextran infusion into the perfusion system tubing without the lung in place. NQO1, NAD(P)H:quinone oxidoreductase 1; NQO1^−/−, Nqo1-null; NQO1^+/+, wild type.

Fig. 2.

Pulmonary venous CoQ₁H₂ efflux rates when CoQ₁ is infused into the pulmonary arterial inflow of NQO1^+/+ or NQO1^−/− lung and effects of rotenone in the lung perfusate or exposure of the mice to hyperoxia. A and B: CoQ₁H₂ efflux rates in NQO1^+/+ and NQO1^−/− lungs. C and D: CoQ₁ concentration ([CoQ₁]) in the perfusion reservoir (infusion concentration) for NQO1^+/+ and NQO1^−/− lungs. E and F: percentage of infused CoQ₁ that appears as [CoQ₁] + CoQ₁H₂ concentration ([CoQ₁H₂]) in the venous effluent for NQO1^+/+ and NQO1^−/− lungs. Values are means ± SE; n = 10 room air-exposed lungs, 4 room air-exposed lungs with rotenone added to the lung perfusate, and 6 hyperoxia-exposed lungs (A, C, and E) and 8 room air-exposed lungs, 4 room air-exposed lungs with rotenone added to the lung perfusate, and 5 hyperoxia-exposed lungs (B, D, and F). Significantly different (by ANOVA and Tukey's honestly significant difference test) within A or within B: *P < 0.05 vs. genotype-matched control room air-exposed lungs; †P < 0.05 vs. genotype-matched rotenone-treated lungs; ‡P < 0.05 vs. hyperoxia-exposed NQO1^+/+ lungs. There were no significant differences (ANOVA) between values within any group (C–F) or between groups (C and D or E and F).

Fig. 3.

Complex I (A) and complex IV (B) activities in mitochondria-enriched lung fractions. Values are means ± SE; n = 4 room air-exposed NQO1^+/+, 4 hyperoxia-exposed NQO1^+/+, 5 room air-exposed NQO1^−/−, and 4 hyperoxia-exposed NQO1^−/− lung mitochondria-enriched fractions. Significantly different (by ANOVA and Tukey's honestly significant difference test): *P < 0.05 vs. genotype-matched control (room air); †P < 0.05 vs. hyperoxia-exposed NQO1^+/+.

Fig. 4.

Effect of KCN on CoQ₁H₂ venous efflux rates vs. infused CoQ₁ concentrations for room air- and hyperoxia-exposed NQO1^+/+ and NQO1^−/− lungs. Values are means ± SE; n = 4 for all conditions in A and n = 4 for NQO1^+/+ and 3 for NQO1^−/− in B. Each value in B is significantly lower than that for the paired condition-CoQ₁ concentration in A. Significantly different (by ANOVA and Tukey's honestly significant difference test): *P < 0.05 vs. paired NQO1^+/+ value within the hyperoxia-exposed group, i.e., within B.

Fig. 5.

CoQ₁ efflux rates vs. infused CoQ₁H₂ concentrations for room air- and hyperoxia-exposed NQO1^−/− lungs. Values are means ± SE; n = 4 room air- and 4 hyperoxia-exposed lungs at each CoQ₁H₂ concentration. There were no significant differences between values for room air- and hyperoxia-exposed lungs at 50 or 200 μM CoQ₁H₂ (P > 0.05, by t-test). There were no statistically significant differences between any of the physiological parameters reported in Tables 2 and 3 (which pertain to the Fig. 2 study) for the animals or lungs in the Fig. 5 study and also no statistically significant differences in the recoveries of infused CoQ₁ as venous effluent CoQ₁ + CoQ₁H₂ in the Fig. 5 study (statistical analysis as described in Table 2 and 3 footnotes and Fig. 2 legend).

Fig. 6.

Pulmonary venous duroquinone (DQ) hydroquinone (DQH₂) efflux rates during DQ infusion into pulmonary arterial inflow of room air- and hyperoxia-exposed NQO1^+/+ mouse lungs, and lung cytosol fraction NQO1 and glucose-6-phosphate dehydrogenase (G-6-PDH) activities. A: steady-state pulmonary venous DQH₂ efflux rates during 50 μM DQ infusion into pulmonary arterial inflow. B: lung cytosol fraction NQO1 activities for lungs in A. DCIP, 2,6-dichlorophenolindophenol. C: lung cytosol fraction G-6-PDH activities for lungs in A. Values are means ± SE; n = 5 room air- and 6 hyperoxia-exposed lungs. There were no statistically significant differences (by t-test) between values within A, B, or C.