Influence of pulmonary arterial endothelial cells on quinone redox status: effect of hyperoxia-induced NAD(P)H:quinone oxidoreductase 1

Abstract

The objective of this study was to examine the impact of chronic hyperoxic exposure (95% O2 for 48 h) on intact bovine pulmonary arterial endothelial cell redox metabolism of 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone, DQ). DQ or durohydroquinone (DQH2) was added to normoxic or hyperoxia-exposed cells in air-saturated medium, and the medium DQ concentrations were measured over 30 min. DQ disappeared from the medium when DQ was added and appeared in the medium when DQH2 was added, such that after approximately 15 min, a steady-state DQ concentration was approached that was approximately 4.5 times lower for the hyperoxia-exposed than the normoxic cells. The rate of DQ-mediated reduction of the cell membrane-impermeant redox indicator, potassium ferricyanide [Fe(CN)6(3-)], was also approximately twofold faster for the hyperoxia-exposed cells. Inhibitor studies and mathematical modeling suggested that in both normoxic and hyperoxia-exposed cells, NAD(P)H:quinone oxidoreductase 1 (NQO1) was the dominant DQ reductase and mitochondrial electron transport complex III the dominant DQH2 oxidase involved and that the difference between the net effects of the cells on DQ redox status could be attributed primarily to a twofold increase in the maximum NQO1-mediated DQ reduction rate in the hyperoxia-exposed cells. Accordingly, NQO1 protein and total activity were higher in hyperoxia-exposed than normoxic cell cytosolic fractions. One outcome for hyperoxia-exposed cells was enhanced protection from cell-mediated DQ redox cycling. This study demonstrates that exposure to chronic hyperoxia increases the capacity of pulmonary arterial endothelial cells to reduce DQ to DQH2 via a hyperoxia-induced increase in NQO1 protein and total activity.

Coenzyme Q1as a probe for mitochondrial complex I activity in the intact perfused hyperoxia-exposed wild-type andNqo1-null mouse lung

“…Thus the sensitivity of complex I activity in the present study is not that surprising, given previous reports of hyperoxic effects on various lung tissue and lung endothelial and epithelial cell mitochondrial functions, including complex I activity (4,7,11,14,41,46,53). We previously observed a hyperoxia-induced depression of complex I activity in the rat lung and bovine pulmonary arterial endothelial cells in culture (4,38). Functional or temporal relationships, if any, of the changes in complex I activity in hyperoxic lung injury on survival times in different mouse strains remain to be seen.…”

“…Hyperoxia was selected because it is a common model of acute lung injury and oxidative stress. Furthermore, our previous studies demonstrate that complex I activity and other mitochondrial functions are affected in rat lung exposed to hyperoxia (85% O 2 for 48 h) and in pulmonary arterial endothelial cells in culture (95% O 2 for 48 h) (4,27,38,39). We also evaluated the impact of the hyperoxic exposure on lung NQO1 activity because of the possibility that NQO1 induction could result in competition between NQO1 and complex I for CoQ 1 reduction.…”

Quantifying mitochondrial and plasma membrane potentials in intact pulmonary arterial endothelial cells based on extracellular disposition of rhodamine dyes

“…It should be emphasized that the effect of hyperoxic exposure on pulmonary endothelial mitochondria would not have been revealed by measuring only the resting ⌬ m or using only a single commonly used CCCP concentration (5 M) to depolarize the mitochondria. Various indications of mitochondrial dysfunction have been reported by us and others in hyperoxiaexposed rat lung, pulmonary endothelial cells in culture, and other cell types (2,3,5,7,8,36,37,51,57). In this context, the present observations are consistent with a hyperoxia-induced mitochondrial deficiency.…”

“…Bovine pulmonary arterial endothelial cells were isolated from segments of calf pulmonary artery obtained from a local slaughterhouse and cultured to confluence on gelatincoated (2% vol/wt) Biosilon (polystyrene) microcarrier beads (230 m mean diameter, 160 cm 2 /ml beads), as previously described (12,41). The control cells were grown in room air; hyperoxia-exposed cells were subjected to 95% O 2-5% CO2 for 48 h, as previously described (36,37).…”

Genetic evidence for NAD(P)H:quinone oxidoreductase 1-catalyzed quinone reduction on passage through the mouse pulmonary circulation

“…Earlier studies also revealed that DQ behaves as an NQO1 probe in intact bovine pulmonary endothelial cells in culture (12,27). In the cell culture studies, DQ was added to the cell culture medium surrounding the cells, and the appearance of DQH 2 in the medium was measured.…”