Effect of chronic hyperoxic exposure on duroquinone reduction in adult rat lungs

Said H. Audi,1,3 Robert D. Bongard,3 Gary S. Krenz,2 David A. Rickaby,3,6 Steven T. Haworth,3 Jessica Eisenhauer,3 David L. Roerig,4,5,6 and Marilyn P. Merker4,5,6

Departments of 1Biomedical Engineering, 2Mathematics, Statistics, and Computer Science, Marquette University; Departments of 3Pulmonary and Critical Care Medicine, 4Anesthesiology, and 5Pharmacology/Toxicology, Medical College of Wisconsin, and 6Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin

Submitted 8 February 2005 ; accepted in final form 23 June 2005


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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NAD(P)H:quinone oxidoreductase 1 (NQO1) plays a dominant role in the reduction of the quinone compound 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone, DQ) to durohydroquinone (DQH2) on passage through the rat lung. Exposure of adult rats to 85% O2 for ≥7 days stimulates adaptation to the otherwise lethal effects of >95% O2. The objective of this study was to examine whether exposure of adult rats to hyperoxia affected lung NQO1 activity as measured by the rate of DQ reduction on passage through the lung. We measured DQH2 appearance in the venous effluent during DQ infusion at different concentrations into the pulmonary artery of isolated perfused lungs from rats exposed to room air or to 85% O2. We also evaluated the effect of hyperoxia on vascular transit time distribution and measured NQO1 activity and protein in lung homogenate. The results demonstrate that exposure to 85% O2 for 21 days increases lung capacity to reduce DQ to DQH2 and that NQO1 is the dominant DQ reductase in normoxic and hyperoxic lungs. Kinetic analysis revealed that 21-day hyperoxia exposure increased the maximum rate of pulmonary DQ reduction, Vmax, and the apparent Michaelis-Menten constant for DQ reduction, Kma. The increase in Vmax suggests a hyperoxia-induced increase in NQO1 activity of lung cells accessible to DQ from the vascular region, consistent qualitatively but not quantitatively with an increase in lung homogenate NQO1 activity in 21-day hyperoxic lungs. The increase in Kma could be accounted for by ~40% increase in vascular transit time heterogeneity in 21-day hyperoxic lungs.

mathematical modeling; NAD(P)H:quinone oxidoreductase; mitochondrial electron transport; pulmonary endothelium; oxidative stress


PULMONARY OXIDATIVE STRESS has been implicated in the pathogenesis of many acute and chronic pulmonary inflammatory diseases (12, 15, 16, 20). Several animal models of hyperoxic lung injury have been developed to investigate the underlying mechanisms of pulmonary oxygen toxicity and the lung response to oxidative stress (1215, 20, 26, 32, 33). Exposure of an adult rat to >95% O2 results in lung injury within 48–60 h and death within 72 h (14, 15). Adult rats exposed to 85% O2 also develop lung injury within 48–72 h, but unlike other species, including mice, guinea pigs, and hamsters, they survive the initial lung injury, and after 7 or more days of exposure to 85% O2, they develop tolerance or adaptation to otherwise lethal hyperoxia (>95% O2) (14, 15). Although the activities of the so-called classic antioxidant enzymes such as superoxide dismutase and catalase are increased in rats adapted to lethal hyperoxia (14, 15, 21), they do not appear to account for all aspects of this adaptive response (13, 20, 32).

Another proposed mechanism involved in rat adaptation to lethal hyperoxia is the induction of phase II detoxification enzymes including NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione-S-transferase, and heme oxygenase-1 (12, 26). The induction occurs via the antioxidant response element mediated by enhanced production of reactive oxygen species (12, 26). Phase II enzymes are, in general, involved in detoxification of reactive electrophilic metabolites including organic peroxides, lipid peroxides, and quinones via conjugation reactions or two-electron reduction that enhance their excretion (9, 12, 17). With respect to NQO1, it has been suggested that additional protective effects include regeneration of endogenous and exogenous antioxidants (8, 17, 31), competition with one-electron quinone reductases, thereby ameliorating the effects of semiquinone formation and subsequent redox cycling (9, 33), and scavenging superoxide (30).

The model quinone, duroquinone (DQ), is one of several redox active compounds that we have used to probe pulmonary endothelial surface and intracellular redox enzymes in the intact lung using indicator dilution methods (1, 2, 4, 6). We have demonstrated that NQO1 plays a dominant role in DQ reduction to its two-electron reduction product durohydroquinone (DQH2) on a single pass through the rat pulmonary circulation (1). The present study examines whether exposure of adult rats to hyperoxia (85% O2 for 48 h or 21 days) affects NQO1 activity in the intact rat lung as measured by the rate of DQ reduction on passage through the lungs. The main approach we took was to measure the rate of appearance of DQH2 in the venous effluent during DQ infusion at different concentrations into the pulmonary artery of isolated perfused lungs from normal rats and from rats that had been exposed to 85% O2 for 48 h or 21 days. We also evaluated the effect of rat exposure to hyperoxia on vascular transit time heterogeneity and measured NQO1 protein and activity in lung homogenate. The results demonstrate increased DQ reduction capacity in lungs of rats exposed to hyperoxia for 21 days and provide insight into the effects of hyperoxia-induced increase in pulmonary vascular transit time heterogeneity on steady-state Michaelis-Menten kinetics.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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Materials

DQ was purchased from Sigma Chemical (St. Louis, MO). Bovine serum albumin (BSA, standard powder) was purchased from Serologicals (Gaithersburg, MD). DQH2 was prepared by reduction of DQ with potassium borohydride (KBH4) as previously described (10). Other chemicals were purchased from Sigma Chemical and were of reagent grade.

Hyperoxic Exposure

Adult Sprague-Dawley rats (Charles River; 250–350 g) were housed in a sealed Plexiglas chamber (13-inch width x 23-inch length x 12-inch height) with ~85% O2 balance N2 for 48 h [final body wt, 326 ± 5 (SE) g, n = 10] or 21 days (final body wt, 290 ± 8 g, n = 14). The total gas flow of ~3.5 l/min was high enough to maintain the chamber CO2 at <0.5%. The animals were maintained on a 12-h light-dark cycle with free access to food and water, which along with bedding, were changed every other day. Control (normoxic) animals (final body wt, 324 ± 6 g, n = 21) were exposed to room air. The protocol was approved by the Institutional Animal Care and Use Committees of the Veterans Affairs Medical Center and Marquette University (Milwaukee, WI).

The 48-h exposure period was chosen to evaluate hyperoxia-induced changes in lung DQ reduction rate and lung NQO1 protein and activity before the development of tolerance to lethal hyperoxia (>95% O2) (14, 15). The 21-day exposure period was chosen because it is long enough to stimulate adaptation to >95% O2 (14, 15) and to reverse the body weight loss that occurs during the first 2 wk of exposure to 85% O2 (14). Lungs of control rats will be referred to as normoxic lungs, and lungs of rats exposed to hyperoxic gas mixture for 48 h or 21 days as 48-h hyperoxic lungs and 21-day hyperoxic lungs, respectively.

Isolated Perfused Lung Experiments

The isolated perfused rat lung preparation has been described previously (1). Briefly, each rat was anesthetized with pentobarbital sodium (40 mg/kg body wt ip), after which the trachea was clamped and the chest opened. Heparin (0.7 IU/g body wt) was injected into the right ventricle. The pulmonary artery and the trachea were cannulated, and the pulmonary venous outflow was accessed via a cannula in the left atrium. The lungs were removed from the chest and attached to a ventilation and perfusion system. The perfusate (referred to as control perfusate) was a physiological salt solution containing (in mM) 4.7 KCl, 2.51 CaCl2, 1.19 MgSO4, 2.5 KH2PO4, 118 NaCl, 25 NaHCO3, 5.5 glucose, and 5% BSA (1).

The single pass perfusion system was primed with control perfusate maintained at 37°C and equilibrated with 15% O2, 6% CO2, balance N2 resulting in perfusate PO2, PCO2, and pH of ~105 Torr, 40 Torr, and 7.4, respectively. Initially, perfusate was pumped (Master Flex roller pump) through the lungs until the lungs were evenly blanched and venous effluent was visually clear of blood. The flow rate was then set at 10 ml/min. The lungs were ventilated (40 breaths/min) with end inspiratory and expiratory pressures of 6 and 3 mmHg, respectively, with the above gas mixture. The pulmonary arterial pressure was referenced to atmospheric pressure at the level of the left atrium and monitored continuously during the course of the experiments. The venous effluent pressure was atmospheric pressure. At the end of each experiment, the lungs were weighed and then dried (60°C) to a constant weight to determine lung dry weight and wet/dry weight ratio. For some lungs, half was homogenized for in vitro determination of NQO1 protein and activity as described below, and the other half was used to determine lung dry weight and wet/dry weight ratio.

To measure the fate of DQ on passage through the lungs, pulse infusion and bolus injection experiments were carried out (1). The pulse infusion experiments provide data in which information about the steady-state aspects of DQ and DQH2 disposition on passage through the lungs is emphasized, whereas the bolus experiments provide data in which information about the transient aspects of DQ and DQH2 disposition is emphasized.

Experimental Protocols

Capacity of lungs for DQ reduction. To determine the DQ reducing capacity of normoxic and hyperoxic lungs, four 60- to 90-s sequential pulse infusions at DQ concentrations of 50, 100, 200, and 400 µM were carried out on each lung. The pump flow rate was set at 10 ml/min. For each pulse infusion, venous effluent samples (~0.5 ml) were collected at 10-s intervals during infusion. Between pulse infusions, the lungs were perfused with ~20 ml of fresh perfusate to wash the perfusion system and the lungs of any remaining traces of DQ and/or DQH2 from the previous pulse. Multiple DQ pulses and high DQ concentrations did not have a significant effect on DQ reduction in subsequent pulses (1).

Inhibitor treatments. To determine the contribution of NQO1 to DQ reduction in normoxic and hyperoxic lungs, DQ pulse infusions were carried out before and after lung treatment with the NQO1 inhibitor dicumarol (25). For each lung, a 90-s pulse infusion of 400 µM DQ was performed, and venous samples were collected as before. This was followed by perfusing the lung with perfusate containing dicumarol (400 µM) for ~5 min. After treatment with dicumarol, another 90-s pulse infusion of DQ (400 µM) plus dicumarol (400 µM) was performed, and venous samples were collected as before. The above concentration of dicumarol was needed to inhibit lung NQO1 activity because dicumarol has a high affinity for plasma proteins (34).

To determine the contribution of DQH2 oxidation to the net effect of the lung on DQ, the above pulse infusion protocol used to determine capacity of lungs for DQ reduction, was repeated in lungs pretreated with cyanide (2 mM) (1). This was accomplished by recirculating perfusate containing cyanide (2 mM) through the lungs for 5 min, a dose and time previously determined to be sufficient to inhibit DQH2 oxidation to DQ on passage through the lungs (1).

Bolus injections. Bolus injection experiments were carried out to evaluate hyperoxia-induced changes in lung tissue volume accessible to DQH2 from the vascular space, lung vascular volume, and vascular perfusion heterogeneity. An injection loop was included in the arterial line to allow introduction of a 0.1-ml bolus into the arterial inflow without altering the flow or perfusion pressure (1). For each lung, perfusate containing cyanide (2 mM) was recirculated for 5 min just before bolus injection. The pump flow rate was set at 10 ml/min. The respirator was stopped at end expiration, and a bolus of the perfusate solution containing either 400 µM DQH2 or 35 µM of the vascular reference indicator fluorescein isothiocyanate dextran (FITC-dex; average mol wt ~43,000) was injected. At the same time that the bolus was introduced into the arterial inflow, the venous outflow was diverted into a modified Gilson Escargot fraction collector for continuous collection of the lung effluent (1). Thirty-five venous effluent samples (0.3 ml each) were collected at 1.8-s intervals. At the end of each experiment, the lungs were removed from the perfusion system, the arterial and venous cannulas were connected directly together, and an additional FITC-dex bolus injection was made. These data were used to obtain the tubing transit time and bolus dispersion needed for calculation of the lung vascular volume and the relative dispersion of vascular transit time distribution (see Data Analysis).

Perfused capillary surface area. To evaluate hyperoxia-induced changes in pulmonary endothelial surface area, the rate of hydrolysis of the peptide N-[3-(2-furyl)acryloyl]-Phe-Gly-Gly (FAPGG), an angiotensin converting enzyme (ACE) substrate, on a single pass through the pulmonary circulation was measured (27). To establish the linearity of the steady-state rate of FAPGG hydrolysis as a function of FAPGG concentration, three pulse infusions with sequentially increasing FAPGG concentrations (100, 200, and 300 µM) were carried out in a normoxic lung. During each infusion, the flow rate was set at 30 ml/min, and venous effluent samples were collected 20 s after the start of the infusion period, which is long enough for the venous FAPGG concentration to reach steady state. For all other normoxic and hyperoxic lungs studied, two 150-µM FAPGG pulse infusions were carried out at the beginning and at the end of each of the above experimental protocols, and venous samples were collected as before. This concentration was determined to fall within the linear range for the steady-state rate of FAPGG hydrolysis on passage through the lungs (see RESULTS). A permeability-surface area product (PS; ml/min), as a measure of the rate constant of ACE-mediated FAPGG hydrolysis on passage through the lungs and an index of perfused capillary surface area, was calculated from the infused arterial FAPGG concentration ([FAPGG]i) and steady state venous effluent FAPGG concentration ([FAPGG]o) using

(1)

(2)
and F is the perfusate flow rate (30 ml/min).

Determination of DQ, DQH2, and FITC-Dex Concentrations in Venous Effluent Samples

The concentrations (in µM) of DQ and DQH2 in the venous effluent samples were determined as previously described (1). Briefly, the venous effluent samples were centrifuged (1.0 min at 5,600 g), after which for each sample 100 µl of the supernatant was pipetted into each of two microcentrifuge tubes, one containing 10 µl of deionized water, and the other containing 10 µl of potassium ferricyanide (1.8–7.2 mM) to oxidize any DQH2 contained in the sample to DQ. Ice-cold absolute ethanol (0.4–0.8 ml) was added to each tube, and the tubes were centrifuged at 5,600 g for 5 min at 4°C. DQ and DQH2 concentrations (in µM) were measured spectrophotometrically at 265 nm using molar extinction coefficients of 21,640 M–1cm–1 for DQ and 1,700 M–1cm–1 for DQH2. FITC-dex concentrations in the venous effluent samples were measured spectrophotometrically at 495 nm using a molar extinction coefficient of 93,478 M–1cm–1. In addition, for the bolus experiments, measured volumes of the injected bolus were added to samples that emerged in the venous effluent before the appearance of the bolus contents. These samples were used as standards for determining the fraction of bolus contents per milliliter of venous effluent sample.

Lung Homogenate NQO1 Activity and NQO1 Immunoblots

At the end of the DQ reduction studies that did not involve using metabolic inhibitors, the lungs were removed from the perfusion system, weighed, and placed on ice. Ice-cold homogenization buffer (5 ml of buffer/g of lung tissue, pH 7.4) containing (in mM) 10 HEPES, 250 sucrose, 3 EDTA, 1 phenylmethylsulfonyl fluoride, and 1% protease inhibitor cocktail (Sigma, cat. no. P8340) was added. The lungs were minced and homogenized on ice using a Polytron tissue homogenizer. The resulting lung homogenate was centrifuged (12,100 g) at 4°C for 30 min. The supernatant was collected and stored on ice, and the protein concentration was determined (Bio-Rad Laboratories, Hercules, CA) using BSA as the standard.

Whole lung NQO1 activity was measured using a modified procedure of Lind et al. (22). Lung homogenate supernatant protein (~10 µg) was added to a semimicro cuvette containing 1 ml of reaction buffer that consisted of Tris·HCl (25 mM, pH 7.4), BSA (0.23 mg/ml), Tween 20 (0.01% vol/vol), 2,6-dichlorophenolindophenol (DCIP, 50 µM), and flavin adenine dinucleotide (5 µM) with and without dicumarol (20 µM). The reaction was initiated by the addition of NADPH (final concentration 200 µM). DCIP reduction (extinction coefficient 21,000 M–1cm–1) was measured spectrophotometrically at 600 nm (25°C). The NQO1 activity was calculated as the difference in the initial rates of DCIP reduction in the absence and presence of dicumarol (20 µM) (23, 33).

To determine the effect of hyperoxic exposure on lung NQO1 protein expression, immunoblot analysis was carried out as previously described (1). A portion of the lung homogenate supernatant (37.5 µg of protein) and purified recombinant human NQO1 protein (0.25 ng) were subjected to electrophoresis using NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA), 4–12% gradient NuPAGE Bis-Tris gel, and MES-SDS running buffer, as previously described (1, 24). The proteins were transferred to a nitrocellulose membrane that was incubated for 1 h in Tris-buffered saline containing 0.1% Tween 20 and 2% BSA, the latter as a blocking agent. The membrane was then incubated sequentially in a 1:10 dilution of tissue culture supernatant containing anti-NQO1 monoclonal antibodies (IgG1) from two mouse hybridoma cell lines (A180 and B771, mixed 1:1; a gift of Dr. David Siegel, Univ. of Colorado Heath Sciences Center, Denver, CO), a 1:7,500 dilution of goat {alpha}-mouse IgG-horseradish peroxidase (Jackson ImmunoResearch Laboratories), and the Supersignal West Pico Chemiluminescent substrate (Pierce). The signal was captured on CL-Xposure Film (Pierce). As a control, nonspecific mouse IgG1 was substituted for the {alpha}-NQO1 monoclonal antibodies. Densitometry was used to quantify band intensities.

Statistical Evaluation of Data

Statistical comparisons among the various experimental conditions were obtained by ANOVA followed by Dunnett's test. P < 0.05 was considered statistically significant.


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Table 1 shows perfusion pressures, wet weights, dry weights, and wet/dry weight ratios of the lungs studied. Exposure to hyperoxia for 21 days increased lung perfusion pressure and wet and dry weights, with small (<9%) but significant increase in wet/dry weight ratios compared with normoxic lungs, indicating a slight edema in hyperoxic lungs. Exposure to hyperoxia for 48 h had no significant effect on any of the above parameters or on any of the parameters described below. Thus the results presented below are limited to comparisons between normoxic lungs and 21-day hyperoxic lungs.


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Table 1. Lung weights and perfusion pressures

 
Lung's DQ Reducing Capacity

Figure 1A shows the effluent concentrations of DQ and DQH2 (as fractions of the total effluent [DQ] + [DQH2]) vs. sampling time obtained during a pulse infusion of 100 µM DQ in a normoxic lung perfused with control perfusate. The venous effluent [DQ] and [DQH2] reached a steady state by ~50 s. Figure 1B shows the fractional steady-state [DQ] and [DQH2] in the venous effluent of a normoxic lung during DQ infusion, expressed as the average of their respective concentrations in samples collected 60 s after the start of DQ infusion. As the infused [DQ] increased, the fractional steady-state [DQ] increased, whereas that of [DQH2] decreased.



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Fig. 1. A: example of the time course for the venous effluent concentrations (as fractions of the respective total effluent [DQ] + [DQH2]) of duroquinone (DQ) and durohydroquinone (DQH2) during a pulse infusion of 100 µM DQ into the pulmonary artery of a normoxic lung perfused with control perfusate at 10 ml/min. B: steady-state venous effluent [DQ] and [DQH2] during DQ infusion at 50, 100, 200, or 400 µM into the pulmonary artery of a normoxic lung perfused with control perfusate at 10 ml/min. Steady-state venous effluent [DQ] and [DQH2] were calculated as the average of their concentrations in samples collected after 60 s of DQ infusion.

 
Table 2 summarizes the DQ pulse infusion steady-state data obtained using control perfusate. The 21-day hyperoxic lungs had a higher steady-state venous effluent [DQH2] than normoxic lungs during the infusion of DQ at 200 and 400 µM.


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Table 2. Steady-state effluent [DQH2] during DQ infusion in lungs perfused with control perfusate or perfusate containing dicumarol

 
Inhibitor Treatments

To evaluate the contribution of NQO1 to DQ reduction, we examined the effect of dicumarol, an NQO1 inhibitor, on DQ reduction (1, 25). Table 2 shows that dicumarol almost completely (>96%) inhibited reduction of 400 µM DQ pulse infusion in normoxic and hyperoxic lungs.

Table 3 summarizes the steady-state venous effluent [DQH2] measured during DQ infusion in normoxic and hyperoxic lungs in the presence of cyanide (2 mM), which blocks DQH2 oxidation to DQ (1). Compared with studies in control perfusate (Table 2), the steady-state venous effluent [DQH2] increased at all four DQ concentrations studied in normoxic lungs and 21-day hyperoxic lungs. Table 3 also shows that cyanide increased steady-state venous effluent [DQH2] during infusion of 400 µM DQ in 21-day hyperoxic lungs compared with normoxic lungs.


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Table 3. Steady-state effluent [DQH2] during DQ infusion in lungs perfused with control perfusate containing 2 mM cyanide

 
The difference in the steady-state venous effluent [DQH2] during DQ infusion with and without cyanide ({Delta}[DQH2]) is proportional to DQH2 oxidation rate. Because DQ pulse infusions with and without cyanide were not carried out in the same lungs, {Delta}[DQH2] was approximated as the difference between the steady-state venous effluent [DQH2] during DQ infusion in the absence of cyanide and the corresponding mean steady-state venous effluent [DQH2] values in the presence of cyanide (Table 3). Figure 2 shows a linear relationship between {Delta}[DQH2] and the infused [DQ] and that this relationship (as measured by the slope of {Delta}[DQH2] vs. infused [DQ]) was not significantly affected by exposure to hyperoxia.



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Fig. 2. Differences in the steady-state venous effluent [DQH2] during DQ infusion in the presence and absence of cyanide, {Delta}[DQH2], are plotted vs. infused [DQ]. Values are means ± SE, n = 7, 4, and 5 for normoxic lungs, 48-h hyperoxic lungs, and 21-day hyperoxic lungs, respectively. Solid lines are linear regression fit to the data.

 
Bolus Injections

Figure 3 shows examples of venous effluent concentrations of FITC-dex, DQ, and DQH2 after bolus injections of DQH2 and FITC-dex in a normoxic lung (Fig. 3A) and a 21-day hyperoxic lung (Fig. 3B). The lungs were treated with cyanide (2 mM) before the bolus injection to inhibit DQH2 oxidation to DQ. Little or no DQ was detected in the venous effluent following the bolus injection of DQH2, consistent with cyanide inhibition of tissue-mediated DQH2 oxidation and the reported slow DQH2 autooxidation rate (1). The FITC-dex concentration vs. time curve indicates what the effluent DQH2 curve would have been had the DQH2 not interacted with the lung as it passed through the pulmonary vessels. The DQH2 curves for normoxic lung and 21-day hyperoxic lung (Fig. 3) are shifted to the right and more dispersed than the FITC-dex curves, consistent with a flow-limited distribution of DQH2 into the tissue (1).



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Fig. 3. Venous effluent concentration (as a fraction of injected amount per milliliter of effluent perfusate) vs. time curves for FITC-dextran, DQ, and DQH2 following the bolus injection of FITC-dextran and DQH2 into the pulmonary artery of a normoxic lung (A) and a 21-day hyperoxic lung (B). The lungs had been treated with cyanide (2 mM) before the bolus injection to inhibit DQH2 oxidation to DQ.

 
Perfused Capillary Surface Area

The steady-state rate of FAPGG hydrolysis in a normoxic lung perfused with control perfusate was linear over a wide range of FAPGG concentrations that included the concentration (150 µM) used in the present study to calculate a PS for ACE-mediated FAPGG hydrolysis. Table 4 shows that exposure to hyperoxia for 21 days decreased PS (ml/min) on average by >70% compared with normoxic lungs. The PS values obtained from FAPGG pulse infusions carried out at the beginning or at the end of a given experimental protocol were not significantly different, indicating that perfusion, multiple DQ pulses, and/or metabolic treatments did not have significant irreversible effects on lung capillary endothelial surface area and/or ACE activity.


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Table 4. Lung angiotensin converting enzyme activity, vascular volumes, and DQH2 extravascular volumes and perfusion heterogeneity

 
Effect of Hyperoxia on NQO1 Activity and Protein

Table 6 shows that exposure to hyperoxia for 21 days significantly increased NQO1 activity in lung homogenate by ~34% compared with normoxic lungs on a per milligram protein basis.


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Table 6. Normalized values of DQ maximum reduction rate, lung homogenate NQO1 activity

 
Figure 4 shows NQO1 immunoblots of normoxic and hyperoxic lung homogenates. Band intensities obtained from 21-day hyperoxic lung homogenates were, on average, more than threefold greater than those from normoxic lung homogenates.



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Fig. 4. NAD(P)H:quinone oxidoreductase 1 (NQO1) immunoblots in lung tissue from a normoxic (A) rat and from rats that had been exposed to hyperoxia for 48 h (B) or 21 days (C). Human recombinant NQO1 (0.25 ng of protein) was used as a standard (D). There were no detectable corresponding bands in the same region of the blots when control, nonspecific IgG1 was used in place of {alpha}-NQO1 antibody. The NQO1 immunoblots shown were chosen for illustrative purposes since their band densities are close to their respective mean values. Band intensities were 2.0 ± 0.3 SE (n = 9) for normoxic lungs, 2.6 ± 0.4 (n = 9) for 48-h hyperoxic lungs, and 6.1 ± 1.4 (n = 5) for 21-day hyperoxic lungs.

 
Data Analysis

Kinetic model. To interpret the experimental results, a kinetic model previously described (1) was modified to describe the fate of DQ on passage through the lungs. The model consists of a capillary volume (Vc) and its surrounding tissue volume (Ve). The free forms (i.e., not albumin bound) of both DQ and DQH2 are assumed to be freely permeable into Ve (1). Within Vc, the model allows for nonspecific rapidly equilibrating interactions of DQ and DQH2 with the perfusate albumin (Pc) (Eq. 3). Within Ve, the model allows for two-electron DQ reduction to DQH2 (Eq. 4), cyanide-sensitive DQH2 oxidation to DQ (Eq. 5), and nonspecific rapidly equilibrating interactions of DQ and DQH2 with lung tissue sites of association (Pe) (Eq. 6). The reduction of DQ to DQH2 is assumed to follow Michaelis-Menten kinetics, where Vmax and Km represent the maximum reduction rate and the Michaelis-Menten constant, respectively. All other reactions are assumed to follow the law of mass action and to proceed with a rate constant ki in the forward direction and, if reversible within the time course of the study, with a rate constant k–i in the reverse direction. Each of the stoichiometric equations may potentially represent multiple parallel and/or series processes that are not explicitly specified. For example, intracellular reduction might occur via parallel enzymes, with different Vmax and Km for DQ, contributing to the total quinone reductase activity.

In perfusate

(3)

In tissue

(4)

(5)

(6)
where EH and E+ are the reduced and oxidized forms, respectively, of intracellular electron donor(s), and the kis represent the reaction rate constants.

The above stoichiometric relationships are expressed as species balance equations

(7)

(8)

(9)
where W = convective transport velocity = L/c; x = 0 and x = L are the capillary inlet and outlet, respectively; c is the capillary mean vascular transit time; [R](x, t) is the concentration of the vascular reference indicator (FITC-dex) at distance x from the capillary inlet and time t; [DQ](x, t) and [DQH2](x, t) are vascular concentrations of free DQ and DQH2 forms, respectively, [] at distance x from the capillary inlet and time t; [] = ([1) and [] = ([DQH2]{alpha}3) are the total (free + protein bound) vascular concentrations of DQ and DQH2, respectively; {alpha}1 = 1 + [Pc] k1/k–1 and {alpha}3 = 1 + [Pc] k2/k–2 are constants that account for the rapidly equilibrating interactions of DQ and DQH2 with perfusate BSA. Kma = {alpha}1Km (µM) is the apparent Michaelis-Menten constant; VF1 = {alpha}2Ve and VF2 = {alpha}4Ve (ml) are the virtual volumes of distribution, where {alpha}2 = 1 + [Pe] k4/k–4 and {alpha}4 = 1 + [Pe] k5/k–5 are constants that account for the rapidly equilibrating interactions of DQ and DQH2 with lung tissue sites of association, respectively; ko = Ve k3[O2]1/2 (ml·min–1) is the tissue-mediated DQH2 oxidation rate constant. To put ko in perspective, if one were to assume Michaelis-Menten kinetics for DQH2 oxidation, ko would be an approximation to the ratio of the maximum oxidation rate over the Michaelis-Menten constant. The [H+], [O2], and [EH] included in some parameter groups are assumed constant during a given sample collection period (1).

For the steady state during the pulse infusion of DQ or DQH2, Eqs. 8 and 9 reduce to

(10)

(11)
Given {alpha}1 and {alpha}3 calculated from the fractions of DQ and DQH2 bound to BSA obtained by ultrafiltration (1), the identifiable model parameters under steady-state conditions are the maximum reduction rate constant Vmax (µmol·min–1); the apparent Michaelis-Menten constant Kma (µM); and the tissue-mediated DQH2 oxidation rate constant ko (ml·min–1).

Under conditions of no DQH2 oxidation to DQ (i.e., ko = 0), Eqs. 10 and 11 simplify to the following uncoupled ordinary differential equations

(12)

(13)
Integrating both sides of Eq. 12 from x = 0 to x = L results in Eq. 14, which relates the rate of DQ reduction, {nu} (µmol·min–1), to the steady-state venous effluent [DQ] and [DQH2], Vmax, and Kma.

(14)
where {nu} is calculated as the product of the effluent steady-state [DQH2] during DQ infusion times perfusate flow rate.

(15)
where [DQ]in is effluent steady-state ([DQ] + [DQH2]) and [DQ]out is effluent steady-state [DQ] during DQ infusion. []log, referred to as the log mean [DQ], is the effective [DQ] in the capillary region during DQ infusion since [DQ] decreases as it passes through the capillary region (1, 7). Thus the values of Vmax and Kma can be estimated by fitting Eq. 14 to {nu} as a function of []log.

Effect of Hyperoxia on DQ Reduction Rate

In the presence of cyanide, DQ reduction rate, {nu}, is equal to the steady-state rate of DQH2 efflux during DQ infusion calculated as the product of the effluent steady-state [DQH2] (Table 3) and perfusate flow rate. Figure 5 shows {nu} as a function of []log for normoxic and hyperoxic lungs. The values of Vmax (µmol/min) and Kma (µM) were determined by fitting Eq. 14 to the data shown in Fig. 5. Table 5 shows that exposure to hyperoxia for 21 days significantly increased Vmax by ~107% and Kma by ~240% compared with normoxic lungs.



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Fig. 5. The relationship between the rate of DQ reduction and the log mean [DQ] during DQ infusion in the presence of cyanide (2 mM) for normoxic and hyperoxic lungs. Values are means ± SE, n = 4, 3, and 4 for normoxic lungs, 48-h hyperoxic lungs, and 21-day hyperoxic lungs, respectively. Solid lines are fits of Eq. 14 to the data.

 

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Table 5. Kinetic parameters descriptive of DQ reduction to DQH2 on passage through the lungs

 
Effects of Hyperoxia on DQH2 Extravascular Volume, Vascular Volume, and Vascular Perfusion Heterogeneity

The extravascular volume of distribution accessible to DQH2 (QD; ml), the vascular volume (QV; ml), and the relative dispersion of the vascular transit time distribution (RDV) werecalculated from the data exemplified by Fig. 3 and FITC-dex tubing data (not shown) using the following equations

(16)

(17)

(18)
where F is the perfusate flow rate (10 ml/min); tR, tD, and tT are the respective mean transit times from bolus injection site to sample collection site for FITC-dex with the lung in place, (DQ + DQH2) with the lung in place, and for FITC-dex with the lung removed; and are the variances of the concentration vs. times FITC-dex outflow curves with and without the lungs connected to the perfusion system, respectively. The different mean transit times and variances were estimated as previously described (3, 5). The QD is a virtual volume that is equal to the product of a physical tissue volume and a tissue-to-perfusate partition coefficient (1). Table 4 shows that exposure to hyperoxia for 21 days decreased QV by >30% and increased RDV by >40% compared with normoxic lungs.


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The results demonstrate that exposure to 85% O2 for 21 days, but not for 48 h, increases lung capacity to reduce DQ to DQH2. This increase is predominantly due to an increase in the rate of DQ reduction to DQH2, with no significant effect on DQH2 oxidation. The results also suggest that NQO1 is the dominant DQ reductase since DQ reduction was dicumarol inhibitable in normoxic and hyperoxic lungs. The increase in wet weights and wet/dry weight ratios of 21-day hyperoxic lungs had no significant effect on the extravascular volume accessible to DQH2. As discussed next, these results are consistent with an increase in NQO1 activity in cells accessible to DQ from the vascular region in 21-day hyperoxic lungs.

Interpretation of the Increase in Vmax, the Maximum Rate of DQ Reduction to DQH2, in 21-Day Hyperoxic Lungs

In addition to increasing Vmax, exposure of rats to hyperoxia for 21 days had increased lung dry weights (Table 1), consistent with an increase in their lung cellular content, presumably due to a large increase in the number of interstitial cells (14). This brought into question whether the increase in Vmax was simply the result of an increase in tissue mass (i.e., more cells) or an induction of NQO1 activity. As shown in Table 6, differences in the values of Vmax can be accounted for by differences in lung dry weights (Table 1). However, normalization to lung dry weight assumes that all lung cells are accessible to DQ on passage through the pulmonary circulation. This would necessitate an increase in the extravascular volume accessible to DQH2, QD, in 21-day hyperoxic lungs proportional to the increase in their dry weights, wherein QD is a measure of tissue volume accessible to lipophilic indicators such as DQ and DQH2. However, the estimated values of QD for normoxic and hyperoxic lungs were not significantly different (Table 4), and hence the values of Vmax/QD for 21-day hyperoxic lungs are larger than those for normoxic lungs (Table 6). These results are consistent with a hyperoxia-induced increase in NQO1 activity in cells accessible to DQ on passage through 21-day hyperoxic lungs, presumably dominated by capillary endothelial cells as discussed later.

The extravascular volume of DQ was not determined since that would require perfusing the lung with 400 µM dicumarol to inhibit DQ reduction. This concentration of dicumarol would interfere with our ability to determine DQ venous effluent concentration spectrophotometrically due to the overlap between the DQ and dicumarol absorbance spectra. Because both DQ and DQH2 are highly lipophilic and have flow-limited access to lung tissue, QD for DQH2 can be thought of as an index of lung tissue volume accessible to lipophilic indicators such as DQ and DQH2. Hence, a condition (hyperoxia) that changes QD for DQH2 would be expected to also proportionately change QD for DQ.

The present study revealed a small increase (<9%) in wet/dry weight ratio of 21-day hyperoxic lungs compared with normoxic lungs indicating slight edema caused by chronic exposure to hyperoxia and/or perfusion. The kinetic model described in Data Analysis suggests that edema would be expected to increase QD, which would result in underestimating the increase in the value of Vmax/QD for hyperoxic lungs (Table 6). Thus the estimated changes in the values of Vmax/QD in Table 6 represent lower bounds on the impact of hyperoxia.

Exposure to hyperoxia for 21 days decreased the PS, which is a measure of the rate constant of ACE-mediated FAPGG hydrolysis (Table 4). Assuming that exposure to hyperoxia for 21 days had no significant effect on ACE activity per unit of surface area, this change in PS can be attributed to a change in perfused capillary surface area (27). The decrease in PS (70% in 21-day hyperoxic lungs) is in fact consistent with the ~60% decrease in pulmonary capillary endothelial surface area in rats following exposure to 85% O2 for 14 days measured morphometrically (14). Normalizing Vmax to PS results in an eightfold increase in NQO1 activity per unit of capillary surface area (Table 6). The loss of capillary endothelial surface area in 21-day hyperoxic lungs is not reflected by a change in DQH2 extravascular volume (Table 4), which may suggest that nonendothelial cell types are also accessible to DQH2. Alternatively, Crapo et al. (14) demonstrated that endothelial cells in lungs of rats exposed to 85% O2 for 14 days undergo hypertrophy, with the net result of no change in the total volume of capillary endothelial cells.

The ~34% increase in lung homogenate NQO1 activity in 21-day hyperoxic lungs is less than one-half the ~85% increase in Vmax/QD (Table 6), which can be taken as a measure of NQO1 activity per unit volume of lung cells accessible to DQ. One reason for this difference may be the complex nature of lung tissue and the additional complexity associated with the cellular changes in lungs of hyperoxia-adapted rats, including the increase in the number of interstitial cells (14). Another possible reason is that key aspects of the intact lung environment that may regulate enzyme activity (e.g., the availability of electron donors and their accessibility to the enzyme) are not preserved in lung homogenate.

Interpretation of the Increase in Kma, the Michaelis-Menten Constant for DQ Reduction to DQH2, in 21-Day Hyperoxic Lungs

Because dicumarol has been shown to inhibit other reductases (28), one possible explanation for an increase in Kma in 21-day hyperoxic lungs (Table 5) might be that dicumarol-inhibitable DQ reductase(s) other than NQO1 are induced.

Another possible explanation is an increase in the capillary transit time heterogeneity in 21-day hyperoxic lungs. The kinetic model described in Data Analysis assumes homogeneous transit time distribution. Table 4 shows that the RDV in 21-day hyperoxic lungs increased by >40%. Most of the pulmonary vascular transit time heterogeneity is attributed to capillary transit time distribution [hc(t)] (1, 5). The kinetic model described in Data Analysis was used to evaluate the effects of hc(t) on Vmax and Kma values estimated under the assumption of homogeneous capillary transit times (Eq. 14) as described below.

A whole organ model that accounts for hc(t) was constructed based on the single capillary element model described in Data Analysis (5). For the analysis, hc(t) was approximated using a random walk function and was accounted for in the whole organ model as previously described (1, 5). To evaluate the effects of hc(t) on the estimated values of Vmax and Kma, two organ models were created to simulate DQ reduction on passage through normoxic lungs (normoxic model) and 21-day hyperoxic lungs (hyperoxic model). For the normoxic model, Vmax was set at 2.0 µmol/min (Table 5); the relative dispersion of hc(t), RDc, was set at 0.7 (Table 4); and Kma was set at 10 µM, which was determined to result in a Kma value estimated using Eq. 14 equal to that for normoxic lungs (Table 5). For the hyperoxic model, Vmax was set at 4.0 µmol/min (Table 5); RDc was set at 1.0 (Table 4); and Kma was set at the same value as for the normoxic model since Kma is an intensive property of NQO1 and would not be expected to be affected by exposure to hyperoxia. The tissue-mediated DQH2 oxidation rate constant, ko, was set at zero for the normoxic and hyperoxic models to simulate lung perfusion with cyanide. For each model, DQ pulse infusions at 50, 100, 200, and 400 µM were simulated, and steady-state venous effluent [DQ] and [DQH2] were determined. Figure 6 shows simulated steady-state rates of DQ reduction to DQH2 as a function of the vascular log mean [DQ], []log. The differences between normoxic and hyperoxic models in Fig. 6 are nearly identical to those between the corresponding data in normoxic lungs and 21-day hyperoxic lungs (Fig. 5). The steady-state rates of DQ reduction (Fig. 6) for the hyperoxic model were larger than those for the normoxic model during the infusion of DQ at 200 and 400 µM, but not at 100 and 50 µM, consistent with an increase in Kma. Table 7 shows the values of Vmax and Kma estimated by fitting Eq. 14 to the simulated DQ reduction rates in Fig. 6 and designated "recovered values," normalized to the corresponding values used in the model simulations and designated "actual values." For Vmax, the recovered values were within 10% of the actual values and were not sensitive to the relative dispersion of hc(t), RDc. However, for Kma, the recovered values for normoxic and hyperoxic models were more than 5-fold and 11-fold larger than the actual value, respectively. Thus the recovered Kma value for the hyperoxic model was more than double that for the normoxic model, although the actual value was the same for both. This suggests that Kma is sensitive to an increase in RDc, which could account for the increase in Kma in 21-day hyperoxic lungs compared with normoxic lungs (Table 5).



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Fig. 6. The relationship between the rate of DQ reduction and the log mean [DQ] obtained from model-simulated infusions of DQ at 4 different concentrations (50, 100, 200, and 400 µM). The normoxic and hyperoxic whole organ models simulate DQ reduction on passage through normoxic and 21-day hyperoxic lungs, respectively. The whole organ models account for capillary transit time distribution, hc(t), and assume different relative dispersions for hc(t) (see text). Solid lines are fits of Eq. 14 to simulation data. Equation 14 assumes homogenous hc(t).

 

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Table 7. The influence of capillary transit time distribution on the estimated values of Vmax and Kma from model simulations

 
The above simulation results are consistent with those of Bass and Robinson (7). They demonstrated that the effect of hc(t) on Michaelis-Menten kinetics is different for different regions of the substrate input concentration range, with its effect being insignificant for high input substrate concentrations compared with the Michaelis-Menten constant (zero order kinetics) since enzyme molecules in all capillaries are saturated by substrate. Hence, Vmax, which is determined mainly by DQ reduction rates at high-input DQ concentrations (relative to Michaelis-Menten constant), is virtually insensitive to hc(t), whereas Kma, which is determined by reduction rates at low- and high-DQ input concentrations, is sensitive to hc(t).

The pulmonary capillary endothelium with its large surface area and direct contact with the perfusate might be expected to be the dominant site of DQ reduction in lungs. Previously, we demonstrated that bovine pulmonary artery endothelial cells in culture are capable of reducing DQ at a rate per cm2 endothelial cell surface area comparable to that measured in lungs of normoxic rats [0.28 nmol/min per cm2 endothelial surface area vs. 0.43 nmol/min per cm2 in the present study, assuming a rat lung capillary surface area of ~4,500 cm2 (14)] (24). On the basis of the fraction of DQ bound to BSA (~96% at 5% BSA) (1), the corresponding free Kma value for DQ reduction in normoxic lungs is ~1.8 µM, which is also consistent with 1.2 µM estimated by our previous studies (24) (Table 2). These results and the fact that the DQ reduction by the cultured pulmonary endothelial cells was dicumarol inhibitable are consistent with the pulmonary endothelium being a dominant site of DQ reduction in lungs. This is potentially important since the pulmonary capillary endothelium is primary target of O2 toxicity and since NQO1 has been shown to confer protection from oxidant stress (8, 9, 17, 30, 31, 33).

The hyperoxia-induced increase in lung NQO1 activity may also have an impact on systemic organs by altering the redox status of compounds (e.g., quinones) passing through the lungs in preparation for entry into the systemic circulation. Depending on the physical and chemical properties of these compounds, the increase in lung NQO1 activity could represent an increased capacity to regenerate plasma antioxidants or production of prooxidant activity with potentially important implications for the physiology and pathophysiology of the systemic vascular system (1, 5, 10, 24).


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This work was supported by National Heart, Lung, and Blood Institute Grants HL-24349 and HL-65537 and the Department of Veterans Affairs.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. H. Audi, Research Service 151, Zablocki VAMC, 5000 W. National Ave., Milwaukee, WI 53295 (e-mail: audis{at}mu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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