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Published ahead of print on December 15, 2005, doi:10.1165/rcmb.2005-0337OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 34, pp. 505-513, 2006
© 2006 American Thoracic Society
DOI: 10.1165/rcmb.2005-0337OC

Impact of Mitochondria and NADPH Oxidases on Acute and Sustained Hypoxic Pulmonary Vasoconstriction

Norbert Weissmann, Stefanie Zeller*, Rolf U. Schäfer*, Carmen Turowski*, Mahmut Ay*, Karin Quanz, Hossein A. Ghofrani, Ralph T. Schermuly, Ludger Fink, Werner Seeger and Friedrich Grimminger

University Giessen Lung Centre (UGLC), Medical Clinic II/V, and Institute of Pathology, Justus-Liebig-University Giessen, Germany

Correspondence and requests for reprints should be addressed to Dr. Norbert Weissmann, University Giessen Lung Centre (UGLC), Medical Clinic II/V, Justus-Liebig-University Giessen, Klinikstrasse 36, 35392 Giessen, Germany. E-mail: Norbert.Weissmann{at}UGLC.de


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion with ventilation to optimize pulmonary gas exchange. However, it remains unclear whether acute HPV (occurring within seconds) and the vasoconstrictor response to sustained alveolar hypoxia (developing over several hours) are triggered by identical mechanisms. We investigated the effect of mitochondrial and NADPH oxidase inhibitors on both phases of HPV in intact rabbit lungs. These studies revealed that the sustained HPV is largely dependent on mitochondrial complex I and totally dependent on complex IV, whereas NADPH oxidase dependence was only observed for acute HPV. These findings were reinforced by an alternative approach employing lungs from mice deficient in the NADPH oxidase subunit p47phox. In these mice (which lack a subunit suggested to be important for the function of most NADPH oxidase isoforms), but not in gp91phox-deficient mice (which represent only one isoform of NADPH oxidases), acute HPV was significantly reduced, while non–hypoxia-induced vasoconstrictions elicited by the thromboxane mimetic U46619 were not affected. We concluded that the acute phase and the sustained phase of HPV are differentially regulated, with NADPH oxidase activity predominating in the acute phase, while a strong dependence on mitochondrial participation was observed for the second phase.

Key Words: hypoxic pulmonary vasoconstriction • mitochondria • mouse • NADPH oxidase • pulmonary hypertension


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung that matches perfusion to ventilation and thus helps to prevent arterial hypoxemia (for review, see Refs. 1, 2). Despite the description of the relevance of this mechanism by von Euler and Liljestrand in 1946 (3), the underlying oxygen sensing and signal transduction processes have not been identified yet. Pathophysiologic conditions such as adult respiratory distress syndrome (ARDS), severe pneumonia, and liver cirrhosis may result in a lack of HPV with deterioration of pulmonary gas exchange. Alternatively, sustained generalized alveolar hypoxia, as it occurs in chronic obstructive and restrictive lung diseases, culminates in the development of pulmonary hypertension characterized by pronounced vascular remodeling.

We have recently demonstrated in isolated ventilated and perfused rabbit and mouse lungs that sustained hypoxic ventilation for at least 120 min results in a biphasic vasoconstrictor response and a loss of posthypoxic vasorelaxation—that is, that the vasoconstrictor response after prolonged hypoxia did not return to baseline levels after reoxygenation. This biphasic kinetics was characterized by an initial vasoconstrictor response peaking at 4–6 min, a pressure nadir at 15–20 min, and a secondary progressive increase in PAP (4, 5). Against this background it remains to be clarified whether the acute HPV (occurring within seconds) and the sustained vasoconstrictor response (occurring during hypoxic ventilation of more than 20 min) are triggered by identical or different oxygen sensing and signal transduction pathways.

In a previous investigation we demonstrated that lung nitric oxide generation and reactive oxygen species contribute to the regulation of both the acute and sustained phase of HPV (4). The flavoprotein inhibitor diphenyleneiodonium (DPI), which inhibits both NADPH oxidases and mitochondria, suppressed the sustained phase of HPV with higher efficacy when compared with the acute phase of HPV. For both the acute and sustained phase of HPV there is ongoing discussion whether mitochondria, NADPH oxidases, or alternative systems are the underlying oxygen sensors and whether they are regulated by identical or by different mechanisms (2, 6–9). Biphasic hypoxic vasoconstrictor responses have repeatedly been shown to occur in isolated vessels, but few studies have focused on the regulation in the intact organ (9–13).

Therefore, we have employed two approaches to determine the possible differential contribution of mitochondria versus NADPH oxidases to the regulation of acute and sustained HPV in intact lungs. First, we used a pharmacologic approach in isolated perfused and ventilated rabbit lungs targeting the different mitochondrial electron transport complexes as well as NADPH oxidases; second, we used a genetic approach with mouse lungs deficient in the NADPH oxidase subunits gp91phox and p47phox, respectively. Essentially, we found that the mitochondrial respiratory chain complex I and, particularly, complex IV, have a much stronger impact on the regulation of the sustained phase of HPV compared with acute HPV, while the impact of nonphagocytic NADPH oxidase function was demonstrated only for the acute phase of HPV.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Reagents
Rotenone, 1-methyl-4-phenylpyridinium iodide (MPP+), 3-nitroproprionic acid (3-NPA), antimycin A, and cyanide were from Sigma-Aldrich (Taufkirchen, Germany). The U46619 was from Paesel+Lorei (Franfurt am Main, Germany). The perfusate was purchased from Serag-Wiessner (Naila, Germany). The NG-monomethyl-L-arginine (L-NMMA) was from by Calbiochem (Bad Soden, Germany). All other biochemicals were purchased from Merck (Munich, Germany).

Animals
For rabbit lung investigations, Chinchilla bastard rabbits of either sex (2.5–3.2 kg) were employed. The isolated mouse lung experiments were performed in mice deficient in either the gp91phox subunit of the NADPH oxidase (B6.129S6-Cybbtm1Din/J [gp91phox–/–]; Jackson Laboratory, Bar Harbor, ME) or for the NADPH oxidase subunit p47phox (p47phox–/–) (14). All animal experiments were approved by the local Governmental Commission. C57BL/6J mice (Jackson Laboratory) were used as controls. Mice weights ranged between 25 and 38 g, and weight-mached pairs of animals (wild-type and transgenic) were used to compensate for weight differences. Mice of either sex were used for the investigation with the same proportion of male and female mice in each group. No sex differences were observed concerning the vasoconstrictor response to hypoxia or the thromboxane mimetic U46619. All animal experiments were approved by the local governmental committee.

Rabbit and Mouse Lung Isolation, Perfusion, and Ventilation
The model of isolated perfused rabbit lungs has been described previously (4, 15). Briefly, the lungs were excised in deep anesthesia while being perfused with Krebs Henseleit buffer (125.0 mmol/liter NaCl, 4.3 mmol/liter KCl, 1.1 mmol/liter KH2PO4, 2.4 mmol/liter CaCl2, 1.3 mmol/liter MgCl2 and 275 mg glucose/100 ml); NaHCO3 was adjusted to result in a constant pH of 7.37–7.40. The total system volume was 250 ml, the buffer flow rate was 150 ml/min, and left atrial pressure was set at 1.5–2.0 mm Hg. The isolated lungs were ventilated with a gas containing 5.3% CO2, 21.0% O2, balanced with N2 (tidal volume, 30 ml; frequency, 30 strokes/min). A positive end-expiratory pressure of 1 cm H2O was chosen. The isolated lungs were freely suspended from a force transducer for continuous monitoring of organ weight. The whole system was heated to 38.5°C. Pressures in the pulmonary artery, the left atrium, and the trachea were continuously registered. The pressure transducers-amplifier system (Combitrans; B. Braun, Melsungen, Germany, and Plugsys 603 including DBA modules; HSE Electronik, March-Hugstetten, Germany) was calibrated before each experiment.

Mouse lungs were isolated from the chest following an analogous protocol, as described previously (5). Positive pressure ventilation was performed with a tidal volume of 250 µl, 90 breaths/min, and 2 cm H2O positive end-expiratory pressure with a gas mixture containing 21% O2, 5.3% CO2, balanced with N2. For perfusion Krebs-Henseleit buffer, containing 120 mmol/liter NaCl, 4.3 mmol/liter KCl, 1.1 mmol/liter KH2PO4, 2.4 mmol/liter CaCl2, 1.3 mmol/liter MgCl2, and 13.32 mmol/liter glucose as well as 5% (wt/vol) hydroxyethylamylopectin (molecular weight 200,000) was employed. The total system volume was 13 ml. The flow rate was set at 2.0 ml/min.

Lungs included in the study were those that had a homogeneous white appearance with no signs of hemostasis, edema, or atelectasis.

Hypoxic Maneuvers
The technique of sequential and sustained hypoxic maneuvers in buffer-perfused mouse and rabbit lungs has been described previously (5, 16, 17). Briefly, a gas mixing chamber (KM 60–3/6MESO, WITT, Witten, Germany) was employed for step changes in the ventilator O2 content (21% vol/vol [alveolar PO2 ~ 160 mm Hg, baseline conditions] to 3% vol/vol [alveolar PO2 ~ 23 mm Hg, hypoxic conditions, rabbit lung], or 1% O2 vol/vol [alveolar PO2 ~ 8 mm Hg, hypoxic conditions, mouse lung]). CO2 at 5.3% (vol/vol) was used throughout, and the percentage of N2 was balanced accordingly.

U46619-Induced Vasocontrictions
To induce a hypoxia-independent vasoconstrictor response, mouse lungs were challenged with a bolus application of U46619 into the pulmonary artery as previously described, resulting in a final concentration of 4.5 nmol/liter in the recirculating buffer fluid (5). In these experiments two hypoxic ventilation maneuvers (10 min) alternating with 15 min periods of normoxic ventilation were performed, followed by the U46619 application. This protocol enabled a direct comparison of the vasoconstrictions to hypoxia and U46619 of each individual lung.

Sustained Alveolar Hypoxia in Rabbit Lungs
After an initial short-term period of hypoxia (10 min duration), which allows assessment of adequate lung response to this challenge (5, 15–17), and a subsequent 15-min period of normoxia, a sustained hypoxic ventilation for 120 min (3% O2) was performed. In the cyanide experiments, a third hypoxic challenge of 10 min duration (3% O2) was started 15 min after cessation of the 120-min period of hypoxia.

Effects of Pharmacologic Agents on 120 min of Alveolar Hypoxia in Isolated Rabbit Lungs
Various pharmacologic agents were investigated for their effects on the pressure responses provoked by sustained alveolar hypoxia of 120 min duration. All of these agents were previously shown to specifically diminish the strength of the acute hypoxic pressure response when investigated during 10-min periods of alveolar hypoxia by comparison with their effect on U46619 (thromboxane analog)-induced vasoconstrictions (17, 18) (except for 3-NPA, because we were not aware of a complex II inhibitor specifically interfering with HPV). To determine possible differences in the responsiveness of the acute hypoxia-elicited pressure elevation (occurring within 10 min of hypoxia) and the secondary (sustained) hypoxia-induced increase in pulmonary artery pressure to the various pharmacologic interventions, the inhibitors were added to the perfusate in a mode that assured constant impact on the strength of acute HPV, as ascertained in separate control experiments. In these experiments, the well characterized mode (16–19) of repetitive 10-min maneuvers of alveolar hypoxia, alternating with periods of normoxic ventilation of 15 min duration, was applied. For inhibition of the acute hypoxic response, the goal was a partial suppression compared with the control response (no inhibitor). Moreover, the dosage protocol of each agent ascertained that such suppressor effect was maintained at a constant level throughout a 145-min observation period as probed by repetitive hypoxia maneuvers during this time period. However, in some cases the "constancy period" did not commence with the first but with a later hypoxic challenge, due to the pharmacokinetics of the individual agent (see below). These preceding control experiments suggested the following protocols to guarantee well-controlled effects of each pharmacologic intervention over the observation period when investigating the impact on sustained hypoxia (all administrations being started 5 min before onset of hypoxia, with concentrations referring to the recirculating perfusate if not stated differently):

MPP+ (mitochondrial complex I inhibitor)—1.25 µmol/liter, followed by continuous infusion for 30 min of a dosage resulting in an increase of 1.0µmol/liter/h, starting 75 min later. Sustained alveolar hypoxia was started corresponding to the timing of the fifth hypoxic challenge.
Rotenone (mitochondrial complex I inhibitor)—40 nmol/liter, followed by repetitive application of 40 nmol/liter every 25 min. Sustained alveolar hypoxia was started corresponding to the timing of the third hypoxic challenge. Due to the pharmacokinetics of rotenone, sustained hypoxia was prolonged in these experiments to 135 min. The rotenone experiments were performed in the presence of L-NMMA (400µM), as this agent was shown to suppress HPV with higher specificity as compared with U46619-induced vasoconstrictions when lung NO synthesis was inhibited (19).
3-NPA (mitochondrial complex II inhibitor)—0.6 mmol/liter (application 15 min before onset of hypoxia), followed by supplementation of 150 ml of the perfusate with fresh buffer 75 min later. Sustained alveolar hypoxia was started corresponding to the timing of the fourth hypoxic challenge.
Antimycin A (mitochondrial complex III inhibitor)—2.0 nmol/liter, followed by application of the following doses every 25 min: 0.5, 0.5, 0.3, 0.3, 0.3, 0.3, 0.3, 0.3, 0.3 nmol/liter. Sustained alveolar hypoxia was started corresponding to the timing of the third hypoxic challenge.
Cyanide (mitochondrial complex IV inhibitor)—Continuous infusion of 50 µmol/liter/h for 15 min, followed by an infusion of 30 µmol/liter/h. Sustained alveolar hypoxia was started corresponding to the timing of the third hypoxic challenge.
4-(2-Aminoethyl)benzenesulfonyl fluoride (AEBSF, NADPH oxidase inhibitor)—500 µmol/liter, followed by continuous infusion for 215 min of a dosage resulting in an increase of 100 µmol/liter/h. Thereafter infusion rate was decreased to 50 µmol/liter/h. Sustained alveolar hypoxia was started corresponding to the timing of the fifth hypoxic challenge. The AEBSF experiments were performed in the presence of L-NMMA (400 µmol/liter) to make them comparable to those from our previous investigation with the application of the NADPH oxidase inhibitor diphenyleneiodonium in the presence of NO synthase inhibition (4, 19). DPI is known to also block lung NO synthesis, and thus NO synthase has to be blocked to avoid interference with this vasodilatory agent.

Sustained Alveolar Hypoxia in Mouse Lungs
After an initial 10-min period of hypoxic ventilation (1% O2) followed by a 15-min period of normoxic ventilation, a 180-min period with hypoxic ventilation (1% O2) was started.

Measurement of Intravascular Superoxide Release by Electronspinresonance Spectroscopy
Intravascular superoxide release was determined as described previously in detail (20). In brief, lungs were perfused with addition of the spin probe 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH, 1 mM) to the buffer fluid. Oxidation of CPH forms the nitroxide CP• radical. The electronspinresonance (ESR) spectrum of the CP• radical was detected by an MS 100 spectrometer (Magnettech, Berlin, Germany). ESR measurements were performed in field scan with the following settings: microwave frequency 9.78 GHz, modulation frequency 100 kHz, modulation amplitude 2 G, microwave power 18 mW. Superoxide radical formation was determined in parallel experiments performed in the presence of SOD (150 U/ml) in the buffer fluid. Samples from the venous outflow of the lung were taken every 2 min in a sequence of two hypoxic ventilation maneuvers (1% O2, 15 min) alternating with normoxic ventilation (21% O2, 15 min), as the ESR data from the first and second hypoxic challenge did not differ significantly. The high ventilation:perfusion ratio (~ 11:1) assured continuous hypoxic conditions during hypoxic ventilation. The continuity of hypoxia was determined by measurement of the buffer PO2 at the outflow of the lung as documented previously for this model (5). Samples were taken in glass capillaries directly from the venous outflow of the lung, sealed immediately, and measured directly in the ESR spectroscope to avoid reoxygenation. CPH oxidation was quantified as increase in signal intensity/min from the low field component of the CP• spectrum.

Statistics
Data are means ± SEM. For comparison of acute and sustained HPV (Figures 2–6GoGoGoGo and 8) a three-factor ANOVA with repeated measures on the factor time with the Student-Neuman-Keuls post hoc test was performed. For comparison of two groups, a Student t test was performed. Data in Figure 7 were analyzed by a one-way ANOVA with the Dunnett post hoc test. Statistical significance was assumed when P ranged < 0.05.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Pharmacologic Interventions Targeting Mitochondrial Complexes I–IV
Hypoxic ventilation of isolated rabbit lungs for 120 min with 3% O2 resulted in a biphasic vasoconstrictor response with a first maximum occurring within ~ 6 min, a pressure nadir at ~ 16 min, and a second, sustained rise in PAP (Figure 1).


Figure 1
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  		Figure 1. Pulmonary artery pressure response during sustained hypoxic ventilation (3% O2) for 120 min. Baseline pulmonary arterial pressure at time point zero was 5.5 ± 0.3 (n = 10). Data are means ± SEM of n = 5 for both groups. A bar indicates significant differences between the hypoxic and the normoxic group. {Delta}PAP: Change in pulmonary arterial pressure as referenced to the starting point of hypoxic ventilation (time set at zero).

 
When applying the mitochondrial complex I inhibitor MPP+ in a mode that reduced acute HPV consistently by ~ 15% in experiments with repetitive hypoxic challenges (data not shown), MPP+ strongly inhibited the sustained phase of HPV with a comparably lesser effect on the acute response (Figure 2A). A similar profile was observed when applying a second complex I inhibitor, rotenone, in a mode that constantly reduced acute HPV by ~ 40% (Figure 2B). The higher response to an acute hypoxic challenge in these experiments resulted from the presence of the NO synthase inhibitor L-NMMA in the perfusate, as we previously demonstrated that rotenone only suppressed acute HPV with high specificity in the presence of L-NMMA (18).


Figure 2
Figure 2
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  		Figure 2. Influence of the complex I inhibitors MPP+ (A) and rotenone (B) on the pressor response to sustained alveolar hypoxia. The initial dosage for MPP+ and rotenone was 1.25 µmol/liter and 40 nmol/liter, respectively (for details see MATERIALS AND METHODS). Changes in pulmonary artery pressure from baseline, referenced at time zero, are given ({Delta}PAP; means ± SEM; n = 5 each). The rotenone experiments were performed in the presence of 400 µmol/liter NG-monomethyl-L-arginine to block nitric oxide synthesis. Hypoxia controls received no MPP+ or rotenone. Normoxia controls were treated in the same way as hypoxic controls but without hypoxic ventilation. Bars indicate significant differences between MPP+ or rotenone-treated lungs and the hypoxia control.

 
The complex II inhibitor 3-NPA affected acute as well as sustained HPV to the same degree when applied in a mode that consistently suppressed acute HPV by ~ 60% (Figure 3). Similarly, the complex III inhibitor antimycin A reduced both the acute phase and the sustained phase of HPV by the same degree when applied in a mode that constantly suppressed acute HPV in experiments with repetitive acute hypoxic ventilation maneuvers (Figure 4).


Figure 3
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  		Figure 3. Influence of the complex II inhibitor 3-NPA on the pressor response to sustained alveolar hypoxia. The initial dosage for 3-NPA was 0.6 mmol/liter (for details see MATERIALS AND METHODS). Changes in pulmonary artery pressure from baseline, referenced at time zero, are given ({Delta}PAP; means ± SEM; n = 5 each). Hypoxia controls received no 3-NPA. Normoxia controls were treated in the same way as hypoxic controls, but without hypoxic ventilation. Bars indicate significant differences between 3-NPA–treated lungs and the hypoxia control.

 

Figure 4
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  		Figure 4. Influence of the complex III antimycin A on the pressor response to sustained alveolar hypoxia. The initial dosage for antimycin A was 2 nmol/liter (for details see MATERIALS AND METHODS). Changes in pulmonary artery pressure from baseline, referenced at time zero, are given ({Delta}PAP; means ± SEM; n = 5 each). Hypoxia controls received no antimycin A. Normoxia controls were treated in the same way as hypoxia controls, but without hypoxic ventilation. Bars indicate significant differences between antimycin A–treated lungs and the hypoxia control.

 
In contrast, application of the complex IV inhibitor cyanide in a mode that mildly reduced acute HPV inhibited the sustained pressure elevation entirely. To demonstrate that this total inhibition of sustained HPV was not caused by a loss of contractility of the lungs in general, we challenged the lungs with an acute hypoxic ventilation period of 10 min, 15 min after cessation of sustained hypoxic ventilation. It is evident from Figure 5 that the cyanide-treated lungs were still responsive to an acute hypoxic challenge in the presence of cyanide. A prerequisite for the application of cyanide was to prove whether it specifically affects acute HPV in comparison to non–hypoxia-induced vasoconstriction as this, in contrast to the other inhibitors applied, has not previously been described. In fact, 25 µM cyanide suppressed acute HPV to 37.8 ± 4.5% of the control response, whereas the vasoconstrictor response to the thromboxane mimetic U46619 was not significantly reduced at this dose of cyanide. Cyanide (80 µmol/liter) totally suppressed acute HPV but reduced U46619-induced vasoconstriction only to 65.5 ± 8.0% of the initial response.


Figure 5
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  		Figure 5. Influence of the complex IV inhibitor cyanide on the pressor response to sustained alveolar hypoxia. The initial dosage for cyanide was 50 µmol/liter/h (for details see MATERIALS AND METHODS). Changes in pulmonary artery pressure from baseline, referenced at time zero, are given ({Delta}PAP; means ± SEM; n = 5 each). Hypoxia controls received no cyanide. Single data points depict reactivity of the cyanide-treated lungs as well as the hypoxic control to an acute hypoxic ventilation of 10 min duration that was started 15 min after cessation of sustained hypoxia. {Delta}PAP values for these data points are referenced to the normoxic value directly before start of hypoxic ventilation. Normoxic PAP before the start of the acute hypoxic ventilation after cessation of sustained hypoxia was 1.1 ± 0.3 mm Hg (hypoxia+cyanide) and 1.7 ± 0.2 mm Hg (hypoxia control). Normoxia controls were treated in the same way as hypoxia controls, but without hypoxic ventilation. Bar indicates significant differences between cyanide-treated lungs and the hypoxia control.

 
Pharmocologic Interventions Targeting NADPH Oxidases
Application of the NADPH oxidase inhibitor AEBSF in a mode that inhibited acute HPV by ~ 70% resulted in only a minor effect on sustained HPV (Figure 6).


Figure 6
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  		Figure 6. Influence of AEBSF on the pressor response to sustained alveolar hypoxia. The initial dosage for AEBSF was 500 µmol/liter (for details see MATERIALS AND METHODS). Changes in pulmonary artery pressure from baseline, referenced at time zero, are given ({Delta}PAP; means ± SEM; n = 5 each). The AEBSF experiments were performed in the presence of 400 µmol/liter NG-monomethyl-L-arginine to block nitric oxide synthesis. Hypoxia controls received no AEBSF. Normoxia controls were treated in the same way as hypoxia controls, but without hypoxic ventilation. Bar indicates significant differences between the AEBSF-treated lungs and the hypoxia control.

 
Acute Hypoxic Vasoconstriction in Mice Deficient for NADPH Oxidase Subunits
When comparing the acute hypoxic vasoconstrictor response to a 10-min hypoxic ventilation period (1% O2) in mice deficient for the NADPH oxidase subunits gp91phox and p47phox, HPV was significantly lower in p47phox-deficient mice (Figure 7A). This reduction was specific for the hypoxic vasoconstrictor response, since vasoconstrictions induced by the thromboxane mimetic U46619 were not significantly reduced in these mice (Figure 7B). In contrast, gp91phox-deficient mice exhibited no difference in the strength of the acute hypoxic vasoconstrictor response when compared with wild-type controls (Figure 7A). Also, the responsiveness to U46619 was not altered in gp91phox-deficient mice when compared with wild-type mice (Figure 7B). Baseline normoxic vascular tone was not different in p47phox or gp91phox mice when compared with wild-type controls (Figure 7C).


Figure 7
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  		Figure 7. Strength of acute hypoxic vasoconstriction (a), U46619-induced vasoconstrictions (b), and baseline vascular tone (c) in mice deficient for the NADPH oxidase subunit gp91phox (gp91phox–/–) or p47phox (gp47phox–/–), and in wild-type controls (wt). Maximum changes in pulmonary artery pressure occurring within a 10-min hypoxic ventilation period (a) or an application of U46619, resulting in a concentration of 4.5 nmol/liter (b) are given. In (c) baseline normoxic vascular tone is given. Data are from at least n = 4 mice each. * Significant differences compared with the wild-type control.

 
Sustained Hypoxic Vasoconstriction in Mice Deficient for the NADPH Oxidase Subunit p47phox
When challenging wild-type mice with a 3-h hypoxic ventilation period (1% O2) HPV displayed a biphasic vasoconstrictor response with a prominent acute vasoconstrictor response and a constant period of sustained vasoconstriction (Figure 8). Whereas acute HPV was again lower in p47phox-deficient mice compared with wild-type mice, no difference was observed for sustained HPV.


Figure 8
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  		Figure 8. Comparison of sustained HPV in mice deficient for p47phox. Changes in pulmonary artery pressure from baseline, referenced at time zero, are given ({Delta}PAP; means ± SEM; at least n = 5 each). Data are for lungs undergoing sustained hypoxia, as well as for normoxically ventilated lungs. Bar indicates significant differences between p47phox–/– mice and wild-type mice in response to hypoxia.

 
Superoxide Release in Acute Hypoxia
Quantification of intravascular superoxide release in wild-type mice during normoxic and acute hypoxic ventilation by ESR spectroscopy revealed a decreased superoxide release during hypoxic ventilation (Figure 9). In p47phox-deficient mice no significant superoxide release was noted during normoxic ventilation. In contrast, acute hypoxic ventilation provoked an increased superoxide release in p47phox-deficient mice when compared with normoxia (Figure 9).


Figure 9
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  		Figure 9. Lung superoxide release during normoxic ventilation and acute hypoxia in wild-type (wt) and p47phox–/– mice. Lungs were perfused with the spin probe CPH. The increase in ESR signal intensity as a measure of CPH oxidation is given for normoxically ventilated lungs (normoxia) and lung challenged with acute hypoxic ventilation (acute hypoxia). To identify the portion of CPH oxidation caused by superoxide, experiments were performed in the presence and the absence of SOD (150 U/ml) in the buffer fluid. Data are mean ± SEM of >= n = 8 measurements from at least four lungs each.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The new findings of the present investigation are: (1) that the acute and sustained phase of the biphasic course of hypoxic vasoconstriction differ with respect to mitochondria and NADPH oxidase dependency; (2) that mitochondrial complex IV in particular has a tremendous, and mitochondrial complex I a strong, impact on the sustained phase of HPV compared with acute HPV; (3) that by investigating two different NADPH oxidase–deficient mouse strains, we provide concrete evidence for the involvement of nonphagocytic NADPH oxidases in the regulation of HPV; (4) that such involvement is observed for the acute, but not for the sustained phase of HPV; and (5) that a nonphagocytic NADPH oxidase may be activated rather than inhibited in acute hypoxia, as normoxic vascular tone was not elevated in p47phox-deficient mice.

General Features of the Biphasic Vasoconstrictor Response in Isolated Lungs
The biphasic kinetics of HPV to an ongoing hypoxic challenge was frequently reported for isolated pulmonary arteries but has less frequently been investigated in intact animals, in in vivo perfused lungs, or in ex vivo perfused lungs (6, 9, 10, 21–23). We recently described that in isolated perfused and ventilated rabbit lungs, a sustained hypoxic ventilation period of 120–180 min provoked a biphasic vasoconstrictor response with an early maximum occurring within 6 min, followed by a pressure nadir and a second, sustained vasoconstrictor phase (4). Moreover, when investigating isolated buffer-perfused mouse lungs totally explanted from the body, we recently showed that the same kinetics as demonstrated for the rabbit lung are also found in lungs from C57BL/6N mice (5).

There is ongoing discussion as to the nature of the oxygen sensor mechanism of HPV, and mitochondria, as well as NADPH oxidases and other mechanisms, have been proposed as potential oxygen sensors with subsequent release of reactive oxygen species being involved in the signal transduction process underlying HPV (2, 8, 24, 25). To elucidate the contribution of these potential oxygen sensing mechanisms to the biphasic course of HPV we here combined (1) a pharmacologic approach by applying inhibitors of the different mitochondrial electron transport complexes and a novel NADPH oxidase inhibitor, AEBSF, in the isolated perfused rabbit lung; and (2) a genetic approach by investigation of mice deficient in phagocytic as well as nonphagocytic NADPH oxidases (14, 26). This dual and complementary approach was undertaken because (1) the isolated rabbit lung is a well-established model for pharmacologic investigations focusing on HPV (4, 15–18, 27); and (2) the sophisticated technique of ex vivo perfused mice lungs presents an opportunity to prove the role of NADPH oxidases for HPV regulation, independent of pharmacologic inhibitors (5).

Pharmacologic Interventions in the Isolated Rabbit Lung
In a pharmacologic approach we investigated whether inhibitors of the mitochondrial electron transport complexes I–IV have different effects on the acute and the sustained phase of HPV. All of these inhibitors were shown in previous investigations from our laboratory, or in the present study, to specifically inhibit acute HPV when compared with their effect on non–hypoxia-induced vasoconstrictions in response to the thromboxane mimetic U46619 (17, 18). The only exception was 3-NPA, which was previously characterized as an unspecific inhibitor of acute HPV when compared with U46619-induced vasoconstriction (18).

However, we are not aware of any complex II inhibitor that specifically inhibits acute HPV in the rabbit lung. While the complex II inhibitor suppressed both phases of HPV to the same degree, the complex I inhibitors strongly suppressed the sustained phase of HPV with only a minor effect on acute HPV in the dosage applied. In contrast, the complex III inhibitor antimycin A inhibited both phases of HPV to a similar extent. Leach and coworkers recently demonstrated that the complex I inhibitor rotenone as well as the complex III inhibitor myxothiazole suppressed both phases of HPV in isolated pulmonary arteries (6). However, no conclusion could be drawn from their experiments on a possible different impact of these agents on the different phases of HPV, as they applied a dosage that fully suppressed both acute and sustained HPV. The fact that acute HPV is inhibitable by proximal electron transport chain inhibitors is in line with the investigation of Waypa and colleagues (28), who found that rotenone and myxothiazol inhibited acute HPV. However, this investigation did not focus on the effect of those inhibitors on sustained HPV.

The most prominent effect in our study was observed with cyanide that fully blocked sustained HPV at a dosage that only slightly reduced acute HPV. This effect was much more prominent than that observed in isolated rat pulmonary arterial vessels (6). Moreover, we demonstrated that the lungs still responded to an acute hypoxic challenge after sustained hypoxia in the presence of cyanide, proving that it was not a lack of energy that is responsible for this effect. This selective effect of cyanide on sustained HPV is in line with the observation of Waypa and coworkers in isolated rat lungs that acute HPV was not affected by cyanide (28). Going beyond these studies that investigated only acute HPV, Wiener and Sylvester investigated in detail the vasodilation and sustained HPV following acute HPV in isolated ferret lungs (9, 13). In these studies, high glucose levels prevented the vasodilation after acute HPV (9, 13), whereas pyruvate did not (9). This suggests that the prevention of the vasodilation by glucose was not dependent on glucose metabolism downstream of pyruvate. However, low glucose concentrations inhibited sustained HPV, an effect that was prevented by pyruvate (9). These data suggest that glucose metabolism beyond pyruvate (i.e., a decreased oxidative phosphorylation or an altered mitochondrial ROS production) is responsible for the inhibition of sustained HPV. This explanation is well in line with our study, as all inhibitors of the mitochondrial electron transport chain are suggested to result in a reduced oxidative phosphorylation in the concentrations applied (29–34). In accordance with the observations of Wiener and Sylvester (9, 13) low glucose also suppressed sustained HPV in isolated rat intrapulmonary arteries (6). However, in this investigation pyruvate did not reverse suppression of sustained HPV, suggesting that glucose facilitated sustained HPV by a mechanism independent from glucose metabolism downstream of pyruvate. This discrepancy may be explained by the fact that the pyruvate concentration applied was much lower than that used by Wiener and Sylvester (9).

As indicated above, alternatively to ATP production, the effects of the mitochondrial inhibitors may be related to mitochondrial ROS production, as previously suggested for acute HPV (2, 28, 35). This explanation is also well in line with our study, as the different inhibitors had different effects on the proportion of acute and sustained HPV. The complex I, and particularly, complex IV inhibitor of the mitochondrial electron transport chain had a much greater impact on the regulation of the sustained phase of HPV when compared with the acute phase of HPV. In contrast, the antimycin A experiments indicated that complex III has a similar effect on the regulation of both phases of HPV.

Concerning the ongoing discussion as to whether mitochondria or NAD(P)H oxidases may play a fundamental role in pulmonary oxygen sensing, we and others previously demonstrated that the NAD(P)H oxidase inhibitors DPI and AEBSF specifically suppressed acute HPV (19, 36). Moreover, investigating the effect of DPI on the acute and sustained phase of HPV, we recently demonstrated that DPI more prominently suppressed sustained HPV (4). As this inhibitor also blocks, for example, complex I of the mitochondrial electron transport chain we, in the present study, investigated the effect of the second structurally unrelated NADPH oxidase inhibitor AEBSF. Interestingly, this agent only affected the acute phase of HPV. These observations, together with the inhibitory effects of the complex I inhibitors rotenone and MPP+, are in line with the interpretation that the effect of DPI observed on the sustained phase of HPV may be related to its inhibitory effect on mitochondrial electron transport complex I.

Investigations in Isolated Perfused Lungs of NADPH Oxidase–Deficient Mice
Concerning the NADPH oxidase hypothesis in the context of HPV, the lack of specificity of NADPH oxidase inhibitors is thought to cause the different opinions between different investigators (2, 8). Thus, we investigated the impact of a deficiency in two different NADPH oxidase subunits (gp91phox and p47phox) in an isolated mouse lung preparation on acute HPV. The unaltered response to acute HPV in gp91phox-deficient mice is in line with a previous observation demonstrating that acute HPV is not critically dependent on the NADPH oxidase subunit gp91phox (37). However, the NADPH oxidase is a multiprotein complex consisting of the membrane-bound subunits gp91phox, p22, and the cytosolic subunits p40phox, p47phox, and p67phox. Moreover, isoforms of gp91phox as well as of p47phox and p67phox have recently been identified and have been shown to play a role as "low-output" NADPH oxidases for physiologic and pathophysiologic processes (38). When comparing acute HPV in mice deficient for p47phox with wild-type mice, HPV was attenuated. This attenuation was specific for HPV, since vasoconstrictions induced by the thromboxane mimetic U46619 were not reduced when compared with wild-type controls. The fact that baseline normoxic vascular tone was not enhanced in p47phox-deficient mice compared with wild-type mice demonstrates that reduction in the strength of HPV in these mice is not induced by some mimicking of hypoxia due to the lack of p47phox. Thus, these findings in genetically engineered mice support the concept that an NADPH oxidase–derived increase in reactive oxygen species release is involved in triggering acute HPV. Our data from the quantification of lung superoxide release are in line with this suggestion. Whereas total normoxic superoxide release was decreased in wild-type mice during acute hypoxia, p47phox-deficient mice released an enhanced amount of superoxide during hypoxic ventilation. In contrast to wild-type mice, no significant superoxide release was noted during normoxic ventilation in p47phox-deficient mice. Speculatively, the hypoxia-induced increase in lung superoxide release triggers HPV and is hidden behind the overall decrease in lung superoxide release observed in wild-type mice. If this concept is correct, the attenuated HPV in p47phox–/– mice may be related to some loss of this increase. Consequently the HPV-specific increase in superoxide release should be higher in wild-type mice than in p47-deficient mice, but is not seen due to the overall decrease in superoxide release in wild-type mice. This interpretation is supported by the fact that baseline PAP was not higher in 47phox–/– mice compared with wild-type mice, as would have been the case if a decreased superoxide release triggers HPV.

The fact that p47phox- but not gp91phox-deficient mice showed a reduced acute HPV points to a nonphagocytic NADPH oxidase playing an important role for regulation of acute HPV. When gp91phox isoforms (e.g., NOX1 and NOX4) were discovered, it was thought that they substitute gp91phox in the low-output NADPH oxidase complex with p47phox as subunit required for function of the NADPH oxidase complex. Thus, in p47phox knockout mice, HPV should be absent if a NADPH oxidase is the oxygen sensor underlying acute HPV. The fact that HPV was only partially reduced in p47phox-deficient mice may be explained by recent investigations that document that p47phox may also be substituted by other homologs, for example, by the recently discovered p47phox homolog NOXO1 (39). On the other hand, possibly both NADPH oxidase and mitochondrial mechanisms may contribute to the regulation of acute HPV. Clarification of this aspect needs further investigation, presumably in knockout mice of the different NADPH oxidase subunits and mitochondrial complexes.

While displaying reduced acute HPV, p47phox-deficient mice fully responded to sustained HPV without any difference from wild-type controls. This demonstrates that the sustained phase of HPV is not critically dependent on p47phox.

In conclusion, we demonstrated in intact lungs that the acute and the sustained phase of HPV are differentially regulated. While mitochondria-dependent pathways, especially those linked to complex I and IV, have a stronger effect on the regulation of sustained HPV, NADPH oxidase–dependent pathways seem to be solely linked to the regulation of acute HPV. Moreover, our investigation provided evidence from a nonpharmacologic approach that the regulation of acute HPV is dependent on nonphagocytic NADPH oxidases. The fact that p47phox-deficient mice have no elevated vascular tone supports the conclusion that a nonphagocytic NADPH oxidase is activated in acute hypoxia.


   Acknowledgments
 
The authors like to thank Dr. Steven M. Holland and Dr. Li Ding, Laboratory of Clinical Infectious Diseases, NIH, Bethesda, MD, for providing the p47phox-deficient mice. They also thank Dr. Rory Morty, Giessen, for linguistic editing of the manuscript.


   Footnotes
 
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 547 project B7). R.U.S. is supported by a predoctoral fellowship from ALTANA Pharma.

* Portions of the doctoral theses of Stefanie Zeller, Carmen Turowski, Rolf U. Schäfer, and Mahmut Ay are incorporated into this report. Back

Originally Published in Press as DOI: 10.1165/rcmb.2005-0337OC on December 15, 2005

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form September 2, 2005

Accepted in final form December 6, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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