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Published ahead of print on June 5, 2003, doi:10.1165/rcmb.2002-0217OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 721-732, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0217OC

Effects of Mitochondrial Inhibitors and Uncouplers on Hypoxic Vasoconstriction in Rabbit Lungs

Norbert Weissmann, Nadine Ebert*, Marit Ahrens*, Hossein A. Ghofrani, Ralph T. Schermuly, Jörg Hänze, Ludger Fink, Frank Rose, Jörg Conzen, Werner Seeger and Friedrich Grimminger

Department of Internal Medicine, Justus-Liebig University Giessen, Giessen, Germany

Address correspondence to: Norbert Weissmann, Ph.D., Department of Internal Medicine, Justus-Liebig-University Giessen, Klinikstrasse 36, 35392 Giessen, Germany. E-mail: Norbert.Weissmann{at}innere.med.uni-giessen.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion to ventilation for optimizing pulmonary gas exchange; however, the underlying mechanism has not yet been fully elucidated. Lung nitric oxide (NO) generation appears to be involved in this process. Recently, mitochondria have been proposed as oxygen sensors, with HPV signaling via a hypoxia-induced increase in the generation of reactive oxygen species derived from mitochondrial complex III and escaping through an anion channel into the cytoplasm. In addition, complex II has been suggested to be specifically involved in hypoxia-dependent generation of reactive oxygen species in the lung. We investigated the effects of several mitochondrial inhibitors and uncouplers on the strength of HPV, and asked for their capacity to mimic HPV during normoxia in isolated buffer-perfused rabbit lungs. Specificity of the agents for HPV was tested by comparison of their effects on non–hypoxia-induced vasoconstriction, elicited by the thromboxane mimetic U46619. Interference with NO metabolism was determined by performing parallel studies with blocked lung NO generation and by measurement of exhaled NO. Rotenone, 3-nitroproprionic acid, and myxothiazol dose-dependently inhibited HPV without being mimics of HPV during normoxia. The inhibitory effect of these agents was only partly specific for HPV by comparison with U46619-induced vasoconstriction. During pre-blocked lung NO synthesis, the selectivity for HPV inhibition was increased for rotenone, but largely lost for myxothiazol. 2-tenoyltrifluoroacetone resulted in an unspecific inhibition of HPV as compared with U46619-induced vasoconstriction. 1-methyl-4-phenylpyridinium iodide and 2-heptyl-4-hydroxyquinoline-N-oxide specifically suppressed HPV and increased normoxic vascular tone. Antimycin A suppressed HPV, an effect being specific in lungs with intact NO synthesis and only partly specific while blocking NO. However, this agent did not mimic HPV during normoxia, as may be expected for interference with the mitochondrial electron transport downstream in complex III. The uncouplers 2,4-dinitrophenol (DNP, 10–200 µM) and carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP, 1–3 µM) induced sustained vasoconstriction during normoxia, with enhancement of HPV by DNP at low and suppression of HPV for both agents at high concentrations. The anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid inhibited HPV and U46619-induced vasoconstriction with identical dose–response curves. These findings suggest that mitochondria are in some manner involved in the regulation of HPV in intact rabbit lungs. The hypothesis that enhanced superoxide leak at complex III of mitochondria represents the underlying mechanism of acute HPV is supported by the rotenone and 2-heptyl-4-hydroxyquinoline-N-oxide data, but partly contradicted by the findings with 1-methyl-4-phenylpyridinium iodide, antimycin A, DNP, and FCCP. Further studies are mandatory to clarify the link between mitochondrial respiratory chain and hypoxic pulmonary vasoconstriction.

Abbreviations: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, FCCP • 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, DIDS • 2,4-dinitrophenol, DNP • 2-heptyl-4-hydroxyquinoline N-oxide, HQNO • pulmonary artery pressure difference, {Delta}PAP • hypoxic pulmonary vasoconstriction, HPV • NG-monomethyl-L-arginine, L-NMMA • 1-methyl-4-phenylpyridinium iodide, MPP+ • pulmonary artery pressure, PAP • 2-tenoyltrifluoroacetone, TTFA • 3-nitroproprionic acid, 3-NPA


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion to ventilation to optimize pulmonary gas exchange (1, 2). The sensor mechanism, cell(s) responsible for O2 sensing, and the pathway(s) of signal transduction leading to contraction of the precapillary vascular smooth muscle cells still remain enigmatic. Nitric oxide (NO) synthesis contributes to this vasoregulatory mechanism (3, 4). Moreover, it has been suggested that the hypoxia signal is forwarded by superoxide and hydrogen peroxide (H2O2). However, there is still controversy about the contribution of reactive oxygen species (ROS), whether decreased or increased superoxide and H2O2 levels may trigger HPV, and from which cellular and intracellular source such reactive oxygen species originate (5–7). Several investigations, including those from our own group, suggested that a NAD(P)H oxidase may function as an O2-sensing complex (8–11), whereas recent investigations support the hypothesis that mitochondrial reactive oxygen species formation is one of the initial steps in the signal cascade underlying HPV (12, 13). In particular, a model has been proposed that hypoxia increases the generation of ROS at the level of complex III by accumulation of electrons "upstream" of complex IV, thereby promoting enhanced superoxide formation at the ubisemiquinone site of complex III, with subsequent egress of superoxide through an anion channel in the inner mitochondrial membrane (6, 14). Moreover, recently complex II was suggested to contribute to hypoxic ROS generation in the lung (7). Downstream events then include acute changes such as membrane depolarization and calcium shifts in HPV, but also chronic events mediated via stabilization of hypoxia inducible factors and subsequent gene regulation (12, 15, 16).

To test this "unifying mitochondrial hypothesis" for the hypoxic pulmonary vasoconstriction in intact rabbit lungs, we investigated the effects of several mitochondrial electron transport chain inhibitors (rotenone, 1-methyl-4-phenylpyridinium iodide [MPP+], 3-nitroproprionic acid [3-NPA],2-tenoyltrifluoroacetone [TTFA], myxothiazol, antimycin A, and 2-heptyl-4-hydroxyquinoline N-oxide [HQNO]), mitochondrial uncouplers (2,4-dinitrophenol [DNP] and carbonyl cyanide p-trifluoromethoxyphenylhydrazone [FCCP]), and an anion channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Dose–effect curves were established for all agents, and specificity for HPV was determined by comparing the effects of the various agents on control vasoconstrictions evoked by the stable thromboxane analog U46619. Moreover, we investigated the interference of these agents with lung NO generation, known to be intimately involved in pulmonary vasoregulation, mitochondrial respiration, and mitochondrial O2--generation (17), by performing the experiments in the absence and in the presence of the NO-inhibitor NG-monomethyl-L-arginine, and by quantifying their impact on NO exhalation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Rotenone, MPP+, 3-NPA, myxothiazol, antimycin A, HQNO, DNP, and DIDS were from Sigma-Aldrich (Taufkirchen, Germany). U46619 came from Paesel+Lorei (Frankfurt/M., Germany) The perfusate was purchased from Serag-Wiessner (Naila, Germany). Acetylsalicylic acid (ASA) was from Bayer (Leverkusen, Germany). NG-monomethyl-L-arginine (L-NMMA) were supplied by Calbiochem (Bad Soden, Germany). FCCP and TTFA were from Fluka (Taufkirchen, Germany). All other biochemicals were purchased from Merck (Munich, Germany).

Lung Isolation, Perfusion, and Ventilation
The model of isolated perfused rabbit lungs has been described previously (18). Briefly, pathogen-free rabbits of either sex (body weight 2.2–3.2 kg) were deeply anesthetized with an intravenous application of a mixture of 80 mg ketamine kg-1 body weight and 20 mg xylazine kg-1 body weight and anticoagulated with heparin (1,000 U kg-1 body weight). The lungs were excised while being perfused with Krebs-Henseleit-buffer through cannulae in the pulmonary artery and the left atrium. The buffer contained 125.0 mM NaCl, 4.3 mM KCl, 1.1 mM KH2PO4, 2.4 mM CaCl2, 1.3 mM MgCl2, and 275 mg glucose per 100 ml; NaHCO3 was adjusted to result in a constant pH range of 7.37–7.40. After rinsing the lungs with at least 1 l of buffer fluid for washout of blood, the perfusion circuit was closed for recirculation (total system volume 250 ml). Meanwhile, the flow was slowly increased from 20 to 150 ml min-1, and left atrial pressure was set at 1.5–2.0 mm Hg to ensure zone III conditions throughout the lung at end expiration. The alternate use of two separate perfusion circuits allowed repeated exchange of buffer fluid. In parallel with the onset of artificial perfusion, ventilation was changed from room air to a mixture of 5.3% CO2, 21.0% O2, balance N2 (tidal volume, 30 ml; frequency, 30 strokes min-1). A positive end-expiratory pressure (PEEP) of 1 cm H2O was chosen (0 referenced at the hilum). The isolated perfused lungs were placed in a temperature-equilibrated housing chamber, freely suspended from a force transducer for continuous monitoring of organ weight. The whole system (perfusate reservoirs, tubing, housing chamber) was heated to 38.5°C. Pressures in the pulmonary artery, the left atrium, and the trachea were registered by means of small-diameter tubing threaded into the perfusion catheters and the trachea and connected to pressure transducers. Lungs included in the study were those that (i) had a homogeneous white appearance with no signs of hemostasis, edema, or atelectasis; (ii) revealed constant mean pulmonary artery and peak ventilation pressure in the normal range; and (iii) were isogravimetric during the initial steady-state period of at least 20 min.

Hypoxic Maneuvers and Pharmacologic Challenges
The technique of successive hypoxic maneuvers in buffer-perfused rabbit lungs has been described previously (18). 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]). 5.3% vol/vol CO2 was used throughout, and the percentage of N2 was balanced accordingly. Sequential hypoxic maneuvers of 10-min duration, interrupted by 15-min periods of normoxia, were performed. The effects of the various pharmacologic agents on pressure responses provoked by alveolar hypoxia (3% O2) were determined within such a sequence of repetitive hypoxic maneuvers. Each agent was added to the buffer fluid 5 min before a hypoxic challenge, starting the addition after accomplishing the second hypoxic maneuver. Cumulative dose–effect curves were established. Controls received the solvent only. For comparison, the influence of the applied agents on U46619-elicited pressor responses was tested in a corresponding time schedule. In these experiments, a mode of repetitive bolus applications of the stable thromboxane analog was employed (addition to the perfusate at 0.5 nM every 25 min) as described previously (19, 20). In each lung preparation the response to the second vasoconstrictor provocation in a sequence of challenges was set at 100% (= reference response). The strength of the following vasoconstrictor responses was related to this reference response. Control experiments were performed without addition of mitochondrial inhibitor or uncoupler. In the experiments with pre-blocked NO-synthesis, 400 µM L-NMMA was present in the perfusate from the beginning of the experiments.

Lung weight was continuously monitored. The total weight gain ranged < 3 g in all experiments, except in the experiments with FCCP, which induced severe edema.

Monitoring of Exhaled NO and Effects of Pharmacologic Intervention on Baseline Pulmonary Artery Pressure
The technique for determination of exhaled NO levels has been described previously (21). Briefly, an aliquot of the mixed expired gas was continuously forwarded to a chemiluminescence NO-analyzer (Sievers 280 NOA; Sievers Instruments, Boulder, CO), and its NO quantity measured in ppb (parts per billion, vol/vol). Measurement of exhaled NO was performed in normoxic ventilated lungs. In these experiments the effect of the mitochondrial inhibitors and uncouplers on normoxic vascular tone was determined in a time-schedule according to the experiments with hypoxic ventilation (application of the agents every 25 min except for DIDS, where reapplication was done after the increase in pulmonary artery pressure [PAP] had reached a maximum).

Statistics
For comparison of multiple groups, ANOVA with the Student-Newman-Keuls post hoc test was performed. For comparison of two groups a paired t test was applied. Statistical significance was assumed when P ranged < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mean PAP values were 7.3 ± 0.2 mm Hg (mean ± SEM, n = 142) under baseline conditions. A hypoxic ventilation with 3% O2 (alveolar PO2 ~ 23 mm Hg) provoked a rapid increase in PAP, with a mean pressure elevation of 2.4 ± 0.2 mm Hg (mean ± SEM, n = 56). Repetitive hypoxic challenges resulted in well reproducible pressure elevations within the same lung as previously described (18). Baseline NO exhalation was 109 ± 6 ppm (n = 32). Hypoxic ventilation resulted in an immediate reduction of NO exhalation by 24.2 ± 1.8% (n = 10) as referenced to baseline NO exhalation. This reduction was fully reversible upon reoxygenation as previously described (4). Addition of 400 µM L-NMMA completely blocked lung NO exhalation, only marginally affected normoxic vascular tone, but increased hypoxic pressor responses throughout the experiments to 5.9 ± 0.3 mm Hg (mean ± SEM, n = 48) as previously described in detail (4). Repetitive bolus application of U46619 provoked well reproducible vasoconstrictor responses under baseline conditions (3.1 ± 0.3 mm Hg; mean ± SEM, n = 56), again enhanced in the presence of L-NMMA (5.2 ± 0.5 mm Hg, mean ± SEM, n = 48).

Rotenone (30–350 nM) dose-dependently suppressed HPV (Figure 1). This inhibition was in part specific for HPV, as determined from the comparison with U46619-induced vasoconstrictions. Under conditions of pre-blocked lung NO synthesis, the selectivity for HPV inhibition was more pronounced, as the effects of rotenone on U46619-induced vasoconstrictions were then only very minor. In corresponding experiments with normoxic ventilation, rotenone induced a transient vasoconstriction at 170 and 350 nM, with some persistent effect noted for 170 nM (Table 1). However, no such persistent elevation was found when lung NO synthesis was pre-blocked (Table 2). Post-hypoxic vasorelaxation was not affected by rotenone (Table 2). During normoxic ventilation, addition of rotenone induced a transient increase in NO exhalation which reached its maximum between 1.8 and 2.2 min, with a subsequent decrease to a level slightly lower than in controls at a time corresponding to the onset of hypoxic ventilation (Table 3).



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  		Figure 1. Effects of rotenone on the strength of hypoxia- and U46619-induced vasoconstrictions during intact (-L-NMMA) and blocked NO synthesis (+L-NMMA). {Delta}PAP, strength of hypoxia or U46619-elicited increase in pulmonary arterial pressure, referenced to the second vasoconstrictive maneuver. Cumulative dose–effect curves are shown. Values are mean ± SEM. Controls received no rotenone. *Significant differences as compared with the corresponding control. **Significant differences in the strength of "HPV rotenone" as compared with "HPV control" and U46619-induced vasoconstrictions in the presence of rotenone. Open circles, HPV control (n = 4); filled circles, HPV rotenone (n = 4); open triangles, U-46619 control (n = 4); filled triangles, U-46619 rotenone (n = 4).

 

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  		TABLE 1 Effects of rotenone, MPP+, 3-NPA, TTFA, myxothiazol, antimycin A, HQNO, DNP, and DIDS on baseline PAP during normoxic ventilation

 

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  		TABLE 2 Effects of rotenone, MPP+, 3-NPA, TTFA, myxothiazol, antimycin A, HQNO, and DNP on baseline pulmonary artery pressure (PAP) and on vasorelaxation induced by posthypoxic reoxygenation during pre-blocked lung NO synthesis

 

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  		TABLE 3 Effects of rotenone, MPP+, 3-NPA, TTFA, myxothiazol, antimycin A, HQNO, and DNP on pulmonary nitric oxide generation during normoxic ventilation

 
MPP+ (1–100 µM) suppressed HPV only at the highest dosage applied (Figure 2). This inhibition was specific for HPV, as U46619-induced vasoconstriction was dose-dependently enhanced by MPP+. The same dose–inhibition profile was found during pre-blocked lung NO synthesis. In corresponding experiments with normoxic ventilation, MPP+ increased normoxic vascular tone (Table 1). The elevation of normoxic vascular tone was lost for the highest dosage applied during conditions of pre-blocked lung NO synthesis (Table 2). During normoxic ventilation, addition of MPP+ resulted in a decrease in NO exhalation (Table 3).



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  		Figure 2. Effects of MPP+ on the strength of hypoxia- and U46619-induced vasoconstrictions during intact (-L-NMMA) and blocked NO synthesis (+L-NMMA). {Delta}PAP, strength of hypoxia, or U46619-elicited increase in PAP, referenced to the second vasoconstrictive maneuver. Cumulative dose–effect curves are shown. Values are mean ± SEM. Controls received no MPP+. *Significant differences as compared with the corresponding control. **Significant differences in the strength of "HPV MPP+" as compared with "HPV control" and U46619-induced vasoconstrictions in the presence of MPP+. Open circles, HPV control (n = 4); filled circles, HPV MPP+ (n = 4); open triangles, U-46619 control (n = 4); filled triangles, U-46619 MPP+ (n = 4).

 
3-NPA (2.5–5.5 mM) dose-dependently inhibited HPV to 12.4 ± 2.8 (n = 4) of the initial response (Figure 3). The strength of U46619-induced vasoconstrictions was also reduced by 3-NPA, however, to a significantly lower degree than the hypoxia-induced vasoconstrictor response. This partial specificity for HPV was reduced during pre-blocked lung NO synthesis. Normoxic vascular tone was not affected by 3-NPA, neither during intact nor after pre-blocked lung NO synthesis (Tables 1 and 2). Post-hypoxic vasorelaxation was also not affected by 3-NPA (Table 2). Exhaled NO levels were slightly decreased after addition of 3-NPA (Table 3).



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  		Figure 3. Effects of 3-NPA on the strength of hypoxia- and U46619-induced vasoconstrictions during intact (-L-NMMA) and blocked NO synthesis (+L-NMMA). {Delta}PAP, strength of hypoxia, or U46619-elicited increase in pulmonary arterial pressure, referenced to the second vasoconstrictive maneuver. Cumulative dose–effect curves are shown. Values are mean ± SEM. Controls received no 3-NPA. *Significant differences as compared with the corresponding control. **Significant differences in the strength of "HPV 3-NPA" as compared with "HPV control" and U46619-induced vasoconstrictions in the presence of 3-NPA. Open circles, HPV control (n = 4); filled circles, HPV 3-NPA (n = 4); open triangles, U-46619 control (n = 4); filled triangles, U-46619 3-NPA (n = 4).

 
TTFA (40–300 µM) dose-dependently reduced HPV to 7.8 ± 5.0 (n = 4, Figure 4). This inhibition was unspecific for HPV by comparison with U46619-induced vasoconstrictions. Pre-blockage of lung NO generation did not affect the impact of TTFA on hypoxia- and U46619-induced vasoconstrictions. No major effects of TTFA on normoxic vascular tone were observed (Table 1). This was also true during pre-blocked NO synthesis (Table 2). Post-hypoxic vasorelaxation was also not affected by TTFA (Table 2). TTFA increased lung NO release with an immediate peak and a slight elevation being persistent for 200 µM (Table 3).



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  		Figure 4. Effects of TTFA on the strength of hypoxia- and U46619-induced vasoconstrictions during intact (-L-NMMA) and blocked NO synthesis (+L-NMMA). {Delta}PAP, strength of hypoxia, or U46619-elicited increase in pulmonary arterial pressure, referenced to the second vasoconstrictive maneuver. Cumulative dose–effect curves are shown. Values are mean ± SEM. Controls received no TTFA. *Significant differences as compared with the corresponding control. Open circles, HPV control (n = 4); filled circles, HPV TTFA (n = 4); open triangles, U-46619 control (n = 4); filled triangles, U-46619 TTFA (n = 4).

 
Myxothiazol (5–80 nM) dose-dependently reduced HPV to 8.4 ± 5.7% of the initial response at 80 nM (n = 4, Figure 5). This inhibition did, however, largely lack specificity, as obvious from comparison with U46619-induced vasoconstrictions. This was all the more true under conditions of pre-blocked NO synthesis, where inhibitory curves on HPV and on U46619-induced vasoconstriction were largely superimposable. Normoxic vascular tone was transiently increased by myxothiazol when applying the highest dose (80 nM, Table 1), with this effect lasting slightly more than 5 min. However, the vasoconstrictor potency during normoxia disappeared when NO synthesis was pre-blocked (Table 2). Post-hypoxic vasorelaxation was not affected by myxothiazol (Table 2). Myxothiazol transiently increased NO exhalation with a peak at 3.2–3.8 min after its addition to the perfusate, with this elevation persisting for more than 5 min (the time corresponding to the starting point of hypoxic ventilation). The myxothiazol-induced increase in NO exhalation then returned to baseline levels with some decrease at 15 and 25 min after myxothiazol application (Table 3).



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  		Figure 5. Effects of myxothiazol on the strength of hypoxia- and U46619-induced vasoconstrictions during intact (-L-NMMA) and blocked NO synthesis (+L-NMMA). {Delta}PAP, strength of hypoxia, or U46619-elicited increase in pulmonary arterial pressure, referenced to the second vasoconstrictive maneuver. Cumulative dose–effect curves are shown. Values are mean ± SEM. Controls received no myxothiazol. *Significant differences as compared with the corresponding control. **Significant differences in the strength of "HPV myxothiazol" as compared with "HPV control" and U46619-induced vasoconstrictions in the presence of myxothiazol. Open circles, HPV control (n = 4); filled circles, HPV myxothiazol (n = 4); open triangles, U-46619 control (n = 4); filled triangles, U-46619 myxothiazol (n = 4).

 
Antimycin A (1–4 nM) dose-dependently inhibited the hypoxic response to 13.8 ± 7.0% (n = 4) upon use of the highest dose (Figure 6). This inhibition was highly specific for the hypoxic response, as vasoconstrictions elicited by the thromboxane analog U46619 were not affected by antimycin A. However, this specifity was largely lost during pre-blockage of lung NO synthesis, with the U46619-induced vasoconstrictions then being also significantly inhibited by antimycin A. Normoxic vascular tone was only transiently elevated at 4 nM antimycin A (Table 1), with this elevation nearly having reached baseline levels at a time corresponding to the onset of hypoxic ventilation according to the routine sequence of maneuvers. In experiments with pre-blocked NO synthesis, no elevation was found at a time corresponding to the onset of hypoxic ventilation (Table 2). Post-hypoxic vasorelaxation was not affected by antimycin A (Table 2). There was a slight increase in exhaled NO levels at 2 nM and 4 nM (Table 3).



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  		Figure 6. Effects of antimycin A on the strength of hypoxia- and U46619-induced vasoconstrictions during intact (-L-NMMA) and blocked NO synthesis (+L-NMMA). {Delta}PAP, strength of hypoxia, or U46619-elicited increase in pulmonary arterial pressure, referenced to the second vasoconstrictive maneuver. Cumulative dose–effect curves are shown. Values are mean ± SEM. Controls received no antimycin A. *Significant differences as compared with the corresponding control. **Significant differences in the strength of "HPV antimycin A" as compared with "HPV control" and U46619-induced vasoconstrictions in the presence of antimycin A. Open circles, HPV control (n = 4); filled circles, HPV antimycin A (n = 4); open triangles, U-46619 control (n = 4); filled triangles, U-46619 antimycin A (n = 4).

 
HQNO (2–5 µM) dose-dependently blocked HPV with a complete inhibition at 5 µM, whereas the U46619-induced vasoconstrictor response was largely unaffected (Figure 7). Complete and specific inhibition was also found during pre-blocked lung NO synthesis. HQNO dose-dependently increased normoxic vascular tone (Table 1) also observed during pre-blocked lung NO synthesis (Table 2). This PAP increase was, however not persistent over the entire observation period of 25 min (Tables 1 and 2). Addition of HQNO to the perfusate transiently increased lung NO exhalation (Table 3).



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  		Figure 7. Effects of HQNO on the strength of hypoxia- and U46619-induced vasoconstrictions during intact (-L-NMMA) and blocked NO synthesis (+L-NMMA). {Delta}PAP, strength of hypoxia, or U46619-elicited increase in pulmonary arterial pressure, referenced to the second vasoconstrictive maneuver. Cumulative dose–effect curves are shown. Values are mean ± SEM. Controls received no HQNO. *Significant differences as compared with the corresponding control. **Significant differences in the strength of "HPV HQNO" as compared with "HPV control" and U46619-induced vasoconstrictions in the presence of HQNO. Open circles, HPV control (n = 4); filled circles, HPV HQNO (n = 4); open triangles, U-46619 control (n = 4); filled triangles, U-46619 HQNO (n = 4).

 
The mitochondrial uncoupler DNP (10–200 µM) specifically blocked the hypoxic response at 100 µM and 200 µM, with a transiently increased response at 10 µM (Figure 8). Suppression of the U46619-induced vasoconstriction was only noted at 200 µM DNP. Virtually the same effects were found during pre-blocked lung NO synthesis. DNP substantially increased normoxic vascular tone during intact and pre-blocked NO-synthesis (Tables 1 and 2). The same effects on HPV and baseline tone were observed for a second uncoupler (FCCP, 1–3 µM). However, as this agent induced severe edema formation, data are not given in detail. Neither DNP nor FCCP affected post-hypoxic vasorelaxation (Table 2). A transient increase in exhaled NO was found upon addition of DNP to the perfusate, which returned to baseline levels at a time point corresponding to the onset of hypoxic ventilation with some further decrease at 15 and 25 min after its application (Table 3).



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  		Figure 8. Effects of DNP on the strength of hypoxia- and U46619-induced vasoconstrictions during intact (-L-NMMA) and blocked NO synthesis (+L-NMMA). {Delta}PAP, strength of hypoxia, or U46619-elicited increase in pulmonary arterial pressure, referenced to the second vasoconstrictive maneuver. Cumulative dose–effect curves are shown. Values are mean ± SEM. Controls received no DNP. *Significant differences as compared with the corresponding control. **Significant differences in the strength of "HPV DNP" as compared with "HPV control" and U46619-induced vasoconstrictions in the presence of DNP. Open circles, HPV control (n = 4); filled circles, HPV DNP (n = 4); open triangles, U-46619 control (n = 4); filled triangles, U-46619 DNP (n = 4).

 
Addition of the anion channel blocker DIDS (50–600 µM) dose-dependently suppressed the hypoxic response (Figure 9). This inhibition was, however, fully unspecific for HPV, because of being nearly superimposable upon U46619-induced vasoconstriction. DIDS decreased normoxic vascular tone at a time corresponding to the initiation of hypoxic ventilation at 200 µM (Table 1).



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  		Figure 9. Effects of DIDS on the strength of hypoxia- and U46619-induced vasoconstrictions. {Delta}PAP, strength of hypoxia, or U46619-elicited increase in pulmonary arterial pressure, referenced to the second vasoconstrictive maneuver. Cumulative dose–effect curves are shown. Values are mean ± SEM. Controls received no DIDS. *Significant differences as compared with the corresponding control. Open circles, HPV control (n = 4); filled circles, HPV DIDS (n = 4); open triangles, U-46619 control (n = 4); filled triangles, U-46619 DIDS (n = 4).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study employed a well established model of HPV in intact rabbit lungs (4, 10, 11, 18–20) to test the "unifying mitochondrial hypothesis" for hypoxic pulmonary vasoconstriction. The advantages of this model are: (i) the excellent reproducibility of sequentially performed hypoxic vasoconstriction maneuvers, allowing the establishment of dose-effect curves within one lung; (ii) the strict control by a non–hypoxia-related pulmonary vasoconstrictor; (iii) the possibility to have a "second view" on all inhibitor effects while blocking NO synthesis, to dissect NO-related and NO-unrelated effects of agents investigated; (iv) the online monitoring of NO exhalation to have a direct readout of the impact of the inhibitors and uncouplers under investigation on NO synthesis; and (v) the independent investigation of whether the inhibitors tested might mimic hypoxic vasoconstriction during normoxia. The mitochondrial hypothesis suggests that hypoxia increases the generation of superoxide at the level of complex III of the precapillary smooth muscle cells, with enhanced O2- egress through an anion channel in the inner mitochondrial membrane and subsequent induction of smooth muscle contraction (6, 14–16). According to this hypothesis, the following effects are to be expected:
  • Rotenone, MPP+, myxothiazol, TTFA, and 3-NPA, inhibitors of the proximal part of the electron transport chain (complex I, complex II, and complex III upstream of ubisemiquinone), should specifically inhibit HPV, exert their effect independent of the NO system, but should not trigger pulmonary vasoconstriction during normoxia (7, 13, 15, 22, 23).
  • Antimycin A, an inhibitor of the mitochondrial electron transport chain downstream of ubisemiquinone, is expected to enhance the hypoxic vasoconstrictor response, even to mimic this response during normoxia, independent of any putative influence on the NO system (13, 15). The same effects are expected for HQNO, which is suggested to bind at a site identical or in the vicinity of the antimycin-binding site (24, 25).
  • The mitochondrial uncouplers DNP and FCCP, which abrogate the proton gradient and thereby maximally increase the mitochondrial electron transport throughput, were noted to reduce the superoxide leak at complex III (26). Thus, these agents are expected to specifically inhibit HPV, to exert their effect independent of the NO system, and to induce no vasoconstriction during normoxia.
  • The same profile is again expected for the anion channel blocker DIDS, due to interference with the O2- egress into the cytoplasm (6, 13).

It is important to determine possible interactions of the inhibitors and uncouplers with lung NO generation because we previously demonstrated that lung NO generation significantly contributes to the regulation of HPV: (i) Hypoxic ventilation results in a rapid decline in exhaled NO levels, whereas intravascularly released NO is not affected by hypoxia. (ii) The decrease in exhaled NO precedes the hypoxia-induced increase in PAP and is fully reversible after cessation of hypoxic ventilation. (iii) The amplification of HPV induced by inhibition of lung NO synthesis is higher than that of non–hypoxia-induced vasonstrictions. These data allow the conclusion that NO does not significantly contribute to the regulation of normoxic vascular tone in rabbits, but that a portion of NO synthesis measured in the exhaled gas plays an important role for the regulation of HPV (4, 21, 27).

Against this background of theoretical considerations, the profiles of the different agents investigated turned out as follows.

Rotenone
This agent indeed caused a dose-dependent inhibition of HPV, which was significantly more prominent than the inhibition of U46619-induced vasoconstriction, and this specifity of action was even increased while blocking the NO synthesis (i.e., it was independent of the NO system). Some HPV mimic during normoxia was observed with an intact NO system, but was absent upon NO blockade. Thus, the rotenone data support the mitochondrial hypothesis of HPV as outlined above. The specificity of the rotenone effects on HPV suppression and the effects on normoxic vascular tone are in accordance with investigations in isolated buffer- and blood-perfused rat lungs, isolated intrapulmonary pulmonary arteries of the rat, and isolated smooth muscle cells from the pulmonary arteries (12, 13, 28, 29). Going beyond these investigations, our study clarified that the specificity of the rotenone effect was not due to an interference with lung NO synthesis.

MPP+
This compound mimicked hypoxia by increasing normoxic vascular tone, and inhibition of HPV was noted at the highest dosage applied. This inhibition was highly specific for HPV by comparison with U46619-induced vasoconstriction. The MPP+ effect on HPV was not dependent on lung NO generation because the presence of L-NMMA did not affect the inhibition of HPV achieved by MPP+ and exhaled NO was decreased by MPP+. However, the increase in normoxic vascular tone was not found for the highest dosage of MPP+ applied during pre-blocked lung NO generation. The increase in normoxic vacular tone by MPP+ is not in accordance with the mitochondrial hypothesis, with complex I inhibitors suggested to inhibit HPV without being a hypoxia mimic. However, the fact that an increase in normoxic vascular tone already occurred at doses which did not affect HPV and was lost during pre-blocked lung NO synthesis at the MPP+ dose which inhibited HPV also offers the explanation that an effect of MPP+ on normoxic vascular tone is unrelated to HPV mechanisms.

3-NPA
This agent attenuated HPV to a higher degree than the U46619-induced vasoconstrictor response. 3-NPA, as expected, did not increase normoxic vascular tone. These effects are thus in principle in accordance with the hypothesis of increased superoxide release from complex III as the underlying mechanism of HPV. Going beyond the hypothesis that complex III is the major site of ROS generation during hypoxia, the 3-NPA data are also in line with the very recent observation that complex II plays an essential role for hypoxic ROS generation (7). However, despite the fact of a stronger inhibition of HPV in comparison with U46619-induced vasoconstrictor responses, a prominent inhibition of the U46619-induced vasoconstriction was also found. This reveals that the major portion of the 3-NPA effect is unspecific for HPV.

TTFA
Even if the effects of TTFA on HPV and normoxic vascular tone are in accordance with the hypothesis that an increase in ROS at complex III triggers HPV, the TTFA experiments do not allow any conclusion about the contribution of mitochondria to the oxygen sensing mechanism underlying HPV, as the U46619-induced vasoconstrictor response was suppressed with an identical inhibition profile. Concerning the recently proposed essential role of complex II for hypoxic ROS generation, the (partial) unspecificity of 3-NPA and TTFA reveal that at least for acute HPV (occurring within minutes) these agents are limited for investigations in intact perfused lung preparations.

Myxothiazol
This agent inhibited HPV only slightly more prominent than U46619-induced vasoconstriction. Moreover, part of this inhibition may be explained by an increase in lung NO synthesis in response to this agent. When assessing the myxothiazol effect during blockade of NO generation, the specifity of the inhibitory effect was entirely lost. Due to this lack of specificity, the myxothiazol data of the present study neither support nor contradict the mitochondrial hypothesis. Myxothiazol was previously used in studies with intrapulmonary arteries of the rat (12) and in a study of Waypa and coworkers (13) in isolated perfused rat lungs as well as in isolated pulmonary artery smooth muscle cells. The absence of a sustained rise of normoxic vascular tone in our investigation is in accordance with both of these studies. But whereas in the first investigation no comparison with a non–hypoxia-induced vasoconstrictor was done, the second study found specific inhibition of HPV by comparison with U46619-induced vasoconstriction. This is in contrast to our investigation, where only a small difference was observed in the inhibitor profile for myxothiazol on HPV and U46619-induced vasoconstriction, which disappeared during pre-blocked lung NO synthesis. Interference with the rat NO system may thus explain the differences to the study of Waypa and colleagues (13).

Antimycin A
This agent specifically inhibited HPV in the absence of L-NMMA, with some specificity still being noted while blocking NO synthesis. It did, however, not mimic HPV during normoxia, in particular not when blocking the influence of this agent on the NO formation. Thus, this profile contradicts the expectation that an inhibition of the electron transport chain downstream of ubisemiquinone should mimic (but not inhibit) HPV. This is in line with previous data in isolated rat lungs, where also only transient vasconstriction by antimycin A during normoxic conditions was found (28, 29). Going beyond these data, the present study excluded that the lack of mimicking HPV during normoxia might be hidden behind some interference with the NO system. Waypa and coworkers (13) recently showed that antimycin A induced contraction in isolated smooth muscle cells from pulmonary arteries. However, data for the effect of antimycin A on HPV and U46619-induced vasoconstrictions were not given, and the same authors found only unspecific inhibition of HPV in isolated perfused rat lungs with only a transient vasoconstrictor effect during normoxic ventilation. Some evidence for HPV specificity by comparison with non–hypoxia-induced vasoconstrictions was forwarded by Archer and colleagues (28) in isolated perfused rat lungs, whereas Rounds and McMurtry (29) only found marginal differences in the inhibition profile of antimycin A concerning hypoxia- and non–hypoxia-related events in isolated perfused rat lungs. A recent investigation demonstrated that antimycin A did not increase normoxic ROS generation, but inhibited hypoxia-induced ROS increase (7). This effect is in accordance with our observation that normoxic vascular tone is not affected by antimycin A but that HPV is inhibited by this agent. Parts of the discrepancies between the various studies may be related to species differences in lung NO metabolism, as this system is clearly affected by antimycin A.

HQNO
This agent is suggested to exert the same effects as antimycin A. However, in contrast to antimycin A, HQNO did not interfere with the lung NO system and specifically suppressed HPV in the presence as well as in the absence of lung NO generation. Additionally, normoxic vascular tone was increased by HQNO. Thus, the HQNO data are fully in agreement with the concept of an increased superoxide generation at complex III as the mechanism triggering HPV.

The Uncouplers DNP and FCCP
Apart from HQNO and MPP+, these were the only agents which forwarded a sustained vasoconstriction during normoxia, in particular in the lower and medium concentration ranges, being even more prominent in the presence of L-NMMA. Though NO exhalation was affected by DNP, this mimicking of HPV during normoxia was thus not attributable to an impact of the uncouplers on the NO system. Starting from an already enhanced level of vascular tone, the hypoxia-induced additional vasoconstriction was then further enhanced at low, and inhibited at high DNP concentrations, the latter findings being significantly different from the impact on U46619-induced vasoconstriction. Both agents thus fulfill the profile of a HPV mimic, which is in contrast to the above-discussed concept that due to reduced superoxide leak at the level of complex III an inhibition of HPV is to be anticipated. This allows two interpretations. Either the assumption of reduced mitochondrial superoxide leak in the presence of these uncouplers is wrong for the pulmonary vascular smooth muscle cells executing HPV (the findings supporting this view do, indeed, originate from other cell types; Ref. 26), or these findings contradict the above-outlined mitochondrial hypothesis of HPV. In line with our investigation, some sustained vasoconstrictor response to DNP was also observed in blood-perfused dog lungs (30). The effect of the uncouplers could, however, also be related to effects of these agents independent from mitochondrial ROS release, as they may also have an impact on mitochondrial pH, cellular pH, and Ca2+ as well as K+ homeostasis (31–34).

The Anion Channel Blocker DIDS
This agent inhibited HPV and U-46619-induced vasoconstriction with superimposable dose-inhibition curves. The data thus do not support the view that interference with the mitochondrial superoxide egress allows selective inhibition of hypoxia-induced as compared with control vasoconstriction. DIDS has been suggested to inhibit the release of superoxide from the mitochondrion and was shown to specifically inhibit HPV without any effect on baseline pulmonary artery pressure in isolated rat lungs and pulmonary artery smooth muscle cells (13), an effect which could not be reproduced in the rabbit lungs of the present study.

Beside the recent studies of Waypa and associates (13), the aspect of lung reactive oxygen species production from mitochondria was formerly addressed in an investigation by Archer and colleagues (28). They found a decrease in ROS release when challenging the lung with hypoxia, rotenone, and antimycin A, measured by lucigenin and luminol-enhanced chemiluminescence. This is in contrast to the current concept of increased ROS as a trigger for HPV (13). However, overall lung ROS generation as measured in isolated rat lung perfusate (28) may not at all be representative for ROS formation in a specific, small compartment such as the precapillary smooth muscle cells. This interpretation is in line with the recent findings of Waypa and coworkers, who demonstrated that DPI, rotenone, and myxothiazole decrease hypoxia-induced Ca2+ release in pulmonary arterial myocytes whereas antimycin A and cyanide did not alter hypoxia-induced Ca2+ increases in these cells (35).

Summarizing all results, the rotenone and HQNO data fully support the hypothesis that enhanced superoxide leak at complex III of the mitochondrial electron transport chain underlies hypoxia-induced pulmonary vasoconstriction. Due to lack of specifity for HPV, the inhibitory effects of myxothiazol, TTFA, and DIDS may not be used to support the concept, and the results with the uncouplers DNP and FCCP, and in part the antimycin A and MPP+ data argue against this hypothesis. At the present stage, we come to the following conclusions:

  • (i) Definite proof for the concept that enhanced superoxide leak at complex III during hypoxia triggers HPV in the intact lung is still missing. The present study provides evidence against as well as in favor of this concept. There are good arguments for the concept of a unifying mitochondrial hypothesis also underlying HPV as recently shown by Waypa and colleagues (35). However, recent findings in airway neuroepithelial bodies (addressing chemoreception; Ref. 36) and in mutant cells that lack mitochondrial respiration (addressing HIF induction; Ref. 37), as well as some portion of the present data in the perfused lungs, are not fully compatible with the unifying mitochondrial hypothesis of hypoxia sensing.
  • (ii) Despite the many inconsistencies described, which may partly be attributable to the lack of specificity of the inhibitors employed and the complexity of the intact organ model, the body of experiments nevertheless signals that mitochondria are in some manner involved in hypoxic pulmonary vasoconstriction. Alternatives to the suggested enhanced superoxide leak at complex III and the suggested contribution of complex II should be elaborated to clarify the link between mitochondrial respiratory chain changes under reduced PO2 conditions and the hypoxia induced changes in lung vasomotor tone.


   Acknowledgments
 
The authors thank K. Quanz for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft, SFB 547, project B7.


   Footnotes
 
* Portions of the doctoral theses of Nadine Ebert and Marit Ahrens are incorporated into this report. Back

Received in original form October 17, 2002

Received in final form May 5, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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