Department of Internal Medicine, Justus-Liebig-University Giessen, 35392 Giessen, Germany
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ABSTRACT |
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It has been suggested that hypoxic pulmonary vasoconstriction (HPV) may mainly proceed via loss of normoxic vasodilation, forwarded by tonic O2-dependent formation of nitric oxide and superoxide (23). Both agents may stimulate guanylate cyclase, the latter via conversion to hydrogen peroxide and formation of compound I with catalase. We probed this hypothesis in perfused rabbit lungs, employing the superoxide scavengers superoxide dismutase (SOD), 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), and nitro blue tetrazolium (NBT) and the catalase inhibitor aminotriazole (AT). NBT turned out to be a potent dose-dependent inhibitor of HPV in a concentration range of 200 nM to 1 µM, and superimposable dose-inhibition curves were obtained when lung synthesis of nitric oxide and vasodilatory prostanoids was preblocked by NG-monomethyl-L-arginine (L-NMMA) and acetylsalicylic acid (ASA). The NBT effect was specific because no inhibition in the vasoconstrictor responses to the stable thromboxane analog U-46619 and angiotensin II was observed. In contrast, SOD and Tiron were ineffective. AT exerted nonspecific inhibition of the hypoxia- and chemical vasoconstrictor-induced pressor responses. When applied under normoxic conditions, however, NBT alone or coapplied with L-NMMA or ASA, both for blockage of parallel vasodilatory pathways, did not mimic the hypoxia-induced vasoconstrictor response. In conclusion, the present study supports an important role for superoxide in the basic mechanism of HPV, but it questions the concept that loss of tonic vasorelaxation via this pathway is the underlying event in rabbit lungs. The mechanisms relating O2 tension-dependent superoxide and hydrogen peroxide generation to the vasoconstrictor event occurring in HPV remain to be further elucidated.
hydrogen peroxide; hypoxic pulmonary vasoconstriction; isolated lung; pulmonary hypertension; superoxide
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INTRODUCTION |
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HYPOXIC PULMONARY VASOCONSTRICTION (HPV) helps prevent arterial hypoxemia by matching perfusion to ventilation (for reviews, see Refs. 14, 35). Since the first description of this mechanism by von Euler and Liljestrand in 1946 (36), great efforts have been made to identify the cell(s) responsible for O2 sensing, the sensor mechanism(s), and the pathway(s) of signal transduction leading to contraction of the vascular smooth muscle cells in the precapillary resistance vessels, the predominant site of HPV. However, the mechanism of HPV has not yet been clarified in detail.
There is good evidence that nitric oxide (NO), a stimulator of intracellular cGMP production, is involved in this mechanism. NO is continuously generated by lung cells, released into both the air space and the vascular compartment. Alveolar hypoxia provokes an immediate drop of NO exhalation, which precedes the pulmonary arterial pressure (PAP) increase and is not observed in response to other vasoconstrictor stimuli (11, 15). Inhibition of lung NO generation exaggerates HPV (3, 15). However, blocking the rabbit lung NO synthesis in the absence of vasoconstrictor agents does not substantially affect the baseline pulmonary vascular tone; i.e., it does not mimic HPV. Hypoxia-induced downregulation of the tonic production of NO thus appears to contribute to HPV but may not fully explain this mechanism.
Preceding studies from different laboratories including our own (2, 16,
22, 34, 38) presented evidence for the involvement of
NAD(P)H oxidase-dependent
O2 radical formation in hypoxia
sensing. Diphenyleneiodonium was shown to inhibit the hypoxia-induced
vasoconstriction but not the pressor response to other vasoactive
stimuli. It was suggested that there is tonic O2-dependent production of
superoxide anion (O2·), which is
then metabolized to hydrogen peroxide
(H2O2),
which reacts with catalase to form compound I, and the latter agent
then provides permanent non-NO-dependent stimulation of guanylate
cyclase and cGMP-dependent vasorelaxation (22, 23). Hypoxia will then lead to decreased superoxide generation and thus loss of cGMP-dependent vasorelaxation.
The present study addressed this hypothesis in a perfused lung model of HPV. We investigated the influence of three superoxide scavengers [superoxide dismutase (SOD), 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), and nitro blue tetrazolium (NBT)] and a catalase inhibitor [aminotriazole (AT)]. We took care that any effects of these agents might not be explained by some additional interference with NO synthesis and asked for specificity with respect to HPV by comparing dose-inhibition curves with those obtained with the use of hypoxia-independent vasoconstrictor agents. In essence, we found that NBT, but not the other agents, specifically inhibited hypoxia-induced pulmonary vasoconstriction, supporting a role for superoxide in the mechanisms of HPV. However, even simultaneous blocking of the postulated NO- and superoxide-H2O2-dependent tonic guanylate cyclase stimulation under normoxic conditions did not mimic the hypoxic stimulus in rabbit lungs.
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MATERIALS AND METHODS |
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Reagents. Tiron, SOD, NBT, AT, ferricytochrome c, and [Asn1,Val5]angiotensin (ANG) II were purchased from Sigma (Munich, Germany). Acetylsalicylic acid (ASA; Aspisol) was obtained from Bayer (Leverkusen, Germany), NG-monomethyl-L-arginine (L-NMMA) was from Calbiochem (Frankfurt, Germany), and U-46619 (stable thromboxane analog) was obtained from Paesel+Lorei (Frankfurt, Germany). The perfusate was provided by Serag-Wiessner (Naila, 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 (28, 29, 37). Briefly, pathogen-free rabbits of either sex (body weight 2.2-3.2 kg) were deeply anesthetized and anticoagulated with heparin (1,000 U/kg body weight). The lungs were excised while being perfused with Krebs-Henseleit buffer through cannulas in the pulmonary artery and 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 of glucose per 100 ml; pH was adjusted with NaHCO3 to result in a constant range of 7.37-7.40. After the lungs were rinsed with at least 1 liter of buffer fluid for the washout of blood, the perfusion circuit was closed for recirculation (total system volume 350 ml). Meanwhile, the flow was slowly increased from 20 to 150 ml/min, and left atrial pressure was set at 1.5-2.0 mmHg 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). A positive end-expiratory pressure of 1 cmH2O was chosen (zero 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, and housing chamber) was heated to 38.5°C. Pressures in the pulmonary artery, left atrium, and trachea were registered by means of small-diameter tubing threaded into the perfusion catheters and trachea and connected to pressure transducers. Lungs included in the study were those that 1) had a homogeneous white appearance with no signs of hemostasis, edema, or atelectasis; 2) revealed constant mean PAP and peak ventilation pressure in the normal range; and 3) were isogravimetric during an initial steady-state period of at least 20 min.
Hypoxic maneuvers and pharmacological challenges. The technique of sequential hypoxic maneuvers in buffer-perfused rabbit lungs has been previously described (37). 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 (PAO2) ~ 160 mmHg; baseline condition] to 3% (vol/vol; PAO2 ~ 23 mmHg; hypoxic condition)}. A CO2 content of 5.3% (vol/vol) was used throughout, and the percentage of N2 was balanced accordingly. Buffer returning from the perfusate reservoir to the lungs passed through a membrane oxygenator (M8Exp, Jostra, Hirrlingen, Germany). By this device, the partial pressure was set at ~40 mmHg for both CO2 and O2 in the postoxygenator buffer fluid entering the pulmonary artery. Sequential hypoxic maneuvers of 10-min duration, interrupted by 15-min periods of normoxia, were performed. The effects of various pharmacological agents on pressure responses provoked by alveolar hypoxia (3% O2) were determined within such a sequence of repetitive hypoxic maneuvers. Each inhibitor was added to the buffer fluid 5 min before a hypoxic challenge, with the addition started after the second hypoxic maneuver was accomplished. Cumulative dose-effect curves were established. For comparison, the influence of these agents on U-46619- and ANG II-elicited pressor responses was tested. In these experiments, a mode of repetitive bolus applications of the stable thromboxane analog U-46619 or of ANG II was employed (addition to the perfusate of 0.5 nM U-46619 or 40 nM ANG II every 25 min) as previously described (15, 16). In each lung preparation, the response to the second vasoconstrictor provocation in a sequence of challenges was set at 100%. The strength of the following vasoconstrictor responses was related to this reference response. Control experiments were performed with use of the vehicle only. Lung weight was continuously monitored, but the total weight gain ranged <3 g in all experiments.
Measurement of NBT effects on lung superoxide release. For evaluation of NBT effects on lung superoxide release, an on-line photometric assay of ferricytochrome c reduction was established as described for repetitive perfusate analysis by Bongard et al. (5). Briefly, 25 µM ferricytochrome c was added to the perfusate throughout, and absorption was continuously measured at the outflow of the lung at 550 nm. Experiments were performed in the absence and presence of NBT. Values are expressed as changes from a baseline concentration of reduced cytochrome (set at 100%) assessed 15 min after perfusate admixture of the dye.
Statistics. Data are means ± SE.
Analysis of variance or a two-sided
t-test was performed; statistical
significance was assumed when P ranged
0.05.
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RESULTS |
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Under baseline conditions, PAP values ranged between 3.6 and 7.6 mmHg in all experiments. A 3% hypoxic challenge (PAO2 ~ 23 mmHg) provoked a rapid increase in PAP, with maximum pressure elevations of 2.9 ± 0.2 mmHg (n = 32 lungs). U-46619 injections into the arterial line produced pressure elevations, with a maximum of 3.3 ± 0.3 mmHg (n = 8 lungs); those elicited by ANG II were 3.2 ± 0.2 mmHg (n = 8 lungs). The HPV maneuvers were readily reproducible within the same lung, with a slight increase in the strength of the pressor response within each sequence of maneuvers (Fig. 1). Reproducibility of the U-46619- and ANG II-provoked pressure elevations was also apparent (Fig. 2).
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Admixture of the superoxide scavengers SOD (100-2,000 U/ml) and Tiron (10 µM to 10 mM) to the perfusate did not affect the strength of the HPV (Fig. 1). In addition, both agents did not change the baseline PAP and did not affect the relaxation induced by posthypoxic reoxygenation (Table 1). In contrast to SOD and Tiron, NBT caused a dose-dependent inhibition of HPV in a concentration range between 200 nM and 1 µM (Fig. 2). The HPV response was reduced by >70% in relation to the reference response. The efficacy of NBT was specific for the hypoxia-induced vasoconstrictor response because the U-46619- and ANG II-elicited pressure elevations were not significantly affected. NBT, however, did not change the baseline PAP and did not affect the relaxation induced by posthypoxic reoxygenation (Table 1).
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Additional experiments were performed while the pulmonary NO generation was blocked by the admixture of 400 µM L-NMMA to the perfusate throughout the experiments. In a previous study from our laboratory (30), this L-NMMA concentration was shown to reduce the rabbit lung NO generation virtually completely. Under these conditions, the baseline PAP did not significantly change, but the strength of the reference hypoxic vasoconstricor response was increased to 7.1 ± 0.6 mmHg (n = 8 lungs). This enhanced pressor response was again dose dependently inhibited by NBT in the same concentration range as employed in the absence of L-NMMA (Figs. 3 and 4). In contrast, vasoconstrictor responses elicited by U-46619 and ANG II in the presence of L-NMMA were not significantly suppressed by NBT. In further studies, the baseline generation of both NO and vasodilatory prostanoids was blocked by the presence of both L-NMMA (400 µM) and ASA (1 mM). Dose-dependent inhibition of HPV by NBT also occurred under these conditions (Fig. 4). In contrast to its influence on the hypoxia-elicited vasoconstrictor response, NBT did not affect the baseline PAP and the pressure decrease induced by posthypoxic reoxygenation in the presence of either L-NMMA or both L-NMMA and ASA (Table 1).
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As shown in Fig. 5, ongoing reduction of cytochrome c was noted in the perfused lungs, with an ~22% increase in absorption at 550 nm within 60 min. This kinetics of cytochrome c reduction was significantly reduced in the presence of NBT. Control experiments performed in the absence of cytochrome c did not show any significant influence of NBT on absorption values at 550 nm (data not shown).
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From a previous study (27) in perfused rabbit lungs, it is known that AT, in a concentration of 50 mM in the perfusion fluid, inhibits ~85% of lung catalase under conditions of H2O2 appearance. Employed in a concentration range between 10 and 150 mM in the present study, AT caused a dose-dependent inhibition of HPV in both the absence (data not shown) and presence of L-NMMA and ASA (Fig. 6). This inhibitory capacity, however, was not specific for the hypoxia-elicited vasoconstrictor response because superimposable dose-inhibition curves were obtained for pressor responses evoked by U-46619 (Fig. 6) and ANG II (data not shown). Baseline PAP was not affected by AT in both the absence and presence of L-NMMA and ASA (Table 1).
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DISCUSSION |
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The present study employed buffer-perfused rabbit lungs to investigate the role of superoxide and H2O2 formation in the mechanisms of HPV in an intact organ model. NBT, an agent that traps superoxide in a manner that prevents H2O2 formation (21, 22), dose dependently inhibited HPV, whereas SOD and Tiron (agents permitting H2O2 appearance; Refs. 21, 22) were ineffective. The NBT effect was specific for the hypoxia-induced vasoconstriction and may not be explained by interference with lung NO synthesis. However, under normoxic conditions, NBT did not affect baseline PAP and thus did not turn out as a hypoxia mimic even when lung NO synthesis and vasodilator prostanoid generation were simultaneously blocked. These data support a role for superoxide and H2O2 in the mechanism of hypoxic vasoconstriction but question the concept that a drop in some tonic H2O2- and NO-related cGMP formation might be a sufficient trigger of the vasoconstrictor response.
As previously described (37), the current perfused lung system employs a membrane oxygenator in the perfusion circuit to keep the PO2 and PCO2 values in the buffer fluid reentering the lung perfectly constant. In this model, excellent reproducibility of both hypoxia- and pharmacologically (U-46619 and ANG II) induced vasoconstriction is achieved, which allows testing of increasing doses of inhibitory agents in a sequential mode. In a very low concentration range of 200 nM to 1 µM, the superoxide scavenger NBT was found to cause strong dose-dependent inhibition of the hypoxia-induced pressor response, whereas the PAP increase provoked by the stable thromboxane analog U-46619 and by ANG II was not affected at all. Impressively, superimposable dose-inhibition curves were again obtained when lung NO synthesis (L-NMMA) or both the generation of NO and the vasodilatory prostanoids (L-NMMA and ASA) were blocked throughout the experiments; i.e., the NBT effect may not be explained by some interference with these important vasoregulatory systems. The efficacy of NBT thus supports a preceding study (22) in isolated calf pulmonary arteries, in which NBT inhibited hypoxic constriction together with superoxide scavenging, however, in a much higher dose range (300 µM). Other NBT effects in addition to scavenging of superoxide are unlikely to contribute to the inhibition of HPV. These might include 1) inhibition of NADPH diaphorase generating NO. Such effects can be excluded because NBT turned out to be fully active in experiments with prior blockage of the NO system; 2) generation of superoxide by reoxidation of the NBT radical (4). This is highly improbable because the lung tissue and tubing system increasingly changed their color to blue, indicating that NBT was continuously reduced to the insoluble blue formazan; 3) generation of molecular O2 due to the addition of NBT (4) that might reduce the strength of hypoxia. However, even entire conversion of the highest NBT dose presently used would generate O2 quantities that are more than four orders of magnitude lower than the quantity of O2 per se transported to the lung during a 10-min period of hypoxia. Thus such a mechanism may not be responsible for the inhibition of HPV by NBT; and 4) reduction of NBT by other than superoxide-generating chemical and enzymatic systems (4, 33). As shown by the data from the ferricytochrome c admixture to the perfusion fluid, the presently used low NBT concentration caused significant quenching of extracellular cytochrome c-reducing capacity. This finding may be assumed to indicate superoxide scavenging by NBT. It has been shown in detail that besides superoxide, only ascorbate release substantially contributes to the cytochrome c-reducing capacity of the rabbit lung vasculature (5). Moreover, there is multiple evidence from biological systems that NBT scavenges superoxide (21, 22).
The fact that the profiles of NBT (assumed to scavenge superoxide) and diphenyleneiodonium (reducing superoxide generation) in rabbit lung HPV are virtually identical strongly favors the assumption that some reduction in the superoxide levels by NBT underlies the HPV inhibitory effect of this agent (22). In contrast to the assumption of Mohazzab-H. and Wolin (22), there is evidence that NBT scavenges extracellular superoxide (12, 33). Recently, Marshall et al. (19) showed that extracellular superoxide is increased during hypoxia in small pulmonary arterial smooth muscle cells, the predominant site of HPV.
In contrast to NBT, the superoxide scavengers SOD and Tiron added to the perfusate did not affect the hypoxia-induced vasoconstriction, even when applied in very high concentrations (2,000 U/ml of SOD; 10 mM Tiron). Three major explanations may underlie these findings.
1) SOD and Tiron might not have sufficient access to the sites of O2 sensing (assumed to be the precapillary smooth muscle cells; Ref. 19) and superoxide generation and efficacy. This explanation may well hold true for SOD because it may not be able to pass the endothelial barrier in sufficient quantities due to its molecular weight of ~31,200. Additionally, steric hindrances and/or electrostatic effects could also be responsible for the ineffectiveness of SOD (13). Moreover, intracellular regions are hardly reached by SOD, which is known to demand vehicle systems such as polyethylene glycol or liposomes for entrance into the intracellular space in substantial quantities (6, 32). In contrast, the low-molecular-weight superoxide scavenger Tiron may enter the cell and has repeatedly been shown to trap even intracellular O2 radicals (18, 20), and such a high concentration as the currently employed 10 mM must thus be assumed to be distributed into all cellular and extracellular compartments related to hypoxia sensing and subsequent signal transduction. Overall, distribution of substantial concentrations of buffer-admixed Tiron into all compartments relevant for superoxide generation and efficacy may be assumed from the basic pharmacological features of this agent, even if the present study may not provide a direct proof for this assumption.
2) Tiron and SOD may not affect superoxide and H2O2 concentrations because of the high rate constant (efficacy) of native SOD or spontaneous dismutation of superoxide.
3) Both SOD and Tiron are scavengers of superoxide that result in the formation of H2O2 from this O2 radical (9, 21). This is in contrast to the effect of NBT, known to trap superoxide without the formation of H2O2 (10). SOD and Tiron on one hand and NBT on the other, although all being scavengers of extracellular superoxide, must thus be assumed to have different effects on the downstream appearance of H2O2.
Notwithstanding the fact that the reasons for the discrepancy between the efficacy of NBT and the ineffectiveness of SOD and Tiron are not fully resolved, the present study supports the notion that changes in the generation of superoxide and H2O2 may be centrally involved in hypoxia sensing and signal transduction to result in vasoconstriction, which is in-line with previous observations. Reactive O2 intermediates have long since been suggested to play a major role in the HPV mechanism (for a review, see Ref. 1). Studies with diphenyleneiodonium in rabbit (16) and rat (34) lungs suggested the contribution of a superoxide-generating NAD(P)H oxidase. The production of superoxide and H2O2 in lungs has been demonstrated (25). Mohazzab-H. and Wolin (22) presented evidence for a role of an NADH-cytochrome b558 oxidoreductase in the vasoconstrictor response of a calf pulmonary artery to hypoxia. Moreover, Marshall et al. (19) recently demonstrated 1) that an enhanced superoxide production via NADPH oxidase occurs during hypoxia in small pulmonary arterial smooth muscle cells (the predominant site of HPV) and 2) that this superoxide is released into the extracellular space.
There is thus good evidence from different systems that in addition to O2 tension-related NO generation, PO2-dependent NAD(P)H oxidase activity with superoxide and possibly H2O2 formation is enrolled in O2 sensing and signaling. Based on observations of reduced lung superoxide generation in hypoxia (22, 25), the concept of tonic normoxic generation of H2O2, forming the intermediate compound I with catalase, which then continuously stimulates cGMP generation for vasorelaxation (7, 23), does possess attractancy: hypoxia will then result in a drop in both NO- and H2O2-catalase-dependent cGMP formation, and this drop causes a loss of normoxic vasodilation, i.e., increase in PAP. This latter part of the concept is, however, challenged by several observations in the rabbit lungs. First, AT, which inhibits catalase by forming an irreversible bond with the catalase intermediate compound I (26), suppressed HPV in rabbit lungs, but, with superimposable dose-inhibition curves, also blocked vasoconstrictor responses to U-46619 and ANG II. This finding may be caused by hitherto unknown non-catalase effects of AT (8, 31) or may be due to the fact that H2O2-dependent pathways are involved in U-46619-induced vasoconstriction, although we are not aware of experimental support for the latter possibility (17, 24). Anyway, because a specific effect of AT on HPV was not found, the use of AT in our rabbit lung model allows no conclusions that elucidate the role of H2O2 or the H2O2-catalase complex in the signaling process underlying HPV. Second, blocking of NAD(P)H oxidase, and thus the suggested sequence of superoxide, H2O2, H2O2-catalase, and cGMP generation, blocked HPV in the rabbit lungs but did not increase the baseline PAP as would be anticipated by a hypoxia mimic (16). Third, the same was true for blocking NO generation by L-NMMA (15), superoxide and H2O2 formation by NBT, and even NO plus superoxide-H2O2 generation at the same time; none of these interventions substantially increased the baseline PAP, and even the posthypoxic vasorelaxation was not substantially affected; thus any mimic of HPV was not achieved. In contrast to the hypothesis of Monaco and Burke-Wolin (23), the recent observation by Marshall et al. (19) of increased superoxide generation during hypoxia in small pulmonary arterial smooth muscle cells is well in-line with our results; if increased superoxide and possibly subsequent H2O2 release during hypoxia triggers vasoconstriction, inhibition of that superoxide (and H2O2) increase should block but not mimic HPV.
In conclusion, the present study provides further evidence for a central role of reactive O2 species formation in the basic mechanism of HPV. The finding that NBT selectively inhibits HPV in low concentrations points to a major contribution of O2 tension-dependent superoxide formation, although actions of NBT other than superoxide scavenging cannot be fully excluded. The fact that pharmacological blockage of NO and superoxide does not mimic HPV questions the concept that loss of tonic guanylate cyclase stimulation via the superoxide-H2O2 axis may sufficiently explain the pressor response to hypoxia but favors the concept of locally increased superoxide and H2O2 concentrations as a trigger for HPV. The mechanisms relating O2 tension-dependent superoxide and H2O2 generation to the vasoconstrictor event occurring in HPV remain to be further elucidated.
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ACKNOWLEDGEMENTS |
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We thank C. Homberger and K. Quanz for excellent technical assistance.
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FOOTNOTES |
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This work was supported by the Deutsche Forschungsgemeinschaft (Klinische Forschergruppe "Respiratorische Insuffizienz" and SFB 547 Project B7).
Portions of the doctoral theses of Jörg Conzen and Robert Voswinckel are incorporated into this report.
Address for reprint requests: W. Seeger, Dept. of Internal Medicine, Justus-Liebig-Univ. Giessen, Klinikstrasse 36, 35392 Giessen, Germany.
Received 12 June 1996; accepted in final form 5 February 1998.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Acker, H.
Mechanisms and meaning of cellular O2 sensing in the organism.
Respir. Physiol.
95:
1-10,
1994[Medline].
2.
Acker, H.,
E. Dufau,
J. Huber,
and
D. Sylvester.
Indications to an NADPH oxidase as a possible PO2 sensor in the rat carotid body.
FEBS Lett.
256:
75-78,
1989[Medline].
3.
Archer, S. L.,
J. P. Tolins,
L. Raij,
and
E. K. Weir.
Hypoxic pulmonary vasoconstriction is enhanced by inhibition of the synthesis of an endothelium derived relaxing factor.
Biochem. Biophys. Res. Commun.
164:
1198-1205,
1989[Medline].
4.
Auclair, C,
and
E. Voisin.
Nitroblue tetrazolium reduction.
In: CRC Handbook of Methods for Oxygen Radical Research, edited by R. A. Greenwald. Boca Raton, FL: CRC, 1985, p. 123-132.
5.
Bongard, R. D.,
D. L. Roerig,
M. S. Johnston,
and
C. A. Dawson.
Perfusate cytochrome c reduction in isolated rabbit lungs.
J. Appl. Physiol.
71:
1705-1713,
1991
6.
Briscoe, P.,
I. Caniggia,
A. Graves,
B. Benson,
L. Huang,
A. K. Tanswell,
and
B. A. Freeman.
Delivery of superoxide dismutase to pulmonary epithelium via pH-sensitive liposomes.
Am. J. Physiol.
268 (Lung Cell. Mol. Physiol. 12):
L374-L380,
1995
7.
Burke-Wolin, T.,
and
M. S. Wolin.
H2O2 and cGMP may function as an O2 sensor in the pulmonary artery.
J. Appl. Physiol.
66:
167-170,
1989
8.
Casteels, M.,
K. Croes,
P. P. Van Veldhoven,
and
G. P. Mannaerts.
Aminotriazole is a potent inhibitor of alpha-oxidation of 3-methyl-substituted fatty acids in rat liver.
Biochem. Pharmacol.
48:
1973-1975,
1994[Medline].
9.
Chance, B.,
H. Sies,
and
A. Boveris.
Hydroperoxide metabolism in mammalian organs.
Physiol. Rev.
59:
527-605,
1979
10.
Clare, D. A.,
M. N. Duong,
D. Darr,
F. Archibald,
and
I. Fridovich.
Effects of molecular oxygen on detection of superoxide radical with nitroblue tetrazolium and on activity stains for catalase.
Anal. Biochem.
140:
532-537,
1984[Medline].
11.
Cremona, G.,
T. Higenbottam,
M. Takao,
L. Hall,
and
E. A. Bower.
Exhaled nitric oxide in isolated pig lungs.
J. Appl. Physiol.
78:
59-63,
1995
12.
DeBari, V. A.,
and
M. A. Needle.
Mechanisms for transport of nitro-blue tetrazolium into viable and non-viable leukocytes.
Histochemistry
56:
155-163,
1978[Medline].
13.
DiGregorio, K. A.,
E. V. Cilento,
and
R. C. Lantz.
A kinetic model of superoxide production from single pulmonary alveolar macrophages.
Am. J. Physiol.
256 (Cell Physiol. 25):
C405-C412,
1989
14.
Fishman, A. P.
Hypoxia on the pulmonary circulation. How and where it acts.
Circ. Res.
38:
683-686,
1976.
15.
Grimminger, F.,
R. Spriestersbach,
N. Weissmann,
D. Walmrath,
and
W. Seeger.
Nitric oxide generation and hypoxic vasoconstriction in buffer-perfused rabbit lungs.
J. Appl. Physiol.
78:
1509-1515,
1995
16.
Grimminger, F.,
N. Weissmann,
R. Spriestersbach,
E. Becker,
S. Rosseau,
and
W. Seeger.
Effects of NADPH oxidase inhibitors on hypoxic vasoconstriction in buffer-perfused rabbit lungs.
Am. J. Physiol.
268 (Lung Cell. Mol. Physiol. 12):
L747-L752,
1995
17.
Jaschonek, K.,
and
C. P. Muller.
Platelet and vessel associated prostacyclin and thromboxane A2/prostaglandin endoperoxide receptors.
Eur. J. Clin. Invest.
18:
1-8,
1988[Medline].
18.
Leffler, C. W.,
R. Mirro,
C. Thompson,
M. Shibata,
W. M. Armstead,
M. Pourcyrous,
and
O. Thelin.
Activated oxygen species do not mediate hypercapnia-induced cerebral vasodilation in newborn pigs.
Am. J. Physiol.
261 (Heart Circ. Physiol. 30):
H335-H342,
1991
19.
Marshall, C.,
A. J. Mamary,
A. J. Verhoeven,
and
B. E. Marshall.
Pulmonary artery NADPH-oxidase is activated in hypoxic pulmonary vasoconstriction.
Am. J. Respir. Cell Mol. Biol.
15:
633-644,
1996[Abstract].
20.
Mellin, M.,
and
H. McLaughlin.
Hydroxyl radical scavengers inhibit human lectin-dependent cellular cytotoxicity.
Immunology
58:
197-202,
1986[Medline].
21.
Mohazzab-H, K. M.,
P. M. Kaminski,
R. P. Fayngersh,
and
M. S. Wolin.
Oxygen-elicited responses in calf coronary arteries: role of H2O2 production via NADH-derived superoxide.
Am. J. Physiol.
270 (Heart Circ. Physiol. 39):
H1044-H1053,
1996
22.
Mohazzab-H, K. M.,
and
M. S. Wolin.
Properties of a superoxide anion-generating microsomal NADH oxidoreductase, a potential pulmonary artery PO2 sensor.
Am. J. Physiol.
267 (Lung Cell. Mol. Physiol. 11):
L823-L831,
1994
23.
Monaco, J. A.,
and
T. Burke-Wolin.
NO and H2O2 mechanisms of guanylate cyclase activation in oxygen-dependent response of rat pulmonary circulation.
Am. J. Physiol.
268 (Lung Cell. Mol. Physiol. 12):
L546-L550,
1995
24.
Negishi, M.,
Y. Sugimoto,
and
A. Ichikawa.
Molecular mechanisms of diverse actions of prostanoid receptors.
Biochim. Biophys. Acta
1259:
109-119,
1995[Medline].
25.
Paky, A.,
J. R. Michael,
T. M. Burke-Wolin,
M. S. Wolin,
and
G. H. Gurtner.
Endogenous production of superoxide by rabbit lungs: effects of hypoxia or metabolic inhibitors.
J. Appl. Physiol.
74:
2868-2874,
1993[Abstract].
26.
Piantadosi, C. A.,
and
L. G. Tatro.
Regional H2O2 concentration in rat brain after hyperoxic convulsions.
J. Appl. Physiol.
69:
1761-1766,
1990
27.
Seeger, W.,
N. Suttorp,
F. Schmidt,
and
H. Neuhof.
The glutathione redox cycle as a defense system against hydrogen-peroxide-induced prostanoid formation and vasoconstriction in rabbit lungs.
Am. Rev. Respir. Dis.
133:
1029-1036,
1986[Medline].
28.
Seeger, W.,
D. Walmrath,
F. Grimminger,
S. Rosseau,
H. Schütte,
H.-J. Krämer,
L. Ermert,
and
L. Kiss.
Adult respiratory distress syndrome: model systems using isolated perfused rabbit lungs.
Methods Enzymol.
233:
549-584,
1994[Medline].
29.
Seeger, W.,
D. Walmrath,
M. Menger,
and
H. Neuhof.
Increased vascular permeability after arachidonic acid and hydrostatic challenge.
J. Appl. Physiol.
61:
1781-1788,
1986
30.
Spriestersbach, R.,
F. Grimminger,
N. Weissmann,
D. Walmrath,
and
W. Seeger.
On-line measurement of nitric oxide generation in buffer-perfused rabbit lungs.
J. Appl. Physiol.
78:
1502-1508,
1995
31.
Svensson, B. E.,
D. Kristina,
S. Lindvall,
and
G. Rydell.
Peroxidase and peroxidase-oxidase activities of isolated human myeloperoxidases.
Biochem. J.
242:
673-680,
1987[Medline].
32.
Tang, G.,
J. E. White,
R. J. Gordon,
P. D. Lumb,
and
M. Tsan.
Polyethylene glycol-conjugated superoxide dismutase protects rats against oxygen toxicity.
J. Appl. Physiol.
74:
1425-1431,
1993[Abstract].
33.
Thayer, W. S.
Superoxide-dependent and superoxide-independent pathways for reduction of nitroblue tetrazolium in isolated rat cardiac myocytes.
Arch. Biochem. Biophys.
276:
139-145,
1990[Medline].
34.
Thomas, H. M., III,
R. C. Carson,
E. D. Fried,
and
R. S. Novitch.
Inhibition of hypoxic pulmonary vasoconstriction by diphenyleneiodonium.
Biochem. Pharmacol.
42:
R9-R12,
1991[Medline].
35.
Voelkel, N. F.
Mechanisms of hypoxic pulmonary vasoconstriction.
Am. Rev. Respir. Dis.
133:
1186-1195,
1986[Medline].
36.
Von Euler, U. S.,
and
G. Liljestrand.
Observations on the pulmonary arterial blood pressure in the cat.
Acta Physiol. Scand.
12:
301-320,
1946.
37.
Weissmann, N.,
F. Grimminger,
D. Walmrath,
and
W. Seeger.
Hypoxic vasoconstriction in buffer-perfused rabbit lungs.
Respir. Physiol.
100:
159-169,
1995[Medline].
38.
Youngsten, C.,
C. Nurse,
H. Yeger,
and
E. Cutz.
Oxygen sensing in airway chemoreceptors.
Nature
365:
153-155,
1993[Medline].