Department of Internal Medicine, Justus-Liebig-University Giessen, 35392 Giessen, Germany
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ABSTRACT |
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Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion with ventilation. Controversy exists whether decreased or increased reactive oxygen species may elicit HPV and from which source such oxygen metabolites are derived. In rabbit lungs, we detected transcripts of a nonphagocytic NADPH oxidase subunit homologous to mitogenic oxidase-1 (Mox1) or NADPH oxidase homolog 1 (NOH-1L). In perfused rabbit lungs, we employed 1) a new NADPH oxidase inhibitor [4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF; 100-600 µM)] and 2) the superoxide dismutase (SOD) inhibitors diethyldithiocarbamic acid (DETC; 100 µM to 10 mM) and triethylenetetramine (TETA; 1-25 mM). Specificity of these agents for HPV was investigated by comparison with U-46619-induced vasoconstrictions. AEBSF induced a transient increase in pulmonary arterial pressure with increased strength of HPV. Subsequent to this initial response, normoxic pulmonary arterial pressure was not affected and HPV was specifically suppressed. Whereas DETC turned out to act in a nonspecific fashion, TETA suppressed HPV specifically. These findings provide evidence of a role for a nonphagocytic NAD(P)H oxidase with superoxide and SOD-related hydrogen peroxide formation in HPV. Because HPV was inhibited but not mimicked by the inhibitors, increased rather than decreased superoxide and/or hydrogen peroxide formation is suggested as the hypoxia-provoked signaling event.
hypoxic pulmonary vasoconstriction; isolated lung; 4-(2-aminoethyl)benzenesulfonyl fluoride; reduced nicotinamide adenine dinucleotide phosphate oxidase; superoxide dismutase; hydrogen peroxide
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INTRODUCTION |
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HYPOXIC PULMONARY VASOCONSTRICTION (HPV) is an essential vasoregulatory mechanism matching lung blood flow with ventilation and thereby optimizing pulmonary gas exchange (13, 35). The sensor mechanism, cell(s) responsible for O2 sensing, and the pathway(s) of signal transduction to the precapillary vascular smooth muscle cells still remain enigmatic. Nitric oxide (NO) synthesis is apparently linked to this vasoregulatory mechanism (14, 27). Recently, evidence has been provided for involvement of superoxide and hydrogen peroxide (H2O2) in HPV. However, there is still controversy whether reactive oxygen species are actually involved in the signal transduction underlying HPV, whether decreased or increased superoxide and H2O2 levels trigger HPV, and from which cellular and intracellular source such reactive oxygen species are derived (2, 5, 6, 15, 23, 25-27, 36, 38). Multiple investigations including our own suggested that a NAD(P)H oxidase may function as a sensing complex (15, 23, 25, 33) and that the sensor is located in the precapillary smooth muscle cells (23, 31). However, the concept of a NAD(P)H oxidase as the oxygen sensor is based largely on the employment of the flavoprotein inhibitor diphenyleneiodonium (DPI), which, besides blocking NADPH oxidase, potentially inhibits all flavoprotein-dependent enzyme systems including the mitochondrial respiratory chain. Taking this into account, a recent study (6) suggested mitochondria as the oxygen sensor. Moreover, the concept of a NADPH oxidase as an oxygen sensor is challenged by the report of unrestricted HPV in the phagocyte NADPH oxidase gp91phox knockout mouse (2). However, this study cannot exclude the possibility that a "low-output" isoform of the phagocyte oxidase may be involved in hypoxia sensing (3, 19, 22, 24, 28, 32).
To investigate further the possible role of NAD(P)H oxidase and derived reactive oxygen species in eliciting hypoxic vasoconstriction in intact lungs, we employed a new specific inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) (10), recently characterized in detail as to its impact on oxygen sensing in the rat carotid body in vitro (22). In addition, the significance of downstream conversion of rising superoxide to H2O2 for the hypoxia-induced vasoconstrictor response was addressed by testing superoxide dismutase (SOD) inhibitors. Portions of the experiments were performed during blocked NO and prostanoid synthesis to exclude interference with these vasodilatory pathways. In addition, the impact of all agents on the pressor responses provoked by the stable thromboxane analog U-46619 was investigated to test for specificity of the effects for the hypoxia-induced vasoconstrictor response. A potential candidate for a hypoxia-sensing oxidase with a subunit identical or homologous with the recently described mitogenic oxidase-1 (Mox1) or NADPH oxidase homolog 1 (NOH-1L) (3, 32) but different from the phagocytic NADPH oxidase containing gp91phox (4, 34) was detected by RT-PCR in rabbit lung tissue and smooth muscle cells isolated from the pulmonary artery and small arterial vessels. In essence, these studies provided further evidence of a central role for the putative oxygen sensor NAD(P)H oxidase in hypoxic vasoconstriction in intact rabbit lungs, with SOD-related conversion of superoxide to H2O2 being suggested as the important downstream signaling event. Because interference with NAD(P)H oxidase and SOD inhibited but did not mimic HPV, superoxide and/or H2O2 generation is suggested to be enhanced rather than decreased under conditions of lowered oxygen tension.
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MATERIALS AND METHODS |
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Reagents.
4-(2-Aminoethyl)benzenesulfonyl fluoride (AEBSF) was provided by Merck
(Munich, Germany). Anti--smooth muscle actin antibody, anti-myosin
antibody, diethyldithiocarbamic acid (DETC), DMEM, triethylenetetramine
dihydrochloride (TETA), phenylmethylsulfonyl fluoride (PMSF), phorbol
12-myristate 13-acetate (PMA), and ferricytochrome c were
obtained from Sigma-Aldrich (Deisenhofen, Germany). U-46619 came from
Paesel and Lorei (Frankfurt-on-Main, Germany) and DMEM nutrient mixture
F-12, Hanks' balanced salt solution, L-glutamine, Moloney murine leukemia virus (MMLV) reverse transcriptase, penicillin, and streptomycin were from GIBCO (Karlsruhe, Germany). The lung perfusate was purchased from Serag-Wiessner (Naila, Germany). Acetylsalicylic acid (ASA) and aprotinin (Trasylol) were from Bayer
(Leverkusen, Germany). The SOD assay kit and
NG-monomethyl-L-arginine
(L-NMMA) were supplied by Calbiochem (Bad Soden, Germany).
Sodium pyruvate was purchased from PAA (Marburg, Germany), anti-smooth
muscle cell antibody was from Boehringer Mannheim (Mannheim,
Germany), fetal calf serum was from Greiner (Frickenhausen, Germany);
RNAzol B was from WAK-Chemie (Bad Homburg, Germany), HotStarTaq
polymerase was from QIAGEN (Hilden, Germany), and collagenase type 2 was from Worthington (Lakewood, NY). All other biochemicals were
purchased from Merck.
Lung isolation, perfusion, and ventilation. The model of isolated perfused rabbit lungs has been previously described (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 wt). 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; NaHCO3 was adjusted to result in a constant pH 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 250 ml). Meanwhile, the flow was slowly increased from 20 to 150 ml/min, and the 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, and the 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, freely suspended from a force transducer, were placed in a temperature-equilibrated housing chamber for continuous monitoring of organ weight. The whole system (perfusate reservoirs, tubing, and housing chamber) was heated to 38.5°C. Pulmonary arterial (PAP), left atrial, and tracheal pressures were registered by means of small-diameter tubing threaded into the perfusion catheters and the trachea and connected to pressure transducers. The 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 the initial steady-state period of at least 20 min.
Hypoxic maneuvers and pharmacological challenges. The technique of successive 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 conditions] to 3% vol/vol (PAO2 ~23 mmHg; hypoxic conditions)}. CO2 at 5.3% (vol/vol) 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 pharmacological 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, the addition starting after the second hypoxic maneuver was accomplished. Cumulative dose-effect curves were established. In the experiments with AEBSF, the applied dosage was increased only with every second hypoxic challenge. For comparison, influence of the applied agents on U-46619-elicited pressor responses was tested in a corresponding time schedule. In these experiments, a mode of repetitive bolus applications of the stable thromboxane analog (0.5 nM added to the perfusate every 25 min) was employed as previously described (15, 36, 38). 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 with use of the vehicle only. In the experiments with preblocked NO synthesis, 400 µM L-NMMA was present in the perfusate from the beginning of the experiments. To determine the effects of the applied inhibitors on normoxic vascular tone, the inhibitors were added according to a time-matched protocol as in the hypoxia experiments. L-NMMA (400 µM) and ASA (1 mM) were present in the perfusate throughout these experiments to avoid masking the inhibitor effects by vasodilatory prostacyclin and NO.
Lung weight was continuously monitored. The total weight gain ranged <3 g in all experiments.Measurement of SOD activity. The activity of SOD was measured with a photometric assay from homogenized lungs. After perfusion, isolated rabbit lungs were washed, minced, and homogenized (1:10 wt/vol) in a solution of ice-cold 0.25 M sucrose and centrifuged (8,500 g for 10 min at 4°C). To 250 µl of the supernatant, 400 µl of ice-cold ethanol-CHCl3 (62.5:37.5 vol/vol) were added. After centrifugation at 3,000 g at 4°C for 10 min, aliquots of the upper aqueous layer were measured for SOD activity with a SOD assay kit (Calbiochem). SOD activity is given in SOD-525 units per milliliter according to the assay manual.
Isolation of alveolar macrophages and measurement of respiratory burst. Lungs from killed rabbits were lavaged with four 50-ml washes with endotoxin-free saline via a tracheal cannula inserted under sterile conditions. Total recovered volume was ~180 ml. The alveolar cells were recovered from the pooled lavage fluid by centrifugation at 300 g for 15 min at room temperature, washed twice, and suspended in Hanks' balanced salt solution. Alveolar macrophages were identified by morphological criteria (air-dried and Giemsa-stained smears) and by esterase staining in each experiment as well as by random electron-microscopic examinations. The percentage of macrophages within the total number of lavaged cells consistently ranged >96%; contaminating polymorphonuclear neutrophils were <1.5% in all preparations. Cell viability in the presence of a stimulus application was assessed via lactate dehydrogenase release. Under all experimental conditions, lactate dehydrogenase release was consistently <3%. Respiratory burst was measured as the SOD-inhibitable reduction of cytochrome c as described elsewhere (7). Briefly, duplicate reaction mixtures containing macrophages (10 × 106 macrophages/ml) and 75 µM ferricytochrome c were incubated at 37°C in the presence and absence of 10 µg/ml of SOD. Stimulation of the respiratory burst was performed by the addition of 2 µM PMA for 30 min at 37°C. AEBSF was added 5 min before stimulation. Control samples received the solvent only. Respiratory burst is given in percent of maximal stimulation elicited by PMA.
Isolation of rabbit pulmonary arterial smooth muscle cells, RNA
extraction, and analysis of Mox1 and NOH-1L homologous transcripts by
RT-PCR.
Smooth muscle cells from rabbit pulmonary artery and small arterial
vessels (diameter ~400 µm) were isolated and cultured by adaptation
of techniques described elsewhere (12, 18, 23). In brief,
the lungs from killed rabbits were flushed free of blood with
Krebs-Henseleit buffer. The pulmonary artery and small arterial vessels
were prepared and freed from connective tissue, the endothelium was
scraped, and media pieces were dissected by fine forceps and scissors
under microscopic control. After digestion with collagenase (1 h at
37°C, 200 U/ml) and centrifugation at 350 g for 5 min, the
sedimented cells were resuspended and cultured until confluent (37°C,
5% CO2). Experiments were performed with cells from
passages 3 to 4. The culture medium was composed
of 16 ml DMEM/100 ml, 64 ml DMEM nutrient mixture F-12/100 ml, and 20 ml fetal calf serum (20%)/100 ml with 100 IU/ml of penicillin, 100 µg/ml of streptomycin, 2 mM L-glutamine, and 1 mM sodium
pyruvate. The cells were characterized 1) morphologically,
2) by their growth pattern, and 3)
immunohistochemically (anti-myosin antibody, anti--smooth muscle
actin antibody, and anti-smooth muscle cell antibody).
Statistics.
For comparison of statistical differences, analysis of variance with
the Student-Newman-Keuls post hoc test was performed. Significance was
assumed when P ranged 0.05. For testing of SOD activity, a
paired t-test was applied.
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RESULTS |
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Under baseline conditions, mean PAP values were 5.1 ± 0.2 (SE) mmHg (n = 64 lungs). A 3% hypoxic challenge (PAO2 ~23 mmHg) consistently provoked a rapid increase in PAP, with a mean pressure of 2.6 ± 0.1 mmHg (n = 40 lungs). Repetitive hypoxic challenges resulted in well-reproducible pressure elevations within the same lung as previously described (38). Prior blockage of NO synthesis with L-NMMA (400 µM) only marginally affected normoxic vascular tone but increased hypoxic pressor responses throughout the experiments; the step increase in PAP in response to hypoxia was increased to 7.0 ± 0.5 mmHg (n = 16 lungs) under these conditions. Similarly, a repetitive bolus application of U-46619 provoked well-reproducible vasoconstrictor responses under baseline conditions (2.8 ± 0.3 mmHg; n = 24 lungs), again enhanced in the presence of L-NMMA (5.2 ± 0.7 mmHg; n = 16 lungs).
The addition of the NADPH oxidase inhibitor AEBSF during normoxic
ventilation transiently increased vascular tone. Under conditions of
preblocked lung NO synthesis and prostanoid generation (400 µM
L-NMMA and 1 mM ASA), this PAP increase reached a time- and dose-dependent maximum of 12.2 ± 0.7 mmHg within 13.1 ± 0.3 min (100 µM AEBSF), 15.6 ± 1.1 mmHg within 5.4 ± 0.9 min
(300 µM AEBSF), and 15.6 ± 1.5 mmHg within 6.2 ± 0.5 min
(600 µM AEBSF). This pressure elevation returned to baseline levels
in normoxic control experiments within 19.8 ± 0.9, 28.1 ± 3.8, and 28.2 ± 3.8 min, respectively (n = 4 lungs each) of AEBSF administration. Performance of hypoxic ventilation
during elevated normoxic vascular tone resulted in increased HPV (Fig.
1A). This amplification was
specific for the hypoxic response because U-46619-induced
vasoconstrictor responses were not increased under these conditions.
Performance of a hypoxic challenge after the PAP increase had returned
to baseline values resulted in inhibition of HPV. This inhibition was
again specific for HPV because vasoconstrictions elicited by U-46619
under identical experimental conditions were not significantly suppressed by AEBSF up to 600 µM (Fig. 1B). The inhibitory
effect of AEBSF on HPV was even more pronounced when lung NO synthesis was preblocked with L-NMMA (Fig. 1C). Thus after
the transient amplification, AEBSF turned out to be a specific
inhibitor of HPV.
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To confirm the NADPH oxidase-inhibitory capacity of AEBSF in the rabbit
system, we measured the effect of AEBSF on the alveolar macrophage
oxidative burst elicited by PMA. Oxidative burst was attenuated by
AEBSF in a dose-dependent manner (Fig.
2).
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To exclude that the effects of AEBSF on HPV are related to the
well-known efficacy of this agent to inhibit serine proteases, two
different protease inhibitors, PMSF and aprotinin, were tested for
their impact on HPV (9). These agents did not
significantly affect HPV up to a concentration of 1 mM for PMSF and
6,000 kallikrein-inhibiting units/ml perfusate for aprotinin
(Fig. 3).
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The SOD inhibitor DETC (100 µM to 10 mM) dose dependently inhibited
HPV. This inhibitory capacity was, however, not specific for HPV
because vasoconstrictor responses elicited by the thromboxane analog
U-46619 were attenuated with corresponding dose-effect curves (Fig.
4). However, the second SOD inhibitor
TETA (1-25 mM) dose dependently inhibited HPV without affecting
the pressor responses elicited by U-46619 (Fig.
5A). Virtually the same
inhibition profile was seen when the experiments after blockage of NO
generation were performed (Fig. 5B). TETA did not affect
normoxic vascular tone, not even during preblocked lung NO synthesis
and prostanoid generation (400 µM L-NMMA and 1 mM ASA;
data not shown). The SOD-inhibitory capacity of TETA was ensured by
measurement of SOD activity from homogenized lungs. SOD activity
decreased to ~44% vs. the control value (Fig.
6).
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With RT-PCR, transcripts of Mox1, NOH-1L, or a strongly homologous
sequence were detected in rabbit lung tissue and isolated cultured
smooth muscle cells of the rabbit pulmonary artery and small arterial
vessels (diameter ~400 µm; Fig. 7).
Additional sequencing of the PCR product (EMBL accession no. AJ271882) showed a homology of ~87% with the human Mox1 or NOH-1L sequence and a weaker homology of ~55% with human gp91phox
(EMBL accession no. NM_000397).
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DISCUSSION |
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The present study focused on the oxygen-sensing mechanism and signal transduction underlying HPV in intact lungs. We provide new evidence 1) for the involvement of a NAD(P)H oxidase as an oxygen sensor for HPV and 2) for the downstream generation of H2O2 being involved in the signaling cascade between hypoxia sensing and vasoconstrictor response. It is suggested that hypoxia results in increased rather than decreased levels of reactive oxygen species, thereby provoking the PAP elevation. These conclusions are based on the following lines of evidence.
HPV was dose dependently inhibited by AEBSF, an agent that has been well characterized as a NADPH oxidase inhibitor on a cellular and molecular level. AEBSF inhibits the assembly of the oxidase subunits and therefore the function of the multiprotein complex of the oxidase (10). This effect is suggested to be related to its function as a heme ligand (11, 22). The inhibitory capacity of AEBSF on the NADPH oxidase was currently ascertained in the rabbit system by measurement of the alveolar macrophage respiratory burst, which was inhibited with dose-effect curves corresponding to those of HPV inhibition. Notably, the vasopressor response to the stable thromboxane analog U-46619 was not affected by AEBSF, thus documenting specificity of this agent for the hypoxia-elicited vasoconstriction. Thus for the first time, our data provide evidence for a specific effect of a NADPH oxidase inhibitor on HPV not related to DPI because all previous investigations (15, 23, 25, 33) supporting the view that a NAD(P)H oxidase is involved in the regulation of HPV were based on the inhibitory profile of the latter agent. The suppressor effect of AEBSF on HPV was clearly independent of lung NO generation as shown in experiments performed in the presence of L-NMMA. This aspect was addressed in view of previous studies (14, 27) suggesting that NO may additionally be involved in the hypoxia-sensing mechanism.
When analyzing the effect of AEBSF on rat carotid body chemoreceptor discharge in detail, Lahiri et al. (22) recently noted that an initial excitation period preceded the silencing effect of this agent. This finding is of interest in view of the current observation that AEBSF admixture to the perfusate first transiently increased the pulmonary vascular tone, concomitant with a specific amplification of HPV, before subsequently blocking the hypoxia-induced vasoconstrictor response while leaving the U-46619 response unaffected. Hypothetically, the transient increase in pulmonary vascular tone and HPV could be caused by the interaction of AEBSF with the iron atom of the NADPH oxidase heme (11, 22) during the initial steps of binding AEBSF. The striking similarity between the AEBSF efficacy profile in the superfused carotid body and the intact lung vasculature further supports the notion that the effect of this agent in the rabbit lung is related to its impact on the oxygen sensor or hypoxic signal transduction. There is no evidence that the efficacy of AEBSF in the lung vasculature might be linked to the only known side effect of this agent to inhibit protease activity because control studies with the protease inhibitors PMSF and aprotinin did not reproduce the AEBSF-elicited changes even when applied in high concentrations.
The recent observation that O2 sensing is preserved in mice lacking the gp91phox subunit of NADPH oxidase strongly argues against a contribution of phagocytic NADPH oxidase to the mechanisms of HPV (2). This observation may, however, not exclude a role of a low-output NAD(P)H oxidase (3, 19, 24-26, 28) in the signaling events underlying HPV. The nonphagocytic low-output NAD(P)H oxidase, occurring in most cell types, is an isoform of the phagocytic high-output enzyme (4), with differences in the amino acid sequence of gp91phox, the polymorphism of the p22phox gene (17), and the involvement of Rac1 or Rac2 (8). Indeed, cloning of the superoxide-generating NADPH oxidase Mox1 or NOH-1L, which encodes a homolog of the catalytic subunit gp91phox of the phagocytic NADPH oxidase, was recently described (3, 32). We detected transcripts from Mox1, NOH-1L, or a strongly homologous sequence in lung tissue and smooth muscle cells isolated from the pulmonary artery and small arterial vessels of the lung. This supports the concept of a NAD(P)H oxidase containing a subunit different from the phagocytic gp91phox as a potential candidate for an oxygen-sensing multiprotein complex.
In addition, mitochondrial reactive oxygen species formation was recently suggested as the central event in hypoxia sensing (6), and the present findings in the lung vasculature might also be compatible with such a concept as far as mitochondrial "NADH oxidase-like activity" may be assumed to be accessible to inhibition with both DPI and AEBSF.
Activated NAD(P)H oxidases produce superoxide, which is rapidly converted to H2O2 by SOD (9, 21, 34). Our group (15, 36) previously provided evidence that increased H2O2 concentrations may be the trigger for HPV. This suggestion is based on the fact that DPI as well as nitro blue tetrazolium, a superoxide scavenger that blocks H2O2 formation, inhibited but did not mimic HPV in perfused rabbit lungs (15, 38). Increased superoxide generation during hypoxia was also found by Marshall et al. (23) for small pulmonary arterial smooth muscle cells originating from fetal calf and by Chandel et al. (6) for wild-type Hep3B cells in vitro. To further address a role of H2O2 generation from superoxide in the transduction mechanisms of HPV in intact lungs, we applied two SOD inhibitors, DETC and TETA (1, 16, 20). DETC nonspecifically inhibited HPV because it was found to similarly suppress the U-46619-elicited PAP increase, thus not allowing any conclusion to be drawn with respect to hypoxia-specific signaling. TETA, in contrast, turned out to be a specific inhibitor of HPV, not affecting vasoconstrictions elicited by the stable thromboxane analog. This finding apparently contrasts to investigations in pulmonary arterial rings of the guinea pig where DETC and TETA amplified hypoxic contraction and slightly increased normoxic vascular tone (1). It has to be kept in mind, however, that these studies differ, focusing on large-artery smooth muscle cells (pulmonary arterial rings) versus small precapillary smooth muscle cells (site of HPV in the intact lungs; 31), and differences between these smooth muscle cell populations have repeatedly been noted (23, 30, 39). The TETA-elicited inhibition of HPV in the present study was not related to some interference with the NO pathway because prior blockage of NO only marginally affected the influence of TETA. The inhibitory capacity of TETA on lung SOD was ascertained by measurement of lung homogenate SOD activity at the end of experiments, with a marked reduction of its activity with TETA being demonstrated.
In conclusion, the present study provides further evidence for involvement of a NAD(P)H oxidase in HPV by 1) detection of transcripts from Mox1, NOH-1L, or a homologous sequence in the rabbit lung tissue, pulmonary arterial smooth muscle cells, and smooth muscle cells from small arterial vessels of the lung; 2) employing a specific NAD(P)H oxidase inhibitor not related to DPI, and 3) by experiments with blockage of lung SOD activity. We propose the following signal cascade for hypoxia sensing and signal transduction in the lung vasculature: hypoxia activates a nonphagocytic NAD(P)H oxidase, and the enhanced superoxide signal is then forwarded via SOD to increase H2O2 levels, with subsequent triggering of vasoconstriction. This proposal is supported by several studies in intact lungs that blocked this pathway at different sites: 1) inhibition of NAD(P)H oxidase activity by DPI (15, 33) and AEBSF (present study), 2) superoxide scavenging by an agent interfering with the appearance of H2O2 (nitro blue tetrazolium) but not by an agent favoring conversion to H2O2 (Tiron) (36), and 3) inhibition of H2O2 formation from superoxide via SOD with TETA (present study). Because all interfering agents referred to inhibited but did not mimic HPV in the intact lung vasculature, increased rather than decreased superoxide and H2O2 formation is assumed to be the underlying signaling event, resulting in precapillary vasoconstriction in response to alveolar hypoxia.
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ACKNOWLEDGEMENTS |
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We thank Dr. H. Acker for fruitful discussions, K. Quanz, G. Dahlem, and C. Homberger for excellent technical assistance, and Dr. R. L. Snipes for linguistic editing of the manuscript.
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FOOTNOTES |
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This work was supported by the Deutsche Forschungsgemeinschaft SFB 547, Project B7.
Portions of the doctoral thesis of André Tadi were
incorporated into this paper.
Address for reprint requests and other correspondence: N. Weissmann, Dept. of Internal Medicine, Justus-Liebig-Univ. Giessen, Klinikstrasse 36, 35392 Giessen, Germany (E-mail: Norbert.Weissmann{at}innere.med.uni-giessen.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 October 1999; accepted in final form 11 April 2000.
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