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 but may also result in chronic pulmonary hypertension. It has not been clarified whether acute HPV and the response to prolonged alveolar hypoxia are triggered by identical mechanisms. We characterized the vascular response to sustained hypoxic ventilation (3% O2 for 120-180 min) in isolated rabbit lungs. Hypoxia provoked a biphasic increase in pulmonary arterial pressure (PAP). Persistent PAP elevation was observed after termination of hypoxia. Total blockage of lung nitric oxide (NO) formation by NG-monomethyl-L-arginine caused a two- to threefold amplification of acute HPV, the sustained pressor response, and the loss of posthypoxic relaxation. This amplification was only moderate when NO formation was partially blocked by the inducible NO synthase inhibitor S-methylisothiourea. The superoxide scavenger nitro blue tetrazolium and the superoxide dismutase inhibitor triethylenetetramine reduced the initial vasoconstrictor response, the prolonged PAP increase, and the loss of posthypoxic vasorelaxation to a similar extent. The NAD(P)H oxidase inhibitor diphenyleneiodonium nearly fully blocked the late vascular responses to hypoxia in a dose that effected a decrease to half of the acute HPV. In conclusion, as similarly suggested for acute HPV, lung NO synthesis and the superoxide-hydrogen peroxide axis appear to be implicated in the prolonged pressor response and the posthypoxic loss of vasorelaxation in perfused rabbit lungs undergoing 2-3 h of hypoxic ventilation.
hypoxic pulmonary vasoconstriction; pulmonary hypertension; nitric oxide
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INTRODUCTION |
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HYPOXIC PULMONARY VASOCONSTRICTION (HPV) matches lung perfusion to ventilation and thus helps prevent arterial hypoxemia (for reviews, see Refs. 6, 23). Since the first description of this mechanism by von Euler and Liljestrand in 1946 (24), a concerted effort has been undertaken 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 precapillary resistance vessels, the predominant site of HPV. However, the mechanism of HPV has not yet been clarified in detail. Pathophysiological entities such as acute respiratory distress syndrome, severe pneumonia, and liver cirrhosis may result in a lack of HPV, with intrapulmonary shunting and arterial hypoxemia. On the other hand, sustained generalized alveolar hypoxia as in chronic obstructive and restrictive lung diseases is believed to lead to pulmonary hypertension and cor pulmonale via generalized HPV. However, the contribution of prolonged vasoconstriction to the vascular remodeling process elicited under these conditions remains enigmatic, and the molecular mechanism underlying development of hypoxia-induced chronic pulmonary hypertension largely remains to be settled. Changes in expression of vascular endothelial growth factor, nitric oxide (NO) synthase (NOS), and endothelin may contribute to the development of hypoxia-driven remodeling events (5, 12). Transcription of these factors is controlled by hypoxia-inducible factor-1, which has been suggested to be regulated by reactive oxygen species. Mechanisms that involve generation of reactive oxygen species may therefore be involved not only in the acute hypoxia-induced vasoconstriction but also in the development of chronic pulmonary hypertension under conditions of prolonged alveolar hypoxia. Time constants underlying the "switch" from sole, fully reversible vasoconstriction to the onset of vascular remodeling during pulmonary hypoxia are, however, largely unknown.
Moreover, it has not been clarified whether the two phases of the biphasic elevation in pulmonary resistance found in isolated lungs or intact animals during prolonged hypoxia are regulated by identical or different mechanisms.
In the present investigation, we therefore characterized the features of the pulmonary vascular response to sustained alveolar hypoxia (120-180 min) in isolated buffer-perfused rabbit lungs. A biphasic pressor response occurred, with an early vasoconstrictor peak corresponding to acute HPV and a subsequent sustained and progressive pulmonary arterial pressure (PAP) increase. Interestingly, in contrast to acute HPV, posthypoxic baseline PAP remained elevated after cessation of these periods of prolonged hypoxia, which might be suggestive of the early onset of the initial steps leading to development of pulmonary hypertension. We then employed this model to ask whether interference with NO synthesis and oxygen radical formation, all previously noted to be involved in acute HPV, similarly affected the prolonged vasoconstrictor response and the loss of posthypoxic vasorelaxation encountered with sustained alveolar hypoxia in the perfused lungs. In essence, both the acute HPV and the sustained vasoconstrictor response and posthypoxic increase in baseline PAP were enhanced by blockage of NO synthesis. Furthermore, superoxide scavenging and the inhibition of hydrogen peroxide (H2O2) appearance by blocking superoxide dismutase (SOD) suppressed acute HPV as well the sustained hypoxic vasoconstrictor response and posthypoxic increase in baseline PAP. NADPH oxidase inhibition blocked the "late" vascular responses to alveolar hypoxia even more impressively than it blocked the acute hypoxic vasoconstrictor response. Thus, as previously described for acute HPV (8, 9, 16, 20, 26, 29), these data suggest a role for NO and also for the sequence of superoxide and H2O2 in the signaling cascade underlying sustained vasoconstriction and elevated posthypoxic vascular tone in this model of 2-3 h of alveolar hypoxia.
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MATERIALS AND METHODS |
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Reagents
NG-monomethyl-L-arginine (L-NMMA), S-methylisothiourea sulfate (SMT), and diphenyleneiodonium (DPI) were from Calbiochem (Frankfurt, Germany), and triethylenetetramine (TETA) was from Fluka (Deisenhofen, 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 previously described (17, 27). 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 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, adjusted with NaHCO3 to a constant pH range of 7.37-7.40. After the lungs were rinsed with at least 1 liter of buffer fluid for washout of the 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-balance N2 (tidal volume 30 ml; frequency 30 strokes/min). A positive end-expiratory pressure of 1 cmH2O was chosen (0 referenced at the hilum). The isolated perfused lungs were placed in a temperature-equilibrated housing chamber and were 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 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 a 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
The technique of sequential hypoxic maneuvers in buffer-perfused rabbit lungs has been previously described (27). 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 mmHg; baseline condition) to 3% vol/vol (alveolar PO2 ~23 mmHg; hypoxic condition)]. CO2 at 5.3% (vol/vol) was used throughout, and the percentage of N2 was balanced accordingly. In portions of the experiments, buffer returning from the perfusate reservoir to the lungs was passed through a membrane oxygenator (M8Exp, Jostra, Hirrlingen, Germany). With this device, the partial pressure was set at ~40 mmHg for both CO2 and O2 in the postoxygenator buffer fluid entering the pulmonary artery. Hypoxic maneuvers were performed by hypoxic ventilation with 3% O2. The perfusate volume was increased in these experiments to 350 ml.Sustained Alveolar Hypoxia
After an initial short-term period of hypoxia (10-min duration), which allows assessment of the adequate lung response to this challenge (26, 27, 30), and a subsequent 15-min period of normoxia, a sustained hypoxic (3% O2) ventilation of 180 or 120 min was performed followed by a period of normoxic (21% O2) ventilation. In a portion of the experiments, a third hypoxic (3% O2) challenge of 10-min duration was started 15 min after cessation of the 120 or 180 min of hypoxia.Measurement of Microvascular Pressures
Microvascular pressures were assessed by the arterial and venous double-occlusion technique. Electromagnetic tube clamping devices were used for the occlusion maneuvers. The occlusion maneuvers were performed in end expiration, synchronized by a digital-to-analog, analog-to-digital converter with a personal computer. Data were collected at a rate of 20 Hz and processed with a spreadsheet program (Microsoft Excel) for calculation of the relative portions of arterial and venous vascular resistance.Effects of Pharmacological Agents on 120 min of Alveolar Hypoxia
Various pharmacological agents were investigated for their effects on the pressure responses provoked by a sustained alveolar hypoxia of 120 min duration and on the normoxic vascular tone after cessation of the prolonged hypoxic period. All of these agents are known 1) to specifically diminish the strength of the acute hypoxic pressure response when investigated during 10-min periods of alveolar hypoxia compared with their effect on U-46619 [thromboxane (Tx) analog]-induced vasoconstriction (9, 26, 29) or, 2) in the case of L-NMMA, to specifically amplify the strength of the acute hypoxic pressure response when investigated during 10-min periods of alveolar hypoxia compared with U-46619 (Tx analog)-induced vasoconstriction (8). Moreover, the inducible NOS (iNOS) inhibitor SMT was investigated. To determine the possible differences in the responsiveness of the acute hypoxia-elicited pressure elevation (occurring within 10 min of hypoxia) and the secondary (sustained) hypoxia-induced increase in PAP to the various pharmacological interventions, the inhibitors were added to the perfusate in a mode that ensured a constant impact on the strength of acute HPV as ascertained in separate control experiments. In these experiments, the well-characterized (9, 26, 27, 30) mode of repetitive 10-min maneuvers of alveolar hypoxia, alternating with periods of normoxic ventilation of 15 min duration, was applied. For agents that inhibit the acute hypoxic response, the goal was a suppression of ~50% of the control response (no inhibitor). Moreover, the dose protocol of each agent ascertained that such a suppressor effect was maintained throughout a 120-min observation period as probed by repetitive hypoxic maneuvers during this time period. However, in some cases, the "constancy period" did not commence with the first but with a later hypoxic challenge due to the pharmacokinetics of the individual agent (see below). Similarly, for agents that amplify the acute vasoconstrictor response to hypoxia (L-NMMA and SMT), constancy of the amplification was ensured in a corresponding protocol of repetitive short-term hypoxic maneuvers. These preceding control experiments suggested the following protocols to guarantee well-controlled effects of each pharmacological intervention over a 120-min observation period when investigating the impact on sustained hypoxia (all administrations being started 5 min before the onset of hypoxia, with concentrations referring to the recirculating perfusate).L-NMMA. The concentration of L-NMMA was 400 µM. Sustained alveolar hypoxia was started corresponding to the time of the second hypoxic challenge with repetitive hypoxic maneuvers in the control lungs.
SMT. The concentration of SMT was 2 µM. Sustained alveolar hypoxia was started corresponding to the time of the third hypoxic challenge.
Nitro blue tetrazolium. The concentration of nitro blue tetrazolium (NBT) was 900 nM followed by a continuous infusion over 75 min of a dose resulting in an increase of 600 nM/h. The infusion rate was then increased to result in an additional drug admixture of 900 nM/h. Sustained alveolar hypoxia was started corresponding to the time of the third hypoxic challenge.
TETA. The concentration of TETA was 25 mM followed by a 1:2 dilution with fresh buffer after 95 min and again after 170 min. Sustained alveolar hypoxia was started corresponding to the time of the third hypoxic challenge. In the TETA experiments performed during preblocked lung NO synthesis, L-NMMA (400 µM) was present in the perfusate from the start of the experiments.
DPI. The concentration of DPI was 1.5 µM followed by an exchange of perfusate (flushed with 500 ml) after 70 min and again after 95 min. Sustained alveolar hypoxia was started corresponding to the time of the fifth hypoxic challenge. To circumvent the interfering effects of the NO-inhibitory effect of DPI, L-NMMA (400 µM) was present in the perfusate throughout the experiment.
Monitoring of Exhaled NO
The technique has been previously described (18). 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 was measured in parts per billion (vol/vol).Measurement of TxA2 and Prostacyclin
TxA2 and prostacyclin were measured from aliquots of the perfusate as their stable hydrolysis products TxB2 and 6-keto-PGF1Statistics
Data are means ± SE. Analysis of variance with the Student-Newman-Keuls post hoc test was used. For comparison of two groups, a two-sided t-test or, if there was a large difference in the number of experiments in each group, a Smith-Satterthwaite test was performed (14, 15). Significance was assumed when P was <0.05. ![]() |
RESULTS |
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Under baseline conditions, the PAP value was 6.8 ± 0.1 mmHg
(n = 117 experiments) in all experiments. A 3%
hypoxic challenge (alveolar PO2 ~23 mmHg) for
180 min provoked a biphasic pressure increase, with an initial pressure
rise of 3.2 ± 0.6 mmHg (n = 4 experiments), which
was reached within 5.5 min, followed by a pressure nadir (change in PAP
from baseline 1.6 ± 0.2 mmHg) at 15.0 ± 0.9 min and a
second ongoing increase in PAP until the cessation of hypoxic
ventilation, which reached a maximum of 4.5 ± 1.0 mmHg after 180 min (Fig. 1A). After
termination of hypoxic ventilation, PAP rapidly decreased to a level
that was significantly elevated compared with normoxic control values.
No differences in the sustained pressor response to hypoxia were
observed by varying the mixed venous PO2 (40 vs. 150 mmHg) in separate experiments (data not shown).
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Measurement of microvascular pressures revealed a predominantly precapillary vasoconstriction for both phases of hypoxia-induced pressure elevation (Fig. 1B).
The elevation in normoxic vascular tone was already evident after a
sustained alveolar hypoxia of 120 min (Fig.
2). The lung weight increase during
2 h of hypoxic ventilation was 2.1 ± 0.6 g
(n = 5 experiments) vs. 2.3 ± 0.3 g
(n = 5 experiments) in normoxic control lungs
during the corresponding time. The exchange of perfusate 20 min after
cessation of hypoxia (flushed with 1 liter of fresh buffer) for the
washout of recirculating metabolites only partially decreased the
elevated posthypoxic vascular tone. Notably, the slight increase in PAP
occurring in time-matched normoxia-ventilated control lungs was
correspondingly decreased by flushing the vasculature with fresh buffer
fluid. The significant elevation in PAP after sustained alveolar
hypoxia was maintained for the investigation period of 40 min after
exchange of the perfusate.
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Measurement of TxA2 and prostacyclin showed no difference between hypoxic ventilation over a 180-min period and normoxia-ventilated control lungs (data not given).
NO
As evident from Fig. 3, exhaled NO decreased immediately on a step change from normoxic to hypoxic ventilation. After a constant level was reached, no further change in the exhaled NO concentration occurred over a hypoxic period of 180 min except for the gradual decline with time, which was also observed in experiments with repetitive performance of short-term hypoxic challenges (10-min periods of hypoxic ventilation alternating with 15-min periods of normoxic ventilation). After the cessation of alveolar hypoxia at 180 min, the step change in NO exhalation was rapidly reversible (Fig. 3).
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Inhibition of lung NO synthesis with 400 µM
L-NMMA (present in the perfusate from the beginning
of the experiments) more than doubled the initial PAP increase in
response to hypoxia (Fig. 4). In
addition, the sustained PAP elevation during prolonged hypoxia was
enhanced in a corresponding fashion (no significant difference in the
relative increase in the acute PAP peak and the sustained PAP rise).
Similarly, a corresponding elevation in the posthypoxic increase in
baseline PAP was noted.
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To investigate a possible contribution of iNOS on the biphasic response, the iNOS inhibitor SMT was applied at a concentration of 2 µM (Fig. 4). This dose prominently decreased exhaled NO from 73 ± 4 to 20 ± 3 parts/billion (n = 5 experiments) but only slightly increased the vasoconstrictor response to 10-min periods of alveolar hypoxia by 0.6 ± 0.1 mmHg from a baseline response of 2.5 ± 0.2mmHg (n = 3 experiments). Correspondingly, a moderate increase in the sustained vasoconstrictor response to 120 min of hypoxia was observed in the presence of SMT. Normoxic vascular tone after cessation of the prolonged hypoxic challenge was elevated to a similar extent.
Reactive Oxygen Species
NBT, an agent that scavenges superoxide and prevents H2O2 formation (16, 26), was applied at a concentration that suppressed the acute hypoxic response by ~50% (Fig. 5). A corresponding inhibitory effect was then also noted for the sustained pressor response to 120 min of hypoxia and for the posthypoxic elevation in baseline PAP. Similarly, the SOD inhibitor TETA, expected to increase superoxide and to decrease H2O2 formation (1), markedly suppressed the acute vasoconstrictor response to hypoxia as well as the sustained PAP elevation provoked by 120 min of hypoxia and the posthypoxic elevation in normoxic vascular tone (Fig. 6). Corresponding results were obtained when the effect of TETA was investigated under conditions of preblocked NO synthesis due to the presence of L-NMMA (Fig. 6). Under these circumstances, all pressure values ranged at higher values; however, the inhibitory effect of TETA was again obvious, with the magnitude of the acute hypoxic vasoconstriction, the sustained PAP elevation, and the posthypoxic elevation in baseline PAP all being reduced to a similar extent.
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The flavoprotein inhibitor DPI was applied under conditions of
preblocked NO synthesis (400 µM L-NMMA) throughout
because this agent is known to inhibit NOS in addition to NAD(P)H
oxidase (8, 25). The dose used ensured inhibition of the
acute vasoconstrictor response to hypoxia to approximately half of the
maximal response (Fig. 7). Under these
conditions, the sustained PAP increase in response to prolonged hypoxia
was nearly fully abrogated as was the elevation in posthypoxic vascular
tone. The inhibitory effect of DPI on the late pressor
responses to hypoxia thus significantly surpassed its suppressor effect
on the immediate hypoxia-elicited PAP increase (P < 0.05 for all values after 40 min of hypoxia compared with the
extent of inhibition of the peak initial pressor response). This
most prominent blockage of the vasoconstrictor events during prolonged
hypoxia in the presence of DPI was, however, not due to some
generalized inhibition of vascular smooth muscle cell contractility;
all DPI-treated lungs still responded to an acute hypoxic challenge
performed 25 min after cessation of sustained alveolar hypoxia, with an
acute pressure elevation in a range corresponding to the initial
hypoxia-elicited pressor response (Fig. 7). In time-matched control
experiments with repetitive short-term hypoxia (10-min periods of
hypoxia), the inhibitory effect of DPI was still present at the
corresponding time point.
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Initial Effects of the Applied Agents on Normoxic PAP
Application of L-NMMA, SMT, or DPI only marginally increased baseline (normoxic) vascular tone within 5 min of addition of either agent (range of PAP increase between 3.7 and 6.6% of baseline PAP), whereas NBT and TETA slightly decreased baseline PAP (<5% change from baseline PAP) within 5 min of their addition. ![]() |
DISCUSSION |
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General Features of Sustained Alveolar Hypoxia
It has previously been found (4, 21, 22, 32) in isolated lungs and in intact and open-chest animals that sustained alveolar hypoxia provokes a prolonged PAP elevation, but the underlying mechanisms have rarely been addressed. Our group (27) previously showed that a biphasic pressor response is regularly elicited by prolonged hypoxia in perfused rabbit lungs, and this was also noted in intact rabbits (21, 22), in vivo perfused lungs of the dog, and intact dogs (4, 31) as well as in isolated ferret lungs (32). A new observation of the present study is the fact that 120-180 min of sustained (uninterrupted) alveolar hypoxia sufficed to cause a persistent elevation in posthypoxic "baseline" PAP. This issue was hitherto only addressed in the study of Welling et al. (31) in dog lungs, but these authors noted unchanged posthypoxic vasorelaxation after 160 min of alveolar hypoxia, with reasons for this discrepancy currently being unknown. The increase in posthypoxic vascular tone was, however, not induced by lung edema formation because no difference was found in the lung weight increase between the hypoxic (2 h of hypoxic ventilation) and normoxic control experiments. In the present investigation, the magnitude of the ongoing PAP elevation after termination of sustained hypoxia was correlated with the strength of the vasoconstrictor response during hypoxia, which is most evident from the studies in the presence of L-NMMA, with a two- to threefold elevation in both the pressor response during hypoxia and the posthypoxic increase in baseline PAP.The elevation in normoxic vascular tone induced by sustained alveolar hypoxia was only partially reversible after exchange of the perfusate in our experiments. The effect of buffer exchange corresponded to that in the normoxic control lungs and may be attributed to the accumulation of some vasoconstrictor agents in the recirculating medium. Clearly, however, even when correcting for buffer exchange, an increase in posthypoxic PAP was consistently noted. As anticipated from previous investigations (2, 11, 28) demonstrating independence of HPV from the prostanoid metabolism, no changes in perfusate TxA2 and prostacyclin levels were encountered in sustained hypoxia, largely excluding a role of these prostanoids in the progressive pressor response to prolonged hypoxia and in the loss of posthypoxic vasorelaxation.
Measurement of the site of vasoconstriction by the double-occlusion technique in the present study uncovered a predominantly precapillary location throughout the prolonged vasoconstrictor response. This is of interest because one may suggest that prolonged hypoxic ventilation as it occurs in high-altitude pulmonary edema may be caused by a postcapillary vasoconstriction. The fact that the course of the biphasic vasoconstrictor response was independent of mixed venous PO2 in our experiments supports the thesis that the location of the O2 sensor is easily accessible from the alveolar but not from the intravascular site. This is in line with a previous investigation (27) in rabbit lungs exploiting the acute vasoconstrictor response to hypoxia.
NO
For the main pulmonary artery and smaller pulmonary arterial vessels of the rat, it has been described (2, 11) that only the first phase of the biphasic response to hypoxia is dependent on the endothelium, which may be the quantitatively dominating source of lung NO synthesis. However, no effect of NO inhibition on the time course of the biphasic response to hypoxia was found in rat pulmonary arteries (2, 10). Unfortunately, no investigations addressing the role of the NO pathway in the biphasic response to sustained hypoxia in intact lungs hitherto exist. Concerning the acute HPV response in rabbit lungs, this was previously noted (8) to be markedly amplified by L-NMMA, significantly surpassing the effect of this agent on pharmacologically induced vasoconstriction. Moreover, Grimminger et al. (8) previously demonstrated that alveolar hypoxia induces an immediate drop in the concentration of exhaled NO that precedes the vasoconstrictor response. When currently analyzed for the sustained hypoxia challenge, the depression in NO exhalation was found to persist over the entire period of hypoxic ventilation. Moreover, virtually complete inhibition of lung NO synthesis by L-NMMA amplified the acute and sustained hypoxic responses to a nearly identical extent, and a corresponding enhancement of the posthypoxic elevation of baseline PAP was noted. In addition, we investigated a possible role of iNOS (NOS II) in the response to sustained alveolar hypoxia. When employing the iNOS inhibitor SMT in a concentration that avoids major suppression of NOS III, marked suppression of exhaled NO was noted, but there was only a moderate increase in the early and protracted pressor responses to alveolar hypoxia. Thus a drop in lung NO synthesis, mostly attributable to NOS III-derived NO, is suggested to contribute to the acute PAP peak and the sustained vasoconstrictor response evoked by alveolar hypoxia. The "remaining" lung NO synthesis during hypoxia nevertheless serves to counterbalance excessive vasoconstrictor events as evident from the exaggerated pressor response in the presence of L-NMMA. These data suggest that the role of NO is comparable in acute HPV and the sustained pressor response to alveolar hypoxia and that in the absence of NO, the loss of posthypoxic vasorelaxation is similarly aggravated.Reactive Oxygen Species
Our laboratory (9, 26, 29, 30) recently provided evidence that superoxide formation, perhaps derived from a NAD(P)H oxidase, and the subsequent generation of H2O2 is the underlying mechanism of acute HPV. We therefore tested 1) NBT, a superoxide scavenger that inhibits H2O2 formation (16, 26), and 2) TETA, a SOD inhibitor (1, 29), each in a concentration that caused a decrease to approximately half of the acute HPV, to determine their impact on the sustained pressor response during prolonged alveolar hypoxia and on posthypoxic vasorelaxation. It was found that the late responses to hypoxia were blocked to a similar extent as the early hypoxic vasoconstrictor responses with both NBT and TETA. This was also true when investigating the effect of TETA under conditions of preblocked lung NO synthesis (presence of L-NMMA). These findings support the hypothesis that the superoxide-H2O2 axis contributes to the signaling events in both acute HPV and the sustained vasoconstrictor phenomena, including the loss of posthypoxic vasorelaxation occurring during prolonged hypoxia.Addressing the same issue, the unspecific NAD(P)H oxidase inhibitor DPI, formerly shown to specifically block the acute hypoxic pressor response compared with pharmacologically provoked vasoconstrictor events (9, 20), was probed at a concentration that partially inhibited the early pressure peak in response to hypoxia. Again, efficacy of this agent was also evident for the sustained hypoxia-driven vasoconstrictor response and the loss of posthypoxic vasorelaxation; most interestingly, however, these late responses to hypoxia were nearly fully (sustained vasoconstriction) or even totally (posthypoxic PAP increase) blocked in the presence of DPI, with the extent of inhibition of these late responses significantly surpassing that of the early vasoconstrictor response to hypoxia. It has to be emphasized that this strong influence of DPI on the late vascular responses to hypoxia was not due to pharmacokinetic reasons, e.g., progressive accumulation of this agent, because the dose regimen was probed in preceding control experiments to ascertain that repetitive acute hypoxic vasoconstrictor reactions were inhibited by ~50% throughout this time period. These control experiments exclude a washout of DPI as the underlying mechanism of the maintenance of the vasoconstrictor response to an acute hypoxic challenge probed 25 min after cessation of sustained alveolar hypoxia. Moreover, the near absence of a PAP increase after 120 min of hypoxia and during the posthypoxic period may not be explained by a general loss in vascular reactivity or an entire loss in hypoxic sensing because the acute responsiveness to alveolar hypoxia, probed after 145 min (25 min after cessation of the sustained hypoxia period), was still in the control range for L-NMMA- and DPI-treated lungs with repetitive short-term hypoxia. These findings again support the view that the superoxide-H2O2 axis is involved in the signal transduction pathways, resulting in both the early vasoconstrictor event and the sustained pressor response, including loss of posthypoxic vasorelaxation to a 2- to 3-h alveolar hypoxic challenge, presuming an inhibitory effect of DPI on NAD(P)H oxidase- or mitochondria-generated superoxide production as suggested by Li and Trush (13). In addition, however, further effects of DPI being operative in the late phase of lung vascular responsiveness to hypoxia are to be assumed. It is currently not known whether such an effect might be related to the nonspecific impact of DPI on a variety of flavoproteins (3, 7, 19, 25) or its suppressor effect on potassium and calcium channels (25). Therefore, further investigations are needed to clarify which of the possible targets of DPI are responsible for the more prominent effect of this agent in the late compared with the early phase of the lung vascular response to hypoxia.
In conclusion, a 2- to 3-h period of alveolar hypoxia in buffer-perfused rabbit lungs is suitable to provoke in a well-reproducible fashion 1) an initial vasoconstrictor response mostly analyzed when addressing acute HPV, 2) a sustained and progressive pressor response commencing after 20-30 min, and 3) a loss of posthypoxic normalization of PAP. We provide evidence that lung NO synthesis and the superoxide-H2O2 axis are operative in both the early and late lung vascular responses to alveolar hypoxia in this model. A decline in NO generation is suggested to contribute to the vasoconstrictor events, but remaining baseline NO is still operative in counterbalancing the exaggerated PAP elevation. The various pharmacological interventions suggest that enhanced rather than reduced formation of superoxide with a subsequent appearance of H2O2 is a decisive signaling event in both the early (acute HPV) and late (sustained pressor response, loss of posthypoxic normalization of PAP) vascular responses to alveolar hypoxia because none of the inhibitors mimicked the hypoxic response. Interestingly, DPI is even more effective in suppressing the late vascular responses to the hypoxic challenge compared with those in acute HPV, the underlying reasons of which remain to be elucidated. The model of sustained alveolar hypoxia in perfused rabbit lungs may thus offer the opportunity to study the signal transduction pathways underlying the transition from early and fully reversible vasoconstriction to a prolonged and only partially reversible PAP increase in sustained alveolar hypoxia. These mechanisms may be identical to the initial steps leading to development of pulmonary hypertension during chronic hypoxia.
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ACKNOWLEDGEMENTS |
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We thank Dr. R. L. Snipes (Justus-Liebig-University Giessen, Giessen, Germany) for linguistic editing of the manuscript.
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FOOTNOTES |
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This work was supported by the Deutsche Forschungsgemeinschaft (Grant SFB 547, Project B7).
Portions of the doctoral thesis of Stefan Winterhalder were incorporated into this report.
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 15 September 2000; accepted in final form 6 November 2000.
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