Contribution of oxygen radicals to altered NO-dependent pulmonary vasodilation in acute and chronic hypoxia

Nikki L. Jernigan, Thomas C. Resta, and Benjimen R. Walker

Vascular Physiology Group, Department of Cell Biology and Physiology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131-0001

Submitted 3 July 2003 ; accepted in final form 10 December 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chronic hypoxia (CH) increases pulmonary arterial endothelial nitric oxide (NO) synthase (NOS) expression and augments endothelium-derived nitric oxide (EDNO)-dependent vasodilation, whereas vasodilatory responses to exogenous NO are attenuated in CH rat lungs. We hypothesized that reactive oxygen species (ROS) inhibit NO-dependent pulmonary vasodilation following CH. To test this hypothesis, we examined responses to the EDNO-dependent vasodilator endothelin-1 (ET-1) and the NO donor S-nitroso-N-acetyl penicillamine (SNAP) in isolated lungs from control and CH rats in the presence or absence of ROS scavengers under normoxic or hypoxic ventilation. NOS was inhibited in lungs used for SNAP experiments to eliminate influences of endogenously produced NO. Additionally, dichlorofluorescein (DCF) fluorescence was measured as an index of ROS levels in isolated pressurized small pulmonary arteries from each group. We found that acute hypoxia increased DCF fluorescence and attenuated vasodilatory responses to ET-1 in lungs from control rats. The addition of ROS scavengers augmented ET-1-induced vasodilation in lungs from both groups during hypoxic ventilation. In contrast, upon NOS inhibition, DCF fluorescence was elevated and SNAP-induced vasodilation diminished in arteries from CH rats during normoxia, whereas acute hypoxia decreased DCF fluorescence, which correlated with augmented reactivity to SNAP in both groups. ROS scavengers enhanced SNAP-induced vasodilation in normoxia-ventilated lungs from CH rats similar to effects of hypoxic ventilation. We conclude that inhibition of NOS during normoxia leads to greater ROS generation in lungs from both control and CH rats. Furthermore, NOS inhibition reveals an effect of acute hypoxia to diminish ROS levels and augment NO-mediated pulmonary vasodilation.

superoxide dismutase; tiron; pulmonary hypertension; S-nitroso-N-acetyl penicillamine; endothelin-1; dichlorofluorescein; nitric oxide


CHRONIC EXPOSURE TO HYPOXIA results in polycythemia, pulmonary arterial remodeling, and pulmonary arterial constriction, all of which contribute to the development of pulmonary hypertension and right ventricular hypertrophy. Endothelial nitric oxide synthase (eNOS), present in the vascular endothelium, synthesizes the potent vasodilatory, nitric oxide (NO), which may reduce the severity of chronic hypoxia (CH)-induced pulmonary hypertension. Indeed, several studies have demonstrated that CH is associated with increased pulmonary eNOS levels, gene expression, and activity in adult rats (7, 14, 37, 39). Furthermore, our laboratory (29, 30, 32) and others (10, 33, 39) have shown that CH augments pulmonary vasodilatory responses to endothelium-derived nitric oxide (EDNO)-dependent vasodilators. However, following inhibition of NOS, vasodilatory responses to several different NO donors are attenuated in isolated lungs from CH rats (11). Although this could potentially be explained by a decrease in smooth muscle sensitivity to NO, this seems unlikely since we have recently shown that the ability to stimulate the NO-dependent vasodilatory signaling pathway via activation of cGMP (11) and PKG (12) is not diminished following CH, but rather, it appears to be enhanced. Although the mechanism(s) for this inconsistency between exogenous and endogenous NO-dependent vasodilatory responsiveness following CH is unclear, some investigators suggest the generation of reactive oxygen species (ROS) during hypoxia-reoxygenation impairs NO formation as well as directly inactivating NO (5, 8, 40). The present study examines the mechanisms responsible for the differential effects of acute and chronic hypoxic exposure on vasodilation to endogenous vs. exogenous NO and the role of ROS on NO-dependent vasoreactivity.

The observation that ROS inhibit NO-dependent vasodilation is not novel. Indeed, even before endothelium-derived relaxing factor (EDRF) was identified as NO, Rubanyi and Vanhoutte (34) demonstrated that EDRF could be inactivated by superoxide and stabilized by superoxide dismutase (SOD). More recent studies have confirmed the ability of antioxidants to improve vasodilatory responses following acute (5) and chronic hypoxia (40), suggesting that reoxygenation following hypoxia may play an important role in ROS production. However, it is equally probable that hypoxia, per se, elevates ROS production (13, 16, 17). Both endothelial cells and vascular smooth muscle cells generate superoxide anion (18, 35, 38), which has been shown to interact with NO producing the cytotoxic oxidant peroxynitrite, thereby preventing NO-induced vasodilation (1, 34); therefore, we hypothesized that increased levels of ROS following CH attenuate NO-dependent pulmonary vasodilation. To test this hypothesis, we assessed both EDNO- and exogenous NO-dependent vasodilatory responses in lungs from control and CH rats during both normoxic ventilation (NV) and hypoxic ventilation (HV). Parallel experiments were conducted in the presence of ROS scavengers to examine the effect of endogenous ROS on NO-dependent reactivity, and ROS levels were measured with the fluorescent indicator 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) in small pulmonary arteries from control and CH rats. Our findings suggest that acute hypoxia increases ROS levels and diminishes EDNO-dependent pulmonary vasodilation in lungs from control rats. In addition, the attenuated vasodilatory response to exogenous NO following CH appears to be due to increased ROS production during NOS inhibition. Finally, NOS inhibition unmasks an acute hypoxia-induced decrease in ROS levels, a response that correlates with normalization of reactivity to exogenous NO between control and CH groups.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
All protocols and surgical procedures employed in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the University of New Mexico School of Medicine (Albuquerque, NM).

Experimental Groups

Female Sprague-Dawley rats (200–280 g, Harlan Industries) were divided into control and CH groups for each experiment. Animals designated for exposure to CH were housed in a hypobaric chamber with barometric pressure maintained at ~380 mmHg for 4 wk. The chamber was opened three times/wk to provide animals with fresh food, water, and clean bedding. On the day of experimentation, rats were removed from the hypobaric chamber and immediately placed in a Plexiglas chamber continuously flushed with a 12% O2-88% N2 gas mixture to reproduce inspired PO2 (~70 mmHg) within the hypobaric chamber. Constant circulation of this gas mixture prevented accumulation of CO2. Age-matched control animals were housed at ambient barometric pressure (~630 mmHg). All animals were maintained on a 12:12-h light-dark cycle.

CH-Induced Right Ventricular Hypertrophy and Polycythemia

Blood samples were obtained by direct cardiac puncture at the time of lung isolation for measurement of hematocrit. Right ventricular hypertrophy was assessed as an index of CH-induced pulmonary hypertension, as previously described (11, 29, 30). Briefly, after each experiment, the atria and major vessels were removed from the ventricles. The right ventricle (RV) was dissected from the left ventricle (LV) and septum, and each was weighed. The degree of right ventricular hypertrophy is expressed as the ratio of RV to total ventricular weight (T). RV/T ratios were similar to previously obtained ratios from freshly isolated hearts (29), suggesting the current ratios were not compromised by ligation during experiments.

Isolated Lung Preparation

Rats from each group were anesthetized with pentobarbital sodium (52 mg ip). After the trachea was cannulated with a 17-gauge needle stub, the lungs were ventilated with a Harvard positive-pressure rodent ventilator (model 683) at a frequency of 55 breaths/min and a tidal volume of 2.5 ml with a warmed and humidified gas mixture (6% CO2-21% O2-balance N2, normoxia; or 6% CO2-balance N2, hypoxia). Inspiratory pressure was set at 9 cmH2O, and positive endexpiratory pressure was set at 3 cmH2O. After a median sternotomy, heparin (100 units) was injected directly into the RV, and the pulmonary artery was cannulated with a 13-gauge needle stub. The preparation was immediately perfused with a physiological saline solution [PSS; (in mM) 129.8 NaCl, 5.4 KCl, 0.83 MgSO4, 19 NaHCO3, 1.8 CaCl2, and 5.5 glucose; all from Sigma] containing 4% (wt/vol) albumin (Sigma) and meclofenamate (30 µM, Sigma) at 0.8 ml/min with a Masterflex microprocessor pump drive (model 7524-10). Meclofenamate was added to minimize potential complicating influences of endogenous prostaglandins on vascular reactivity. This dose of meclofenamate is approximately threefold higher than that previously shown to provide effective inhibition of prostaglandin synthesis in this preparation (11).

The LV was cannulated with a plastic tube (4-mm outer diameter), and the heart and lungs were removed en bloc and suspended in a humidified chamber maintained at 38°C. The perfusion rate was gradually increased to 30 ml·min-1·kg body wt (BW)-1 and maintained at this rate for the duration of the experiment. Twenty milliliters of perfusate were washed through the lungs and discarded before recirculation was initiated with 40 ml. Experiments were performed with lungs in zone 3 conditions, achieved by elevating the perfusate reservoir until pulmonary venous pressure (Pv) was 3–4 mmHg. Pulmonary arterial pressure (Pa) and Pv were measured with Spectramed model P23XL pressure transducers and recorded on a Gould RS 3400 chart recorder. Data were stored and processed with a computer-based data acquisition/analysis system (AT-CODAS, Dataq Instruments).

After a 30-min stabilization period, the thromboxane analog 9,11-dideoxy-9{alpha},11{alpha}-epoxymethanoprostaglandin F2{alpha} (U-46619, Cayman Chemical) was added to the perfusate reservoir until a stable arterial pressor response of ~10 mmHg was achieved. U-46619 provides consistent and stable pressor responses in this preparation (29, 30, 32), allowing assessment of subsequent vasodilatory responses as outlined in the following protocols.

Isolated Lung Experiments

Effect of NOS inhibition on endothelin-1-mediated vasodilation. To determine the contribution of NO to endothelin-1 (ET-1)-mediated vasodilation, we examined vasodilatory responses to ET-1 (1 nM) in U-46619-constricted lungs from control and CH rats in the presence or absence of the NOS inhibitor N{omega}-nitro-L-arginine (L-NNA, 300 µM). This concentration of ET-1 was determined to result in EDNO-dependent vasodilation in previous experiments (32).

Effect of soluble guanylyl cyclase inhibition on ET-1-mediated vasodilation. The contribution of endogenous cGMP in mediating vasodilatory responses to ET-1 in each group of rats was assessed with a heme site-specific soluble guanylyl cyclase (sGC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3-{alpha}]quinoxalin-1-one (ODQ, Sigma). ODQ (50 µM) or its vehicle (DMSO, 75 µl) was added to the recirculating reservoir (40 ml) immediately after lung isolation and was present throughout the experiment. This dose of ODQ has been previously employed by our laboratory (11) to inhibit sGC in this preparation. Responses to ET-1 (1 nM) were then determined in control and CH lungs as described above.

Vasodilatory responses to ET-1 and S-nitroso-N-acetyl penicillamine during hypoxic ventilation. To determine the influence of acute HV on EDNO-dependent vasodilation, we isolated and ventilated lungs from control and CH rats with a normoxic gas mixture (6% CO2, balance air). After a 15-min equilibration, the lungs were either maintained in normoxia for an additional 30 min before administration of U-46619 or switched to HV (6% CO2, balance N2) for the remainder of the experiment. Hypoxic vasoconstriction is transient in this preparation, and pulmonary vascular resistance was allowed to return to baseline before the administration of U-46619 (~30 min). Responses to ET-1 (1 nM) were then determined in U-46619-constricted lungs from control and CH rats.

A cumulative concentration-response relationship to the NO-donor S-nitroso-N-acetyl penicillamine (SNAP; 0.5, 1.0, 10 µM) was assessed in a separate set of U-46619-constricted lungs from both groups during NV and HV. For these and all other SNAP experiments, 300 µM L-NNA was added to minimize the potential complicating influences of endogenous NO on vascular reactivity (11). A stable vasodilatory response to each dose of SNAP was allowed to develop before administration of subsequent doses. Due to concerns with the longevity of stable U-46619 constriction, responses to only three concentrations of SNAP were assessed in each lung.

Vasodilatory responses to ET-1 and SNAP in the presence of ROS scavengers. The contribution of oxygen radicals in altering vasodilatory responses to NO in each group of rats was determined with SOD and catalase (a H2O2 reductase) or 4,5-dihydroxy-1,3-benzene-disulfonic acid (tiron). Catalase was used to reduce H2O2, since H2O2 has been shown to influence cGMP-mediated relaxation upon alterations in PO2 (19, 24). Either SOD (150 U/ml) plus catalase (1,200 U/ml) or tiron (10 mM) was added to the recirculating reservoir (40 ml) immediately after lung isolation and was present throughout the experiment. These doses of SOD/catalase (5, 40) and tiron (36) have been previously employed by other investigators to scavenge oxygen radicals in this preparation. Responses to SNAP (0.5, 1.0, and 10 µM) and ET-1 (1 nM) were then determined in each group during both NV and HV as described above.

ROS Production in Isolated, Pressurized Small Pulmonary Arteries

Rats were anesthetized with pentobarbital sodium (32.5 mg ip), and the heart and lungs were exposed by midline thoracotomy. Heparin (100 units in 0.1 ml) was injected into the RV. The left lung was removed and immediately placed in ice cold PSS. The lung lobe was pinned out in iced PSS in a Silastic-coated dissection dish. A fourthorder intrapulmonary artery (~100- to 300-µm inner diameter) of ~1 mm length was dissected free and transferred to a vessel chamber (Living Systems, CH-1) containing aerated PSS. The proximal end of the artery was cannulated with a tapered glass pipette, secured in place with a single strand of silk ligature, and gently flushed to remove any blood from the lumen. Next, the distal end of the vessel was cannulated, and the artery was stretched longitudinally to approximate its in vivo length and pressurized to 12 mmHg. The vessel chamber was transferred to the stage of a Nikon Diaphot 300 microscope equipped with a x10 Nikon fluorescence objective (numerical aperture 0.30). Vessels were superfused at 37°C for 30 min with PSS aerated with either normoxic (21% O2, 6% CO2, balance N2) or hypoxic gas (6% CO2, balance N2). A cover was placed over the chamber so that the gas mixture also flowed over the top of the chamber bath. Samples of superfusate were periodically drawn for measurement of PO2, PCO2, and pH with a blood-gas analyzer (Radiometer). After the 30-min equilibration, the pressurized pulmonary arteries were loaded with the cell-permeant ROS-sensitive fluorescent indicator CM-H2DCFDA (Molecular Probes). CM-H2DCFDA was dissolved in anhydrous DMSO at a concentration of 50 µg/ml. Immediately before loading, CM-H2DCFDA was mixed with a 20% solution of pluronic acid in DMSO, and this mixture was diluted with PSS to yield a final concentration of 5 µM CM-H2DCFDA and 0.05% pluronic acid. Vessels were incubated in this solution for 30 min at room temperature in the dark. The diluted CM-H2DCFDA solution was aerated with either normoxic or hypoxic gas mixtures during the loading period. We obtained fluorescent images using a standard FITC filter before loading the vessel (for background subtraction) and after the 30-min incubation with CM-H2DCFDA. Images were generated with a cooled, digital charge-coupled device camera (SenSys 1400) and processed with MetaFluor 4.5 software (Universal Imaging). Normalized fluorescence intensity is defined as average gray scale values for all pixels in the field above background. Because isolated lung experiments examining responses to exogenous NO were conducted in the presence of L-NNA, ROS production was measured in separate sets of vessels treated with L-NNA (300 µM).

To demonstrate changes in CM-H2DCFDA fluorescence in response to agents known to produce or scavenge ROS, we prepared a separate set of normoxic control arteries as above and treated it with xanthine (2 mM) and xanthine oxidase (5 mU/ml) to stimulate ROS production. In separate experiments, vessels were treated with tiron (10 mM) before the addition of xanthine/xanthine oxidase.

Calculations and Statistics

Pulmonary vascular resistance was calculated as the difference between Pa and Pv divided by flow (30 ml·min-1·kg BW-1). Vasodilatory responses were calculated as percent reversal of U-46619-induced vasoconstriction. All data are expressed as means ± SE, and values of n refer to the number of animals in each group. Data were analyzed with two-way ANOVA, and if differences were detected, individual groups were compared with the Student-Newman-Keuls test. A probability of P <= 0.05 was accepted as significant for all comparisons.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CH-Induced Right Ventricular Hypertrophy and Polycythemia

RV/T ratios were greater in CH rats (0.312 ± 0.003, n = 86) compared with control rats (0.208 ± 0.003, n = 84), thus demonstrating right ventricular hypertrophy indicative of pulmonary hypertension. Further, CH rats exhibited polycythemia as indicated by a significantly greater hematocrit in CH rats (60.8 ± 0.3%, n = 86) compared with control rats (42.6 ± 0.2%, n = 84).

Isolated Lung Experiments

Baseline vascular resistances and responses to U-46619. All baseline vascular resistances, regardless of treatment, were greater in lungs from CH rats compared with controls (Table 1). Because the pulmonary circulation exhibits no detectable tone in this preparation (31), these data provide functional evidence for CH-induced vascular remodeling. The concentration of U-46619 required to elicit comparable vasoconstriction between groups was significantly less in lungs from CH animals compared with controls (Table 1). Furthermore, in the presence of L-NNA, less U-46619 was required for similar vasoconstriction in both groups, suggesting that endogenous NO attenuates U-46619-induced vasoconstriction in both groups.


View this table:
[in this window]
[in a new window]
 
Table 1. Baseline vascular resistance, concentration of U-46619 administered to induce an ~10 mmHg pressor response, and change in resistance with the administration of U-46619 in isolated lungs from control and CH rats in the presence or absence of L-NNA

 

Effect of NOS inhibition on ET-1-mediated vasodilation. Vasodilatory responses to ET-1 (1 nM), in the absence of L-NNA, were augmented in lungs from CH rats compared with control rats (Fig. 1A) as previously reported (32). The presence of L-NNA abolished vasodilation in lungs from both control and CH rats (Fig. 1A), supporting a primary role for NO in ET-1-dependent pulmonary vasodilation.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. Vasodilatory responses (% of U-46619-induced tone) in lungs from control and chronically hypoxic (CH) rats to endothelin-1 (ET-1) in the presence of N{omega}-nitro-L-arginine (L-NNA, 300 µM, n = 5) or vehicle (saline, n = 7; A) and 1H-[1,2,4]oxadiazolo[4,3-{alpha}]quinoxalin-1-one (ODQ, 50 µM, n = 5) or vehicle (DMSO, n = 5; B). Experiments were conducted in the presence of meclofenamate (30 µM). Values are means ± SE. *P <= 0.05 vs. control vehicle group; #P <= 0.05 vs. corresponding vehicle-treated group.

 

Effect of sGC inhibition on ET-1-mediated vasodilation. The sGC inhibitor ODQ effectively blocked vasodilatory responses to ET-1 (1 nM, Fig. 1B) in lungs from both CH and control rats. These data further suggest ET-1-dependent vasodilation is mediated through cGMP and are consistent with the previously reported inhibition of pulmonary vasodilation to exogenous NO by ODQ in this preparation (11).

Vasodilatory responses to ET-1 during HV. Vasodilatory responses to ET-1 were augmented in lungs from CH rats vs. controls under both NV and HV (Fig. 2). Furthermore, ventilation with hypoxic gas significantly attenuated vasodilatory responses to ET-1 in lungs from control but not CH rats.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2. Vasodilatory responses in lungs from control and CH rats to ET-1 (1 nM, n = 5) during normoxic ventilation (NV; 6% CO2, balance air) or hypoxic ventilation (HV; 6% CO2, balance N2). Experiments were conducted in the presence of meclofenamate (30 µM). Values are means ± SE. *P <= 0.05 vs. corresponding control group; #P <= 0.05 vs. control-NV group.

 

Saline perfusate gas data collected during NV and HV for both ET-1 and SNAP experiments are shown in Table 2. HV had no effect on pH or PCO2 compared with NV; however, as expected, HV resulted in a lower perfusate PO2 in both control and CH groups. Furthermore, there were no differences in perfusate gases between control and CH groups within NV and HV treatments. We observed transient hypoxic vasoconstriction during the first 20 min of HV; however, this constriction was allowed to return to baseline before the administration of U-46619 (Table 3). The change in pulmonary resistance during the peak hypoxic pressor responses was not different between control and CH groups.


View this table:
[in this window]
[in a new window]
 
Table 2. Saline perfusate pH, PCO2, and PO2 in isolated lungs from control and CH rats during normoxic and hypoxic ventilation

 

View this table:
[in this window]
[in a new window]
 
Table 3. Normoxic baseline resistance, peak change in resistance to hypoxia, and hypoxic baseline resistance in isolated lungs from control and CH rats in the presence or absence of L-NNA

 

Vasodilatory responses to ET-1 in the presence of ROS scavengers. Whereas vasodilatory responses to ET-1 in both control and CH groups were unaffected by administration of either SOD/catalase or tiron during NV (Fig. 3A), oxygen radical scavengers greatly augmented ET-1-mediated vasodilation in control lungs ventilated with hypoxic gas (Fig. 3B). A similar effect of oxygen radical scavengers to enhance reactivity to ET-1 was observed for CH lungs during HV, although significance was achieved only for SOD/catalase (Fig. 3B).



View larger version (46K):
[in this window]
[in a new window]
 
Fig. 3. Vasodilatory responses in lungs from control and CH rats to ET-1 (1 nM) in the presence of the oxygen-radical scavengers superoxide dismutase (SOD, 150 U/ml) plus catalase (1,200 U/ml) or tiron (10 mM) or vehicle during NV (A) and HV (n = 5/group, B). Experiments were conducted in the presence of meclofenamate (30 µM). Values are means ± SE. *P <= 0.05 vs. corresponding control group; #P <= 0.05 vs. vehicle in corresponding group.

 

Vasodilatory responses to SNAP during HV. In contrast to ET-1 responses, vasodilatory responses to 0.5 and 1.0 µM SNAP in lungs from CH rats were significantly attenuated compared with those of control rats during NV (Fig. 4), although diminished reactivity was not observed at the highest concentration of SNAP (10 µM). Interestingly, HV augmented vasodilatory responses to 0.5 and 1.0 µM SNAP in lungs from both control and CH rats and normalized reactivity between these groups. Vasodilatory responses to 10 µM SNAP were significantly greater in lungs from CH rats compared with controls during HV (Fig. 4).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 4. Vasodilatory responses in lungs from control and CH rats to S-nitroso-N-acetyl penicillamine (SNAP; 0.5, 1.0, 10 µM; n = 5/group) during NV (6% CO2, balance air) or HV (6% CO2, balance N2). Experiments were conducted in the presence of L-NNA (300 µM) and meclofenamate (30 µM). Values are means ± SE. *P <= 0.05 vs. corresponding control group; #P <= 0.05 vs. NV in corresponding group.

 

Vasodilatory responses to SNAP in the presence of ROS scavengers. Similar to effects of HV, administration of SOD/catalase or tiron significantly augmented vasodilatory responses to 0.5 and 1.0 µM SNAP in lungs from CH rats during NV (Fig. 5, A and B). Although a similar tendency for ROS scavengers to augment SNAP-induced vasodilation was observed for control lungs, significance was achieved only for tiron at the 0.5 µM concentration of SNAP (Fig. 5B). No differences between groups were noted at the highest concentration of SNAP (1 and 10 µM).



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5. Vasodilatory responses in lungs from control and CH rats to SNAP (0.1, 0.5, and 1.0 µM) in the presence of the oxygen-radical scavengers SOD (150 U/ml) and catalase (1,200 U/ml, A) or tiron (10 mM, B) or vehicle during normoxic ventilation (n = 5/group). Experiments were conducted in the presence of L-NNA (300 µM) and meclofenamate (30 µM). Values are means ± SE. *P <= 0.05 CH- vs. control-vehicle group; {dagger}P <= 0.05 CH- vs. control-treated group; #P <= 0.05 vehicle vs. treated in CH group; and $P <= 0.05 vehicle vs. treated in control group.

 

In contrast to effects of ROS scavengers in NV lungs, there was little or no effect of either SOD/catalase or tiron on vasodilatory responses to SNAP in lungs from either group of rats when ventilated with a hypoxic gas mixture (Fig. 6, A and B), although a significantly greater vasodilation to 0.5 µM SNAP was observed in control lungs treated with tiron vs. vehicle (Fig. 6B). Furthermore, NO-dependent vasodilation was augmented in lungs from CH rats vs. controls at the 10 µM dose of SNAP.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 6. Vasodilatory responses in lungs from control and CH rats to SNAP (0.1, 0.5, and 1.0 µM) in the presence of the oxygen-radical scavengers SOD (150 U/ml) and catalase (1,200 U/ml, A) or tiron (10 mM, B) or vehicle during HV (n = 5/group). Experiments were conducted in the presence of L-NNA (300 µM) and meclofenamate (30 µM). Values are means ± SE. *P <= 0.05 CH- vs. control-vehicle group; {dagger}P <= 0.05 CH- vs. control-treated group; #P <= 0.05 vehicle vs. treated in CH group; and $P <= 0.05 vehicle vs. treated in control group.

 

ROS Production in Isolated, Pressurized Small Pulmonary Arteries

We validated the CM-H2DCFDA signal by monitoring fluorescence obtained from treatment with xanthine-xanthine oxidase in the presence or absence of tiron in pulmonary vessels from control rats under normoxic conditions. Treatment with xanthine-xanthine oxidase in the absence of tiron resulted in greater DCF average fluorescent intensity (988 + 76 gray scale values) compared with xanthine-xanthine oxidase in the presence of tiron (234 + 48 gray scale values), thus demonstrating our ability to measure ROS changes in small pulmonary arteries. In the absence of L-NNA, hypoxia increased DCF fluorescence in vessels from control animals but had no effect in vessels from CH animals (Fig. 7A). However, in the presence of L-NNA, hypoxia greatly attenuated DCF fluorescence in both groups (Fig. 7B). Furthermore, ROS production was significantly greater in vessels from CH rats compared with controls under normoxia (Fig. 7B). These data suggest that acute hypoxia may decrease ROS production in the absence of an intact NOS system; however, ROS levels are largely influenced by NOS activity. Superfusate gas data collected during normoxia and hypoxia are shown in Table 4.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 7. 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) fluorescence (average gray scale values above background) in isolated small pulmonary arteries from control and CH rats under normoxic or hypoxic conditions in the absence (A) or presence of L-NNA (300 µM) (B). Values are means ± SE. *P < 0.05 vs. corresponding control group; # P < 0.05 vs. corresponding normoxic group.

 

View this table:
[in this window]
[in a new window]
 
Table 4. Saline perfusate pH, PCO2, and PO2 in isolated small pulmonary arteries from control and CH rats during normoxic and hypoxic superfusion

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The major findings from this study are 1) ET-1-induced pulmonary vasodilation is mediated by NO/cGMP; 2) compared with NV, HV attenuates ET-1-mediated vasodilation in lungs from control, but not CH, rats. This correlates with increased ROS production in pulmonary arteries from control rats during hypoxia. Additionally, ROS scavengers augment ET-1-dependent vasodilation in lungs from both groups under HV, and 3) HV augments vasodilatory responses to SNAP in L-NNA-treated lungs from both control and CH rats and normalizes NO-dependent vasoreactivity between groups. Similar to HV, ROS scavengers augment SNAP-mediated vasodilation in NV lungs from CH rats but have little influence in lungs ventilated with hypoxic gas. Consistent with these observations, ROS production is greater in both groups under normoxic conditions compared with hypoxia in the presence of L-NNA; however, this augmented ROS generation is significantly enhanced in pulmonary arteries from CH rats compared with controls. We conclude that inhibition of NOS during normoxia leads to greater ROS generation in lungs from both control and CH rats. Furthermore, NOS inhibition reveals an effect of acute hypoxia to diminish ROS levels and augment NO-mediated pulmonary vasodilation. Finally, impaired NO-dependent pulmonary vasodilation following CH appears to be mediated at least in part by greater ROS production during NOS inhibition. A summary of these findings is provided in Fig. 8.



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 8. Summary of major findings. A: effects of acute hypoxia on reactive oxygen species (ROS) generation and endothelium-dependent nitric oxide (EDNO)-dependent vasodilation in the presence or absence of nitric oxide synthase (NOS) inhibition in lungs/vessels from control rats. Directional arrows reflect changes vs. normoxia. ROS, reactive oxygen species. B: effects of CH on ROS production and EDNO-dependent vasodilation following acute restoration of normoxia or maintained hypoxia in lungs/vessels with an intact NOS system. Directional arrows indicate changes vs. control animals. C: effects of CH on ROS production and NO-dependent vasodilation following acute restoration of normoxia or maintained hypoxia in NOS-inhibited lungs/vessels. Directional arrows reflect changes vs. control animals.

 

Consistent with previous studies from our laboratory (29, 30, 32), the current study demonstrates that CH augments ET-1-mediated pulmonary vasodilation (Fig. 8B). It is possible the upregulation of eNOS mRNA and protein levels (6, 14, 29, 30, 37, 39) following CH contributes to this increased dilation. Indeed, ET-1-mediated vasodilation appears to be entirely dependent on NO/cGMP in this preparation, given that vasodilation was abolished by pretreatment with either the NOS inhibitor L-NNA or the sGC inhibitor ODQ (Fig. 1). Moreover, several investigators have shown NO synthesis to be enhanced following CH (7, 10, 20, 39), although eNOS expression and NO production may be diminished in other species in response to CH (15).

The EDNO-dependent vasodilatory response to ET-1 was attenuated during HV in lungs from control, but not CH, animals (Fig. 2). Although this observation could be explained by diminished NO production resulting from decreased eNOS activity in response to acute hypoxia (15), our finding that acute hypoxia was without effect on ET-1-dependent vasodilation in lungs from CH animals is inconsistent with this possibility. An alternative explanation is that HV differentially alters ROS production and thus NO bioavailability between groups. In agreement with this hypothesis, we found that acute hypoxia resulted in increased ROS levels in control arteries but was without a significant effect on ROS levels in arteries from CH rats (Fig. 7A) with an intact NOS system. Consistent with these observations, several investigators have demonstrated an increase in ROS with acute exposure to hypoxia (13, 16, 17). The mechanism by which CH impairs acute hypoxic increases in ROS generation is not presently understood but could potentially be explained by a chronic change in the redox status of the cytosol to a more reduced state in lungs from CH rats as previously reported (28).

In contrast to an inhibitory effect of acute hypoxia on EDNO-dependent reactivity to ET-1, acute hypoxia enhanced vasodilatory responsiveness to exogenous NO in L-NNA-treated lungs (Fig. 4). Similar to these effects of HV, ROS scavengers augmented reactivity to SNAP during NV (Fig. 5). Furthermore, the combination of hypoxia and ROS scavengers did not appear to be additive and tended to normalize reactivity between groups (Fig. 6), suggesting that hypoxia and ROS scavengers are acting to augment reactivity through a common mechanism to reduce ROS. Because L-NNA was administered for those experiments examining SNAP-induced vasodilation to minimize complicating influences of endogenous NO on vascular reactivity, we additionally examined ROS production from arteries in the presence of L-NNA. Interestingly, we found that inhibiting NO synthesis dramatically increased ROS levels under normoxic conditions in both groups and further revealed an effect of acute hypoxia to diminish ROS production (Figs. 7B and 8A). Such decreased ROS generation during acute hypoxic exposure likely results in increased NO bioavailability and could explain our present observations of enhanced reactivity to SNAP during HV.

Although the mechanism by which NOS inhibition augments ROS generation in pulmonary arteries under normoxic conditions is not clear, these results are consistent with previous findings in human systemic vessels and porcine pulmonary arteries (9, 21). Exogenous NO has additionally been shown to reduce aortic ROS levels (26), implying NO plays a protective role to scavenge ROS. However, such an effect of NO to scavenge ROS does not likely explain the increased DCF fluorescence observed following NOS inhibition in our preparation, since DCF also detects peroxynitrite, the product of superoxide/NO interaction (22). Furthermore, inhibition of NOS enhanced ROS production only during normoxia but resulted in decreased ROS production with hypoxia when O2 availability was limited. Also unlikely is the possibility that elevated levels of ROS following administration of L-NNA result from uncoupling of eNOS activity, since ROS production from eNOS has been recently demonstrated to be inhibitable by the similar L-arginine analog N{omega}-nitro-L-arginine methyl ester (L-NAME) (25). Alternatively, NO could be having a direct effect to inhibit ROS-generating enzymes. Cote et al. (4) demonstrated that the activity of the ROS-generating enzyme xanthine oxidase was decreased by L-arginine and increased by L-NAME in pulmonary artery endothelial cells, suggesting that NO negatively regulates this enzyme. Furthermore, NO can inhibit NADPH oxidase and cytochrome P-450 oxidase activity (3, 27), thus reducing ROS formation by these enzymes. Such an effect of NO to inhibit the activity of ROS-generating enzymes could explain our current findings that NOS inhibition leads to an apparent elevation in ROS production during normoxia. Furthermore, the ability of each of these enzymes to produce ROS is highly dependent on the availability of O2, which may therefore account for the observed reduction in ROS generation during exposure to acute hypoxia.

Previous work from our laboratory suggests that attenuated pulmonary vasodilation to exogenous NO following CH is mediated in part by increased degradation of cGMP by phosphodiesterases (11). However, the present study suggests that elevated vascular production of ROS in pulmonary arteries from CH rats following return to normoxia may additionally contribute to this altered vasoresponsiveness (Fig. 8C). Further support for a role for increased ROS in attenuating reactivity to NO following long-term hypoxia is provided by the observation that both ROS scavengers and HV tended to normalize vasodilatory responses to NO between groups (Fig. 8C). Such an effect of CH to stimulate ROS synthesis could result from increased activity of ROS-generating enzymes or rather compromised mechanisms of ROS scavenging. Consistent with this possibility, pulmonary hypertension in fetal lambs also leads to increased ROS production and diminished NO-dependent vasoreactivity, apparently resulting from a reduction in pulmonary arterial SOD activity as well as elevated NADPH oxidase expression (2). Furthermore, Nakanishi et al. (23) similarly demonstrated that lung SOD enzyme levels and activity are reduced in CH rats, which could contribute to elevated ROS levels and thus decreased reactivity to NO under normoxic conditions in lungs from CH rats.

In conclusion, the present study demonstrates the importance of altered ROS generation during acute and chronic hypoxia as a determinant of the vasodilatory effectiveness of NO in the pulmonary circulation and the role of NOS in regulating ROS synthesis. Specifically, our findings suggest that NOS inhibition unmasks an effect of acute hypoxia to diminish ROS generation in small intrapulmonary arteries and to augment NO-dependent vasodilation (Fig. 8A), presumably by increasing NO bioavailability. NOS inhibition was additionally found to increase ROS levels during normoxia in arteries from both control and CH. Finally, impaired NO-dependent pulmonary vasodilation following CH appears to be mediated at least in part by greater ROS production during NOS inhibition (Fig. 8C). Further investigation is needed to develop a better understanding of the cellular and enzymatic sources of ROS and how they are influenced by hypoxia and NOS inhibition to influence NO vasoreactivity.


    ACKNOWLEDGMENTS
 
GRANTS

This work was supported by an American Heart Association Scientist Development Grant (T. C. Resta) and by National Institutes of Health Grants RR-16480 (T. C. Resta), HL-58124 (B. R. Walker), and HL-63207 (B. R. Walker). T. C. Resta is a Parker B. Francis Fellow in Pulmonary Research.


    FOOTNOTES
 

Address for reprint requests and other correspondence: N. L. Jernigan, Dept. of Cell Biology and Physiology, MSC08 4750, 1 Univ. of New Mexico, Albuquerque, NM 87131-0001 (E-mail: njernigan{at}salud.unm.edu).

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.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Beckman JS, Beckman TW, Chen J, Marshall PA, and Freeman BA. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 87: 1620-1624, 1990.[Abstract]
  2. Brennan LA, Steinhorn RH, Wedgwood S, Mata-Greenwood E, Roark EA, Russell JA, and Black SM. Increased superoxide generation is associated with pulmonary hypertension in fetal lambs: a role for NADPH oxidase. Circ Res 92: 683-691, 2003.[Abstract/Free Full Text]
  3. Clancy RM, Leszczynska-Piziak J, and Abramson SB. Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide anion production via a direct action on the NADPH oxidase. J Clin Invest 90: 1116-1121, 1992.[ISI][Medline]
  4. Cote CG, Yu FS, Zulueta JJ, Vosatka RJ, and Hassoun PM. Regulation of intracellular xanthine oxidase by endothelial-derived nitric oxide. Am J Physiol Lung Cell Mol Physiol 271: L869-L874, 1996.[Abstract/Free Full Text]
  5. Eddahibi S, Raffestin B, Tayarani Y, Carville C, and Adnot S. Hypoxia-reoxygenation impairs NO-mediated vasodilation in rat lungs. Am J Physiol Lung Cell Mol Physiol 271: L441-L449, 1996.[Abstract/Free Full Text]
  6. Fagan KA, Fouty BW, Tyler RC, Morris KG Jr, Hepler LK, Sato K, LeCras TD, Abman SH, Weinberger HD, Huang PL, McMurtry IF, and Rodman DM. The pulmonary circulation of homozygous or heterozygous eNOS-null mice is hyperresponsive to mild hypoxia. J Clin Invest 103: 291-299, 1999.[Abstract/Free Full Text]
  7. Fagan KA, Morrissey B, Fouty BW, Sato K, Harral JW, Morris KG Jr, Hoedt-Miller M, Vidmar S, McMurtry IF, and Rodman DM. Upregulation of nitric oxide synthase in mice with severe hypoxia-induced pulmonary hypertension. Respir Res 2: 306-313, 2001.[CrossRef][Medline]
  8. Gryglewski RJ, Palmer RM, and Moncada S. Superoxide anion is involved in the breakdown of endothelium-derived vascular relaxing factor. Nature 320: 454-456, 1986.[ISI][Medline]
  9. Guzik TJ, West NE, Pillai R, Taggart DP, and Channon KM. Nitric oxide modulates superoxide release and peroxynitrite formation in human blood vessels. Hypertension 39: 1088-1094, 2002.[Abstract/Free Full Text]
  10. Isaacson T, Hampl V, Weir E, Nelson D, and Archer S. Increased endothelium-derived NO in hypertensive pulmonary circulation of chronically hypoxic rats. J Appl Physiol 76: 933-940, 1994.[Abstract/Free Full Text]
  11. Jernigan NL and Resta TC. Chronic hypoxia attenuates cGMP-dependent pulmonary vasodilation. Am J Physiol Lung Cell Mol Physiol 282: L1366-L1375, 2002.[Abstract/Free Full Text]
  12. Jernigan NL, Walker BR, and Resta TC. Pulmonary PKG-1 is upregulated following chronic hypoxia. Am J Physiol Lung Cell Mol Physiol 285: L634-L642, 2003.[Abstract/Free Full Text]
  13. Killilea DW, Hester R, Balczon R, Babal P, and Gillespie MN. Free radical production in hypoxic pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 279: L408-L412, 2000.[Abstract/Free Full Text]
  14. Le Cras T, Xue C, Rengasamy A, and Johns R. Chronic hypoxia upregulates endothelial and inducible NO synthase gene and protein expression in rat lung. Am J Physiol Lung Cell Mol Physiol 270: L164-L170, 1996.[Abstract/Free Full Text]
  15. Le Cras TD and McMurtry IF. Nitric oxide production in the hypoxic lung. Am J Physiol Lung Cell Mol Physiol 280: L575-L582, 2001.[Abstract/Free Full Text]
  16. Liu JQ, Sham JS, Shimoda LA, Kuppusamy P, and Sylvester JT. Hypoxic constriction and reactive oxygen species in porcine distal pulmonary arteries. Am J Physiol Lung Cell Mol Physiol 285: L322-L333, 2003.[Abstract/Free Full Text]
  17. Marshall C, Mamary AJ, Verhoeven AJ, and Marshall BE. Pulmonary artery NADPH-oxidase is activated in hypoxic pulmonary vasoconstriction. Am J Respir Cell Mol Biol 15: 633-644, 1996.[Abstract]
  18. Matsubara T and Ziff M. Superoxide anion release by human endothelial cells: synergism between a phorbol ester and a calcium ionophore. J Cell Physiol 127: 207-210, 1986.[ISI][Medline]
  19. Mohazzab KM, Fayngersh RP, Kaminski PM, and Wolin MS. Potential role of NADH oxidoreductase-derived reactive O2 species in calf pulmonary arterial PO2-elicited responses. Am J Physiol Lung Cell Mol Physiol 269: L637-L644, 1995.[Abstract/Free Full Text]
  20. Muramatsu M, Tyler R, Rodman D, and McMurtry I. Thapsigargin stimulates increased NO activity in hypoxic hypertensive rat lungs and pulmonary arteries. J Appl Physiol 80: 1336-1344, 1996.[Abstract/Free Full Text]
  21. Muzaffar S, Jeremy JY, Angelini GD, Stuart-Smith K, and Shukla N. Role of the endothelium and nitric oxide synthases in modulating superoxide formation induced by endotoxin and cytokines in porcine pulmonary arteries. Thorax 58: 598-604, 2003.[Abstract/Free Full Text]
  22. Myhre O, Andersen JM, Aarnes H, and Fonnum F. Evaluation of the probes 2',7'-dichlorofluorescein diacetate, luminol, and lucigenin as indicators of reactive species formation. Biochem Pharmacol 65: 1575-1582, 2003.[CrossRef][ISI][Medline]
  23. Nakanishi K, Tajima F, Nakamura A, Yagura S, Ookawara T, Yamashita H, Suzuki K, Taniguchi N, and Ohno H. Effects of hypobaric hypoxia on antioxidant enzymes in rats. J Physiol 489: 869-876, 1995.[Abstract]
  24. Omar HA, Mohazzab KM, Mortelliti MP, and Wolin MS. O2-dependent modulation of calf pulmonary artery tone by lactate: potential role of H2O2 and cGMP. Am J Physiol Lung Cell Mol Physiol 264: L141-L145, 1993.[Abstract/Free Full Text]
  25. Ou J, Ou Z, Ackerman AW, Oldham KT, and Pritchard KA Jr. Inhibition of heat shock protein 90 (hsp90) in proliferating endothelial cells uncouples endothelial nitric oxide synthase activity. Free Radic Biol Med 34: 269-276, 2003.[CrossRef][ISI][Medline]
  26. Pagano PJ, Tornheim K, and Cohen RA. Superoxide anion production by rabbit thoracic aorta: effect of endothelium-derived nitric oxide. Am J Physiol Heart Circ Physiol 265: H707-H712, 1993.[Abstract/Free Full Text]
  27. Puntarulo S and Cederbaum AI. Production of reactive oxygen species by microsomes enriched in specific human cytochrome P450 enzymes. Free Radic Biol Med 24: 1324-1330, 1998.[CrossRef][ISI][Medline]
  28. Reeve HL, Michelakis E, Nelson DP, Weir EK, and Archer SL. Alterations in a redox oxygen sensing mechanism in chronic hypoxia. J Appl Physiol 90: 2249-2256, 2001.[Abstract/Free Full Text]
  29. Resta TC, Chicoine LG, Omdahl JL, and Walker BR. Maintained upregulation of pulmonary eNOS gene and protein expression during recovery from chronic hypoxia. Am J Physiol Heart Circ Physiol 276: H699-H708, 1999.[Abstract/Free Full Text]
  30. Resta TC, Kanagy NL, and Walker BR. Estradiol-induced attenuation of pulmonary hypertension is not associated with altered eNOS expression. Am J Physiol Lung Cell Mol Physiol 280: L88-L97, 2001.[Abstract/Free Full Text]
  31. Resta TC, Sanders TC, Eichinger MR, Crowley MR, and Walker BR. Segmental vasodilatory effectiveness of inhaled NO in lungs from chronically hypoxic rats. Respir Physiol 114: 161-173, 1998.[CrossRef][ISI][Medline]
  32. Resta TC and Walker BR. Chronic hypoxia selectively augments endothelium-dependent pulmonary arterial vasodilation. Am J Physiol Heart Circ Physiol 270: H888-H896, 1996.[Abstract/Free Full Text]
  33. Roos C, Frank D, Xue C, Johns R, and Rich G. Chronic inhaled nitric oxide: effects on pulmonary vascular endothelial function and pathology in rats. J Appl Physiol 80: 252-260, 1996.[Abstract/Free Full Text]
  34. Rubanyi GM and Vanhoutte PM. Superoxide anions and hyperoxia inactivate endothelium-derived relaxing factor. Am J Physiol Heart Circ Physiol 250: H822-H827, 1986.[Abstract/Free Full Text]
  35. Schinetti ML, Sbarbati R, and Scarlattini M. Superoxide production by human umbilical vein endothelial cells in an anoxia-reoxygenation model. Cardiovasc Res 23: 76-80, 1989.[ISI][Medline]
  36. Seki S, Flavahan NA, Smedira NG, and Murray PA. Superoxide anion scavengers restore NO-mediated pulmonary vasodilation after lung transplantation. Am J Physiol Heart Circ Physiol 276: H42-H46, 1999.[Abstract/Free Full Text]
  37. Shaul P, North A, Brannon T, Ujiie K, Wells L, Nisen P, Lowenstein C, Snyder S, and Star R. Prolonged in vivo hypoxia enhances nitric oxide synthase type I and type III gene expression in adult rat lung. Am J Respir Cell Mol Biol 13: 167-174, 1995.[Abstract]
  38. Terada LS, Guidot AM, Leff JA, Willingham IR, Hanley ME, Piermatei D, and Repine JE. Hypoxia injures endothelial cells by increasing endogenous xanthine oxidase activity. Proc Natl Acad Sci USA 89: 3362-3366, 1992.[Abstract]
  39. Tyler RC, Muramatsu M, Abman SH, Stelzner TJ, Rodman DM, Bloch KD, and McMurtry IF. Variable expression of endothelial NO synthase in three forms of rat pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 276: L297-L303, 1999.[Abstract/Free Full Text]
  40. Wanstall JC, Kaye JA, and Gambino A. The in vitro pulmonary vascular effects of FK409 (nitric oxide donor): a study in normotensive and pulmonary hypertensive rats. Br J Pharmacol 121: 280-286, 1997.[Abstract]