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Am J Physiol Lung Cell Mol Physiol 290: L570-L578, 2006. First published October 14, 2005; doi:10.1152/ajplung.00262.2005
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Hypoxia-induced reactive oxygen species downregulate ETB receptor-mediated contraction of rat pulmonary arteries

Xiaohua Wang,1 Mei Tong,1 Shashi Chinta,3 J. Usha Raj,3 and Yuansheng Gao2,3

1Department of Physiology and Pathophysiology, Peking University Health Science Center, 2Key Laboratory of Molecular Cardiovascular Sciences (Peking University), Ministry of Education, Beijing 100083, China; and 3Division of Neonatology, Harbor-UCLA Medical Center, Geffen School of Medicine at University of California, Los Angeles, and Los Angeles Biomedical Research Institute, Los Angeles, California

Submitted 17 June 2005 ; accepted in final form 6 October 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Production of reactive oxygen species (ROS) may be increased during hypoxia in pulmonary arteries. In this study, the role of ROS in the effect of hypoxia on endothelin (ET) type B (ETB) receptor-mediated vasocontraction in lungs was determined. In rat intrapulmonary (~0.63 mm ID) arteries, contraction induced by IRL-1620 (a selective ETB receptor agonist) was significantly attenuated after 4 h of hypoxia (30 mmHg PO2) compared with normoxic control (140 mmHg PO2). The effect was abolished by tiron, a scavenger of superoxide anions, but not by polyethylene glycol (PEG)-conjugated catalase, which scavenges H2O2. The hypoxic effect on ETB receptor-mediated vasoconstriction was also abolished by endothelium denudation but not by nitro-L-arginine and indomethacin. Exposure for 4 h to exogenous superoxide anions, but not H2O2, attenuated the vasoconstriction induced by IRL-1620. Confocal study showed that hypoxia increased ROS production in pulmonary arteries that were scavenged by PEG-conjugated SOD. In endothelium-intact pulmonary arteries, the ETB receptor protein was reduced after 4 h of exposure to hypoxia, exogenous superoxide anions, or ET-1. BQ-788, a selective ETB receptor antagonist, prevented these effects. ET-1 production was stimulated in endothelium-intact arteries after 4 h of exposure to hypoxia or exogenous superoxide anions. This effect was blunted by PEG-conjugated SOD. These results demonstrate that exposure to hypoxia attenuates ETB receptor-mediated contraction of rat pulmonary arteries. A hypoxia-induced production of superoxide anions may increase ET-1 release from the endothelium and result in downregulation of ETB receptors on smooth muscle.

superoxide; endothelin

REACTIVE OXYGEN SPECIES (ROS), such as superoxide, H2O2, and hydroxyl radical, are by-products of cellular metabolism (51, 55). Under physiological conditions, they are present in the picomolar-to-low nanomolar range and may act as essential participants in cell signaling through oxidative modification of proteins. Under pathological conditions, ROS production may increase and reach much higher levels, at which ROS are injurious to the tissues. However, increasing evidence suggests that, depending on concentration, duration, and location, ROS may act as signaling molecules to induce adaptive cellular responses to counteract the pathological stimuli (51, 55). For instance, increased ROS production caused by mild ischemia or hypoxia may protect tissue by activating distinct signal pathways and by upregulating a number of enzymes such as SOD, glutathione peroxidase, and nitric oxide (NO) synthase (5, 7).

Endothelin (ET)-1, a potent pulmonary vasoconstrictor, acts via two receptor subtypes, ETA and ETB (8). Although it is believed that the ETA receptor is the principal subtype for ET-1-induced pulmonary vasoconstriction, emerging evidence indicates that ETB receptors may also play a major role in mediating ET-1-induced constriction of intrapulmonary conduit and resistance arteries in the human and the rat (19, 33, 34, 40, 48). Because some studies showed that hypoxia increases ROS production in pulmonary artery smooth muscle and endothelial cells (18, 27, 32, 34, 43, 53, 56) and that ETB receptor expression in pulmonary arteries may be modulated by hypoxia (11, 17, 31, 39), we hypothesized that hypoxia may affect ETB receptor-mediated responses of pulmonary arteries through ROS.

To test this hypothesis, the response of rat intrapulmonary arteries to IRL-1620, a highly selective ETB receptor agonist (19, 44, 49), was studied. Previous studies showed that ET-1-induced contraction of these vessels is mainly mediated by the ETB receptor and that ETB receptors exist predominantly in smooth muscle, with lower expression in the endothelium (19, 48). Our results show that ROS, principally superoxide anions, may act as the signaling molecules in the hypoxia-induced downregulation of ETB receptor-mediated contraction of rat pulmonary arteries.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Male Sprague-Dawley rats (250–300 g body wt) were killed with an overdose of pentobarbital sodium (100 mg/kg im). Animal handling and study protocols were reviewed and approved by the Animal Care and Use Review Committees of Peking University Health Science Center and the Los Angeles Biomedical Research Institute.

The lungs were immediately removed, and the fourth- and fifth-generation pulmonary arteries (0.63 ± 0.01 mm ID, n = 38) were dissected free of parenchyma and cut into rings (~5 mm long). We mechanically removed endothelium from some vessels by inserting the tips of a watchmaker's forceps into the lumen of the vessel and rolling the vessel back and forth on saline-saturated filter paper. Removal of the endothelium was confirmed by lack of relaxation to 10–5 M acetylcholine (15).

Vessel tension study. Vessel rings were suspended in organ chambers filled with 15 ml of modified Krebs-Ringer bicarbonate solution (in mM: 118.3 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3, and 11.1 glucose) maintained at 37 ± 0.5°C and aerated with 20% O2-5% CO2 (pH 7.4). Each ring was suspended via two stirrups that were passed through the lumen: one stirrup was anchored to the bottom of the organ chamber, and the other was connected to a strain gauge. The isometric tension was measured with a recording-and-analysis system (model ML785 PowerLab/8sp, ADInstruments, Castle Hill, Australia).

At the beginning of the experiment, each vessel ring was stretched to its optimal resting tension by stretching, in 0.1-g increments, until the active contraction of the vessel ring to 100 mM KCl reached a plateau. The optimal resting tension of pulmonary arteries was 0.62 ± 0.01 g (n = 38).

Vessel rings were then exposed to hypoxia (30 mmHg PO2) or normoxia (140 mmHg PO2) for 1, 2, and 4 h. Adjustment of the proportions of aerating O2 and N2 maintained PO2 in the solution. PCO2 was kept constant at 36 mmHg and pH at 7.4. PO2, PCO2, and pH were measured with a pH/blood gas analyzer (Stat Profile Plus 3, Nova Biomedical, Waltham, MA). To determine whether exogenous ROS could produce the same effect as hypoxia, in some experiments the vessels were incubated in normoxia for 4 h with exogenous superoxide anions [xanthine oxidase (100 mU/ml) + hypoxanthine (10–4 M)] or with H2O2 (10–4 M). The stability of H2O2 under the experimental conditions was assessed in the presence of tissues with the use of a peroxide assay kit (PeroxiDetect Kit, Sigma). Under these conditions, H2O2 administered at 10–4 M was 6.2 ± 0.4 x 10–5 M after 4 h of incubation (n = 8, P < 0.05). The oxidant stress of the tissues after incubation with H2O2 was determined using dichlorofluorescin diacetate as a probe (50). The intracellular dichlorofluorescin diacetate fluorescence of pulmonary arteries after 4 h of incubation with 10–4 M H2O2 was 192.9 ± 19.5% of control (n = 5 for each group, P < 0.05).

After incubation, the vessels were washed with modified Krebs-Ringer bicarbonate solution three times, each for 5 min, and allowed to recover to normoxic conditions. Then the concentration-response curves to IRL-1620, a selective ETB receptor agonist (19, 44, 49), were constructed in a cumulative fashion.

ROS. The oxidative fluorescent dye dihydroethidium (DHE) was used to evaluate ROS production in isolated rat pulmonary arteries. DHE is freely permeable to cells and, in the presence of ROS, superoxide anion in particular, is oxidized to ethidium bromide (EtBr), which is trapped by intercalation with the DNA. DHE conversion to EtBr was used to determine intracellular ROS levels, with DHE detected separately using an excitation wavelength of 380 nm and an emission wavelength of 445 nm. EtBr was excited at 510 nm, and signals were collected through a 605-nm filter.

Isolated pulmonary arteries were preloaded with 10–5 M DHE and incubated under normoxic or hypoxic conditions for 1 h as described above in a light-protected fashion in the presence or absence of polyethylene glycol (PEG)-conjugated SOD (PEG-SOD, 150 U/ml). The vessels were then washed three times in modified Krebs-Ringer bicarbonate solution to remove excess dye. The vessels were frozen, cut into 7-µm-thick sections, and placed on glass coverslips coated with tissue adhesive. Images were obtained with a confocal microscope (Leica Microsystems). The area of the vessel wall was selected as the investigational subject target, and a polygon was drawn onto the digitized image. The fluorescence intensity of DHE and EtBr in the selected area was separately recorded. Change in ROS level was expressed as the ratio of EtBr to DHE fluorescence values. Vessels subjected to different treatments were processed and imaged in parallel at identical settings.

Western blot analysis of ETB receptor. Isolated pulmonary arteries were incubated for 4 h under hypoxia as described above. Some vessels were incubated under normoxic conditions with exogenous superoxide anions [xanthine oxidase (100 mU/ml) + hypoxanthine (10–4 M)] or 10–9 M ET-1. For each measurement, six to eight artery segments (50–60 mg wet wt) from different rats were used. At the end of incubation, pulmonary arteries were rapidly frozen in liquid nitrogen and homogenized in six volumes of ice-cold buffer containing 34.67 mM SDS, 1 mM sodium orthovandate, and 10 mM Tris base (pH 7.4). The homogenate was centrifuged at 500 g for 10 min at 4°C. The supernatant was centrifuged at 100,000 g for 1 h at 4°C, and the resulting pellets were resuspended in a buffer similar to that used for tissue homogenization. Protein concentrations of the preparations were measured by the Bradford method, with bovine serum albumin used as the standard.

The membrane preparations, each containing 20 µg of protein, were subjected to SDS-PAGE and electrotransferred to nitrocellulose. A polyclonal antibody that recognizes human, mouse, rat, and sheep ETB receptors on SDS-PAGE immunoblots (Biodesign International) was used. Nonspecific binding of antibodies was blocked by washing with Tris-buffered saline (TBS) containing 10% milk for 1 h. The blot was then subjected to two brief washes with TBS + 0.5% Tween 20 and incubated in TBS + 0.1% Tween 20 and a 1:300 dilution of ETB receptor antibody for 1 h. After two more washes in TBS + 0.1% Tween 20, the blot was incubated for 40 min in a 1:2,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Sigma Chemical), washed, and developed using SuperSignal West Pico Chemiluminescent Substrate (Pierce). The amount of ETB receptor protein in blots was quantified by densitometry with an Eagle Eye II Still Video System (Stratagene, La Jolla, CA). The blot was subsequently stripped and reprobed with antiactin antibody, to which ETB receptor values were normalized.

ET-1 radioimmunoassay. Pulmonary arteries were incubated under normoxic or hypoxic conditions in the presence or absence of PEG-SOD (150 U/ml). For each measurement, four to five artery segments (25–30 mg wet wt) from different rats were used. After 4 h of incubation, the tissues were rapidly frozen and homogenized in 1 ml of 50 mM Tris buffer (pH 7.4). The homogenate was mixed with 1.5 ml of extraction solvent (40:1:5 acetone-1 N HCl-water) and centrifuged for 20 min at 2,000 g at 4°C. The supernatant was lyophilized and reconstituted in 200 µl of the assay buffer. ET-1 was measured using an ET-1 radioimmunoassay kit (Peninsula Laboratories, Belmont, CA). The content of ET-1 was expressed as femtomoles per milligram of protein.

Drugs. IRL-1620 was obtained from Alexis Biochemicals, nitro-L-arginine, indomethacin, N-acetyl-L-cysteine, tiron, PEG-conjugated catalase, xanthine oxidase, hypoxanthine, and PEG-SOD from Sigma Chemical, and BQ-788 from Alexis Biochemicals.

Indomethacin (10–5 M) was prepared in equal-molar Na2CO3. Hypoxanthine was dissolved in 0.01 M NaOH. Such concentrations of Na2CO3 or NaOH did not significantly affect the pH of the solution in the organ chamber. IRL-1620 was dissolved in DMSO (final concentration <0.01%). Preliminary experiments showed that <0.01% DMSO had no effect on contraction to IRL-1620. The other drugs were prepared using distilled water. In all experiments, the inhibitors were administered ≥30 min before their effects were examined.

Data analysis. Values are means ± SE. When mean values of two groups were compared, Student's t-test for unpaired observations was used. When mean values of the same group before and after stimulation were compared, Student's t-test for paired observations was used. Comparison of mean values of more than two groups was performed with one-way ANOVA with Student-Newman-Keuls test for post hoc testing of multiple comparisons. Statistical significance was accepted at P (2-tailed) < 0.05. In all experiments, n represents the number of animals.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Organ chamber studies. There was no significant difference between the amount of contraction of rat pulmonary arteries evoked by high concentrations of potassium in vessels exposed to hypoxia (30 mmHg PO2) for 4 h and in vessels exposed to normoxia (140 mmHg PO2): maximal increase in tension = 1.62 ± 0.1 vs. 1.64 ± 0.1 g, and EC50 = 40.1 ± 2.6 vs. 40.0 ± 1.2 mM (n = 6, P > 0.05). IRL-1620, a specific ETB receptor agonist (49), caused a contraction of the pulmonary arteries that was not significantly affected by BQ-123, an ETA receptor antagonist (42), but was markedly inhibited by BQ-788, an ETB receptor antagonist (52) (Fig. 1). The contraction induced by IRL-1620 was not affected by 1 h of exposure to hypoxia but was significantly attenuated by 2 and 4 h of exposure to hypoxia (Fig. 2). Contraction of pulmonary arteries evoked by 10–8 M ET-1 in the presence of 3 x 10–6 M BQ-788 was augmented after 4 h of exposure to hypoxia compared with normoxia: 1.16 ± 0.09 vs. 0.86 ± 0.10 g (n = 6, P < 0.05).


Figure 1
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  		Fig. 1. Effects of BQ-123 (3 x 10–6 M) and BQ-788 (3 x 10–6 M) on contraction of rat pulmonary arteries to IRL-1620. Values are means ± SE; n = 4. *Significantly different from control and BQ-123 (P < 0.05).

 

Figure 2
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  		Fig. 2. Response of rat pulmonary arteries to IRL-1620 after 1, 2, and 4 h of normoxia or hypoxia. Vasoconstriction is expressed as percentage of contraction to 100 mM KCl. Values are means ± SE; n = 6–9. *Significantly different from normoxia (P < 0.05).

 
In vessels with endothelium but not those without endothelium IRL-1620-induced contraction was significantly less after 4 h of hypoxia than normoxia. The differential response between hypoxic and normoxic exposure in vessels with endothelium was not prevented by 10–4 M nitro-L-arginine, an inhibitor of NO synthase (38), plus 10–5 M indomethacin, an inhibitor of cyclooxygenase (14) (Fig. 3).


Figure 3
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  		Fig. 3. IRL-1620-induced contractions of endothelium-intact (E+) and endothelium-denuded (E–) rat pulmonary arteries (left) or endothelium-intact vessels treated with nitro-L-arginine (10–4 M, NLA) + indomethacin (10–5 M, IND; right) after 4 h of hypoxia or normoxia. Vasoconstriction is expressed as percentage of contraction to 100 mM KCl. Values are means ± SE; n = 6. *Significantly different from normoxia (P < 0.05).

 
The inhibition of IRL-1620-induced vasoconstriction by 4 h of hypoxia was also blunted by addition of 20 mM N-acetyl-L-cysteine, a nonspecific scavenger of ROS (41), and 10 mM tiron, a scavenger of superoxide anion (23), but not PEG-conjugated catalase (2,000 U/ml), a cell membrane-permeable scavenger of H2O2 (6), to the incubation solution (Fig. 4).


Figure 4
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  		Fig. 4. Response of rat pulmonary arteries to IRL-1620 after 4 h of normoxia or hypoxia in the presence of N-acetyl-L-cysteine (20 mM), tiron (10 mM), or polyethylene glycol (PEG)-conjugated catalase (PEG-catalase, 2,000 U/ml). Vasoconstriction is expressed as percentage of contraction to 100 mM KCl. Values are means ± SE; n = 6–9. *Significantly different from normoxia (P < 0.05).

 
In endothelium-intact, but not endothelium-denuded, rat pulmonary arteries, incubation for 4 h with xanthine oxidase (100 mU/ml) + hypoxanthine (10–4 M) significantly blunted contraction of the vessels to IRL-1620 compared with vessels incubated with control solution. The effect of xanthine oxidase + hypoxanthine on contractions was abrogated by addition of 10 mM tiron to the incubation solution. Incubation for 4 h with 10–4 M H2O2 had no significant effect on IRL-1620-induced contraction (Fig. 5).


Figure 5
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  		Fig. 5. IRL-1620-induced contractions of endothelium-intact and endothelium-denuded rat pulmonary arteries after 4 h of incubation with exogenous superoxide anion [xanthine oxidase (XO)], XO + tiron (10 mM), H2O2 (10–4 M), or control solvent. Exogenous superoxide anion was generated by hypoxanthine (10–4 M) + xanthine oxidase (100 mU/ml). Vasoconstriction is expressed as percentage of contraction to 100 mM KCl. Values are means ± SE; n = 6. *Significantly different from control (P < 0.05).

 
ROS production. Conversion of the fluorescent dye DHE to EtBr shows that ROS production in the endothelial and smooth muscle layer of rat pulmonary arteries was significantly greater after 1 h of exposure to hypoxia than in normoxia: EtBr-to-DHE ratio = 1.31 ± 0.08 vs. 0.81 ± 0.06 (n = 6, P < 0.05) for the endothelial layer and 1.59 ± 0.09 vs. 0.95 ± 0.05 for the smooth muscle layer (n = 6, P < 0.05). In the presence of PEG-SOD (150 U/ml), a membrane-permeable specific scavenger of superoxide anions (6), ROS production in the vessels exposed to hypoxia was not significantly different from that in the vessels exposed to normoxia: EtBr-to-DHE ratio = 0.73 ± 0.08 vs. 0.78 ± 0.09 for the smooth muscle layer (n = 6, P < 0.05; Fig. 6). The hypoxia-induced increased in ROS production was also observed in endothelium-denuded pulmonary arteries: EtBr-to-DHE ratio = 1.26 ± 0.12 and 0.89 ± 0.09 for hypoxia and normoxia, respectively (n = 4, P < 0.05; Fig. 7).


Figure 6
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  		Fig. 6. Confocal microscopic images of reactive oxygen species (ROS) production in isolated pulmonary arteries [preloaded with dihydroethidium (DHE, 10–5 M)] after 1 h of normoxia or hypoxia in the presence or absence of PEG-conjugated SOD (PEG-SOD, 150 U/ml). ROS production was determined by conversion of DHE to ethidium bromide (EtBr). Tissues with different treatments were processed and imaged in parallel at identical settings. Magnification x40.

 

Figure 7
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  		Fig. 7. Confocal microscopic images showing of ROS production in isolated endothelium-denuded pulmonary arteries (preloaded with 10–5 M DHE) after 1 h of normoxia or hypoxia. ROS production was determined by conversion of DHE to EtBr. Tissues with different treatments were processed and imaged in parallel at identical settings. Magnification x40.

 
ETB receptor protein expression. Exposure to hypoxia or incubation with xanthine oxidase (100 mU/ml) + hypoxanthine (10–4 M) for 4 h significantly suppressed ETB receptor protein level in endothelium-intact, but not endothelium-denuded, rat pulmonary arteries (Fig. 8). The effect was blocked by the inclusion of PEG-SOD (150 U/ml) or BQ-788 (3 x 10–6 M), a selective ETB receptor antagonist (42), in the incubation solution (Fig. 9).


Figure 8
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  		Fig. 8. Top: Western blot analysis of ETB receptor in membrane preparations of rat pulmonary arteries after 4 h of normoxia (140 mmHg PO2) or hypoxia (30 mmHg PO2) or treatment with exogenous superoxide anion [xanthine oxidase (100 mU/ml) + hypoxanthine (10–4 M)]. Bottom: results from densitometric scanning of ETB receptor normalized to actin. Values are means ± SE; n = 4. *Significantly different from vessels without endothelium (P < 0.05).

 

Figure 9
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  		Fig. 9. Western blot analysis of ETB receptor in membrane preparations of rat pulmonary arteries after 4 h of normoxia or hypoxia or treatment with exogenous superoxide anion [xanthine oxidase (100 mU/ml) + hypoxanthine (10–4 M)]. Lanes 1 and 4, control; lanes 2 and 5, PEG-SOD (150 U/ml); lanes 3 and 6, 3 x 10–6 M BQ-788. Bottom: results from densitometric scanning of ETB receptor normalized to actin. Values are means ± SE; n = 4–5. *Significantly different from normoxia/basal (P < 0.05).

 
A reduced ETB receptor protein level in pulmonary arteries was also observed after 4 h of incubation with 10–9 M ET-1. The effect of ET-1 on ETB receptor protein level was prevented by addition of BQ-788 (3 x 10–6 M) to the incubation solution (Fig. 10).


Figure 10
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  		Fig. 10. Top: Western blot analysis of ETB receptor in membrane preparations of rat pulmonary arteries after 4 h of incubation under control conditions or treatment with ET-1 (10–9 M). Lanes 1 and 3, control; lanes 2 and 4, 3 x 10–6 M BQ-788. Bottom: results from densitometric scanning of ETB receptor normalized to actin. Values are means ± SE; n = 4–5. *Significantly different from basal (P < 0.05).

 
ET-1. The ET-1 level was significantly elevated in endothelium-intact pulmonary arteries subjected to 4 h of hypoxia or treated with exogenous superoxide anion [xanthine oxidase (100 mU/ml) + hypoxanthine (10–4 M)]. The effect was prevented by PEG-SOD (150 U/ml). Hypoxia and exogenous superoxide anions had no significant effect on ET-1 in endothelium-denuded pulmonary arteries (Fig. 11).


Figure 11
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  		Fig. 11. ET-1 production in pulmonary arteries after 4 h of incubation under normoxia or hypoxia or treatment with exogenous superoxide anion [xanthine oxidase (100 mU/ml) + hypoxanthine (10–4 M)]. Values are means ± SE; n = 6–9. *Significantly different from normoxia (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, exposure to hypoxia for 2 and 4 h, but not 1 h, attenuated contractions of rat intrapulmonary arteries to IRL-1620, a selective ETB receptor agonist (49), and also downregulated ETB receptors in the arteries. The vasoconstriction to IRL-1620 was inhibited by BQ-788, a selective ETB receptor antagonist (42), but was not affected by BQ-123, a selective ETA receptor antagonist (52). Hence, a reduction in ETB receptor expression may in part contribute to decreased contractions induced by IRL-1620 after hypoxic exposure. We found that the contraction of pulmonary arteries evoked by ET-1 after the blockade of ETA receptor with BQ-788 was augmented. Therefore, the suppression of ETB receptor-mediated contraction by hypoxia is not nonspecific. Our results also show that the effect of hypoxia was endothelium dependent and was not prevented by nitro-L-arginine and indomethacin, inhibitors of NO synthase and cyclooxygenase (14, 38), respectively. It appears that an endothelium-derived factor that is neither NO nor a dilator prostaglandin may act as a mediator.

Hypoxia has been shown to increase the production of ROS, such as superoxide and H2O2, in pulmonary artery smooth muscle of the rat, rabbit, pig, and calf (27, 32, 34, 43, 53), as well as in pulmonary vascular endothelial cells of the mouse, rat, and pig (18, 43, 56). In the present study, using DHE as the probe, we found that hypoxia increased ROS production in the endothelial and smooth muscle layers of intact pulmonary arteries, as well as in the smooth muscle layer of endothelium-denuded pulmonary arteries. DHE is considered to be a more specific probe for superoxide anions (50). Moreover, the effect of hypoxia was prevented by PEG-SOD, a membrane-permeable specific scavenger of superoxide anions (6). Therefore, it is likely that production of superoxide anions increased in rat pulmonary arteries during hypoxia. Although a number of studies reported an increased production of ROS in pulmonary artery smooth muscle and endothelial cells during hypoxia (18, 27, 32, 34, 43, 53, 56), some studies found otherwise (36, 37). Many factors may contribute to this difference: tissue or cell preparations, ROS probes, and experimental protocols. The reasons remain to be determined (37, 54).

Our study indicates that an increased production of ROS, in particular superoxide anions, may contribute to the attenuation of ETB receptor-mediated pulmonary vasoconstriction after exposure to hypoxia. Such a notion is based on several lines of evidence. 1) The effect of hypoxia on contraction of rat pulmonary arteries to IRL-1620 was prevented by N-acetyl-L-cysteine, a nonspecific scavenger of ROS (41), and tiron, a scavenger of superoxide anions (23), but not by PEG-catalase, a cell membrane-permeable scavenger of H2O2 (6). 2) The hypoxic effect could also be obtained with exogenous superoxide anions, but not with exogenous H2O2. Xanthine oxidase + hypoxanthine was used to generate superoxide anions (26). The effect of xanthine oxidase + hypoxanthine was abolished by tiron (23). 3) Western blot analysis showed that the decrease in protein level of ETB receptors induced by hypoxia and by exogenous superoxide anions was prevented by PEG-SOD (6). The effects of hypoxia and exogenous superoxide anions on IRL-1620-induced contraction and on ETB receptor protein expression depended on the presence of the endothelium, suggesting that the effect of ROS is not directly on smooth muscle cells but, rather, indirectly through the endothelium.

In the present study, the expression of ETB receptors in pulmonary arteries was suppressed not only by hypoxia and exogenous superoxide anions but also by 4 h of incubation with ET-1 at a low concentration (10–9 M). Furthermore, the effects of hypoxia and exogenous superoxide anions on ETB receptor protein expression were blocked by BQ-788, a specific antagonist of ETB receptors (42). Hence, it is possible that an increased release of ET evoked by the elevated production of ROS during hypoxia may act on ETB receptors and cause the downregulation of the receptor. An increased production of ET-1 and reduced ETB receptor protein expression in the lungs have been found in a number of disease conditions such as pulmonary hypertension and congestive heart failure (3, 21, 28, 30). In rat brains, dexamethasone produced an increase in ET-1 mRNA that preceded the decrease in ETB receptor mRNA levels in the same region of the brain (46). In cultured human fibroblasts and in tumorigenic HeLa cells, incubation with ET-1 for 2 h results in downregulation of ETB receptors. This effect was inhibited by BQ-788 (9). These studies support a causal relation between an enhanced production of ET-1 and the depressed ETB receptor levels.

Increased production of ET-1 has been observed in hypoxia-exposed animal lung tissues and pulmonary arteries (1, 2, 10, 12, 47), as well as in patients with hypoxic pulmonary hypertension (16). In the vascular system, ET-1 is produced predominantly by endothelial cells and released in response to various stimuli such as cytokines, growth factors, and shear stress, as well as by hypoxia (45). An increased ET-1 expression has also been found in human umbilical vein endothelial cells stimulated with exogenous superoxide anions (24, 25). In the present study, the stimulated production of ET-1 by hypoxia and exogenous superoxide anions was observed in endothelium-intact, but not endothelium-denuded, rat pulmonary arteries. The elevation in ET-1 was blunted by SOD. These results suggest that hypoxia may downregulate ETB receptors by increasing the production of ET-1 from the endothelium through ROS, in particular superoxide anions. In the present study, we found that ROS production increased not only in the endothelial layer but also in the smooth muscle layer of the vessel wall under hypoxia. It was not clear whether ROS produced in the smooth muscle layer had an effect on the hypoxia-induced ET-1 production from the endothelium, because we had no means to selectively block the ROS production in the smooth muscle layer.

ROS has long been thought to be detrimental to cells and tissues. In recent years, there has been increasing evidence that individual ROS may act as signaling molecules to modulate various cellular functions through oxidative modification of proteins and enzymes (51, 55). In pulmonary arteries, it is postulated that ROS may serve as the O2 sensor in hypoxia-induced vasoconstriction (37, 54). Acute hypoxic pulmonary vasoconstriction is a physiological response whereby circulating blood is diverted from hypoxic alveoli to optimize the matching of perfusion with ventilation. However, prolonged hypoxia-induced vasoconstriction may lead to pulmonary vascular remodeling and pulmonary arterial hypertension. Our present results suggest that the increased production of superoxide anions may play a protective role by suppressing ETB receptor-mediated pulmonary vasoconstriction during prolonged hypoxia. It is not clear why superoxide, rather than other ROS, such as H2O2 or hydroxyl radicals, mediates this effect. It has been shown that, in signal transduction mediated by reversible regulation of protein-tyrosine phosphatase 1B, superoxide is a kinetically more efficient and a chemically more specific oxidant than H2O2 for the inhibition of the phosphatases. It is thought that the specificity of superoxide over H2O2 for this effect may be related to the presence of positively charged residues surrounding the site of action (4). The mechanism by which superoxide increases the production of ET-1 by pulmonary vascular endothelium has yet to be elucidated.

The present study demonstrated for the first time that a distinct type of ROS, namely, superoxide anion, may act to modulate ETB receptor-mediated pulmonary vasoconstriction during hypoxia. Activation of ETB receptor may cause vasoconstriction by directly acting on smooth muscle cells and cause vasodilation through the release of NO and prostaglandins (3). The distinct effects of this receptor type vary depending on the species and vascular bed (3, 13, 20, 29). In human and rat resistance pulmonary arteries, ETB is the principal subtype of ET-1 receptor (19, 33, 35, 40, 48). Because production of ET-1 increases during hypoxia and the major site of action of this constrictor peptide is the small pulmonary artery (3), the present findings that superoxide anions mediated the suppression of ETB receptor-mediated contraction of intrapulmonary arteries may represent a protective mechanism during prolonged hypoxia. Because the present results were obtained in isolated rat small intrapulmonary arteries that were stimulated with short-term hypoxia, they may not reflect the role of the ETB receptor in other segments of the pulmonary vascular tree and its role in the regulation of pulmonary circulation under in vivo conditions. For instance, in a genetic rat model of ETB receptor deficiency, studies in vivo show that ETB receptor-deficient rats exhibit an exaggerated pulmonary hypertensive response to hypoxia and ET-1 stimulation. The underlying mechanism is not well understood. It may result not only from the loss of the ETB receptors in the pulmonary vasculature but also from other changes in this model, such as elevated plasma ET-1 levels and diminished endothelial NO synthase protein content and NO production in the lungs (21, 22).


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This study was supported in part by National Natural Science Foundation of China Grants 30370523 and 30470629, Research Fund for Doctorial Programs of Higher Education, Ministry of Education, China, Grant 20030001031, and National Heart, Lung, and Blood Institute Grants HL-059435 and HL-075187.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Gao, Dept. of Physiology and Pathophysiology, Peking Univ. Health Science Center, 38 Xue Yuan Rd., Beijing 100083, China (e-mail: ygao{at}bjmu.edu.cn)

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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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