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J Appl Physiol 92: 1152-1158, 2002. First published November 9, 2001; doi:10.1152/japplphysiol.00829.2001
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Vol. 92, Issue 3, 1152-1158, March 2002

Hypoxia-induced pulmonary endothelin-1 expression is unaltered by nitric oxide

Scott Earley, Leif D. Nelin, Louis G. Chicoine, and Benjimen R. Walker

Vascular Physiology Group, Departments of Cell Biology and Physiology, and Pediatrics, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (NO) attenuates hypoxia-induced endothelin (ET)-1 expression in cultured umbilical vein endothelial cells. We hypothesized that NO similarly attenuates hypoxia-induced increases in ET-1 expression in the lungs of intact animals and reasoned that potentially reduced ET-1 levels may contribute to the protective effects of NO against the development of pulmonary hypertension during chronic hypoxia. As expected, hypoxic exposure (24 h, 10% O2) increased rat lung ET-1 peptide and prepro-ET-1 mRNA levels. Contrary to our hypothesis, inhaled NO (iNO) did not attenuate hypoxia-induced increases in pulmonary ET-1 peptide or prepro-ET-1 mRNA levels. Because of this surprising finding, we also examined the effects of NO on hypoxia-induced increases in ET peptide levels in cultured cell experiments. Consistent with the results of iNO experiments, administration of the NO donor S-nitroso-N-acetyl-penicillamine to cultured bovine pulmonary endothelial cells did not attenuate increases in ET peptide levels resulting from hypoxic (24 h, 3% O2) exposure. In additional experiments, we examined the effects of NO on the activity of a cloned ET-1 promoter fragment containing a functional hypoxia inducible factor-1 binding site in reporter gene experiments. Whereas moderate hypoxia (24 h, 3% O2) had no effect on ET-1 promoter activity, activity was increased by severe hypoxic (24 h, 0.5% O2) exposure. ET-1 promoter activity after S-nitroso-N-acetyl-penicillamine administration during severe hypoxia was greater than that in normoxic controls, although activity was reduced compared with that in hypoxic controls. These findings suggest that hypoxia-induced pulmonary ET-1 expression is unaffected by NO.

rat; gene expression; inhaled nitric oxide; pulmonary hypertension


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CHRONIC HYPOXIA IS A POWERFUL physiological stimulus resulting in hypoxic pulmonary vasoconstriction and pulmonary arterial remodeling, thereby increasing vascular resistance in this normally low-pressure circuit. Prolonged hypoxic exposure induces right ventricular hypertrophy and can lead to right heart failure secondary to the resultant pulmonary hypertension. Endothelial cells appear to be involved in the pulmonary vascular response to hypoxia through the release of vasoactive factors, such as nitric oxide (NO) (19, 25) and endothelin (ET)-1 (11). NO is a potent vasodilator and inhibits the proliferation of vascular smooth muscle cells (7), whereas ET-1 is a powerful vasoconstrictor (27) and vascular smooth muscle cell mitogen (8). Considering these divergent activities, NO and ET-1 may play opposing roles in the development of pulmonary hypertension.

In addition to its vasodilatory and antimitogenic effects, NO may influence hypoxia-induced ET-1 expression. For example, administration of the NO donor sodium nitroprusside (SNP) has been shown to attenuate increased ET-1 production in response to hypoxia in human umbilical vein endothelial cells (HUVEC) (12). Other reports suggest that NO attenuates hypoxia-induced increases in gene expression (10, 20) by antagonizing the activity of hypoxia-inducible factor-1 (HIF-1) (18, 23, 24). Because the rat ET-1 promoter reportedly contains a functional binding site for HIF-1 (9), elevated endothelial cell production of ET-1 during hypoxia may be due to increased gene expression acting through this transcription factor. Taken together, these findings suggest that NO may attenuate hypoxia-induced increases in ET-1 expression by interfering with HIF-1 activity.

Studies examining the effects of NO on hypoxia-induced increases in pulmonary ET-1 gene expression in intact animals have yielded conflicting results. Blumberg et al. (1) demonstrated that administration of the NO donor molsidomine attenuated hypoxia-induced increases in prepro-ET-1 mRNA levels in rat lung. In contrast, Ozaki et al. (15) showed that plasma and pulmonary ET-1 peptide levels were increased by hypoxic exposure in transgenic mice overexpressing the endothelial NO synthase gene. Thus the purpose of this study was to examine the effects of NO on pulmonary ET-1 production within the lungs of hypoxic rats. We hypothesized that NO administration would attenuate hypoxia-induced increases in ET-1 expression in this setting. To address this hypothesis, we examined the combined effects of inhaled NO (iNO) and hypoxia on pulmonary ET-1 peptide and prepro-ET-1 mRNA production in vivo. Because of our surprising finding that NO had no effect on hypoxia-induced increases in ET-1 production in rat lungs, we also investigated the effects of NO donor administration on ET peptide production and ET-1 promoter activity during hypoxic exposure in cultured pulmonary artery endothelial cells.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In Vivo iNO Experiments

To investigate the effects of NO on hypoxia-induced increases in ET levels, we examined the combined effects of iNO and hypoxia on ET-1 peptide and prepro-ET-1 mRNA levels within the lungs of intact rats.

Experimental animal groups. All protocols involving animals employed in this study were reviewed and approved by the Institutional Animal Care Committee of the University of New Mexico School of Medicine. Male Sprague-Dawley rats (250-400 g; Harlan Industries) were randomly divided into normoxic control (n = 6), normoxic + 10 parts/million (ppm) iNO (n = 6), normoxic + 100 ppm iNO (n = 6), hypoxic (24 h, 10% O2) control (n = 6), hypoxic + 10 ppm iNO (n = 6), and hypoxic + 100 ppm iNO (n = 8) groups. The level of hypoxia employed was selected based on previous studies (1, 4) investigating hypoxic induction of ET-1 expression in rats. Rats were housed in Plexiglas chambers during exposure and were provided with food, water, and clean bedding. O2 and CO2 levels were constantly monitored in all chambers using an Ametek CD-3A CO2 analyzer and an Ametek S-3A/I O2 analyzer. Soda lime was used to absorb CO2. NO and NO2 levels were also continuously observed using an electrochemical NO and NO2 analyzer (14) during iNO administration. Normobaric hypoxia was maintained by flushing chambers with compressed air and nitrogen. Flow rates of these gases were adjusted to achieve an O2 concentration of 10%. Normoxic control and normoxic iNO chambers were flushed with compressed air at flow rates similar to those used to maintain hypoxic conditions. Rats were anesthetized with pentobarbital sodium (32.5 mg ip) at the end of the 24-h exposure period. Blood was collected by cardiac puncture, and the lungs were removed and snap-frozen in liquid nitrogen. Nitrite/nitrate (NOx-) and ET peptide levels were measured in plasma samples, and pulmonary ET-1 peptide and prepro-ET-1 mRNA levels were determined.

Measurement of ET peptide in plasma and lung tissue. Plasma was stored at -80°C until assay and was then thawed and centrifuged briefly to clarify before analysis. Acidified plasma was extracted using reverse-phase Amprep C2 columns (Amersham-Pharmacia). Plasma ET peptide levels were determined using a RIA kit (Amersham-Pharmacia) and were expressed as picograms of ET per milliliter of plasma. The antibody included with this kit has 1,305% cross-reactivity with ET-2, <0.001% cross-reactivity with ET-3, and a limit of detection of 1.25 pg/ml, according to the manufacturer. Because this assay is sensitive for both ET-1 and ET-2, results are expressed as ET peptide levels. Snap-frozen lung tissue was homogenized using a Polytron blender in ice-cold methanol. After brief centrifugation, the extract was purified and concentrated using reverse-phase Amprep C2 columns. ET-1 peptide levels were more concentrated in lung extracts than in plasma, which allowed us to use a more specific, but less sensitive RIA kit (Peninsula Laboratories) for these experiments. ET-1 levels in the lung were expressed as picograms of ET-1 per milligram of extracted tissue. The antibody included with this kit has 7% cross-reactivity with both ET-2 and ET-3.

Ribonuclease protection assay for prepro-ET-1 mRNA. RNA isolation, ribonuclease protection assay (RPA), and data analysis procedures were performed largely as previously described (16). The RPA probe template for ET-1 was constructed using PCR primers 5'-GAACTCCGAGCCCAAAGTAC-3' (forward) and 5'-CTTGCTAAGATCCCAGCCA-3' (reverse), based on a published rat ET-1 mRNA sequence (GenBank Accession M64711). 18S rRNA was used as a constitutively expressed internal control for RNA quantity and quality. ET-1 mRNA abundance was determined by dividing volumes for ET-1 bands by those of corresponding 18S rRNA bands. Additional hybridizations containing 2.5, 5, or 10 µg of RNA from hypoxic rat lung were performed and subjected to RPA to demonstrate the linearity of the relationship between ET-1 and 18S rRNA band volumes and input RNA quantity.

NOx- assay. Plasma samples were assayed spectrophotometrically in duplicate for NOx- as previously described (22). Fifty microliters of reduced NAPD (0.8 mg/ml) and 10 µl of nitrate reductase (5 U/ml) were added to 500 µl of plasma, and the mixture was incubated for 3 h at room temperature. Then, 300 µl of Griess reagent [1% sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride, and 2.5% phosphoric acid] were added and incubated for 10 min at room temperature. Absorbance was read at 546 nm by using a blank solution with added reduced NADP, nitrate reductase, and the Griess reagent. Absorbance values were compared with a standard curve generated by adding known amounts of NaNO3 to a blank solution and assaying as described above.

Cultured Cell Experiments

In contrast to a previous study employing cultured HUVEC (12), NO administration did not attenuate hypoxia-induced ET-1 expression in rat lungs. However, in the intact setting, endothelial cell responses to NO may be influenced by circulating factors or by interactions with other cell types. Furthermore, it is possible that HUVECs are phenotypically distinct from pulmonary vascular endothelial cells and may demonstrate different responses to NO. We, therefore, tested the effects of NO administration on hypoxia-induced ET expression in cultured pulmonary artery endothelial cells, rather than umbilical vein endothelial cells. Although the previous study on HUVECs used the NO donor SNP (11), the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) was employed for these studies because of concerns about NO-independent effects of SNP (6). NO-independent effects of SNAP have not been reported, and, furthermore, acetyl-penicillamine (AP), a compound structurally similar to SNAP that does not donate NO, was used in these studies to control for potential NO-independent effects (5).

Cell culture. Bovine pulmonary artery endothelial cells (BPAECs) (Clonetics) were cultured at 37°C, 6% CO2, balance air, in humidified incubators in endothelial growth medium (EGM) (Clonetics) supplemented with 2% fetal bovine serum. Cells were passaged with 0.025% trypsin-EDTA when confluent. Cells between passages 3 and 8 were used for these studies. Hypoxic (24 h; 6% CO2, 3% O2, balance N2) exposures were performed in a Napco 7000 series three-gas incubator at 37°C. The O2 concentration and duration of hypoxic exposure were based on previous reports (11, 12). Chamber O2 concentration was verified by using an Ametek model S-3A/I O2 analyzer.

Measurement of ET peptide in culture supernatants. Culture supernatants were sampled from endothelial cells incubated under hypoxic (24 h; 3% O2) or normoxic conditions in six-well plates. Supernatants were also obtained from normoxic and hypoxic cultures that had been treated with SNAP (100 µM). ET peptide levels in culture supernatants were much more concentrated than levels present in plasma samples and lung homogenates. Therefore, ET levels in cell culture supernatants were determined by using a nonradioactive enzyme immunoassay kit (Cayman Chemical) and were expressed as picograms of ET per milliliter of culture supernatant. The antibody included with this kit has 100% cross-reactivity with ET-2 and ET-3 and a limit of detection of >= 50 pg/ml as stated by the manufacturer. Because this assay is sensitive for ET-2 and ET-3 as well as ET-1, results are expressed as ET peptide levels.

Reporter Gene Experiments

Hypoxic induction of ET-1 gene expression is reportedly mediated by the transcription factor HIF-1 (9). Although several reports demonstrate that NO attenuates hypoxia-induced gene expression in transformed hepatoma cell lines by interfering with HIF-1 (10, 20), this effect of NO has not been examined in nontransformed endothelial cells. Our findings demonstrated that NO did not attenuate hypoxia-induced ET-1 expression in either whole lungs or cultured pulmonary artery endothelial cells, suggesting that HIF-1-dependent increases in ET-1 gene expression were unaffected by NO. To resolve these potentially conflicting observations, we examined the effects of NO on hypoxia-induced increases in pulmonary endothelial cell ET-1 gene expression in reporter gene experiments.

Plamid construction. A segment of the prepro-ET-1 promoter containing a functional hypoxia response element (HRE) and other response elements was cloned from rat genomic DNA into a luciferase reporter vector. A 745-bp promoter fragment was amplified by PCR using primers 5'-TAGGATGTGCCTGACGAAAC-3' (forward) and 5'-AGACCCAGTCAGGCTCTCAG-3' (reverse) based on a published sequence (GenBank accession S76970). This fragment contains a functional HRE that binds HIF-1 in gel-shift assays (9). The amplified fragment was cloned into the Srf I site of pPCR-Script-Amp+ (Stratagene), and the orientation of the insert was determined by restriction mapping. The Sst I-Hind III fragment containing the ET-1 promoter was cloned into the Sst I-Hind III sites of the firefly luciferase reporter vector pGL2-basic (Promega), and the resulting plasmid was designated pGL2-ET1P. The insert was confirmed by sequencing.

Site-directed mutagenesis. Site-directed mutation of the HRE (5'-ACGTGC-3') within the cloned ET-1 promoter fragment was performed using a Quick-change site-directed mutagenesis kit (Stratagene). The sequence of the mutagenic primer was 5'-GGGTCTTATCTCCGGCTGCATACTGCCTGTGGGTGACTAATC-3'. Incorporation of this sequence by Pyrococcus furiosus PCR into pGL2-ET1P followed by Dpn I digestion (to remove template DNA) altered the sequence of the ET-1 promoter HRE to 5'-ATACGC-3'. A previous report shows that HIF-1 does not bind this sequence in gel-shift assays (9). The mutation was confirmed by sequencing.

Transfections and reporter gene assays. Transfections of BPAECs were performed by using Superfect transfection reagent (Qiagen). BPAECs were cultured as described above, and cells were split into six-well plates at a density of 100,000 per well. On the next day, cells were washed and exposed to a total of 1 µg of pGL2 DNA and 5 µl of Superfect reagent in 600 µl of EGM. Plasmid pRL-TK, which expresses Renilla reniformis luciferase under the control of the minimal herpes simplex virus thymidine kinase promoter, was used as an internal control for cell viability and transfection efficiency. pRL-TK DNA was cotransfected with pGL2 DNA at ratios of 1:5. These ratios were selected on the basis of pilot experiments as the minimum amount of pRL-TK DNA required to produce reproducible and measurable Renilla luciferase activity. Cells were washed, and fresh EGM was added after a 4-h incubation period. Transfected cells were incubated for an additional 24 h before exposure to experimental treatments. Cells were exposed to experimental treatments for 24 h before passive lysis and luciferase assay. ET-1 promoter activity was determined by the dual-luciferase assay (Promega). Luciferase measurements were performed using a Turner Designs model 20 luminometer. ET-1 promoter activity in cell lysates was determined by dividing the luminescence observed after the addition of firefly luciferase substrate by that obtained after quenching firefly luciferase activity and adding the substrate for Renilla luciferase. The mean background luminescence from six mock-transfected samples was determined for each experiment and was subtracted from each sample before ratio calculation.

Reporter Gene Experimental Protocols

Effects of SNAP on hypoxia-induced ET-1 promoter activity. Cells transfected with the ET-1 promoter reporter gene construct were cultured under normoxic or hypoxic conditions. The effects of hypoxia and SNAP (100 µM) administration on ET-1 promoter activity were evaluated. SNAP (RPI or Sigma Chemical) and AP (Sigma Chemical) were dissolved in ethanol on the day of experimentation. In experiments involving exposure to SNAP, all groups that were not treated with SNAP were treated with AP (100 µM) to control for NO-independent effects of SNAP (5). Hypoxic (24 h; 6% CO2, 3% O2, or 0.5% O2, balance N2) exposures were performed in a Napco 7000 series three-gas incubator at 37°C. Chamber O2 concentration was verified by using an Ametek model S-3A/I O2 analyzer.

Effects of HRE mutation on hypoxia-induced ET-1 promoter activity. To test the assumption that hypoxia-induced increases in ET-1 promoter activity are mediated by HIF-1, we mutated the HRE within the cloned ET-1 promoter fragment to a sequence that does not bind HIF-1 (9). Cells transfected with the HRE-mutant ET-1 reporter vector (Delta HRE) were cultured under normoxia or hypoxia, and promoter activity was measured. The effect of SNAP (100 µM) administration on Delta HRE ET-1 promoter activity during hypoxia was also determined.

Effects of 8-bromo-cGMP ET-1 promoter activity. NO is a potent activator of soluble guanylyl cyclase and, consequently, increases intracellular concentrations of the second messenger molecule cGMP. Experiments were conducted to determine whether increases in cGMP affected hypoxia-induced ET-1 promoter activity. Cells transfected with the ET-1 promoter construct were cultured under hypoxic or normoxic conditions, and the effects of administration of the membrane-permeant cGMP analog 8-bromo-cGMP (1 mM) on ET-1 promoter activity was determined. 8-bromo-cGMP (Sigma Chemical) was dissolved in water and sterilized by filtration before administration.

Calculations and Statistics

All data are expressed as means ± SE. Values of n refer to the number of animals or the number of replicate cultures in each group. One-way ANOVA was used to make comparisons. If differences were detected by ANOVA, individual groups were compared by using the Student-Newman-Keuls test for all pairwise comparisons. All data expressed as percentages were normalized using arcsine transformation before statistical analysis. A level of P <=  0.05 was accepted as statistically significant for all comparisons.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In Vivo iNO Experiments

Effects of hypoxia and iNO on ET levels in plasma and lung tissue. ET peptide levels in plasma isolated from normoxic rats treated with 10 ppm iNO were less than those of the normoxic control group (11.28 ± 0.29 vs. 12.86 ± 0.47 pg/ml). Plasma ET peptide levels were not different compared with normoxic controls after hypoxic exposure (11.74 ± 0.56 pg/ml) or hypoxia + 10 ppm iNO (13.10 ± 0.37 pg/ml).

In contrast to the lack of effect of hypoxia on circulating ET levels, ET-1 peptide levels in lung tissue isolated from hypoxic rats were about fourfold greater than those of normoxic controls (Fig. 1). Contrary to our hypothesis, ET-1 peptide levels in lung tissue isolated from rats treated with iNO during hypoxic exposure were not different from those of hypoxic rat lungs that were not exposed to iNO (Fig. 1), suggesting that NO does not attenuate hypoxia-induced increases in pulmonary ET-1 levels in intact rats. Interestingly, pulmonary ET-1 peptide levels for normoxic rats that were administered 10 or 100 ppm iNO were greater than those of controls (Fig. 1).


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  		Fig. 1.   Pulmonary endothelin (ET)-1 peptide levels for normoxic control (n = 6) rats, normoxic rats exposed to 10 parts/million (ppm) (n = 6) or 100 ppm (n = 6) inhaled nitric oxide (iNO), hypoxic (24 h, 10% O2, n = 6) rats, and hypoxic rats exposed to 10 ppm iNO (n = 6) or 100 ppm iNO (n = 8). Values are means ± SE. * P <=  0.05 vs. normoxic control.

Effects of hypoxia and iNO on prepro-ET-1 mRNA levels in rat lung. Consistent with the effects of hypoxia on ET peptide levels in whole lung, pulmonary prepro-ET-1 mRNA levels were also increased in tissues isolated from hypoxic rats compared with controls (Fig. 2). Lung prepro-ET-1 mRNA levels for hypoxic rats treated with 10 ppm iNO did not differ from those of hypoxic rats that were not administered iNO (Fig. 2), suggesting that NO does not alter hypoxia-induced prepro-ET-1 gene expression. Pulmonary prepro-ET-1 mRNA levels for hypoxic rats treated with 10 ppm iNO were not different compared with untreated hypoxic rats or normoxic controls. Prepro-ET-1 mRNA levels in lungs from normoxic rats that were administered 10 ppm iNO were not different from those of normoxic controls (Fig. 2), suggesting that iNO-induced increases in ET peptide levels during normoxia may result from a nontranscriptional mechanism.


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  		Fig. 2.   Prepro-ET-1 mRNA levels in lungs from normoxic rats, normoxic rats exposed to 10 ppm iNO, hypoxic (24 h, 10% O2) rats, and hypoxic rats exposed to 10 ppm iNO. Values are means ± SE; n = 6 for all groups. * P <=  0.05 vs. normoxic control.

Effects of hypoxia and iNO on plasma NOx- levels. Administration of iNO to either normoxic or hypoxic rats resulted in increased plasma NOx- levels compared with those in controls (Fig. 3). Furthermore, NOx- levels were greater in rats treated with 100 ppm iNO compared with rats treated with 10 ppm iNO for both normoxic and hypoxic groups (Fig. 3).


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  		Fig. 3.   Plasma nitrate/nitrite (NO<UP><SUB>x</SUB><SUP>−</SUP></UP>) levels from normoxic (n = 6) rats and normoxic rats exposed to 10 ppm (n = 6) or 100 ppm (n = 6) iNO. Also shown are data from hypoxic rats (24 h, 10% O2, n = 6) and hypoxic rats exposed to 10 ppm iNO (n = 6) or 100 ppm iNO (n = 8). Values are means ± SE. * P <=  0.05 vs. normoxic control; # P <=  0.05 vs. normoxia + 10 ppm iNO.

Cultured Cell Experiments

Effects of NO on hypoxia-induced ET peptide levels in BPAECs. Consistent with the effects of hypoxia on ET levels in whole lungs, ET peptide levels in BPAEC supernatants after hypoxic exposure were greater than those in normoxic controls (Fig. 4). In contrast to a previous report demonstrating that administration of SNP attenuated hypoxia-induced increases in ET-1 peptide levels in HUVECs (12), ET peptide levels were not different in supernatants from hypoxic cultures after administration of SNAP compared with those in hypoxic controls (Fig. 4). These data are consistent with the finding of the iNO studies (Fig. 1) and suggest that NO does not attenuate hypoxia-induced increases in ET production in pulmonary artery endothelial cells. ET peptide levels in normoxic culture supernatants treated with SNAP were not different from those in normoxic controls (Fig. 4).


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  		Fig. 4.   ET peptide levels in bovine pulmonary artery endothelial cell (BPAEC) culture supernatants from cells cultured under hypoxic (24 h, 3% O2) or normoxic conditions and treated with S-nitroso-N-acetyl-penicillamine (SNAP; 100 µM). Values are means ± SE; n = 6 for all groups. * P <=  0.05 vs. normoxia; # P <=  0.05 vs. normoxia + SNAP.

Reporter Gene Experiments

Effects of SNAP on ET-1 promoter activity. Hypoxic (24 h, 3% O2) exposure did not alter ET-1 promoter activity relative to that of normoxic controls (data not shown). In contrast, severe hypoxic exposure (24 h, 0.5% O2) resulted in increased ET-1 promoter activity compared with that of normoxic controls (Fig. 5A). ET-1 promoter activity in hypoxic cultures treated with SNAP was reduced compared with that of hypoxic controls but was still greater than that of normoxic controls (Fig. 5A). This effect may not be specific to hypoxia-induced ET-1 promoter activity because administration of SNAP to normoxic cultures transfected with the ET-1 promoter construct also resulted in decreased ET-1 promoter activity compared with that of normoxic controls (Fig. 5B).


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  		Fig. 5.   A: ET-1 promoter activity for BPAECs cultured under normoxia, hypoxia (24 h, 0.5% O2), or hypoxia + SNAP (100 µM). B: ET-1 promoter activity for BPAECs cultured under normoxia or normoxia + SNAP (100 µM). Values are means ± SE; n = 6 for all groups. * P <=  0.05 vs. vehicle; # P <=  0.05 vs. hypoxia.

Effects of HRE mutation on hypoxia-induced ET-1 promoter activity. Delta HRE-ET-1 promoter activity for cells cultured under hypoxic conditions was not increased compared with that of normoxic cells (Fig. 6), suggesting that induction of ET-1 promoter activity by hypoxia is HIF dependent. Surprisingly, hypoxic cultures treated with SNAP demonstrated greater Delta HRE-ET-1 promoter activity relative to that of normoxic controls (Fig. 6).


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  		Fig. 6.   Hypoxia response element-mutated (Delta HRE) ET-1 promoter activity for BPAECs cultured under normoxia, hypoxia (24 h, 0.5% O2), or hypoxia + SNAP (100 µM). Values are means ± SE; n = 6 for all groups. * P <=  0.05 vs. normoxia.

Effects of 8-bromo-cGMP on hypoxia-induced ET-1 promoter activity. Treatment of cells transfected with the ET-1 promoter construct with 8-bromo-cGMP did not alter ET-1 promoter activity compared with vehicle-treated cells under both normoxic and hypoxic conditions (Fig. 7). These results suggest that ET-1 promoter activity is independent of elevated cGMP levels.


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  		Fig. 7.   ET-1 promoter activity for BPAECs cultured under normoxia or hypoxia (24 h, 0.5% O2) and treated with 8-bromo-cGMP (1 mM) or vehicle. Values are means ± SE; n = 6 for all groups. * P <=  0.05 vs. normoxia.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study examined the effects of iNO on hypoxia-induced ET-1 gene expression and ET-1 peptide levels in rat lung, as well as the effects of a NO donor on hypoxia-induced increases in ET peptide levels and ET-1 promoter activity in cultured endothelial cells. The major findings of this study are as follows. 1) iNO does not attenuate hypoxia-induced increases in ET-1 peptide or prepro-ET-1 mRNA levels in rat lungs. 2) Similarly, administration of the NO donor SNAP did not attenuate hypoxia-induced increases in ET peptide production in cultured bovine pulmonary endothelial cells. 3) In reporter gene experiments, hypoxia-induced ET-1 promoter activity in cells treated with SNAP was greater than that in normoxic controls, although activity was somewhat less than that in hypoxic controls. 4) ET-1 promoter activity was not altered by administration of the membrane-permeable cGMP analog 8-bromo-cGMP. Contrary to previously reported results (12), but in agreement with a recent study (15), these findings demonstrate that NO does not influence hypoxia-induced increases in ET-1 gene expression or peptide production in the lungs of intact animals or cultured pulmonary artery endothelial cells.

The vasoconstrictor and mitogenic activities of ET-1 may contribute to the development of pulmonary hypertension. For example, endothelin A-receptor blockade attenuates the development of hypoxia-induced pulmonary arterial remodeling (3). In contrast, NO may attenuate the development of hypoxia-induced pulmonary hypertension (13, 17). Because increased ET-1 production is correlated with hypoxia-induced pulmonary hypertension, we reasoned that, if hypoxia-induced ET-1 gene expression is diminished by NO, then this attenuation of increased ET-1 peptide production may contribute to NO-dependent attenuation of pulmonary hypertension. Contrary to our hypothesis, we consistently found that NO did not alter hypoxia-induced increases in ET-1 peptide levels or gene expression in various experimental settings. For example, administration of iNO did not attenuate hypoxia-induced increases in pulmonary ET peptide levels (Fig. 1) or prepro-ET-1 mRNA levels (Fig. 2). Similarly, administration of a NO donor did not diminish hypoxia-induced increases in ET peptide production in pulmonary artery endothelial cell cultures (Fig. 4). In contrast, it was recently reported (1) that administration of the NO donor molsidomine to rats during long-term hypoxic exposure (4 wk, 10% O2) attenuated hypoxia-induced pulmonary hypertension and ET-1 gene expression. A recent study by Ozaki et al. (15) demonstrated that the development of hypoxia (3 wk, 10% O2)-induced pulmonary hypertension is attenuated in transgenic mice that overexpress the endothelial NO synthase gene. In agreement with our results (Fig. 1), pulmonary ET-1 peptide levels were increased by hypoxia in these animals, despite increased NO production. The report by Ozaki et al. and our findings (Fig. 1) suggest that NO may not attenuate the development of pulmonary hypertension by reducing hypoxia-induced increases in local ET-1 peptide levels.

Several differences in experimental procedures could account for the discrepancies between our data demonstrating that increased ET-1 expression during hypoxia is unaffected by NO (Fig. 4) and an earlier report describing an inhibitory effect of a NO donor on hypoxia-induced ET-1 production in cultured cells (12). For example, we studied this response in BPAECs and in vivo in intact rat lung, whereas the previous work (12) employed HUVEC. Endothelial cells isolated from venous and arterial segments have well-described phenotypic differences (21). Furthermore, different NO donors were employed. The previous study (12) used the NO donor SNP and did not control for potential NO-independent effects of this compound, such as activation of ATP-sensitive potassium channels (6). In contrast, we employed the NO donor SNAP in cultured cell experiments and used AP, a structurally similar compound that does not donate NO, as a control for NO-independent effects (5). Thus divergent responses of endothelial cells isolated from different vascular segments to NO or nonspecific effects of SNP could account for the contrary results.

Studies employing transformed hepatoma cell lines have demonstrated that NO donors attenuate hypoxia-induced gene expression by antagonizing the activity of HIF-1 (10, 20). In contrast, in nontransformed, low-passage-number pulmonary artery endothelial cells, our findings demonstrate minimal effects of NO on hypoxia-induced ET-1 promoter activity. Although administration of a NO donor partially attenuated hypoxia-induced responses (Fig. 5A), ET-1 promoter activity of hypoxic SNAP-treated cultures was still greater than that of normoxic controls. SNAP administration also slightly decreased ET-1 promoter activity under normoxic conditions (Fig. 5B), suggesting that the effects of NO are not hypoxia specific. The divergent effects of NO on hypoxia-induced promoter activity observed in our study and an earlier report (12) may be due to the different cell type employed. We attempted to test this possibility by examining ET-1 promoter activity in the transformed hepatoma cell line Hep3B. However, reporter gene activity in Hep3B cells was very low (data not shown), possibly because of the presence of endothelial cell-specific response elements (2) within the ET-1 promoter fragment, confounding our attempts to examine the effects of NO on hypoxia-induced promoter activity in these cells. In agreement with previous reports (9, 26), mutation of the HRE within the cloned ET-1 promoter fragment abolished hypoxic induction of promoter activity (Fig. 6), indicating that this response is HIF dependent. The results of the reporter gene experiments are consistent with the whole animal (Figs. 1 and 2) and cultured cell (Fig. 4) data in demonstrating that hypoxia-induced increases in ET peptide levels, prepro-ET-1 mRNA levels, and ET-1 promoter activity remain elevated compared with that in normoxic controls after NO administration.

In summary, we have demonstrated that, contrary to our hypothesis and previous reports, hypoxia-induced increases in ET peptide levels in rat lungs and cultured pulmonary endothelial cells do not appear to be attenuated by NO.


    ACKNOWLEDGEMENTS

We thank Heather E. Nash and Minerva Murphy for technical assistance, and the Center for Genetics in Medicine of the University of New Mexico Department of Biochemistry and Molecular Biology for DNA sequencing services and PCR primer synthesis.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-58124 (to B. R. Walker) and HL-04050 (to L. G. Chicoine) and by a Grant-in-Aid from the Desert-Mountain Affiliate of the American Heart Association (to L. D. Nelin).

Address for reprint requests and other correspondence: S. Earley, Vascular Physiology Group, Dept. of Cell Biology and Physiology, Univ. of New Mexico HSC, 915 Camino de Salud, N.E., Albuquerque, NM 87131-5218 (E-mail: searley{at}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.

10.1152/japplphysiol.00829.2001

Received 6 August 2001; accepted in final form 6 November 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Blumberg, FC, Wolf K, Sandner P, Lorenz C, Riegger GA, and Pfeifer M. The NO donor molsidomine reduces endothelin-1 gene expression in chronic hypoxic rat lungs. Am J Physiol Lung Cell Mol Physiol 280: L258-L263, 2001[Abstract/Free Full Text].

2.   Bu, X, and Quertermous T. Identification of an endothelial cell-specific regulatory region in the murine endothelin-1 gene. J Biol Chem 272: 32613-32622, 1997[Abstract/Free Full Text].

3.   Chen, SJ, Chen YF, Opgenorth TJ, Wessale JL, Meng QC, Durand J, DiCarlo VS, and Oparil S. The orally active nonpeptide endothelin A-receptor antagonist A-127722 prevents and reverses hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling in Sprague-Dawley rats. J Cardiovasc Pharmacol 29: 713-725, 1997[ISI][Medline].

4.   Elton, TS, Oparil S, Taylor GR, Hicks PH, Yang RH, Jin H, and Chen YF. Normobaric hypoxia stimulates endothelin-1 gene expression in the rat. Am J Physiol Regulatory Integrative Comp Physiol 263: R1260-R1264, 1992[Abstract/Free Full Text].

5.   Emery, CJ. Vasodilator action of the S-nitrosothiol, SNAP, in rat isolated perfused lung. Physiol Res 44: 19-24, 1995[ISI][Medline].

6.   Gambone, LM, Fujiwara Y, and Murray PA. Endothelium-dependent pulmonary vasodilation is selectively attenuated during isoflurane anesthesia. Am J Physiol Heart Circ Physiol 272: H290-H298, 1997[Abstract/Free Full Text].

7.   Garg, UC, and Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest 83: 1774-1777, 1989.

8.   Hirata, Y, Takagi Y, Fukuda Y, and Marumo F. Endothelin is a potent mitogen for rat vascular smooth muscle cells. Atherosclerosis 78: 225-228, 1989[ISI][Medline].

9.   Hu, J, Discher DJ, Bishopric NH, and Webster KA. Hypoxia regulates expression of the endothelin-1 gene through a proximal hypoxia-inducible factor-1 binding site on the antisense strand. Biochem Biophys Res Commun 245: 894-899, 1998[ISI][Medline].

10.   Huang, LE, Willmore WG, Gu J, Goldberg MA, and Bunn HF. Inhibition of hypoxia-inducible factor 1 activation by carbon monoxide and nitric oxide. Implications for oxygen sensing and signaling. J Biol Chem 274: 9038-9044, 1999[Abstract/Free Full Text].

11.   Kourembanas, S, Marsden PA, McQuillan LP, and Faller DV. Hypoxia induces endothelin gene expression and secretion in cultured human endothelium. J Clin Invest 88: 1054-1057, 1991.

12.   Kourembanas, S, McQuillan LP, Leung GK, and Faller DV. Nitric oxide regulates the expression of vasoconstrictors and growth factors by vascular endothelium under both normoxia and hypoxia. J Clin Invest 92: 99-104, 1993.

13.   Kouyoumdjian, C, Adnot S, Levame M, Eddahibi S, Bousbaa H, and Raffestin B. Continuous inhalation of nitric oxide protects against development of pulmonary hypertension in chronically hypoxic rats. J Clin Invest 94: 578-584, 1994.

14.   Nelin, LD, Christman NT, Morrisey JF, and Dawson CA. Electrochemical nitric oxide and nitrogen dioxide analyzer for use with inhaled nitric oxide. J Appl Physiol 81: 1423-1429, 1996[Abstract/Free Full Text].

15.   Ozaki, M, Kawashima S, Yamashita T, Ohashi Y, Rikitake Y, Inoue N, Hirata KI, Hayashi Y, Itoh H, and Yokoyama M. Reduced hypoxic pulmonary vascular remodeling by nitric oxide from the endothelium. Hypertension 37: 322-327, 2001[Abstract/Free Full Text].

16.   Resta, TC, O'Donaughy TL, Earley S, Chicoine LG, and Walker BR. Unaltered vasoconstrictor responsiveness after iNOS inhibition in lungs from chronically hypoxic rats. Am J Physiol Lung Cell Mol Physiol 276: L122-L130, 1999[Abstract/Free Full Text].

17.   Roos, CM, Frank DU, Xue C, Johns RA, and Rich GF. 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].

18.   Semenza, GL, and Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol 12: 5447-5454, 1992[Abstract/Free Full Text].

19.   Shaul, PW, North AJ, Brannon TS, Ujiie K, Wells LB, Nisen PA, Lowenstein CJ, Snyder SH, and Star RA. 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].

20.   Sogawa, K, Numayama-Tsuruta K, Ema M, Abe M, Abe H, and Fujii-Kuriyama Y. Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia. Proc Natl Acad Sci USA 95: 7368-7373, 1998[Abstract/Free Full Text].

21.   Wagner, WH, Henderson RM, Hicks HE, Banes AJ, and Johnson G, Jr. Differences in morphology, growth rate, and protein synthesis between cultured arterial and venous endothelial cells. J Vasc Surg 8: 509-519, 1988[ISI][Medline].

22.   Walker, BR, Resta TC, and Nelin LD. Nitric oxide-dependent pulmonary vasodilation in polycythemic rats. Am J Physiol Heart Circ Physiol 279: H2382-H2389, 2000[Abstract/Free Full Text].

23.   Wang, GL, and Semenza GL. Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. J Biol Chem 268: 21513-21518, 1993[Abstract/Free Full Text].

24.   Wang, GL, and Semenza GL. General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia. Proc Natl Acad Sci USA 90: 4304-4308, 1993[Abstract/Free Full Text].

25.   Xu, XP, Pollock JS, Tanner MA, and Myers PR. Hypoxia activates nitric oxide synthase and stimulates nitric oxide production in porcine coronary resistance arteriolar endothelial cells. Cardiovasc Res 30: 841-847, 1995[ISI][Medline].

26.   Yamashita, K, Discher DJ, Hu J, Bishopric NH, and Webster KA. Molecular regulation of the endothelin-1 gene by hypoxia. Contributions of hypoxia-inducible factor-1, activator protein-1, GATA-2, AND p300/CBP. J Biol Chem 276: 12645-12653, 2001[Abstract/Free Full Text].

27.   Yanagisawa, M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, and Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411-415, 1988[Medline].

J APPL PHYSIOL 92(3):1152-1158
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