Divisions of 1 Respiratory, Endothelin-1
(ET-1) is a pluripotent mediator that modulates vascular tone and
influences the inflammatory response. Patients with inflammatory lung
disorders frequently have elevated circulating ET-1 levels. Because
these pathophysiological conditions generate reactive oxygen species
that can regulate gene expression, we investigated whether the level of
oxidant stress influences ET-1 production in cultured rat pulmonary
arterial endothelial cells (RPAEC). Treatment with the antioxidant
1,3-dimethyl-2-thiourea (10 mM) or the iron chelator deferoxamine (1.8 µM) doubles basal ET-1 release. Conversely, exposing cells to
H2O2
generated by glucose and glucose oxidase (0.1-10 mU/ml) for 4 h
causes a concentration-dependent decrease in ET-1 release. This effect
occurs at concentrations of glucose oxidase that do not affect
[3H]leucine
incorporation or specific 51Cr
release from RPAEC. Catalase prevents the decrease in ET-1 synthesis
caused by glucose and glucose oxidase. Glucose and glucose oxidase
decrease not only ET-1 generation but also ET-1 mRNA as assessed by
semiquantitative polymerase chain reaction. Our results indicate that
changes in oxidative stress can either up- or downregulate basal ET-1
generation by cultured pulmonary endothelial cells.
hydrogen peroxide; messenger ribonucleic acid; glucose oxidase; 1,3-dimethyl-2-thiourea; deferoxamine
ENDOTHELIN-1 (ET-1) is a cytokine that can
potently modulate pulmonary vascular tone, lung smooth muscle growth,
and inflammatory processes. Proinflammatory effects of ET-1 include
priming neutrophils, activating mast cells, stimulating oxygen radical
production by macrophages, releasing growth factors from smooth muscle
cells, and increasing adhesion molecule expression on endothelial cells (19). ET-1 also stimulates monocytes to produce interleukin-6, interleukin-8, and prostaglandin
E2, all important in modulating immune responses (17). Besides the proinflammatory effects of ET-1, its
ability to regulate vascular and airway tone, augment airway and
vascular smooth muscle cell growth, and stimulate fibroblast growth,
migration, and collagen synthesis indicate its potential to participate
in the tissue reaction to ongoing inflammation (19).
Patients with a variety of inflammatory lung diseases, including the
acute respiratory distress syndrome, have evidence for increased ET-1
production and reactive oxygen species generation in their lungs (7,
13, 21, 32). Because reactive oxygen species can regulate gene
expression, we investigated the effect of oxidant stress on ET-1
production by cultured pulmonary endothelial cells.
Reagents
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-32P]GTP was
from ICN Biomedicals (Costa Mesa, CA); 2-mercaptoethanol was from
Bio-Rad Laboratories (Richmond, CA); phenol saturated solution was from
Amresco (Solon, OH); chloroform and calcium chloride were from
Mallinckrodt (Paris, KY); ethyl alcohol was from Quantum Chemical
(Tuscola, IL); and RNase-free presiliconized microcentrifuge tubes were
from Intermountain Scientific (Bountiful, UT). All other reagents and
chemicals were purchased from Sigma Chemical unless otherwise stated.
Cell Culture
Rat pulmonary artery endothelial cells (RPAEC) were initially isolated with microcarrier beads as previously described and were generously provided by Dr. Una Ryan (28). The isolated cells have a cobblestone morphology by light and electron microscopy and have been identified as endothelial cells by the presence of factor VIII antigen, by the expression of angiotensin-converting enzyme activity, and by the uptake of acetylated low-density lipoproteins. The cells were maintained in monolayer culture at 37°C and 5% CO2, using Ryan's red medium (medium 199, 6.7% bovine calf serum, 3.3% fetal calf serum, 10Protocols for Measurement of ET-1
RPAEC were exposed to various treatments for 4 h in serum-free PRF-DMEM. To test the effect of antioxidants or an iron chelator on basal ET-1 release, cells were incubated with DMTU (10 mM), ascorbic acid (1 mM), deferoxamine, (1.8 µM), and urea (10 mM) for 4 h. To study the effect of H2O2 on ET-1 production, cells were exposed to varying concentrations of glucose oxidase (0, 0.1, 1, 2.5, 5, and 10 mU/ml) in the presence of 5.6 mM glucose. We also studied the effect of treatment with catalase (2,000 U/ml), DMTU (10 mM), or urea (10 mM) in the presence of glucose and glucose oxidase (10 mU/ml) for 4 h.ET-1 Assay
After incubation, the supernatants were removed, and ET-1 was measured using a commercially available RIA kit as previously described (15). The lower limit of sensitivity for ET-1 detection was 2 pg. Intra-assay variation was <9%; interassay variation was <15%. After extraction in 1 ml of 0.1 N NaOH, the protein level in each well was measured from an aliquot of the solubilized cells using the BCA protein assay reagents. ET-1 measurements were expressed as picograms ET-1 per milligram total cell protein.Protocol for Measuring Intracellular cGMP
We tested the effect of glucose and glucose oxidase exposure for 15 min and 4 h on intracellular cGMP levels. In the experiments with 15 min of exposure to glucose and glucose oxidase, confluent monolayers of pulmonary endothelial cells were equilibrated with Krebs buffer containing 0.1 mM 3-isobutyl-1-methylxanthine (IBMX) at 37°C for 30 min before adding the glucose oxidase. The Krebs buffer contained (in mM) 145 NaCl, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 5 glucose, 5 KCl, 1 Na2HPO4, 2.5 CaCl2, and 1.8 MgSO4-7H2O at pH 7.3. The cells were then incubated in Krebs buffer with or without 10 mU/ml glucose oxidase for 15 min at 37°C. Additional studies were performed in which the cells were incubated in media without IBMX and were exposed to varying concentrations of glucose oxidase (0, 2.5, 5, and 10 mU/ml) for 4 h. At the end of the experiments, the buffer was removed, and 1 ml of 100% ethanol-HCl solution (50 ml of 100% ethanol and 2 drops of concentrated HCl) was left on the cells overnight at 4°C.cGMP Levels
cGMP levels were measured using a commercially available RIA kit. The cell homogenate was dried in a Speed-Vac (Savant, Farmingdale, NY). The pellet was resuspended in 500 µl of RIA assay buffer, and a 50-µl aliquot was incubated with 50 µl of 125I-labeled cGMP and 50 µl of anti-cGMP antiserum overnight at 4°C. Amerlex-M donkey anti-rabbit serum (200 µl) was then added for 10 min at room temperature. The samples were centrifuged at 3,000 revolutions/min for 10 min at 4°C, and the counts per minute in the pellet were determined. Total protein measurements were performed as outlined above, and the results were expressed as cGMP in femtomoles per milligram cell protein.Measurement of ET-1 and -Actin mRNA by RT-PCR
Amplification of RNA
RT-PCR Amplification of RNA
RT-PCR amplification of RNA was performed as previously described by this laboratory (16). Five micrograms of total RNA from each sample were reverse transcribed by incubation with 250 pmol random hexamers, 400 units Moloney murine leukemia virus RT, 80 units RNasin, 2 mM deoxynucleotide triphosphates, 0.5 mM dithiothreitol, 75 mM KCl, 3 mM MgCl2, and 50 mM Tris · HCl (pH 8.3, final volume 50 µl) for 1 h at 37°C. The RT was inactivated by heating for 10 min at 94°C. The cDNA was stored at 4°C.The cDNA was amplified by PCR. Each sample was measured for ET-1 and
-actin in separate tubes using specific primers. The upstream and
downstream primers for ET-1 were
5'-GCCAAGCAGACAAAGAACTCCGAG-3' and
5'-GCTCTGTAGTCAATGTGCTCGGTT-3', respectively. These give a 247-bp fragment that is complementary to position 371-618 in rat ET-1 cDNA (29). PCR of rat genomic DNA yields a single 1,300-bp product, indicating that these primers span an intron. The upstream and
downstream primers for
-actin were
5'-TGGAGAAGAGCTATGAGCTGCCTG-3' and
5'-GTGCCACCAGACAGCACTGTGTTG-3', respectively, which
produces a single band corresponding to a 201-bp cDNA fragment. PCR of rat genomic DNA with the
-actin primers yields a 289-bp product that
is complementary to position 2499-2788 in the
-actin gene, confirming that this primer set spans an intron. Finally, the ET-1 and
-actin product sequences were verified by Margaret Robinson in Dr.
Ray White's laboratory at the University of Utah, using fluoresceinated primer ends and cycle sequencing.
PCR was performed by incubating 5 µl of sample cDNA with 50 mM KCl,
10 mM Tris · HCl, 1.5 mM
MgCl2, 0.01% gelatin, 200 µM total dNTP, 2 units Taq DNA
polymerase, 2.5% formamide, 0.15 µCi [32P]dCTP, and
100 pmol ET-1 or -actin primers in 50 µl final volume (final pH
8.3 at room temperature). PCR using
-actin primers was carried out
for 25 cycles (15 s at 94°C, 15 s at 65°C, 30 s at 72°C)
after 1 min of early DNA denaturation at 94°C using a Perkin-Elmer
Cetus 9600 GeneAMP PCR System. PCR using ET-1 was carried out for 30 cycles under identical conditions. Different primers were never
combined in the same tube. Twenty microliters of the final PCR reaction
were electrophoresed on a 7% nondenaturing polyacrylamide gel.
Gels were stained with ethidium bromide, and the bands corresponding to
the cDNA product were excised, mixed with scintillation cocktail, and
the counts per minute were determined on a Beckman beta counter.
ET-1 and -actin cDNA obtained from PCR of reverse transcribed RNA
were used to generate standard curves. The cDNA was amplified by PCR,
and the resultant amplified product was divided into small fractions
that were, in turn, reamplified. After removal of primers using Magic
PCR Prep (Promega), the purity of the final product was confirmed by
electrophoresis. At the end of the purification, the amount of standard
cDNA was quantified spectrophotometrically. Standard curves for
-actin or ET-1 were made by simultaneously amplifying sample cDNA
and, in separate tubes, standard cDNA
(10
1 to
10
8 ng/tube). Every PCR
amplification included a standard curve. All PCR consisted of
simultaneous amplification (in separate tubes) of cDNA for ET-1 and
-actin. All results are expressed as femtograms ET-1 cDNA per
picogram
-actin cDNA to control for the amount of RNA initially
reverse transcribed. The accuracy of this semiquantitative PCR
technique has been described previously in detail (8, 12).
51Cr Release
When monolayers of RPAEC were 70-80% confluent in six-well culture plates, they were incubated in 1.5 ml/well Ryan's medium containing 1 µCi/ml 51Cr for 16-18 h. After labeling, the monolayers were washed three times with 1.5 ml/well PRF-DMEM, followed by incubation in 2 ml/well PRF-DMEM in the absence or presence of varying concentrations of glucose oxidase (0, 1, 2.5, 5, and 10 mU/ml). All of the experiments were performed at 37°C in an environment containing 5% CO2 for 4 h unless otherwise indicated. The supernate was then removed and centrifuged, and the cell-free supernate was used. The cells were then dissolved in 2 ml/well of 0.1 N NaOH. The samples were counted for 1 min in a Packard A5530 gamma counter (Packard Instrument, Downers Grove, IL). The percent 51Cr release was calculated as follows: {(SLeucine Incorporation Assay
Protein synthetic rate was estimated by [3H]leucine incorporation into trichloroacetic acid (TCA)-precipitable protein. Confluent cultures of RPAEC in 24-well plates were exposed to PRF-DMEM alone or containing 1, 2.5, 5, or 10 mU/ml glucose oxidase for 4 h at 37°C in 5% CO2. [3H]leucine (1 µCi/ml) in PRF-DMEM was added for 10 min at 37°C, and the cells were rinsed five times with phosphate-buffered saline and were solubilized with 0.1% sodium dodecyl sulfate. Proteins were precipitated in 10% TCA at 4°C for 2 h in the presence of 2 mg bovine serum albumin. The precipitate was centrifuged and rinsed two times in 10% TCA, and the counts per minute were determined in a Beckman LS6000Measurement of H2O2 Produced by Glucose and Glucose Oxidase
The amount of H2O2 produced by glucose and glucose oxidase in PRF-HBSS was determined. Experiments were performed in six-well plates. A 0.2-ml aliquot of the sample was mixed with 0.8 ml of 100 mM potassium phosphate buffer (pH 7.0). The final concentrations of the reagents in the 1 ml reaction mixture were 20 U/ml horseradish peroxidase (type II, 200 purpurogallin U/mg), 1.5 mM 4-aminoantipyrine, and 0.11 M phenol. Absorbance was measured at 510 nm with a Hitachi U-3210 spectrophotometer (Hitachi, Tokyo, Japan). The concentration of H2O2 was calculated using a molar extinction coefficient of 6.58 mMStatistical Analysis
Data were analyzed by the Mann-Whitney test, unpaired Student's t-test, or analysis of variance. Statistical significance was taken as P < 0.05. Values are presented as means ± SE. ![]() |
RESULTS |
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Effect of Antioxidants on Basal ET-1 Release
Treatment of RPAEC with the antioxidant DMTU or the iron chelator deferoxamine doubles ET-1 production (Fig. 1). In contrast, ascorbate does not affect ET-1 release (Fig. 1). Because DMTU is a urea derivative, we used urea as a control, and it did not alter ET-1 production (Fig. 1).
|
Effect of Oxidant Stress on Basal ET-1 Release
Glucose and glucose oxidase selectively generate H2O2 in a dose-dependent fashion. Glucose oxidase (10 mU/ml) and glucose (5.6 mM) produce ~195 µM H2O2 over 4 h. Exposure of cells to glucose oxidase and glucose causes a concentration-dependent decrease in ET-1 production (Fig. 2). The decrease in ET-1 synthesis occurs at doses that do not affect [3H]leucine incorporation into protein or specific 51Cr release (Table 1).
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|
Catalase completely prevents the decrease in ET-1 production caused by glucose oxidase (Fig. 3). Exposing cells to both DMTU and glucose oxidase results in ET-1 levels that are intermediate between the effects of DMTU alone and glucose plus glucose oxidase by themselves [DMTU 209 ± 28% (SE) control, glucose and glucose oxidase 58 ± 8% control, and DMTU + glucose oxidase 121 ± 6% control]. Urea does not prevent the inhibitory effect of glucose oxidase on ET-1 release (Fig. 3).
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Oxidant exposure not only decreases ET-1 release (Fig. 2) but also ET-1 mRNA as assessed by semiquantitative PCR (Fig. 4). We tested the possibility that the H2O2 generated by glucose and glucose oxidase might increase intracellular cGMP levels and thereby reduce ET-1 release. Exposure of RPAEC to glucose and glucose oxidase for 15 min or 4 h, however, does not affect intracellular cGMP levels (Fig. 5).
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DISCUSSION |
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The current study demonstrates that changes in oxidant stress regulate ET-1 generation. Treatment with the antioxidant DMTU or the iron chelator deferoxamine substantially increases baseline endothelial ET-1 release (Fig. 1), implying that basal oxidant stress tonically reduces ET-1 production. In contrast, increasing oxidant stress with glucose oxidase reduces in a dose-dependent manner ET-1 production by rat pulmonary arterial endothelial cells. H2O2 mediates this effect of glucose and glucose oxidase since catalase, which converts H2O2 to water and oxygen, completely abrogates this inhibitory effect on pulmonary endothelial ET-1 release. Previous work indicates that the superoxide anion or hydroxyl radical may reduce the level of immunoreactive ET-1 by inducing structural changes in the molecule (9, 34). We and others, however, have demonstrated that H2O2 by itself does not alter ET-1 immunoreactivity (9, 34). Although H2O2 may cause cytotoxicity, we found no evidence of generalized cell damage or dysfunction (as assessed by [3H]leucine incorporation into protein and 51Cr release) at concentrations of glucose oxidase that substantially reduce ET-1 production. Additionally, exposure to glucose and glucose oxidase causes a concomitant decrease in ET-1 mRNA as assessed by semiquantitative PCR. Our finding that both endogenous and exogenous oxidants reduce ET-1 synthesis without frank cell damage provides strong evidence that short-term oxidative stress can inhibit pulmonary endothelial ET-1 production.
Catalase, which eliminates H2O2, completely prevents the effect of glucose and glucose oxidase (Fig. 3). Unlike catalase, DMTU does not alter H2O2 levels but likely scavenges subsequent radicals, such as the hydroxyl radical. Combining DMTU, glucose, and glucose oxidase results in ET-1 levels that are intermediate between the effects of DMTU alone versus glucose plus glucose oxidase by themselves. The intermediate ET-1 level seen with DMTU and glucose oxidase may reflect their net effect on intracellular oxidant stress. The ability of catalase, but not DMTU, to completely block the glucose oxidase-mediated decrease in ET-1 suggests that H2O2 by itself can lead to a decrease in ET-1 synthesis.
Relatively little data exist on oxidant regulation of ET-1 production.
H2O2
(0.1-20 mM) has been reported to reduce ET-1 release by human
umbilical vein endothelial cells (22). This study, however, did not
assess cytotoxicity. In contrast, another group detected no effect of
exogenous superoxide anion or
H2O2
on ET-1 release from bovine pulmonary artery endothelial cells (24). These investigators found, however, that exogenous hydroxyl radical production augmented ET-1 release, but at concentrations that elicited
cell damage (24). This report did not measure ET-1 mRNA levels. Other
studies suggest that
H2O2
may increase ET-1 mRNA levels in bovine aortic endothelial cells (ET-1
release was not measured; see Ref. 25) and ET-1 release by, and mRNA
levels in, human renal mesangial cells (9). The reasons for these disparate results are unknown but may relate to differences in cell
type, duration, or concentration of oxidant exposure, technique (most
studies have failed to closely assess cytotoxicity), or other factors.
As an example of the potential for cell-specific differences, one
mechanism by which
H2O2
may regulate gene expression is by activating transcription factors,
such as nuclear factor (NF)-B (18, 30). The ability of
H2O2
to activate NF-
B, however, depends on the cell type studied (18,
30). Additionally, the capacity of
H2O2
to induce NF-
B in endothelial cells varies with the vascular bed
studied (1, 2).
H2O2,
for example, activates NF-
B in porcine aortic endothelial cells (1)
but not in human umbilical vein endothelial cells (2).
The observation that oxidants can modulate vasoactive factor production has precedence. Reactive oxygen species, for example, can activate phospholipase A2 and enhance arachidonic acid release and mediator production from pulmonary endothelium (5), stimulate thromboxane A2 production in alveolar macrophages (31), differentially alter the synthesis of prostacyclin and 15-hydroxyeicosatetraenoic acid in coronary artery endothelium (4), increase platelet-activating factor synthesis in pulmonary endothelial cells (14), and augment cytokine-induced nitric oxide synthesis (20). Additionally, superoxide anion can combine with nitric oxide, thereby inactivating nitric oxide.
The mechanisms by which oxidants regulate ET-1 production remain
speculative. Relatively few factors decrease ET-1 synthesis, and,
frequently, they appear to act by enhancing cGMP accumulation (11).
This mechanism does not explain the findings in the current study since
glucose oxidase had no effect on RPAEC cGMP levels. Reactive oxygen
species can affect a multitude of signaling pathways involved in
regulating gene expression, including factors interacting with promoter
elements, such as the antioxidant response element (27),
CC(A/T)6GG sequences (6), and
consensus binding sites for transcription factors like heat shock
factor (3), activator protein (AP)-1 (18), AP-1/E26 virus
transformation-specific protein (26), and NF-B (30).
Which, if any, of these factors contribute to
H2O2
regulation of ET-1 production by RPAEC remains to be determined.
Elevated circulating levels of ET-1 have been observed in patients with the acute respiratory distress syndrome (7, 13, 21), a condition associated with increased H2O2 production by the lung (32). Plasma ET-1 levels in these patients average five- to eightfold higher than in control subjects. Animal models of acute lung injury associated with increased oxidant production, such as ischemia-reperfusion (23, 33), also elevate ET-1 levels. Pulmonary ischemia followed by reperfusion increases circulating ET-1 (23), ET-1 release from the lung (33), and ET-1 mRNA in pulmonary tissue (23). Phosphoramidon, an inhibitor of endothelin-converting enzyme, prevents ET-1 release and markedly reduces the increase in pulmonary insufflation pressure in a model of lung ischemia-reperfusion (33). Additionally, in a rat model of unilateral warm ischemia and reperfusion lung injury, an endothelin subtype A (ETA) receptor blocker reduces the fall in arterial oxygenation, the gain in wet/dry lung weight, and the extent of neutrophil influx that develops 90 min after reperfusion (23). Blockade of ETA receptors also prevents the increase in pulmonary capillary permeability caused by ischemia-reperfusion in rat lungs (10). Thus injury to the pulmonary vascular bed in both animals and humans significantly elevates circulating ET-1 levels, which may contribute to the pathogenesis. The mechanisms underlying the increase in circulating (7, 13, 21, 23) and pulmonary ET-1 (23, 33) are unknown.
Although oxidant exposure appears to increase ET-1 synthesis in the lung, the oxidant species involved or what cells produce ET-1 is unknown. Our results indicate that the level of oxidant stress can regulate ET-1 production by cultured pulmonary endothelial cells. Treatment with an antioxidant or an iron chelator increases basal ET-1 synthesis. Conversely, short-term exposure to exogenous H2O2 decreases ET-1 synthesis. This observation has important implications regarding the mechanisms responsible for the enhanced ET-1 generation in oxidant lung injury. It implies that other oxidant species or additional factors, such as the presence of inflammatory mediators, are necessary to augment pulmonary endothelial ET-1 synthesis or that other lung cells distinct from endothelial cells serve as the source of the increased ET-1 production.
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ACKNOWLEDGEMENTS |
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This research was supported by Department of Veterans Affairs Medical Research Funds (to D. E. Kohan and J. R. Michael) and by National Institutes of Health Grants R29 DK-44440 (to D. E. Kohan) and the University of Utah Specialized Center of Research on Acute Lung Injury Grant IP 50 HL-50153 (to J. R. Michael). B. A. Markewitz was supported by an American Lung Association Research Training Fellowship Award, an Edward P. Stiles Trust Grant from the Louisiana State University Medical Center at Shreveport, and the Board of Regents of the State of Louisiana through the Louisiana Education Quality Support Fund (1996-99)-RD-A-20.
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FOOTNOTES |
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Address for reprint requests: J. R. Michael, Div. of Respiratory, Critical Care and Occupational Pulmonary Medicine, 725 Wintrobe Bldg, 50 North Medical Dr., University of Utah Medical Center, Salt Lake City, UT 84132.
Received 5 November 1996; accepted in final form 20 June 1997.
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