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ABSTRACT |
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INTRODUCTION
METHODS
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Recently, we have shown a substantial increase in the endothelin-1 (ET-1) concentration in bronchoalveolar fluid (BALF) during an experimental eosinophilic airway inflammation. Moreover, we observed a significant inhibition of the inflammatory response after treatment with an endothelin receptor antagonist. This indicates that ET-1 may have proinflammatory properties and play a key role in eosinophilic inflammations, such as bronchial asthma. Accordingly, we hypothesized that the synthesis and release of ET-1 precedes the inflammatory response, and that the bronchial epithelium is the site of ET-1 synthesis in the lungs. An eosinophilic airway inflammation was induced by intratracheal Sephadex instillation in rats, and the animals were evaluated after 15 min, 30 min, 1, 2, 3, 6, 12, and 48 h. The ET-1 mRNA synthesis, assessed by Northern and slot blot analyses, was significantly increased 15 min after Sephadex challenge, peaking at 30 min with a 4.7-fold increase, before any signs of inflammation in the BALF could be observed. The increased synthesis was mainly located to the bronchial epithelium and macrophages at sites of inflammation as determined by in situ hybridization. A significant increase in tissue ET-1 was observed 3 h after provocation, and the recruitment of eosinophils followed a substantial release of ET-1 peptide in BALF peaking at 24 h with a 13-fold increase. Therefore, the rapid ET-1 mRNA synthesis and the considerable increase in the level of ET-1 indicate that this peptide plays an important role in the initiation of an eosinophilic airway inflammation.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Endothelin-1 (ET-1), originally isolated from cultured endothelial cells, is among the most potent bronchoconstrictors yet described (1, 2). In addition, ET-1 may stimulate mucus secretion (3) and edema formation in airways (4). There have also been indications that ET-1 is involved in inflammatory reactions in the airways (5), but the exact role of ET-1 in inflammation is not yet defined. However, a key role for ET-1 in the pathogenesis of bronchial asthma, a predominantly eosinophilic inflammation, has been suggested (8, 10).
We have recently demonstrated a substantial increase in the ET-1 concentration in bronchoalveolar fluid (BALF) during an experimental eosinophilic airway inflammation (9). In rats evaluated between Days 1 and 14 after induced inflammation, we found a 20-fold increase in the ET-1 concentration. Additionally, that was the first study to report an anti-inflammatory effect of endothelin receptor antagonism in airway inflammation (9), indicating that ET-1 could play a pivotal role in the development of an eosinophilic airway inflammation, such as bronchial asthma.
Because ET-1 may exert proinflammatory properties, we hypothesized that the ET-1 synthesis and release precedes the inflammatory response. If ET-1 really plays a pivotal role in the pathogenesis of airway inflammation, the early events leading to airway inflammation, as measured by the influx of inflammatory cells to the airway lumen, should involve ET-1 in the initial step. We also hypothesized that the bronchial epithelium is the main source of ET-1 synthesis because the bronchial epithelium is the first target to be attacked by inhaled agents and has a large surface area. To test these two hypotheses, we have investigated the very early phase of an experimental eosinophilic airway inflammation in rats following intratracheal Sephadex challenge. Rats have an endogenous hypersensitivity to dextrans such as Sephadex, which results in an eosinophilic inflammatory response (11). We evaluated the animals at 15 min, 30 min, 1, 2, 3, 6, 12, 24, and 48 h after instillation of either Sephadex or phosphate-buffered saline (PBS) intratracheally. The BALF was analyzed for cell counts and concentrations of mature ET-1; additionally, ET-1 was measured in homogenized lung tissues. Northern and slot blot analyses were used to assess the ET-1 messenger RNA (mRNA) in the lungs during airway inflammation. To localize the site of ET-1 synthesis, the in situ hybridization technique was employed.
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ABSTRACT
INTRODUCTION
METHODS
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Experimental Procedure
A total of 213 male Wistar rats age 11 wk with an average weight of 310 g were used in the study. The experiments were approved by the Norwegian Ethics Committee for Animal Research, and performed according to National Institutes of Health (NIH) guidelines.
Animals were evaluated at 15 min, 30 min, 1, 2, 3, 6, 12, 24, and
48 h after induced inflammation. An eosinophilic airway inflammation was provoked by intratracheal instillation of Sephadex particles
(G-200 Superfine; Pharmacia & Upjohn, Uppsala, Sweden) dissolved
in PBS (5.0 mg · ml
1) given intratracheally through a cannula in a
volume of 1.0 ml · kg1 body weight as previously described (9). Control animals receiving PBS (n = 3 in each group) were examined at
identical time points. Six animals in each group were used for Northern blot analyses. Additionally, tissue specimens for in situ hybridization were obtained at 30 min, 3 h, and 24 h after induced inflammation
as well as from control animals at identical time points (n = 3 in each
group). During intratracheal challenge with Sephadex or PBS alone
(controls), the animals were anesthetized with a mixture of 30/70%
O2/N2O, and 4% halothane (Fluothane; Zeneca, Macclesfield Cheshire,
UK). Atropine sulfate (Atropin; Nycomed Pharma, Oslo, Norway),
1.0 ml · kg1 body weight, was injected intraperitoneally immediately
before the challenge to reduce saliva production. Later, the animals were killed by a mixture of midazolam (Dormicum; Hoffman-La
Roche, Basel, Switzerland) and fluanison (Hypnorm; Janssen Pharmaceutica BV, Beerse, Belgium).
Bronchoalveolar lavage was performed as previously described (9), by instillation of 3 + 2 + 2 ml PBS in the right stem bronchus, distal to the upper lobe, and the procedure was repeated on the left side. For determination of ET-1 content in lung tissues as well as Northern and slot blot analyses, animals were anesthetized as described, the lungs immediately removed and frozen in liquid nitrogen. The specimens for in situ hybridization were fixed for 8 h in freshly prepared 4% paraformaldehyde, then washed with PBS, and subsequently preserved in PBS with 15% sucrose and 0.01% Na azide until processed.
Cell and ET-1 Analysis in BALF and Lung Tissues
The lavage fluid was collected into prechilled tubes containing EDTA
and kept on ice until centrifuged at 800 g for 10 min at 4° C. The cell
pellet from the BALF was resuspended in PBS, pH 7.4, and the total
number of cells was counted in a Bürker hemocytometer. Cytospin
preparations were obtained using a cytocentrifuge (Cytospin Shandon, Southern Ltd, Runcorn, UK) operated at 100 g for 5 min. The
slides were stained using the May-Grünwald-Giemsa staining procedure and at least 400 nonepithelial cells were counted to determine
the proportion of pulmonary alveolar macrophages, eosinophils, neutrophils, and lymphocytes. The supernatants from the BALF were immediately stored at 
70° C until analyzed. ET-1 was determined using
a radioimmuno-linked Endothelin 1-21 specific [125I] assay system
(RPA 555) from Amersham International (Cardiff, UK). Prior to ET-1
analysis, BALF samples were extracted in duplicate from 2 ml as previously described (9). The ET-1 assay has a limit of detection equivalent to 1.6 pg · ml1. For the samples with ET-1 concentrations below
this limit, the values were set to 1.6 pg · ml1. For determination of tissue ET-1, frozen lung tissue samples were cut into small pieces and
heated at 95° C in 20 volumes of 1 M acetic acid for 10 min. The samples were subsequently chilled in an ice-water bath, homogenized, and
centrifuged for 15 min at 3,200 g at 4° C. The supernatants from tissues were lyophilized, and stored at 20° C until analysis by an ET-1-specific ELISA (R&D Systems, Oxon, UK). Total protein content
was measured with a colorimetric method, using bicinchoninic acid
(BCA) (Pierce b.v., BA oud-Beijerland, Netherlands).
Because there were no time-dependent effects of intratracheal PBS instillation, these data were pooled in one group, herein referred to as controls.
Northern and Slot Blot Analysis of ET-1 mRNA
Messenger RNA was extracted from homogenized lung tissue using oligo-deoxythymidine (dT)-conjugated paramagnetic beads according to the manufacturer's instructions (Dynal A/S, Oslo, Norway). The poly A+ RNA was denatured in a solution containing 60% formamide, 7.2% formaldehyde, 24 mM HEPES, 6 mM sodium acetate, and 1.2 mM EDTA, subsequently size-fractionated on a formaldehyde- agarose gel using 15 µg poly A+ RNA per lane, transferred to a Biotrans nylon membrane (ICN Biomedicals Inc., OH) by capillary blotting, and hybridized with radiolabeled complementary DNA (cDNA) probes for prepro-ET-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). For slot blot analyses, 0.5, 1.0, and 2.0 µg poly A+ RNA was loaded onto the nylon membrane of a Minifold II (Schleicher & Schuell, Dassel, Germany), respectively. The nylon membranes were prehybridized at 42° C for 3 h in a solution containing 5× standard saline citrate (SSC), 5× Denhardt's solution, and 0.1% sodium dodecyl sulfate (SDS) and were then hybridized with 32P-labeled cDNA probes in the same solution at 42° C for 17 h. The filters were finally washed twice in 2× SSC with 0.1% SDS at room temperature for 5 × 5 min before washing twice with 0.1× SSC at 60° C for 15 min and then subjected to autoradiography. The prepro-ET-1 cDNA used as a probe in the present study was generously provided by Dr. Takashi Miyauchi, and is described elsewhere (12). The same membranes were rehybridized with a GAPDH cDNA probe as internal control which allows correction for any variations in loading of the blots with RNA.
Autoradiography of the filters was carried out in a storage phosphorscreen and analyzed by densitometric scanning analysis using ImageQuant Version 3.3 software (Molecular Dynamics Lab., Sunnyvale, CA). To estimate the ET-1 mRNA tissue levels, the ET-1 mRNA to GAPDH ratios were determined in each sample.
In situ Hybridization
Cryostat sections (10 µm thick) from paraformaldehyde-fixed tissues were hybridized with 35S-labeled ET-1 complementary RNA probe for 14 h at 42° C (1 × 106 counts/min per section) as described by Giaid and coworkers (13). Sections were subsequently washed in sodium citrate, and unhybridized probe was removed by incubation in a solution of ribonuclease A (RNAse A). The sections were dried, dipped in autoradiographic emulsion (Amersham International plc, Buckinghamshire, UK), and exposed for 7 to 14 d at 4° C. Consecutive sections were stained with hematoxylin for histological evaluation of in situ hybridization results. Sections from Sephadex-treated airways and controls were hybridized with a probe with a sequence identical to that of the coding ET-1 mRNA (sense probe), or treated with RNAse A solution before hybridization with the ET-1 complementary RNA probe.
Statistical Analysis
All values are expressed as means ± SEM. Statistical analyses were performed using scientific statistical software (SigmaStat version 2.0; Jandel Scientific GmbH, Ekrath, Germany). The groups were compared using Kruskal-Wallis test followed by Dunn's test for multiple comparisons. A p value of less than 0.05 was considered statistically significant.
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Cell Profile in BALF after Induced Inflammation
A slight increase in the total cell count in BALF was observed
6 h after intratracheal Sephadex instillation (Figure 1A). From
this time point, the total cell count rose further, peaking at 24 h
with a threefold increase. At 48 h a decline in total cell count
was noted, compared with 24 h. The number of neutrophils in
BALF increased from 2.3 · 102 · ml1 to 48.5 · 102 · ml1 1 h after intratracheal Sephadex challenge (Figure 1B), representing 0.4% and 8.1% of the total cell count, respectively. The
number of neutrophils continued to increase until 6 h, when
46.2% of the BALF cells were neutrophils, and thereafter decreased to 7.6% at 48 h. The increase in eosinophilic granulocytes was delayed compared with the neutrophils (Figure 1C).
At 6 h the fraction of eosinophils was 3.5% as compared with
control level of 0.1%. At 12 h the fraction had increased to
22.1% and peaked at 33.4% 48 h after Sephadex instillation.
The absolute number of macrophages increased insignificantly during the period studied (Figure 1D).
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Endothelin Synthesis and Release in the Airways
Northern and slot blot analysis revealed the presence of ET-1 mRNA synthesis in homogenized lung and airway tissue in all rats studied. Northern blot analysis showed a single band of ~ 2.3 kb, demonstrating the specificity of the probe. Slot blot analysis revealed a marked upregulation of ET-1 mRNA during the very early phase of inflammation (Figure 2). Fifteen minutes after Sephadex instillation, the expression of ET-1 mRNA was significantly increased compared with control animals. The highest signal was observed 30 min after challenge, when a 4.7-fold increase was measured (Figure 3A). At 1 h the ET-1 mRNA levels were still increased, but declined rapidly, being on average 2-fold higher throughout the period studied until 48 h (NS).
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In situ hybridization analyses of control animals revealed ET-1 mRNA signals in the airway epithelium and smooth muscle of the bronchi and blood vessels, as well as in single alveolar macrophages (Figure 4A). After Sephadex challenge, there was a stronger expression of ET-1 mRNA in macrophages at the inflammatory sites, i.e., peribronchially as well as in close connection to the Sephadex beads (Figure 4B). Macrophages in the alveoli of regions with no histological signs of inflammation were not different from controls. In addition, an increase in signals from the bronchial epithelium (Figure 4C) and smooth muscle was observed, but the difference between Sephadex and control animals was not as obvious as the marked changes related to the macrophages.
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The tissue levels of ET-1 peptide increased significantly at 3 h compared with the values measured in control samples, 15 min and 30 min after Sephadex challenge (Figure 3B). In BALF a small increase in the concentrations of ET-1 was observed during the first 3 h after induced inflammation (Figure 3C). After 6 h a threefold increase occurred, and the highest concentration was measured at 24 h. Comparing the time course of the increase in ET-1 concentrations in BALF with the course of neutrophils and eosinophils, the increase in ET-1 peptide paralleled the increase in neutrophils until 3 h after which the neutrophils rose more rapidly than ET-1 (Figure 5). The increase in the number of eosinophils followed the increase in ET-1 peptide (Figure 5).
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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The present study demonstrates a very early and marked increase in ET-1 mRNA synthesis and ET-1 peptide release during the initial phase of an experimental eosinophilic airway inflammation. The increased synthesis was mainly located to macrophages at sites of inflammation, in addition to the bronchial epithelium and smooth muscle. The marked ET-1 mRNA synthesis and tissue ET-1 peptide preceded the influx of inflammatory cells to the airway lumen. The development of the eosinophilic airway inflammation followed the release of ET-1 after a transient increase in neutrophils.
The ET-1 mRNA synthesis in the lung increased significantly as early as 15 min after Sephadex challenge and peaked
at 30 min with a 4.7-fold increase. To our knowledge, this represents the most rapid increase in gene expression in vivo of
any cytokine or mediator of relevance to asthma so far reported. There are few studies on ET-1 synthesis in airways in
vivo, however a relatively rapid induction of ET-1 mRNA has
been demonstrated by other investigators as well, employing a
model of hypoxic exposure of rat lungs (14). In that work,
there was a tendency toward increased ET-1 mRNA transcript levels after 24 h of hypoxic exposure, but 48 h of hypoxia was needed to demonstrate a twofold increase (14). In vitro, ET-1 synthesis has been reported in pulmonary endothelial cells (15) and in airway epithelial cells (16) upon
exposure to proinflammatory stimuli like lipopolysaccharide
(LPS), tumor necrosis factor alpha (TNF-
), and interleukin-1
(IL-1) (15, 16). In our study, the very rapid ET-1 mRNA synthesis in the lungs occurred before any signs of inflammation
could be observed in BALF. Neither have any histological
changes been noted at this stage during previous work with
this model in our laboratory.
In situ hybridization analyses showed an abundant expression of ET-1 mRNA located to macrophages at inflammatory sites, i.e., peribronchially and surrounding Sephadex beads, as well as surface epithelium and smooth muscle. This indicates that activated macrophages may be partly responsible for increased ET-1 synthesis, although the absolute number of macrophages increased insignificantly during the first 48 h. ET-1 synthesis by macrophages has previously been demonstrated (17). Because we recently reported increased ET-like immunostaining in the bronchial epithelium during Sephadex-induced airway inflammation (9), demonstrating the presence of ET-1 peptide, we anticipated ET-1 mRNA signals located to the epithelium. Increased ET-1 mRNA synthesis in the bronchial epithelium was confirmed, but the most striking differences between Sephadex and control animals were located to macrophages and not to the epithelial lining. Other investigators have found increased ET-1 mRNA expression in the airway epithelium of patients with the inflammatory disease cryptogenic fibrosing alveolitis (13). In bronchial asthma as well, increased ET-1 immunoreactivity has been demonstrated in the epithelial cells (18). Due to the large surface of the bronchial epithelial lining, it is likely that even a minor increase in ET-1 mRNA synthesis would contribute significantly to the increased production of ET-1 in the lungs. This synthesis of ET-1 by both epithelial cells and macrophages is consistent with the finding of a very rapid induction of ET-1 mRNA synthesis, because these cells are among the first to be stimulated by inhaled agents.
The release of ET-1 peptide in lung tissue was significantly increased as early as 3 h after Sephadex provocation. In comparison, the ET-1 release into BALF was somewhat delayed. It is believed that the secretion of ET-1 from the endothelium is mainly abluminal (19, 20). There is only one study addressing ET-1 release from epithelial cells. This recently published study demonstrates that more than 90% of the ET-1 peptide release from cultured airway epithelial cells occurs toward the submucosal side (21). This is in line with the present in vivo study demonstrating a more rapid increase in the ET-1 peptide concentration in lung tissue than in BALF. We believe that the ET-1 peptide response in lung tissue and BALF is mainly due to a de novo synthesis within lung tissue. This is supported both by increased transcription signals, increased immunostaining in inflamed airway epithelium and macrophages (9), and the fact that the plasma levels of ET-1 remain unchanged during airway inflammation (9).
The marked synthesis of ET-1 mRNA and the increase in
tissue ET-1 preceded the influx of inflammatory cells into the
airways. The ET-1 peptide concentration in BALF was twofold increased at 2 h, but the increase did not reach statistical
significance until 6 h, coinciding with the increase in eosinophils. Taking into account the close relationship between the
kinetics of ET-1 peptide and the time-dependent recruitment
of inflammatory cells, particularly eosinophils, chemotactic
properties of ET-1 seem probable. This interpretation is in
agreement with other investigations, demonstrating that ET-1
may promote cell adhesion and migration of leukocytes into
the alveoli (22). In addition, we have recently shown that treatment with an endothelin receptor antagonist inhibited the influx of inflammatory cells to the airways in the rat model employed in the present study (9). Whether ET-1 has chemotactic
properties per se or acts through the release of other cytokines
is not known. In vitro, ET-1 has been shown to stimulate the
release of metabolites of the arachidonic acid cascade (23),
as well as the cytokines TNF-, IL-1
, IL-6, and IL-8 (24),
all substances with chemotactic properties. Thus, both direct
chemotactic properties of ET-1 and an interaction between
ET-1 and other mediators could possibly initiate both the
transient neutrophilic cell influx and the subsequent profound
eosinophilic response, a BALF cell pattern similar to what is
seen in patients with asthma exacerbation (27) and allergen-induced acute asthma (28). Because ET-1 is found to be increased in these conditions (6, 29), a pathogenic role of ET-1
is possible. The mechanism underlying neutrophilia as a prelude to the recruitment of eosinophils is not clear. Recently it
has been reported that a leukotriene B4 antagonist in asthmatics inhibits the early neutrophilic influx, but no physiological
benefit could be measured (30). This questions the functional
role of the neutrophils in the pathophysiology of allergen-induced asthma (30).
In conclusion, the present study demonstrates a very early increase in ET-1 mRNA synthesis during the initial phase of an experimental eosinophilic airway inflammation. This increase preceded the influx of inflammatory cells into the airway lumen and was mainly located to macrophages at sites of inflammation, in addition to the bronchial epithelium. Due to the rapid ET-1 mRNA synthesis and ET-1 peptide release, taken together with our previous findings of an inhibitory effect on cell influx of ET-receptor antagonism, this study is consistent with a hypothesis of ET-1 playing an important role in the initiation of an eosinophilic airway inflammation. Whether ET-1 plays a significant role in the pathogenesis of bronchial asthma is an intriguing, but as yet unresolved question.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Finn Finsnes, M.D., Institute for Experimental Medical Research, Ullevål Hospital, 0407 Oslo, Norway.
(Received in original form July 17, 1997 and in revised form March 16, 1998).
Acknowledgments: The authors thank Annlaug Ødegaard, Thea Sandsbråten Solum, Unni Lie Henriken, Trude Asplin, and Sonja Flagestad for excellent technical assistance.
Supported by the Anders Jahre's Fund for the Promotion of Science, the Professor Carl Semb's Medical Research Fund, Research Forum Ullevål Hospital, and The Norwegian Research Council.
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References |
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REFERENCES
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  R. McWhinnie, D. V. Pechkovsky, D. Zhou, D. Lane, A. J. Halayko, D. A. Knight, and T. R. Bai Endothelin-1 induces hypertrophy and inhibits apoptosis in human airway smooth muscle cells Am J Physiol Lung Cell Mol Physiol, January 1, 2007; 292(1): L278 - L286. [Abstract] [Full Text] [PDF]
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 M. Metz, V. Lammel, B. F. Gibbs, and M. Maurer Inflammatory Murine Skin Responses to UV-B Light Are Partially Dependent on Endothelin-1 and Mast Cells Am. J. Pathol., September 1, 2006; 169(3): 815 - 822. [Abstract] [Full Text] [PDF]
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 J. L. Wright, H. Tai, and A. Churg Vasoactive mediators and pulmonary hypertension after cigarette smoke exposure in the guinea pig J Appl Physiol, February 1, 2006; 100(2): 672 - 678. [Abstract] [Full Text] [PDF]
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P. J. Henry, T. S. Mann, A. C. D'Aprile, G. J. Self, and R. G. Goldie An endothelin receptor antagonist, SB-217242, inhibits airway hyperresponsiveness in allergic mice Am J Physiol Lung Cell Mol Physiol, November 1, 2002; 283(5): L1072 - L1078. [Abstract] [Full Text] [PDF]
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 M. T. Borchers, P. J. Justice, T. Ansay, V. Mancino, M. P. McGarry, J. Crosby, M. I. Simon, N. A. Lee, and J. J. Lee Gq Signaling Is Required for Allergen-Induced Pulmonary Eosinophilia J. Immunol., April 1, 2002; 168(7): 3543 - 3549. [Abstract] [Full Text] [PDF]
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 M. J. Shaw, H. Shennib, N. Bousette, E. H. Ohlstein, and A. Giaid Effect of endothelin receptor antagonist on lung allograft apoptosis and NOSII expression Ann. Thorac. Surg., August 1, 2001; 72(2): 386 - 390. [Abstract] [Full Text] [PDF]
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 L. Chen, H. He, E. F. Mondejar, F. Freden, P. Wiklund, K. Alving, and G. Hedenstierna Endothelin-1 and nitric oxide synthase in short rebound reaction to short exposure to inhaled nitric oxide Am J Physiol Heart Circ Physiol, July 1, 2001; 281(1): H124 - H131. [Abstract] [Full Text] [PDF]
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F. Finsnes, T. Lyberg, G. Christensen, and O. H. Skjonsberg Effect of endothelin antagonism on the production of cytokines in eosinophilic airway inflammation Am J Physiol Lung Cell Mol Physiol, April 1, 2001; 280(4): L659 - L665. [Abstract] [Full Text] [PDF]
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 J. D. Pomonis, S. D. Rogers, C. M. Peters, J. R. Ghilardi, and P. W. Mantyh Expression and Localization of Endothelin Receptors: Implications for the Involvement of Peripheral Glia in Nociception J. Neurosci., February 1, 2001; 21(3): 999 - 1006. [Abstract] [Full Text] [PDF]
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 M. Salmon, Y.-C. Liu, J. C. W. Mak, J. Rousell, T.-J. Huang, T. Hisada, P. L. Nicklin, and K. Fan Chung Contribution of Upregulated Airway Endothelin-1 Expression to Airway Smooth Muscle and Epithelial Cell DNA Synthesis after Repeated Allergen Exposure of Sensitized Brown-Norway Rats Am. J. Respir. Cell Mol. Biol., November 1, 2000; 23(5): 618 - 625. [Abstract] [Full Text]
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