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Montreal Heart Institute and University of Montreal, Montreal
Institut National de la Recherche ScientifiqueeCInstitut Armand Frappier Universitee du Queebec, Pointe-Claire, Quebec, Canada
ABSTRACT
Although local tissue activation of the endothelin (ET) system contributes to the development of pulmonary hypertension, the impact of isolated chronic plasma hyperendothelinemia on the pulmonary circulation is unknown. Methods: Mini-osmotic pumps were implanted in rats to deliver ET-1 during 7 or 28 days. After in vivo hemodynamics, the lungs were isolated to derive pressureeCflow relations. Small pulmonary arteries ( 250 e) were mounted on an isometric myograph to study their reactivity. Results: Plasma ET-1 approximately doubled (p < 0.05) after 7 and 28 days. Lung tissue ET-1 level increased fourfold after 7 days (p < 0.001) but was no longer significantly elevated after 28 days. Right ventricular systolic pressure was unaffected. The pulmonary pressureeCflow relation shifted upward with a steeper slope (p < 0.05) at 7 days, but not after 28 days. Maximum dilatations to both acetylcholine (p < 0.01) and sodium nitroprusside (p < 0.001) were greatly reduced by approximately 50% after 28 days and were normalized by the addition of the nitric oxide synthase inhibitor L-NNA and the antioxidant N-acetyl-L-cysteine, respectively. Conclusion: Chronic hyperendothelinemia reduces the pulmonary vasodilator reserve in response to nitric oxide. Correction by an antioxidant and L-NNA suggests that this relates to increased production of reactive oxygen species, which may have clinical relevance for conditions associated with chronic increase of ET. Further studies are required to determine if, in the long term, this could contribute to the development of pulmonary hypertension.
Key Words: acetylcholine endothelium pulmonary circulation pulmonary hypertension smooth muscle
Circulating endothelin (ET) levels are elevated in animal models of pulmonary hypertension (PH) as well as in human PH of various causes (1). In many studies, the increase in ET levels correlates well with the severity of the underlying disease process (2eC7). Whether this increase in ET levels is merely a marker of the PH that reflects an increased pulmonary ET production, with or without reduced plasma clearance of this peptide, or actually contributes to the development and maintenance of PH is still a subject of debate (8). Chronic elevation of circulating ET levels could modulate vascular reactivity by direct action on ET receptors, and indirectly by modifying receptor expression and/or sensitivity. It could also alter smooth muscle cell properties to vasoactive agents as previously demonstrated in small rat cerebral arteries (9).
Knowledge of the impact of chronically elevated ET levels on the pulmonary circulation is important not only to broaden our understanding of the physiopathology of PH but also to appreciate the potential impact on pulmonary vascular reactivity in other conditions associated with increased ET levels. Cirrhosis of the liver, for example, is associated with increased plasma ET levels (10) caused in part to a reduction in liver clearance of this peptide (11). Whether this increase in ET levels in hepatic outflow could modify pulmonary vascular reactivity and contribute to the rare occurrence of porto-PH is unknown.
The present study was therefore designed to evaluate the impact of isolated acute and chronic hyperendothelinemia, mimicking usual physiopathologic levels, on pulmonary vascular reactivity and right ventricular function. We determined the effect of increased levels of circulating ET-1 in rats for a duration of 7 and 28 days on in vivo hemodynamics, on isolated lung vascular resistive properties, on isolated small pulmonary artery reactivity ( 250 e), and on their expression of ET-1, ETA, and ETB receptors measured by confocal microscopy. Some of the results of these studies have been previously reported in the form of an abstract (12).
METHODS
The animal ethics committee of our institution approved the study, which was performed on male Wistar rats (Charles River, St. Constant, Quebec) weighing 250 to 300 g. Rats were anesthetized with halothane; the left jugular vein was exposed, and a subcutaneous pouch was formed. A polyethylene catheter connected to a mini-osmotic pump (Alzet, Cupertino, CA) was inserted into the jugular vein and securely tied in place. The control animals were subjected to the same procedure except for the insertion of the pumps. The mini-osmotic pump was filled with an aqueous solution of ET-1 to be delivered at a rate of 10 ng/kg/minute for a period of 7 or 28 days. Four experimental groups were thus created: (1) control, 7 days; (2) ET, 7 days; (3) control, 28 days; and (4) ET, 28 days.
Determination of ET-1 Stability and Biological Activity
ET-1 was incubated at 37°C for durations of 7 and 28 days. Stability was evaluated with analytic reverse-phase HPLC using a Phenomenex Jupiter C18 (300 , 5 mm, 250 x 4.6 mm) column (Torrance, CA) connected to a Beckman 128 solvent module and a Beckman 168 PDA detector (Mississauga, ON, Canada), with a flow rate maintained at 1 ml/minute. Peptide elution was performed with a linear gradient from 20 to 60% acetonitrile in aqueous TFA (Sigma-Aldrich, Oakville, ON, Canada) 0.06%, and peaks were resolved at 230 nm. Biological activity was evaluated by cumulative concentrationeCresponse curves of isolated small pulmonary vessels to the incubated ET-1 according to the isometric recording of tension methods described later.
In Vivo Hemodynamic Measurements and PressureeCFlow Relationship in Isolated Lungs
The rats were anesthetized with a mixture of ketamine and xylazine followed by intraperitoneal injection of 2,000 U of heparin. The right jugular vein and right carotid artery were isolated, incised, and cannulated with polyethylene catheters to measure central venous, right ventricular, systemic arterial, and left ventricular pressures. All measurements were recorded and monitored on a Gould TA 400 polygraph (Gould Electronics, Valley View, OH). Finally, 3 ml of blood were sampled for plasma ET-1 measurement.
The trachea was then isolated, cannulated, and connected to a rodent ventilator. The lungs were ventilated with a tidal volume of 1 ml at 60 cycles/minute with 2 cm H2O positive end-expiratory pressure. After a midline sternotomy, the pulmonary artery was rapidly cannulated through an incision in the right ventricle. The lung perfusion was initiated by infusing Krebs solution with heparin (100 U/ml) at 2 ml/minute. The Krebs solution had the following composition (in mmol/L): NaCl, 120; NaHCO3, 25; KCl, 4.7; KH2PO4, 1.18; MgSO4, 1.17; CaCl2, 2.5, and glucose, 5.5. Before each experiment, the Krebs solution was passed through a 0.22-e filter (Sarstedt, Newton, NC) and adjusted to a pH of 7.4. The lungs were then rapidly removed and suspended in a water-jacketed chamber at 37°C to be perfused at a constant flow rate (5 ml/minute) using a Masterflex roller pump (Cole-Palmer Instruments Co., Vernon Hills, IL) with Krebs solution supplemented with 3% albumin. The pulmonary flow rate was constantly monitored with a flow probe (Transonic, Ithaca, NY).
After 10 minutes of equilibration, the relationship between perfusion pressure and flow rate was first obtained during baseline conditions and after treatment with the nonselective nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (L-NNA; 100 eol/L). The flow rate was increased in the range of 5 to 25 ml/minute, and the corresponding perfusion pressure was measured. At the end of each experiment, the right ventricle and the left ventricle plus septum weight were determined to calculate the right ventricle/(left ventricle plus septum) ratio. The wet and dry weights of the pulmonary right lower lobe were determined. The remaining lung tissues were rapidly frozen at eC80°C to measure tissue ET-1 levels.
Isometric Recording of Tension of Isolated Microvessels
Rat pulmonary arteries (270 ± 10 e) were gently dissected and placed in Krebs solution with ethylenediaminetetraacetic acid (0.026 mmol/L) and aerated with 12% O2, 5% CO2, and 83% N2 (pH 7.4). Segments 2 mm in length were mounted on 20-e tungsten wires in microvessel myographs (IMF; University of Vermont, Burlington, VT). Vessels were equilibrated for 30 minutes at their optimal tension and then challenged twice with 40 mmol/L KCl followed by the addition of 100 eol/L of the endothelium-dependent vasodilator acetylcholine (Ach) to test the endothelium integrity. The arteries' reactivity was then evaluated with two different protocols, one for vasoconstriction and one for vasodilatation. DoseeCresponse constricting curves were performed using U-46619 (0.1 nmol/L to 1 eol/L) and ET-1 (1 pmol/L to 3 eol/L). To evaluate vasodilatation, arteries were preconstricted with U-46619 (100 nmol/L) before each doseeCresponse curve with Ach (1 nmol/L to 100 eol/L) and the endothelium-independent vasodilator sodium nitroprusside (SNP; 1 nmol/L to 10 eol/L). For the ET 28-day group, responses to Ach were also assessed in the presence of 100 mM of L-NNA, and responses to SNP were obtained in the presence of 10 mM of the antioxydant N-acetyl-L-cysteine; both solutions were preincubated with the arteries for 30 minutes before the stimulation protocols.
ET-1 Levels
Plasma and lung tissue homogenate samples were passed on Sep-Pak C18 columns (Waters, Milford, MA) before determination of ET-1 levels by ELISA according to the manufacturer's instructions (Biomedica, distributed by Medicorp, Montreal, PQ, Canada). Tissue protein content was determined by a Lowry assay.
Confocal Imaging, Deconvolution, and Fluorescence Quantification of ET-1, ETA, and ETB Receptors
Rat lungs were removed, snap-frozen in liquid nitrogen, and immersed in 2-methyl-butane (Sigma-Aldrich). The lungs were oriented to cross-section the arteries of interest and 14-e cryocuts were performed. Tissues were fixed with fresh 4% paraformadehyde, pH 7.2, and blocked in 2% normal donkey serum (Jackson, West Grove, PA) and 0.5% Triton X-100 (Sigma-Aldrich). Anti-ETA receptor antibody (rabbit; Alomone, Jerusalem, Israel) and anti-ETB receptor antibody (rabbit; Alomone) were incubated respectively with anti- smooth muscle actin antibody (mouse; Sigma-Aldrich). They were diluted in 1% normal donkey serum and 0.1% Triton X-100. For the determination of ET-1 expression, slides were blocked with 2% bovine serum albumin (Sigma-Aldrich) and 0.5% Triton X-100 and then were incubated with antieCET-1 antibody (rabbit; Phoenix Pharmaceuticals, Belmont, CA) diluted in 1% bovine serum albumin and 0.1% Triton X-100. Primary antibody incubation was performed overnight at 4°C. Antirabbit Alexa 555 (donkey; Molecular Probes, Eugene, Oregon) and antimouse Alexa 647 (donkey; Molecular Probes) in their respective antibody diluents were then applied. Slides were mounted using 0.2% 1.4-diazabicyclo(2.2.2)-octane (Sigma-Aldrich) diluted with glycerol (1:5). We used phosphate-buffered saline (NaCl, 137 mM; KCl, 2.7 mM; Na2HPO4, 4.3 mM; KH2PO4, 1.4 mM) for all the washes that followed fixation and primary and secondary antibody incubations.
Slides were analyzed using a Zeiss LSM 510 confocal microscope (North York, ON, Canada). Images were collected with a Plan-Apochromat 63x/1.4 oil differential interference contrast (DIC) objective. HeNe1 (543 nm) and HeNe2 (633 nm) lasers were used for excitation of the antirabbit Alexa 555 and antimouse Alexa 647 antibody, respectively. Internal elastic and external elastic lamina autofluorescence was obtained with the argon laser line (488 nm) and collected between 505 and 530 nm. Z stacks of each tissue were performed, and images were taken at every 0.16 e (top to bottom) to respect the Nyquist criteria in Z-sampling. The Z stacks were then deconvolved using the maximum likelihood estimation algorithm of the Huygens Pro software (version 2.4.1; Scientific Volume Imaging). Transparent projections (in face view) were applied to each Z stack using the projection tool of the LSM 510 software. Images were saved in TIFF file format. To quantify fluorescence intensity of ETA, ETB, and ET-1, we used internal and external elastic lamina autofluorescence to identify the limits of the media and the endothelium. Using the "close free shape curve" tool of the LSM image software, the endothelium or the media was isolated by masking the remaining image. Mean fluorescence intensity was calculated over the nonmasked region by the LSM 510 software. This operation was executed at every five images of each Z stack. The mean fluorescence intensity of all the images in a Z stack was then averaged.
Study Drugs
The thromboxane A2 mimetic U-46619, L-NNA, Ach, SNP, and N-acetyl-L-cysteine were purchased from Sigma Chemicals, and ET-1 was purchased from American Peptide (Sunnyvale, CA). All drugs were dissolved in nanopure water except for U-46619, which was dissolved in 95% ethanol. All solutions were kept at eC20°C at different concentrations of 1 pmol/L to 100 eol/L, except for L-NNA, which was freshly prepared.
Statistical Analysis
Results are expressed as mean ± SEM. Hemodynamics, gravimetric parameters, and ET-1 levels compared by analysis of variance followed by multiple groups comparisons using the Bonferroni correction. Differences in the isolated lungs' pressureeCflow relations were evaluated by repeated-measures analysis of variance followed by multiple groups comparisons with Bonferroni correction. The individual pressureeCflow relationships for each group were fitted by linear regression to determine their slope and intercept. Vasoconstriction of pulmonary vessels was expressed as a percentage of the maximal response (Emax) obtained in the presence of 127 mmol/L KCl; vasodilatations are expressed as the percentage of inhibition of the preconstricting tone induced by 100 nmol/L U46619. The EC50 and the Emax were measured from each individual doseeCresponse curve using a five-parameter logistic function with SigmaPlot curve-fitting software. The pD2 value reported is the negative log of the EC50. For these parameters, differences between the ET 7-day or ET 28-day groups and their respective control groups were evaluated with a two-tailed unpaired Student's t test. Statistical significance was assumed at p < 0.05.
RESULTS
Stability and Biological Activity of ET-1 after 7 and 28 Days
After in vitro incubation for 7 and 28 days, there was no degradation of ET-1 as measured by HPLC (data not shown). The biological activity of ET-1, as assessed from its vasoconstrictive action on isolated small pulmonary vessels, was also preserved (Figure 1). The maximal constriction and pD2 values for nonincubated ET-1 were 90.8 ± 3.4% and 8.06 ± 0.12%. The same parameters for ET-1 incubated for 7 days were 90.8 ± 6.6% and 8.14 ± 0.22%, respectively, and 90.2 ± 8.0% and 7.50 ± 0.29%, respectively, for the ET-1 incubated for 28 days.
Effect of ET-1 Infusion on Hemodynamic and Gravimetric Parameters
Infusion of ET-1 almost doubled plasma levels after both 7 and 28 days (Figure 2A). This finding was accompanied by an increase in lung tissue ET-1 levels after 7 days, and levels remained increased, although not significantly, after 28 days (Figure 2B).
The mean arterial pressure was nonsignificantly higher in the ET 28-day group (105.5 ± 3.1 mm Hg) compared with the control group (98.3 ± 2.8 mm Hg; Table 1). The ET-1eCperfused rats did not develop PH as demonstrated by similar right ventricular systolic pressure values. The only significant hemodynamic change was a higher rate of relaxation of the left ventricle (LV-dP/dT) in both the ET 7- and 28-day groups compared with the control group. At Days 7 and 28, the body weight of the ET-infused rats was significantly lower (p < 0.05) compared with control rats (Table 1). There were no differences in the right ventricle/(left ventricle plus septum) weight and the lung dry/wet weight ratios between the four experimental groups.
Pulmonary PressureeCFlow Relationships
The pressureeCflow relationship was shifted upward (p < 0.05) in the ET 7-day group compared with the control group (Figure 3). Furthermore, the slope of this relationship was higher in the ET 7-day group (0.53 ± 0.02 mm Hg/ml/second) compared with the 7-day control group (0.47 ± 0.01 mm Hg/ml/second; p < 0.05), but there was no difference for the intercept. On the other hand, the 28-day groups did not display any significant difference. The administration of L-NNA did not modify the pressureeCflow relationships in any group (Figure 4).
Pulmonary Vascular Reactivity
The maximal vasodilator response to acetylcholine (Ach) was greatly reduced in the ET 28-day group (23.7 ± 4.0%) compared with 28-day control group (53.6 ± 6.1%, p < 0.01) but without any difference in the pD2 values (Figure 5A; Table 2). ET infusion for 7 days did not modify the maximal relaxation to Ach, but mildly increased the sensitivity of the small pulmonary arteries as evidenced by a higher pD2 value when compared with control animals (7.24 ± 0.27 vs. 6.52 ± 0.28; p < 0.05).
The doseeCresponse curves to SNP were nearly identical for the 7-day groups (Figure 5B; Table 2). The maximal endothelium-independent vasodilator response of small arteries from the ET 28-day group, however, was greatly reduced from 64.8 ± 4.2% in control animals to 35.1 ± 4.0% (p < 0.001).
All four experimental groups demonstrated similar vasoconstriction when challenged with the thromboxane A2 mimetic U-46619 (Figure 6A; Table 3), except for a greater maximal vasoconstriction in the ET 28-day group (77.5 ± 6.3% vs. 60.4 ± 2.0%; p < 0.05). The vasoconstrictor response to ET-1 was also unaffected in the experimental groups (Figure 6B; Table 3), except for a reduced sensitivity in the ET 7-day groups as evidenced by a slightly lower pD2 value (8.00 ± 0.03) compared with the control group (8.38 ± 0.08; p < 0.001).
To further evaluate the mechanisms responsible for the reduced vasodilator response to Ach and SNP in the ET 28-day group, experiments were performed with the addition of L-NNA and N-acetyl-L-cysteine (Figure 7). The addition of L-NNA normalized the response to Ach and increased the maximum vasodilator response from 23.7 ± 4.0% to 54.6 ± 8.8% (p = 0.013). The addition of N-acetyl-L-cysteine also normalized the response to SNP from 35.1 ± 4% to 69.5 ± 13.9% (p = 0.029).
ET-1, ETA, and ETB Receptors in Small Pulmonary Arteries
Examples of composite Z-stacked images obtained with antibodies to ET-1, the ETA, and ETB receptors and smooth muscle actin are shown in Figure 8. Autofluorescence of the internal and external elastic lamina enables easy demarcation of the endothelium from the media. As expected, there was no detectable ETA receptor on the endothelium. ET-1 and the ETB receptor were present on both the endothelium and in the media in similar proportion. Fluorescence intensity revealed an approximate threefold increase in ETB receptor in both the intima and media of small pulmonary arteries in the ET 7-day group (p < 0.01; Figure 8), which returned to control levels after 28 days. There was no significant modification of the ETA receptor and of ET-1.
DISCUSSION
This study was designed to evaluate the impact of chronically elevated plasma ET-1 levels on pulmonary vascular reactivity. We found that increased ET levels for a period of up to 4 weeks did not cause PH or right ventricular hypertrophy (RVH) but was associated with modifications of pulmonary vascular reactivity characterized by a markedly reduced response to NO.
Effects of ET-1 Infusion on Plasma and Tissue Levels
Chronic ET-1 perfusion almost doubled plasma levels after 7 and 28 days. The levels attained are similar to those reported in many animal models of PH as well as in human subjects with PH. This finding was accompanied by an increase in lung tissue ET-1 levels after 7 days, which was less marked and no longer significant after 28 days. The pulmonary circulation is an important site for ET-1 removal from the circulation that is mediated by the endothelial ETB receptor (13). In isolated rat lungs, close to 60% of injected ET is removed within a single transit time (14). It is thus possible that the increased tissue levels were a direct consequence of ET-1 uptake and internalization. This may have been facilitated by the measured increase in endothelium ETB that we found after 7 days of ET-1 infusion. In isolated porcine endothelial cells, binding of exogenous ET-1 to the ETB receptor has also been shown to modulate ET-1 synthesis through a negative feedback loop (15, 16). This second mechanism together with the reduction of endothelium ETB receptor may explain the lowering of lung tissue ET-1 levels after 28 days of ET-1 infusion.
It has also been demonstrated that cultured peripheral lung microvascular smooth muscle cells can synthesize ET-1, and that ET-1 itself can stimulate prepro-ET-1 expression in these cells through stimulation of both the ETA and ETB receptors (17). The increased expression of ET-1 in cultured sheep endothelial cells after ET-1 stimulation is transient, however (17). Our findings of normal total lung tissue ET-1 levels after 28 days, and of unchanged small pulmonary artery ET-1 expression in the media after both 7 and 28 days, suggest a lack of in vivo effect of increased plasma ET-1 levels on vascular smooth muscle cell ET-1 production.
Effects of ET-1 Infusion on Hemodynamics
Except for an improvement in the rate of relaxation of the left ventricle, chronic ET-1 infusion did not significantly modify in vivo hemodynamics in the systemic and pulmonary circulations. ET-1 is a potent inotropic agent and a negative lusitropic agent when used at pharmacologic doses in vitro. ET-1 also has a tonic positive chronotropic effect in normal subjects, which can be unmasked by selective ETA receptor blockade, but opposite negative inotropic effects in subjects with dilated cardiomyopathy (18). It is therefore difficult to determine if the positive lusitropic effects observed in the present study represent a compensatory mechanism or a direct effect of the ET infusion on the heart. Overall, the lack of important effects of isolated higher plasma ET levels on hemodynamics suggests a greater importance of local tissue production of ET-1 in pathologic conditions because ET-1 is principally a paracrine substance. The secondary increase in circulating ET levels may, however, contribute to alterations in vascular reactivity of more distant organs, such as the lung.
Effects of ET Infusion on Pulmonary Vascular Reactivity
The isolated lung pressureeCflow relationship was mildly but significantly shifted with a steeper slope after 7 days of ET infusion, suggesting an increase in pulmonary vascular resistance. This finding was no longer apparent after 28 days of infusion, possibly because of compensatory mechanisms. The more than threefold increase in medial ETB receptor that we found after 7 days of ET infusion, with the subsequent reduction after 28 days, may represent such a mechanism. Among the possible mediators that could be modified by ET infusion, we evaluated the role of basal NO production and found that it did not modulate isolated pulmonary vascular tone in either control or ET-infused animals.
We further explored the impact of chronically elevated plasma ET levels by studying isolated small pulmonary arteries. The most striking finding of these experiments was an important reduction of both endothelium-dependent and -independent vasodilatation to Ach and SNP, respectively, supporting either reduced smooth muscle sensitivity to NO or reduced biovavailability of produced NO. Although increased exposure of pulmonary artery smooth muscle cells to NO can reduce soluble guanylate cyclase mRNA and enzyme activity (19), this mechanism is unlikely in the present study. Furthermore, the lack of effect of NO synthase inhibition on pulmonary pressureeCflow relationships does not support a compensatory increase in basal NO production after ET infusion. Another possible explanation for our findings could stem from demonstrated effects of ET-1 on smooth muscle cell properties. It has been demonstrated in cerebral vessels that ET-1 can desensitize smooth muscle cell responsiveness to NO by a protein kinase CeCindependent pathway (20).
We believe, however, that the most likely explanation for our findings resides in ET-1eCinduced augmentation of reactive oxygen species production in endothelial cells and smooth muscle cells, which would contribute to a reduction in the bioavailability of NO (21eC23). This hypothesis is strongly supported by the normalization of SNP-induced vasodilatation by the addition of the antioxidant N-acetyl-L-cysteine. The apparently paradoxic normalization of the vasodilator response to Ach by the addition of the nonselective NO synthase inhibitor L-NNA may also be explained by increased reactive oxygen species production. Although highly speculative at this point, a possible explanation could reside in an increased superoxide formation through a modification of NO synthase isoforms and/or their substrates (24, 25), whereas maintained vasodilatation could occur by the release of endothelium-derived hyperpolarizing factor (26). The previous demonstrations that chronic ET receptor blockade could improve endothelium-dependent vasodilation to Ach in isolated rat lungs from animals with monocrotaline-induced PH as well as in lungs from rats with venous PH after myocardial infarction also support a deleterious effect of ET on endothelium-dependent relaxation (27, 28).
Implications of These Findings
Chronic elevations of circulating ET levels are found in numerous pathologic conditions, such as congestive heart failure, liver disease, hypertension, and atherosclerosis. Because PH from all causes is associated with elevated ET levels and because some conditions cited previously (e.g., congestive heart failure and liver disease) may be associated with secondary PH, it becomes relevant to determine if elevated ET levels could contribute to altered pulmonary vascular reactivity. The impact of elevated levels on lung circulation may be particularly important because this organ is a primary site for circulating ET-1 clearance (29).
Our major finding of an important reduction in the pulmonary vasodilator reserve in response to NO may therefore have significant pathologic implications in that context.
Conclusions
One month of chronic hyperendothelinemia failed to induce PH or right ventricular hypertrophy. However, this condition causes an important modification of pulmonary vascular reactivity characterized by a reduced response of smooth muscle to NO. These findings suggest that conditions associated with increased circulating ET levels may reduce the pulmonary vasodilatory reserve in relation to an increased production of reactive oxygen species. Further studies are needed to determine if, in the long term, this situation could contribute to the development of PH.
REFERENCES
Michel RP, Langleben D, Dupuis J. The endothelin system in pulmonary hypertension. Can J Physiol Pharmacol 2003;81:542eC554.
Cacoub P, Dorent R, Nataf P, Carayon A, Riquet M, Noe E, Piette JC, Godeau P, Gandjbakhch I. Endothelin-1 in the lungs of patients with pulmonary hypertension. Cardiovasc Res 1997;33:196eC200.
Cody RJ, Haas GJ, Binkley PF, Capers Q, Kelley R. Plasma endothelin correlates with the extent of pulmonary hypertension in patients with chronic congestive heart failure. Circulation 1992;85:504eC509. [Published erratum appears in Circulation 1993;87(3):1064.]
Kumar P, Kazzi NJ, Shankaran S. Plasma immunoreactive endothelin-1 concentrations in infants with persistent pulmonary hypertension of the newborn. Am J Perinatol 1996;13:335eC341.
Langleben D, DeMarchie M, Laporta D, Spanier AH, Schlesinger RD, Stewart DJ. Endothelin-1 in acute lung injury and the adult respiratory distress syndrome. Am Rev Respir Dis 1993;148:1646eC1650.
Nootens M, Kaufmann E, Rector T, Toher C, Judd D, Francis GS, Rich S. Neurohormonal activation in patients with right ventricular failure from pulmonary hypertension: relation to hemodynamic variables and endothelin levels. J Am Coll Cardiol 1995;26:1581eC1585.
Yamakami T, Taguchi O, Gabazza EC, Yoshida M, Kobayashi T, Kobayashi H, Yasui H, Ibata H, Adachi Y. Arterial endothelin-1 level in pulmonary emphysema and interstitial lung disease. Relation with pulmonary hypertension during exercise. Eur Respir J 1997;10:2055eC2060.
Stewart DJ, Levy RD, Cernacek P, Langleben D. Increased plasma endothelin-1 in pulmonary hypertension: marker or mediator of disease Ann Intern Med 1991;114:464eC469.
Thorin E, Cernacek P, Dupuis J. Endothelin-1 regulates tone of isolated small arteries in the rat: effect of hyperendothelinemia. Hypertension 1998;31:1035eC1041.
Tsai YT, Lin HC, Yang MC, Lee FY, Hou MC, Chen LS, Lee SD. Plasma endothelin levels in patients with cirrhosis and their relationships to the severity of cirrhosis and renal function. J Hepatol 1995;23:681eC688.
Tran Duc AT, Schwab AJ, Simard A, Villeneuve L, Dupuis J. Reduction in hepatic endothelin-1 clearance in cirrhosis. Clin Sci (Lond) 2003;105:227eC234.
Migneault A, Leblanc N, Dupuis J. Effects of isolated hyperendothelinemia on the pulmonary circulation . FASEB J 2002;16:A71.
Dupuis J, Goresky CA, Fournier A. Pulmonary clearance of circulating endothelin-1 in dogs in vivo: exclusive role of ETB receptors. J Appl Physiol 1996;81:1510eC1515.
Dupuis J, Jasmin JF, Prie S, Cernacek P. Importance of local production of endothelin-1 and of the ET(B) receptor in the regulation of pulmonary vascular tone. Pulm Pharmacol Ther 2000;13:135eC140.
Boulanger C, Luscher TF. Release of endothelin from the porcine aorta. Inhibition by endothelium-derived nitric oxide. J Clin Invest 1990;85:587eC590.
Sanchez R, MacKenzie A, Farhat N, Nguyen TD, Stewart DJ, Mercier I, Calderone A, Thorin E. Endothelin B receptor-mediated regulation of endothelin-1 content and release in cultured porcine aorta endothelial cell. J Cardiovasc Pharmacol 2002;39:652eC659.
Chen D, Balyakina EV, Lawrence M, Christman BW, Meyrick B. Cyclooxygenase is regulated by ET-1 and MAPKs in peripheral lung microvascular smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2003;284:L614eCL621.
MacCarthy PA, Grocott-Mason R, Prendergast BD, Shah AM. Contrasting inotropic effects of endogenous endothelin in the normal and failing human heart: studies with an intracoronary ET(A) receptor antagonist. Circulation 2000;101:142eC147.
Scott WS, Nakayama DK. Sustained nitric oxide exposure decreases soluble guanylate cyclase mRNA and enzyme activity in pulmonary artery smooth muscle. J Surg Res 1998;79:66eC70.
Gilbert P, Tremblay J, Thorin E. Endothelium-derived endothelin-1 reduces cerebral artery sensitivity to nitric oxide by a protein kinase C-independent pathway. Stroke 2001;32:2351eC2355.
Duerrschmidt N, Wippich N, Goettsch W, Broemme HJ, Morawietz H. Endothelin-1 induces NAD(P)H oxidase in human endothelial cells. Biochem Biophys Res Commun 2000;269:713eC717.
Wedgwood S, Dettman RW, Black SM. ET-1 stimulates pulmonary arterial smooth muscle cell proliferation via induction of reactive oxygen species. Am J Physiol Lung Cell Mol Physiol 2001;281:L1058eCL1067.
Wedgwood S, McMullan DM, Bekker JM, Fineman JR, Black SM. Role for endothelin-1-induced superoxide and peroxynitrite production in rebound pulmonary hypertension associated with inhaled nitric oxide therapy. Circ Res 2001;89:357eC364.
Xia Y, Dawson VL, Dawson TM, Snyder SH, Zweier JL. Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury. Proc Natl Acad Sci U S A 1996;93:6770eC6774.
Xia Y, Tsai AL, Berka V, Zweier JL. Superoxide generation from endothelial nitric-oxide synthase. A Ca2+/calmodulin-dependent and tetrahydrobiopterin regulatory process. J Biol Chem 1998;273:25804eC25808.
Chen G, Suzuki H, Weston AH. Acetylcholine releases endothelium-derived hyperpolarizing factor and EDRF from rat blood vessels. Br J Pharmacol 1988;95:1165eC1174.
Prie S, Stewart DJ, Dupuis J. EndothelinA receptor blockade improves nitric oxide-mediated vasodilation in monocrotaline-induced pulmonary hypertension. Circulation 1998;97:2169eC2174.
Lucas M, Jasmin JF, Dupuis J. Effect of ET(A) receptor antagonist on pulmonary hypertension and vascular reactivity in rats with congestive heart failure. Pulm Pharmacol Ther 2001;14:307eC314.
Dupuis J, Stewart DJ, Cernacek P, Gosselin G. Human pulmonary circulation is an important site for both clearance and production of endothelin-1. Circulation 1996;94:1578eC1584.