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Am J Physiol Heart Circ Physiol 284: H480-H490, 2003. First published October 24, 2002; doi:10.1152/ajpheart.00493.2002
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Vol. 284, Issue 2, H480-H490, February 2003

Alterations in ET-1, not nitric oxide, in 1-week-old lambs with increased pulmonary blood flow

Boaz Ovadia1, Olaf Reinhartz2, Robert Fitzgerald1, Janine M. Bekker1, Michael J. Johengen1, Anthony Azakie2, Stephan Thelitz2, Stephen M. Black3, and Jeffrey R. Fineman1,4

Departments of 1 Pediatrics and 2 Cardiothoracic Surgery, University of California, San Francisco, 94143; 3 Department of Pediatrics, Northwestern University, Chicago, Illinois 60611; and 4 Cardiovascular Research Institute, San Francisco, California 94143


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Altered pulmonary vascular reactivity is a source of morbidity and mortality for children with congenital heart disease and increased pulmonary blood flow. Nitric oxide (NO) and endothelin (ET)-1 are important mediators of pulmonary vascular reactivity. We hypothesize that early alterations in endothelial function contribute to the altered vascular reactivity associated with congenital heart disease. The objective of this study was to characterize endothelial function in our lamb model of increased pulmonary blood flow at 1 wk of life. Eleven fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt) and were studied 7 days after delivery. The pulmonary vasodilator response to both intravenous ACh (endothelium dependent) and inhaled NO (endothelium independent) was similar in shunted and control lambs. In addition, tissue NOx, NO synthase (NOS) activity, and endothelial NOS protein levels were similar. Conversely, the vasodilator response to both ET-1 and 4Ala-ET-1 (an ETB receptor agonist) were attenuated in shunted lambs, and tissue ET-1 concentrations were increased (P < 0.05). Associated with these changes were an increase in ET-converting enzyme-1 protein and a decrease in ETB receptor protein levels (P < 0.05). These data demonstrate that increased pulmonary blood flow induces alterations in ET-1 signaling before NO signaling and suggest an early role for ET-1 in the altered vascular reactivity associated with increased pulmonary blood flow.

endothelin; pulmonary hypertension; pulmonary circulation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE DEVELOPMENT of pulmonary hypertension and its associated increased vascular reactivity commonly accompanies congenital heart disease with increased pulmonary blood flow (8, 21, 44). Recent evidence suggests that normal pulmonary vascular reactivity and vascular smooth muscle cell proliferation are regulated by a complex interaction of vasoactive substances, such as nitric oxide (NO) and endothelin (ET)-1, that are produced locally by the vascular endothelium (7, 13, 22, 46). Endothelial injury secondary to increased pulmonary blood flow and/or pressure disrupts these regulatory mechanisms and is a potential factor in the development of pulmonary hypertension.

NO is an endothelium-derived relaxing factor synthesized from L-arginine after activation of NO synthase (NOS) (9, 30, 32, 36). After NO is synthesized, it activates soluble guanylate cyclase and induces vasodilation via a cGMP-dependent mechanism (23, 25, 31). Endothelium-derived NO (and the resulting cGMP) is an important mediator of resting pulmonary vascular tone, a modulator of pulmonary vasoconstriction, and an inhibitor of platelet aggregation and smooth muscle mitogenesis (7, 13, 16). ET-1 is a 21-amino acid polypeptide produced by vascular endothelial cells from a 203-amino acid peptide precursor (preproET-1), which is then cleaved to form proendothelin-1 (Big ET-1). Big ET-1 is then cleaved by the metalloprotease ET-converting enzyme-1 (ECE-1) into its functional form (26). The complex pulmonary vasoactive effects of ET-1, which may include pulmonary vasoconstriction and/or pulmonary vasodilation, are mediated by at least two different receptors, ETA and ETB. ETA receptors are located on vascular smooth muscle cells and mediate vasoconstriction. ETB receptors are primarily located on vascular endothelial cells and mediate vasodilation (1, 38). A second subpopulation of ETB receptors are located on smooth muscle cells and mediate vasoconstriction (39). In addition, the ETB receptor is involved in the clearance of circulating ET-1 within the lung (15).

Several studies demonstrate aberrations in the NO-cGMP and ET-1 cascades in humans with pulmonary hypertension. For example, children and adults with increased pulmonary blood flow secondary to congenital heart disease have selective impairment of endothelium-dependent pulmonary vasodilation and increased plasma concentrations of ET-1 (10, 48). In addition, adults with advanced pulmonary hypertension have decreased endothelial NOS (eNOS) gene expression and increased preproET-1 expression within pulmonary vascular endothelial cells (12, 17, 18). However, because most patients who undergo histological and physiological evaluation have more advanced pulmonary hypertension, it has been difficult to investigate early alterations in endothelial dysfunction and their potential role in the development of pulmonary hypertension secondary to increased pulmonary blood flow.

To better understand the role of endothelial dysfunction in the early pathophysiology of pulmonary hypertension, we established a unique animal model of increased pulmonary blood flow and pulmonary hypertension utilizing in utero placement of an aortopulmonary vascular graft in the fetal lamb (33). We showed previously that these shunted lambs have physiological and molecular alterations in both the NO-cGMP and ET-1 cascades as early as 4 wk of age. For example, at 4 wk of age shunted lambs display a selective impairment of endothelium-dependent pulmonary vasodilation, which persists at 8 wk of age (4, 34, 40). This is associated with an early upregulation of basal NO activity, which decreases by 8 wk of age (4, 6). In addition, shunted lambs have increased ET-1 levels at both 4 and 8 wk of age, loss of ETB receptor-mediated pulmonary vasodilation at 4 wk of age, and the emergence of ETB receptor-mediated vasoconstriction at 8 wk of age (3, 5, 45).

We hypothesize that early alterations in endothelial function contribute to the altered vascular reactivity associated with pulmonary hypertension induced by increased pulmonary blood flow. Therefore, the purpose of the present study was to better define the timing and sequence of endothelial dysfunction associated with increased pulmonary blood flow. To this end, we characterized potential alterations in the NO and ET-1 cascades induced by increased pulmonary blood flow at 1 wk of life. To characterize potential early alterations in the NO cascade, the response to endothelium-dependent and -independent vasodilators were studied in 1-wk-old shunted and age-matched control lambs. In addition, total NOS activity, lung tissue NOx concentrations, and eNOS protein levels were studied and compared. To determine potential early alterations in the ET-1 cascade, the responses to ET-1 and 4Ala-ET-1 (an ETB receptor agonist) were studied in 1-wk-old shunted and age-matched control lambs. In addition, tissue ET-1 concentrations and preproET-1, ECE-1, ETA receptor, and ETB receptor protein levels were compared.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Surgical Preparations and Care

Ewes. Eleven pregnant mixed-breed Western ewes (135-140 days gestation; term = 145 days) were operated on under sterile conditions as previously described (33). Through a left lateral fetal thoracotomy, an 8.0-mm Gore-Tex vascular graft (~2-mm length; W. L. Gore, Milpitas, CA) was anastomosed between the ascending aorta and main pulmonary artery of the fetus with 7.0 proline (Ethicon, Somerville, NJ) by a continuous suture technique as previously described (33). After recovery from anesthesia, the ewe was returned to the cage with free access to food and water. Antibiotics (1 g cefazolin and 100 mg gentamicin sulfate) were administered to the ewe during surgery and daily thereafter until 2 days after spontaneous delivery of the lamb.

Lambs. After spontaneous delivery, antibiotics (106 U penicillin G potassium and 25 mg gentamicin sulfate im) were administered to the 11 shunted lambs and 13 twin or age-matched controls for the first 2 days of life. The lambs were weighed daily, and the respiratory rate and heart rates were obtained. Furosemide (1 mg/kg im) was administered daily, and elemental iron (50 mg im) was given on the first day of life to all lambs. At 1 wk of age, the lambs were anesthetized with ketamine hydrochloride (15 mg/kg im). Under additional local anesthesia with 1% lidocaine hydrochloride, polyvinyl catheters were placed in an artery and a vein of one hind leg. These catheters were advanced to the descending aorta and the inferior vena cava, respectively. The lambs were then anesthetized with ketamine hydrochloride (~0.3 mg · kg-1 · min-1), diazepam (0.002 mg · kg-1 · min-1), and fentanyl citrate (1.0 µg · kg-1 · h-1), intubated with a 5.0- to 6.0-mm-outer diameter endotracheal tube, and mechanically ventilated with a Healthdyne (Marietta, GA) pediatric time-cycled, pressure-limited ventilator. An intravenous infusion of lactated Ringer solution and 5% dextrose (7 5 ml/h) was begun and continued throughout the study period. Succinylcholine chloride (2 mg · kg-1 · dose-1) was given intermittently for muscle relaxation. Heart rate and systemic blood pressure were monitored continuously to ensure adequate anesthesia. Ventilation with 21% oxygen was adjusted to maintain a PaCO2 between 35 and 45 Torr. A midsternotomy incision was performed, and the pericardium was incised. Two single-lumen polyurethane catheters were inserted into the left and right atrium, respectively. A double-lumen polyurethane catheter was placed in the main pulmonary artery distal to the vascular graft. An ultrasonic flow probe (Transonics Systems, Ithaca, NY) was placed around the left pulmonary artery to measure left pulmonary blood flow. After a 30-min recovery from surgery, blood was obtained from the left and right atria, distal pulmonary artery, right ventricle, and descending aorta for hemoglobin and oxygen saturation determinations. The midsternotomy incision was then temporarily closed with towel clamps, and the baseline hemodynamic variables were obtained.

Thirteen lambs (6 shunted and 7 age-matched controls) then underwent a hemodynamic study as described in Experimental Protocol. After the last protocol, the lambs were killed by an intravenous injection of pentobarbital sodium (Euthanasia CII; Central City Medical, Union City, CA). Eleven of the lambs (5 shunted and 6 controls) were killed without undergoing any hemodynamic study. These lungs were removed and prepared for tissue NOx, NOS activity, ET-1 concentrations, and Western blot analysis.

All protocols and procedures were approved by the Committee on Animal Research of the University of California, San Francisco. All animals were euthanized with appropriate methods as described in the NIH Guide for the Care and Use of Laboratory Animals.

Experimental Protocol

The responses to ET-1 and 4Ala-ET-1 were determined in control lambs at rest and in shunted lambs at rest with the vascular graft open. However, the responses to vasodilating agents may be dependent on the resting tone of the vascular bed studied (37). Therefore, to ensure that response differences were not tone dependent, the vasodilator responses in control lambs were also studied during an intravenous infusion of U-46619 (a thromboxane A2 mimic) and shunted lambs were also studied after the vascular graft was closed, when pulmonary blood flow is similar to that in controls.

Control lambs. After a 45-min recovery period from surgery, baseline measurements of the hemodynamic variables (pulmonary and systemic arterial pressure, heart rate, left pulmonary blood flow, left and right atrial pressures) and systemic arterial blood gases and pH were measured. ACh (1.0 µg/kg), inhaled NO (40 ppm), ET-1 (250 µg/kg), or 4Ala-ET-1 (1,275 ng/kg) was then administered in random order. ACh, ET-1, and 4Ala-ET-1 were injected into the pulmonary artery over 1 min; inhaled NO was delivered through the ventilator for 15 min. The hemodynamic variables were measured continuously, and systemic arterial blood gases and pH were obtained when a new steady state was achieved. At least 20 min were allowed for the hemodynamic variables to return to preinjection values. All measurements were then repeated, and the other agent was given. After recovery from the last agent, infusion of U-46619 (a thromboxane A2 mimic) was then begun into the inferior vena cava. After 15 min of steady-state pulmonary hypertension, baseline measurements were again obtained, the vasoactive stimuli were administered, and the hemodynamic variables were again measured.

Shunted lambs. After a 45-min recovery period from surgery, baseline measurements of the hemodynamic variables and systemic arterial blood gases and pH were made. ACh, ET-1, 4Ala-ET-1, and inhaled NO were then administered as described in Control lambs. The vascular graft was then closed. After a 60-min recovery, the responses to the vasoactive stimuli were repeated.

Measurements

Pulmonary and systemic arterial and right and left atrial pressures were measured with Sorenson neonatal transducers (Abbott Critical Care Systems, Chicago, IL). Mean pressures were obtained by electrical integration. Heart rate was measured by a cardiotachometer triggered from the phasic systemic arterial pressure pulse wave. Left pulmonary blood flow was measured on an ultrasonic flowmeter (Transonic Systems). All hemodynamic variables were recorded continuously on a Gould (Cleveland, OH) multichannel electrostatic recorder. Systemic arterial blood gases and pH were measured on a Radiometer (Copenhagen, Denmark) ABL5 pH-blood gas analyzer. Hemoglobin concentration and oxygen saturation were measured by a hemoximeter (model 270; Ciba-Corning). The ratio of pulmonary to systemic blood flow (Qp/Qs) was calculated with the Fick equation. Pulmonary vascular resistance was calculated with standard formulas.

Drug Preparation

ACh chloride (Iolab, Claremont, CA) was diluted in sterile 0.9% saline. Inhaled NO (40 ppm) was delivered in nitrogen into the inspiratory limb of the ventilator (Inovent; Ohmeda, Liberty, NJ). The inspired concentrations of NO and NO2 were continuously quantified by electrochemical methodology (Inovent, Ohmeda). ET-1 (0.5 mg, mol wt 2491.1; Peptides International, Louisville, KY) was resuspended in 10 ml of sterile water and stored at -20°C. 4Ala-ET-1 (Ala1,3,11,15ET-1, mol wt 2367.7; Peptides International) was resuspended in 2% DMSO in sterile water. Immediately before administration, each dose of ET-1 and 4Ala-ET-1 was measured and diluted to 1 ml with 0.9% saline. 9,11-Dideoxy-11alpha ,9alpha -epoxymethanoprostaglandin F2alpha (U-46619; Sigma, St. Louis, MO) dissolved in 95% ethanol was stored at -20°C. Immediately before the study, 100 µg was dissolved in 20 ml of 0.9% saline. All solutions were prepared on the day of the study.

NOx Determinations

Lung tissue was homogenized in 6% TCA at 4°C to give a 10% (wt/vol) homogenate and centrifuged at 3,000 rpm for 15 min at 4°C. The supernate was recovered and used immediately for analysis. In solution, NO reacts with molecular oxygen to form nitrite and with oxyhemoglobin and superoxide anion to form nitrate. The nitrite and nitrate were reduced with vanadium(III) and hydrochloric acid at 90°C. NO was then purged from solution, resulting in a peak of NO. Therefore, this value represents a combination of total NO, nitrite, and nitrate (NOx). This peak was then detected by chemiluminescence (NOA 280; Sievers Instruments, Boulder, CO). The detection limit is 1 nM/ml of nitrate (49).

Assay for NOS activity

This was performed with the conversion of 3H-labeled arginine to [3H]citrulline as a measure of NOS activity essentially as described by Bush et al. (9). Briefly, peripheral lung tissues and isolated fifth-generation pulmonary arteries were homogenized in NOS assay buffer (50 mM Tris · HCl, pH 7.5, containing 0.1 mM EDTA and 0.1 mM EGTA) with a protease inhibitor cocktail. Enzyme reactions were carried out at 37°C in the presence of total lung protein extracts (500 µg), 1 mM NADPH, 14 µM tetrahydrobiopterin, 100 µM FAD, 1 mM MgCl2, 5 µM unlabeled L-arginine, 15 nM [3H]arginine, 25 U calmodulin, and 5 mM calcium to produce conditions that drive the reaction at maximal velocity. Duplicate assays were run in the presence of the NOS inhibitor (L-NAME). Assays were incubated for 60 min at 37°C such that no more than 20% of the [3H]arginine was metabolized, to ensure that the substrate was not limiting. The reactions were stopped by the addition of iced stop buffer (in mM: 20 sodium acetate, pH 5, 1 L-citrulline, 2 EDTA, and 0.2 EGTA) and then applied to columns containing 1 ml of Dowex AG50W-X8 resin, Na+ form, preequilibrated with 1 N NaOH. [3H]citrulline was then quantitated by scintillation counting.

ET-1 Determinations

Lung tissues were homogenized in 1 M acetic acid containing 10 µg/ml pepstatin (Peptide International) and immediately boiled for 10 min. The homogenates were centrifuged at 25,000 g for 30 min at 4°C, and the supernates were stored at -30°C before being assayed for immunoreactive (ir) ET-1. ET-1 standard, 125I-labeled ET-1, anti-ET antibody, and secondary antibody were purchased from Peninsula Laboratories (Belmont, CA). Cross-reactivity for measured human and bovine ET-1 antiserum is 100% for human ET-1, 7% for human ET-2 and ET-3, and 0% for bovine ET-2 and ET-3. Interassay and intra-assay variabilities were 10 and 4%, respectively. Each sample was assayed in duplicate. This assay was modified from a previously published method (3).

Tissue Preparation

The heart and lungs were removed en bloc. Isolated fifth-generation pulmonary arteries and peripheral lung were dissected with care to preserve the integrity of the vascular endothelium. Two- to three-gram sections from each lobe of the lung were removed. These tissues were snap-frozen in liquid nitrogen and stored at -70°C until used.

For protein isolation, the snap-frozen lung tissue was allowed to thaw on ice and then homogenized with a Tissuemizer with Triton lysis buffer (20 mM Tris · HCl, pH 7.6, 0.5% Triton X-100, and 20% glycerol) supplemented with protease inhibitors. The supernatant was removed for protein determination and Western blot analysis.

Preparation of Protein Extracts and Western Blot Analysis

Lung protein extracts were prepared as described in Tissue Preparation. Supernatants were quantitated for protein concentration with the Bradford reagent (Bio-Rad, Richmond, CA) and then used for Western blot analysis. Western blot analysis was performed as previously described (3, 5, 6). Briefly, protein extracts (25 µg) were separated on 7.5% denaturing polyacrylamide gels for eNOS and ECE-1alpha , 10% denaturing polyacrylamide gels for ETA and ETB receptors, and 4-20% denaturing polyacrylamide gradient gels for preproET-1. All gels were electrophoretically transferred to Hybond polyvinylidene difluoride membranes (Amersham, Arlington Heights, IL). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween. After blocking was completed, the membranes were incubated at room temperature with the appropriate dilution of the antiserum of interest (1:2,500 for eNOS, 1:1,000 for ECE-1alpha , 1:1,000 for ETA and ETB, or 1:500 for preproET-1), washed with TBS containing 0.1% Tween, and then incubated with a either a goat anti-mouse IgG-horseradish peroxidase conjugate (for eNOS and preproET-1) or a goat anti-rabbit IgG-horseradish peroxidase conjugate (for ECE-1alpha and ETA and ETB receptors). After washing was completed, chemiluminescence was used to detect the protein bands.

The eNOS antiserum was obtained from Transduction Laboratories (Lexington, KY). The ETA receptor antiserum was generated as previously described (3). The ETB receptor antiserum was obtained from Maine Biotechnology Services (Portland, ME). The preproET-1 antibody was obtained from Affinity Bioreagents (Golden, CO). The specificity of the preproET-1 antibody was verified with a preincubation step with purified ET-1 (50 ng ET-1/15 µl of antiserum) protein. The purified ET-1 was purchased from Sigma. ECE-1alpha antiserum was generated as previously described (27). The methodology and exposure times used were those that we demonstrated previously (3, 4, 6) to be within the linear range of the autoradiographic film and able to detect changes in lung protein expression.

Statistical Analysis

Means ± SD were calculated for the baseline hemodynamic variables, systemic arterial blood gases and pH, tissue NOx, ET-1 concentrations, and NOS activity. The general hemodynamic variables, systemic arterial blood gases and pH, tissue NOx, ET-1 concentrations, and tissue NOS activity were compared between study groups by the unpaired t-test. The effects of each vasoactive agent were compared with their previous steady-state condition by the paired t-test with the Bonferroni correction when necessary. The percent change in pulmonary arterial pressure and left pulmonary vascular resistance induced by these agents was compared between study groups by the unpaired t-test or ANOVA for repeated measures with multiple comparison testing.

Quantitation of autoradiographic results was performed by scanning (Hewlett-Packard SCA Jet IICX; Hewlett-Packard, Palo Alto, CA) the bands of interest into an imageediting software program (Adobe Photoshop; Adobe Systems, Mountain View, CA). Band intensities from Western blot analysis were analyzed densitometrically. For Western blot analysis, to ensure equal protein loading, duplicate polyacrylamide gels were run. One was stained with Coomassie blue. Means ± SD were calculated for the relative protein. Results from control lungs were assigned the value of 1 (relative protein). Comparisons between control and shunted lambs were made by the unpaired t-test. A P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

All ewes spontaneously delivered viable fetuses 5-10 days after surgery. At 1 wk of age, all shunted lambs had an audible continuous murmur and an increase in oxygen saturation between the right ventricle and distal pulmonary artery. Qp/Qs was 2.3 ± 0.6. Mean pulmonary arterial pressure was increased (23.0 ± 4.1 vs. 18.4 ± 4.0 mmHg; P < 0.05) to 53% of systemic values. This was associated with an increase in left pulmonary blood flow and left atrial pressure and a decrease in mean systemic arterial pressure (P < 0.05). The calculated left pulmonary vascular resistance was decreased (P < 0.05). Heart rate, right atrial pressure, systemic arterial blood gases, and pH were not significantly different between the two groups (Table 1).

                              
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  		Table 1.   General hemodynamics

Vasoactive Responses

Control lambs. At rest, intrapulmonary injection of ACh decreased mean pulmonary arterial pressure (from 21.0 ± 3.3 to 19.3 ± 2.3 mmHg), left pulmonary vascular resistance (from 0.243 ± 0.09 to 0.199 ± 0.07 mmHg · ml-1 · kg · min), and mean systemic arterial pressure (from 51.0 ± 14.0 to 29.0 ± 3.9 mmHg) (P < 0.05). Left pulmonary blood flow, heart rate, and left and right atrial pressures were unchanged. Inhaled NO decreased mean pulmonary arterial pressure (from 20.8 ± 1.6 to 17.2 ± 2.1 mmHg) and left pulmonary vascular resistance (from 0.228 ± 0.04 to 0.175 ± 0.04 mmHg · ml-1 · kg · min) (P < 0.05). Left pulmonary blood flow, mean systemic arterial pressure, heart rate, and left and right atrial pressure were unchanged. Similarly, during steady-state pulmonary hypertension induced by the infusion of U-46619, both ACh and inhaled NO decreased mean pulmonary arterial pressure (ACh, -13.5%; NO, -30.7%; P < 0.05) and left pulmonary vascular resistance (ACh, -26.8%; NO, -38.3%; P < 0.05).

At rest, the intrapulmonary injection of ET-1 decreased mean pulmonary arterial pressure (from 19.8 ± 2.2 to 17.9 ± 2.2 mmHg) and left pulmonary vascular resistance (from 0.196 ± 0.05 to 0.175 ± 0.04 mmHg · ml-1 · kg · min) (P < 0.05). Mean systemic arterial pressure increased (from 45.6 ± 8.4 to 48.70 ± 10.8 mmHg; P < 0.05). Left pulmonary blood flow, heart rate, and left and right atrial pressures were unchanged. 4Ala-ET-1 decreased mean pulmonary arterial pressure (from 19.7 ± 1.5 to 17.7 ± 1.3 mmHg) and left pulmonary vascular resistance (from 0.193 ± 0.04 to 0.175 ± 0.04 mmHg · ml-1 · kg · min) (P < 0.05). Mean systemic arterial pressure, left pulmonary blood flow, heart rate, and left and right atrial pressures were unchanged. Similarly, during steady-state pulmonary hypertension induced by the infusion of U-46619, both ET-1 and 4Ala-ET-1 decreased mean pulmonary arterial pressure (ET-1, -15.3%; 4Ala-ET-1 -16.7%; P < 0.05) and left pulmonary vascular resistance (ET-1, -21.6%; 4Ala-ET-1, -12.2%; P < 0.05).

Shunted lambs. In shunted lambs, the intrapulmonary injection of ACh decreased mean pulmonary arterial pressure (from 23.0 ± 5.1 to 19.5 ± 4.6 mmHg), left pulmonary vascular resistance (from 0.113 ± 0.03 to 0.092 ± 0.02 mmHg · ml-1 · kg · min), mean systemic arterial pressure (from 46.3 ± 15.9 to 31.7 ± 7.8 mmHg), and left atrial pressure (from 8.3 ± 2.3 to 7.6 ± 1.9 mmHg) (P < 0.05). Left pulmonary blood flow, heart rate, and right atrial pressure were unchanged. Inhaled NO decreased mean pulmonary arterial pressure (from 23.0 ± 7.5 to 17.6 ± 5.1 mmHg), left pulmonary vascular resistance (from 0.119 ± 0.04 to 0.081 ± 0.03 mmHg · ml-1 · kg · min), and mean systemic arterial pressure (from 46.3 ± 16.6 to 43.27 ± 17.0 mmHg) (P < 0.05). Left pulmonary blood flow, heart rate, and left and right atrial pressures were unchanged. Similarly, after shunt closure, both ACh and inhaled NO decreased mean pulmonary arterial pressure (ACh, -9.3%; NO, -20.2%) and left pulmonary vascular resistance (ACh, -11.3%; NO, -38.2%) (P < 0.05).

The intrapulmonary injection of ET-1 did not change mean pulmonary arterial pressure, left pulmonary vascular resistance, left pulmonary blood flow, heart rate, or right atrial pressure. Mean systemic arterial pressure (from 48.3 ± 17.2 to 52.5 ± 20.3 mmHg) and left atrial pressure (from 8.8 ± 2.0 to 10.0 ± 2.8 mmHg) increased (P < 0.05). 4Ala-ET-1 did not change mean pulmonary and systemic arterial pressure, left pulmonary vascular resistance, left pulmonary blood flow, heart rate, or left atrial pressure. Right atrial pressure increased (from 5.2 ± 2.3 to 6.0 ± 2.5 mmHg; P < 0.05). Similarly, after shunt closure, neither ET-1 nor 4Ala-ET-1 decreased mean pulmonary arterial pressure (ET-1, +3.4%; 4Ala-ET-1, +6.1%) or left pulmonary vascular resistance (ET-1, +4.9%; 4Ala-ET-1, +2.7%).

The responses to both ACh (an endothelium-dependent vasodilator) and inhaled NO (an endothelium-independent vasodilator) were similar in both groups (Fig. 1). However, compared with control lambs, the pulmonary vasodilating response to both ET-1 and 4Ala-ET-1 was significantly attenuated in 1-wk-old shunted lambs (Fig. 2).


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  		Fig. 1.   In shunted lambs, the decrease in mean pulmonary arterial pressure (MPAP; A) and left pulmonary vascular resistance (LPVR; B) in response to both ACh (endothelium-dependent vasodilator) and inhaled nitric oxide (NO; endothelium-independent vasodilator) are similar to control lambs. Values are means ± SD of 7 control lambs at rest and 6 shunted lambs with the vascular graft open. *P < 0.05 vs. baseline.



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  		Fig. 2.   In control lambs, endothelin (ET)-1 and 4Ala-ET-1 (ETB receptor agonist) decrease both MPAP (A) and LPVR (B); in shunted lambs, ET-1 and 4Ala-ET-1 do not. Values are means ± SD of 7 control lambs at rest and 6 shunted lambs with the vascular graft open. *P < 0.05 vs. baseline.

NO Cascade

Total peripheral lung (1.16 ± 0.5 vs. 0.883 ± 0.7 pmol · min-1 · mg-1) and isolated pulmonary artery (1.87 ± 0.9 vs. 2.36 ± 0.3 pmol · min-1 · mg-1) NOS activity was similar in shunt and twin control lambs (Fig. 3). In addition, peripheral lung tissue NOx concentrations (7.4 ± 1.8 vs. 8.8 ± 1.0 µmol/ml; n = 4), an indirect determination of NO production, were similar in both groups.


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  		Fig. 3.   Top: NO synthase (NOS) activity (pmol · min-1 · mg protein-1) was measured by the conversion of [3H]arginine to [3H]citrulline in peripheral lung tissue (A) and pulmonary arteries (B) isolated from control and shunted lambs. In shunted lambs, NOS activity was unchanged from control lambs. Values are means ± SD of 6 control and 5 shunted lambs. Bottom: Western blot analysis for endothelial NOS (eNOS) protein in peripheral lung tissue and isolated pulmonary arteries from 1-wk-old lambs. Representative Western blot from protein extracts (50 µg) prepared from peripheral lung tissue (C) and pulmonary arteries (D) isolated from four 1-wk-old lambs (2 control and 2 shunt) were separated on a 7.5% SDS-polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analyzed with a specific antiserum raised against eNOS. Densitometric values for relative eNOS protein (normalized to control values) obtained from peripheral lung (E) and pulmonary arteries (F) are also shown. In shunted lambs, eNOS protein is unchanged from that in control lambs. Values are means ± SD for 6 control and 5 shunted lambs.

Western blot analysis also demonstrated no differences in eNOS protein levels in either peripheral lung or isolated pulmonary arteries obtained from shunted and twin control lambs (Fig. 3).

ET-1 Cascade

Peripheral lung tissue irET-1 concentrations were greater in shunted lambs than control lambs (P < 0.05; Fig. 4). To determine whether the alterations in ET-1 levels and physiological responses were associated with changes in gene expression, we performed Western blot analysis. Compared with age-matched control lambs, the levels of preproET-1 protein and ETA receptors were unchanged in both the peripheral lung and isolated pulmonary arteries of shunted lambs. ETB receptor protein was decreased in both the peripheral lung and isolated pulmonary arteries of shunted lambs (P < 0.05). ECE-1alpha protein was increased in the peripheral lung of shunted lambs, but these trends did not reach statistical significance in isolated pulmonary arteries (Figs. 5 and 6).


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  		Fig. 4.   Peripheral lung tissue concentrations of immunoreactive (ir)ET-1 are increased in 1-wk-old shunted lambs compared with age-matched controls. Values are means ± SD; n = 6 control and 5 shunted lambs. *P < 0.05 vs. control.



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  		Fig. 5.   Western blot analysis for preproendothelin-1 ET-converting enzyme-1alpha (ECE-1alpha ) in lung tissue from 1-wk-old lambs. Top: protein extracts (50 µg) prepared from peripheral lung tissue (left) and isolated pulmonary arteries (right) from two 1-wk-old lambs (1 control and 1 shunted) were separated on a 6% denaturing polyacrylamide gradient gel, electrophoretically transferred to Hybond membranes, and analyzed with a specific antiserum. Bottom: densitometric values for relative ECE-1alpha (normalized to control) from 6 control and 5 shunted lambs. In peripheral lung (left), relative ECE-1alpha protein was increased in shunted lambs. The increase in pulmonary arteries (right) did not reach statistical significance. Values are means ± SD. *P < 0.05 vs. control lambs.



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  		Fig. 6.   Western blot analysis for the ETB receptor in lung tissue from 1-wk-old lambs. Top: protein extracts (50 µg) prepared from peripheral lung tissue (left) and isolated pulmonary arteries (right) from two 1-wk-old lambs (1 control and 1 shunted) were separated on a 10% denaturing polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analyzed with a specific antiserum raised against the ETB receptor. Bottom: densitometric values for relative ETB receptor protein (normalized to control) from 6 control and 5 shunted lambs. In both peripheral lung (left) and isolated pulmonary arteries (right), relative ETB receptor protein was decreased in shunted lambs. Values are means ± SD. *P < 0.05 vs. control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Endothelial injury and the resulting aberration in endothelial function have been implicated in the pathophysiology of pulmonary hypertension and its associated increased vascular reactivity. For example, lungs from adults with advanced pulmonary vascular disease display impaired endothelium-dependent pulmonary relaxation, decreased eNOS expression, and increased ET-1 expression (12, 17, 18). In addition, earlier endothelial dysfunction, as displayed by impaired endothelium-dependent pulmonary vasodilation and increased plasma ET-1 levels, has also been described in children with increased pulmonary blood flow within the first years of life, before the development of significant vascular remodeling (10, 48). To study potential early alterations in endothelial function secondary to increased pulmonary blood flow, we developed a model of pulmonary hypertension with increased pulmonary blood flow in the lamb using in utero placement of an aorta-to-pulmonary vascular graft (33). Previously, we demonstrated (3, 34) that at 4 wk of age, these lambs display a selective impairment in endothelium-dependent pulmonary vasodilation and increased ET-1 levels. In addition, this was associated with loss of ETB receptor-mediated pulmonary vasodilation and increased ETA-mediated vasoconstriction (3, 45). However, in contrast to advanced pulmonary vascular disease, we found evidence of increased basal NO activity in the lungs of 4-wk-old shunted lambs, which may represent an early adaptive response of the pulmonary circulation (6). The purpose of the present study was to better identify the timing and sequence of endothelial alterations secondary to increased pulmonary blood flow. To this end, we used our lamb model to study alterations in NO and ET-1 signaling at 1 wk of life. We found that agonist-induced and basal NO signaling were intact at 1 wk of life, as demonstrated by preserved endothelium-dependent vasodilation and normal tissue levels of NOx, NOS activity, and eNOS protein. However, ET-1 signaling was already altered, as demonstrated by increased tissue ET-1 levels and the loss of ETB receptor-mediated pulmonary vasodilation.

To investigate potential differences in agonist-induced NO responses in vivo, we studied the vasoactive effects of the endothelium-dependent vasodilator ACh and the endothelium-independent vasodilator inhaled NO (14, 35). In intact control lambs, both ACh and inhaled NO produced significant pulmonary vasodilation. Similar to control lambs, and in contrast to 4-wk-old shunted lambs, we found that both ACh and inhaled NO produced significant pulmonary vasodilation in 1-wk-old shunt lambs, suggesting that agonist-induced NO activity was intact.

To determine potential alterations in basal NO activity, we also determined and compared lung tissue NOx concentrations, total NOS activity, and eNOS protein levels. In the present study, we found no differences in lung tissue NOx concentrations between shunted and control lambs. In addition, we determined total NOS activity in peripheral lung by the conversion of [3H]arginine to [3H]citrulline and found no differences between shunted and control lambs. However, it should be noted that this assay determines all NOS isoforms, and potential differences in the endothelial isoform may be difficult to detect. Therefore, to better define potential changes in eNOS induced by 1 wk of increased pulmonary blood flow and/or pressure, we also determined NOS activity in isolated pulmonary arteries and eNOS protein levels by Western blot analysis in both peripheral lung and isolated pulmonary arteries. As noted in Fig. 3, we detected no differences in pulmonary artery NOS activity or eNOS protein levels between shunted and control lambs. Together, these data suggest that basal NO activity is intact in 1-wk-old shunted lambs.

The vasoactive properties of ET-1 are mediated by at least two distinct receptor populations, ETA and ETB receptors. In both the fetal and the postnatal lamb, the pulmonary vasodilating effects of ET-1 are produced by ETB receptor activation (13, 26). This vasodilation is mediated in part by endothelium-derived NO production and potassium channel activation (13, 26). In 4-wk-old shunted lambs, we previously found that exogenous ET-1 induced pulmonary vasoconstriction and there was loss of ETB receptor-mediated vasodilation, because 4Ala-ET-1 did not change pulmonary hemodynamics. This was associated with increased ETA receptor protein levels and decreased ETB receptor protein levels (3, 45). In the present study, ET-1 and 4Ala-ET-1 produced pulmonary vasodilation in control lambs both at rest and during pulmonary hypertension induced by U-46619. In contrast, neither ET-1 nor 4Ala ET-1 changed pulmonary hemodynamics in 1-wk-old shunted lambs with increased pulmonary blood flow, and this was consistent after pulmonary blood flow was reduced to normal with shunt closure. This was associated with normal ETA receptor protein levels but decreased ETB receptor protein. Together, these data suggest that loss of ETB receptor-mediated pulmonary vasodilation is the initial event leading to loss of ET-1-induced vasodilation. From our data in 4-wk-old lambs, we can suggest that the subsequent upregulation of ETA receptors results in the emergence of ET-1-induced pulmonary vasoconstriction noted at 4 wk of age.

Similar to our previous studies in 4- and 8-wk-old lambs, we found that 1-wk-old shunted lambs have increased ET-1 levels (3, 5). This is also consistent with studies in humans and other animal models of pulmonary hypertension, but the mechanisms may differ. For example, in humans with advanced pulmonary hypertension and in animal models of persistent pulmonary hypertension of the newborn, increased ET levels are associated with an increase in preproET-1 expression, suggesting that the increase in ET-1 is secondary to increased preproET-1 (18, 24). However, several studies suggest that ECE-1 activity may also regulate tissue and plasma ET-1 levels. For example, plasma molar levels of Big ET-1 (the inactive precursor to ET-1) are much higher than those of ET-1 in humans and animals (41). In addition, in rats, congestive heart failure significantly increases plasma levels of ET-1 while Big ET-1 levels remain unchanged, suggesting that the majority of Big ET-1 within vascular endothelial cells is not converted to ET-1 (42). Because preproET-1 protein is unchanged in 1-wk-old shunted lambs, the upregulation of ECE-1 is most likely responsible for the increased tissue concentrations of ET-1 noted in these lambs. In addition, decreased ETB receptor-mediated clearance may participate.

Similar to our data from 4-wk-old lambs, the current results demonstrate that ETB receptor protein is downregulated in 1-wk-old lambs with increased pulmonary blood flow. The downregulation of ETB receptors has also been demonstrated in pulmonary hypertension produced by monocrotaline and by in utero ligation of the ductus arteriosus (24, 47). In vitro data suggests that increased ET-1 exposure downregulates ETB receptor gene expression (11). Increased circulating levels of ET-1 are found in lambs with increased pulmonary blood flow, as well as in rats after monocrotaline injections and lambs after in utero ligation of the ductus arteriosus (3, 24, 47). Therefore, increased ET-1 production may contribute to the downregulation of the ETB receptor in these models of pulmonary hypertension. Conversely, because the ETB receptor participates in the clearance of ET-1, downregulation of the ETB receptor may contribute to the increase in ET-1 levels secondary to decreased clearance (15). Our recent findings (52) in 8-wk-old lambs and a recent report (2) in humans with advanced pulmonary hypertension suggest that after an initial downregulation of endothelial ETB receptors there is a subsequent upregulation of vasoconstricting smooth muscle ETB receptors. Therefore, these changes in ETB receptor expression may have important implications in the pathophysiology of pulmonary hypertension.

The mechanisms of the changes in either ETB receptor or ECE-1 expression noted in this study are unclear. During the first week of life, the pulmonary vasculature of shunted lambs is exposed to altered mechanical forces, such as increased pressure and flow. In vitro data do suggest that mechanical forces alter ET-1 signaling, although the results are inconsistent and dependent on the type and duration of stimulus as well as the cell type used (20, 29). Therefore, mechanical forces may be the stimulus for the alterations in ET signaling noted in this study, but these mechanisms are speculative and require further study. Other potential factors include growth factors, cytokines, vasoactive substances, and vascular injury.

Within the lung, ET-1 may be synthesized by a variety of cell types, which include bronchial and alveolar epithelial cells, neuroendocrine cells, macrophages, smooth muscle cells, and endothelial cells (28). Therefore, protein levels were determined in both peripheral lung and isolated fifth-generation pulmonary arteries. In pulmonary arteries, the decrease in ETB receptor protein noted in the lung of shunted lambs persisted, suggesting that the differences were predominantly within the vascular endothelium. The increase in ECE-1 protein noted in the lung of shunted lambs tended to increase in isolated pulmonary arteries as well, but these values did not reach statistical significance secondary to large variability. In addition, it is noteworthy that the changes in gene expression correlated with the physiological alterations previously noted within the pulmonary circulation, suggesting that the changes occurred, at least in part, within the vasculature.

In the present study, we demonstrate that alterations in ET-1 signaling occur within the first week of life, whereas NO activity appears intact. These data suggest an important role for early ET-1 alterations in the development of pulmonary hypertension secondary to increased pulmonary blood flow. Although early surgical repair of many congenital heart defects has decreased the incidence of perioperative pulmonary hypertension, subsets of children may still suffer significant perioperative morbidity and mortality from enhanced pulmonary vascular reactivity even within the first few months of life (19). We speculate that early preservation of NO signaling may be responsible, in part, for the overall decreased incidence of perioperative morbidity in young neonates. However, we further speculate that early alterations in ET-1 signaling are responsible for the altered perioperative pulmonary vascular reactivity noted in some young neonates. Further investigation of these changes and their mechanisms may lead to important prevention and treatment strategies for pulmonary hypertension secondary to increased pulmonary blood flow.


    FOOTNOTES

Address for reprint requests and other correspondence: J. R. Fineman, Univ. of California, San Francisco, 505 Parnassus Ave., Box 0106, M-680, San Francisco, CA 94143-0106 (E-mail: jfineman{at}pedcard.ucsf.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.

First published October 24, 2002;10.1152/ajpheart.00493.2002

Received 13 June 2002; accepted in final form 17 October 2002.


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DISCUSSION
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