AJP - Heart Myographs and Tissue organ baths
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 290: H1922-H1932, 2006. First published December 9, 2005; doi:10.1152/ajpheart.01091.2005
0363-6135/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/5/H1922    most recent
01091.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Oishi, P.
Right arrow Articles by Fineman, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Oishi, P.
Right arrow Articles by Fineman, J. R.

Nitric oxide-endothelin-1 interactions after surgically induced acute increases in pulmonary blood flow in intact lambs

Peter Oishi,1 Anthony Azakie,2 Cynthia Harmon,1 Robert K. Fitzgerald,1 Albert Grobe,4 Jie Xu,5 Karen Hendricks-Munoz,5 Stephen M. Black,4 and Jeffrey R. Fineman1,3

1Department of Pediatrics, 2Department of Surgery, and 3Cardiovascular Research Institute, University of California, San Francisco, California; 4Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, Montana; and 5Department of Pediatrics, New York University, New York, New York

Submitted 17 October 2005 ; accepted in final form 7 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Several congenital heart defects require surgery that acutely increases pulmonary blood flow (PBF). This can lead to dynamic alterations in postoperative pulmonary vascular resistance (PVR) and can contribute to morbidity and mortality. Thus the objective of this study was to determine the role of nitric oxide (NO), endothelin (ET)-1, and their interactions in the alterations of PVR after surgically induced increases in PBF. Twenty lambs underwent placement of an aortopulmonary vascular graft. Lambs were instrumented to measure vascular pressures and PBF and studied for 4 h. Before and after shunt opening, lambs received an infusion of saline (n = 9), tezosentan, an ETA- and ETB-receptor antagonist (n = 6), or N{omega}-nitro-L-arginine (L-NNA), a NO synthase (NOS) inhibitor (n = 5). In control lambs, shunt opening increased PBF by 117.8% and decreased PVR by 40.7% (P < 0.05) by 15 min, without further changes thereafter. Plasma ET-1 levels increased 17.6% (P < 0.05), and total NOS activity decreased 61.1% (P < 0.05) at 4 h. ET-receptor blockade (tezosentan) prevented the plateau of PBF and PVR, such that PBF was increased and PVR was decreased compared with controls at 3 and 4 h (P < 0.05). These changes were associated with an increase in total NOS activity (+61.4%; P < 0.05) at 4 h. NOS inhibition (L-NNA) after shunt placement prevented the sustained decrease in PVR seen in control lambs. In these lambs, PVR decreased by 15 min (P < 0.05) but returned to baseline by 2 h. Together, these data suggest that surgically induced increases in PBF are limited by vasoconstriction, at least in part by an ET-receptor-mediated decrease in lung NOS activity. Thus NO appears to be important in maintaining a reduction in PVR after acutely increased PBF.

pulmonary circulation; congenital heart disease

ACUTE CHANGES in pulmonary blood flow (PBF) accompany the surgical repair of a number of congenital heart defects, including tetralogy of Fallot, pulmonary and tricuspid atresia, and other single ventricular heart defects. Dynamic changes in pulmonary vascular resistance (PVR) after surgery can be a significant source of morbidity and mortality, because the balance between systemic vascular resistance (SVR) and PVR is critical in maintaining adequate cardiac output and systemic arterial oxygen saturation in these patients (5, 30). Indeed, postoperative therapeutic interventions are often aimed at altering PVR to achieve an appropriate circulatory balance (4, 45). However, despite this clinical focus, little is known about the mechanisms that regulate pulmonary vascular tone after surgically induced alterations in PBF.

Mounting data demonstrate the importance of vasoactive factors, such as nitric oxide (NO) and endothelin-1 (ET-1), in the regulation of vascular tone in both health and disease (17). NO is produced in the vascular endothelium by the enzyme endothelial NO synthase (eNOS) from the precursor L-arginine (32). Once formed, NO diffuses into the adjacent smooth muscle cell, where it activates soluble guanylate cyclase, catalyzing the conversion of GTP to cGMP (25). Increased cGMP levels ultimately result in smooth muscle cell relaxation and vasodilatation. ET-1 is likewise produced in the vascular endothelium by the endothelin-converting enzyme (ECE-1) from the precursors preproET and proendothelin-1 (Big-ET) (51). ET-1-induced pulmonary vasoactive responses, which include both vasoconstriction and/or vasodilation, are mediated by at least two receptor populations: the ETA and ETB receptors. The ETA receptor is located on smooth muscle cells and mediates vasoconstriction. There are two ETB-receptor subpopulations, one on endothelial cells that mediate vasodilation via NO production and another, less well characterized, population on smooth muscle cells, that likely mediate vasoconstriction (3, 27, 39, 43). Alterations in NO and ET-1 signaling are known to contribute to the pathophysiology of a wide array of pulmonary hypertensive disorders, including congenital heart disease with increased PBF (2, 9, 10, 19, 21, 22, 46).

Alterations in NO and ET-1 during acute increases in PBF have been examined in the fetal lamb after mechanical constriction of the ductus arteriosus (1, 31). In this model, acute constriction of the ductus arteriosus is associated with an initial sharp increase in PBF, which is subsequently reversed by profound pulmonary vasoconstriction (1, 31). Recent studies indicate that this increase in pulmonary vascular tone is associated with increased ET-1 levels and ET-receptor-mediated decreases in NO signaling (31). However, the role of NO and ET-1 in regulating vascular tone after acute changes in PBF in the postnatal pulmonary circulation has not been determined. Therefore, the purpose of this study was to examine the role of NO, ET-1, and their potential interactions in regulating pulmonary vascular tone after surgically induced increases in PBF in the intact juvenile lamb. We hypothesized that acute increases in PBF would result in increased ET-1 signaling and in ET-receptor-mediated decreases in NO signaling, with a net result of increasing PVR over time.

To address this hypothesis, we established a model of increased PBF by placing an 8-mm aortopulmonary vascular graft in intact juvenile (4–6 wk old) lambs. Lambs were instrumented to measure hemodynamic variables and PBF and studied over a 4-h period. To determine potential alterations in NO and ET-1, we determined lung tissue NOS activity (both calcium dependent and independent), plasma ET-1 levels, ECE-1 activity, and protein levels of eNOS, inducible NOS (iNOS), neuronal NOS (nNOS), preproET-1, ECE-1, and ETA and ETB receptors before and 4 h after shunt placement. To elucidate potential differential roles of ET-1 and NO, these parameters were compared with two additional groups of lambs: one group treated with tezosentan, a combined ETA- and ETB-receptor antagonist, and a second group treated with N{omega}-nitro-L-arginine (L-NNA), a NOS inhibitor, both before and after aortopulmonary shunt placement. Finally, to elucidate potential changes related to the study preparation, an additional group of lambs was studied after a sham surgical procedure.

We found that surgically induced acute increases in PBF are accompanied by increases in plasma ET-1 levels, independent of changes in ECE-1 activity and protein levels, and by ET-receptor-mediated decreases in eNOS activity. These alterations correlate with increases in PVR and act to limit increases in PBF over time.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Twenty-four juvenile lambs (4–6 wk of age) were fasted for 24 h, with free access to water. The lambs were then anesthetized with ketamine hydrochloride (15 mg/kg im). While the animals were under additional local anesthesia with 2% lidocaine hydrochloride, polyurethane catheters were placed in an artery and vein of each 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 7.0-mm-outer diameter cuffed endotracheal tube, and mechanically ventilated with a VIP Bird Sterling (Palm Springs, CA) time-cycled, pressure-limited ventilator. Pancuronium bromide (0.1 mg·kg–1·dose–1) was given intermittently for muscle relaxation. With the use of strict aseptic technique, a midsternotomy incision was then performed, and the pericardium was incised. With the use of side-biting vascular clamps, an 8.0-mm Gore-Tex vascular graft (~2-mm length; Gore and Associates, Milpitas, CA), which was occluded by vascular clips, was anastomosed between the ascending aorta and main pulmonary artery with 7.0 proline (Ethicon, Somerville, NJ), using a continuous suture technique. With the use of a purse-string suture technique, polyurethane catheters were placed directly into the right and left atrium and main pulmonary artery. An ultrasonic flow probe (Transonics Systems, Ithaca, NY) was placed around the left pulmonary artery to measure PBF. The midsternotomy incision was then temporarily closed with towel clamps. An intravenous infusion of lactated Ringer solution and 5% dextrose (75 ml/h) was begun and continued throughout the study period. Cefazolin (500 mg iv) and gentamicin (3 mg/kg iv) were administered before the first surgical incision. The lambs were maintained normothermic (39°C) with a heating blanket.

Experimental protocol. After a 30-min recovery period, baseline measurements of the hemodynamic variables (pulmonary and systemic arterial pressure, heart rate, left PBF, and left and right atrial pressures) and systemic arterial blood gases and pH were measured. Blood was collected from the femoral artery for plasma ET-1, and a peripheral lung wedge biopsy was obtained for NOS activity and eNOS, iNOS, nNOS, preproET-1, ECE-1, ETA- and ETB-receptor protein determinations. A side-biting vascular clamp was utilized to isolate peripheral lung tissue from a randomly selected lobe, and the incision was cauterized. Approximately 300 mg of peripheral lung were obtained for each biopsy.

In control (group 1, n = 9) and sham animals (group 2, n = 4), an infusion of normal saline (vehicle) was initiated and continued throughout the study period. In group 3 (n = 6), an infusion of tezosentan (a combined ETA- and ETB-receptor antagonist) was initiated at 0.5 mg·kg–1·h–1 and continued throughout the study period. The dose of tezosentan was determined from previous work that demonstrates physiologically significant blockade of ET-1-induced vascular alterations (18). In group 4 (n = 5), an infusion of L-NNA was initiated at 6 mg·kg–1·h–1 and continued throughout the study period. The dose of L-NNA was determined from previous studies that demonstrate complete blockade of acetylcholine-induced vasodilation (16).

One hour after the initiation of the infusion (vehicle, tezosentan, or L-NNA), the vascular clips were removed from the aortopulmonary graft, opening the shunt, in groups 1, 3, and 4. In the sham group (group 2), the procedure was identical, except that the graft was not opened. Hemodynamic variables were monitored continuously for 4 h. At 4 h after shunt opening, blood was again taken for plasma ET-1 determinations, and a lung biopsy was obtained for NOS activity and eNOS, iNOS, nNOS, preproET-1, ECE-1, and ETA- and ETB-receptor protein determinations.

At the end of the protocol, all lambs were killed with a lethal injection of pentobarbital sodium, followed by bilateral thoracotomy as described in Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996). All protocols and procedures were approved by the Committee on Animal Research of the University of California, San Francisco.

Drug preparation. Tezosentan (molecular weight 649.6, Actelion Pharmaceuticals, Allschwil, Switzerland) was diluted in sterile 0.9% saline to a final concentration of 1 mg/ml and used immediately. L-NNA (Sigma Chemical, St Louis, MO) was diluted in sterile 0.9% saline to a final concentration of 2 mg/ml and used immediately.

Measurements. Pulmonary and systemic arterial pressures and right and left atrial pressures were measured using Sorenson neonatal transducers (Abbott Critical Care Systems, North 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 PBF was measured on an ultrasonic flowmeter (Transonic Systems). All hemodynamic variables were measured continuously with a Gould Ponemah Physiology Platform (version 4.2) and Acquisition Interface (model ACG-16; Gould, Cleveland, OH) and recorded with a Dell Inspiron 5160 computer (Dell, Round Rock, TX). Blood gases and pH were measured on a Radiometer ABL5 pH/blood gas analyzer (Radiometer, Copenhagen, Denmark). Hemoglobin concentration and oxygen saturation were measured by a co-oximeter (model 682, Instrumentation Laboratory, Lexington, MA). PVR was calculated using standard formulas. Body temperature was monitored continuously with a rectal temperature probe.

Assay for NOS activity. NOS activity was determined by using the conversion of L-[3H]arginine to L-[3H]citrulline as described by Bush et al. (12). Briefly, peripheral lung tissues 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 tetrahydro-biopterin, 100 µM FAD, 1 mM MgCl2, 5 µM unlabeled L-arginine, 15 nM L-[3H]arginine, calmodulin (25 units), 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-NNA to detect nonspecific production of [3H]citrulline. This value was then subtracted to obtain the final activity value. 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 [20 mM sodium acetate (pH 5), 1 mM L-citrulline, 2 mM EDTA, and 0.2 mM EGTA] then applied to columns containing 1 ml of Dowex AG50W-X8 resin, Na+ form, preequilibrated with 1 N NaOH. L-[3H]citrulline was then quantitated by scintillation counting. All activities were normalized to the amount of protein in each lysate. To determine the potential contribution of iNOS to total NOS activity, assays were repeated without calcium supplementation.

Plasma ET-1 determinations. Three milliliters of systemic arterial blood were collected and placed in iced polypropylene tubes containing 330 µl aprotinin and 100 µl EDTA. The tubes were immediately centrifuged at 4,000 g for 20 min. Collected plasma was treated with equal volumes of 0.1% trifluoroacetic acid and stored at –70°C. The acidified supernatant was centrifuged at 1,000 g for 20 min and loaded on a 3 x 18 C18 Sep-Pak column (Peninsula Laboratories, Belmont, CA) equilibrated with 0.1% trifluoroacetic acid. The adsorbed material was eluted with 3 ml of 0.1% trifluoroacetic acid-60% acetronitrile. The eluant was dried in a Savant speed vac and stored at –70°C or assayed immediately for immunoreactive ET-1. ET-1 standard, 125I-labeled ET-1, anti-ET antibody, and secondary antibody were purchased from Peninsula Laboratories. 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. Inter- and intra-assay variabilities were 10 and 4%, respectively. Each sample was assayed in duplicate. This assay was modified from a previously published method (50).

Assay for ECE activity. Tissues (snap frozen and stored at –80°C) were minced in 5x of extraction buffer [25 mM sodium phosphate buffer (pH 6.8) and 0.1% Triton X-100] and then sonicated for 10 s on ice at 35% amplitude using a High Intensity Ultrasonic Processor (Autotune series A; Daigger, Vernon Hills, IL). The samples were centrifuged at 20,000 g for 10 min at 4°C, the supernatants were collected, and the protein concentration was determined using the Bio-Rad DC Protein Assay (Bio-Rad Laboratories, Hercules, CA). Aliquots of the tissue extracts were stored at –80°C until assayed.

To determine ECE-1 activity, 1 µg of total protein was incubated in the presence or absence of 10 µM disodium salt (Phosphoramidon; Calbiochem/EMD Biosciences, La Jolla, CA) in 0.1 M sodium phosphate buffer (pH 6.8), 0.5 M NaCl in a total reaction volume of 50 µl for 30 min at room temperature. An equal volume of 20 µM (final 10 µM concentration) fluorescent peptide V (R&D Systems, Minneapolis, MN) was added, and the relative fluorescence units were measured with the 320 nm excitation/405 nm emission filter pair, 300-ms integration time for 10 readings over 390 s (Fluoroskan Ascent FL; Thermo Electron, Pittsburgh, PA). The change in fluorescence was used to calculate the specific activity over time. Mca-Pro-Leu-OH, the fluorescent cleavage product of FP5 (Bachem California, Torrance, CA), was used to generate the fluorescence standards for the calculation of the enzyme specific activity.

Preparation of protein extracts and Western blot analysis. Lung protein extracts were prepared by homogenizing peripheral lung tissues in Triton lysis buffer [50 mM Tris·HCl (pH 7.6), 0.5% Triton X-100, and 20% glycerol] containing a protease inhibitor cocktail. Extracts were then clarified by centrifugation (15,000 g x 10 min at 4°C). Supernatant fractions were then assayed for protein concentration using the Bradford reagent (Bio-Rad, Richmond, CA) and used for Western blot analysis. Western blot analysis was performed as previously described (7, 29, 47). Briefly, protein extracts (25 µg) were separated on 7.5% denaturing polyacrylamide gels for eNOS, iNOS, nNOS, and ECE-1{alpha}, 10% denaturing polyacrylamide gels for ETA, 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 being blocked, the membranes were incubated at room temperature with the appropriate dilution of the antiserum of interest (1:2,500 for eNOS, iNOS, and nNOS; 1:1,000 for ECE-1{alpha}; 1:1,000 for ETA; and ETB, 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, iNOS, nNOS, and preproET-1) or a goat anti-rabbit IgG-horseradish peroxidase conjugate (for ECE-1{alpha} and ETA and ETB receptors). In addition, for the studies examining eNOS, iNOS, and nNOS expression, all blots were coincubated with a monoclonal antibody raised to beta-actin (1:10,000 dilution; Sigma). After the membranes were washed, the protein bands were visualized with chemiluminescence using a Kodak Digital Science Image Station (NEN) and analyzed using the KED-1 software. All captured and analyzed images were determined to be in the dynamic range of the system.

The eNOS, nNOS, and iNOS antiserum was obtained from Transduction Laboratories (Lexington, KY). The ETA-receptor antiserum was generated as previously described (7). 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-1{alpha} antisera were generated as previously described (29).

Positive controls were run to demonstrate antibody specificity. The methodology and exposure times used were those that we have previously demonstrated to be within the linear range of the autoradiographic film and able to detect changes in lung protein expression (7, 9). In addition, to ensure that equal protein was loaded in each lane for the experiments in which preproET-1, ECE-1{alpha}, ETA- and ETB-receptor expression was investigated, all blots were stripped and reprobed for beta-actin. Blots were stripped in 100 mM beta-mercapto-ethanol, 2% SDS, and 62.5 mM Tris·HCl (pH 6.8) for 30 min at 50°C. The membranes were blocked again by using 5% nonfat dry milk in TBS containing 0.1% Tween and then incubated at room temperature with a monoclonal antibody to beta-actin (1:10,000 dilution, Sigma). After being washed with TBS containing 0.1% Tween, the blots were incubated with a goat anti-mouse IgG-horseradish peroxidase conjugate and the protein bands were visualized as described above.

All data from the Western blot analysis are reported as the ratio of densitometric value of the protein of interest divided by the densitometric value of the beta-actin band to generate a relative expression for each protein.

Immunohistochemistry for iNOS and nNOS. Snap frozen lung tissue samples stored at –80°C were embedded in Tissue-Tek OCT Compound (Sakura Finetek USA, Torrance, CA), cryosectioned at 5 µm, collected onto Superfrost Plus slides (VWR Scientific, West Chester, PA), allowed to air-dry at room temperature, and stored at –80°C until needed. For staining, slides were removed from –80°C and blocked in PBS containing 0.5% Tween 20 and 5% normal goat serum (PBS-T/NGS) at room temperature (2 h). Slides were probed for iNOS or nNOS (0.5 µg/ml, BD Transduction Labs) in PBS-T/NGS for 1 h at room temperature in a moist chamber and washed with PBS-T; goat anti-mouse Alexafluor-546 in PBS-T/NGS (1:1,000, Molecular Probes, Eugene, OR) was added for 1 h at room temperature in the dark. The slides were rinsed extensively with PBS-T, coverslipped and imaged with an Olympus IX51 inverted microscope, and analyzed with Image-ProPlus software (Media Cybernetics, Silver Spring, MD). Mean integrated optical density values were collected from multiple blood vessels per sample and adjusted to mean ± 2 SD to remove outliers.

Statistical analysis. The means ± SD and SE were calculated for the baseline hemodynamic variables, systemic arterial blood gases and pH, and ET-1 concentrations. The general hemodynamic variables and systemic arterial blood gases and pH were compared within each group over time by ANOVA for repeated measures. Student-Newman-Keuls post hoc testing was performed. Plasma ET-1 levels, before and 4 h after shunt opening, were compared by the paired t-test. Comparisons of PBF and PVR between study conditions were made by ANOVA. Quantitation of autoradiographic results was performed by scanning (Hewlett-Packard SCA Jet IICX, Hewlett-Packard, Palo Alto, CA) the bands of interest into an image-editing software program (Adobe Photoshop; Adobe Systems, Mountain View, CA). Band intensities from Western blot analysis were analyzed densitometrically on a Dell Inspiron 5160 computer using the public domain Scion program (developed at NIH and available at http://rsb.info.nih.gov/nih-image). To normalize for protein loading in the Western blot analyses, blots were reprobed with the housekeeping protein beta-actin. Relative protein expression was then determined as a ratio of the protein to beta-actin signals. Densitometric values for each protein of interest were compared before and 4 h after shunt opening by the paired t-test. A P < 0.05 was considered statistically significant.

All the values presented in the text and tables are means ± SD to allow the reader to appreciate the dispersion of the raw data. We elected to use means ± SE in the figures to portray the confidence of the estimation of the mean.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
There were no differences in gestational age, weight (mean 14.5 ± 3.3 kg), sex distribution, or baseline hemodynamic variables between control, sham, tezosentan-treated, or L-NNA-treated lambs (data not shown).

In control lambs, opening of the aortopulmonary vascular graft resulted in a rapid increase in PBF (from 37.7 ± 11.7 to 82.1 ± 21.83 ml·min–1·kg–1 at 15 min; P < 0.05) and decrease in left PVR (LPVR; from 0.27 ± 0.12 to 0.16 ± 0.06 mmHg·ml–1·min·kg at 15 min; P < 0.05), which did not change further over the subsequent 4-h study period (Fig. 1). Pulmonary artery pressure increased, and mean and diastolic systemic arterial pressure decreased at 15 min after shunt opening and did not change further over the 4-h study period (P < 0.05; Table 1). Left atrial pressure increased over baseline at 1 h after shunt opening (P < 0.05; Table 1). Opening of the shunt was not associated with changes in systolic systemic arterial pressure, heart rate, or right atrial pressure (Table 1). Systemic arterial pH (from 7.43 ± 0.06 to 7.39 ± 0.04), PCO2 (from 33.0 ± 7.0 to 39.1 ± 6.7 Torr), and PaO2 (from 87.1 ± 18.7 to 80.1 ± 17.1 Torr) did not change.


Figure 1
View larger version (14K):
[in this window]
[in a new window]
  		Fig. 1. Pulmonary blood flow (PBF) and left pulmonary vascular resistance (LPVR) after aortopulmonary shunt opening. Opening of aortopulmonary vascular graft results in a sharp increase in PBF (A) and decrease in LPVR (B) by 15 min. PBF and LPVR then plateau, without significant changes thereafter. Values are means ± SE; n = 9. *P < 0.05 vs. baseline.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Hemodynamic changes after aortopulmonary shunt opening in vehicle-treated (control) lambs

 
Four hours after shunt opening, plasma ET-1 levels increased (from 9.33 ± 1.0 to 11.00 ± 2.1 pg/ml; P < 0.05). This increase was independent of changes in ECE-1 activity (0.165 ± 0.04 vs. 0.160 ± 0.01 pmol·min–1·µg–1) and protein levels (relative to beta-actin) of ECE (0.167 ± 0.063 vs. 0.192 ± 0.042), PreproET (0.203 ± 0.125 vs. 0.133 ± 0.062), and ETA (0.203 ± 0.120 vs. 0.266 ± 0.147) and ETB (0.099 ± 0.052 vs. 0.1 ± 0.056) receptors (data expressed as mean densitometric values ± SD for baseline vs. 4 h after shunt opening). Furthermore, total NOS activity decreased by 61.1% compared with baseline at 4 h after shunt opening (P < 0.05; Fig. 2A). Before shunt opening, calcium-independent NOS activity consisted of 18.9% of total NOS activity and decreased by 36.8% at 4 h after shunt opening (P < 0.05). The levels of eNOS protein were unchanged over the 4-h study period (Fig. 2, B and C). Detected iNOS protein levels were minimal, and nNOS protein levels were not detectable (Fig. 2, C and D). Because Western blot analysis did not detect significant iNOS and nNOS protein levels, we performed immunohistochemistry. As seen in Fig. 3, low levels of both iNOS and nNOS were detected in the lamb lungs, without significant changes noted after 4 h of shunt opening.


Figure 2
View larger version (60K):
[in this window]
[in a new window]
  		Fig. 2. Lung tissue nitric oxide (NO) synthase (NOS) activity and Western blot analysis for NOS protein in lung tissue before and 4 h after aortopulmonary shunt opening. A: total lung tissue NOS activity is decreased at 4 h after opening of aortopulmonary shunt. B: endothelial NOS (eNOS) protein levels do not change by 4 h after shunt opening when compared with baseline. B, top: representative Western blots are shown for protein extracts prepared from lung tissue separated on a 7.5% SDS-polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analyzed by using a specific antiseruma raised against eNOS (equal protein loading is shown by signal obtained from beta-actin). B, bottom: densitometric values for eNOS protein from control lambs (normalized to beta-actin). Values are means ± SE; n = 9. *P < 0.05 vs. baseline. C and D: detected inducible NOS (iNOS) protein levels were minimal. Equal protein loading is shown by signal obtained from beta-actin, and neuronal NOS (nNOS) protein levels were not detected either at baseline or at 4 h after aortopulmonary shunt opening, although we again demonstrate equal protein loading with the beta-actin signal.

 

Figure 3
View larger version (34K):
[in this window]
[in a new window]
  		Fig. 3. Immunohistochemical detection of iNOS and nNOS protein in juvenile lamb lungs. Lung tissue samples cryosectioned at 5 µm were blocked and probed for iNOS or nNOS as described in MATERIALS AND METHODS. Bound antibody was detected using a goat anti-mouse Alexafluor-546, and sections were imaged by fluorescence. Both iNOS (A) and nNOS (B) protein levels are detected by immunohistochemistry but are unchanged in acute shunt (top) or sham-operated (bottom) lambs. Representative vessels are shown. Scale bar = 50 µm.

 
The infusion of tezosentan resulted in a decrease in diastolic and mean systemic arterial pressure (P < 0.05; Table 2). There were no changes in PBF, LPVR, heart rate, systolic systemic arterial pressure, or right and left atrial pressure.


View this table:
[in this window]
[in a new window]
  		Table 2. Hemodynamic changes after aortopulmonary shunt opening in Tezo-treated lambs

 
In tezosentan-treated lambs, opening of the vascular graft resulted in a rapid increase in PBF (from 41.03 ± 10.9 to 71.4 ± 21.83 ml·min–1·kg–1 at 15 min; P < 0.05) and decrease in LPVR (from 0.36 ± 0.15 to 0.2 ± 0.06 mmHg·ml–1·min·kg at 15 min; P < 0.05; Fig. 4). However, unlike control lambs, PBF and LPVR did not reach a plateau but rather continued to change from 15 min to 4 h after shunt opening (ANOVA; P < 0.05; Fig. 4). Furthermore, by 3 h, PBF was higher and LPVR was lower than in vehicle-treated control lambs (P < 0.05; Fig. 4). Pulmonary artery pressure and left atrial pressure increased, mean and diastolic systemic arterial pressure decreased, and right atrial pressure did not change after shunt opening. Heart rate increased and systolic systemic arterial pressure decreased by 4 h after shunt opening (P < 0.05; Table 2). Systemic arterial pH (from 7.38 ± 0.09 to 7.37 ± 0.10), PCO2 (from 39.7 ± 7.0 to 44.3 ± 10.0 Torr), and PaO2 (from 77.5 ± 4.5 to 67.5 ± 13.5 Torr) did not change.


Figure 4
View larger version (15K):
[in this window]
[in a new window]
  		Fig. 4. PBF and LPVR after opening of aortopulmonary shunt in tezosentan (Tezo)-treated and N{omega}-nitro-L-arginine (L-NNA)-treated lambs. A: PBF increases sharply by 15 min after opening of aortopulmonary shunt in all groups. PBF continues to increase in Tezo-treated lambs (dashed line) but not L-NNA-treated (dotted line) or control (solid line) lambs over the 4-h study period. In Tezo-treated lambs, PBF is higher than in control-treated lambs at 3 and 4 h. B: LPVR decreases sharply by 15 min after opening of aortopulmonary shunt in all groups. In L-NNA-treated lambs (dotted line), LPVR then increases, returning to baseline by 2 h, unlike Tezo-treated (dashed line) or control (solid line) lambs. In L-NNA-treated lambs, LPVR is significantly higher than in control lambs at 4 h. In Tezo-treated lambs, LPVR continues to decrease after aortopulmonary shunt opening over the 4-h study period. In Tezo-treated lambs, LPVR is significantly lower than in control lambs at 3 and 4 h. Control, n = 9; Tezo-treated, n = 6; L-NNA-treated, n = 5. Values are means ± SE. *P < 0.05 vs. baseline; {dagger}P < 0.05 vs. controls.

 
Interestingly, total NOS activity increased by 61.4% (from 0.34 ± 0.14 to 0.49 ± 0.14 pmol·min–1·mg protein–1; P < 0.05) at 4 h after shunt opening, as opposed to the decrease in control animals (Fig. 5A). Like control lambs, calcium-independent NOS activity consisted of 21.0% of total NOS activity, and decreased by 57.1% (from 0.07 ± 0.02 to 0.03 ± 0.02 pmol·min–1·mg protein–1; P < 0.05) at 4 h after shunt opening (P < 0.05). Similar to control lambs, these changes in NOS activity were independent of changes in eNOS protein levels (Fig. 5B).


Figure 5
View larger version (14K):
[in this window]
[in a new window]
  		Fig. 5. Lung tissue NOS activity and eNOS protein levels. A: relative lung tissue NOS activity (normalized to baseline) increases at 4 h in Tezo-treated lambs as opposed to the decrease seen in control animals. B: eNOS protein levels in lung tissue do not change before and 4 h after opening of aortopulmonary shunt in Tezo-treated lambs. B, top: representative Western blots are shown for protein extracts prepared from lung tissue separated on a 7.5% SDS-polyacrylamide gel, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against eNOS (equal protein loading is shown by signal obtained from beta-actin). B, bottom: densitometric values for eNOS protein (normalized to beta-actin) from Tezo-treated lambs. eNOS protein levels do not change in Tezo-treated lambs by 4 h after aortopulmonary shunt placement. Control, n = 9; Tezo-treated, n = 6. Values are means ± SE. *P < 0.05 vs. baseline.

 
The infusion of L-NNA resulted in an increase in systemic systolic, diastolic, and mean arterial pressures (P < 0.05; Table 3). Pulmonary arterial pressure, right and left atrial pressure, PBF, LPVR, and heart rate did not change.


View this table:
[in this window]
[in a new window]
  		Table 3. Hemodynamic changes after aortopulmonary shunt opening in L-NNA -treated lambs

 
In L-NNA-treated lambs, opening of the vascular graft resulted in a rapid increase in PBF (from 29.14 ± 7.7 to 79.98 ± 18.49 ml·min–1·kg–1 at 15 min; P < 0.05), which did not change further over the 4-h study period (Fig. 4A). Shunt opening resulted in a rapid decrease in LPVR (from 0.26 ± 0.09 to 0.14 ± 0.02 mmHg·ml–1·min·kg at 15 min; P < 0.05; Fig. 4B). However, unlike in control and tezosentan-treated lambs, LPVR increased from 15 min to 4 h after shunt opening, returning to baseline values by 2 h (ANOVA; P < 0.05; Fig. 4B). Furthermore, by 4 h, LPVR was higher than in control lambs (P < 0.05; Fig. 4B). Pulmonary artery pressure and left atrial pressure increased, and systolic, mean, and diastolic systemic arterial pressure decreased after shunt opening, whereas heart rate did not change (Table 3). Systemic arterial pH (from 7.41 ± 0.04 to 7.38 ± 0.07), PCO2 (from 40.0 ± 3.5 to 44.5 ± 11.6 Torr), and PaO2 (from 97.2 ± 8.7 to 88.1 ± 15.6 Torr) did not change. NOS activity was not detectable in L-NNA-treated lungs.

In sham-operated lambs, PBF, LPVR, mean pulmonary artery pressure, left and right atrial pressure, systemic arterial pressure, and heart rate did not change significantly during the 4-h study period (Table 4). Systemic arterial pH (from 7.41 ± 0.04 to 7.39 ± 0.07), PCO2 (from 46.0 ± 3.2 to 45.5 ± 2.3 Torr), and PaO2 (from 95.8 ± 11.3 to 99.1 ± 29.5 Torr) did not change. Plasma ET-1 levels (from 6.02 ± 1.1 to 5.78 ± 2.2 pg/ml) and tissue ECE-1 levels (0.140 ± 0.01 vs. 0.18 ± 0.03 pmol·min–1·µg–1) were also unchanged. In addition, both total (from 0.58 ± 0.20 to 0.48 ± 0.12 pmol·min–1·mg protein–1) and calcium-independent (from 0.08 ± 0.08 to 0.09 ± 0.04 pmol·min–1·mg protein–1) NOS activity was unchanged, and eNOS (from 0.38 ± 0.09 to 0.34 ± 0.09, densitometric values) protein levels were unchanged. iNOS protein levels were minimally detectable, and nNOS protein levels were not detectable by Western blot analysis. Immunohistochemistry did detect low levels of both iNOS and nNOS, which did not change during the 4-h study period (Fig. 3).


View this table:
[in this window]
[in a new window]
  		Table 4. Hemodynamics in sham-operated lambs

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Vascular dysfunction is integral to the pathophysiology of a wide array of disease processes, including congenital heart disease (11, 13, 14). In these patients, pulmonary vascular dysfunction contributes to the dynamic alterations in PVR that complicate the postoperative period. Postoperative pulmonary vascular dysfunction may be secondary to a number of factors, including abnormal pulmonary mechanical forces, ischemia-reperfusion injury, and cardiopulmonary bypass (6, 34, 36, 37, 44). The current study suggests that the abnormal forces associated with acute increases in PBF alone are sufficient to produce altered ET-1 and NO signaling in the intact lamb. Specifically, we found that surgically induced increases in PBF are associated with increases in plasma ET-1 levels and ET-receptor-mediated decreases in lung tissue NOS activity. Together, these changes tend to increase PVR postoperatively.

Acute increases in PBF have been examined in the fetal lamb after mechanical constriction of the ductus arteriosus. In this model, acute increases in PBF and decreases in PVR are reversed within 2–4 h by intense pulmonary vasoconstriction (1, 31). Recent data demonstrate that increased plasma ET-1 levels and ETA-receptor-mediated decreases in lung tissue NOS activity are associated with these changes (31). In the current study, acute increases in PBF were induced by the surgical placement of a large (8 mm) aortopulmonary shunt in intact juvenile (4–6 wk) lambs. Shunt placement resulted in a rapid sharp increase in PBF and decrease in PVR. However, very rapidly, PBF and PVR reached a plateau such that neither was significantly different at 4 h compared with 15 min after shunt opening. In isolation, it is unclear whether this plateau represents maximal flow through the pulmonary circulation or a limitation in response to an increase in pulmonary vascular tone. However, the fact that plasma ET-1 levels were increased and lung tissue NOS activity was decreased at 4 h after shunt opening suggests that pulmonary vascular tone may be increased.

To elucidate the role of ET-1 signaling in these animals, a group of lambs was treated with tezosentan, a combined ETA- and ETB-receptor antagonist, before and after shunt placement. Treatment with tezosentan augmented the increase in PBF and decrease in PVR, preventing a sustained plateau, such that PBF was higher and PVR was lower than controls at 3 and 4 h. Furthermore, lung tissue NOS activity was increased in tezosentan-treated lambs as opposed to the decrease observed in control lambs. These data indicate that ET-receptor activation may modulate a flow- and/or pressure-induced decrease in eNOS activity that occurs with an acute increase in PBF. Moreover, these data suggest that PBF after shunt placement is limited, at least in part, by active vasoconstriction because treatment with tezosentan allowed for a continued increase in PBF and decrease in PVR over time.

To elucidate the role of NO signaling in these animals, another group of lambs was treated with L-NNA, an inhibitor of NOS. Treatment with L-NNA did not affect the rapid increase in PBF or decrease in PVR observed after shunt opening. However, unlike vehicle or tezosentan-treated lambs, L-NNA-treated lambs demonstrated an increase in PVR over time, reaching baseline values by 2 h. These data suggest that whereas mechanical forces (i.e., vessel recruitment and vascular elasticity) may account for much of the decrease in PVR observed immediately after shunt placement, NO may play an important role in maintaining a low PVR once PBF is increased. Unlike PVR, PBF did not differ between L-NNA-treated lambs and vehicle-treated lambs, despite the fact that NOS activity was completely blocked. The reasons for PBF not further decreasing in L-NNA-treated lambs are unclear. However, in the presence of an aortopulmonary communication, PBF is dependent on several factors, which include the ratio of pulmonary to systemic vascular resistance. Indeed, recently published studies (15, 24) on the postoperative management of patients with single-ventricle physiology advocate a pharmacological decrease in SVR, as opposed to increased PVR, to decrease pulmonary overcirculation and maintain adequate systemic oxygen delivery. In this study, infusion of L-NNA was associated with an increase in systemic blood pressure, suggesting an increase in SVR, which may have affected the ratio of pulmonary to systemic vascular resistance to favor the maintenance of PBF in the face of an increasing PVR. The instrumentation utilized in the current study did not allow for direct measurements of SVR without significant assumptions. However, L-NNA has been shown to increase SVR in other studies at the dose utilized in this study (16).

The acute increase in PBF was associated with a decrease in NOS activity under normal conditions and an increase in NOS activity during ET-receptor blockade. In the vasculature, NO may be produced by three major NOS isoforms: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). Both nNOS and eNOS are constitutive and calcium dependent, whereas iNOS is inducible and calcium independent. All three isoforms have been identified in the developing sheep lung (42). To determine the potential contributions of these isoforms in our studies, NOS activity assays were performed with and without calcium. We found that the majority of total NOS activity was calcium dependent and the protein expression of nNOS was minimal, suggesting that changes in eNOS activity were dominant. In fact, Western blot analysis could not detect nNOS protein levels, so we utilized immunohistochemistry to confirm the presence of nNOS in our lambs, as previously described (42). A large body of evidence indicates that eNOS can be regulated at the transcriptional and posttranslational levels (23, 33). For example, laminar shear stress has been shown to increase eNOS transcription, and a number of factors, including intracellular location, protein-protein interactions, phosphorylation, and substrate and cofactor availability, have all been shown to regulate eNOS activity (23, 33, 35, 40). In the current study, the change in NOS activity was not associated with changes in protein levels, suggesting a posttranslational modification. Previously, we have demonstrated posttranslational modifications of eNOS in lambs secondary to chronic exposure to inhaled NO (8, 29, 47). Interestingly, both exogenous inhaled NO and increased PBF induced by acute ductal constriction result in alterations in NOS activity via an ET-receptor-dependent mechanism (29, 31, 47). The current study also demonstrates a link between ET-receptor activation and alterations in NOS activity, because NOS activity did not decrease, but actually increased, in tezosentan-treated lambs. It is also noteworthy that calcium-independent NOS activity, although a small contributor to total NOS activity, also decreased during acute increases in PBF. iNOS can be regulated at many levels, including transcriptional, pretranslational, and posttranslational (38, 52). The potential mechanism for the decrease in iNOS activity in our study requires further study. However, because calcium-independent NOS activity also decreased during ET-receptor blockade, our data suggest that contrary to eNOS activity, the decrease in iNOS activity during acute increases in PBF is independent of ET signaling. Lastly, it should be noted that determinations of both NOS activity and protein levels were made on peripheral lung tissue. Therefore, potential changes of these parameters within particular cell types could not be determined in this in vivo investigation.

Increased plasma ET-1 levels may result from increased production, increased release, and/or decreased ET-1 clearance. ET-1 is produced in endothelial cells from the precursor preproET-1, which is then cleaved to Big-ET. The metalloprotease ECE-1 catalyzes the conversion of Big-ET-1 to the active ET-1 (51). There are at least two known receptors for ET-1, the ETA and the ETB receptors (3, 39). ETA receptors are found on smooth muscle cells, and their activation results in vasoconstriction. ETB receptors have two separate subpopulations; one is located on smooth muscle cells and mediates vasoconstriction, and the other is located on endothelial cells and mediates vasodilation via NO signaling (27, 43). Furthermore, ETB receptors are involved in the clearance of ET-1 (20). In the present study, plasma ET-1 levels increased by 4 h after shunt opening. This increase was independent of changes in ECE-1 activity and preproET, ECE-1, and ETA and ETB protein levels. ET-1 release from intracellular granules has been demonstrated in response to various stimuli, including stretch, and could explain the increase in ET-1 in response to acute increases in PBF in the present study (28). However, changes in ETB-receptor binding affinity with decreased ET-1 clearance could also explain this result (20).

Whereas NO-ET-1 interactions are implicated in the pulmonary vasoconstriction observed in this study, the relative roles of NO and ET-1 remain unclear. In the mature circulation, ET-1 is an extremely potent vasoconstrictor, but its effects in the developing circulation are more varied (26, 49, 51). In fact, exogenous ET-1 given to the newborn pulmonary circulation results in vasodilation (48). In the juvenile lamb, we have demonstrated that ET-1 has a negligible effect on resting pulmonary vascular tone but results in vasodilation if the pulmonary vasculature is pharmacologically constricted before ET-1 exposure (49). Thus, whereas combined ET-receptor antagonism resulted in an increase in PBF and decrease in PVR in the present study, it is possible that this effect resulted more from the inhibition of an ET-receptor-mediated decrease in NOS activity than from inhibition of direct ET-1-mediated vasoconstriction. Indeed, NOS activity increased by 61.4% in tezosentan-treated lambs, raising the possibility that the persistent fall in PVR and increase in PBF represents active NO-mediated vascular relaxation. L-NNA treatment prevented a sustained decrease in PVR after shunt opening but did not alter the initial reduction in PVR and increase in PBF. In addition, PBF did not decrease as PVR increased from 15 min to 4 h in L-NNA-treated lambs. These data suggest that NO is critical in maintaining the reduction in PVR that follows acutely increased PBF but that it does not account for all of the hemodynamic manifestations that follow shunt placement. Mechanical forces and the balance between PVR and SVR are likely also critical.

Several important limitations of the present study are noteworthy. First, only one age group was studied. The pulmonary circulation undergoes significant developmental changes. Thus we cannot assume that the NO-ET-1 interactions described in the juvenile lamb would be the same in other age groups. Second, the model utilized in this study employs a very large (8 mm) aortopulmonary vascular graft. In practice, systemic to pulmonary shunts placed in patients with congenital heart disease are generally <4.5 mm. Furthermore, such a large shunt may obfuscate important changes in PBF that would otherwise accompany changes in PVR. Third, our model involved the placement of a shunt in an intact lamb with previously normal PBF. Clinically, systemic to pulmonary shunts are usually placed in the setting of significant abnormalities in PBF. Whereas more complex models are warranted to examine the effects of acute alterations in PBF in the setting of abnormal pulmonary circulations, it is critical to first establish the effects of increased PBF alone. The present study contributes to this effort. Fourth, a combined ET-receptor antagonist was utilized, which prevents insight into potential receptor-specific mechanisms. Future studies are needed to delineate the role of ETA and ETB receptors in the interactions described in the present study. Fifth, we did not directly measure systemic blood flow in this preparation, which would have provided important additional information, because these determinations would have necessitated the placement of two additional flow probes and the risk of vascular compression. However, by determining the pulmonary-to-systemic blood flow ratio (Qp/Qs) utilizing the Fick equation and assuming that left PBF is 40% of total flow, we can estimate systemic blood flow by dividing the total PBF by the Qp/Qs. With the use of these parameters, estimated systemic blood flow did not change over the study period in any of the study groups (data not shown). Finally, our study describes alterations over a 4-h period. Important vascular alterations likely manifest later after acute changes in PBF, and the changes we describe may lose relevance over time.

In summary, we found that surgically induced acute increases in PBF in the intact juvenile lamb result in increased plasma ET-1 levels and ET-receptor-mediated decreased NOS activity. These endothelial alterations favor active vasoconstriction, which limits PBF postoperatively. Interestingly, decreases in NO signaling and increases in ET-1 signaling are associated with pulmonary hypertension in a number of clinical settings as well as experimental models (2, 9, 10, 19, 21, 22, 46). Furthermore, the NO-ET-1 interactions described in the present study recapitulate alterations demonstrated with acute mechanical constriction of the ductus arteriosus in fetal lambs and chronic inhaled NO therapy in juvenile lambs (8, 29, 31). A better understanding of these novel endothelial interactions may result in postoperative strategies that improve the care of neonates, infants, and children with congenital heart disease and may have important implications for a number of pulmonary and systemic vascular disorders.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported in part by Grants HL-60190 (to S. M. Black), HL-67841 (to S. M. Black), HL-72123 (to S. M. Black), HL-70061 (to S. M. Black), HD-047349 (to P. Oishi), and HL-61284 (to J. R. Fineman), all from National Institutes of Health, and a Transatlantic Network Development Grant from the LeDuq Foundation (to S. M. Black and J. R. Fineman).


   ACKNOWLEDGMENTS
 
We thank Michael Johengen for his expert technical assistance in the completion of this study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. R. Fineman, Dept. of Pediatrics, Univ. of California, San Francisco, 505 Parnassus Ave., Box 0106, San Francisco, CA 94143-0106 (e-mail: jeff.fineman{at}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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abman SH and Accurso FJ. Acute effects of partial compression of ductus arteriosus on fetal pulmonary circulation. Am J Physiol Heart Circ Physiol 257: H626–H634, 1989.[Abstract/Free Full Text]
  2. Apostolopoulou SC, Rammos S, Kyriakides ZS, Webb DJ, Johnston NR, Cokkinos DV, and Kremastinos DT. Acute endothelin A receptor antagonism improves pulmonary and systemic haemodynamics in patients with pulmonary arterial hypertension that is primary or autoimmune and related to congenital heart disease. Heart 89: 1221–1226, 2003.[Abstract/Free Full Text]
  3. Arai H, Hori S, Aramori I, Ohkubo H, and Nakanishi S. Cloning and expression of a cDNA encoding an endothelin receptor. Nature 348: 730–732, 1990.[CrossRef][Medline]
  4. Austin EH III. Postoperative management after the Norwood procedure. Semin Thorac Cardiovasc Surg Pediatr Card Surg Annu 1: 109–122, 1998.[Medline]
  5. Barnea O, Austin EH, Richman B, and Santamore WP. Balancing the circulation: theoretic optimization of pulmonary/systemic flow ratio in hypoplastic left heart syndrome. J Am Coll Cardiol 24: 1376–1381, 1994.[Abstract]
  6. Beghetti M, Silkoff PE, Caramori M, Holtby HM, Slutsky AS, and Adatia I. Decreased exhaled nitric oxide may be a marker of cardiopulmonary bypass-induced injury. Ann Thorac Surg 66: 532–534, 1998.[Abstract/Free Full Text]
  7. Black SM, Bekker JM, Johengen MJ, Parry AJ, Soifer SJ, and Fineman JR. Altered regulation of the ET-1 cascade in lambs with increased pulmonary blood flow and pulmonary hypertension. Pediatr Res 47: 97–106, 2000.[ISI][Medline]
  8. Black SM, Heidersbach RS, McMullan DM, Bekker JM, Johengen MJ, and Fineman JR. Inhaled nitric oxide inhibits NOS activity in lambs: potential mechanism for rebound pulmonary hypertension. Am J Physiol Heart Circ Physiol 277: H1849–H1856, 1999.[Abstract/Free Full Text]
  9. Black SM, Johengen MJ, and Soifer SJ. Coordinated regulation of genes of the nitric oxide and endothelin pathways during the development of pulmonary hypertension in fetal lambs. Pediatr Res 44: 821–830, 1998.[ISI][Medline]
  10. Black SM, Mata-Greenwood E, Dettman RW, Ovadia B, Fitzgerald RK, Reinhartz O, Thelitz S, Steinhorn RH, Gerrets R, Hendricks-Munoz K, Ross GA, Bekker JM, Johengen MJ, and Fineman JR. Emergence of smooth muscle cell endothelin B-mediated vasoconstriction in lambs with experimental congenital heart disease and increased pulmonary blood flow. Circulation 108: 1646–1654, 2003.[Abstract/Free Full Text]
  11. Budhiraja R, Tuder RM, and Hassoun PM. Endothelial dysfunction in pulmonary hypertension. Circulation 109: 159–165, 2004.[Free Full Text]
  12. Bush PA, Gonzalez NE, and Ignarro LJ. Biosynthesis of nitric oxide and citrulline from L-arginine by constitutive nitric oxide synthase present in rabbit corpus cavernosum. Biochem Biophys Res Commun 186: 308–314, 1992.[CrossRef][ISI][Medline]
  13. Celermajer DS, Cullen S, and Deanfield JE. Impairment of endothelium-dependent pulmonary artery relaxation in children with congenital heart disease and abnormal pulmonary hemodynamics. Circulation 87: 440–446, 1993.[Abstract/Free Full Text]
  14. Davignon J and Ganz P. Role of endothelial dysfunction in atherosclerosis. Circulation 109: III27–III32, 2004.
  15. De Oliveira NC and Van Arsdell GS. Practical use of alpha blockade strategy in the management of hypoplastic left heart syndrome following stage one palliation with a Blalock-Taussig shunt. Semin Thorac Cardiovasc Surg Pediatr Card Surg Annu 7: 11–15, 2004.[Medline]
  16. Fineman JR, Heymann MA, and Soifer SJ. N{omega}-nitro-L-arginine attenuates endothelium-dependent pulmonary vasodilation in lambs. Am J Physiol Heart Circ Physiol 260: H1299–H1306, 1991.[Abstract/Free Full Text]
  17. Fineman JR, Soifer SJ, and Heymann MA. Regulation of pulmonary vascular tone in the perinatal period. Annu Rev Physiol 57: 115–134, 1995.[CrossRef][ISI][Medline]
  18. Fitzgerald RK, Oishi P, Ovadia B, Ross GA, Reinhartz O, Johengen MJ, and Fineman JR. Tezosentan, a combined parenteral endothelin receptor antagonist, produces pulmonary vasodilation in lambs with acute and chronic pulmonary hypertension. Pediatr Crit Care Med 5: 571–577, 2004.[CrossRef][Medline]
  19. Fratz S, Geiger R, Kresse H, Roemer G, Hennig M, Sebening W, and Hess J. Pulmonary blood pressure, not flow, is associated with net endothelin-1 production in the lungs of patients with congenital heart disease and normal pulmonary vascular resistance. J Thorac Cardiovasc Surg 126: 1724–1729, 2003.[Abstract/Free Full Text]
  20. Fukuroda T, Fujikawa T, Ozaki S, Ishikawa K, Yano M, and Nishikibe M. Clearance of circulating endothelin-1 by ETB receptors in rats. Biochem Biophys Res Commun 199: 1461–1465, 1994.[CrossRef][ISI][Medline]
  21. Giaid A and Saleh D. Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med 333: 214–221, 1995.[Abstract/Free Full Text]
  22. Giaid A, Yanagisawa M, Langleben D, Michel RP, Levy R, Shennib H, Kimura S, Masaki T, Duguid WP, and Stewart DJ. Expression of endothelin-1 in the lungs of patients with pulmonary hypertension. N Engl J Med 328: 1732–1739, 1993.[Abstract/Free Full Text]
  23. Govers R and Rabelink TJ. Cellular regulation of endothelial nitric oxide synthase. Am J Physiol Renal Physiol 280: F193–F206, 2001.[Abstract/Free Full Text]
  24. Hoffman GM, Tweddell JS, Ghanayem NS, Mussatto KA, Stuth EA, Jaquis RD, and Berger S. Alteration of the critical arteriovenous oxygen saturation relationship by sustained afterload reduction after the Norwood procedure. J Thorac Cardiovasc Surg 127: 738–745, 2004.[Abstract/Free Full Text]
  25. Ignarro LJ, Ross G, and Tillisch J. Pharmacology of endothelium-derived nitric oxide and nitrovasodilators. West J Med 154: 51–62, 1991.[ISI][Medline]
  26. Ivy DD, le Cras TD, Parker TA, Zenge JP, Jakkula M, Markham NE, Kinsella JP, and Abman SH. Developmental changes in endothelin expression and activity in the ovine fetal lung. Am J Physiol Lung Cell Mol Physiol 278: L785–L793, 2000.[Abstract/Free Full Text]
  27. Luscher TF, Yang Z, Tschudi M, von Segesser L, Stulz P, Boulanger C, Siebenmann R, Turina M, and Buhler FR. Interaction between endothelin-1 and endothelium-derived relaxing factor in human arteries and veins. Circ Res 66: 1088–1094, 1990.[Abstract/Free Full Text]
  28. Macarthur H, Warner TD, Wood EG, Corder R, and Vane JR. Endothelin-1 release from endothelial cells in culture is elevated both acutely and chronically by short periods of mechanical stretch. Biochem Biophys Res Commun 200: 395–400, 1994.[CrossRef][ISI][Medline]
  29. McMullan DM, Bekker JM, Johengen MJ, Hendricks-Munoz K, Gerrets R, Black SM, and Fineman JR. Inhaled nitric oxide-induced rebound pulmonary hypertension: role for endothelin-1. Am J Physiol Heart Circ Physiol 280: H777–H785, 2001.[Abstract/Free Full Text]
  30. Nakano T, Kado H, Shiokawa Y, Fukae K, Nishimura Y, Miyamoto K, Tanoue Y, Tatewaki H, and Fusazaki N. The low resistance strategy for the perioperative management of the Norwood procedure. Ann Thorac Surg 77: 908–912, 2004.[Abstract/Free Full Text]
  31. Ovadia B, Bekker JM, Fitzgerald RK, Kon A, Thelitz S, Johengen MJ, Hendricks-Munoz K, Gerrets R, Black SM, and Fineman JR. Nitric oxide-endothelin-1 interactions after acute ductal constriction in fetal lambs. Am J Physiol Heart Circ Physiol 282: H862–H871, 2002.[Abstract/Free Full Text]
  32. Palmer RM, Ashton DS, and Moncada S. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 333: 664–666, 1988.[CrossRef][Medline]
  33. Papapetropoulos A, Rudic RD, and Sessa WC. Molecular control of nitric oxide synthases in the cardiovascular system. Cardiovasc Res 43: 509–520, 1999.[Abstract/Free Full Text]
  34. Pearl JM, Nelson DP, Wellmann SA, Raake JL, Wagner CJ, McNamara JL, and Duffy JY. Acute hypoxia and reoxygenation impairs exhaled nitric oxide release and pulmonary mechanics. J Thorac Cardiovasc Surg 119: 931–938, 2000.[Abstract/Free Full Text]
  35. Pritchard KA, Jr, Ackerman AW, Gross ER, Stepp DW, Shi Y, Fontana JT, Baker JE, and Sessa WC. Heat shock protein 90 mediates the balance of nitric oxide and superoxide anion from endothelial nitric-oxide synthase. J Biol Chem 276: 17621–17624, 2001.[Abstract/Free Full Text]
  36. Reddy VM, Hendricks-Munoz KD, Rajasinghe HA, Petrossian E, Hanley FL, and Fineman JR. Post-cardiopulmonary bypass pulmonary hypertension in lambs with increased pulmonary blood flow. A role for endothelin 1. Circulation 95: 1054–1061, 1997.[Abstract/Free Full Text]
  37. Reddy VM, Wong J, Liddicoat JR, Johengen M, Chang R, and Fineman JR. Altered endothelium-dependent responses in lambs with pulmonary hypertension and increased pulmonary blood flow. Am J Physiol Heart Circ Physiol 271: H562–H570, 1996.[Abstract/Free Full Text]
  38. Ricciardolo FL, Sterk PJ, Gaston B, and Folkerts G. Nitric oxide in health and disease of the respiratory system. Physiol Rev 84: 731–765, 2004.[Abstract/Free Full Text]
  39. Sakurai T, Yanagisawa M, Takuwa Y, Miyazaki H, Kimura S, Goto K, and Masaki T. Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature 348: 732–735, 1990.[CrossRef][Medline]
  40. Shaul PW. Regulation of endothelial nitric oxide synthase: location, location, location. Annu Rev Physiol 64: 749–774, 2002.[CrossRef][ISI][Medline]
  41. Sheehy AM, Burson MA, and Black SM. Nitric oxide exposure inhibits endothelial NOS activity but not gene expression: a role for superoxide. Am J Physiol Lung Cell Mol Physiol 274: L833–L841, 1998.[Abstract/Free Full Text]
  42. Sherman TS, Chen Z, Yuhanna IS, Lau KS, Margraf LR, and Shaul PW. Nitric oxide synthase isoform expression in the developing lung epithelium. Am J Physiol Lung Cell Mol Physiol 276: L383–L390, 1999.[Abstract/Free Full Text]
  43. Shetty SS, Okada T, Webb RL, DelGrande D, and Lappe RW. Functionally distinct endothelin B receptors in vascular endothelium and smooth muscle. Biochem Biophys Res Commun 191: 459–464, 1993.[CrossRef][ISI][Medline]
  44. Steinhorn RH and Fineman JR. The pathophysiology of pulmonary hypertension in congenital heart disease. Artif Organs 23: 970–974, 1999.[CrossRef][ISI][Medline]
  45. Tweddell JS and Hoffman GM. Postoperative management in patients with complex congenital heart disease. Semin Thorac Cardiovasc Surg Pediatr Card Surg Annu 5: 187–205, 2002.[CrossRef][Medline]
  46. Tworetzky W, Moore P, Bekker JM, Bristow J, Black SM, and Fineman JR. Pulmonary blood flow alters nitric oxide production in patients undergoing device closure of atrial septal defects. J Am Coll Cardiol 35: 463–467, 2000.[Abstract/Free Full Text]
  47. Wedgwood S, McMullan DM, Bekker JM, Fineman JR, and Black SM. Role for endothelin-1-induced superoxide and peroxynitrite production in rebound pulmonary hypertension associated with inhaled nitric oxide therapy. Circ Res 89: 357–364, 2001.[Abstract/Free Full Text]
  48. Wong J, Vanderford PA, Fineman JR, Chang R, and Soifer SJ. Endothelin-1 produces pulmonary vasodilation in the intact newborn lamb. Am J Physiol Heart Circ Physiol 265: H1318–H1325, 1993.[Abstract/Free Full Text]
  49. Wong J, Vanderford PA, Fineman JR, and Soifer SJ. Developmental effects of endothelin-1 on the pulmonary circulation in sheep. Pediatr Res 36: 394–401, 1994.[ISI][Medline]
  50. Xuan YT, Whorton AR, Shearer-Poor E, Boyd J, and Watkins WD. Determination of immunoreactive endothelin in medium from cultured endothelial cells and human plasma. Biochem Biophys Res Commun 164: 326–332, 1989.[CrossRef][ISI][Medline]
  51. Yanagisawa M, Kurihara H, Kimura S, Tomobe Y, Kobayashi M, Mitsui Y, Yazaki Y, Goto K, and Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332: 411–415, 1988.[CrossRef][Medline]
  52. Zhang W, Kuncewicz T, Yu ZY, Zou L, Xu X, and Kone BC. Protein-protein interactions involving inducible nitric oxide synthase. Acta Physiol Scand 179: 137–142, 2003.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
P.-H. Rolland, P. de Lagausie, E. Stathopoulos, O. Lepretre, G. Viudes, G. Gorincour, G. Hery, C. de Magnee, O. Paut, and J.-M. Guys
Phasic hemodynamics and reverse blood flows in the ao