Endothelial alterations during inhaled NO in lambs with pulmonary hypertension: implications for rebound hypertension

Gregory A. Ross,1,* Peter Oishi,1,* Anthony Azakie,2 Sohrab Fratz,1 Robert K. Fitzgerald,1 Michael J. Johengen,1 Cynthia Harmon,1 Karen Hendricks-Munoz,3 Jie Xu,4 Stephen M. Black,4 and Jeffrey R. Fineman1,5

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

Submitted 20 April 2004 ; accepted in final form 30 August 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Clinically significant increases in pulmonary vascular resistance (PVR) have been noted upon acute withdrawal of inhaled nitric oxide (iNO). Previous studies in the normal pulmonary circulation demonstrate that iNO increases endothelin-1 (ET-1) levels and decreases endogenous nitric oxide synthase (NOS) activity, implicating an endothelial etiology for the increase in resistance upon iNO withdrawal. However, the effect of iNO on endogenous endothelial function in the clinically relevant pulmonary hypertensive circulation is unknown. The objective of this study was to determine the effects of iNO on endogenous NO-cGMP and ET-1 signaling in lambs with preexisting pulmonary hypertension secondary to increased pulmonary blood flow. Eight fetal lambs underwent in utero placement of an aortopulmonary vascular graft (shunt lambs). After delivery (4 wk), the shunt lambs were mechanically ventilated with iNO (40 ppm) for 24 h. After 24 h of inhaled NO, plasma ET-1 levels increased by 34.8% independently of changes in protein levels (P < 0.05). Contrary to findings in normal lambs, total NOS activity did not decrease during iNO. In fact, Western blot analysis demonstrated that tissue endothelial NOS protein levels decreased by 43% such that NOS activity relative to protein levels actually increased during iNO (P < 0.05). In addition, the {beta}-subunit of soluble guanylate cyclase decreased by 70%, whereas phosphodiesterase 5 levels were unchanged (P < 0.05). Withdrawal of iNO was associated with an acute increase in PVR, which exceeded baseline PVR by 45%, and a decrease in cGMP concentrations to levels that were below baseline. These data suggest that the endothelial response to iNO and the potential mechanisms of rebound pulmonary hypertension are dependent upon the underlying pulmonary vasculature.

endothelium-derived factors; pulmonary heart disease; nitric oxide; endothelin-1


EXOGENOUSLY ADMINISTERED INHALED NO is currently utilized as an adjuvant therapy for a number of pulmonary hypertensive disorders, including persistent pulmonary hypertension of the newborn (PPHN) and perioperative pulmonary hypertension after repair of congenital heart disease (4, 12, 14). One of the more important issues regarding inhaled NO therapy is the safety of acute withdrawal. Several studies have noted a potentially life-threatening increase in pulmonary vascular resistance upon acute withdrawal of inhaled NO (1, 15). This "rebound pulmonary hypertension" is manifested by an increase in pulmonary vascular resistance, compromised cardiac output, and/or severe hypoxemia (1, 15). Recent data in normal animals demonstrate that exogenous NO exposure inhibits endogenous endothelial nitric oxide synthase (eNOS) activity and increases plasma endothelin (ET)-1 levels (8, 24). In addition, in vitro data suggest that the decrease in NOS activity during inhaled NO is mediated by an ET-1-induced increase in superoxide production, which results in preoxynitrite formation and the subsequent nitration and inactivation of eNOS (34, 42).

These studies in the normal pulmonary vasculature suggest that alterations in endogenous endothelial function during inhaled NO exposure mediate the rebound pulmonary hypertension associated with its acute withdrawal. However, clinically, inhaled NO is most often administered to patients with pulmonary vascular disorders that have associated preexisting alterations in endogenous pulmonary vascular endothelial function. For example, alterations in both endogenous NO-cGMP and ET-1 signaling have been demonstrated in newborns with persistent pulmonary hypertension and infants and children with increased pulmonary blood flow secondary to congenital heart disease (13, 22, 45). The potential effect of inhaled NO on endogenous endothelial function in the clinically relevant altered pulmonary vasculature has not been studied.

Previously, we developed a model of congenital heart disease with increased pulmonary blood flow in the lamb utilizing in utero placement of an aortopulmonary vascular graft (28). At 4 wk of age, these lambs have a mean pulmonary arterial pressure that is 35–75% of systemic values, a pulmonary-to-systemic blood flow ratio of ~2.5:1, and morphometric abnormalities of the pulmonary vasculature that include medial hypertrophy, abnormal extension of muscle to the periphery, and increased vessel density. In addition, these lambs have altered endothelial function, which include impaired endothelium-dependent pulmonary vasodilation, increased NOS activity and eNOS gene expression, increased ET-1 levels, decreased ETB receptor-mediated pulmonary vasodilation and protein levels, and increased ETA receptor-mediated vasoconstriction and protein levels (6, 7, 29, 43).

We hypothesized that, compared with the normal vasculature, the endothelial response to inhaled NO exposure would be altered in the pulmonary hypertensive vasculature because of preexisting endothelial alterations. Therefore, the purpose of this study was to investigate the effects of inhaled NO on endogenous NO-cGMP and ET-1 signaling in the pulmonary hypertensive vasculature. To this end, inhaled NO (40 ppm) was administered to eight 4-wk-old shunt lambs for 24 h. To determine the effects of inhaled NO on endogenous NO-cGMP signaling, sequential plasma samples were taken for cGMP concentrations. In addition, sequential peripheral lung biopsies were taken for NOS activity determinations and protein determinations of eNOS, inducible NOS (iNOS), neuronal NOS (nNOS), soluble guanylate cyclase (sGC), and phosphodiesterase (PDE) 5 by Western blot analysis. To determine the effects of inhaled NO on endogenous ET-1 signaling, sequential plasma samples were taken for ET-1 concentrations, and sequential peripheral lung biopsies were taken for protein determinations of prepro-ET-1, endothelin converting enzyme (ECE)-1{alpha}, ETA receptors, and ETB receptors.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Surgical preparation. Twelve mixed-breed Western pregnant ewes (137–141 days gestation, term = 145 days) were operated on under sterile conditions with the use of local anesthesia (2% lidocaine hydrochloride) and inhalational anesthesia (1–3% isoflurane). A midline incision was made in the ventral abdomen, and the pregnant horn of the uterus was exposed. Through a small uterine incision, the left fetal forelimb and chest were exposed, and a left lateral thoracotomy was performed in the third intercostal space. Additional fetal anesthesia consisted of local anesthesia with 1% lidocaine hydrochloride and ketamine hydrochloride (20 mg im). With the use of side-biting vascular clamps, an 8.0-mm Gore-tex vascular graft (~2 mm length; Gore, Milpitas, CA) was anastomosed between the ascending aorta and main pulmonary artery with 7.0 proline (Ethicon, Somerville, NJ), using a continuous suture technique. The thoracotomy incision was then closed in layers. This procedure is previously described in detail (28). After surgery, antibiotics (1 million units penicillin G potassium and 100 mg gentamicin sulfate im) were administered to the ewe for 3 days. After spontaneous delivery, the lambs were weighed daily, and the respiratory rate and heart rate were obtained. Furosemide (1 mg/kg im) was administered daily. Elemental iron (50 mg im) was given weekly.

At 4 wk of age, the lambs were fasted for 24 h, with free access to water. The lambs were then anesthetized with ketamine hydrochloride (15 mg/kg im). Under additional local anesthesia with 1% lidocaine hydrochloride, polyurethane catheters were placed in an artery and vein of a hindleg. 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 Healthdyne pediatric time-cycled, pressure-limited ventilator. Pancuronium bromide (0.1 mg·kg–1·dose–1) was given intermittently for muscle relaxation. With the use of a strict aseptic technique, a midsternotomy incision was then performed, and the pericardium was incised. With the purse-string suture technique, polyurethane catheters were placed directly in 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 pulmonary blood flow. The midsternotomy incision was then temporarily closed with towel clamps. An intravenous infusion of Lactated Ringer 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, and every 8 h thereafter. The lambs were maintained normothermic (39°C) with a heating blanket.

Experimental protocol. After a 60-min recovery, 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 (pre-NO). Blood was collected from the femoral artery for plasma ET-1 and cGMP determinations, and a peripheral lung wedge biopsy was obtained for NOS activity and eNOS, iNOS, nNOS, sGC, PDE-5, prepro-ET-1, ECE-1, and ETA and ETB receptor protein determinations. A side-biting vascular clamp was used to isolate peripheral lung tissue from a randomly selected lobe, and the incision was cauterized. Approximately 300 mg peripheral lung were obtained for each biopsy.

In eight of the lambs, inhaled NO (40 ppm) was then delivered in nitrogen in the inspiratory limb of the ventilator (Inovent; Ohmeda, Liberty, NJ) and continued for 24 h. The inspired concentrations of NO and nitrogen dioxide were continuously quantified by electrochemical methodology (Inovent; Ohmeda). The hemodynamic variables were monitored continuously. Systemic arterial blood gases were determined intermittently, and ventilation was adjusted to achieve a PaCO2 between 35 and 45 Torr and a PaO2 >50 Torr. Sodium bicarbonate was administered intermittently to maintain a pH >7.30. Normal saline was administered intermittently to maintain stable atrial pressures throughout the study period. Peripheral lung wedge biopsies were performed, and blood was obtained after 24 h of therapy. The inhaled NO was then stopped, and the hemodynamic variables were monitored for two additional hours. Blood and lung tissue was obtained 120 min after discontinuation of inhaled NO. All blood losses were replaced with maternal blood (~15 ml/kg over the study period). Four additional shunt lambs underwent the identical protocol without inhaled NO administration.

At the end of the protocol, all lambs were killed with a lethal injection of pentobarbital sodium followed by bilateral thoracotomy, as described in the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals. All protocols and procedures were approved by the Committee on Animal Research of the University of California, San Francisco.

Measurements. Pulmonary and systemic arterial and right and left atrial pressures were measured using Sorenson Neonatal Transducers (Abbott Critical Care Systems, N. 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 multichannel electrostatic recorder (Gould, Cleveland, OH). Systemic arterial blood gases and pH were measured on a Radiometer ABL5 pH/blood gas analyzer (Radiometer, Copenhagen, Denmark). Hb concentration and oxygen saturation were measured by a hemoximeter (model 270; Ciba-Corning). Pulmonary vascular resistance was calculated using standard formulas. Body temperature was monitored continuously with a rectal temperature probe.

Plasma cGMP determinations. Plasma samples were assayed with a cGMP 125I RIA kit (Amersham International) according to the manufacturer's instructions. Cross-reactivity for other nucleotides is <0.001.

Assay for NOS activity. This was performed using the conversion of [3H]arginine to [3H]citrulline as a measure of NOS activity, essentially as described by Bush et al. (11). 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 units 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 NG-nitro-L-arginine methyl ester. 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) and then applied to columns containing 1 ml Dowex AG50W-X8 resin, Na+ form, preequilibrated with 1 N NaOH. [3H]citrulline was then quantitated by scintillation counting.

Plasma ET-1 determinations. Systemic arterial blood (4 ml) was 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. 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 (44).

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 (6, 7, 9, 24, 34, 42). 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 and ETB receptors, sGC, and PDE5, and 4–20% denaturing polyacrylamide gradient gels for prepro-ET-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, nNOS, 1:1,000 for ECE-1{alpha}, 1:1,000 for ETA and ETB, 1:500 for prepro-ET-1, 1:1,000 for the {alpha}1-subunit of guanylate cyclase, 1:10,000 for the {beta}1-subunit of guanylate cyclase, or 1:2,000 for PDE5), washed with TBS containing 0.1% Tween, and then incubated with a either a goat anti-mouse IgG-horseradish peroxidase conjugate (for eNOS and prepro-ET-1) or a goat anti-rabbit IgG-horseradish peroxidase conjugate (for ECE-1{alpha} and ETA and ETB receptors). After being washed, the protein bands were visualized with chemiluminescence using a Kodak Digital Science Image Station (NEN) and analyzed using KED-1 software. All captured and analyzed images were determined to be in the dynamic range of the system.

The eNOS and iNOS antiserum was obtained from Transduction Laboratories (Lexington, KY). The ETA receptor antiserum was generated as previously described (6). The nNOS antiserum was prepared as previously described (34). The ETB receptor antiserum was obtained from Maine Biotechnology Services (Portland, ME). The prepro-ET-1 antibody was obtained from Affinity Bioreagents (Golden, CO). The specificity of the prepro-ET-1 antibody was verified with a preincubation step with purified ET-1 (50 ng ET-1/15 µl antiserum) protein. The purified ET-1 was purchased from Sigma (St. Louis, MO). ECE-1{alpha} antiserum was generated as previously described (24). The antiserum for the {alpha}1-subunit and the {beta}1-subunit of guanylate cyclase was a gift from Dr. Peter Yuen, and the antiserum for PDE5 was a gift from Dr. Stefan Janssens, University Hospital Gasthuisberg, Lueven, Belgium.

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.

Statistical analysis. The means ± SD and SE were calculated for the baseline hemodynamic variables, systemic arterial blood gases and pH, and plasma cGMP and ET-1 concentrations. The general hemodynamic variables, systemic arterial blood gases and pH, and cGMP and ET-1 concentrations were compared over time by ANOVA for repeated measures. 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, Mt. View, CA). Band intensities from Western blot analysis were analyzed densitometrically on a Macintosh computer (model 9500; Apple Computer, Cupertino, CA) using the public domain NIH Image program (developed at the National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image). For Western blot analysis, to ensure equal protein loading, duplicate polyacrylamide gels were run. One was stained with Coomassie blue. In the expected molecular weight range of each protein of interest, the density of the Coomassie blue bands was determined and used to normalize the Western blot band intensities. The means ± SE were calculated for the relative protein at each time point after the start of inhaled NO therapy. Comparisons over time were made by the paired t-test. P < 0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In shunt lambs, inhaled NO (40 ppm) rapidly decreased mean pulmonary arterial pressure (P < 0.05). Left pulmonary blood flow, left pulmonary vascular resistance, mean systemic arterial pressure, heart rate, right and left atrial pressures, and systemic arterial blood gases and pH were all unchanged. During the 24-h treatment course, pulmonary arterial pressure remained below pre-NO values. Systemic arterial pressure, pulmonary blood flow, heart rate, and systemic arterial PO2 all decreased slightly, whereas left and right atrial pressures increased (Table 1). Upon discontinuation of inhaled NO, there was a rapid increase in both mean pulmonary arterial pressure and left pulmonary vascular resistance (P < 0.05; Table 1 and Fig. 1). Left pulmonary blood flow, mean systemic arterial pressure, heart rate, left and right atrial pressures, and systemic arterial PO2, PCO2, and pH remained unchanged from 24-h NO values (Table 1). Pulmonary vascular resistance remained above pre-NO values for the remaining 2-h study period (Fig. 1).


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Table 1. Hemodynamic changes during and after 24 h of inhaled NO in shunt lambs

 


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Fig. 1. Changes in left pulmonary vascular resistance before, during, and after 24 h of inhaled nitric oxide (NO; 40 ppm) therapy; n = 8 shunt lambs. Values are means ± SE. *P < 0.05 vs. 0 h (ANOVA). LPVR, left pulmonary vascular resistance.

 
To determine the effects of inhaled NO on endogenous NO-cGMP signaling, we determined plasma cGMP concentrations, total NOS activity, and eNOS, iNOS, nNOS, sGC, and PDE5 protein levels. We found that plasma cGMP levels increased during inhaled NO, but rapidly decreased after NO withdrawal and remained below pre-NO values for the remaining 2-h study period (Fig. 2). As opposed to our findings in normal lambs, total NOS activity did not decrease in shunt lambs during NO exposure. In fact, eNOS protein levels decreased by 43% such that NOS activity relative to protein levels actually increased during inhaled NO by 227% (P < 0.05). These changes returned to pre-NO values 2 h after NO withdrawal (Fig. 3). iNOS and nNOS protein levels were not detectable during the study period. The {alpha}-sGC subunit and PDE5 protein levels were unchanged during inhaled NO, but the {beta}-sGC subunit decreased by 70% (P < 0.05; Fig. 4).



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Fig. 2. Changes in plasma cGMP concentrations before, during, and after 24 h of inhaled NO (40 ppm) therapy; n = 8 shunt lambs. Values are means ± SE. *P < 0.05 vs. pre-NO.

 


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Fig. 3. A: changes in total nitric oxide synthase (NOS) activity during inhaled NO in shunt lambs. Values are means ± SE; n = 8. NOS activity is unchanged during inhaled NO exposure. B: Western blot analysis for endothelial NOS (eNOS) protein in lung tissue before and after 24 h of inhaled NO (40 ppm) therapy. Top: representative Western blot. Bottom: densitometric values for eNOS protein. Values are means ± SE; n = 8. eNOS protein expression is decreased during inhaled NO therapy. C: NOS activity relative to eNOS protein changes during inhaled NO. Because inducible NOS (iNOS) and neuronal NOS (nNOS) protein was undetectable, eNOS protein values represent total protein. Values are normalized to pre-NO values. Values are means ± SE; n = 8. NOS activity relative to protein levels increases during inhaled NO. *P < 0.05 vs. pre-NO.

 


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Fig. 4. A: Western blot analysis for the {alpha}1-subunit of soluble guanylate cyclase (sGC) protein in lung tissue from shunt lambs before and after inhaled NO. Top: representative Western blot. Bottom: densitometric values for {alpha}1-sGC normalized to pre-NO. Values are means ± SE; n = 6. sGC {alpha}1-protein expression is unchanged during inhaled NO therapy. B: Western blot analysis for the {beta}1-subunit of sGC protein in lung tissue from shunt lambs before and after inhaled NO. Top: representative Western blot. Bottom: densitometric values for {beta}1-sGC normalized to pre-NO. Values are means ± SE; n = 6. *P < 0.05. sGC {beta}1-protein expression is decreased during inhaled NO therapy. C: Western blot analysis for phosphodiesterase (PDE) 5 protein in lung tissue from shunt lambs before and after inhaled NO. Top: representative Western blot. Bottom: densitometric values for PDE5 normalized to pre-NO. Values are means ± SE; n = 6. PDE5 protein expression is unchanged during inhaled NO therapy.

 
To determine the effects of inhaled NO on endogenous ET-1 production, we determined plasma ET-1 concentrations and lung protein levels. We found that plasma ET-1 concentrations were increased after 24 h of inhaled NO but returned to pre-NO values 2 h after discontinuation (P < 0.05; Fig. 5). In addition, Western blot analysis demonstrated no change in prepro- ET-1, ECE-1{alpha}, ETA receptor, or ETB receptor protein levels throughout the study period (Fig. 6).



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Fig. 5. Changes in plasma endothelin (ET)-1 concentrations before, during, and after 24 h of inhaled NO (40 ppm) therapy; n = 8 shunt lambs. Values are means ± SE. *P < 0.05 vs. pre-NO.

 


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Fig. 6. A: Western blot analysis for prepro-ET-1 protein in lung tissue from shunt lambs before and after inhaled NO. Top: representative Western blot. Bottom: densitometric values for prepro-ET-1 normalized to pre-NO. Values are means ± SE; n = 7. Prepro-ET-1 protein expression is unchanged during inhaled NO therapy. B: Western blot analysis for ECE-1{alpha} protein in lung tissue from shunt lambs before and after inhaled NO. Top: representative Western blot. Bottom: densitometric values for endothelin converting enzyme (ECE)-1{alpha} normalized to pre-NO. Values are means ± SE; n = 7. *P < 0.05. ECE-1{alpha} protein expression is unchanged during inhaled NO therapy. C: Western blot analysis for ETA receptor protein in lung tissue from shunt lambs before and after inhaled NO. Top: representative Western blot. Bottom: densitometric values for ETA receptor normalized to pre-NO. Values are means ± SE; n = 7. ETA receptor protein expression is unchanged during inhaled NO therapy. D: Western blot analysis for ETB receptor protein in lung tissue from shunt lambs before and after inhaled NO. Top: representative Western blot. Bottom: densitometric values for ETB receptor normalized to pre-NO. Values are means ± SE; n = 7. ETB receptor protein expression is unchanged during inhaled NO therapy.

 
In four additional shunt lambs, 24 h of mechanical ventilation alone, without inhaled NO therapy, did not change mean pulmonary arterial pressure. Similar to NO-treated lambs, mean systemic arterial pressure, left pulmonary blood flow, and heart rate decreased (Table 2; P < 0.05). Left and right atrial pressures and systemic arterial blood gases and pH were not changed (Table 2). Plasma cGMP levels, tissue NOS activity, and eNOS, iNOS, and nNOS protein levels were unchanged. In addition, plasma ET-1 levels were unchanged after 24 h of ventilation without inhaled NO. This was associated with no changes in prepro-ET-1, ECE-1{alpha}, ETA receptor, or ETB receptor protein levels (data not shown).


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Table 2. Hemodynamic changes during and after 24 h of ventilation without inhaled NO

 
The lambs required 1–3 meq/kg bicarbonate during the study period to maintain normal acid-base status and 30–50 ml/kg volume to maintain atrial pressures. There were no differences between NO-treated lambs and vehicle-treated lambs in the amount of volume, buffer, or ventilatory support required.


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Exogenous inhaled NO is increasingly utilized as an adjunct therapy for pediatric pulmonary hypertension disorders, including infants and children with congenital heart disease (4). It produces potent, selective pulmonary vasodilation that is independent of endothelial cell function. Therefore, it has been employed in the preoperative assessment of pulmonary hypertension, the perioperative management of pulmonary hypertension, and the postoperative assessment of pulmonary hypertension (2, 4, 5). Although many studies demonstrate a clear benefit in patient outcome with inhaled NO use, several safety concerns remain, including unpredictable and nonsustained responses to inhaled NO and a clinically significant rapid increase in pulmonary vascular resistance upon its acute withdrawal (1, 1517). Recent data suggest that alterations in endogenous endothelial function mediate this rebound pulmonary hypertension. Indeed, recent in vitro and in vivo animal data in the normal pulmonary circulation suggest that exogenous NO decreases endogenous NOS activity via an ET-1-mediated increase in superoxide production. Superoxide then binds to available NO, resulting in peroxynitrite production, which phosphorylates and inactivates NOS (8, 24, 34, 42). The net result of these changes, increased ET-1 activity and decreased NOS activity, may mediate the clinically significant increases in pulmonary vascular resistance noted upon inhaled NO withdrawal. However, the potential effects of inhaled NO on endogenous NO-cGMP and ET-1 signaling have not been studied previously in the pulmonary hypertensive circulation. In the present study, we found that, similar to normal lambs, plasma levels of ET-1 increased in shunt lambs during inhaled NO independent of changes in protein levels (24). However, as opposed to normal lambs in which NOS activity decreased and eNOS protein levels were unchanged during NO exposure, NOS activity did not change in shunt lambs, and eNOS protein levels decreased, suggesting a posttranslational increase in NOS activity (8). These data suggest that the effects of inhaled NO on endogenous endothelial function, and the potential mechanisms of the rebound pulmonary hypertension associated with NO withdrawal, are dependent upon the preexisting status of the pulmonary circulation.

Although initially considered to be a constitutively expressed enzyme, an increasing body of literature demonstrates that eNOS is dynamically regulated at the transcriptional and posttranslational levels (20, 25). For example, laminar shear stress increases eNOS transcription, whereas factors such as intracellular location, protein-protein interactions (e.g., calmodulin, caveolin, and heat shock protein 90), phosphorylation, oxidant stress, and substrate and cofactor availability may all dynamically regulate eNOS activity (20, 25, 27, 33). The effect of exogenous NO exposure on endogenous NO gene expression has been previously studied in vitro and has yielded conflicting results. For example, in fetal intrapulmonary vascular endothelial cells, exposure to NO donor compounds increased eNOS protein and mRNA levels, whereas in pulmonary endothelial cells isolated from the fetal main pulmonary artery, exposure to NO donors did not change eNOS protein or mRNA (34, 46). In the normal 4-wk-old lamb, we have previously demonstrated that eNOS protein levels were unchanged during inhaled NO exposure (8). However, in the present study inhaled NO significantly decreases eNOS protein levels in 4-wk-old shunt lambs. The disparity in the regulation in eNOS gene expression with NO exposure may be explained, in part, by differences in basal eNOS protein and mRNA levels between normal and shunt lambs. For example, we have previously demonstrated that, under conditions of high pulmonary blood flow and pressure, shunt lambs have increased eNOS protein and mRNA levels compared with normal control lambs (7). We speculate that these conditions alter the regulatory response of eNOS to exogenous NO. However, these and potential other regulatory mechanisms will require further investigation.

Previously, we have also demonstrated a posttranslational decrease in NOS activity during NO exposure in the normal lamb secondary to ET-1-mediated superoxide production and subsequent peroxynitrite production (8). However, in the present study, total NOS activity was unchanged during inhaled NO. In fact, relative to the decrease in eNOS protein, NOS activity actually increased during NO exposure. Because total NOS activity may represent changes in eNOS, iNOS, and nNOS activity, we determined iNOS and nNOS protein levels and found no detectable changes in either during the treatment protocol, suggesting that the preservation of NOS activity represented a posttranslational increase in total NOS activity. We have previously demonstrated that baseline NOS activity is significantly increased in shunt lambs compared with controls (7). In addition, we have previously demonstrated that several known regulators of NOS activity, such as oxidant stress and cofactor availability, are altered in shunt lambs. For example, shunt lambs have decreased plasma levels of L-arginine (the substrate for NOS) and increased superoxide levels (28, 36). We speculate that these altered baseline conditions secondary to the chronic stimulus of increased flow and pressure have altered the posttranslational response of NOS to exogenous NO in shunt lambs. The exact mechanisms for these differences are unclear and warrant further study.

NO induces vasodilation by activating sGC. sGC is a heterodimeric enzyme comprising two subunits termed {alpha} and {beta} that generate cGMP from GTP (21). Although much focus has been placed on potential alterations in NOS expression in pulmonary hypertensive disorders, data also suggest that changes in sGC expression and activity may be important in the pathophysiology of pulmonary hypertension. For example, decreased sGC protein levels and activity have been demonstrated in animal models of PPHN (37). In vitro, this is associated with impaired pulmonary vasodilation in response to NO donors and may play a role in the decreased responsiveness of some newborns with PPHN (40). Decreased sGC gene expression and activity have also been implicated in other vascular disorders, including essential hypertension (30). Although little data are available on the regulation of sGC, limited in vitro studies suggest that exogenous NO does alter sGC expression and activity. For example, in rat medullary interstitial cells, NO donor compounds decreased mRNA levels of both {alpha}1- and {beta}1-sGC subunits (41). In addition, in rat pulmonary artery smooth muscle cells, exposure to NO donors induced a cGMP-dependent decrease in sGC mRNA and protein levels (18). Previously, we demonstrated that inhaled NO decreases pulmonary {alpha}1- and {beta}1-sGC protein levels in normal lambs (38). In the current study, inhaled NO decreased the {beta}1-subunit protein levels by 70%, whereas the {alpha}1-subunit protein levels were unchanged. At baseline, both the {alpha}1- and {beta}1-sGC subunits are upregulated in shunt lambs compared with age-matched controls (10). The potential mechanisms for the differential response to inhaled NO between shunt and control lambs may be secondary, in part, to these differences in basal regulation, but require further investigation.

Intracellular cGMP concentrations are not simply determined by the accumulation of cGMP, but rather by a balance between synthesis and degradation. Cyclic nucleotide PDEs are the enzymes responsible for cGMP degradation (3). In the mammalian lung, there are a number of PDEs, but the cGMP-specific PDE, PDE5, is prevalent, especially early in development (32). As with sGC, little is known about the regulation of PDE5 gene expression. However, developmental regulation and alterations in animal models of pulmonary hypertension have been demonstrated (31, 32). In this study, we have demonstrated that the expression of PDE5 is unchanged during inhaled NO therapy. During the 24-h study period, cGMP levels were increased despite a decrease in {beta}1-sGC protein levels. This is most likely secondary to the increase in enzyme substrate provided during inhaled NO treatment. However, the net effect of decreased sGC without a compensatory decrease in PDE5 will result in decreased cGMP levels after the withdrawal of exogenous NO. This decrease in cGMP levels below baseline values demonstrated in Fig. 2 may contribute to the physiological increase in pulmonary vascular resistance noted upon NO withdrawal.

Although the effects of NO exposure on ET-1 regulation in vitro have yielded conflicting results, previous reports in vivo have demonstrated that inhaled NO increases ET-1 levels. For example, in both children with pulmonary hypertension after cardiac surgery and normal 4-wk-old lambs, plasma ET-1 concentrations increased during inhaled NO exposure (24, 26). Similarly, in the current study, plasma ET-1 concentrations increased in lambs with preexisting increased pulmonary blood flow during inhaled NO exposure. Increases in plasma ET-1 levels may result from increases in ET-1 production, ET-1 release, and/or decreased ET-1 clearance. The production of ET-1 begins with the cleavage of the translational product prepro-ET-1 into a nonactive 38-amino-acid residue known as big ET-1. Big ET-1 is then cleaved into its functional form, ET-1, by the endopeptidase ECE-1 (39). ECE-1 exists in two isoforms, ECE-1{alpha} and ECE-1{beta}, with ECE-1{alpha} considered to be the most biologically important (35). Because many studies suggest that ET-1 production is regulated at the transcriptional level of prepro-ET-1 and/or ECE-1, we performed sequential lung biopsies to determine potential changes in prepro-ET-1 and ECE-1{alpha} protein levels. We found that both prepro-ET-1 and ECE-1{alpha} protein levels were unchanged during inhaled NO therapy, suggesting that the increased plasma concentrations are independent of changes in gene expression. In addition, the ETB receptor has been implicated in the clearance of ET-1 from the circulation, but we found no changes in protein levels of the ETB receptor during inhaled NO (19). Rapid ET-1 release from intracellular secretory granules has been demonstrated after such stimuli as cytokines and stretch (23). Therefore, the increase in plasma ET-1 induced by inhaled NO may represent an increase in ET-1 release. However, potential changes in ECE-1 activity, NO-induced displacement of ET-1 from its receptors, and/or potential changes in ETB binding affinity represent additional potential mechanisms that were not studied but warrant investigation. These findings are similar to those seen in our previous study in the normal pulmonary vasculature (24). However, as opposed to normal lambs, exogenous ET-1 induces potent pulmonary vasoconstriction in shunt lambs secondary to increased ETA receptor expression and/or decreased ETB receptor expression (6, 43). Therefore, a similar increase in plasma ET-1 levels in shunt lambs may have greater physiological significance than control lambs and suggests that ET-1-induced vasoconstriction may play a greater role in the increase in pulmonary vascular resistance after NO withdrawal in shunt lambs.

After the 24-h treatment period, mean systemic arterial pressure was decreased. The etiology of this hypotension is unclear. However, it was associated with a decrease in pulmonary blood flow and heart rate, suggesting a decrease in cardiac output secondary to long cumulative doses of anesthetics. Identical hemodynamic changes occurred in those lambs ventilated without inhaled NO, suggesting that these changes were independent of inhaled NO treatment. Furthermore, because nontreated lambs did not undergo the biochemical and protein changes noted in NO-treated lambs, despite having similar decreases in systemic arterial pressure, blood flow, and heart rate, the biochemical and protein changes were likely secondary to inhaled NO treatment and independent of these physiological changes.

Three limitations of the current study are noteworthy. Only one dose of inhaled NO (40 ppm) and one treatment duration (24 h) were studied. Further investigations are needed to determine the potential of different doses and treatment durations on endogenous ET-1. In addition, these studies were performed in 21% oxygen. Clinically, inhaled NO is most often administered with supplemental oxygen. We performed our studies in 21% oxygen to isolate the changes to NO, but further studies are warranted to determine the potential effect of supplemental oxygen. Last, these studies were performed in shunt lambs with the vascular graft left open. Although inhaled NO is used in the preoperative evaluation of pulmonary hypertension secondary to increased pulmonary blood flow, the majority of its use occurs in the postoperative period after pulmonary blood flow has been decreased by surgical repair (4). We did perform preliminary data that demonstrate a similar preservation of NOS activity when inhaled NO was initiated after graft closure (n = 2; data not shown). This suggests that the changes we observed are independent of graft patency and rather the chronic underlying endothelial function. However, further studies are warranted to better determine potential alterations in the endothelial response to inhaled NO when administered after normalization of pulmonary blood flow.

The present study is the first in vivo investigation of the effects of inhaled NO therapy on endogenous endothelial function in the pulmonary hypertensive circulation. We found that alterations in the ET-1 cascade induced by inhaled NO, increased plasma ET-1 levels that occurred independently of changes in protein levels, were similar to those found in the normal circulation. However, the response of endogenous eNOS to inhaled NO in the pulmonary hypertensive circulation differed from the response in the normal circulation. As opposed to a decrease in NOS activity without changes in protein levels, eNOS protein levels decreased during NO in shunt lambs, whereas activity increased from a presumed posttranslational modification. Similar to normal lambs, sGC decreased, PDE5 was unchanged during NO exposure, and cGMP concentrations decreased in shunt lambs after NO withdrawal. These data suggest that the decreases in cGMP levels are predominantly secondary to changes in sGC in shunt lambs as opposed to normal lambs where both decreases in NOS activity and sGC protein may contribute. In addition, the increases of ET-1 in shunt lambs likely have a greater primary role in inducing pulmonary vasoconstriction upon inhaled NO withdrawal in shunt lambs in which ETA receptor protein levels are increased, especially given the net increase in NOS activity in these lambs (6). We conclude that the endothelial response to inhaled NO therapy is dependent on the baseline endothelial function of the pulmonary vasculature. Therefore, the pathophysiology of rebound pulmonary hypertension may be different in the normal vs. the abnormal pulmonary circulation. A better understanding of these different mechanisms may lead to improved treatment strategies for rebound pulmonary hypertension and the preservation of endothelial function during chronic NO usage in pulmonary vascular disease states.


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This research was supported by National Heart, Lung, and Blood Institute Grants HL-61284 (J. R. Fineman), HL-60190 (S. M. Black), HL-067841 (S. M. Black), HL-072123 (S. M. Black), and HL-070061 (S. M. Black).


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. R. Fineman, Medical Center at UC San Francisco, 505 Parnassus Ave., Box 0106, 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.

* G. A. Ross and P. Oishi contributed equally to this work. Back


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